Microorganisms that produce useful substances and production methods

Abstract

One or more embodiments of the present invention provide a microbial strain having an improved ability to produce γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione.　The present specification discloses a microbial strain in which [1] a gene encoding γ-glutamyltransferase and [2] a gene encoding phosphoglycerate mutase are deleted, and in which the expression of [3] a gene encoding glutamate-cysteine ligase and / or a gene encoding glutathione synthetase, or [4] a gene encoding bifunctional glutathione synthetase, is enhanced. The present specification also discloses a method for culturing the microbial strain to thereby produce the substance.

Description

[Technical Field]

[0001] One or more embodiments of the present invention relate to novel microbial strains.

[0002] One or more other embodiments of the present invention relate to a method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione. [Background technology]

[0003] Glutathione is a peptide composed of three amino acids: L-cysteine, L-glutamic acid, and glycine. It is present not only in the human body but also in many other living organisms, including other animals, plants, and microorganisms. It is an important compound for the body, with functions such as scavenging active oxygen, detoxification, and amino acid metabolism.

[0004] Glutathione exists in the body 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.

[0005] Known methods for producing glutathione include a method of producing it by fermentation using yeast (Patent Document 1) and a method of producing it by using a microorganism to produce γ-glutamylcysteine ​​synthetase or glutathione synthetase and enzymatically linking L-glutamic acid, L-cysteine, and glycine (Patent Documents 2 and 3).

[0006] Furthermore, Patent Document 4 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 recovering glutathione or γ-glutamylcysteine ​​from the culture. Example 4 of Patent Document 4 describes that when an Escherichia coli strain in which the gshA gene, a glutamate cysteine ​​ligase gene derived from Escherichia coli, and the gshB gene, a glutathione synthetase gene, were overexpressed, the glutathione concentration in the medium was 160 mg / L.

[0007] 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]

[0008] [Patent Document 1] International Publication WO2016 / 140349 [Patent Document 2] Japanese Patent Application Publication No. 60-27396 [Patent Document 3] Japanese Patent Publication No. 60-27397 [Patent Document 4] International Publication WO2008 / 126784 [Non-patent literature]

[0009] [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]

[0010] One or more embodiments of the present invention aim to solve an issue to be resolved by improving the productivity of glutathione or a related substance thereof, specifically, γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione, through fermentation by a microorganism such as a bacterium. [Means for solving the problem]

[0011] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that glutathione productivity is significantly improved in a microbial strain lacking a gene encoding phosphoglycerate mutase, and have completed the following embodiments of the present invention. [I] A microbial strain lacking the genes [1] and [2] and with enhanced expression of the gene [3] or [4]: [1] The gene encoding γ-glutamyltransferase (EC:3.4.19.13); [2] a gene encoding phosphoglycerate mutase (EC:5.4.2.11 or EC:5.4.1.12); [3] A gene encoding glutamate-cysteine ​​ligase (EC: 6.3.2.2) and / or a gene encoding glutathione synthetase (EC: 6.3.2.3); [4] A gene encoding bifunctional glutathione synthetase. [II] The microbial strain according to [I], which contains one or more genetic modifications selected from [5] to

[12] : [5] Defects in the gene encoding tryptophanase (EC:4.1.99.1); [6] Defects in the gene encoding tripeptide peptidase (EC:3.4.11.4); [7] Defects in the gene encoding glutathione reductase (EC:1.8.1.7); [8] Defects in genes encoding proteins involved in glutathione uptake; [9] enhanced expression of genes encoding proteins involved in putrescine excretion;

[10] Defects in genes encoding proteins involved in putrescine uptake;

[11] Defects in genes encoding proteins involved in putrescine synthesis;

[12] Enhanced expression of the gene encoding serine-O-acetyltransferase (EC:2.3.1.30). [III] The microbial strain according to [I] or [II], which is a bacterial transformant. [IV] The microbial strain described in [III], which is a transformant of an enterobacteria. [V] The microbial strain according to [III], which is a transformant of a Gram-negative bacterium. [VI] The microbial strain according to [III], which is a transformant of Escherichia coli. [VII] A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione, which comprises culturing the microbial strain according to any one of [I] to [VI].

[0012] This specification includes the disclosure of Japanese Patent Application No. 2020-209478, from which this application claims priority. [Effects of the Invention]

[0013] The microbial strains according to one or more embodiments of the present invention have high fermentative productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione. The production method according to one or more embodiments of the present invention is capable of efficiently producing the target substance. DETAILED DESCRIPTION OF THE INVENTION

[0014] <Host microorganism> According to one or more embodiments of the present invention, the host (parent strain) of a microbial strain having a predetermined genetic modification is preferably a prokaryotic microorganism, more 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, more preferably an Escherichia bacterium, and particularly preferably Escherichia coli.

[0015] The E. coli used as a host is not particularly limited, but is preferably a K12 strain or an E. coli strain derived from the K12 strain, such as DH10B, BW25113, DH5α, MG1655, JM109, or W3110.

[0016] A microbial strain according to one or more embodiments of the present invention may be a transformant obtained by deleting a specific gene in a host strain and retaining the specific gene.

[0017] 1. γ-Glutamyltransferase γ-Glutamyltransferase (EC: 3.4.19.13) is an enzyme that hydrolyzes γ-glutamyl peptides such as glutathione.

[0018] A "gene encoding γ-glutamyltransferase (EC:3.4.19.13)" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of γ-glutamyltransferase, and may be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene encoding γ-glutamyltransferase exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0019] Specific examples of γ-glutamyltransferase include: (1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22; (1B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22 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 C-terminus), which has γ-glutamyltransferase activity; (1C) a polypeptide consisting of 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: 22, and having γ-glutamyltransferase activity; or (1D) A fragment of any one of the polypeptides (1A) to (1C) having γ-glutamyltransferase activity. It can be.

[0020] In (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 amino acids. Furthermore, conservative amino acid substitutions are desirable for amino acid substitutions. "Conservative amino acid substitutions" refer to substitutions 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), and aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine). Hereinafter, the term "conservative amino acid substitution" will be used in this sense.

[0021] In (1C), "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein set forth in SEQ ID NO: 22, when two amino acid sequences are aligned, with gaps introduced as necessary, to maximize the degree of amino acid identity between the two. Sequence identity can be calculated using protein search systems such as BLAST or FASTA (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, S. F. et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, W. R. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Hereinafter, the term "sequence identity" of amino acid sequences will be used in the same sense.

[0022] In (1D) above, 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, more preferably 550 or more.

[0023] 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 wild-type microorganisms, the nucleotide sequence of SEQ ID NO: 21 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 21, or the nucleotide sequence of SEQ ID NO: 21 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0024] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of γ-glutamyltransferase include: (1E) the 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 the amino acid sequence of 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 the amino acid sequence of a polypeptide having γ-glutamyltransferase activity; A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having γ-glutamyltransferase activity, which is any one of the nucleotide sequences (1H), (1E), and (1G). (1I) A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences of (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 the middle. Examples include:

[0025] In (1G), "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in the nucleotide sequence of SEQ ID NO: 21, when two nucleotide sequences are aligned, with gaps introduced as necessary, to maximize the degree of amino acid identity between the two. Sequence identity can be calculated using BLAST or FASTA nucleotide sequence search systems (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, S. F. et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, W. R. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Hereinafter, the term "sequence identity" of nucleotide sequences will be used in the same sense.

[0026] In the above (1F) and (1I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0027] 2. Phosphoglycerate mutase Phosphoglycerate mutase (EC: 5.4.2.11 or EC: 5.4.1.12) is an enzyme that catalyzes the isomerization of 3-phosphoglycerate (3PG) to produce 2-phosphoglycerate (2PG). Phosphoglycerate mutases include 2,3-phosphoglycerate-dependent phosphoglycerate mutases (gpmA or gpmB) assigned to EC: 5.4.2.11 and 2,3-phosphoglycerate-independent phosphoglycerate mutases (gpmI) assigned to EC: 5.4.1.12.

[0028] A "gene encoding phosphoglycerate mutase (EC:5.4.2.11 or EC:5.4.1.12)" refers to a nucleic acid (preferably DNA) encoding the amino acid sequence of phosphoglycerate mutase, and may be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene encoding phosphoglycerate mutase has higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0029] Specific examples of 2,3-phosphoglycerate-dependent phosphoglycerate mutases include: (2-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20; (2-1B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20 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), which has 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; (2-1C) A polypeptide consisting of 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, wherein the polypeptide has 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; or (2-1D) A fragment of any one of the polypeptides (2-1A) to (2-1C) having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity. It can be. Any of the polypeptides (2-1A) to (2-1D) is an example of GpmA.

[0030] In the above (2-1B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0031] In the above (2-1D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 230 or more amino acids.

[0032] An example of DNA encoding the amino acid sequence of 2,3-phosphoglycerate-dependent phosphoglycerate mutase derived from Escherichia coli shown in SEQ ID NO: 20 is shown in SEQ ID NO: 19. However, in the genomic DNA of wild-type microorganisms, the nucleotide sequence of SEQ ID NO: 19 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 19, or the nucleotide sequence of SEQ ID NO: 19 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0033] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of 2,3-phosphoglycerate-dependent phosphoglycerate mutase include: (2-1E) the nucleotide sequence shown in SEQ ID NO: 19; (2-1F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 19 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 19), which encodes the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; (2-1G) 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: 19, which nucleotide sequence encodes the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity, which is any one of the nucleotide sequences (2-1H)(2-1E) to (2-1G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (2-1I) (2-1E) to (2-1H); (2-1J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (2-1A) to (2-1D); or A base sequence in which any of the base sequences (2-1K) (2-1E) to (2-1J) is an exon sequence and one or more intron sequences are interposed in the middle. Examples include: Any of the nucleotide sequences (2-1E) to (2-1K) is an example of the nucleotide sequence of the gpmA gene.

[0034] In the above (2-1F) and (2-1I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0035] Other specific examples of 2,3-phosphoglycerate-dependent phosphoglycerate mutases include: (2-2A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 78; (2-2B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 78 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 78 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), which has 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; (2-2C) A polypeptide consisting of 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: 78, wherein the polypeptide has 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; or (2-2D) A fragment of any one of the polypeptides (2-2A) to (2-2C) having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity. It can be. Any of the polypeptides (2-2A) to (2-2D) is an example of GpmB.

[0036] In the above (2-2B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0037] In the above (2-2D), the fragment may be a polypeptide having preferably 150 or more amino acids, more preferably 200 or more amino acids.

[0038] An example of DNA encoding the amino acid sequence of 2,3-phosphoglycerate-dependent phosphoglycerate mutase derived from Escherichia coli shown in SEQ ID NO: 78 is shown in SEQ ID NO: 77. However, in the genomic DNA of wild-type microorganisms, the nucleotide sequence of SEQ ID NO: 77 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 77, or the nucleotide sequence of SEQ ID NO: 77 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0039] That is, another specific example of the base sequence of the gene encoding the amino acid sequence of 2,3-phosphoglycerate-dependent phosphoglycerate mutase is: (2-2E) the nucleotide sequence shown in SEQ ID NO: 77; (2-2F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 77 (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: 77), which nucleotide sequence encodes the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; (2-2G) 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 set forth in SEQ ID NO: 77, which nucleotide sequence encodes the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-dependent phosphoglycerate mutase activity, which is any one of the nucleotide sequences (2-2H)(2-2E) to (2-2G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (2-2I), (2-2E), and (2-2H); (2-2J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (2-2A) to (2-2D); or A base sequence in which any of the base sequences (2-2K) (2-2E) to (2-2J) is an exon sequence and one or more intron sequences are interposed in the middle. Examples include: Any of the nucleotide sequences (2-2E) to (2-2K) is an example of the nucleotide sequence of the gpmB gene.

[0040] In the above (2-2F) and (2-2I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0041] Specific examples of 2,3-phosphoglycerate-independent phosphoglycerate mutases include: (2-3A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 80; (2-3B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 80 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 78 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), which has 2,3-phosphoglycerate-independent phosphoglycerate mutase activity; (2-3C) A polypeptide consisting of 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: 80, wherein the polypeptide has 2,3-phosphoglycerate-independent phosphoglycerate mutase activity; or (2-3D) A fragment of any one of the polypeptides (2-3A) to (2-3C) having 2,3-phosphoglycerate-independent phosphoglycerate mutase activity. It can be. Any of the polypeptides (2-3A) to (2-3D) is an example of GpmI.

[0042] In the above (2-3B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0043] In the above (2-3D), the fragment may be a polypeptide having preferably 300 or more amino acids, more preferably 400 or more, and more preferably 500 or more.

[0044] An example of DNA encoding the amino acid sequence of 2,3-phosphoglycerate-independent phosphoglycerate mutase derived from Escherichia coli shown in SEQ ID NO: 80 is shown in SEQ ID NO: 79. However, in the genomic DNA of wild-type microorganisms, the nucleotide sequence of SEQ ID NO: 79 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 79, or the nucleotide sequence of SEQ ID NO: 79 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0045] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of 2,3-phosphoglycerate-independent phosphoglycerate mutase include: (2-3E) the nucleotide sequence shown in SEQ ID NO: 79; (2-3F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 79 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 79), which encodes the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-independent phosphoglycerate mutase activity; (2-3G) 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 set forth in SEQ ID NO: 79, which nucleotide sequence encodes the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-independent phosphoglycerate mutase activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having 2,3-phosphoglycerate-independent phosphoglycerate mutase activity, which is any one of the nucleotide sequences (2-3H)(2-3E) to (2-3G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (2-3I), (2-3E), and (2-3H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (2-3J) (2-3A) to (2-3D); or A base sequence in which any of the base sequences (2-3K) (2-3E) to (2-3J) is an exon sequence and one or more intron sequences are interposed in the middle Examples include: Any of the base sequences (2-3E) to (2-3K) is an example of the base sequence of the gpmI gene.

[0046] In the above (2-3F) and (2-3I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0047] As the phosphoglycerate mutase, 2,3-phosphoglycerate-dependent phosphoglycerate mutase is particularly preferred, with GpmA being particularly preferred. As the 2,3-phosphoglycerate-dependent phosphoglycerate mutase, any one of the polypeptides (2-1A) to (2-1D) above is particularly preferred, and its base sequence is not limited, but any one of the base sequences (2-1A) to (2-1D) above can be exemplified.

[0048] <3-1. 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 the reaction of producing γ-glutamylcysteine ​​by binding it to L-glutamic acid. There are no particular limitations on its origin, structure, etc., as long as it has the activity. In this specification, this activity is referred to as glutamate-cysteine ​​ligase activity. 1 U of this activity means the activity of producing 1 μmol of γ-glutamylcysteine ​​per minute at 30°C, and is measured under the following conditions.

[0049] (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. The reaction is stopped by adding 6N hydrochloric acid. γ-Glutamylcysteine ​​in the reaction solution is quantified using high-performance liquid chromatography.

[0050] 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

[0051] The glutamic acid-cysteine ​​ligase to be used preferably has a glutamic acid-cysteine ​​ligase activity (specific activity) of 0.5 U or more per 1 mg of protein.

[0052] A "gene encoding glutamate-cysteine ​​ligase (EC:6.3.2.2)" refers to a nucleic acid (preferably DNA) encoding the amino acid sequence of glutamate-cysteine ​​ligase. A microbial strain in which expression of glutamate-cysteine ​​ligase is enhanced exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0053] The origin of glutamate-cysteine ​​ligase is not particularly limited, and glutamate-cysteine ​​ligase derived from microorganisms, animals, plants, etc. is preferred. Glutamate-cysteine ​​ligase derived from microorganisms is particularly preferred, and glutamate-cysteine ​​ligase derived from enterobacteria such as Escherichia coli, bacteria such as coryneform bacteria, eukaryotic microorganisms such as yeast, etc.

[0054] 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: 73 and SEQ ID NO: 74, respectively.

[0055] The glutamate-cysteine ​​ligase is not limited to the glutamate-cysteine ​​ligase consisting of the amino acid sequence set forth in SEQ ID NO: 74, but other polypeptides having glutamate-cysteine ​​ligase activity, such as its active mutants and orthologs from other species, can also be used. 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 using the glutamate-cysteine ​​ligase consisting of the amino acid sequence set forth in SEQ ID NO: 74 under the activity measurement conditions described above.

[0056] Specific examples of glutamate-cysteine ​​ligases include: (3-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 74; (3-1B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 74 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 74 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), which has glutamate-cysteine ​​ligase activity; (3-1C) a polypeptide consisting of 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: 74, wherein the polypeptide has 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.

[0057] 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.

[0058] 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0059] The term "glutamate-cysteine ​​ligase-encoding gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of glutamate-cysteine ​​ligase.

[0060] An example of DNA encoding the amino acid sequence of glutamate-cysteine ​​ligase derived from Escherichia coli shown in SEQ ID NO: 74 is shown in SEQ ID NO: 73. The base sequence of the nucleic acid encoding the amino acid sequence of glutamate-cysteine ​​ligase may be codon-optimized to suit the host.

[0061] That is, a specific example of the base sequence of a gene encoding the amino acid sequence of glutamate-cysteine ​​ligase is: (3-1E) the nucleotide sequence shown in SEQ ID NO: 73; (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: 73 (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: 73), which encodes the amino acid sequence of 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 set forth in SEQ ID NO: 73, which nucleotide sequence encodes the amino acid sequence of a polypeptide having glutamate-cysteine ​​ligase activity; 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 (3-1H) (3-1E) to (3-1G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (3-1I) (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:

[0062] In the above (3-1F) and (3-1I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0063] <3-2. 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 the reaction of producing glutathione by binding it to glycine. Its origin, structure, etc., are not particularly limited as long as it has the activity. In the specification, this activity is referred to as glutathione synthetase activity. 1 U of this activity means the activity of producing 1 μmol of glutathione per minute at 30°C, and is measured under the following conditions:

[0064] (Measurement conditions) The enzyme solution is added to 50 mM Tris-HCl 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. The reaction is stopped by adding 6N hydrochloric acid. Glutathione in the reaction solution is quantified using high-performance liquid chromatography.

[0065] The high performance liquid chromatography conditions are the same as those described above for the method for measuring glutamate-cysteine ​​ligase activity.

[0066] The glutathione synthetase to be used preferably has a glutathione synthetase activity (specific activity) of 0.5 U or more per 1 mg of protein.

[0067] A "gene encoding glutathione synthetase (EC:6.3.2.3)" refers to a nucleic acid (preferably DNA) encoding the amino acid sequence of glutathione synthetase. A microbial strain in which expression of glutathione synthetase is enhanced exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0068] The glutathione synthetase is not particularly limited and can be derived from microorganisms, animals, plants, etc. Glutathione synthetases derived from microorganisms are preferred, and glutathione synthetases derived from enterobacteria such as Escherichia coli, bacteria such as coryneform bacteria, eukaryotic microorganisms such as yeast, microorganisms belonging to the family Hydrogenophilales, etc. are particularly preferred.

[0069] 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, and particularly preferably a glutathione synthetase derived from the Thiobacillus denitrificans ATCC25259 strain.

[0070] (Preferred embodiment of glutathione synthetase derived from Escherichia coli or a mutant thereof) Specific examples of the nucleotide sequence of glutathione synthetase derived from Escherichia coli and the amino acid sequence encoded by said nucleotide sequence are shown in SEQ ID NO: 75 and SEQ ID NO: 76, respectively.

[0071] The glutathione synthetase is not limited to the glutathione synthetase consisting of the amino acid sequence set forth in SEQ ID NO: 76, but other polypeptides having glutathione synthetase activity, such as active mutants thereof and orthologs from other species, 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 observed when using the glutathione synthetase consisting of the amino acid sequence set forth in SEQ ID NO: 76 under the activity measurement conditions described above.

[0072] 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: 76; (3-2B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 76 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 76 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), which has glutathione synthetase activity; (3-2C) a polypeptide consisting of 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: 76, and having glutathione synthetase activity; or (3-2D) A fragment of any one of the polypeptides (3-2A) to (3-2C) having glutathione synthetase activity. It can be.

[0073] In the above (3-2D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0074] 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0075] The term "glutathione synthetase-encoding gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of glutathione synthetase.

[0076] An example of DNA encoding the amino acid sequence of glutathione synthetase derived from Escherichia coli shown in SEQ ID NO: 76 is shown in SEQ ID NO: 75. The base sequence of the nucleic acid encoding the amino acid sequence of glutathione synthetase may be codon-optimized to suit the host.

[0077] 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: (3-2E) the nucleotide sequence shown in SEQ ID NO: 75; (3-2F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 75 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 75), which encodes the amino acid sequence of 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 set forth in SEQ ID NO: 75, which nucleotide sequence encodes the amino acid sequence of a polypeptide having glutathione synthetase activity; a partial nucleotide sequence encoding the 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); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (3-2I), (3-2E), and (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 the middle Examples include:

[0078] In the above (3-2F) and (3-2I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0079] (Preferred embodiment of glutathione synthetase derived from Thiobacillus denitrificans or a mutant thereof) Another preferred example of the glutathione synthetase is a wild-type glutathione synthetase derived from Thiobacillus denitrificans ATCC25259 strain or an active mutant thereof. Specific examples of the nucleotide sequence of the wild-type glutathione synthetase from Thiobacillus denitrificans ATCC25259 strain and the amino acid sequence encoded by the nucleotide sequence are shown in SEQ ID NOs: 67 and 68, 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 a wild-type glutathione synthetase consisting of the amino acid sequence shown in SEQ ID NO: 68 under the above-mentioned activity measurement conditions.

[0080] Specific examples of the glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain or a mutant thereof include: (3-3A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 68; (3-3B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 68 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 68 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), which has glutathione synthetase activity; (3-3C) A polypeptide consisting of 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: 68, and having glutathione synthetase activity; or (3-3D) A fragment of any one of the polypeptides (3-3A) to (3-3C) having glutathione synthetase activity. It can be.

[0081] In the above (3-3D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0082] 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0083] An example of DNA encoding the amino acid sequence of glutathione synthetase from Thiobacillus denitrificans ATCC25259 strain shown in SEQ ID NO: 68 is shown in SEQ ID NO: 67. The base sequence of the nucleic acid encoding the amino acid sequence of glutathione synthetase may be codon-optimized to suit the host.

[0084] Specifically, specific examples of the nucleotide sequence of the gene encoding the amino acid sequence of the glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain or a mutant thereof include: (3-3E) the nucleotide sequence shown in SEQ ID NO: 67; (3-3F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 67 (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: 67), which encodes the amino acid sequence of 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: 67, which nucleotide sequence encodes the amino acid sequence of a polypeptide having glutathione synthetase activity; a partial nucleotide sequence encoding the 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); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (3-3I), (3-3E), and (3-3H); A nucleotide 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 the middle Examples include:

[0085] In the above (3-3F) and (3-3I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0086] (Preferred embodiment of an active mutant of glutathione synthetase from Thiobacillus denitrificans) Another preferred example of 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: 68, and the polypeptide described in International Publication WO2018 / 084165 is particularly preferred.

[0087] The active mutant specifically includes: (3-4A) The following group of amino acid sequences represented by SEQ ID NO: 68: 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 glutathione synthetase activity, which is an amino acid sequence obtained by adding, deleting, or substituting one or more amino acids among the amino acids at positions other than the amino acid positions in the amino acid sequence 3-4A (particularly preferably, a polypeptide having an amino acid sequence obtained by substituting, deleting, and / or adding, preferably deleting and / or adding, one or more amino acids in total at either or both of the N-terminus and the C-terminus of the amino acid sequence 3-4A); (3-4C) A polypeptide having glutathione synthetase activity, which is composed of 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 other sites; or (3-4D) A fragment of any one of the polypeptides (3-4A) to (3-4C) having glutathione synthetase activity. It can be.

[0088] In (3-4D) above, the fragment to be used is a polypeptide having preferably 150 or more amino acids, more preferably 200 or more amino acids, and even more preferably 300 or more amino acids.

[0089] 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0090] The amino acid sequence 3-4A is more preferably selected from the group consisting of the following in the amino acid sequence shown in SEQ ID NO: 68: 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.

[0091] The amino acid sequence 3-4A is particularly preferably the following amino acid sequence (1) to (35) of the amino acid sequence shown in SEQ ID NO: 68: (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, 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) 39th is threonine, 260th is alanine, (7) 70th position is serine, 260th position is alanine, (8) 78th position is leucine, 278th position is alanine, (9) The 101st residue is asparagine. (10) The 101st amino acid is glutamine. (11) The 101st base is serine. (12) The 101st position is serine, the 260th position is alanine, (13) The 101st amino acid is threonine. (14) The 125th amino acid is valine, the 249th amino acid is glutamic acid, (15) The 125th amino acid is valine, the 152nd amino acid is glutamine, (16) 136th amino acid is threonine, (17) Alanine at position 138, glutamine at position 149, histidine at position 241, glutamine at position 263, (18) Asparagine at position 154 and arginine at position 246. (19) 155th position is leucine, 239th position is serine, (20) The 197th amino acid is glutamine. (21) The 200th residue is serine, the 260th residue is alanine, (22) Arginine at position 226 and alanine at position 260. (23) The 227th position is serine, the 260th position is alanine, (24) Aspartic acid at position 254 and alanine at position 260. (25) The 260th residue is alanine. (26) Alanine at position 260, glycine at position 278, and valine at position 307. (27) Alanine at position 260 and alanine at position 299. (28) Alanine at position 260 and glycine at position 305. (29) Alanine at position 260 and threonine at position 310. (30) The 260th residue is cysteine. (31) The 260th residue is glycine. (32) The 260th amino acid is glutamine. (33) The 260th amino acid is threonine. (34) 262nd amino acid is cysteine, (35) The 270th residue is isoleucine. It is an amino acid sequence into which an amino acid substitution represented by any one of the following has been introduced:

[0092] A nucleotide sequence encoding the amino acid sequence of any of the polypeptides (3-4A) to (3-4D) above can be used as a "gene encoding glutathione synthetase."

[0093] An example of a nucleotide sequence encoding an active mutant (SEQ ID NO: 70) of the glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain shown in SEQ ID NO: 68, in which valine at position 260 is replaced with alanine, is shown in SEQ ID NO: 69. The nucleotide sequence of a nucleic acid encoding the active mutant of the glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain may be codon-optimized for a host. For example, SEQ ID NO: 69 shows a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 70 that has been codon-optimized for expression in Escherichia coli.

[0094] <4. Bifunctional glutathione synthetase> Bifunctional glutathione synthetase is an enzyme that has both the activity of recognizing L-cysteine ​​as a substrate in the presence of ATP and catalyzing a reaction to produce γ-glutamylcysteine ​​by binding with L-glutamic acid, and the activity of recognizing γ-glutamylcysteine ​​as a substrate in the presence of ATP and catalyzing a reaction to produce glutathione by binding with glycine, and is not particularly limited in terms of its origin, structure, etc., as long as it has these activities. In this specification, this activity is referred to as bifunctional glutathione synthetase activity. 1 U of this activity means the activity of producing 1 μmol of glutathione per minute at 30°C, and is measured under the following measurement conditions.

[0095] (Measurement conditions) The enzyme solution is added to 50 mM Tris-HCl 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. The reaction is stopped by adding 6N hydrochloric acid. Glutathione in the reaction solution is quantified using high-performance liquid chromatography.

[0096] The high performance liquid chromatography conditions are the same as those described above for the method for measuring glutamate-cysteine ​​ligase activity.

[0097] The bifunctional glutathione synthetase to be used preferably has a bifunctional glutathione synthetase activity (specific activity) of 0.5 U or more per mg of protein.

[0098] The term "gene encoding bifunctional glutathione synthetase" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of bifunctional glutathione synthetase. A microbial strain in which the expression of bifunctional glutathione synthetase is enhanced exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0099] The origin of the bifunctional glutathione synthetase is not particularly limited, and those derived from microorganisms, animals, plants, etc. Bifunctional glutathione synthetase derived from microorganisms is preferred. Particularly preferred is a bifunctional glutathione synthetase derived from bacteria, 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; or bacteria of the genus Listeria innocua. Preferred is a bifunctional glutathione synthetase derived from at least one species selected from the group consisting of bacteria of the genus Listeria, such as Listeria innocua and Listeria monocytogenes; bacteria of the genus Enterococcus, such as Enterococcus faecalis and Enterococcus faecium; bacteria of the genus Pasteurella, such as Pasteurella multocida; bacteria of the genus Mannheimia, such as Mannheimia succiniciprodecens; and bacteria of the genus Haemophilus, such as Haemophilus somnus.

[0100] 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: 71 and SEQ ID NO: 72, respectively. The nucleotide sequence of SEQ ID NO: 71 is a nucleotide sequence encoding the bifunctional glutathione synthetase derived from Streptococcus agalactiae consisting of the amino acid sequence shown in SEQ ID NO: 72, and is a nucleotide sequence adapted to the codon usage frequency in Escherichia coli.

[0101] The bifunctional glutathione synthetase is not limited to the bifunctional glutathione synthetase consisting of the amino acid sequence set forth in SEQ ID NO: 72, but other polypeptides having bifunctional glutathione synthetase activity, such as its active mutants and orthologs from other species, 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 observed when a bifunctional glutathione synthetase consisting of the amino acid sequence set forth in SEQ ID NO: 72 is used under the above-mentioned activity measurement conditions.

[0102] Specific examples of bifunctional glutathione synthetases include: (4A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 72; (4B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 72 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 72 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), which has bifunctional glutathione synthetase activity; (4C) a polypeptide consisting of 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: 72, said polypeptide having bifunctional glutathione synthetase activity; or (4D) A fragment of any one of the polypeptides (4A) to (4C) having bifunctional glutathione synthetase activity. It can be.

[0103] 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, more preferably 730 or more.

[0104] In (4B) above, "plurality" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0105] The term "gene encoding bifunctional glutathione synthetase" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of a bifunctional glutathione synthetase.

[0106] Specific examples of the base sequence of a gene encoding the amino acid sequence of a bifunctional glutathione synthetase include: (4E) the base sequence shown in SEQ ID NO: 71; (4F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 71 (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: 71), which encodes the amino acid sequence of 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: 71, wherein the nucleotide sequence encodes the amino acid sequence of 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), and (4G). A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (4I) (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 the middle. Examples include:

[0107] In the above (4F) and (4I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0108] 5. Tryptophanase Tryptophanase (EC: 4.1.99.1) is an enzyme protein that has the activity of decomposing cysteine. An example of tryptophanase in microorganisms is TnaA, and the gene encoding the amino acid sequence of TnaA is tnaA. A "gene encoding tryptophanase (EC:4.1.99.1)" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of tryptophanase, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene encoding tryptophanase exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0109] Specific examples of TnaA proteins include: (5A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 36; (5B) A polypeptide having tryptophanase activity, which is an amino acid sequence obtained by adding, deleting, or substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO: 36 (particularly preferably, a polypeptide having an amino acid sequence obtained by substituting, deleting, and / or adding, preferably deleting and / or adding, one or more amino acids in total at either or both of the N-terminus and the C-terminus of the amino acid sequence shown in SEQ ID NO: 36); (5C) a polypeptide consisting of 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: 36, and having tryptophanase activity; or (5D) A fragment of any one of the polypeptides (5A) to (5C) having tryptophanase activity. It can be.

[0110] In (5D) above, the fragment may preferably have 200 or more amino acids, more preferably 300 or more, more preferably 400 or more, and more preferably 450 or more amino acids.

[0111] In (5B) above, "plurality" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0112] The term "tnaA gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of TnaA, and is contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0113] An example of DNA encoding the amino acid sequence of TnaA derived from Escherichia coli shown in SEQ ID NO: 36 is shown in SEQ ID NO: 35. However, in the genomic DNA of wild-type microbial strains, the nucleotide sequence of SEQ ID NO: 35 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 35, or the nucleotide sequence of SEQ ID NO: 35 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0114] That is, specific examples of the gene encoding the amino acid sequence of TnaA or the nucleotide sequence of the tnaA gene are: (5E) the base sequence shown in SEQ ID NO: 35; (5F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 35 (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: 35), which encodes the amino acid sequence of a polypeptide having tryptophanase 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: 35, which nucleotide sequence encodes the amino acid sequence of a polypeptide having tryptophanase activity; A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having tryptophanase activity, which is any one of the nucleotide sequences (5H) (5E) to (5G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (5I) (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:

[0115] In the above (5F) and (5I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0116] <6. 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.

[0117] A "gene encoding a tripeptide peptidase (EC:3.4.11.4)" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of a tripeptide peptidase, and may be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene encoding tripeptidase exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0118] Specific examples of tripeptide peptidases include: (6A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24; (6B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24 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 C-terminus), which has tripeptide peptidase activity; (6C) a polypeptide consisting of 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, and having tripeptide peptidase activity; or (6D) A fragment of any one of the polypeptides (6A) to (6C) having tripeptide peptidase activity. It can be.

[0119] In (6D) above, the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more amino acids, and even more preferably 350 or more amino acids.

[0120] In (6B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0121] 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 wild-type microorganisms, the nucleotide sequence of SEQ ID NO: 23 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 23, or the nucleotide sequence of SEQ ID NO: 23 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0122] That is, a specific example of the base sequence of a gene encoding the amino acid sequence of tripeptide peptidase is: (6E) the nucleotide sequence shown in SEQ ID NO: 23; (6F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 23 (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 3'-end of the nucleotide sequence shown in SEQ ID NO: 23), which encodes the amino acid sequence of a polypeptide having tripeptide peptidase activity; (6G) 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, which nucleotide sequence encodes the amino acid sequence of a polypeptide having tripeptide peptidase activity; A partial base sequence encoding the amino acid sequence of a polypeptide having tripeptide peptidase activity, which is any one of the base sequences (6H) (6E) to (6G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (6I) (6E) to (6H); (6J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (6A) to (6D); or A base sequence in which any of the base sequences (6K)(6E) to (6J) is an exon sequence and one or more intron sequences are interposed in the middle. Examples include:

[0123] In the above (6F) and (6I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0124] 7. 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.

[0125] The term "gene encoding glutathione reductase (EC:1.8.1.7)" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of glutathione reductase, and may be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene encoding glutathione reductase exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0126] Specific examples of glutathione reductase include: (7A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26; (7B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26 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), which has glutathione reductase activity; (7C) a polypeptide consisting of 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 (7D) A fragment of any one of the polypeptides (7A) to (7C) having glutathione reductase activity. It can be.

[0127] In (7D) above, the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more amino acids, and even more preferably 400 or more amino acids.

[0128] In (7B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0129] 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 wild-type microorganisms, the nucleotide sequence of SEQ ID NO: 25 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 25, or the nucleotide sequence of SEQ ID NO: 25 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0130] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of glutathione reductase include: (7E) the nucleotide sequence shown in SEQ ID NO: 25; (7F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 25 (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: 25), which encodes the amino acid sequence of a polypeptide having glutathione reductase activity; (7G) 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 the amino acid sequence of a polypeptide having glutathione reductase activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having glutathione reductase activity, which is any one of the nucleotide sequences (7H), (7E), and (7G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (7I), (7E), and (7H); (7J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (7A) to (7D); or A base sequence in which any of the base sequences (7K)(7E) to (7J) is an exon sequence and one or more intron sequences are interposed in the middle. Examples include:

[0131] In the above (7F) and (7I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0132] 8. Proteins involved in glutathione uptake Proteins involved in glutathione uptake are proteins that have the function of taking extracellular glutathione into cells.

[0133] Examples of proteins involved in glutathione uptake in microorganisms include one or more selected from YliA (glutathione transport system ATP-binding protein), YliB (glutathione transport system substrate-binding protein), YliC (glutathione transport system permease protein), and YliD (glutathione transport system permease protein). The genes encoding the amino acid sequences of YliA, YliB, YliC, and YliD are yliA, yliB, yliC, and yliD, respectively. yliA, yliB, yliC, and yliD form an operon on the genomic DNA of the microorganism, and their expression is controlled by a promoter located upstream of yliA. The YliA, YliB, YliC, and YliD proteins may be collectively referred to as "YliABCD," and the yliA, yliB, yliC, and yliD genes may be collectively referred to as "yliABCD."

[0134] A "gene encoding a protein involved in glutathione uptake" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of a protein involved in glutathione uptake, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene has higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione than a wild-type microbial strain.

[0135] In the microbial strain according to one or more embodiments of the present invention described below, preferably, one or more genes selected from yliA, yliB, yliC, and yliD are deleted, and more preferably, the yliA, yliB, yliC, and yliD genes are deleted.

[0136] Specific examples of YliA proteins (glutathione transport system ATP-binding proteins) include: (8-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28; (8-1B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28 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 C-terminus), which has activity as YliA; (8-1C) a polypeptide consisting of 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: 28, wherein the polypeptide has activity as YliA; or (8-1D) A fragment of any one of the polypeptides (8-1A) to (8-1C) having YliA activity. It can be.

[0137] In the above (8-1B) to (8-1D) and the following (8-1F) to (8-1H), "having activity as YliA" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28, in particular, the glutathione transport system ATP binding activity.

[0138] In the above (8-1D), the fragment may be a polypeptide having preferably 400 or more amino acids, more preferably 500 or more, and even more preferably 600 or more.

[0139] In the above (8-1B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0140] The term "yliA gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of YliA, and is contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0141] An example of DNA encoding the amino acid sequence of YliA derived from Escherichia coli shown in SEQ ID NO: 28 is shown in SEQ ID NO: 27. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 27 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 27, or the nucleotide sequence of SEQ ID NO: 27 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0142] That is, specific examples of the gene encoding the amino acid sequence of YliA or the nucleotide sequence of the yliA gene are: (8-1E) the nucleotide sequence shown in SEQ ID NO: 27; (8-1F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 27 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 27), which encodes the amino acid sequence of a polypeptide having activity as YliA; (8-1G) 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 set forth in SEQ ID NO: 27, which nucleotide sequence encodes the amino acid sequence of a polypeptide having YliA activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having YliA activity, which is any one of the nucleotide sequences (8-1H) (8-1E) to (8-1G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (8-1I), (8-1E), and (8-1H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (8-1J) (8-1A) to (8-1D); or A base sequence in which any of the base sequences (8-1K)(8-1E) to (8-1J) is an exon sequence and one or more intron sequences are interposed in the middle Examples include:

[0143] In the above (8-1F) and (8-1I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0144] Specific examples of YliB proteins (glutathione transport system substrate-binding proteins) include: (8-2A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30; (8-2B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30, in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30, 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 C-terminus), which has activity as YliB; (8-2C) a polypeptide consisting of 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: 30, and having activity as YliB; or (8-2D) A fragment of any one of the polypeptides (8-2A) to (8-2C) having YliB activity. It can be.

[0145] In the above (8-2B) to (8-2D) and the following (8-2F) to (8-2H), "having activity as YliB" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30, in particular, glutathione transport system substrate binding activity.

[0146] In the above (8-2D), the fragment may be a polypeptide having preferably 300 or more amino acids, more preferably 400 or more, and more preferably 500 or more.

[0147] In the above (8-2B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0148] The term "yliB gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of YliB, and is contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0149] An example of DNA encoding the amino acid sequence of YliB derived from Escherichia coli shown in SEQ ID NO: 30 is shown in SEQ ID NO: 29. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 29 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 29, or the nucleotide sequence of SEQ ID NO: 29 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0150] That is, specific examples of the gene encoding the amino acid sequence of YliB or the nucleotide sequence of the yliB gene are: (8-2E) the nucleotide sequence shown in SEQ ID NO: 29; (8-2F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence of SEQ ID NO: 29 (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 of SEQ ID NO: 29), which encodes the amino acid sequence of a polypeptide having activity as YliB; (8-2G) 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 set forth in SEQ ID NO: 29, which nucleotide sequence encodes the amino acid sequence of a polypeptide having activity as YliB; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having YliB activity, which is any one of the nucleotide sequences (8-2H) (8-2E) to (8-2G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (8-2I), (8-2E), and (8-2H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (8-2J) (8-2A) to (8-2D); or A base sequence consisting of any of the base sequences (8-2K)(8-2E) to (8-2J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0151] In the above (8-2F) and (8-2I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0152] Specific examples of YliC proteins (glutathione transport system permease proteins) include: (8-3A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32; (8-3B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32 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), which has activity as YliC; (8-3C) a polypeptide consisting of 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: 32, wherein the polypeptide has activity as YliC; or (8-3D) A fragment of any of the polypeptides (8-3A) to (8-3C) having YliC activity. It can be.

[0153] In the above (8-3B) to (8-3D) and the following (8-3F) to (8-3H), "having activity as YliC" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32, in particular, glutathione transport system permease activity.

[0154] In the above (8-3D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0155] In the above (8-3B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0156] The term "yliC gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of YliC, and is contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0157] An example of DNA encoding the amino acid sequence of YliC derived from Escherichia coli shown in SEQ ID NO: 32 is shown in SEQ ID NO: 31. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 31 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 31, or the nucleotide sequence of SEQ ID NO: 31 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0158] That is, specific examples of the gene encoding the amino acid sequence of YliC or the nucleotide sequence of the yliC gene are: (8-3E) the nucleotide sequence shown in SEQ ID NO: 31; (8-3F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 31 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 31), which encodes the amino acid sequence of a polypeptide having activity as YliC; (8-3G) 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: 31, and encoding the amino acid sequence of a polypeptide having activity as YliC; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having YliC activity, which is any one of the nucleotide sequences (8-3H)(8-3E) to (8-3G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (8-3I), (8-3E), and (8-3H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (8-3J) (8-3A) to (8-3D); or A base sequence consisting of any of the base sequences (8-3K)(8-3E) to (8-3J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0159] In the above (8-3F) and (8-3I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0160] Specific examples of YliD proteins (glutathione transport system permease proteins) include: (8-4A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34; (8-4B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34, in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34, 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 C-terminus), which has activity as YliD; (8-4C) a polypeptide consisting of 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: 34, wherein the polypeptide has activity as YliD; or (8-4D) A fragment of any one of the polypeptides (8-4A) to (8-4C) having YliD activity. It can be.

[0161] In the above (8-4B) to (8-4D) and the below-described (8-4F) to (8-4H), "having activity as YliD" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34, in particular, glutathione transport system permease activity.

[0162] In the above (8-4D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0163] In the above (8-4B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0164] The term "yliD gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of YliD, and is contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0165] An example of DNA encoding the amino acid sequence of YliD derived from Escherichia coli shown in SEQ ID NO: 34 is shown in SEQ ID NO: 33. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 33 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 33, or the nucleotide sequence of SEQ ID NO: 33 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0166] That is, specific examples of the gene encoding the amino acid sequence of YliD or the nucleotide sequence of the yliD gene are: (8-4E) the nucleotide sequence shown in SEQ ID NO: 33; (8-4F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 33 (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: 33), which encodes the amino acid sequence of a polypeptide having activity as YliD; (8-4G) 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: 33, and encoding the amino acid sequence of a polypeptide having activity as YliD; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having YliD activity, which is any one of the nucleotide sequences (8-4H)(8-4E) to (8-4G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (8-4I), (8-4E), and (8-4H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (8-4J) (8-4A) to (8-4D); or A base sequence consisting of any of the base sequences (8-4K)(8-4E) to (8-4J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0167] In the above (8-4F) and (8-4I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0168] 9. Proteins involved in putrescine excretion Putrescine is a compound having the following structure and is biosynthesized in microbial cells. [ka]

[0169] Putrescine is known to promote protein synthesis and cell growth in microbial cells. However, the relationship between the putrescine concentration in microbial cells and the productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione has not been investigated.

[0170] A "gene encoding a protein involved in putrescine excretion" refers to a nucleic acid (preferably DNA) encoding the amino acid sequence of a protein involved in putrescine excretion. A microbial strain in which the expression of one or more genes encoding proteins involved in putrescine excretion is enhanced has higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain. This genetic modification is presumed to reduce the intracellular putrescine concentration.

[0171] The protein involved in putrescine excretion is a protein that has the function of excreting putrescine present in cells to the outside of the cells.

[0172] Proteins involved in putrescine excretion in microorganisms include one or more proteins selected from cationic peptide transport system substrate-binding proteins, cationic peptide transport system permease proteins, and cationic peptide transport system ATP-binding proteins. By enhancing the expression of one or more genes encoding any protein involved in putrescine excretion, including but not limited to these, the productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione by a microbial strain can be increased.

[0173] An example of a cationic peptide transport system substrate-binding protein is SapA. SapA is a protein derived from Escherichia coli. The cationic peptide transport system substrate-binding protein is not limited to proteins similar in amino acid sequence or three-dimensional structure to SapA, as long as it has cationic peptide transport system substrate-binding activity and is involved in putrescine excretion.

[0174] Examples of cationic peptide transport system permease proteins include SapB and SapC. SapB and SapC are proteins derived from Escherichia coli. The cationic peptide transport system permease protein is not limited to those similar in amino acid sequence or three-dimensional structure to SapB or SapC, and may be any protein that has cationic peptide transport system permease activity and is involved in putrescine excretion.

[0175] Examples of cationic peptide transport system ATP-binding proteins include SapD and SapF. SapD and SapF are proteins derived from Escherichia coli. The cationic peptide transport system ATP-binding protein is not limited to those similar in amino acid sequence or three-dimensional structure to SapD or SapF, and may be any protein that has cationic peptide transport system ATP-binding activity and is involved in putrescine excretion.

[0176] The protein involved in putrescine excretion in a microorganism is preferably one or more selected from SapA, SapB, ​​SapC, SapD, and SapF. Genes encoding the amino acid sequences of SapA, SapB, ​​SapC, SapD, and SapF are sapA, sapB, sapC, sapD, and sapF, respectively. sapA, sapB, sapC, sapD, and sapF form an operon on the genomic DNA of the microorganism, and their expression is controlled by a promoter located upstream of sapA. The SapA, SapB, ​​SapC, SapD, and SapF proteins may be collectively referred to as "SapABCDF," and the sapA, sapB, sapC, sapD, and sapF genes may be collectively referred to as "sapABCDF."

[0177] "A gene encoding a protein involved in putrescine excretion" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of a protein involved in putrescine excretion, and may be contained in the genomic DNA on the chromosome of a microbial strain.

[0178] In the microbial strain according to one or more embodiments of the present invention described below, preferably, the expression of one or more genes selected from sapA, sapB, sapC, sapD, and sapF is enhanced, and more preferably, the expression of all of the genes sapA, sapB, sapC, sapD, and sapF is enhanced.

[0179] Specific examples of SapA proteins (cationic peptide transport system substrate binding proteins) include: (9-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 38; (9-1B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 38 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 38 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 C-terminus), which has activity as SapA; (9-1C) a polypeptide consisting of 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: 38, and having activity as SapA; or (9-1D) A fragment of any one of the polypeptides (9-1A) to (9-1C) having SapA activity. It can be.

[0180] In the above (9-1B) to (9-1D) and the following (9-1F) to (9-1H), "having the activity of SapA" means having the function of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 38, in particular, the cationic peptide transport system substrate binding activity.

[0181] In the above (9-1D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more, more preferably 400 or more, and more preferably 500 or more.

[0182] In the above (9-1B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0183] The term "sapA gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SapA, and may be contained in the genomic DNA on the chromosome of a microbial strain.

[0184] An example of DNA encoding the amino acid sequence of SapA derived from Escherichia coli shown in SEQ ID NO: 38 is shown in SEQ ID NO: 37. However, in the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 37 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 37, or the nucleotide sequence of SEQ ID NO: 37 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0185] That is, specific examples of the gene encoding the amino acid sequence of SapA or the nucleotide sequence of the sapA gene are: (9-1E) the nucleotide sequence shown in SEQ ID NO: 37; (9-1F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 37 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 37), which encodes the amino acid sequence of a polypeptide having activity as SapA; (9-1G) 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 set forth in SEQ ID NO: 37, and encoding the amino acid sequence of a polypeptide having activity as SapA; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having SapA activity, which is any one of the nucleotide sequences (9-1H), (9-1E), and (9-1G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (9-1I), (9-1E), and (9-1H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (9-1J) (9-1A) to (9-1D); or A base sequence in which any of the base sequences (9-1K) (9-1E) to (9-1J) is an exon sequence and one or more intron sequences are interposed in the middle Examples include:

[0186] In the above (9-1F) and (9-1I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0187] Specific examples of SapB proteins (cationic peptide transport system permease proteins) include: (9-2A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 40; (9-2B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 40 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 40 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), which has activity as SapB; (9-2C) a polypeptide consisting of 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: 40, and having activity as SapB; or (9-2D) A fragment of any one of the polypeptides (9-2A) to (9-2C) having SapB activity. It can be.

[0188] In the above (9-2B) to (9-2D) and the following (9-2F) to (9-2H), "having activity as SapB" means having the function of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 40, in particular, cationic peptide transport system permease activity.

[0189] In the above (9-2D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0190] In the above (9-2B), "plurality" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0191] The term "sapB gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SapB, ​​and may be contained in the genomic DNA on the chromosome of a microbial strain.

[0192] An example of DNA encoding the amino acid sequence of SapB derived from Escherichia coli shown in SEQ ID NO: 40 is shown in SEQ ID NO: 39. However, in the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 39 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 39, or the nucleotide sequence of SEQ ID NO: 39 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0193] That is, specific examples of the gene encoding the amino acid sequence of SapB or the nucleotide sequence of the sapB gene are: (9-2E) the nucleotide sequence shown in SEQ ID NO: 39; (9-2F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence of SEQ ID NO: 39 (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 either or both of the 5' end and the 3' end of the nucleotide sequence of SEQ ID NO: 39), which encodes the amino acid sequence of a polypeptide having activity as SapB; (9-2G) 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 set forth in SEQ ID NO: 39, and encoding the amino acid sequence of a polypeptide having activity as SapB; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having SapB activity, which is any one of the nucleotide sequences (9-2H), (9-2E), and (9-2G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (9-2I), (9-2E), and (9-2H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (9-2J) (9-2A) to (9-2D); or A base sequence in which any of the base sequences (9-2K) (9-2E) to (9-2J) is an exon sequence and one or more intron sequences are interposed in the middle Examples include:

[0194] In the above (9-2F) and (9-2I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0195] Specific examples of SapC proteins (cationic peptide transport system permease proteins) include: (9-3A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 42; (9-3B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 42 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 42 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), which has activity as SapC; (9-3C) a polypeptide consisting of 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: 42, and having activity as SapC; or (9-3D) A fragment of any one of the polypeptides (9-3A) to (9-3C) having SapC activity. It can be.

[0196] In the above (9-3B) to (9-3D) and the following (9-3F) to (9-3H), "having activity as SapC" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 42, in particular, cationic peptide transport system permease activity.

[0197] In the above (9-3D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids.

[0198] In the above (9-3B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0199] The term "sapC gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SapC, and may be contained in genomic DNA on the chromosome of a microbial strain.

[0200] An example of DNA encoding the amino acid sequence of SapC derived from Escherichia coli shown in SEQ ID NO: 42 is shown in SEQ ID NO: 41. However, in the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 41 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 41, or the nucleotide sequence of SEQ ID NO: 41 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0201] That is, specific examples of the gene encoding the amino acid sequence of SapC or the nucleotide sequence of the sapC gene are: (9-3E) the nucleotide sequence shown in SEQ ID NO: 41; (9-3F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence of SEQ ID NO: 41 (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 of SEQ ID NO: 41), which encodes the amino acid sequence of a polypeptide having activity as SapC; (9-3G) 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 set forth in SEQ ID NO: 41, and encoding the amino acid sequence of a polypeptide having activity as SapC; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having SapC activity, which is any one of the nucleotide sequences (9-3H) (9-3E) to (9-3G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (9-3I), (9-3E), and (9-3H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (9-3J) (9-3A) to (9-3D); or A base sequence in which any of the base sequences (9-3K) (9-3E) to (9-3J) is an exon sequence and one or more intron sequences are interposed in the middle Examples include:

[0202] In the above (9-3F) and (9-3I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0203] Specific examples of SapD proteins (cationic peptide transport system ATP-binding proteins) include: (9-4A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 44; (9-4B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 44 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 44 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), which has activity as SapD; (9-4C) a polypeptide consisting of 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: 44, and having activity as SapD; or (9-4D) A fragment of any one of the polypeptides (9-4A) to (9-4C) having SapD activity. It can be.

[0204] In the above (9-4B) to (9-4D) and the following (9-4F) to (9-4H), "having activity as SapD" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 44, in particular, the ATP-binding activity of a cationic peptide transport system.

[0205] In the above (9-4D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0206] In the above (9-4B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0207] The term "sapD gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SapD, and may be contained in the genomic DNA on the chromosome of a microbial strain.

[0208] An example of DNA encoding the amino acid sequence of SapD derived from Escherichia coli shown in SEQ ID NO: 44 is shown in SEQ ID NO: 43. However, in the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 43 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 43, or the nucleotide sequence of SEQ ID NO: 43 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0209] That is, specific examples of the gene encoding the amino acid sequence of SapD or the nucleotide sequence of the sapD gene are: (9-4E) the nucleotide sequence shown in SEQ ID NO: 43; (9-4F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 43 (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: 43), which encodes the amino acid sequence of a polypeptide having activity as SapD; (9-4G) 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 set forth in SEQ ID NO: 43, and encoding the amino acid sequence of a polypeptide having SapD activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having SapD activity, which is any one of the nucleotide sequences (9-4H) (9-4E) to (9-4G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (9-4I), (9-4E), and (9-4H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (9-4J) (9-4A) to (9-4D); or A base sequence consisting of any of the base sequences (9-4K)(9-4E) to (9-4J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0210] In the above (9-4F) and (9-4I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0211] Specific examples of SapF proteins (cationic peptide transport system ATP-binding proteins) include: (9-5A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 46; (9-5B) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 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 C-terminus), which has activity as SapF; (9-5C) a polypeptide consisting of 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 set forth in SEQ ID NO: 46, and having activity as SapF; or (9-5D) A fragment of any one of the polypeptides (9-5A) to (9-5C) having SapF activity. It can be.

[0212] In the above (9-5B) to (9-5D) and the following (9-5F) to (9-5H), "having activity as SapF" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 46, in particular, the ATP-binding activity of a cationic peptide transport system.

[0213] In the above (9-5D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids.

[0214] In (9-5B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0215] The term "sapF gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SapF, and may be contained in the genomic DNA on the chromosome of a microbial strain.

[0216] An example of DNA encoding the amino acid sequence of SapF derived from Escherichia coli shown in SEQ ID NO: 46 is shown in SEQ ID NO: 45. However, in the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 45 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 45, or the nucleotide sequence of SEQ ID NO: 45 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0217] That is, specific examples of the gene encoding the amino acid sequence of SapF or the nucleotide sequence of the sapF gene are: (9-5E) the nucleotide sequence shown in SEQ ID NO: 45; (9-5F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence of SEQ ID NO: 45 (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 either or both of the 5' end and the 3' end of the nucleotide sequence of SEQ ID NO: 45), which encodes the amino acid sequence of a polypeptide having activity as SapF; (9-5G) 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 set forth in SEQ ID NO: 45, and encoding the amino acid sequence of a polypeptide having SapF activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having SapF activity, which is any one of the nucleotide sequences (9-5H)(9-5E) to (9-5G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (9-5I), (9-5E), and (9-5H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (9-5J) (9-5A) to (9-5D); or A base sequence consisting of any of the base sequences (9-5K)(9-5E) to (9-5J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0218] In the above (9-5F) and (9-5I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0219] 10. Proteins involved in putrescine uptake The protein involved in putrescine uptake is a protein that has the function of taking up extracellular putrescine into cells.

[0220] The term "gene encoding a protein involved in putrescine uptake" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of a protein involved in putrescine uptake, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene has higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione than a wild-type microbial strain.

[0221] Proteins involved in putrescine uptake in microorganisms include one or more proteins selected from putrescine transport system substrate-binding proteins, putrescine transport system ATP-binding proteins, putrescine transport system permease proteins, putrescine importers, and putrescine ornithine antiporters. Without limitation to these, any protein involved in putrescine uptake can be increased in productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione by a microbial strain by deleting one or more genes encoding the protein.

[0222] An example of a putrescine transport system substrate-binding protein is PotF. PotF is a protein derived from Escherichia coli. The putrescine transport system substrate-binding protein is not limited to those having an amino acid sequence or three-dimensional structure similar to PotF, and may be any protein that has putrescine transport system substrate-binding activity and is involved in putrescine uptake.

[0223] An example of a putrescine transport system ATP-binding protein is PotG. PotG is a protein derived from Escherichia coli. The putrescine transport system ATP-binding protein is not limited to those similar in amino acid sequence or three-dimensional structure to PotG, and may be any protein that has putrescine transport system ATP-binding activity and is involved in putrescine uptake.

[0224] Examples of putrescine transport system permease proteins include PotH and PotI. PotH and PotI are proteins derived from Escherichia coli. The putrescine transport system permease protein is not limited to those similar in amino acid sequence or three-dimensional structure to PotH or PotI, and may be any protein that has putrescine transport system permease activity and is involved in putrescine uptake.

[0225] An example of a putrescine importer is PuuP. PuuP is a protein derived from Escherichia coli. The putrescine importer is not limited to one similar in amino acid sequence or three-dimensional structure to PuuP, and may be any protein that has putrescine importer activity and is involved in putrescine uptake.

[0226] An example of a putrescine ornithine antiporter is PotE. PotE is a protein derived from Escherichia coli. The putrescine ornithine antiporter is not limited to those having an amino acid sequence or a three-dimensional structure similar to PotE, and may be any protein that has putrescine ornithine antiporter activity and is involved in putrescine uptake.

[0227] The protein involved in putrescine uptake in a microorganism is preferably one or more selected from PotF, PotG, PotH, PotI, PuuP, and PotE. The genes encoding the amino acid sequences of PotF, PotG, PotH, PotI, PuuP, and PotE are potF, potG, potH, potI, puuP, and potE, respectively. Of these, potF, potG, potH, and potI form an operon on the genomic DNA of the microorganism, and their expression is controlled by a promoter located upstream of potF. The PotF, PotG, PotH, and PotI proteins may be collectively referred to as "PotFGHI," and the potF, potG, potH, and potI genes may be collectively referred to as "potFGHI."

[0228] The term "gene encoding a protein involved in putrescine uptake" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of a protein involved in putrescine uptake, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0229] In the microbial strain according to one or more embodiments of the present invention described below, preferably, a gene encoding one or more selected from a putrescine transport system substrate-binding protein, a putrescine transport system ATP-binding protein, and a putrescine transport system permease protein is deleted, and more preferably, all of these genes are deleted.

[0230] The microbial strains according to one or more embodiments of the present invention described below are preferably deleted in the gene encoding the putrescine importer.

[0231] The microbial strains according to one or more embodiments of the present invention described below are preferably deleted in the gene encoding the putrescine ornithine antiporter.

[0232] In the microbial strain according to one or more embodiments of the present invention described below, preferably, one or more genes selected from potF, potG, potH, potI, puuP, and potE are deleted, and more preferably, the potF, potG, potH, potI, and puuP genes are deleted, or the puuP gene is deleted, or the potE gene is deleted.

[0233] Specific examples of PotF proteins (putrescine transport system substrate binding proteins) include: (10-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 54; (10-1B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 54, in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 54, 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 C-terminus), which has activity as PotF; (10-1C) a polypeptide consisting of 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: 54, wherein the polypeptide has activity as PotF; or (10-1D) A fragment of any one of the polypeptides (10-1A) to (10-1C) having PotF activity. It can be.

[0234] In the above (10-1B) to (10-1D) and the below-described (10-1F) to (10-1H), "having PotF activity" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 54, in particular, the putrescine transport system substrate binding activity.

[0235] In the above (10-1D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more amino acids, and even more preferably 350 or more amino acids.

[0236] In the above (10-1B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0237] The term "potF gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of PotF, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0238] An example of DNA derived from Escherichia coli that encodes the amino acid sequence of PotF shown in SEQ ID NO: 54 is shown in SEQ ID NO: 53. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 53 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 53, or the nucleotide sequence of SEQ ID NO: 53 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0239] That is, specific examples of the gene encoding the amino acid sequence of PotF or the base sequence of the potF gene are: (10-1E) the nucleotide sequence shown in SEQ ID NO: 53; (10-1F) A nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 53 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 53), which encodes the amino acid sequence of a polypeptide having activity as PotF; (10-1G) 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 set forth in SEQ ID NO: 53, which nucleotide sequence encodes the amino acid sequence of a polypeptide having PotF activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having PotF activity, which is any one of the nucleotide sequences (10-1H) (10-1E) to (10-1G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (10-1I) (10-1E) to (10-1H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (10-1J) (10-1A) to (10-1D); or A base sequence consisting of any of the base sequences (10-1K)(10-1E) to (10-1J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0240] In (10-1F) and (10-1I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0241] Specific examples of PotG proteins (putrescine transport system ATP-binding proteins) include: (10-2A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 56; (10-2B) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 56 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 56 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 C-terminus), which has activity as PotG; (10-2C) a polypeptide consisting of 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: 56, said polypeptide having PotG activity; or (10-2D) A fragment of any one of the polypeptides (10-2A) to (10-2C) having PotG activity. It can be.

[0242] In the above (10-2B) to (10-2D) and the following (10-2F) to (10-2H), "having PotG activity" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 56, in particular, the putrescine transport system ATP binding activity.

[0243] In the above (10-2D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 300 or more amino acids, and even more preferably 350 or more amino acids.

[0244] In (10-2B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0245] The term "potG gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of PotG, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0246] An example of DNA derived from Escherichia coli that encodes the amino acid sequence of PotG shown in SEQ ID NO: 56 is shown in SEQ ID NO: 55. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 55 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 55, or the nucleotide sequence of SEQ ID NO: 55 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0247] That is, specific examples of the gene encoding the amino acid sequence of PotG or the base sequence of the potG gene are: (10-2E) the nucleotide sequence shown in SEQ ID NO: 55; (10-2F) A nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence of SEQ ID NO: 55 (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 either or both of the 5' end and the 3' end of the nucleotide sequence of SEQ ID NO: 55), which encodes the amino acid sequence of a polypeptide having activity as PotG; (10-2G) 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 set forth in SEQ ID NO: 55, wherein the nucleotide sequence encodes the amino acid sequence of a polypeptide having PotG activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having PotG activity, which is any one of the nucleotide sequences (10-2H)(10-2E) to (10-2G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (10-2I), (10-2E), and (10-2H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (10-2J) (10-2A) to (10-2D); or A base sequence consisting of any of the base sequences (10-2K)(10-2E) to (10-2J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0248] In (10-2F) and (10-2I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0249] Specific examples of PotH proteins (putrescine transport system permease proteins) include: (10-3A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 58; (10-3B) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 58 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 58 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), which has activity as PotH; (10-3C) a polypeptide consisting of 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: 58, wherein the polypeptide has activity as PotH; or (10-3D) A fragment of any one of the polypeptides (10-3A) to (10-3C) having PotH activity. It can be.

[0250] In the above (10-3B) to (10-3D) and the following (10-3F) to (10-3H), "having activity as PotH" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 58, in particular, putrescine transport system permease activity.

[0251] In the above (10-3D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0252] In (10-3B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0253] The term "potH gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of PotH, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0254] An example of DNA encoding the amino acid sequence of PotH derived from Escherichia coli shown in SEQ ID NO: 58 is shown in SEQ ID NO: 57. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 57 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 57, or the nucleotide sequence of SEQ ID NO: 57 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0255] That is, specific examples of the gene encoding the amino acid sequence of PotH or the base sequence of the potH gene are: (10-3E) the nucleotide sequence shown in SEQ ID NO: 57; (10-3F) A nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 57 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 57), which encodes the amino acid sequence of a polypeptide having activity as PotH; (10-3G) 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 set forth in SEQ ID NO: 57, which nucleotide sequence encodes the amino acid sequence of a polypeptide having activity as PotH; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having PotH activity, which is any one of the nucleotide sequences (10-3H)(10-3E) to (10-3G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (10-3I), (10-3E), and (10-3H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (10-3J) (10-3A) to (10-3D); or A base sequence consisting of any of the base sequences (10-3K)(10-3E) to (10-3J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0256] In (10-3F) and (10-3I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0257] Specific examples of PotI proteins (putrescine transport system permease proteins) include: (10-4A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 60; (10-4B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 60, in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 60, 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 C-terminus), which has activity as PotI; (10-4C) a polypeptide consisting of 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 set forth in SEQ ID NO: 60, said polypeptide having activity as PotI; or (10-4D) A fragment of any one of the polypeptides (10-4A) to (10-4C) having PotI activity. It can be.

[0258] In the above (10-4B) to (10-4D) and the following (10-4F) to (10-4H), "having PotI activity" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 60, in particular, putrescine transport system permease activity.

[0259] In the above (10-4D), the fragment may be a polypeptide having preferably 150 or more amino acids, more preferably 200 or more amino acids, and even more preferably 250 or more amino acids.

[0260] In (10-4B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0261] The term "potI gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of PotI, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0262] An example of DNA derived from Escherichia coli that encodes the amino acid sequence of PotI shown in SEQ ID NO: 60 is shown in SEQ ID NO: 59. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 59 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 59, or the nucleotide sequence of SEQ ID NO: 59 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0263] That is, specific examples of the gene encoding the amino acid sequence of PotI or the base sequence of the potI gene are: (10-4E) the nucleotide sequence shown in SEQ ID NO: 59; (10-4F) A nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence of SEQ ID NO: 59 (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 either or both of the 5' end and the 3' end of the nucleotide sequence of SEQ ID NO: 59), which encodes the amino acid sequence of a polypeptide having activity as PotI; (10-4G) 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 set forth in SEQ ID NO: 59, which nucleotide sequence encodes the amino acid sequence of a polypeptide having PotI activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having PotI activity, which is any one of the nucleotide sequences (10-4H)(10-4E) to (10-4G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (10-4I), (10-4E), and (10-4H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (10-4J) (10-4A) to (10-4D); or A base sequence consisting of any of the base sequences (10-4K)(10-4E) to (10-4J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0264] In the above (10-4F) and (10-4I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0265] Specific examples of PuuP proteins (putrescine importers) include: (10-5A) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 62; (10-5B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 62 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 62 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), which has activity as PuuP; (10-5C) a polypeptide consisting of 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 set forth in SEQ ID NO: 62, said polypeptide having activity as PuuP; or (10-5D) A fragment of any one of the polypeptides (10-5A) to (10-5C) having PuuP activity. It can be.

[0266] In the above (10-5B) to (10-5D) and the following (10-5F) to (10-5H), "having activity as PuuP" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 62, in particular, putrescine importer activity.

[0267] In the above (10-5D), the fragment may be a polypeptide having preferably 300 or more amino acids, more preferably 400 or more amino acids, and even more preferably 450 or more amino acids.

[0268] In the above (10-5B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0269] The term "puuP gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of PuuP, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0270] An example of DNA derived from Escherichia coli that encodes the amino acid sequence of PuuP shown in SEQ ID NO: 62 is shown in SEQ ID NO: 61. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 61 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 61, or the nucleotide sequence of SEQ ID NO: 61 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0271] That is, specific examples of the gene encoding the amino acid sequence of PuuP or the base sequence of the puuP gene are: (10-5E) the nucleotide sequence shown in SEQ ID NO: 61; (10-5F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 61 (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: 61), which nucleotide sequence encodes the amino acid sequence of a polypeptide having activity as PuuP; (10-5G) 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 set forth in SEQ ID NO: 61, and encoding the amino acid sequence of a polypeptide having activity as PuuP; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having PuuP activity, which is any one of the nucleotide sequences (10-5H)(10-5E) to (10-5G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (10-5I), (10-5E), and (10-5H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (10-5J) (10-5A) to (10-5D); or A base sequence consisting of any of the base sequences (10-5K)(10-5E) to (10-5J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0272] In the above (10-5F) and (10-5I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0273] Specific examples of PotE proteins (putrescine ornithine antiporters) include: (10-6A) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 64; (10-6B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 64, in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 64, 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), which has activity as PotE; (10-6C) a polypeptide consisting of 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: 64, wherein the polypeptide has activity as PotE; or (10-6D) A fragment of any one of the polypeptides (10-6A) to (10-6C) having PotE activity. It can be.

[0274] In the above (10-6B) to (10-6D) and the following (10-6F) to (10-6H), "having PotE activity" means having the function of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 64, in particular, putrescine ornithine antiporter activity.

[0275] In the above (10-6D), the fragment may be a polypeptide having preferably 300 or more amino acids, more preferably 400 or more amino acids, and even more preferably 420 or more amino acids.

[0276] In the above (10-6B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0277] The term "potE gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of PotE, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0278] An example of DNA encoding the amino acid sequence of PotE derived from Escherichia coli shown in SEQ ID NO: 64 is shown in SEQ ID NO: 63. However, in the genomic DNA of a wild-type microbial strain before the gene is deleted, the nucleotide sequence of SEQ ID NO: 63 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 63, or the nucleotide sequence of SEQ ID NO: 63 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0279] That is, specific examples of the gene encoding the amino acid sequence of PotE or the base sequence of the potE gene are: (10-6E) the nucleotide sequence shown in SEQ ID NO: 63; (10-6F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 63 (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: 63), which encodes the amino acid sequence of a polypeptide having activity as PotE; (10-6G) 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: 63, and encoding the amino acid sequence of a polypeptide having activity as PotE; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having PotE activity, which is any one of the nucleotide sequences (10-6H)(10-6E) to (10-6G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (10-6I), (10-6E), and (10-6H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (10-6J) (10-6A) to (10-6D); or A base sequence consisting of any of the base sequences (10-6K)(10-6E) to (10-6J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0280] In the above (10-6F) and (10-6I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0281] 11. Proteins involved in putrescine synthesis Putrescine is biosynthesized intracellularly in microbial strains, and multiple enzyme proteins are known to be involved. The term "gene encoding a protein involved in putrescine synthesis" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of a protein involved in putrescine synthesis, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted. A microbial strain deleted in the gene has higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione than a wild-type microbial strain. In microorganisms, proteins known to be involved in putrescine synthesis include the enzyme protein EC: 4.1.1.19 (arginine decarboxylase), the enzyme protein EC: 3.5.3.11 (agmatinase), and the enzyme protein EC: 4.1.1.17 (ornithine decarboxylase). Not limited to these, as long as the protein is involved in putrescine synthesis, the productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione by a microbial strain can be increased by deleting one or more of the genes encoding the protein.

[0282] A specific example of an enzyme protein in EC:4.1.1.19 (arginine decarboxylase) is SpeA. SpeA is a protein derived from Escherichia coli. The enzyme protein in EC:4.1.1.19 is not limited to those similar in amino acid sequence or three-dimensional structure to SpeA, but may be any protein that has the enzyme activity defined in EC:4.1.1.19 and is involved in putrescine synthesis.

[0283] A specific example of an enzyme protein (agmatinase) in EC:3.5.3.11 is SpeB. SpeB is a protein derived from Escherichia coli. The enzyme protein in EC:3.5.3.11 is not limited to those similar in amino acid sequence or three-dimensional structure to SpeB, but may be any protein that has the enzyme activity defined in EC:3.5.3.11 and is involved in putrescine synthesis.

[0284] An example of an enzyme protein (ornithine decarboxylase) in EC:4.1.1.17 is SpeC. SpeC is a protein derived from Escherichia coli. The enzyme protein in EC:4.1.1.17 is not limited to those similar in amino acid sequence or three-dimensional structure to SpeC, but may be any protein that has the enzyme activity defined in EC:4.1.1.17 and is involved in putrescine synthesis.

[0285] The enzyme proteins in EC:4.1.1.19 and EC:3.5.3.11 are enzyme proteins that catalyze reactions in the pathway for synthesizing putrescine from arginine. The enzyme protein in EC:4.1.1.17 is an enzyme protein that catalyzes reactions in the pathway for synthesizing putrescine from ornithine. The genes encoding the amino acid sequences of SpeA, SpeB, and SpeC are speA, speB, and speC, respectively.

[0286] Specific examples of SpeA proteins include: (11-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 48; (11-1B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 48 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 48 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), which has an enzyme activity defined in EC: 4.1.1.19; (11-1C) A polypeptide consisting of 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: 48, wherein the polypeptide has an enzyme activity defined in EC: 4.1.1.19; or (11-1D) A fragment of any of the polypeptides (11-1A) to (11-1C) having the enzymatic activity defined in EC: 4.1.1.19. It can be.

[0287] In the above (11-1B) to (11-1D) and the below-described (11-1F) to (11-1H), the "enzyme activity defined in EC:4.1.1.19" refers to arginine decarboxylase activity, and refers to, for example, the activity of catalyzing a reaction catalyzed by a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:48.

[0288] In the above (11-1D), 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, more preferably 600 or more.

[0289] In the above (11-1B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0290] The term "speA gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SpeA, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0291] An example of DNA encoding the amino acid sequence of SpeA derived from Escherichia coli shown in SEQ ID NO: 48 is shown in SEQ ID NO: 47. However, in the genomic DNA of wild-type microbial strains, the nucleotide sequence of SEQ ID NO: 47 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 47, or the nucleotide sequence of SEQ ID NO: 47 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0292] That is, specific examples of the gene encoding the amino acid sequence of SpeA or the nucleotide sequence of the speA gene are: (11-1E) the nucleotide sequence shown in SEQ ID NO: 47; (11-1F) A nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 47 (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: 47), which encodes the amino acid sequence of a polypeptide having an enzyme activity defined in EC: 4.1.1.19; (11-1G) 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: 47, wherein the nucleotide sequence encodes the amino acid sequence of a polypeptide having an enzyme activity defined in EC: 4.1.1.19; a partial nucleotide sequence of any one of the nucleotide sequences (11-1H) (11-1E) to (11-1G) encoding the amino acid sequence of a polypeptide having the enzyme activity defined in EC: 4.1.1.19; A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (11-1I), (11-1E), and (11-1H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (11-1J) (11-1A) to (11-1D); or A base sequence in which any of the base sequences (11-1K) (11-1E) to (11-1J) is an exon sequence and one or more intron sequences are interposed in the middle. Examples include:

[0293] In the above (11-1F) and (11-1I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0294] Specific examples of SpeB proteins include: (11-2A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 50; (11-2B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 50, in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 50, 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), which has an enzyme activity defined in EC:3.5.3.11; (11-2C) A polypeptide consisting of 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: 50, wherein the polypeptide has an enzyme activity defined in EC: 3.5.3.11; or (11-2D) A fragment of any of the polypeptides (11-2A) to (11-2C) having the enzymatic activity defined in EC: 3.5.3.11. It can be.

[0295] In the above (11-2B) to (11-2D) and the below-described (11-2F) to (11-2H), the "enzyme activity defined by EC:3.5.3.11" refers to agmatinase activity, and refers to, for example, the activity of catalyzing a reaction catalyzed by a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:50.

[0296] In the above (11-2D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0297] In the above (11-2B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0298] The term "speB gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SpeB, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0299] An example of DNA encoding the amino acid sequence of SpeB derived from Escherichia coli shown in SEQ ID NO: 50 is shown in SEQ ID NO: 49. However, in the genomic DNA of wild-type microbial strains, the nucleotide sequence of SEQ ID NO: 49 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 49, or the nucleotide sequence of SEQ ID NO: 49 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0300] That is, specific examples of the gene encoding the amino acid sequence of SpeB or the nucleotide sequence of the speB gene are: (11-2E) the nucleotide sequence shown in SEQ ID NO: 49; (11-2F) A nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 49 (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: 49), which encodes the amino acid sequence of a polypeptide having an enzyme activity defined in EC: 3.5.3.11; (11-2G) 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 set forth in SEQ ID NO: 49, wherein the nucleotide sequence encodes the amino acid sequence of a polypeptide having an enzyme activity defined in EC: 3.5.3.11; a partial nucleotide sequence of any of the nucleotide sequences (11-2H) (11-2E) to (11-2G) encoding the amino acid sequence of a polypeptide having the enzyme activity defined in EC:3.5.3.11; A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (11-2I), (11-2E), and (11-2H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (11-2J) (11-2A) to (11-2D); or A base sequence consisting of any of the base sequences (11-2K)(11-2E) to (11-2J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0301] In the above (11-2F) and (11-2I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0302] Specific examples of SpeC proteins include: (11-3A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 52; (11-3B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 52 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 52 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), which has an enzyme activity defined in EC: 4.1.1.17; (11-3C) A polypeptide consisting of 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: 52, wherein the polypeptide has an enzyme activity defined in EC: 4.1.1.17; or (11-3D) A fragment of any of the polypeptides (11-3A) to (11-3C) having the enzymatic activity defined in EC: 4.1.1.17. It can be.

[0303] In the above (11-3B) to (11-3D) and the below-described (11-3F) to (11-3H), the "enzyme activity defined in EC:4.1.1.17" refers to ornithine decarboxylase activity, and refers to, for example, the activity of catalyzing a reaction catalyzed by a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:52.

[0304] In the above (11-3D), 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, more preferably 600 or more, more preferably 700 or more.

[0305] In the above (11-3B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0306] The term "speC gene" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of SpeC, and can be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted.

[0307] An example of DNA encoding the amino acid sequence of SpeC derived from Escherichia coli shown in SEQ ID NO: 52 is shown in SEQ ID NO: 51. However, in the genomic DNA of wild-type microbial strains, the nucleotide sequence of SEQ ID NO: 51 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 51, or the nucleotide sequence of SEQ ID NO: 51 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0308] That is, specific examples of the gene encoding the amino acid sequence of SpeC or the nucleotide sequence of the speC gene are: (11-3E) the nucleotide sequence shown in SEQ ID NO: 51; (11-3F) A nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 51 (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 either or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 51), which encodes the amino acid sequence of a polypeptide having an enzyme activity defined in EC: 4.1.1.17; (11-3G) 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: 51, wherein the nucleotide sequence encodes the amino acid sequence of a polypeptide having an enzyme activity defined in EC: 4.1.1.17; a partial nucleotide sequence of any one of the nucleotide sequences (11-3H) (11-3E) to (11-3G) encoding the amino acid sequence of a polypeptide having the enzyme activity defined in EC: 4.1.1.17; A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (11-3I), (11-3E), and (11-3H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (11-3J) (11-3A) to (11-3D); or A base sequence consisting of any of the base sequences (11-3K) (11-3E) to (11-3J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0309] In the above (11-3F) and (11-3I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0310] 12. Serine-O-acetyltransferase Serine-O-acetyltransferase (EC: 2.3.1.30) is an enzyme that catalyzes the reaction of acetylating L-serine in a CoA-dependent manner to produce O-acetylcysteine, and its origin, structure, etc. are not particularly limited as long as it has the activity.

[0311] The origin of serine-O-acetyltransferase is not particularly limited, and serine-O-acetyltransferase derived from microorganisms, animals, plants, etc. is preferably derived from microorganisms, and particularly preferably from enterobacteria such as Escherichia coli, bacteria such as coryneform bacteria, or eukaryotic microorganisms such as yeast.

[0312] A "gene encoding serine-O-acetyltransferase (EC:2.3.1.30)" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of serine-O-acetyltransferase. A microbial strain in which the expression of serine-O-acetyltransferase is enhanced exhibits higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0313] Specific examples of the base sequence of serine-O-acetyltransferase derived from Escherichia coli and the amino acid sequence encoded by said base sequence are shown in SEQ ID NO: 65 and SEQ ID NO: 66, respectively.

[0314] The serine-O-acetyltransferase is not limited to the serine-O-acetyltransferase consisting of the amino acid sequence set forth in SEQ ID NO: 66, but also includes other polypeptides having serine-O-acetyltransferase activity, such as its active mutants and orthologs from other species. The other polypeptides having serine-O-acetyltransferase activity are 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 of catalyzing the reaction of acetylating L-serine in a CoA-dependent manner to produce O-acetylcysteine, as compared to when the serine-O-acetyltransferase consisting of the amino acid sequence set forth in SEQ ID NO: 66 is used.

[0315] Specific examples of serine-O-acetyltransferases include: (12A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 66; (12B) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 66 in which one or more amino acids have been added, deleted, or substituted (particularly preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 66 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), which has serine-O-acetyltransferase activity; (12C) A polypeptide consisting of 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: 66, said polypeptide having serine-O-acetyltransferase activity; or (12D) A fragment of any one of the polypeptides (12A) to (12C) having serine-O-acetyltransferase activity. It can be.

[0316] In the above (12D), the fragment may be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids.

[0317] In (12B), "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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0318] The term "gene encoding serine-O-acetyltransferase (EC:2.3.1.30)" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of serine-O-acetyltransferase.

[0319] An example of DNA encoding the amino acid sequence of serine-O-acetyltransferase derived from Escherichia coli shown in SEQ ID NO: 66 is shown in SEQ ID NO: 65. The nucleotide sequence of the nucleic acid encoding the amino acid sequence of serine-O-acetyltransferase may be codon-optimized to suit the host. In the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 65 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 65, or the nucleotide sequence of SEQ ID NO: 65 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0320] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of serine-O-acetyltransferase include: (12E) the nucleotide sequence shown in SEQ ID NO: 65; (12F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 65 (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: 65), which encodes the amino acid sequence of a polypeptide having serine-O-acetyltransferase activity; (12G) 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 set forth in SEQ ID NO: 65, wherein the nucleotide sequence encodes the amino acid sequence of a polypeptide having serine-O-acetyltransferase activity; a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having serine-O-acetyltransferase activity, which is any one of the nucleotide sequences (12H)(12E) to (12G); A nucleotide sequence in which one or more silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (12I), (12E), and (12H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (12J) (12A) to (12D); or A base sequence consisting of any of the base sequences (12K)(12E) to (12J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0321] In the above (12F) and (12I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0322] <13. Enolase> Enolase (phosphopyruvate hydratase) (EC: 4.2.1.11) is an enzyme that catalyzes the conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate. The enolase-catalyzed reaction is downstream of the glycolytic reaction that converts 2PG from 3PG, which is catalyzed by phosphoglycerate mutase.

[0323] A "gene encoding enolase (phosphopyruvate hydratase) (EC:4.2.1.11)" refers to a nucleic acid (preferably DNA) that encodes the amino acid sequence of enolase, and may be contained in the genomic DNA on the chromosome of a wild-type microbial strain before the gene is deleted or weakened. A microbial strain in which the gene encoding enolase has been deleted or weakened has higher productivity of γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione compared to a wild-type microbial strain.

[0324] Specific examples of enolases include: (13A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 82; (13B) A polypeptide having an enolase activity, which is 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: 82 (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 C-terminus of the amino acid sequence shown in SEQ ID NO: 82); (13C) A polypeptide having enolase 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: 82; or (13D) A fragment of any one of the polypeptides (13A) to (13C) having enolase activity. It can be.

[0325] In (13B), "plurality" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution.

[0326] In the above (13D), the fragment may be a polypeptide having preferably 300 or more amino acids, more preferably 400 or more amino acids.

[0327] An example of DNA encoding the amino acid sequence of enolase derived from Escherichia coli shown in SEQ ID NO: 82 is shown in SEQ ID NO: 81. However, in the genomic DNA of wild-type microorganisms, the nucleotide sequence of SEQ ID NO: 81 does not necessarily exist as is, but may exist as a mutant sequence of the nucleotide sequence of SEQ ID NO: 81, or the nucleotide sequence of SEQ ID NO: 81 or its mutant sequence may be an exon sequence with one or more intron sequences intervening therein.

[0328] That is, a specific example of the base sequence of the gene encoding the amino acid sequence of enolase is: (13E) the nucleotide sequence shown in SEQ ID NO: 81; (13F) a nucleotide sequence in which one or more nucleotides have been added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 81 (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: 81), which encodes the amino acid sequence of a polypeptide having enolase activity; (13G) 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: 81, which nucleotide sequence encodes the amino acid sequence of a polypeptide having enolase activity; A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having enolase activity, which is any one of the nucleotide sequences (13H)(13E) to (13G); A nucleotide sequence in which one to several silent mutations (nucleotide substitutions that do not change the encoded amino acid residues) have been introduced into any of the nucleotide sequences (13I), (13E), and (13H); A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides (13J) (13A) to (13D); or A base sequence consisting of any of the base sequences (13K)(13E) to (13J) as an exon sequence, with one or more intron sequences intervening in the middle. Examples include:

[0329] In the above (13F) and (13I), "plurality" refers to, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0330] <Microbial strain according to the present invention> One or more embodiments of the present invention may include: A microbial strain lacking the genes [1] and [2] and with enhanced expression of the gene [3] or [4]: [1] The gene encoding γ-glutamyltransferase (EC:3.4.19.13); [2] a gene encoding phosphoglycerate mutase (EC:5.4.2.11 or EC:5.4.1.12); [3] A gene encoding glutamate-cysteine ​​ligase (EC: 6.3.2.2) and / or a gene encoding glutathione synthetase (EC: 6.3.2.3); [4] A gene encoding bifunctional glutathione synthetase Regarding.

[0331] The microbial strain has a high ability to produce γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione by fermentation.

[0332] The microbial strain preferably has a higher ability to produce γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione by fermentation than the ability of the host strain (wild-type strain or parent strain) to produce the substances before the introduction of the specified genetic modification.

[0333] When the microbial strain is used for producing γ-glutamylcysteine, bis-γ-glutamylcystine, and / or γ-glutamylcystine, it preferably has the genetic modification [3] of the genetic modifications [3] and [4]. In this case, the genetic modification [3] is preferably enhanced expression of a gene encoding glutamate-cysteine ​​ligase.

[0334] When the microorganism is used to produce reduced glutathione and / or oxidized glutathione, it may have either or both of the genetic modifications [3] and [4]. In this case, the genetic modification [3] may be enhancement of expression of only one of the gene encoding glutamate-cysteine ​​ligase and the gene encoding glutathione synthetase, but it is more preferable that the expression of both be enhanced.

[0335] More preferably, the microbial strain further comprises one or more of the following genetic modifications [5] to

[12] . [5] Defects in the gene encoding tryptophanase (EC:4.1.99.1); [6] Defects in the gene encoding tripeptide peptidase (EC:3.4.11.4); [7] Defects in the gene encoding glutathione reductase (EC:1.8.1.7); [8] Defects in genes encoding proteins involved in glutathione uptake; [9] enhanced expression of genes encoding proteins involved in putrescine excretion;

[10] Defects in genes encoding proteins involved in putrescine uptake;

[11] Defects in genes encoding proteins involved in putrescine synthesis;

[12] Enhanced expression of the gene encoding serine-O-acetyltransferase (EC:2.3.1.30).

[0336] A microbial strain having one or more of the genetic modifications [5] to

[12] can produce γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione in excess compared to the host strain. Therefore, when combined with genetic modifications including deletion of the genes [1] and [2] and enhanced expression of the genes [3] or [4], the substances can be produced particularly efficiently.

[0337] In a more preferred embodiment of the microbial strain, the microbial strain has preferably two or more, more preferably three or more, more preferably four or more, and more preferably six or more of the genetic modifications [5] to

[12] . In a more preferred embodiment of the microbial strain, the microbial strain has one or more of the genetic modifications [9] to

[11] , and even more preferably has two or more, more preferably three or more, more preferably four or more, and more preferably all of the genetic modifications [5], [6], [7], [8], and

[12] , and has one or more (particularly preferably at least

[11] ) of the genetic modifications [9] to

[11] .

[0338] More preferably, the microbial strain further comprises the following genetic modification

[13] :

[13] Weakening of the gene encoding enolase (phosphopyruvate hydratase) (EC:4.2.1.11).

[0339] Microorganisms that serve as hosts for the microbial strains according to one or more embodiments of the present invention have been described above.

[0340] Preferred examples of genes to be deleted or whose expression is enhanced in the microbial strain according to one or more embodiments of the present invention are as described above.

[0341] In accordance with one or more embodiments of the present invention, enhanced expression of a given gene in a microbial strain is described.

[0342] One or more genes whose expression is to be enhanced as specified in [3], [4], [9], and

[12] above may be referred to as "expression-enhanced genes." The following explanation can be applied independently to each gene whose expression is to be enhanced. A microbial strain in which the expression of an expression-enhanced gene is enhanced encompasses both cases in which, when a host strain (wild-type strain or parent strain) of the microbial strain originally expresses the expression-enhanced gene, the expression level of the expression-enhanced gene is increased compared to the host strain, and cases in which, when a host strain does not originally express the expression-enhanced gene, the ability to express the expression-enhanced gene has been imparted to the host strain.

[0343] The expression level of the expression-enhanced gene can be increased by replacing the promoter that controls the expression of the expression-enhanced gene on the genomic DNA of the microbial cell with a stronger expression promoter, or by increasing the copy number of the expression-enhanced gene within the microbial cell.

[0344] When the promoter of the expression-enhancing gene is replaced with a stronger expression promoter on the genomic DNA of a microbial cell, preferred examples of the expression promoter include the ompF promoter, tac promoter, trc promoter, ompA promoter, cysK promoter, and lpp promoter. An example of a nucleotide sequence in which the ompF promoter and SD sequence are linked is shown in SEQ ID NO: 7. An example of a nucleotide sequence in which the tac promoter and SD sequence are linked is shown in SEQ ID NO: 14. An example of a nucleotide sequence in which the trc promoter and SD sequence are linked is shown in SEQ ID NO: 15. An example of a nucleotide sequence in which the ompA promoter and SD sequence are linked is shown in SEQ ID NO: 16. An example of a nucleotide sequence in which the cysK promoter and SD sequence are linked is shown in SEQ ID NO: 17. An example of a nucleotide sequence in which the lpp promoter and SD sequence are linked is shown in SEQ ID NO: 18.

[0345] An inducible promoter may be used as the expression promoter, and the above expression promoter may be functionally linked to an operator sequence to form an inducible promoter.

[0346] Examples of inducible promoters include isopropyl-β-thiogalactopyranoside (IPTG)-inducible promoters, light-inducible promoters that induce gene expression under light irradiation, the araBAD promoter (arabinose-inducible), the rhaBAD promoter (rhamnose-inducible), the tet promoter (drug-inducible), the penP promoter (drug-inducible), the cspA promoter (temperature-inducible promoter that responds to low temperatures), and promoters containing the tetO or lacO operator as an operator sequence, with IPTG-inducible promoters, the araBAD promoter, the rhaBAD promoter, the tet promoter, the penP promoter, the cspA promoter, and promoters containing the tetO or lacO operator as an operator sequence being preferred.

[0347] Specific examples of IPTG-inducible promoters include the lacUV5 promoter, lac promoter, lacT5 promoter, lacT7 promoter, and the T5 promoter, T7 promoter, tac promoter, etc., which are operably linked to an operator sequence to make them IPTG-inducible. As the inducible promoter, an IPTG-inducible promoter is particularly preferred, and among IPTG-inducible promoters, the T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, and tac promoter are particularly preferred.

[0348] The promoter may be a highly active version of a conventional promoter modified by using various reporter genes. For example, promoter activity can be increased by adjusting the -35 and -10 regions of the promoter region to a consensus sequence (International Publication No. 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)).

[0349] Increasing the copy number of the expression-enhancing gene in the cells of the microbial strain to enhance expression of the expression-enhancing gene includes: (A) introducing an expression vector containing the expression-enhancing gene into cells of a microbial strain; or (B) introducing the expression-enhancing gene into the genomic DNA of cells of the microbial strain. This can be achieved by:

[0350] The expression vector used in the above aspect (A) can 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 a predetermined protein and a promoter operably linked to a position where the DNA can be transcribed. The expression vector can be configured to be capable of expressing the expression-enhancing gene in the cells of the microbial strain. The expression vector is preferably recombinant DNA that is autonomously replicable in microbial cells and contains a base sequence composed of a promoter, a ribosome binding sequence, the base sequence of the expression-enhancing gene, and a transcription termination sequence.

[0351] The microbial strain according to one or more embodiments of the present invention preferably harbors an expression vector comprising a nucleotide sequence encoding the expression-enhancing gene.

[0352] 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); and pAC (available from Nippon Gene Co., Ltd.), pUCN18 (which can be prepared by modifying pUC18 (available from Takara Bio Inc.)), pSTV28 (available from Takara Bio Inc.), and pUCNT (WO 94 / 03613).

[0353] The expression vector preferably contains a promoter that controls the transcription of the expression-enhancing gene, and more preferably contains an inducible promoter. Preferred examples of the promoter are as described above.

[0354] When an expression vector containing the expression-enhancing gene is introduced into the cells of a microbial strain, the copy number of the expression vector in the cells 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.

[0355] When increasing the expression levels of two or more of the expression-enhancing genes in cells of a microbial strain, two or more genes may be contained in a single expression vector, in which case the two or more genes may be placed under the control of a single expression promoter, or each of the two or more genes may be placed under the control of a different expression promoter. Alternatively, the two or more genes may each be contained in a separate expression vector.

[0356] According to the aspect (B), when the expression-enhancing gene is introduced into the genomic DNA of the cells of the microbial strain, homologous recombination can be used. The expression-enhancing gene is preferably introduced into the genomic DNA as DNA containing a promoter, a ribosome binding sequence, the base sequence of the expression-enhancing gene, and a transcription termination sequence. The DNA containing the base sequence of the expression-enhancing gene can be configured so that the expression-enhancing gene can be expressed in the cells of the microbial strain under the control of the promoter.

[0357] In a microbial strain according to one or more embodiments of the present invention, the degree of expression enhancement (increase in 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 corresponding to the expression-enhanced gene extracted from cells. This mRNA-based expression level is preferably expressed as a relative value to the amount of mRNA encoding an appropriate internal standard protein.

[0358] Next, the deletion of a specific gene in a microbial strain according to one or more embodiments of the present invention will be described.

[0359] In the above [1], [2], [5], [6], [7], [8],

[10] , and

[11] , the gene to be deleted may be referred to as the "gene to be deleted." The "deletion" of the gene to be deleted means that the activity of the protein encoded by the gene to be deleted is reduced compared to the host strain, including the complete loss of activity. The following explanation can be applied independently to each gene to be deleted. Microbial strains according to one or more embodiments of the present invention are those in which the function of the gene to be deleted is lost or the function is reduced. Specific examples include microbial strains in which the expression levels of mRNA, which is the transcription product of the gene to be deleted, or protein, which is the translation product, of the gene to be deleted are reduced, or in which the mRNA, which is the transcription product of the gene to be deleted, or protein, which is the translation product of the gene to be deleted, do not function normally as mRNA or protein.

[0360] The deletion of the target gene can be achieved, for example, by artificially modifying the gene of the host strain by, for example, mutation treatment, genetic recombination technology, gene expression suppression treatment using RNAi, gene editing, etc.

[0361] Examples of mutation treatments include ultraviolet irradiation and treatment with mutagens commonly used in mutation treatments, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0362] Known techniques for genetic recombination can be used (e.g., FEMS Microbiology Letters 165 (1998) 335-340, JOURNAL OF BACTERIOLOGY, December 1995, p. 7171-7177, Curr Genet 1986; 10(8):573-578, WO98 / 14600, etc.).

[0363] The gene encoding the predetermined protein described in [1], [2], [5], [6], [7], [8],

[10] , and

[11] above 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.

[0364] The deletion of the target gene is more preferably a deletion of the target gene in the genomic DNA of the microbial strain. The deletion of the target gene may be a deletion of part or all of an expression regulatory sequence, or a deletion of part or all of the coding region of the amino acid sequence of each of the proteins. Here, "deletion" means a deletion or damage, and preferably a deletion.

[0365] The entire gene may be deleted in the genomic DNA of the host strain, including the sequences before and after the gene to be deleted. When deleting part or all of the coding region of the amino acid sequence of the protein encoded by the gene to be deleted, any coding region, such as the N-terminal region, internal region, or C-terminal region, may be deleted as long as the activity of the protein is reduced. Generally, the longer the deleted region, the more reliably the gene can be inactivated. Furthermore, it is preferable that the sequences before and after the deleted region do not have the same reading frame. In a preferred embodiment, the microbial strain has deleted in its genomic DNA at least a portion of the coding region of the amino acid sequence and / or expression regulatory sequence of the gene to be deleted, 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, or more than 100% of the total number of bases in the coding region and / or expression regulatory sequence. Particularly preferred is a microbial strain having deleted in its genomic DNA at least the region from the start codon to the stop codon of the gene to be deleted.

[0366] Other examples of deletions of the gene to be deleted that result in reduced protein activity include damage to the gene to be deleted, such as introducing an amino acid substitution (missense mutation) into the amino acid sequence coding region of the gene to be deleted on the genomic DNA, introducing a stop codon (nonsense mutation), or introducing a frameshift mutation that adds or deletes 1 to 2 bases.

[0367] Furthermore, the deletion of the target gene, which reduces the activity of the protein, 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 in any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. Furthermore, 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, but examples include marker genes and genes useful for producing target substances such as glutathione.

[0368] Deleting the target gene in genomic DNA as described above can be achieved, for example, by creating an inactive gene by modifying the target gene so that it does not produce a normally functioning protein, transforming a host strain with recombinant DNA containing the inactive gene, and causing homologous recombination between the inactive gene and the gene in the genomic DNA, thereby replacing the target gene in the genomic DNA with the inactive gene. In this case, incorporating a marker gene into the recombinant DNA according to the host's nutrient requirements and other traits can facilitate manipulation. Furthermore, linearizing the recombinant DNA by, for example, restriction enzyme digestion can efficiently obtain strains in which the recombinant DNA has been integrated into the genomic DNA. Even if a protein encoded by the inactive gene is produced, it will have a different three-dimensional structure from the wild-type protein, resulting in reduced or lost function.

[0369] Alternatively, for example, a microorganism can be transformed with a linear DNA containing an arbitrary sequence, the arbitrary sequence being provided at both ends with sequences upstream and downstream of the site to be replaced (typically, part or all of the gene to be deleted) on the genomic DNA, or with a linear DNA directly linking the upstream and downstream sequences of the site to be replaced on the genomic DNA, and homologous recombination can be induced upstream and downstream of the site to be replaced in the genomic DNA of the host strain, thereby replacing the site to be replaced with the sequence of the linear DNA in a single step. The arbitrary sequence may include, for example, a marker gene sequence. The marker gene may then be removed, if necessary. When removing the marker gene, sequences for homologous recombination may be added to both ends of the marker gene to enable efficient removal of the marker gene.

[0370] The deficiency of the target gene in the microbial strain can be confirmed by a decrease in the activity of the protein encoded by the target gene, which can be confirmed by measuring the activity of the protein.

[0371] The reduction in the transcription level of the target gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the host 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% of that of the host strain.

[0372] The reduction in the amount of the protein 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 microbial strain according to one or more embodiments of the present invention, the amount of the protein 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 host strain.

[0373] In

[13] above, "attenuated expression" of a gene means that although the strain has enolase activity, the enolase activity is reduced compared to that of the host strain. Microbial strains according to one or more embodiments of the present invention are microbial strains in a state in which the function of the enolase gene is reduced, specifically, microbial strains in a state in which the expression levels of mRNA, which is a transcription product of the enolase gene, or protein, which is a translation product of the enolase gene, are reduced, or microbial strains in which the mRNA, which is a transcription product of the enolase gene, or protein, which is a translation product of the enolase gene, are mRNA encoding an enolase with reduced activity or enolase with reduced activity.

[0374] The enolase gene can be attenuated by, for example, artificially modifying the host strain's genes, for example, by mutation, genetic engineering, RNAi-based gene silencing, gene editing, or the like.

[0375] In the above

[13] , the gene encoding enolase 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.

[0376] An example of weakening the enolase gene so as to reduce the activity of the protein is to introduce a mutation into the amino acid sequence coding region of the enolase gene on the genomic DNA so as to encode the amino acid sequence of an enolase with reduced activity.

[0377] The enolase gene in genomic DNA can be deleted as described above by, for example, modifying the enolase gene so that it encodes an enolase with reduced activity to create an attenuated enolase gene, transforming a host strain with recombinant DNA containing the attenuated enolase gene, and inducing homologous recombination between the attenuated enolase gene and the enolase gene in the genomic DNA, thereby replacing the enolase gene in the genomic DNA with the attenuated enolase gene. In this case, incorporating a marker gene into the recombinant DNA according to the host's nutrient requirements and other traits can facilitate manipulation. Furthermore, linearizing the recombinant DNA by, for example, cleaving it with a restriction enzyme can efficiently obtain strains in which the recombinant DNA has been integrated into the genomic DNA.

[0378] The attenuation of the enolase gene in a microbial strain can be confirmed by a decrease in enolase activity.

[0379] The reduction in the transcription level of the enolase gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the host strain. The method for evaluating the amount of mRNA is as described above. The amount of mRNA is preferably reduced to, for example, 90% or less or 60% or less compared to that of the host strain.

[0380] The reduction in the amount of the protein encoded by the enolase gene can be confirmed by Western blotting using an antibody. In the microbial strain according to one or more embodiments of the present invention, the amount of the protein encoded by the enolase gene is preferably reduced, for example, by 90% or less or 60% or less compared to the host strain.

[0381] <Method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione> One or more further embodiments of the present invention comprise: Cultivating a microbial strain according to one or more embodiments of the present invention as described above, The present invention relates to a method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione.

[0382] This method makes it possible to produce γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione at low cost. This method provides high productivity of the target substance (γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione, and / or oxidized glutathione). In one aspect of this method, the yield of the target substance relative to the sugar raw material supplied to the medium is high (high sugar yield). In another aspect, the target substance can be secreted into the medium at a high concentration.

[0383] When this method is a method for producing reduced glutathione and / or oxidized glutathione, the microbial strain used is deficient in the genes [1] and [2] and has enhanced expression of the gene [3] or [4], and it is preferable that [3] is both a gene encoding glutamate-cysteine ​​ligase and a gene encoding glutathione synthetase.

[0384] When this method is a method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, and / or γ-glutamylcystine, it is preferable that the microbial strain used is deficient in the genes [1] and [2] and has enhanced expression of the gene [3] or [4], wherein [3] is a gene encoding glutamate-cysteine ​​ligase.

[0385] The microbial strain according to one or more embodiments of the present invention can be cultured in an appropriate medium. The medium may be either a synthetic medium or a natural medium, as long as it contains nutrients necessary for the growth of the microbial strain and for the biosynthesis of the target substance, such as a carbon source, a nitrogen source, inorganic salts, and vitamins. Preferably, M9 medium is used.

[0386] Any carbon source can be used as long as it can be assimilated by the microorganism used, and examples thereof include carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0387] 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.

[0388] Examples of vitamins include biotin, thiamine, etc. Furthermore, if necessary, substances required for growth by the microbial strain according to one or more embodiments of the present invention (for example, required amino acids in the case of an amino acid-requiring microbial strain) can be added.

[0389] 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, 10 mM to 2000 mM. The concentration of the sulfur source added to the medium is, for example, 10 mM to 2000 mM.

[0390] As the sulfur source, one or more inorganic sulfur compounds such as sulfuric acid, thiosulfuric acid, sulfite, hyposulfite, sulfide, or salts thereof can be added. The sulfuric acid, thiosulfuric acid, sulfite, hyposulfite, or sulfide 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.

[0391] Glycine may be in the free form, in the form of a salt, or in any mixture thereof. The salt is not particularly limited, and examples thereof include sulfate and hydrochloride.

[0392] 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, continuously, or intermittently.

[0393] The sulfur source and / or glycine may be contained in the medium throughout the entire culture period, or only during a portion of the culture period. For example, the amounts of sulfur source and glycine added do not need to be within the above-mentioned ranges throughout the entire stage of producing and accumulating the target substance. The sulfur source and / or glycine may be added to the medium during the culture so that the content falls within the above-mentioned ranges, and the sulfur source and / or glycine content may decrease over the course of the culture period. Furthermore, the sulfur source and / or glycine may be added continuously or intermittently. The concentrations of medium components other than the sulfur source and / or glycine may also be varied or added during the culture period.

[0394] Cultivation 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 cultivation is 5 to 9, preferably 6 to 7.5. The cultivation time is 3 hours to 5 days, preferably 5 hours to 3 days.

[0395] The target substance accumulated in the culture (medium and / or microbial strain) can be collected by a conventional purification method. For example, after the culture is completed, the target substance can be collected from the culture or a disrupted culture by purification treatment such as column chromatography, concentration, crystallization fractionation, etc. If necessary, the culture or disrupted culture may be subjected to solid-liquid separation such as centrifugation to remove bacterial cells and solid matter, followed by purification treatment. [Example]

[0396] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0397] The genetic manipulations described below can be carried out with reference to the descriptions in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). The enzymes, cloning hosts, and other enzymes 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.

[0398] (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 4.6 mm x 250 mm (Nomura Chemical) Mobile phase: Dissolve 30.5 g of potassium dihydrogen phosphate and 18 g of sodium heptanesulfonate in 4.5 L of distilled water, then adjust the pH to 3 with phosphoric acid. Add 250 mL of methanol, then adjust the pH to 3 again with phosphoric acid. Flow rate: 1mL / min Detection: UV detector λ=210nm Column temperature: 40℃ Injection volume: 10μL To analyze 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 the culture supernatant. The obtained culture supernatant was diluted 10-fold with distilled water and subjected to HPLC.

[0399] (Production Example 1) Construction of BW25113 Δggt strain First, a plasmid vector was constructed to disrupt the ggt (γ-glutamylcysteine ​​transferase) gene (SEQ ID NO: 21). A DNA fragment (SEQ ID NO: 1) containing the upstream and downstream sequences of the ggt gene on the chromosome was obtained by PCR using synthetic oligonucleotides. The resulting 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.

[0400] Next, the BW25113 Δggt strain was constructed using pTH18cs1-ggt-UD. pTH18cs1-ggt-UD was introduced into E. coli BW25113 by electroporation, and the resulting transformants were plated onto LB agar plates containing 10 μg / mL chloramphenicol and cultured at 30°C. The resulting transformants were cultured overnight at 30°C with shaking in LB liquid medium containing 10 μg / mL chloramphenicol. The culture was then plated onto LB agar plates containing 10 μg / mL chloramphenicol and cultured at 42°C to obtain transformants. The resulting transformants were then cultured overnight in LB liquid medium at 42°C and plated onto LB agar plates to obtain colonies. The resulting colonies were replicated onto LB agar plates and LB agar plates containing 10 μg / mL chloramphenicol, and chloramphenicol-sensitive transformants were selected. From the selected transformants, we isolated a strain lacking the ggt gene from its initiation codon to its termination codon by PCR and DNA sequencing, and named this gene disruptant BW25113 Δggt.

[0401] The BW25113 Δggt 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.

[0402] (Production Example 2) Preparation of BW25113 Δggt ΔpepT strain First, a plasmid vector was constructed to disrupt the pepT (tripeptide peptidase) gene (SEQ ID NO: 23). PCR using synthetic oligo DNA yielded a DNA fragment (SEQ ID NO: 2) containing the upstream and downstream sequences of the pepT gene on the chromosome. The resulting fragment was digested with XbaI and HindIII and ligated to a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation High Ver. 2 to yield the plasmid vector pTH18cs1-pepT-UD.

[0403] Next, using the BW25113 Δggt strain prepared in Production Example 1 as the parent strain, one strain in which the pepT gene on the chromosome from the initiation codon to the termination codon was deleted was isolated using pTH18cs1-pepT-UD in the same manner as in Production Example 1. This gene-disrupted strain was designated BW25113 Δggt ΔpepT strain.

[0404] The BW25113 Δggt ΔpepT strain is a bacterial strain that uses the E. coli BW25113 strain as a host and lacks the ggt gene and the pepT gene on the chromosome from the initiation codon to the termination codon.

[0405] (Production Example 3) Construction of BW25113 Δggt ΔpepT Δgor strain First, a plasmid vector for disrupting the gor (glutathione reductase) gene was constructed. A DNA fragment (SEQ ID NO: 3) containing the upstream and downstream sequences of the gor gene on the chromosome 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 pTH18cs1 with XbaI and HindIII using Ligation High Ver. 2 to obtain the plasmid vector pTH18cs1-gor-UD.

[0406] Next, using the BW25113 Δggt ΔpepT strain prepared in Production Example 2 as the parent strain, one strain in which the gor gene on the chromosome from the initiation codon to the termination codon was deleted was isolated using pTH18cs1-gor-UD in the same manner as in Production Example 1. This gene-disrupted strain was designated BW25113 Δggt ΔpepT Δgor strain.

[0407] (Production Example 4) Construction of BW25113 Δggt ΔpepT Δgor ΔyliABCD strain First, a plasmid vector was constructed to disrupt the chromosomal yliABCD genes, which form an operon consisting of the yliA (glutathione transport system ATP-binding protein) gene (SEQ ID NO: 27), yliB (glutathione transport system substrate-binding protein) gene (SEQ ID NO: 29), yliC (glutathione transport system permease protein) gene (SEQ ID NO: 31), and yliD (glutathione transport system permease protein) gene (SEQ ID NO: 33). PCR using synthetic oligonucleotides yielded a DNA fragment (SEQ ID NO: 4) containing the upstream sequence of the yliA gene and the downstream sequence of the yliD gene on the chromosome. The resulting fragment was digested with XbaI and HindIII and ligated to a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation High Ver. 2 to obtain the plasmid vector pTH18cs1-yliABCD-UD.

[0408] Next, using the BW25113 Δggt ΔpepT Δgor strain prepared in Production Example 3 as the parent strain, one strain in which the region from the initiation codon to the termination codon of the yliABCD gene on the chromosome was deleted was isolated using pTH18cs1-yliABCD-UD in the same manner as in Production Example 1. This gene-disrupted strain was designated BW25113 Δggt ΔpepT Δgor ΔyliABCD strain.

[0409] (Production Example 5) Construction of BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA strain First, a plasmid vector was constructed to disrupt the tnaA (tryptophanase) gene (SEQ ID NO: 35). A DNA fragment (SEQ ID NO: 5) containing the upstream and downstream sequences of the tnaA gene on the chromosome was obtained by PCR using synthetic oligo DNA. The resulting fragment was digested with XbaI and HindIII, and ligated to a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation High Ver. 2 to obtain the plasmid vector pTH18cs1-tnaA-UD.

[0410] Next, using the BW25113 Δggt ΔpepT Δgor ΔyliABCD strain prepared in Production Example 4 as the parent strain, one strain in which the region from the initiation codon to the termination codon of the tnaA gene on the chromosome was deleted was isolated using pTH18cs1-tnaA-UD in the same manner as in Production Example 1. This gene-disrupted strain was designated BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA strain.

[0411] (Production Example 6) Construction of BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC strain First, a plasmid vector for disrupting the speC gene (SEQ ID NO: 51) was constructed. A DNA fragment (SEQ ID NO: 6) containing the upstream and downstream sequences of the speC gene on the chromosome 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 pTH18cs1 with XbaI and HindIII using Ligation High Ver. 2 to obtain the plasmid vector pTH18cs1-speC-UD.

[0412] Next, using the BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA strain prepared in Production Example 5 as the parent strain, one strain in which the region from the initiation codon to the termination codon of the speC gene on the chromosome was deleted was isolated using pTH18cs1-speC-UD in the same manner as in Production Example 1. This gene-disrupted strain was designated BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC strain.

[0413] (Production Example 7) Construction of BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE strain First, a plasmid vector was constructed to enhance cysE gene expression by inserting the ompF promoter and SD sequence (SEQ ID NO: 7) upstream of the cysE gene (SEQ ID NO: 65) on the chromosome. PCR using synthetic oligonucleotides yielded a DNA fragment (SEQ ID NO: 8) containing the upstream sequence of the cysE gene on the chromosome, the ompF promoter and SD sequence, and a 500-bp sequence from the start codon of the cysE gene. The resulting fragment was digested with XbaI and HindIII and ligated to a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation High Ver. 2 to obtain the plasmid vector pTH18cs1-PompF-cysE-UD.

[0414] Next, using the BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC strain prepared in Production Example 6 as the parent strain, one strain was isolated in which the ompF promoter and SD sequence were inserted upstream of the cysE gene on the chromosome using pTH18cs1-PompF-cysE-UD in the same manner as in Production Example 1. This strain was designated BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE strain.

[0415] (Production Example 8) Construction of BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE ΔgpmA strain First, a plasmid vector was constructed to disrupt the gpmA (phosphoglycerate mutase A) gene (SEQ ID NO: 19). A DNA fragment (SEQ ID NO: 9) containing the upstream and downstream sequences of the gpmA gene in the chromosome 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 pTH18cs1 with XbaI and HindIII using Ligation High Ver. 2 to obtain the plasmid vector pTH18cs1-gpmA-UD.

[0416] Next, using the BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE strain prepared in Production Example 7 as the parent strain, one strain in which the gpmA gene on the chromosome from the initiation codon to the termination codon was deleted was isolated using pTH18cs1-gpmA-UD in the same manner as in Production Example 1. This gene-disrupted strain was designated BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE ΔgpmA strain.

[0417] (Production Example 9) Preparation of pQEK1-PT5-ABTd*-term First, to construct a vector for gene introduction into E. coli, the drug resistance marker was changed to a tetracycline resistance gene using pQE-80L (QIAGEN) as a base to construct the pQEK1 vector shown in SEQ ID NO: 10. 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: 11.

[0418] Next, a DNA fragment (SEQ ID NO: 12) consisting of the T5 promoter, the E. coli-derived gshA gene (SEQ ID NO: 73), and the Thiobacillus denitrificans-derived gshB gene (harboring the V260A mutation) (SEQ ID NO: 69) 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*-term shown in SEQ ID NO: 13.

[0419] (Production Example 10) Construction of BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE / pQEK1-PT5-ABTd*-term strain The pQEK1-PT5-ABTd*-term prepared in Production Example 9 was electroporated into the BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE strain prepared in Production Example 7, and transformants were selected by plating on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain carrying pQEK1-PT5-ABTd*-term was isolated by PCR analysis. This strain was designated the BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE / pQEK1-PT5-ABTd*-term strain.

[0420] (Production Example 11) Construction of BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE ΔgpmA / pQEK1-PT5-ABTd*-term strain The pQEK1-PT5-ABTd*-term gene, prepared in Production Example 9, was introduced into the BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE ΔgpmA strain prepared in Production Example 8 by electroporation, and transformants were selected by plating on an LB agar plate containing 20 μg / mL of tetracycline. From the selected transformants, one strain carrying pQEK1-PT5-ABTd*-term was isolated by PCR analysis. This strain was designated the BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE ΔgpmA / pQEK1-PT5-ABTd*-term gene.

[0421] Example 1: Fermentative production of glutathione by BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE ΔgpmA / pQEK1-PT5-ABTd*-term strain The BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE ΔgpmA / pQEK1-PT5-ABTd*-term strain obtained in Production Example 11 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. One 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-HCl, 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). After 18 hours of culture, 20 mL of the 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 Bioengine Bioneer-Neo). During cultivation, a 50% (w / v) glucose solution was added as needed to maintain the glucose concentration in the system above 15 g / L. After 6 hours of cultivation, 0.1 mM isopropyl-β-thiogalactopyranoside was added, along with glycine and sodium sulfate to a final concentration of 100 mM. After 48 hours of cultivation, an appropriate amount of the culture medium was sampled and centrifuged to separate the cells and the supernatant. The supernatant was appropriately diluted with distilled water, and GSH and GSSG were quantified by HPLC analysis. The quantitative results are shown in Table 1.

[0422] (Comparative Example 1) Fermentative production of glutathione by BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE / pQEK1-PT5-ABTd*-term strain The BW25113 Δggt ΔpepT Δgor ΔyliABCD ΔtnaA ΔspeC PompF-cysE / pQEK1-PT5-ABTd*-term strain obtained in Production Example 10 was cultured under the same conditions as in Example 1 to produce GSH and GSSG. The results are shown in Table 1.

[0423] [Table 1]

[0424] <Consideration> Comparing the results of Example 1 and Comparative Example 1 in Table 1, it can be seen that deletion of the gpmA gene significantly increases glutathione productivity (GSH + GSSG), demonstrating that deletion of the gene encoding phosphoglycerate mutase is effective in glutathione fermentation production.

[0425] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A microbial strain lacking the genes [1] and [2] and in which expression of the gene [3] or [4] is enhanced: [1] A gene encoding γ-glutamyltransferase (EC: 3.4.19.13); [2] A gene encoding phosphoglycerate mutase (EC: 5.4.2.11 or EC: 5.4.1.12); [3] A gene encoding glutamate-cysteine ​​ligase (EC: 6.3.2.2) and / or a gene encoding glutathione synthetase (EC: 6.3.2.3); [4] A gene encoding a bifunctional glutathione synthetase.

2. The microbial strain according to claim 1, comprising any one or more genetic modifications of [5] to [12]: [5] A deletion of the gene encoding tryptophanase (EC: 4.1.99.1); [6] A deletion of a gene encoding tripeptide peptidase (EC: 3.4.11.4); [7] A deletion of the gene encoding glutathione reductase (EC: 1.8.1.7); [8] a deficiency in a gene encoding a protein involved in glutathione uptake; [9] Enhancement of expression of genes encoding proteins involved in putrescine excretion; [10] A deficiency in a gene encoding a protein involved in putrescine uptake; [11] A deficiency in a gene encoding a protein involved in putrescine synthesis; [12] Enhancement of expression of the gene encoding serine-O-acetyltransferase (EC:2.3.1.30).

3. The microbial strain according to claim 1 or 2, which is a bacterial transformant.

4. The microbial strain of claim 3, which is a transformant of an enterobacterium.

5. The microbial strain of claim 3, which is a transformant of a Gram-negative bacterium.

6. The microbial strain of claim 3, which is a transformant of Escherichia coli.

7. A method for producing γ-glutamylcysteine, bis-γ-glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione, which comprises culturing the microbial strain according to any one of claims 1 to 6.

Images