Methods for producing useful substances

Abstract

The present description discloses: a method for producing a peptide such as glutathione; and a microorganism which can be used in the method.　At least one embodiment of a first disclosure relates to a method for producing a peptide such as glutathione, the method comprising culturing a prokaryotic microorganism strain of which the expression amount of at least one gene selected from gshA gene, gshB gene and gshF gene is increased compared with that in a wild-type strain thereof in a culture medium in which the total concentration of cysteine and cystine is 0.5 g / L or less. A second disclosure relates to a microorganism in which γ-glutamyltransferase gene and glutathione reductase gene are deleted and the expression of gshA gene and gshB or the expression of gshF gene is enhanced.

Description

[Technical field]

[0001] One or more embodiments of the first disclosure relate to a method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione.

[0002] One or more further embodiments of the first disclosure relate to prokaryotic microbial strains capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione.

[0003] The second disclosure relates to a microorganism that produces glutathione and is deficient in the glutathione reductase gene, and a method for producing glutathione using the microorganism. [Background technology]

[0004] Glutathione is known to exist in reduced and oxidized forms. Reduced glutathione is a peptide consisting of three amino acids: L-cysteine, L-glutamic acid, and glycine. Oxidized glutathione is a compound in which the thiol groups of two molecules of reduced glutathione are disulfide-bonded. It is present not only in the human body but also in many other living organisms, including other animals, plants, and microorganisms, and is an important compound for living organisms, such as for eliminating active oxygen, detoxifying, and metabolizing amino acids. For this reason, it has attracted attention in the pharmaceutical, food, and cosmetics industries. In recent years, it has also been discovered that oxidized glutathione has the effect of promoting plant growth, and it is expected to be used in various fields, including agriculture.

[0005] In the body, glutathione exists in one of two forms: reduced glutathione (hereinafter sometimes referred to as "GSH"), in which the thiol group of the L-cysteine ​​residue is reduced to form SH, and oxidized glutathione (hereinafter sometimes referred to as "GSSG"), in which the thiol group of the L-cysteine ​​residue is oxidized to form a disulfide bond between two glutathione molecules.

[0006] Known methods for producing glutathione include a method of producing it by fermentation using yeast or Escherichia coli (Patent Document 1), and a method of producing it by enzymatically linking L-glutamic acid, L-cysteine, and glycine using microorganisms to produce γ-glutamylcysteine ​​synthetase or glutathione synthetase (Patent Documents 3 and 4).

[0007] For example, Patent Document 1 describes a method for producing glutathione, which is characterized by culturing in a medium a yeast having increased thiol oxidase activity compared to a parent strain to produce glutathione, and then recovering glutathione from the resulting culture medium.

[0008] Patent Document 2 also describes a method for producing glutathione or γ-glutamylcysteine, which comprises culturing in a medium a microorganism in which the activity of a protein having glutathione transport activity and the activity of a protein involved in the biosynthesis of glutathione or γ-glutamylcysteine ​​are higher than those of a parent strain, producing and accumulating glutathione or γ-glutamylcysteine ​​in the medium, and collecting glutathione or γ-glutamylcysteine ​​from the culture. Example 4 of Patent Document 2 describes that when an Escherichia coli strain in which the gshA gene, which is a glutamate-cysteine ​​ligase gene derived from Escherichia coli, and the gshB gene, which is a glutathione synthetase gene, are overexpressed in a medium supplemented with amino acids, the glutathione concentration in the medium was 160 mg / L.

[0009] Non-Patent Document 1 describes a method for producing glutathione by culturing Escherichia coli transformed with an expression vector containing the bifunctional glutathione synthetase gshF gene placed under the control of a constitutive promoter in a medium supplemented with L-cysteine, L-glutamic acid, and glycine, which are the constituent amino acids of glutathione. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication WO2016 / 140349 [Patent Document 2] International Publication WO2008 / 126784 [Patent Document 3] Japanese Patent Application Publication No. 60-27396 [Patent Document 4] Japanese Patent Publication No. 60-27397 [Non-patent literature]

[0011] [Non-Patent Document 1] Journal of Biotechnology (2018), https: / / doi.org / 10.1016 / j.jbiotec.2018.11.001 Summary of the Invention [Problem to be solved by the invention]

[0012] First, there is a need for new aspects of prokaryotic microbial strains capable of producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione, and methods for producing the above peptides using the same. Secondly, there is a need for a new embodiment of a microorganism capable of producing glutathione and a method for producing glutathione using the same. [Means for solving the problem]

[0013] The first disclosure of this specification includes the following embodiments (1) to (14).

[0014] (1) A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione, comprising the steps of: A method comprising culturing a prokaryotic microbial strain in which the expression level of one or more genes selected from a glutamate-cysteine ​​ligase gene, a glutathione synthetase gene, and a bifunctional glutathione synthetase gene is increased compared to a wild-type strain, in a medium having a total concentration of cysteine ​​and cystine of 0.5 g / L or less. (2) The method according to (1), wherein the prokaryotic microbial strain is capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione by inducible expression of the one or more genes. (3) A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione, comprising the steps of: The method includes culturing in a medium a prokaryotic microbial strain in which the expression level of one or more genes selected from a glutamate-cysteine ​​ligase gene, a glutathione synthetase gene, and a bifunctional glutathione synthetase gene is increased compared to a wild-type strain, The method does not include adding cysteine ​​or cystine to the medium. (4) The method according to (3), wherein the prokaryotic microbial strain is capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione by inducible expression of the one or more genes. (5) A prokaryotic microbial strain comprising one or more genes selected from a glutamate-cysteine ​​ligase gene, a glutathione synthetase gene, and a bifunctional glutathione synthetase gene, all of which are operably linked to a promoter, the promoter is a promoter that increases the transcription amount of the glutamate-cysteine ​​ligase gene by the prokaryotic microbial strain by 20 times or more compared to that of a wild-type strain when the one or more genes are glutamate-cysteine ​​ligase genes; A prokaryotic microbial strain capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione. (6) The prokaryotic microbial strain according to (5), wherein the promoter is an inducible promoter. (7) The prokaryotic microbial strain according to (6), wherein the inducible promoter is an IPTG-inducible promoter, a light-inducible promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing the tetO or lacO operator as an operator sequence. (8) The prokaryotic microbial strain according to (7), wherein the inducible promoter is a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter, a tac promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing a tetO or lacO operator as an operator sequence. (9) The prokaryotic microbial strain according to (8), wherein the inducible promoter is a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter, or a tac promoter. (10) The prokaryotic microbial strain according to (9), wherein the inducible promoter is a T5 promoter. (11) The prokaryotic microbial strain according to any one of (5) to (10), which is a transformant of an enterobacteria. (12) The prokaryotic microbial strain according to any one of (5) to (10), which is a transformant of Escherichia coli. (13) The method according to (1) or (2), wherein the prokaryotic microbial strain is the prokaryotic microbial strain according to any one of (5) to (12). (14) The method according to (3) or (4), wherein the prokaryotic microbial strain is the prokaryotic microbial strain according to any one of (5) to (12).

[0015] The second disclosure of the present specification includes the following embodiments (15) to (22).

[0016] (15) A microorganism that is deficient in the following genes [1] and [2] and has enhanced expression of the gene [3] or the gene [4]: [1] The gene encoding γ-glutamyltransferase (EC:2.3.2.2); [2] A gene encoding glutathione reductase (EC:1.8.1.7); [3] A gene encoding glutamate-cysteine ​​ligase (EC:6.3.2.2) and a gene encoding glutathione synthetase (EC:6.3.2.3); [4] A gene encoding bifunctional glutathione synthetase. (16) The microorganism according to (15), which is deleted from the following gene [5]: [5] A gene encoding tripeptide peptidase (EC:3.4.11.4). (17) The microorganism according to (15) or (16), which is a bacterial transformant. (18) The microorganism according to (15) or (16), which is a transformant of an enterobacteria. (19) The microorganism according to (15) or (16), which is a transformant of a gram-negative bacterium. (20) The microorganism according to (15) or (16), which is a transformant of Escherichia coli. (21) A method for producing glutathione, comprising culturing the microorganism according to any one of (15) to (20) in a medium.

[0017] This specification includes the disclosures of Japanese Patent Application No. 2019-211477 and Japanese Patent Application No. 2020-002363, which are priority documents of this application. Effect of the Invention

[0018] According to the method of the first disclosure of the present specification, since a step of adding cysteine ​​or cystine is not required, γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione can be produced at low cost.

[0019] The prokaryotic microbial strain according to the first disclosure of the present specification can efficiently produce γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione.

[0020] The microorganism according to the second disclosure of the present specification has high productivity of glutathione through fermentation.

[0021] According to the method for producing glutathione according to the second disclosure of the present specification, it is possible to efficiently produce glutathione. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Preferred embodiments of the first and second disclosures of this specification will be specifically described below, but the scope of the first and second disclosures of this specification is not limited to these embodiments.

[0023] <1. Enzymes> 1.1. γ-Glutamyltransferase γ-Glutamyltransferase (EC: 2.3.2.2) is an enzyme that hydrolyzes γ-glutamyl peptides such as glutathione.

[0024] "γ-glutamyltransferase" is also referred to as "γ-glutamyltranspeptidase" or "Ggt." In this specification, "γ-glutamyltransferase," "γ-glutamyltranspeptidase," and "Ggt" are interchangeable.

[0025] Specific examples of γ-glutamyltransferase include: (1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22; (1B) a polypeptide having an amino acid sequence in which one or more amino acids have been added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO:22 (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or added, preferably deleted and / or added, in total, at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO:22), and having γ-glutamyltransferase activity; (1C) a polypeptide having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 22, and having γ-glutamyltransferase activity; or (1D) A fragment having γ-glutamyltransferase activity of any one of the polypeptides (1A) to (1C). It can be.

[0026] In the above (1D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more, more preferably 400 or more, more preferably 500 or more, and more preferably 550 or more.

[0027] Each of the above polypeptides may be appropriately chemically modified.

[0028] In the above (1B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" refers to a substitution between amino acids with similar properties such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties can be classified into, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), and the like.

[0029] In the above (1C), "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the protein shown in SEQ ID NO: 22 when the two amino acid sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid identity between the two. Sequence identity can be calculated using a protein search system such as BLAST or FASTA (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, SF et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Hereinafter, in this specification, "sequence identity" of amino acid sequences is used in the same sense.

[0030] "A gene encoding γ-glutamyltransferase (EC:2.3.2.2)" refers to a nucleic acid (DNA or RNA, preferably DNA) that encodes the amino acid sequence of γ-glutamyltransferase, and is contained in the genomic DNA on the chromosome of a wild-type microorganism before γ-glutamyltransferase is deleted.

[0031] An example of DNA encoding the amino acid sequence of γ-glutamyltransferase derived from Escherichia coli shown in SEQ ID NO: 22 is shown in SEQ ID NO: 21. However, in the genomic DNA of a wild-type microorganism, the nucleotide sequence of SEQ ID NO: 21 does not necessarily exist as is, and the nucleotide sequence of SEQ ID NO: 21 may be an exon sequence with one or more intron sequences intervening therein.

[0032] That is, a specific example of the base sequence of a gene encoding the amino acid sequence of γ-glutamyltransferase is: (1E) a nucleotide sequence shown in SEQ ID NO: 21; (1F) a nucleotide sequence in which one or more nucleotides have been added, deleted or substituted in the nucleotide sequence shown in SEQ ID NO:21 (particularly preferably, a nucleotide sequence in which one or more nucleotides have been substituted, deleted and / or added, preferably deleted and / or added, in total at one or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO:21), which encodes a polypeptide having γ-glutamyltransferase activity; (1G) a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 21, and encoding a polypeptide having γ-glutamyltransferase activity; (1H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having γ-glutamyltransferase activity, which is any one of the nucleotide sequences (1E) to (1G); (1I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) has been introduced into any of the nucleotide sequences (1E) to (1H); (1J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (1A) to (1D); or A base sequence in which any of the base sequences (1K)(1E) to (1J) is an exon sequence and one or more intron sequences are interposed in between. Examples include:

[0033] In the above (1G), "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in SEQ ID NO: 21 when the two base sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid identity between the two. Sequence identity can be calculated using a BLAST or FASTA base sequence search system (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, SF et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Hereinafter, in this specification, the "sequence identity" of a base sequence is used in the same sense.

[0034] <1.2. Glutathione reductase> Glutathione reductase (EC: 1.8.1.7) is an enzyme that catalyzes the reaction of reducing oxidized glutathione (glutathione disulfide) in the presence of NADPH to produce reduced glutathione.

[0035] Examples of glutathione reductase include: (2A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26; (2B) a polypeptide having an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:26 (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added, preferably deleted and / or added, in total, at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO:26), and having glutathione reductase activity; (2C) a polypeptide having an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 26, and having glutathione reductase activity; or (2D) A fragment of any one of the polypeptides (2A) to (2C) having glutathione reductase activity. It can be.

[0036] In the above (2D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more, and more preferably 400 or more.

[0037] In the above (2B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. The "conservative amino acid substitution" is as explained in the section <1.1. γ-glutamyltransferase> regarding (1B).

[0038] The "sequence identity" in (2C) above is as explained in (1C) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (2C) above refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the protein shown in SEQ ID NO: 26 when two amino acid sequences are aligned and gaps are introduced, if necessary, to maximize the degree of amino acid identity between the two.

[0039] "A gene encoding glutathione reductase (EC:1.8.1.7)" refers to a nucleic acid (DNA or RNA, preferably DNA) that encodes the amino acid sequence of glutathione reductase, and is contained in the genomic DNA on the chromosome of a wild-type microorganism before glutathione reductase is deleted.

[0040] An example of DNA encoding the amino acid sequence of glutathione reductase derived from Escherichia coli shown in SEQ ID NO: 26 is shown in SEQ ID NO: 25. However, in the genomic DNA of a wild-type microorganism, the nucleotide sequence of SEQ ID NO: 25 does not necessarily exist as is, and the nucleotide sequence of SEQ ID NO: 25 may be an exon sequence with one or more intron sequences intervening therein.

[0041] That is, a specific example of the base sequence of a gene encoding the amino acid sequence of glutathione reductase is: (2E) a nucleotide sequence shown in SEQ ID NO: 25; (2F) a base sequence in which one or more bases have been added, deleted or substituted in the base sequence shown in SEQ ID NO:25 (particularly preferably, a base sequence in which one or more bases have been substituted, deleted and / or added, preferably deleted and / or added, in total at one or both of the 5' end and the 3' end of the base sequence shown in SEQ ID NO:25), which encodes a polypeptide having glutathione reductase activity; (2G) a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 25, and encoding a polypeptide having glutathione reductase activity; (2H) A partial base sequence encoding the amino acid sequence of a polypeptide having glutathione reductase activity, which is any of the base sequences (2E) to (2G). (2I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) has been introduced into any of the nucleotide sequences (2E) to (2H); (2J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (2A) to (2D); or A base sequence in which any of the base sequences (2K)(2E) to (2J) is an exon sequence and one or more intron sequences are interposed in between. Examples include:

[0042] The "sequence identity" in (2G) is as explained in (1G) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (2G) refers to the ratio (%) of identical bases to the total number of bases of SEQ ID NO: 25 when the two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two sequences.

[0043] <1.3. Tripeptide peptidase> Tripeptide peptidase (EC: 3.4.11.4) is an enzyme that catalyzes the reaction of releasing the N-terminal amino acid residue from a tripeptide. "Tripeptide peptidase" is also referred to as "peptidase T" or "PepT." In this specification, "tripeptide peptidase," "peptidase T," and "PepT" are interchangeable.

[0044] Specific examples of tripeptide peptidases include: (5A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24; (5B) a polypeptide having an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:24 (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added, preferably deleted and / or added, in total, at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO:24), and having tripeptide peptidase activity; (5C) a polypeptide having tripeptide peptidase activity, comprising an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 24; or (5D) A fragment having tripeptide peptidase activity of any one of the polypeptides (5A) to (5C). It can be.

[0045] In the above (5D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more, and more preferably 350 or more.

[0046] Each of the above polypeptides may be appropriately chemically modified. In the above (5B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. The "conservative amino acid substitution" is as explained in the section <1.1. γ-glutamyltransferase> regarding (1B).

[0047] The "sequence identity" in (5C) above is as explained in (1C) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (5C) above refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the protein shown in SEQ ID NO: 24 when two amino acid sequences are aligned and gaps are introduced, if necessary, to maximize the degree of amino acid identity between the two.

[0048] The term "gene encoding tripeptide peptidase (EC: 3.4.11.4)" refers to a nucleic acid (DNA or RNA, preferably DNA) that encodes the amino acid sequence of tripeptide peptidase, and is contained in the genomic DNA on the chromosome of a wild-type microorganism before the tripeptide peptidase is deleted.

[0049] An example of DNA encoding the amino acid sequence of tripeptide peptidase derived from Escherichia coli shown in SEQ ID NO: 24 is shown in SEQ ID NO: 23. However, in the genomic DNA of a wild-type microorganism, the nucleotide sequence of SEQ ID NO: 23 does not necessarily exist as is, and the nucleotide sequence of SEQ ID NO: 23 may be an exon sequence with one or more intron sequences intervening therein.

[0050] That is, a specific example of the base sequence of a gene encoding the amino acid sequence of tripeptide peptidase is (5E) a nucleotide sequence shown in SEQ ID NO: 23; (5F) a base sequence in which one or more bases have been added, deleted or substituted in the base sequence shown in SEQ ID NO:23 (particularly preferably, a base sequence in which one or more bases have been substituted, deleted and / or added, preferably deleted and / or added, in total at one or both of the 5'-terminus and the 3'-terminus of the base sequence shown in SEQ ID NO:23), which encodes a polypeptide having tripeptide peptidase activity; (5G) a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 23, and encoding a polypeptide having tripeptide peptidase activity; A partial base sequence encoding an amino acid sequence of a polypeptide having tripeptide peptidase activity, which is any one of the base sequences (5H) (5E) to (5G); (5I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) has been introduced into any of the nucleotide sequences of (5E) to (5H); (5J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (5A) to (5D); or A base sequence in which any of the base sequences (5K)(5E) to (5J) is an exon sequence and one or more intron sequences are interposed in the middle Examples include:

[0051] The "sequence identity" in (5G) is as explained in (1G) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (5G) refers to the ratio (%) of identical bases to the total number of bases in SEQ ID NO: 23 when the two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two sequences.

[0052] <1.4. Glutamate-cysteine ​​ligase> Glutamate-cysteine ​​ligase (EC: 6.3.2.2) is an enzyme that recognizes L-cysteine ​​as a substrate in the presence of ATP and catalyzes a reaction to generate γ-glutamylcysteine ​​by binding with L-glutamic acid, and its origin, structure, etc. are not particularly limited as long as it has the activity. In this specification, the activity is referred to as glutamate-cysteine ​​ligase activity. 1 U of the activity means the activity to generate 1 μmol of γ-glutamylcysteine ​​per minute at 30° C., and is measured under the following measurement conditions.

[0053] "Glutamic acid-cysteine ​​ligase" is also referred to as "glutamic acid cysteine ​​ligase" or "GshA." In this specification, "glutamic acid-cysteine ​​ligase," "glutamic acid cysteine ​​ligase," and "GshA" are interchangeable.

[0054] (Measurement conditions) The enzyme solution is added to 50 mM Tris hydrochloride buffer (pH 8.0) containing 10 mM ATP, 15 mM L-glutamic acid, 15 mM L-cysteine, and 10 mM magnesium sulfate, and the reaction is carried out by incubating at 30°C, and the reaction is stopped by adding 6N hydrochloric acid. The amount of γ-glutamylcysteine ​​in the reaction solution is quantified using high performance liquid chromatography.

[0055] The conditions for the high performance liquid chromatography are as follows: Under these conditions, reduced glutathione (GSH), γ-glutamylcysteine ​​(γ-GC), bis-γ-glutamylcystine (oxidized γ-GC), and oxidized glutathione (GSSG) are eluted in this order. [HPLC conditions] Column: ODS-HG-3 (4.6 mmφ×150 mm, Nomura Chemical Co., Ltd.); Eluent: 12.2 g of potassium dihydrogen phosphate and 3.6 g of sodium heptanesulfonate were dissolved in 1.8 L of distilled water, and the solution was adjusted to pH 2.8 with phosphoric acid, and 186 ml of methanol was added to dissolve the solution; Flow rate: 1.0ml / min; Column temperature: 40°C; Measurement wavelength: 210nm

[0056] The glutamate-cysteine ​​ligase to be used preferably has a glutamate-cysteine ​​ligase activity (specific activity) per 1 mg of protein of 0.5 U or more, preferably 1 U or more, more preferably 5 U or more, and most preferably 10 U or more.

[0057] The origin of the glutamate-cysteine ​​ligase is not particularly limited, and it can be derived from microorganisms, animals, plants, etc. Glutamate-cysteine ​​ligase derived from microorganisms is preferred, and glutamate-cysteine ​​ligase derived from enterobacteria such as Escherichia coli, bacteria such as coryneform bacteria, eukaryotic microorganisms such as yeast, etc. is particularly preferred.

[0058] Specific examples of the nucleotide sequence of glutamate-cysteine ​​ligase derived from Escherichia coli and the amino acid sequence encoded by said nucleotide sequence are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

[0059] The glutamate-cysteine ​​ligase is not limited to the glutamate-cysteine ​​ligase consisting of the amino acid sequence shown in SEQ ID NO: 13, but may also be other polypeptides having glutamate-cysteine ​​ligase activity, such as its active mutants and other orthologs. The other polypeptides having glutamate-cysteine ​​ligase activity are preferably polypeptides that exhibit 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, and even more preferably 90% or more of the activity observed when the glutamate-cysteine ​​ligase consisting of the amino acid sequence shown in SEQ ID NO: 13 is used under the above activity measurement conditions.

[0060] Examples of glutamate-cysteine ​​ligases include: (3-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13; (3-1B) A polypeptide having an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 13 (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added, preferably deleted and / or added, in total, at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO: 13), which has glutamate-cysteine ​​ligase activity; (3-1C) a polypeptide having an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 13, and having glutamate-cysteine ​​ligase activity; or (3-1D) A fragment of any one of the polypeptides (3-1A) to (3-1C) having glutamate-cysteine ​​ligase activity. It can be.

[0061] In the above (3-1D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more, more preferably 400 or more, more preferably 450 or more, more preferably 500 or more.

[0062] Each of the above polypeptides may be appropriately chemically modified.

[0063] In the above (3-1B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. The "conservative amino acid substitution" is as explained in the section <1.1. γ-glutamyltransferase> with respect to (1B).

[0064] The "sequence identity" in (3-1C) above is as explained in (1C) in the section <1.1. γ-glutamyltransferase>. That is, in (3-1C) above, the "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the protein shown in SEQ ID NO: 13 when two amino acid sequences are aligned and gaps are introduced, if necessary, to maximize the degree of amino acid identity between the two.

[0065] The term "gene encoding glutamate-cysteine ​​ligase (EC: 6.3.2.2)" refers to a nucleic acid (DNA or RNA, preferably DNA) that encodes the amino acid sequence of glutamate-cysteine ​​ligase.

[0066] An example of DNA encoding the amino acid sequence of glutamate-cysteine ​​ligase derived from Escherichia coli shown in SEQ ID NO: 13 is shown in SEQ ID NO: 12. The base sequence of the nucleic acid encoding the amino acid sequence of glutamate-cysteine ​​ligase may be codon-optimized according to the host.

[0067] That is, a specific example of the base sequence of a gene encoding the amino acid sequence of glutamate-cysteine ​​ligase is: (3-1E) a nucleotide sequence shown in SEQ ID NO: 12; (3-1F) a nucleotide sequence in which one or more nucleotides have been added, deleted or substituted in the nucleotide sequence shown in SEQ ID NO: 12 (particularly preferably, a nucleotide sequence in which one or more nucleotides have been substituted, deleted and / or added, preferably deleted and / or added, in total at one or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 12), which encodes a polypeptide having glutamate-cysteine ​​ligase activity; (3-1G) a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 12, and encoding a polypeptide having glutamate-cysteine ​​ligase activity; (3-1H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having glutamate-cysteine ​​ligase activity, which is any one of the nucleotide sequences (3-1E) to (3-1G); (3-1I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) has been introduced into any of the nucleotide sequences (3-1E) to (3-1H); (3-1J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (3-1A) to (3-1D); or A base sequence in which any of the base sequences (3-1K)(3-1E) to (3-1J) is an exon sequence and one or more intron sequences are interposed in the middle Examples include:

[0068] The "sequence identity" in (3-1G) is as explained in (1G) in the section <1.1. γ-glutamyltransferase>. That is, in (3-1G), the "sequence identity" refers to the ratio (%) of identical bases to the total number of bases in SEQ ID NO: 12 when two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two sequences.

[0069] <1.5. Glutathione synthetase> Glutathione synthetase (EC: 6.3.2.3) is an enzyme that recognizes γ-glutamylcysteine ​​as a substrate in the presence of ATP and catalyzes a reaction to generate GSH by binding it with glycine, and its origin, structure, etc. are not particularly limited as long as it has the activity. In this specification, the activity is referred to as glutathione synthetase activity. 1 U of the activity means the activity to generate 1 μmol of glutathione per minute at 30° C., and is measured under the following measurement conditions.

[0070] "Glutathione synthetase" is also referred to as "GshB." In this specification, "glutathione synthetase" and "GshB" are interchangeable.

[0071] (Measurement conditions) The enzyme solution is added to 50 mM Tris hydrochloride buffer (pH 8.0) containing 10 mM ATP, 15 mM γ-glutamylcysteine, 15 mM glycine, and 10 mM magnesium sulfate, and the reaction is carried out by incubating at 30°C, and the reaction is stopped by adding 6N hydrochloric acid. Glutathione in the reaction solution is quantified using high performance liquid chromatography.

[0072] The conditions for high performance liquid chromatography are the same as those described above for the method for measuring glutamate-cysteine ​​ligase activity.

[0073] The glutathione synthetase to be used preferably has a glutathione synthetase activity (specific activity) per 1 mg of protein of 0.5 U or more, preferably 1 U or more, more preferably 5 U or more, and most preferably 10 U or more.

[0074] The glutathione synthetase is not particularly limited, and may be derived from a microorganism, an animal, a plant, etc. A glutathione synthetase derived from a microorganism is preferable, and a glutathione synthetase derived from an intestinal bacterium such as Escherichia coli, a bacterium such as a coryneform bacterium, a eukaryotic microorganism such as yeast, a microorganism belonging to the family Hydrogenophilales, or the like is particularly preferable.

[0075] The glutathione synthetase derived from a microorganism belonging to the family Hydrogenophilales is preferably a glutathione synthetase derived from a microorganism belonging to the genus Thiobacillus, more preferably a glutathione synthetase derived from a microorganism belonging to Thiobacillus denitrificans. In particular, the glutathione synthetase derived from the Thiobacillus denitrificans ATCC25259 strain is preferred.

[0076] (Preferred embodiment of glutathione synthetase derived from Escherichia coli or a mutant thereof) Specific examples of the base sequence of glutathione synthetase derived from Escherichia coli and the amino acid sequence encoded by the base sequence are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0077] The glutathione synthetase is not limited to the glutathione synthetase consisting of the amino acid sequence shown in SEQ ID NO: 15, but other polypeptides having glutathione synthetase activity, such as active mutants and other orthologs thereof, can also be used. The other polypeptides having glutathione synthetase activity are preferably polypeptides that exhibit 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, and even more preferably 90% or more of the activity when the glutathione synthetase consisting of the amino acid sequence shown in SEQ ID NO: 15 is used under the above activity measurement conditions.

[0078] Specific examples of glutathione synthetase derived from Escherichia coli or its mutants include: (3-2A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15; (3-2B) A polypeptide having an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 15 (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added, preferably deleted and / or added, in total, at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO: 15), which has glutathione synthetase activity; (3-2C) a polypeptide having an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 15, and having glutathione synthetase activity; or (3-2D) A fragment having glutathione synthetase activity of any one of the polypeptides (3-2A) to (3-2C). It can be.

[0079] In the above (3-2D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more, and more preferably 300 or more.

[0080] Each of the above polypeptides may be appropriately chemically modified.

[0081] In the above (3-2B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. The "conservative amino acid substitution" is as explained in the section <1.1. γ-glutamyltransferase> with respect to (1B).

[0082] The "sequence identity" in (3-2C) above is as explained in (1C) in the section <1.1. γ-glutamyltransferase>. That is, in (3-2C) above, the "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the protein shown in SEQ ID NO: 15, when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid identity between the two.

[0083] The term "gene encoding glutathione synthetase (EC: 6.3.2.3)" refers to a nucleic acid (DNA or RNA, preferably DNA) that encodes the amino acid sequence of glutathione synthetase.

[0084] An example of DNA encoding the amino acid sequence of glutathione synthetase derived from Escherichia coli shown in SEQ ID NO: 15 is shown in SEQ ID NO: 14. The base sequence of the nucleic acid encoding the amino acid sequence of glutathione synthetase may be codon-optimized according to the host.

[0085] Specifically, specific examples of the base sequence of a gene encoding the amino acid sequence of glutathione synthetase derived from Escherichia coli or a mutant thereof include the following: (3-2E) a base sequence shown in SEQ ID NO: 14; (3-2F) a base sequence in which one or more bases have been added, deleted or substituted in the base sequence shown in SEQ ID NO: 14 (particularly preferably, a base sequence in which one or more bases have been substituted, deleted and / or added, preferably deleted and / or added, in total, at one or both of the 5' end and the 3' end of the base sequence shown in SEQ ID NO: 14), which encodes a polypeptide having glutathione synthetase activity; (3-2G) a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 14, and encoding a polypeptide having glutathione synthetase activity; A partial nucleotide sequence encoding an amino acid sequence of a polypeptide having glutathione synthetase activity, which is any one of the nucleotide sequences (3-2H) (3-2E) to (3-2G); (3-2I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) has been introduced into any of the nucleotide sequences (3-2E) to (3-2H); (3-2J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (3-2A) to (3-2D); or A base sequence in which any of the base sequences (3-2K)(3-2E) to (3-2J) is an exon sequence and one or more intron sequences are interposed in between. Examples include:

[0086] The "sequence identity" in (3-2G) is as explained for (1G) in the section <1.1. γ-glutamyltransferase>. That is, in (3-2G), the "sequence identity" refers to the ratio (%) of identical bases to the total number of bases in SEQ ID NO: 14 when the two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two sequences.

[0087] (Preferred embodiment of glutathione synthetase derived from Thiobacillus denitrificans or a mutant thereof) Another preferred specific example of glutathione synthetase is a wild-type glutathione synthetase derived from Thiobacillus denitrificans ATCC25259 strain or an active mutant thereof. Specific examples of the base sequence of the wild-type glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain and the amino acid sequence encoded by the base sequence are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively. The active mutant of the wild-type glutathione synthetase is preferably a polypeptide that exhibits 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, and even more preferably 90% or more of the activity of the wild-type glutathione synthetase consisting of the amino acid sequence shown in SEQ ID NO: 17 under the above activity measurement conditions.

[0088] Specific examples of the glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain or its mutants include (3-3A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 17; (3-3B) A polypeptide having an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 17 (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added, preferably deleted and / or added, in total, at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO: 17), which has glutathione synthetase activity; (3-3C) a polypeptide having an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 17, and having glutathione synthetase activity; or (3-3D) A fragment having glutathione synthetase activity of any of the polypeptides (3-3A) to (3-3C). It can be.

[0089] In the above (3-3D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more, and more preferably 300 or more.

[0090] Each of the above polypeptides may be appropriately chemically modified.

[0091] In the above (3-3B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. The "conservative amino acid substitution" is as explained in the section <1.1. γ-glutamyltransferase> with respect to (1B).

[0092] The "sequence identity" in (3-3C) is as explained in (1C) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (3-3C) refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the protein shown in SEQ ID NO: 17 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid identity between the two.

[0093] An example of DNA encoding the amino acid sequence of glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain shown in SEQ ID NO: 17 is shown in SEQ ID NO: 16. The base sequence of the nucleic acid encoding the amino acid sequence of glutathione synthetase may be codon-optimized according to the host.

[0094] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of the glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain or a mutant thereof are as follows: (3-3E) a nucleotide sequence shown in SEQ ID NO: 16; (3-3F) a base sequence in which one or more bases have been added, deleted or substituted in the base sequence shown in SEQ ID NO: 16 (particularly preferably, a base sequence in which one or more bases have been substituted, deleted and / or added, preferably deleted and / or added, in total at one or both of the 5' end and the 3' end of the base sequence shown in SEQ ID NO: 16), which encodes a polypeptide having glutathione synthetase activity; (3-3G) a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 16, and encoding a polypeptide having glutathione synthetase activity; A partial nucleotide sequence encoding an amino acid sequence of a polypeptide having glutathione synthetase activity, which is any one of the nucleotide sequences (3-3H) (3-3E) to (3-3G); (3-3I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) has been introduced into any of the nucleotide sequences (3-3E) to (3-3H); A base sequence encoding the amino acid sequence of any one of the polypeptides (3-3J) (3-3A) to (3-3D); or A base sequence in which any of the base sequences (3-3K)(3-3E) to (3-3J) is an exon sequence and one or more intron sequences are interposed in between. Examples include:

[0095] The "sequence identity" in (3-3G) is as explained in (1G) in the section <1.1. γ-glutamyltransferase>. That is, in (3-3G), the "sequence identity" refers to the ratio (%) of identical bases to the total number of bases in SEQ ID NO: 16 when two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two sequences.

[0096] (Preferred embodiment of active mutant of glutathione synthetase from Thiobacillus denitrificans) Another preferred example of a glutathione synthetase is an active mutant of the wild-type glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain comprising the amino acid sequence shown in SEQ ID NO: 17, and the polypeptide described in International Publication WO2018 / 084165 is particularly preferred.

[0097] Specifically, the active mutant is (3-4A) The following group of amino acid sequences shown in SEQ ID NO: 17: a polypeptide consisting of the amino acid sequence 3-4A in which one or more amino acids selected from positions 13, 17, 20, 23, 39, 70, 78, 101, 113, 125, 126, 136, 138, 149, 152, 154, 155, 197, 200, 215, 226, 227, 230, 239, 241, 246, 249, 254, 260, 262, 263, 270, 278, 299, 305, 307 and 310 have been substituted; (3-4B) A polypeptide having an amino acid sequence in which one or more amino acids other than the amino acid sites in the amino acid sequence 3-4A have been added, deleted or substituted (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids in total have been substituted, deleted and / or added, preferably deleted and / or added, in one or both of the N-terminus and the C-terminus of the amino acid sequence 3-4A), and having glutathione synthetase activity; (3-4C) a polypeptide having an amino acid sequence that is identical to the amino acid sequence 3-4A at the amino acid site and has 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity at the portion other than the amino acid site, and has glutathione synthetase activity; or (3-4D) A fragment having glutathione synthetase activity of any one of the polypeptides (3-4A) to (3-4C). It can be.

[0098] In the above (3-4D), the fragment to be used may be a polypeptide having preferably 150 or more amino acids, more preferably 200 or more, and even more preferably 300 or more.

[0099] Each of the above polypeptides may be appropriately chemically modified.

[0100] In the above (3-4B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. The "conservative amino acid substitution" is as explained in the section <1.1. γ-glutamyltransferase> with respect to (1B).

[0101] The "sequence identity" in (3-4C) is as explained in (1C) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (3-4C) refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues other than the amino acid site of the amino acid sequence 3-4A when the two amino acid sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid identity between the two.

[0102] More preferably, the amino acid sequence 3-4A is selected from the group consisting of the following amino acid sequence in the amino acid sequence shown in SEQ ID NO: 17: The 13th is serine, the 17th is glutamic acid, the 20th is threonine, the 23rd is leucine, the 39th is threonine, the 70th is serine, the 78th is leucine, the 101st is asparagine, glutamine, serine, threonine, the 113th is histidine, the 125th is valine, the 126th is asparagine, the 136th is threonine, the 138th is alanine, the 149th is glutamine, the 152nd is glutamine, the 154th is asparagine, the 155th is leucine, the 197th is glutamine, the 200th is serine, the 215th is alanine. aspartic acid, arginine at position 226, serine at position 227, proline at position 230, serine at position 239, histidine at position 241, arginine at position 246, glutamic acid at position 249, aspartic acid at position 254, alanine, cysteine, glycine, glutamine, threonine at position 260, cysteine ​​at position 262, arginine at position 263, isoleucine at position 270, glycine, alanine at position 278, alanine at position 299, alanine at position 305, glycine at position 307, valine at position 310, and threonine at position 310. It is an amino acid sequence into which one or more amino acid substitutions selected from the following have been introduced.

[0103] The amino acid sequence 3-4A is particularly preferably the following amino acid sequence (1) to (35) among the amino acid sequences shown in SEQ ID NO: 17: (1) The 13th is serine. (2) 17th amino acid is glutamic acid, 113th amino acid is histidine, and 230th amino acid is proline. (3) The 20th amino acid is threonine and the 215th amino acid is aspartic acid. (4) 20th amino acid is threonine, 241st amino acid is histidine, (5) 23rd position is leucine, 126th position is asparagine, (6) The 39th amino acid is threonine and the 260th amino acid is alanine. (7) The 70th residue is serine and the 260th residue is alanine. (8) 78th amino acid is leucine, 278th amino acid is alanine, (9) The 101st position is asparagine. (10) The 101st amino acid is glutamine. (11) The 101st base is serine. (12) The 101st position is serine and the 260th position is alanine. (13) The 101st amino acid is threonine. (14) 125th amino acid is valine, 249th amino acid is glutamic acid, (15) 125th amino acid is valine, 152nd amino acid is glutamine, (16) 136th amino acid is threonine, (17) 138th amino acid is alanine, 149th amino acid is glutamine, 241st amino acid is histidine, and 263rd amino acid is glutamine. (18) Asparagine at position 154 and arginine at position 246. (19) Leucine at position 155 and serine at position 239. (20) 197th amino acid is glutamine. (21) The 200th residue is serine and the 260th residue is alanine. (22) Arginine at position 226 and alanine at position 260. (23) The 227th position is serine and the 260th position is alanine. (24) Aspartic acid at position 254 and alanine at position 260. (25) The 260th residue is alanine. (26) The 260th amino acid is alanine, the 278th amino acid is glycine, and the 307th amino acid is valine. (27) The 260th amino acid is alanine, the 299th amino acid is alanine, (28) Alanine at position 260 and glycine at position 305. (29) Alanine at position 260 and threonine at position 310. (30) Cysteine ​​at position 260; (31) The 260th residue is glycine. (32) 260th amino acid is glutamine. (33) The 260th amino acid is threonine. (34) Cysteine ​​at position 262, (35) The 270th amino acid is isoleucine. It is an amino acid sequence into which an amino acid substitution represented by any one of the following has been introduced:

[0104] A base sequence encoding the amino acid sequence of any one of the polypeptides (3-4A) to (3-4D) can be used as a "gene encoding glutathione synthetase (EC: 6.3.2.3)".

[0105] An example of a nucleotide sequence encoding an amino acid sequence of an active mutant of glutathione synthetase from Thiobacillus denitrificans ATCC25259 strain, in which valine at position 260 in the amino acid sequence of glutathione synthetase from Thiobacillus denitrificans ATCC25259 strain shown in SEQ ID NO: 17 has been substituted with alanine, is shown in SEQ ID NO: 18. The nucleotide sequence of a nucleic acid encoding the amino acid sequence of an active mutant of glutathione synthetase from Thiobacillus denitrificans ATCC25259 strain may be codon-optimized according to the host.

[0106] <1.6. Bifunctional glutathione synthetase> Bifunctional glutathione synthetase is an enzyme that has both an activity of catalyzing a reaction in which L-cysteine ​​is recognized as a substrate in the presence of ATP and γ-glutamic acid is bound to it to produce γ-glutamylcysteine, and an activity of catalyzing a reaction in which γ-glutamylcysteine ​​is recognized as a substrate in the presence of ATP and glycine is bound to it to produce GSH, and the origin, structure, etc. of the enzyme are not particularly limited as long as it has the activity. In this specification, the activity is referred to as bifunctional glutathione synthetase activity. 1 U of the activity means the activity of producing 1 μmol of GSH per minute at 30° C., and is measured under the following measurement conditions.

[0107] "Bifunctional glutathione synthetase" is also referred to as "GshF." In this specification, "bifunctional glutathione synthetase" and "GshF" are interchangeable.

[0108] (Measurement conditions) The enzyme solution is added to 50 mM Tris hydrochloride buffer (pH 8.0) containing 10 mM ATP, 15 mM L-glutamic acid, 15 mM L-cysteine, 15 mM glycine, and 10 mM magnesium sulfate, and the reaction is carried out by incubating at 30°C, and the reaction is stopped by adding 6N hydrochloric acid. Glutathione in the reaction solution is quantified using high performance liquid chromatography.

[0109] The conditions for high performance liquid chromatography are the same as those described above for the method for measuring glutamate-cysteine ​​ligase activity.

[0110] It is preferable to use a bifunctional glutathione synthetase having a bifunctional glutathione synthetase activity (specific activity) per 1 mg of protein of 0.5 U or more, preferably 1 U or more, more preferably 5 U or more, and most preferably 10 U or more.

[0111] The origin of the bifunctional glutathione synthetase is not particularly limited, and it may be derived from a microorganism, an animal, a plant, etc. A bifunctional glutathione synthetase derived from a microorganism is preferred. In particular, a bifunctional glutathione synthetase derived from a bacteria is preferable, and specifically, bacteria of the genus Streptococcus such as Streptococcus agalactiae, Streptococcus mutans, Streptococcus suis, and Streptococcus thermophilus; bacteria of the genus Lactobacillus such as Lactobacillus plantarum; bacteria of the genus Desulfotalea such as Desulfotalea psychrophila; bacteria of the genus Clostridium such as Clostridium perfringens; Preferably, the bifunctional glutathione synthetase is derived from at least one species selected from the group consisting of Listeria bacteria of the genus, such as Listeria innocua and Listeria monocytogenes; Enterococcus bacteria of the genus, such as Enterococcus faecalis and Enterococcus faecium; Pasteurella bacteria of the genus, such as Pasteurella multocida; Mannheimia bacteria of the genus, such as Mannheimia succiniciprodecens; and Haemophilus bacteria of the genus, such as Haemophilus somnus.

[0112] Specific examples of the nucleotide sequence of the bifunctional glutathione synthetase derived from Streptococcus agalactiae and the amino acid sequence encoded by the nucleotide sequence are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively. The nucleotide sequence of SEQ ID NO: 19 is a nucleotide sequence that encodes the bifunctional glutathione synthetase derived from Streptococcus agalactiae, which consists of the amino acid sequence shown in SEQ ID NO: 20, and is a nucleotide sequence adapted to the codon usage frequency in Escherichia coli.

[0113] The bifunctional glutathione synthetase is not limited to the bifunctional glutathione synthetase consisting of the amino acid sequence shown in SEQ ID NO: 20, but other polypeptides having bifunctional glutathione synthetase activity, such as its active mutants and other orthologs, can also be used. The other polypeptides having bifunctional glutathione synthetase activity are preferably polypeptides that exhibit 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, and even more preferably 90% or more of the activity when the bifunctional glutathione synthetase consisting of the amino acid sequence shown in SEQ ID NO: 20 is used under the above activity measurement conditions.

[0114] Specific examples of bifunctional glutathione synthetases include: (4A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20; (4B) a polypeptide having an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:20 (particularly preferably, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added, preferably deleted and / or added, in total, at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO:20), and having bifunctional glutathione synthetase activity; (4C) a polypeptide having a bifunctional glutathione synthetase activity, comprising an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 20; or (4D) A fragment of any one of the polypeptides (4A) to (4C) having bifunctional glutathione synthetase activity. It can be.

[0115] In (4D) above, the fragment to be used may be a polypeptide having preferably 400 or more amino acids, more preferably 500 or more, more preferably 600 or more, more preferably 700 or more, and more preferably 730 or more.

[0116] Each of the above polypeptides may be appropriately chemically modified. In the above (4B), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, the amino acid substitution is preferably a conservative amino acid substitution. The "conservative amino acid substitution" is as explained in the section <1.1. γ-glutamyltransferase> regarding (1B).

[0117] The "sequence identity" in (4C) above is as explained in (1C) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (4C) above refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the protein shown in SEQ ID NO: 20 when two amino acid sequences are aligned and gaps are introduced, if necessary, to maximize the degree of amino acid identity between the two.

[0118] The term "gene encoding bifunctional glutathione synthetase" refers to a nucleic acid (DNA or RNA, preferably DNA) that encodes the amino acid sequence of a bifunctional glutathione synthetase.

[0119] Specific examples of the base sequence of a gene encoding the amino acid sequence of a bifunctional glutathione synthetase include: (4E) a nucleotide sequence shown in SEQ ID NO: 19; (4F) a base sequence in which one or more bases have been added, deleted or substituted in the base sequence shown in SEQ ID NO: 19 (particularly preferably, a base sequence in which one or more bases have been substituted, deleted and / or added, preferably deleted and / or added, in total, at one or both of the 5' end and the 3' end of the base sequence shown in SEQ ID NO: 19), which encodes a polypeptide having bifunctional glutathione synthetase activity; (4G) a nucleotide sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 19, which encodes a polypeptide having bifunctional glutathione synthetase activity; A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having bifunctional glutathione synthetase activity, which is any one of the nucleotide sequences (4H) (4E) to (4G); (4I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) has been introduced into any of the nucleotide sequences (4E) to (4H); (4J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (4A) to (4D); or A base sequence in which any of the base sequences (4K)(4E) to (4J) is an exon sequence and one or more intron sequences are interposed in between.

[0120] The "sequence identity" in (4G) is as explained in (1G) in the section <1.1. γ-glutamyltransferase>. That is, the "sequence identity" in (4G) refers to the ratio (%) of identical bases to the total number of bases in SEQ ID NO: 19 when the two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two sequences.

[0121] <2. First Disclosure> The first disclosure of the present specification describes a method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione, and a prokaryotic microbial strain. <2.1. Prokaryotic Microbial Strain According to the First Disclosure of the Present Specification> One or more embodiments of the first disclosure relate to a prokaryotic microbial strain in which the expression level of one or more genes selected from a glutamate-cysteine ​​ligase gene, a glutathione synthetase gene, and a bifunctional glutathione synthetase gene is increased compared to a wild-type strain, and which is capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione.

[0122] The prokaryotic microbial strain according to one or more embodiments of the first disclosure can be used for the purpose of efficiently producing the peptide, since it can overproduce γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione and accumulate them in the medium by culturing in the medium. The prokaryotic microorganism according to one or more embodiments of the first disclosure can produce the peptide even when cultured in a medium having a total concentration of cysteine ​​and cystine of 0.5 g / L or less, or in a medium prepared without adding cysteine ​​or cystine, thereby reducing the production cost of the peptide.

[0123] The prokaryotic microbial strain according to one or more embodiments of the first disclosure is preferably a prokaryotic microbial strain capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione by inducible expression of the one or more genes. The prokaryotic microbial strain according to one or more embodiments of the first disclosure can be cultured in a medium and overproduced by inducible expression of the one or more genes, and can accumulate γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione in the medium, and can be used for the purpose of efficiently producing the peptide.

[0124] The enzyme whose gene expression level is increased in the prokaryotic microbial strain according to one or more embodiments of the first disclosure may be one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase. Specific examples of each enzyme are as described above. When the prokaryotic microbial strain is used for the purpose of producing γ-glutamylcysteine, bis-γ-glutamylcystine, and / or γ-glutamylcystine, it is preferable that the gene expression level of glutamate-cysteine ​​ligase and / or bifunctional glutathione synthetase is increased compared to a wild-type strain. When the prokaryotic microbial strain is used for the purpose of producing reduced glutathione and / or oxidized glutathione, it is preferable that the gene expression level of glutamate-cysteine ​​ligase and glutathione synthetase is increased compared to a parent strain, or the gene expression level of bifunctional glutathione synthetase is increased compared to a wild-type strain.

[0125] In one or more embodiments of the first disclosure, the prokaryotic microbial host strain may be a bacterium, particularly a microbial cell belonging to the genus Escherichia, Bacillus, Brevibacterium, or Corynebacterium, and is particularly preferably a microbial cell belonging to the genus Escherichia, and most preferably a cell of Escherichia coli. The prokaryotic microbial host strain may also be an enterobacterium. The prokaryotic microbial host strain according to one or more embodiments of the first disclosure may be a transformant in which a prokaryotic microorganism is allowed to carry a specific gene.

[0126] The term "wild strain" refers to a host strain before introduction of one or more genes selected from the glutamate-cysteine ​​ligase gene, the glutathione synthetase gene, and the bifunctional glutathione synthetase gene, and can also be referred to as a "parent strain."

[0127] Here, "the expression level of one or more genes selected from the glutamate-cysteine ​​ligase gene, the glutathione synthetase gene, and the bifunctional glutathione synthetase gene is increased compared to that of the wild strain" encompasses both a case in which the expression level of the one or more genes is increased compared to that of the wild strain when the wild strain originally expresses the one or more genes, and a case in which the wild strain does not originally express the one or more genes, in which the ability to express the one or more genes is conferred to the wild strain.

[0128] The expression level of the one or more genes can be increased by increasing the copy number of the one or more genes in the cells of the prokaryotic microbial strain, or by replacing the promoter that controls the expression of the one or more genes on the genomic DNA of the cells of the prokaryotic microbial strain with a stronger expression promoter.

[0129] Increasing the copy number of the one or more genes in the cell of a prokaryotic microorganism includes (1) introducing an expression vector containing one or more of the genes into a cell of a prokaryotic microorganism; or (2) introducing said one or more genes into the genomic DNA of a cell of a prokaryotic microorganism; This can be achieved by:

[0130] The expression vector used in the above embodiment (1) may be a plasmid vector or the like containing one or more of the genes. The expression vector is preferably capable of autonomous replication in a prokaryotic microbial cell. The expression vector preferably contains a DNA encoding one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase, and a promoter operably linked to a position where the DNA can be transcribed. The expression vector is preferably a recombinant DNA that is capable of autonomous replication in a prokaryotic microbial cell and contains a base sequence composed of a promoter, a ribosome binding sequence, a base sequence encoding the amino acid sequence of the one or more enzymes, and a transcription termination sequence.

[0131] Suitable plasmid vectors include pQEK1, pCA24N (DNA RESEARCH, 12, 191-299 (2005)), pACYC177, pACYC184 (available from Nippon Gene Co., Ltd.), pQE30, pQE60, pQE70, pQE80 and pQE9 (available from Qiagen); pTipQC1 (available from Qiagen or Hokkaido System Science), pTipRT2 (available from Hokkaido System Science); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, p Examples include NH18A and pNH46A (available from Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (available from Addgene); pRSF (available from MERCK); as well as pAC (available from Nippon Gene Co., Ltd.), pUCN18 (which can be prepared by modifying pUC18 (available from Takara Bio Co., Ltd.)), pSTV28 (available from Takara Bio Co., Ltd.), pUCNT (WO 94 / 03613), and the like.

[0132] The expression vector preferably comprises a promoter that controls the transcription of the one or more genes.

[0133] In one or more embodiments of the first disclosure, the promoter is preferably an inducible promoter.

[0134] Examples of inducible promoters include isopropyl-β-thiogalactopyranoside (IPTG)-inducible promoters, light-inducible promoters that induce gene expression under light irradiation, araBAD promoters (arabinose-inducible), rhaBAD promoters (rhamnose-inducible), tet promoters (drug-inducible), penP promoters (drug-inducible), cspA promoters (temperature-inducible promoters that respond to low temperatures), and promoters containing the tetO or lacO operator as an operator sequence, with IPTG-inducible promoters, araBAD promoters, rhaBAD promoters, tet promoters, penP promoters, cspA promoters, or promoters containing the tetO or lacO operator as an operator sequence being preferred.

[0135] Specific examples of IPTG-inducible promoters include T5 promoter, lacUV5 promoter, lac promoter, T7 promoter, lacT5 promoter, lacT7 promoter, tac promoter, etc. As the inducible promoter, IPTG-inducible promoters are particularly preferred, and among IPTG-inducible promoters, T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, or tac promoter are particularly preferred.

[0136] The promoter may be a highly active promoter obtained by modifying a conventional promoter using various reporter genes. For example, promoter activity can be increased by making the -35 and -10 regions in the promoter region closer to the consensus sequence (International Publication WO00 / 18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)) and elsewhere.

[0137] When an expression vector containing the one or more genes is introduced into a cell of a prokaryotic microorganism, the copy number of the expression vector in the cell is preferably 2 or more, more preferably 3 or more, more preferably 5 or more, more preferably 10 or more, more preferably 15 or more, and more preferably 20 or more.

[0138] When increasing the expression levels of two or more of the above genes in a prokaryotic microbial cell, the two or more genes may be contained in one expression vector, in which case the two or more genes may be placed under the control of one expression promoter, or the two or more genes may be contained in separate expression vectors.

[0139] When the one or more genes are introduced into the genomic DNA of a prokaryotic microorganism cell according to the above aspect (2), homologous recombination can be utilized.

[0140] When the promoter of one or more genes on the genomic DNA of a prokaryotic microorganism cell is replaced with a stronger expression promoter, the same promoter as that of the expression vector, more preferably an inducible promoter, can be used as the expression promoter. Specific examples of preferred promoters are as described above.

[0141] In the prokaryotic microbial strain according to one or more embodiments of the first disclosure, the degree of increase in the expression level of the one or more genes is not particularly limited. The expression level of the one or more genes can be expressed as the amount of mRNA (i.e., mRNA encoding the amino acid sequence of one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase) corresponding to the one or more genes extracted from a cell. The expression level based on this mRNA is preferably expressed as a relative value to the amount of mRNA encoding an appropriate internal standard protein. In an embodiment in which the expression level of the glutamate-cysteine ​​ligase gene is increased, the expression level of the glutamate-cysteine ​​ligase in the prokaryotic microbial strain (preferably a relative value obtained by dividing the amount of mRNA of glutamate-cysteine ​​ligase in the prokaryotic microbial strain by the amount of mRNA encoding an internal standard protein in the same strain) is preferably 5 times or more, more preferably 10 times or more, and more preferably 20 times or more than the expression level of the glutamate-cysteine ​​ligase in a wild-type strain (preferably a relative value obtained by dividing the amount of mRNA of glutamate-cysteine ​​ligase in the wild-type strain by the amount of mRNA encoding an internal standard protein in the same strain). In an embodiment in which the expression level of the glutathione synthetase gene is increased, the expression level of glutathione synthetase in a prokaryotic microbial strain (preferably, a relative value obtained by dividing the amount of glutathione synthetase mRNA in a prokaryotic microbial strain by the amount of mRNA encoding an internal standard protein in the same strain) is preferably 5 times or more, more preferably 10 times or more, and more preferably 20 times or more than the expression level of glutathione synthetase in a wild strain (preferably, a relative value obtained by dividing the amount of glutathione synthetase mRNA in a wild strain by the amount of mRNA encoding an internal standard protein in the same strain). An example of the internal standard protein is a protein encoded by hcaT (SEQ ID NO: 27), which is known as a housekeeping gene.

[0142] In the prokaryotic microbial strain according to one or more embodiments of the first disclosure, more preferably, the expression level of a gene for an enzyme having an activity of degrading cysteine, γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, or oxidized glutathione, or a glutathione uptake transporter gene is lower than that of a wild-type strain, or expression of the gene is lost. When such a prokaryotic microbial strain is cultured, the peptide is likely to accumulate in the medium.

[0143] Examples of the enzyme having the activity of decomposing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, or oxidized glutathione include γ-glutamyltransferase and tripeptide peptidase. An example of the gene for the enzyme having the activity of decomposing cysteine ​​is the tryptophanase gene tnaA. An example of the glutathione uptake transporter gene is yliABCD.

[0144] Specific examples of γ-glutamyltransferase are as described above.

[0145] Specific examples of tripeptide peptidases are as described above.

[0146] A prokaryotic microbial strain in which the expression level of a gene encoding an enzyme having activity of decomposing cysteine, γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, or oxidized glutathione, or a glutathione uptake transporter gene is lower than that of a wild-type strain, or in which expression of the gene has been lost, can be produced by a method of introducing a deletion, substitution, or addition of a base into the base sequence encoding the enzyme on the genomic DNA of the prokaryotic microbial strain. Examples of such a method include a method utilizing homologous recombination, and specifically, the method described in JP-A-2004-344029 can be used.

[0147] A more preferred embodiment of the prokaryotic microbial strain according to one or more embodiments of the first disclosure described above comprises: A prokaryotic microbial strain comprising one or more genes selected from a glutamate-cysteine ​​ligase gene, a glutathione synthetase gene, and a bifunctional glutathione synthetase gene, all of which are operably linked to an inducible promoter, the inducible promoter is an inducible promoter that, when the one or more genes are glutamate-cysteine ​​ligase genes, causes the transcription amount of the glutamate-cysteine ​​ligase gene by the prokaryotic microbial strain to be 20 times or more higher than that of a wild-type strain; The prokaryotic microbial strain is capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione by inducible expression of the one or more genes.

[0148] In the prokaryotic microbial strain according to this more preferred embodiment, the one or more genes operably linked to an inducible promoter may be maintained in a state in which they are contained in a part of the genomic DNA of the prokaryotic microbial strain, or may be maintained in a state in which they are contained in an expression vector present in the prokaryotic microbial strain. Specific examples of the expression vector are as described above.

[0149] The inducible promoter is not particularly limited as long as it makes the amount of transcription of the glutamate-cysteine ​​ligase gene by the prokaryotic microbial strain 20 times or more than that of a wild-type strain when the one or more genes are glutamate-cysteine ​​ligase genes. The amount of transcription can be evaluated based on the amount of mRNA. The glutamate-cysteine ​​ligase gene used to confirm that the inducible promoter has a predetermined expression ability is preferably the same type as the glutamate-cysteine ​​ligase gene of the wild-type strain.

[0150] Such a high expression inducible promoter can be selected from the above-mentioned examples of inducible promoters, and preferably includes an IPTG inducible promoter, a light inducible promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing a tetO or lacO operator as an operator sequence. As a high expression inducible promoter, an IPTG inducible promoter is particularly preferred, and among IPTG inducible promoters, a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter, or a tac promoter is particularly preferred.

[0151] As the high expression inducible promoter, an inducible promoter that has been modified to a highly active form by using various reporter genes as described above can also be used.

[0152] <2.2. Method for producing a useful substance by cell culture according to the first disclosure of the present specification> One or more further embodiments of the first disclosure include: A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione (hereinafter referred to as "target peptide"), comprising the steps of: The present invention relates to a method comprising culturing a prokaryotic microbial strain in which the expression level of one or more genes selected from a glutamate-cysteine ​​ligase gene, a glutathione synthetase gene, and a bifunctional glutathione synthetase gene is increased compared to a wild-type strain, in a medium having a total concentration of cysteine ​​and cystine of 0.5 g / L or less. The prokaryotic microbial strain used in this method is preferably a prokaryotic microbial strain capable of overproducing said peptide of interest by inducible expression of said one or more genes.

[0153] Another further embodiment of the first disclosure includes: The method for producing a target peptide, The method includes culturing in a medium a prokaryotic microbial strain in which the expression level of one or more genes selected from a glutamate-cysteine ​​ligase gene, a glutathione synthetase gene, and a bifunctional glutathione synthetase gene is increased compared to a wild-type strain, The method does not include adding cysteine ​​or cystine to the medium. The prokaryotic microbial strain used in this method is preferably a prokaryotic microbial strain capable of overproducing said peptide of interest by inducible expression of said one or more genes.

[0154] This method is based on the unexpected finding that the prokaryotic microbial strain according to one or more embodiments of the first disclosure described above can accumulate the target peptide containing cysteine ​​or cystine as a constituent amino acid in a medium even when cultured in a medium with a low total concentration of cysteine ​​and cystine of 0.5 g / L or less, or in a medium prepared without a step of adding cysteine ​​or cystine. According to this method, the target peptide can be produced at low cost.

[0155] The medium used in the method may be either a synthetic medium or a natural medium, so long as it contains nutrients necessary for the growth of the microorganism used in the first disclosure and for the biosynthesis of the target peptide, such as a carbon source, a nitrogen source, inorganic salts, and vitamins.

[0156] The carbon source may be any carbon source that can be assimilated by the microorganism used, and examples of the carbon source include carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0157] Examples of the nitrogen source include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate.

[0158] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0159] Examples of vitamins include biotin, thiamine, etc. Furthermore, if necessary, a substance required for the growth of the microorganism of the first disclosure (for example, a required amino acid in the case of an amino acid-requiring microorganism) can be added.

[0160] It is preferable to add at least one of a sulfur source and glycine, preferably both, to the medium. The concentration of glycine added to the medium is, for example, 100 mM to 2000 mM, preferably 400 mM to 1200 mM. The concentration of the sulfur source added to the medium is, for example, 100 mM to 2000 mM, preferably 400 mM to 1200 mM.

[0161] As the sulfur source, one or more inorganic sulfur compounds such as sulfuric acid, thiosulfuric acid, sulfurous acid, hyposulfurous acid, sulfides, or salts thereof can be added. Sulfuric acid, thiosulfuric acid, sulfurous acid, hyposulfurous acid, or sulfides may be in a free form, a salt, or any mixture thereof. The salt is not particularly limited, and examples thereof include sodium salts, calcium salts, ammonium salts, and potassium salts.

[0162] Glycine may be in a free form, a salt, or any mixture thereof. The salt is not particularly limited, and examples thereof include sulfate and hydrochloride.

[0163] The sulfur source and / or glycine can be added to the medium at the start of the culture or during the culture. The sulfur source and / or glycine can be added to the medium all at once, or can be added to the medium continuously or intermittently.

[0164] The sulfur source and / or glycine may be contained in the medium throughout the entire culture period, or may be contained in the medium only during a part of the culture period. For example, the amount of the sulfur source and glycine added does not need to be within the above range throughout the entire period of the stage of producing and accumulating the target peptide, and the sulfur source and / or glycine may be added to the medium during the culture so that the content is within the above range, and the sulfur source and / or glycine content may decrease with the passage of culture time. In addition, the sulfur source and / or glycine may be additionally added continuously or intermittently. The concentrations of medium components other than the sulfur source and / or glycine may also be changed or additionally added during the culture period.

[0165] The culture is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is 20 to 50° C., preferably 20 to 42° C., and more preferably 28 to 38° C. The pH during culture is 5 to 9, preferably 6 to 7.5. The culture time is 3 hours to 5 days, and preferably 5 hours to 3 days.

[0166] The target peptide accumulated in the culture can be collected by a conventional purification method, for example, after the culture is completed, the cells and solid matter in the culture are removed by centrifugation or the like, and the target peptide can be collected by ion exchange, concentration, or crystallization fractionation.

[0167] <3. Second Disclosure> The microorganism and the method for producing glutathione according to the second disclosure of the present specification will be described below. In the present specification, "glutathione" may be reduced glutathione, oxidized glutathione, or a mixture of reduced glutathione and oxidized glutathione. In the present specification, "glutathione" and "reduced glutathione and / or oxidized glutathione" are mutually interchangeable.

[0168] <3.1.Host microorganisms> According to one or more embodiments of the second disclosure, the microorganism serving as a host (parent strain) of the microorganism lacking the genes [1] and [2] below and having enhanced expression of the gene [3] or the gene [4] is preferably a bacterium. The bacterium may be an enterobacterium. The bacterium may be a gram-negative bacterium such as Escherichia or Pantoea, or a gram-positive bacterium such as Bacillus, Brevibacterium, or Corynebacterium, but is preferably a gram-negative bacterium, and is particularly preferably Escherichia coli.

[0169] A microorganism according to one or more embodiments of the second disclosure may be a transformant obtained by deleting a specific gene in a microorganism and retaining the specific gene in an expressible manner. <3.2. Microorganisms according to the second disclosure of the present specification> One or more embodiments of the second disclosure are directed to a microorganism that is deficient in the following genes [1] and [2], and in which expression of the following gene [3] or gene [4] is enhanced: [1] The gene encoding γ-glutamyltransferase (EC:2.3.2.2); [2] A gene encoding glutathione reductase (EC:1.8.1.7); [3] A gene encoding glutamate-cysteine ​​ligase (EC:6.3.2.2) and a gene encoding glutathione synthetase (EC:6.3.2.3); [4] A gene encoding a bifunctional glutathione synthetase, Regarding.

[0170] The microorganism has a high ability to produce glutathione by fermentation, and is therefore suitable for producing glutathione. The microorganism can produce glutathione by culturing in a medium.

[0171] The above-mentioned microorganism is more preferably a microorganism further lacking the gene described in [5] below. [5] A gene encoding tripeptide peptidase (EC:3.4.11.4).

[0172] Furthermore, the above-mentioned microorganism lacking the tripeptide peptidase gene is preferable because it has a particularly high glutathione-producing ability.

[0173] The microorganisms that serve as hosts for the microorganisms according to one or more embodiments of the second disclosure are as described above.

[0174] Deletion of a predetermined gene in a microorganism according to one or more embodiments of the second disclosure is described.

[0175] In the microorganism according to one or more embodiments of the second disclosure, the "deficiency" of the gene encoding γ-glutamyltransferase and the gene encoding glutathione reductase, or further the gene encoding tripeptide peptidase (hereinafter sometimes referred to as "target genes for deficiency") means that the activity of the enzyme encoded by the target genes for deficiency is reduced compared to the parent strain, including the case where the activity is completely lost. The microorganism according to one or more embodiments of the second disclosure is a microorganism in a state where the function of the target genes for deficiency is lost or the function is reduced, specifically, a microorganism in a state where the expression level of mRNA, which is the transcription product of the target genes for deficiency, or protein, which is the translation product, is reduced, or a microorganism in a state where the mRNA, which is the transcription product of the target genes for deficiency, or protein, which is the translation product, does not function normally as mRNA or protein.

[0176] The deletion of the target gene can be achieved, for example, by artificially modifying the gene of the parent strain of the microorganism. Such modification can be achieved, for example, by mutation treatment, gene recombination technology, gene expression suppression treatment using RNAi, gene editing, etc.

[0177] Examples of the mutation treatment include ultraviolet light irradiation and treatment with a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), or methyl methanesulfonate (MMS), which are commonly used in mutation treatments.

[0178] As gene recombination techniques, for example, known techniques (FEMS Microbiology Letters 165 (1998) 335-340, JOURNAL OF BACTERIOLOGY, Dec. 1995, p7171-7177, Curr Genet 1986; 10(8):573-578, WO 98 / 14600, etc.) can be used.

[0179] The gene encoding γ-glutamyltransferase, glutathione reductase, or tripeptide peptidase refers not only to the coding region of the amino acid sequence of each protein, but also to its expression regulatory sequence (promoter sequence, etc.), exon sequence, intron sequence, etc. When the expression regulatory sequence is modified, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases are modified in the expression regulatory sequence.

[0180] The deletion of the target gene in the second disclosure is more preferably a deletion of the target gene in the genomic DNA of a microorganism. The deletion of the target gene may be a deletion of a part or the whole of an expression regulatory sequence, or a deletion of a part or the whole of a coding region of the amino acid sequence of the enzyme. Here, "deletion" means a deletion or damage, and is preferably a deletion.

[0181] The entire gene may be deleted in the genomic DNA of the parent strain of the microorganism, including the sequences before and after the gene to be deleted. When a part or all of the coding region of the amino acid sequence of the enzyme encoded by the gene to be deleted is deleted, any region such as the N-terminal region, the internal region, or the C-terminal region may be deleted as long as the enzyme activity can be reduced. Usually, the longer the region to be deleted, the more reliably the gene can be inactivated. In addition, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. In a preferred embodiment, the microorganism is one in which at least a part of the coding region of the amino acid sequence of the gene to be deleted and / or the expression regulatory sequence in the genomic DNA, for example, a region consisting of preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and more preferably 100% of the total number of bases of the coding region and / or the expression regulatory sequence, is deleted. Particularly preferred is a microorganism in which the region from the start codon to the stop codon of the gene to be deleted is deleted in the genomic DNA.

[0182] Other examples of deletions of the target gene that result in reduced enzyme activity include damage to the gene, such as introducing an amino acid substitution (missense mutation) into the amino acid sequence coding region of the target gene on the genomic DNA, introducing a stop codon (nonsense mutation), or introducing a frameshift mutation that adds or deletes 1 to 2 bases.

[0183] In addition, the deletion of the target gene to be deleted, which reduces the enzyme activity, can also be achieved, for example, by inserting another sequence into the expression regulatory sequence or amino acid sequence coding region of the target gene on the genomic DNA. The insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. In addition, it is preferable that the sequences before and after the insertion site do not have the same reading frame. The other sequence is not particularly limited as long as it reduces or eliminates the function of the encoded protein, and examples thereof include marker genes and genes useful for producing γ-glutamyl compounds such as glutathione.

[0184] The gene to be deleted on the genomic DNA can be deleted as described above, for example, by creating an inactive gene by modifying the gene to be deleted so that it does not produce a protein that functions normally, transforming a microorganism with recombinant DNA containing the inactive gene, and causing homologous recombination between the inactive gene and the gene on the genomic DNA, thereby replacing the gene on the genomic DNA with the inactive gene. In this case, the recombinant DNA can be easily manipulated by including a marker gene according to the host's characteristics such as nutritional requirements. In addition, if the recombinant DNA is linearized by cutting with a restriction enzyme, a strain in which the recombinant DNA is integrated into the genomic DNA can be efficiently obtained. Even if a protein encoded by an inactive gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost.

[0185] In addition, for example, a linear DNA containing an arbitrary sequence, which has sequences upstream and downstream of a site to be replaced on a genomic DNA (typically, a part or all of the gene to be deleted) on both ends of the arbitrary sequence, or a linear DNA in which the sequences upstream and downstream of the site to be replaced on the genomic DNA are directly linked, is used to transform a microorganism, and homologous recombination is caused upstream and downstream of the site to be replaced on the genomic DNA of the microorganism, respectively, thereby replacing the site to be replaced with the sequence of the linear DNA in one step. The arbitrary sequence may include, for example, a marker gene sequence. The marker gene may then be removed as necessary. When removing a marker gene, sequences for homologous recombination may be added to both ends of the marker gene so that the marker gene can be efficiently removed.

[0186] The deficiency of the target gene in the microorganism can be confirmed by a decrease in the activity of the enzyme encoded by the target gene. The decrease in the activity of the enzyme can be confirmed by measuring the activity of the enzyme. For example, the activity of glutathione reductase can be measured by a known method (such as Glutathione Reductase Assay Kit product number 7510-100-K manufactured by Cosmo Bio Co., Ltd.).

[0187] The reduced transcription level of the deleted target gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the parent strain. Methods for evaluating the amount of mRNA include Northern hybridization and RT-PCR (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the parent strain.

[0188] The reduction in the amount of the enzyme encoded by the gene to be deleted can be confirmed by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). In the microorganism according to one or more embodiments of the second disclosure, the amount of the enzyme encoded by the gene to be deleted is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the parent strain.

[0189] This is followed by a description of the enhancement of expression of a given gene in a microorganism according to one or more embodiments of the second disclosure.

[0190] A microorganism in which "expression is enhanced" of a specific expression-enhanced gene (glutamate-cysteine ​​ligase gene and glutathione synthetase gene, or bifunctional glutathione synthetase gene) includes both cases in which, when the parent strain (wild strain) of the microorganism originally expresses the expression-enhanced gene, the expression level of the expression-enhanced gene is increased compared to that of the parent strain, and cases in which, when the parent strain does not originally express the expression-enhanced gene, the parent strain has been conferred the ability to express the expression-enhanced gene.

[0191] The expression level of the enhanced expression gene can be increased by increasing the copy number of the enhanced expression gene in the microbial cell, or by replacing the promoter that controls the expression of the enhanced expression gene on the genomic DNA of the microbial cell with a stronger expression promoter.

[0192] Increasing the copy number of the expression-enhancing gene in the cell of the microorganism (A) introducing an expression vector containing the expression-enhancing gene into a cell of a microorganism; or (B) introducing the expression enhancing gene into the genomic DNA of a cell of a microorganism. This can be achieved by:

[0193] The expression vector used in the embodiment (A) may be a plasmid vector or the like containing the expression enhancing gene. The expression vector is preferably capable of autonomous replication in microbial cells. The expression vector preferably contains DNA encoding one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase, and a promoter operably linked to a position where the DNA can be transcribed. The expression vector is preferably a recombinant DNA that is autonomously replicable in microbial cells and contains a base sequence composed of a promoter, a ribosome binding sequence, a base sequence encoding the amino acid sequence of the one or more enzymes, and a transcription termination sequence. The microorganism according to one or more embodiments of the second disclosure preferably carries an expression vector including a base sequence encoding the expression-enhancing gene, and the microorganism is capable of expressing the expression-enhancing gene from the expression vector.

[0194] Suitable plasmid vectors include pQEK1, pCA24N (DNA RESEARCH, 12, 191-299 (2005)), pACYC177, pACYC184 (available from Nippon Gene Co., Ltd.), pQE30, pQE60, pQE70, pQE80 and pQE9 (available from Qiagen); pTipQC1 (available from Qiagen or Hokkaido System Science), pTipRT2 (available from Hokkaido System Science); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, p Examples include NH18A and pNH46A (available from Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (available from Addgene); pRSF (available from MERCK); as well as pAC (available from Nippon Gene Co., Ltd.), pUCN18 (which can be prepared by modifying pUC18 (available from Takara Bio Co., Ltd.)), pSTV28 (available from Takara Bio Co., Ltd.), pUCNT (WO 94 / 03613), and the like.

[0195] The expression vector preferably comprises a promoter that controls the transcription of the expression enhancing gene.

[0196] In one or more embodiments of the second disclosure, the promoter is preferably an inducible promoter.

[0197] Examples of inducible promoters include isopropyl-β-thiogalactopyranoside (IPTG)-inducible promoters, light-inducible promoters that induce gene expression under light irradiation, araBAD promoters (arabinose-inducible), rhaBAD promoters (rhamnose-inducible), tet promoters (drug-inducible), penP promoters (drug-inducible), cspA promoters (temperature-inducible promoters that respond to low temperatures), and promoters containing the tetO or lacO operator as an operator sequence, with IPTG-inducible promoters, araBAD promoters, rhaBAD promoters, tet promoters, penP promoters, cspA promoters, or promoters containing the tetO or lacO operator as an operator sequence being preferred.

[0198] Specific examples of IPTG-inducible promoters include T5 promoter, lacUV5 promoter, lac promoter, T7 promoter, lacT5 promoter, lacT7 promoter, tac promoter, etc. As the inducible promoter, IPTG-inducible promoters are particularly preferred, and among IPTG-inducible promoters, T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, or tac promoter are particularly preferred.

[0199] The promoter may be a highly active promoter obtained by modifying a conventional promoter using various reporter genes. For example, the activity of the promoter can be increased by making the -35 and -10 regions in the promoter region closer to the consensus sequence (International Publication WO00 / 18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent Application 2006134574). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0200] When an expression vector containing the expression-enhancing gene is introduced into a microbial cell, the copy number of the expression vector in the cell is preferably 2 or more, more preferably 3 or more, more preferably 5 or more, more preferably 10 or more, more preferably 15 or more, and more preferably 20 or more.

[0201] When increasing the expression levels of two or more of the expression-enhancing genes in a microbial cell, two or more genes may be contained in one expression vector, in which case the two or more genes may be placed under the control of one expression promoter, or the two or more genes may be contained in separate expression vectors.

[0202] According to the embodiment (B), when the expression-enhancing gene is introduced into the genomic DNA of a microbial cell, homologous recombination can be used.

[0203] When the promoter of the gene for enhanced expression is replaced with a stronger promoter on the genomic DNA of the cell of the microorganism, the promoter may be the same as that of the expression vector, more preferably an inducible promoter. Specific examples of preferred promoters are as described above.

[0204] In the microorganism according to one or more embodiments of the second disclosure, the degree of enhanced expression (increased expression level) of the expression-enhanced gene is not particularly limited. The expression level of the expression-enhanced gene can be expressed as the amount of mRNA (i.e., mRNA encoding the amino acid sequence of one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase) corresponding to the expression-enhanced gene extracted from a cell. The expression level based on this mRNA is preferably expressed as a relative value to the amount of mRNA encoding an appropriate internal standard protein. In one or more embodiments of the microorganism in which expression of a glutamate-cysteine ​​ligase gene is enhanced, the expression amount of glutamate-cysteine ​​ligase in the microorganism (preferably a relative value obtained by dividing the amount of mRNA of glutamate-cysteine ​​ligase in the microorganism by the amount of mRNA encoding an internal standard protein in the same strain) is preferably at least 5 times, more preferably at least 10 times, and more preferably at least 20 times the expression amount of glutamate-cysteine ​​ligase in a parent strain (preferably a relative value obtained by dividing the amount of mRNA of glutamate-cysteine ​​ligase in a wild-type strain by the amount of mRNA encoding an internal standard protein in the same strain). In one or more embodiments of the microorganism in which expression of the glutathione synthetase gene is enhanced, the expression level of glutathione synthetase in the microorganism (preferably the relative value obtained by dividing the amount of glutathione synthetase mRNA in the microorganism by the amount of mRNA encoding the internal standard protein in the same strain) is preferably 5 times or more, more preferably 10 times or more, and more preferably 20 times or more than the expression level of glutathione synthetase in a wild strain (preferably the relative value obtained by dividing the amount of glutathione synthetase mRNA in the wild strain by the amount of mRNA encoding the internal standard protein in the same strain). An example of the internal standard protein is a protein encoded by the hcaT gene, known as a housekeeping gene.

[0205] <3.3. Method for Producing Glutathione According to the Second Disclosure of the Present Specification> One or more further embodiments of the second disclosure include: Cultivating a microorganism according to one or more embodiments of the second disclosure in a medium, The present invention relates to a method for producing glutathione.

[0206] According to the method for producing glutathione according to this embodiment, it is possible to efficiently produce glutathione.

[0207] The medium may be either a synthetic medium or a natural medium, so long as it contains nutrients necessary for the growth of the microorganism used in the second disclosure and for the biosynthesis of glutathione, such as a carbon source, a nitrogen source, inorganic salts, and vitamins. Preferably, M9 medium is used.

[0208] The carbon source may be any carbon source that can be assimilated by the microorganism used, and examples of the carbon source include carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0209] Examples of the nitrogen source include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate. Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0210] Examples of vitamins include biotin, thiamine, etc. Furthermore, if necessary, a substance required for growth by the microorganism according to one or more embodiments of the second disclosure (for example, a required amino acid in the case of an amino acid-requiring microorganism) can be added.

[0211] It is preferable to add at least one of a sulfur source and glycine, preferably both, to the medium. The concentration of glycine added to the medium is, for example, 100 mM to 2000 mM, preferably 400 mM to 1200 mM. The concentration of the sulfur source added to the medium is, for example, 100 mM to 2000 mM, preferably 400 mM to 1200 mM.

[0212] As the sulfur source, one or more inorganic sulfur compounds such as sulfuric acid, thiosulfuric acid, sulfurous acid, hyposulfurous acid, sulfides, or salts thereof can be added. Sulfuric acid, thiosulfuric acid, sulfurous acid, hyposulfurous acid, or sulfides may be in a free form, a salt, or any mixture thereof. The salt is not particularly limited, and examples thereof include sodium salts, calcium salts, ammonium salts, and potassium salts.

[0213] Glycine may be in a free form, a salt, or any mixture thereof. The salt is not particularly limited, and examples thereof include sulfate and hydrochloride.

[0214] The sulfur source and / or glycine can be added to the medium at the start of the culture or during the culture. The sulfur source and / or glycine can be added to the medium all at once, or can be added to the medium continuously or intermittently.

[0215] The sulfur source and / or glycine may be contained in the medium throughout the entire culture period, or may be contained in the medium only during a part of the culture period. For example, the amount of sulfur source and glycine added does not need to be within the above range throughout the entire period of the stage of producing and accumulating glutathione, and the sulfur source and / or glycine may be added to the medium during the culture so that the content is within the above range, and the sulfur source and / or glycine content may decrease with the passage of culture time. In addition, the sulfur source and / or glycine may be additionally added continuously or intermittently. The concentration of medium components other than the sulfur source and / or glycine may also be changed or additionally added during the culture period.

[0216] The culture is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is 20 to 50° C., preferably 20 to 42° C., and more preferably 28 to 38° C. The pH during culture is 5 to 9, preferably 6 to 7.5. The culture time is 3 hours to 5 days, and preferably 5 hours to 3 days.

[0217] The glutathione accumulated in the culture can be collected by a conventional purification method. For example, after the culture is completed, the bacteria and solid matter in the culture can be removed by centrifugation or the like, and then the glutathione can be collected by ion exchange, concentration, or crystallization fractionation. EXAMPLES

[0218] The first and second disclosures of the present specification will be described in more detail below with reference to examples, but the first and second disclosures of the present specification are not limited to these examples.

[0219] The genetic manipulations described below can be carried out with reference to the descriptions in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, and the like used in the genetic manipulations can be purchased from commercial suppliers and used according to their instructions. The enzymes are not particularly limited as long as they can be used in genetic manipulations.

[0220] <First Disclosure> Experimental results to illustrate the first disclosure of this specification are presented below.

[0221] [Example 1-1] Construction of glutathione synthesis gene expression vector (1) A T5 promoter, an E. coli-derived gshA gene (SEQ ID NO: 12), and an E. coli-derived gshB gene (SEQ ID NO: 14) were inserted between the SmaI site and the HindIII site of the plasmid vector pQEK1-term described in SEQ ID NO: 4. Primers were designed based on the instructions for NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and vector construction was performed according to the procedure described. The constructed plasmid vector is called pQEK1-PT5-ABEc-term. pQEK1-PT5-ABEc-term may also be written as pQEK1-PT5-gshA-gshB.

[0222] [Example 1-2] Construction of glutathione synthesis gene expression vector (2) Between the SmaI site and the HindIII site of the plasmid vector pQEK1-term described in SEQ ID NO: 4, the T5 promoter, the gshA gene derived from Escherichia coli (SEQ ID NO: 12), and the TDgshB (V260A) gene (SEQ ID NO: 18) encoding a mutant enzyme of glutathione synthase derived from the sulfur bacterium Thiobacillus denitrificans (WO2018 / 084165) were inserted. Primers were designed based on the instructions for NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and vector construction was performed according to the procedure described. The constructed plasmid vector is referred to as pQEK1-PT5-ABTd (V260A)-term. pQEK1-PT5-ABTd (V260A)-term may also be referred to as pQEK1-PT5-gshA-TDgshB (V260A).

[0223] [Example 1-3] Construction of glutathione synthesis gene expression vector (3) A T5 promoter and a SAgshF gene (SEQ ID NO: 19) encoding a bifunctional glutathione synthetase gene derived from Streptococcus agalactiae were inserted between the SmaI site and the HindIII site of the plasmid vector pQEK1-term described in SEQ ID NO: 4. Primers were designed based on the instructions for NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and vector construction was performed according to the procedure described. The constructed plasmid vector is called pQEK1-PT5-FSa-term. pQEK1-PT5-AFSa-term may also be written as pQEK1-PT5-SAgshF.

[0224] [Example 1-4] Construction of glutathione synthesis gene expression vector (4) Between the SmaI site and the HindIII site of the plasmid vector pQEK1-term described in SEQ ID NO: 4, the lac promoter, the gshA gene derived from Escherichia coli (SEQ ID NO: 12), and the TDgshB (V260A) gene (SEQ ID NO: 18) encoding a mutant enzyme of glutathione synthase derived from the sulfur bacterium Thiobacillus denitrificans (WO2018 / 084165) were inserted. Primers were designed based on the instructions for NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and vector construction was performed according to the procedure described. The constructed plasmid vector is called pQEK1-Plac-ABTd (V260A)-term. pQEK1-Plac-ABTd (V260A)-term may also be written as pQEK1-Plac-gshA-TDgshB (V260A).

[0225] [Example 1-5] Construction of glutathione synthesis gene expression vector (5) Between the SmaI site and the HindIII site of the plasmid vector pQEK1-term described in SEQ ID NO: 4, the lacUV5 promoter, the gshA gene derived from Escherichia coli (SEQ ID NO: 12), and the TDgshB (V260A) gene (SEQ ID NO: 18) encoding a mutant enzyme of glutathione synthase derived from the sulfur bacterium Thiobacillus denitrificans (WO2018 / 084165) were inserted. Primers were designed based on the instructions for NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and vector construction was performed according to the procedure described. The constructed plasmid vector is referred to as pQEK1-PlacUV5-ABTd (V260A)-term. pQEK1-PlacUV5-ABTd (V260A)-term may also be referred to as pQEK1-PlacUV5-gshA-TDgshB (V260A).

[0226] [Examples 1-6] Preparation of host strain A strain lacking the γ-glutamyltransferase gene (sequence number 21) and the tripeptide peptidase gene (sequence number 23) was prepared using the Escherichia coli strain BW25113 obtained from the Institute of Genetics, National University, and the plasmid pTH18cs1 obtained from the same institute, in the same manner as the method for preparing a cytosine deaminase-deficient strain disclosed in JP 2004-344029 A.

[0227] [Example 1-7] Preparation of a strain with enhanced glutathione synthesis genes Competent cells of the host strain prepared in Example 1-6 were prepared by a conventional method, and transformed with the plasmid vectors prepared in Examples 1-1 to 1-5 to obtain respective transformants.

[0228] [Example 1-8] Evaluation of fermentative production of glutathione The host strain (without plasmid) prepared in Example 1-6 or the glutathione synthesis gene enhanced strain prepared in Example 1-7 was inoculated into 5 mL LB medium (containing 20 μg / mL tetracycline) and cultured with shaking at 300 rpm and 30° C. for 8 hours. 1 mL of this culture was inoculated into 100 mL of M9 medium (6 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 1 mM magnesium sulfate, 0.001% thiamine-hydrochloric acid, 0.1 mM calcium chloride, 2% glucose) supplemented with 20 μg / mL tetracycline. The culture after inoculation was cultured for 18 hours at 34° C., pH 6.5, stirring at 1000 rpm, and aeration at 100 mL / min using a culture device (Able Bio Jr.8). After 18 hours of culture, 20 mL of the culture solution was inoculated into 2 L of M9 medium supplemented with 20 μg / mL tetracycline. The culture solution after the second inoculation was further cultured at 34 ° C, pH 6.7, stirring at 600 rpm, and aeration at 4 L / min using a culture device (Bioneer-Neo manufactured by Marubishi Bioengineering Co., Ltd.). During the culture, 50 w / v% glucose solution was added at any time to adjust the glucose concentration in the system so that it did not fall below 15 g / L. After 6 hours of culture, 0.1 mM isopropyl-β-thiogalactopyranoside was added, and 780 mM glycine and 780 mM sodium sulfate were also added at the same time. An appropriate amount of the culture solution was sampled 24 hours after culture, and the cells and the supernatant were separated by centrifugation. The supernatant was appropriately diluted with distilled water, and GSH and GSSG in the culture supernatant were quantified by the method described in WO2016 / 002884 to determine the total concentration. The quantitative results of the total concentration of GSH and GSSG in the culture supernatant are shown in Table 1. The culture medium used in the above experiments was substantially free of cysteine ​​and cystine, with their total concentration being less than 0.5 g / L.

[0229] [Table 1]

[0230] [Examples 1-9] Transcriptional analysis of glutamate-cysteine ​​ligase gene and glutathione synthetase gene The expression level of genes overexpressed using the plasmid was analyzed by real-time PCR. In the culture described in Example 1-8, the culture solution after the second inoculation was cultured for 6 hours, 0.1 mM isopropyl-β-thiogalactopyranoside was added, and an appropriate amount was sampled 1 hour later. RNA was extracted using NucleoSpin RNA purchased from Takara Bio Inc. according to the attached instructions. Each RNA sample was adjusted to a concentration of 50 ng / μL by diluting with water. Reverse transcription was performed using PrimeScript RT reagent Kit (Perfect Real Time) purchased from Takara Bio Inc. according to the attached instructions, and cDNA was synthesized from RNA. gshA, TDgshB (V260A), gshB, and SAgshF in each sample were quantified using TB Green Premix Ex Taq II (Tli RNaseH Plus) purchased from Takara Bio Inc. and QuantStudio 3 real-time PCR system manufactured by Thermo Fisher Scientific Co., Ltd. As an internal standard, hcaT (SEQ ID NO: 27), known as a housekeeping gene, was used. For hcaT, gshA, and gshB, the host E. coli genomic DNA was used to perform real-time PCR reactions simultaneously with the samples, and a standard curve was prepared. The amount of each gene contained in each cDNA was quantified based on the standard curve. For TDgshB(V260A), a standard curve was prepared using the plasmid pTDGSH2m15 described in WO2018 / 084165, and for SAgshF, a standard curve was prepared using the plasmid pNGSHF described in WO2016 / 017631. In the same sample, the quantitative value of each gene was divided by the quantitative value of the internal standard hcaT to standardize the expression level of each gene.The forward primer used to amplify hcaT was SEQ ID NO: 28, the reverse primer was SEQ ID NO: 29, the forward primer used to amplify gshA was SEQ ID NO: 30, the reverse primer was SEQ ID NO: 31, the forward primer used to amplify TDgshB(V260A) was SEQ ID NO: 32, the reverse primer was SEQ ID NO: 33, the forward primer used to amplify gshB was SEQ ID NO: 34, the reverse primer was SEQ ID NO: 35, and the forward primer used to amplify SAgshF was SEQ ID NO: 36, and the reverse primer was SEQ ID NO: 37.

[0231] The expression level of the E. coli-derived gshA gene by the transformants into which pQEK1-PT5-ABTd(V260A)-term, pQEK1-Plac-ABTd(V260A)-term, pQEK1-PlacUV5-ABTd(V260A)-term, or pQEK1-PT5-ABEc-term, which are plasmid vectors containing the E. coli-derived gshA gene, was introduced (expression level standardized by dividing the quantitative value of the expression level of the gshA gene of each transformant by the quantitative value of the expression level of hcaT in the same transformant) was calculated as a relative amount to the expression level of the E. coli-derived gshA gene by the untransformed host strain prepared in Example 1-6 (expression level standardized by dividing the quantitative value of the expression level of the gshA gene of the host strain by the quantitative value of the expression level of hcaT in the same host strain), which was set to 1. The relative amount was evaluated as 5 or more and less than 10 as "+", 10 or more and less than 20 as "++", and 20 or more as "+++". Similarly, the expression level of the E. coli-derived gshB gene by the transformant into which pQEK1-PT5-ABEc-term, a plasmid vector containing the E. coli-derived gshB gene, was introduced (expression level standardized by dividing the quantitative value of the expression level of the gshB gene of each transformant by the quantitative value of the expression level of hcaT in the same transformant) was calculated as a relative amount to the expression level of the E. coli-derived gshB gene by the untransformed host strain (expression level standardized by dividing the quantitative value of the expression level of the gshB gene of the host strain by the quantitative value of the expression level of hcaT in the same host strain), which was set to 1. The relative amount was evaluated as "+" when it was 5 or more and less than 10, "++" when it was 10 or more and less than 20, and "+++" when it was 20 or more. The results are shown in Table 2.

[0232] [Table 2]

[0233] The expression levels of the TDgshB (V260A) gene or the SAgshF gene by the transformants into which the plasmid vectors pQEK1-PT5-ABTd (V260A)-term, pQEK1-Plac-ABTd (V260A)-term, pQEK1-PlacUV5-ABTd (V260A)-term or pQEK1-PT5-FSa-term containing the TDgshB (V260A) gene or the SAgshF gene that the host strain, which is Escherichia coli, does not originally have were standardized by dividing the quantitative value of the expression level of each gene by the quantitative value of the hcaT gene in the same sample. As a negative control, the gene expression level was also determined in the same manner for the untransformed host strain (without plasmid). The results are shown in Table 3.

[0234] [Table 3]

[0235] <Second Disclosure> Next, experimental results for illustrating the second disclosure of this specification will be shown below.

[0236] (Analysis of glutathione concentration in culture medium) The glutathione concentration in the culture medium was measured using a high performance liquid chromatograph (HPLC, Shimadzu Corporation). The HPLC analysis conditions are as follows. Column: Develosil ODS-HG-3 4.6mm x 250mm (Nomura Chemical) Mobile phase: 30.5 g of potassium dihydrogen phosphate and 18 g of sodium heptanesulfonate were dissolved in 4.5 L of distilled water, and then the pH was adjusted to 3 with phosphoric acid. After adding 250 mL of methanol, the pH was adjusted to 3 again with phosphoric acid. Flow rate: 1mL / min Detection: UV detector, λ=210nm Column temperature: 40℃ Injection volume: 10μL

[0237] When analyzing the glutathione concentration in the culture medium, the cells were removed by centrifugation, and the supernatant was passed through a syringe filter (Advantec, φ=0.2 μm) to obtain a culture supernatant. The culture supernatant obtained was diluted 10-fold with distilled water and subjected to HPLC.

[0238] (Production Example 2-1) Preparation of BW25113Δggt strain First, a plasmid vector for disrupting the ggt (γ-glutamyltransferase) gene (SEQ ID NO: 21) was prepared. A DNA fragment (SEQ ID NO: 1) having the upstream and downstream sequences of the ggt gene was obtained by PCR using synthetic oligo DNA. The obtained fragment was digested with XbaI and HindIII, and ligated to a fragment obtained by digesting the temperature-sensitive plasmid pTH18cs1 (GenBank accession number AB019610) [Hashimoto-Gotoh, T., Gene, 241, 185-191 (2000)] with XbaI and HindIII using Ligation high Ver.2 (Toyobo) to obtain the plasmid vector pTH18cs1-ggt-UD.

[0239] Next, the BW25113Δggt strain was prepared using pTH18cs1-ggt-UD. pTH18cs1-ggt-UD was introduced into the E. coli BW25113 strain by electroporation, and the resulting transformant was spread on an LB agar plate containing 10 μg / mL of chloramphenicol and cultured at 30 ° C. The obtained transformant was shake-cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / mL of chloramphenicol, and the culture was spread on an LB agar plate containing 10 μg / mL of chloramphenicol and cultured at 42 ° C. to obtain a transformant. The obtained transformant was cultured overnight in an LB liquid medium at 42 ° C., and then spread on an LB agar plate to obtain a colony. The obtained colonies were replicated on an LB agar plate and an LB agar plate containing 10 μg / mL of chloramphenicol, and a transformant showing chloramphenicol sensitivity was selected. From the selected transformants, we isolated one strain that lacked the initiation codon to termination codon of the ggt gene on the chromosome by PCR and DNA sequence analysis, and named this gene disruptant BW25113Δggt strain. The BW25113Δggt strain is a bacterial strain that uses the E. coli BW25113 strain as a host and lacks the portion from the initiation codon to the termination codon of the ggt gene on the chromosome.

[0240] (Production Example 2-2) Preparation of BW25113ΔggtΔpepT strain First, a plasmid vector was prepared to disrupt the pepT (tripeptide peptidase) gene (SEQ ID NO: 23). A DNA fragment (SEQ ID NO: 2) having the upstream and downstream sequences of the pepT gene was obtained by PCR using synthetic oligo DNA. The obtained fragment was digested with XbaI and HindIII, and ligated with a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver.2 to obtain the plasmid vector pTH18cs1-pepT-UD.

[0241] Next, using the BW25113Δggt strain prepared in Production Example 2-1 as a parent strain, one strain in which the initiation codon to termination codon of the pepT gene on the chromosome was deleted was isolated using pTH18cs1-pepT-UD in the same manner as in Production Example 2-1. This gene-disrupted strain was named BW25113ΔggtΔpepT strain.

[0242] The BW25113ΔggtΔpepT strain is a bacterial strain that uses the E. coli BW25113 strain as a host and lacks the ggt gene and the pepT gene from the initiation codon to the termination codon on the chromosome.

[0243] (Production Example 2-3) Preparation of pQEK1-PT5-ABTd(V260A)-term First, to construct a vector for gene introduction into E. coli, the drug resistance marker was changed to a tetracycline resistance gene based on pQE-80L (QIAGEN) to construct the pQEK1 vector shown in SEQ ID NO: 3. Furthermore, a terminator sequence derived from lambda phage was inserted into the HindIII locus of pQEK1 to construct the pQEK1-term vector shown in SEQ ID NO: 4.

[0244] Next, a DNA fragment (SEQ ID NO: 5) consisting of a T5 promoter, an E. coli-derived gshA gene, and a Thiobacillus denitrificans-derived gshB gene (having a V260A mutation) was obtained by PCR using synthetic oligo DNA. The obtained fragment was ligated to a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) to obtain pQEK1-PT5-ABTd(V260A)-term shown in SEQ ID NO: 6.

[0245] (Production Example 2-4) Preparation of BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term strain The pQEK1-PT5-ABTd(V260A)-term prepared in Production Example 2-3 was introduced into the BW25113ΔggtΔpepT strain prepared in Production Example 2-2 by electroporation, and the transformants were selected by spreading on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain into which pQEK1-PT5-ABTd(V260A)-term was introduced was isolated by PCR analysis. This strain was named the BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term strain.

[0246] (Production Example 2-5) Preparation of BW25113ΔggtΔpepTΔgor strain First, a plasmid vector was prepared to disrupt the gor (glutathione reductase) gene. A DNA fragment (SEQ ID NO: 7) having the upstream and downstream sequences of the gor gene was obtained by PCR using synthetic oligo DNA. The obtained fragment was digested with XbaI and HindIII, and ligated with a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver.2 to obtain the plasmid vector pTH18cs1-gor-UD.

[0247] Next, using the BW25113ΔggtΔpepT strain prepared in Production Example 2-2 as a parent strain, one strain in which the start codon to the stop codon of the gor gene on the chromosome was deleted was isolated using pTH18cs1-gor-UD in the same manner as in Production Example 2-1. This gene-disrupted strain was named BW25113ΔggtΔpepTΔgor strain.

[0248] (Production Example 2-6) Preparation of BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term strain The pQEK1-PT5-ABTd(V260A)-term prepared in Production Example 2-3 was introduced into the BW25113 ΔggtΔpepTΔgor strain prepared in Production Example 2-5 by electroporation, and the transformants were selected by spreading on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain into which pQEK1-PT5-ABTd(V260A)-term was introduced was isolated by PCR analysis. This strain was named the BW25113 ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term strain.

[0249] (Production Example 2-7) Preparation of pQEK1-PT5-ABEc-term A DNA fragment (SEQ ID NO: 8) consisting of the T5 promoter, the E. coli-derived gshA gene, and the E. coli-derived gshB gene was obtained by PCR using synthetic oligo DNA. The obtained fragment was ligated to a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix to obtain pQEK1-PT5-ABEc-term shown in SEQ ID NO: 9.

[0250] (Production Example 2-8) Preparation of BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term strain The pQEK1-PT5-ABEc-term prepared in Production Example 2-7 was introduced into the BW25113ΔggtΔpepT strain prepared in Production Example 2-2 by electroporation, and the transformants were selected by spreading on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain into which pQEK1-PT5-ABEc-term had been introduced was isolated by PCR analysis. This strain was named the BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term strain.

[0251] (Production Example 2-9) Preparation of BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term strain The pQEK1-PT5-ABEc-term prepared in Production Example 2-3 was introduced into the BW25113ΔggtΔpepTΔgor strain prepared in Production Example 2-5 by electroporation, and the transformants were selected by spreading on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain into which pQEK1-PT5-ABEc-term had been introduced was isolated by PCR analysis. This strain was named the BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term strain.

[0252] (Production Example 2-10) Preparation of pQEK1-PT5-FSa-term A DNA fragment (SEQ ID NO: 10) consisting of the T5 promoter and the gshF gene derived from Streptococcus agalactiae was obtained by PCR using synthetic oligo DNA. The obtained fragment was ligated to a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix to obtain pQEK1-PT5-FSa-term shown in SEQ ID NO: 11.

[0253] (Production Example 2-11) Preparation of BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term strain The pQEK1-PT5-FSa-term prepared in Production Example 2-10 was introduced into the BW25113ΔggtΔpepT strain prepared in Production Example 2-2 by electroporation, and the transformants were selected by spreading on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain into which pQEK1-PT5-FSa-term was introduced was isolated by PCR analysis. This strain was named the BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term strain.

[0254] (Production Example 2-12) Preparation of BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term strain The pQEK1-PT5-FSa-term prepared in Production Example 2-10 was introduced into the BW25113ΔggtΔpepTΔgor strain prepared in Production Example 2-5 by electroporation, and the transformants were selected by spreading on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain into which pQEK1-PT5-FSa-term had been introduced was isolated by PCR analysis. This strain was named the BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term strain.

[0255] (Example 2-1) Fermentative production of glutathione by BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term strain The BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term strain obtained in Production Example 2-6 was cultured under the following conditions to produce GSH and GSSG: The strain was inoculated into 5 mL of LB medium (containing 20 μg / mL tetracycline) and cultured with shaking at 300 rpm at 30° C. for 8 hours. 1 mL of this culture was inoculated into 100 mL of M9 medium (6 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 1 mM magnesium sulfate, 0.001% thiamine-hydrochloric acid, 0.1 mM calcium chloride, 2% glucose) supplemented with 20 μg / mL tetracycline, and cultured for 18 hours at 34 ° C, pH 6.5, stirring at 1000 rpm, and aeration at 100 mL / min using a culture device (Able Bio Jr. 8). 20 mL of the culture after 18 hours of culture was inoculated into 2 L of M9 medium supplemented with 20 μg / mL tetracycline, and cultured at 34 ° C, pH 6.7, stirring at 600 rpm, and aeration at 4 L / min using a culture device (Marubishi Bio Engineering Bioneer-Neo). During the culture, a 50% (w / v) glucose solution was added at any time to maintain the glucose concentration in the system at 15 g / L or higher. After 6 hours of culture, 0.1 mM isopropyl-β-thiogalactopyranoside was added, and glycine and sodium sulfate were added at the same time to a final concentration of 100 mM. After 30 hours of culture, an appropriate amount of the culture medium was sampled, and the cells and the supernatant were separated by centrifugation. The supernatant was appropriately diluted with distilled water, and GSH and GSSG were quantified by HPLC analysis. The quantitative results are shown in Table 4.

[0256] (Comparative Example 2-1) Fermentative production of glutathione by the BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term strain The BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term strain obtained in Production Example 2-4 was cultured under the same conditions as in Example 2-1 to produce GSH and GSSG. The results are shown in Table 4.

[0257] [Table 4]

[0258] <Consideration> Comparing the results of Example 2-1 and Comparative Example 2-1 in Table 4, it can be seen that the gor gene disruption significantly increases glutathione productivity (GSH+GSSG), demonstrating that the disruption of the gor gene is effective in glutathione fermentation production.

[0259] (Example 2-2) Fermentative production of glutathione by BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term strain The BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term strain obtained in Production Example 2-9 was cultured under the same conditions as in Example 2-1 to produce GSH and GSSG. The results are shown in Table 5.

[0260] (Comparative Example 2-2) Fermentative production of glutathione by BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term strain The BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term strain obtained in Production Example 2-8 was cultured under the same conditions as in Example 2-1 to produce GSH and GSSG. The results are shown in Table 5.

[0261] [Table 5]

[0262] <Consideration> Comparing the results of Example 2-2 and Comparative Example 2-2 in Table 5, it can be seen that the gor gene disruption significantly increases glutathione productivity (GSH+GSSG), demonstrating that the disruption of the gor gene is effective in glutathione fermentation production.

[0263] (Example 2-3) Fermentative production of glutathione by BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term strain The BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term strain obtained in Production Example 2-12 was cultured under the same conditions as in Example 2-1 to produce GSH and GSSG. The results are shown in Table 6.

[0264] (Comparative Example 2-3) Fermentative production of glutathione by BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term strain The BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term strain obtained in Production Example 2-11 was cultured under the same conditions as in Example 2-1 to produce GSH and GSSG. The results are shown in Table 6.

[0265] [Table 6]

[0266] <Consideration> Comparing the results of Example 2-3 and Comparative Example 2-3 in Table 6, it can be seen that the gor gene disruption significantly increases glutathione productivity (GSH+GSSG), demonstrating that the disruption of the gor gene is effective in glutathione fermentation production.

[0267] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione, comprising the steps of: The expression level of one or more genes selected from genes encoding glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase is increased compared to that of a wild-type strain, and The gene encoding γ-glutamyltransferase and the gene encoding glutathione reductase are deleted, The method comprises culturing in a medium Escherichia coli capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione, The method does not include adding cysteine ​​or cystine to the medium other than at the time of inoculation.

2. The E. coli comprises one or more genes selected from genes encoding glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase, all of which are operably linked to an inducible promoter; the inducible promoter is an inducible promoter that, when the one or more genes are genes encoding glutamate-cysteine ​​ligase, causes the expression level of the gene encoding glutamate-cysteine ​​ligase by the E. coli to be 20-fold or more higher than that of a wild-type strain; The method of claim 1.

3. The method of claim 2, wherein the inducible promoter is an IPTG-inducible promoter, a light-inducible promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing the tetO or lacO operator as an operator sequence.

4. The method of claim 3, wherein the inducible promoter is a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter, a tac promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing a tetO or lacO operator as an operator sequence.

5. The method of claim 4, wherein the inducible promoter is a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter, or a tac promoter.

6. A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione, comprising the steps of: The expression level of one or more genes selected from genes encoding glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase is increased compared to that of a wild-type strain, and Defective in the gene encoding γ-glutamyltransferase; The method comprises culturing in a medium Escherichia coli capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione, The E. coli comprises one or more genes selected from genes encoding glutamate-cysteine ​​ligase, glutathione synthetase, and bifunctional glutathione synthetase, all of which are operably linked to an inducible promoter; the inducible promoter is a T5 promoter; The method does not include adding cysteine ​​or cystine to the medium other than at the time of inoculation.

7. The method according to any one of claims 1 to 6, comprising continuously or intermittently adding glycine to a medium for culturing E. coli.

8. E. coli lacking the following genes [1] and [2] and in which expression of the gene [3] or the gene [4] is enhanced: [1] A gene encoding γ-glutamyltransferase (EC: 2.3.2.2); [2] A gene encoding glutathione reductase (EC: 1.8.1.7); [3] A gene encoding glutamate-cysteine ​​ligase (EC: 6.3.2.2) and a gene encoding glutathione synthetase (EC: 6.3.2.3); [4] A gene encoding a bifunctional glutathione synthetase.

9. The E. coli according to claim 8, which is deleted of the following gene [5]: [5] A gene encoding tripeptide peptidase (EC: 3.4.11.4).

10. A method for producing glutathione, comprising culturing the Escherichia coli according to claim 8 or 9 in a medium.

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