Microorganisms for producing useful substances, and production methods

Abstract

The present invention addresses the problem, to be solved, of improving the productivity of glutathione fermentation by microbes.　The present invention pertains to a microbe strain which is configured for enhanced expression of a gene that codes for serine-O-acetyltransferase (EC:2.3.1.30) and which is capable of excessively expressing γ-glutamylcysteine, bis- γ- glutamylcystine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione. The present invention also pertains to a method that uses the microbe strain.

Description

Technical Field

[0001] One or more embodiments of the present invention relate to microbial strains capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione.

[0002] Another one or more embodiments of the present invention relate to methods for producing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione.

Background Art

[0003] Glutathione is a peptide composed of three amino acids, L-cysteine, L-glutamic acid, and glycine. It exists in many living organisms such as humans, other animals, plants, and microorganisms, and is an important compound for living organisms, such as the scavenging of reactive oxygen species, detoxification, and amino acid metabolism.

[0004] In vivo, glutathione exists in either the reduced form (hereinafter sometimes referred to as "GSH") in which the thiol group of the L-cysteine residue is reduced to the SH form, or the oxidized form (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] As methods for producing glutathione, a method of producing by fermentation using yeast (Patent Document 1), a method of producing γ-glutamylcysteine synthase and glutathione synthase using microorganisms and enzymatically linking L-glutamic acid, L-cysteine, and glycine (Patent Documents 2 and 3), etc. are known.

[0006] Patent Document 4 also describes a method for producing glutathione or γ-glutamylcysteine, which comprises culturing, in a medium, a microorganism having higher activities of a protein having glutathione transport activity and a protein involved in the biosynthesis of glutathione or γ-glutamylcysteine than the parent strain, generating and accumulating glutathione or γ-glutamylcysteine in the medium, and collecting glutathione or γ-glutamylcysteine from the culture. In Example 4 of Patent Document 4, it is described that when an Escherichia coli strain overexpressing the gshA gene, which is a glutamate cysteine ligase gene derived from Escherichia coli, and the gshB gene, which is a glutathione synthase gene, was cultured, 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 a bifunctional glutathione synthase gshF gene placed under the control of a constitutive promoter in a medium supplemented with L-cysteine, L-glutamate, and glycine, which are constituent amino acids of glutathione.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0009]

Non-Patent Document 1

[0010] As another example of glutathione fermentation using microorganisms such as bacteria, Non-Patent Document 2 is known. However, since L-cysteine, which is an expensive substrate, is added to the culture solution, it is difficult to put into practical use. As an example of the fermentative production of glutathione under the condition of non-addition of L-cysteine by bacteria, Patent Document 5 has been reported. However, the glutathione productivity is low (<1 g / L), and since the practicality is poor, improvement has been demanded.

[0011] Therefore, the present invention aims to solve the problem of improving the productivity of glutathione and its related substances, specifically, γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione, by fermentation of microorganisms such as bacteria. [Means for Solving the Problems]

[0012] As a result of intensive studies to solve the above problems, the present inventors have found that the productivity of glutathione and its related substances is significantly improved in microorganisms in which the expression of the gene encoding serine-O-acetyltransferase (EC: 2.3.1.30) is enhanced, and have completed the present invention.

[0013] Specifically, the present invention includes the following inventions. [I] A microbial strain having the gene modification of [1] and capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione: [1]Enhancement of the expression of the gene encoding serine-O-acetyltransferase (EC: 2.3.1.30). [II] The microbial strain according to [I], having the gene modification of [2] and one or more gene modifications selected from [3] and [4]: [2]Deletion of the gene encoding γ-glutamyltransferase (EC: 3.4.19.13); [3]Enhancement of the expression of the gene encoding glutamate-cysteine ligase (EC: 6.3.2.2) and / or the gene encoding glutathione synthetase (EC: 6.3.2.3); [4]Enhancement of the expression of the gene encoding bifunctional glutathione synthetase. [III] The microbial strain according to [I] or [II], having one or more gene modifications selected from [5], [6], [7], [8] and [9]: [5]Deletion of the gene encoding tryptophanase (EC: 4.1.99.1); [6]Deletion of the gene encoding tripeptide peptidase (EC: 3.4.11.4); [7]Deletion of one or more of the genes encoding proteins involved in glutathione uptake; [8]Deletion of the gene encoding glutathione reductase (EC: 1.8.1.7); [9]Enhancement of the expression of one or more of the genes encoding proteins involved in putrescine excretion. [IV] The microbial strain according to any one of [I] to [III], which is a bacterial transformant. [V] The microbial strain according to [IV], which is an enterobacterial transformant. [VI] The microbial strain according to [IV], which is a Gram-negative bacterial transformant. [VII] The microbial strain according to [IV], which is an Escherichia coli transformant. [VIII] A method for producing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione, which comprises culturing a microbial strain according to any one of [I] to [VII]. This specification incorporates the disclosure of Japanese Patent Application No. 2020-079058, which is the basis of the priority of this application.

Effect of the Invention

[0014] The microbial strains according to one or more embodiments of the present invention have high productivity of γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione by fermentation. The production method according to one or more embodiments of the present invention can efficiently produce the target substance.

Mode for Carrying Out the Invention

[0015] <Host Microorganism> The microbial strain serving as the host (parent strain) of the microbial strain having a predetermined gene modification according to one or more embodiments of the present invention 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 a bacterium belonging to the genus Escherichia or a bacterium belonging to the genus Pantoea, or a gram-positive bacterium such as a bacterium belonging to the genus Bacillus, a bacterium belonging to the genus Brevibacterium, or a bacterium belonging to the genus Corynebacterium, but is preferably a gram-negative bacterium, and particularly preferably Escherichia coli.

[0016] The microbial strains according to one or more embodiments of the present invention can be transformants in which a predetermined gene is deleted in the host strain and a predetermined gene is retained.

[0017] <1. 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 such activity.

[0018] The origin of serine - O - acetyltransferase is not particularly limited, and those derived from microorganisms, animals, plants, etc. can be used. Serine - O - acetyltransferase derived from microorganisms is preferred, and particularly preferred are serine - O - acetyltransferases derived from enterobacteria such as Escherichia coli, bacteria such as coryneform bacteria, and eukaryotic microorganisms such as yeast.

[0019] Specific examples of the nucleotide sequence of serine - O - acetyltransferase derived from Escherichia coli and the amino acid sequence encoded by the nucleotide sequence are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0020] As serine - O - acetyltransferase, it is not limited to the serine - O - acetyltransferase consisting of the amino acid sequence shown in SEQ ID NO: 22, and other polypeptides having serine - O - acetyltransferase activity, such as active mutants and heterologous orthologs thereof, can also be used. Other polypeptides having serine - O - acetyltransferase activity preferably show an activity of 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, and still more preferably 90% or more when using the serine - O - acetyltransferase consisting of the amino acid sequence shown in SEQ ID NO: 22 under the above activity measurement conditions.

[0021] Specific examples of serine - O - acetyltransferase include (1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22; (1B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 22 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 22), and having serine-O-acetyltransferase 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 serine-O-acetyltransferase activity; or (1D) A fragment of any of the polypeptides of (1A) to (1C) having serine-O-acetyltransferase activity It can be.

[0022] In the above (1D), the fragment can preferably be a polypeptide having 200 or more, more preferably 250 or more amino acids.

[0023] In the above (1B), "a plurality" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In the above (1B) and (2B), (3-1B), (3-2B), (3-3B), (3-4B), (4B), (5B), (6B), (7-1B), (7-2B), (7-3B), (7-4B), (8B), (9-1B), (9-2B), (9-3B), (9-4B), and (9-5B) described below, "one or more amino acids are added, deleted, or substituted" means that the total number of added, deleted, or substituted amino acids is one or more. Also, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" means a substitution between amino acids with similar properties such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties can be classified, for example, into basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. Hereinafter, in this specification, the term "conservative amino acid substitution" is used in this meaning.

[0024] In the above (1C), "sequence identity" refers to the percentage (%) of identical amino acid residues with respect to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 22 when two amino acid sequences are aligned (aligned), introducing gaps as necessary so that the amino acid identity between the two is maximized. Sequence identity can be calculated using protein search systems such as BLAST and 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, in this specification, "sequence identity" of an amino acid sequence is used in the same meaning.

[0025] The "gene encoding serine-O-acetyltransferase (EC: 2.3.1.30)" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of serine-O-acetyltransferase.

[0026] An example of DNA encoding the amino acid sequence shown in SEQ ID NO: 22 of serine-O-acetyltransferase derived from Escherichia coli is shown in SEQ ID NO: 21. The nucleotide sequence of the nucleic acid encoding the amino acid sequence of serine-O-acetyltransferase may be codon-optimized according to the host. In the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 21 does not necessarily exist as it is, and the nucleotide sequence of SEQ ID NO: 21 may be an exon sequence with one or more intron sequences intervening therein.

[0027] That is, specific examples of the nucleotide sequence of the gene encoding the amino acid sequence of serine-O-acetyltransferase include

[0028] (1E) the nucleotide sequence shown in SEQ ID NO: 21; (1F) A nucleotide sequence in which one or more nucleotides are added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 21 (particularly preferably, a nucleotide sequence in which a total of one or more nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5'-end and 3'-end of the nucleotide sequence shown in SEQ ID NO: 21), and which encodes a polypeptide having serine-O-acetyltransferase activity; (1G) A nucleotide sequence having a sequence identity of 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 with the nucleotide sequence shown in SEQ ID NO: 21, and which encodes a polypeptide having serine-O-acetyltransferase activity; (1H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having serine-O-acetyltransferase activity of any one of the nucleotide sequences of (1E) to (1G); (1I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) is introduced in any one of the nucleotide sequences of (1E) to (1H); (1J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides of (1A) to (1D); or (1K) A nucleotide sequence having one or more intron sequences intervening therein, with any one of the nucleotide sequences of (1E) to (1J) as an exon sequence may be mentioned.

[0029] In the above (1G), "sequence identity" refers to the percentage (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 21 when two base sequences are aligned (aligned), and gaps are introduced as necessary so that the base match degree between the two is the highest. Sequence identity can be calculated using a base sequence search system 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, in this specification, "sequence identity" of a base sequence is used in the same meaning.

[0030] In the above (1F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3. In the above (1F) and (2F), (3-1F), (3-2F), (3-3F), (3-4F), (4F), (5F), (6F), (7-1F), (7-2F), (7-3F), (7-4F), (8F), (9-1F), (9-2F), (9-3F), (9-4F), and (9-5F) described below, "one or more bases are added, deleted, or substituted" means that the total number of added, deleted, or substituted bases is one or more.

[0031] <2. γ-Glutamyltransferase> γ-Glutamyltransferase (EC: 3.4.19.13 or 2.3.2.2) is an enzyme that hydrolyzes γ-glutamyl peptides such as glutathione.

[0032] Specific examples of γ-glutamyltransferase include (2A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24; (2B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 24 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 24), and having γ-glutamyltransferase activity; (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: 24, and having γ-glutamyltransferase activity; or (2D) A fragment of any of the polypeptides of (2A) to (2C) having γ-glutamyltransferase activity It can be such.

[0033] In the above (2D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 300 or more amino acids, more preferably 400 or more amino acids, more preferably 500 or more amino acids, more preferably 550 or more amino acids.

[0034] In the above (2B), "a plurality" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0035] In the above (2C), "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 24 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between them.

[0036] The "gene encoding γ-glutamyltransferase" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of γ-glutamyltransferase, and is contained in the genomic DNA on the chromosome of a wild-type microorganism before deletion of γ-glutamyltransferase.

[0037] An example of DNA encoding the amino acid sequence shown in SEQ ID NO: 24 of γ-glutamyltransferase derived from Escherichia coli is shown in SEQ ID NO: 23. However, in the genomic DNA of a wild-type microorganism, the base sequence of SEQ ID NO: 23 does not necessarily exist as it is, and the base sequence of SEQ ID NO: 23 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0038] That is, specific examples of the base sequence of the gene encoding the amino acid sequence of γ-glutamyltransferase include (2E) the base sequence shown in SEQ ID NO: 23; (2F) a base sequence in which one or more bases are added, deleted, or substituted in the base sequence shown in SEQ ID NO: 23 (particularly preferably, a total of one or more bases are substituted, deleted, and / or added at one or both of the 5'-end and 3'-end of the base sequence shown in SEQ ID NO: 23, preferably a base sequence with deletion and / or addition), and encoding a polypeptide having γ-glutamyltransferase activity; (2G) a base sequence having a sequence identity of 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 with the base sequence shown in SEQ ID NO: 23, and encoding a polypeptide having γ-glutamyltransferase activity; (2H) a partial base sequence encoding the amino acid sequence of a polypeptide having γ-glutamyltransferase activity of any of the base sequences of (2E) to (2G); (2I) a base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (2E) to (2H); A base sequence encoding the amino acid sequence of any one of the polypeptides of (2J), (2A) to (2D); or, A base sequence having, as an exon sequence, any one of the base sequences of (2K), (2E) to (2J), with one or more intron sequences intervening therein may be mentioned.

[0039] In the above (2F), the term "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0040] In the above (2G), the "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 23 when two base sequences are aligned and gaps are introduced as necessary to maximize the base match between the two.

[0041] <3-1. Glutamate-Cysteine Ligase> Glutamate-cysteine ligase (EC: 6.3.2.2) is an enzyme that catalyzes the reaction of generating γ-Glu-Cys by recognizing L-cysteine (L-Cys) as a substrate in the presence of ATP and binding it to L-glutamate (L-Glu). As long as it has such activity, its origin, structure, etc. are not particularly limited. In the present invention, such activity is referred to as glutamate-cysteine ligase activity. 1 U of such activity means the activity of generating 1 μmol of γ-glutamylcysteine per minute at 30°C, and it is measured under the following measurement conditions.

[0042] (Measurement conditions) The reaction is carried out by adding an enzyme solution to a 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM ATP, 15 mM L-glutamate, 15 mM L-cysteine, and 10 mM magnesium sulfate and incubating at 30°C, and the reaction is stopped by adding 6N hydrochloric acid. High-performance liquid chromatography is used to quantify γ-glutamylcysteine in the reaction solution.

[0043] The conditions for the above high performance liquid chromatography are as follows. Under these conditions, glutathione (GSH), γ-glutamylcysteine (γ-GC), bis-γ-glutamylcysteine (oxidized γ-GC), and oxidized glutathione (GSSG) elute in this order. [HPLC Conditions] Column: ODS-HG-3 (4.6 mm φ × 150 mm, manufactured by Nomura Chemical Co., Ltd.); Eluent: A solution prepared by dissolving 12.2 g of potassium dihydrogen phosphate and 3.6 g of sodium heptanesulfonate in 1.8 L of distilled water, adjusting the pH of the solution to 2.8 with phosphoric acid, and adding 186 ml of methanol and dissolving; Flow rate: 1.0 ml / min; Column temperature: 40 °C; Measurement wavelength: 210 nm

[0044] As the glutamate-cysteine ligase, it is preferable to use one having a glutamate-cysteine ligase activity (specific activity) of 0.5 U or more per 1 mg of protein.

[0045] The origin of the glutamate-cysteine ligase is not particularly limited, and those derived from microorganisms, animals, plants, etc. can be used. Glutamate-cysteine ligase derived from microorganisms is preferable, and in particular, glutamate-cysteine ligase derived from enterobacteria such as Escherichia coli, bacteria such as coryneform bacteria, and eukaryotic microorganisms such as yeast is preferable.

[0046] Specific examples of the base sequence of glutamate-cysteine ligase derived from Escherichia coli and the amino acid sequence encoded by the base sequence are shown in SEQ ID NO: 55 and SEQ ID NO: 56, respectively.

[0047] As glutamate-cysteine ligase, not only the glutamate-cysteine ligase consisting of the amino acid sequence shown in SEQ ID NO: 56, but also other polypeptides having glutamate-cysteine ligase activity, such as active mutants and heterologous orthologs thereof, can be used. Other polypeptides having glutamate-cysteine ligase activity preferably exhibit an activity of 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, and still more preferably 90% or more of the glutamate-cysteine ligase consisting of the amino acid sequence shown in SEQ ID NO: 56 under the above activity measurement conditions.

[0048] Specific examples of glutamate-cysteine ligase include (3-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 56; (3-1B) a polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 56 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 56) and having 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: 56 and having glutamate-cysteine ligase activity; or (3-1D) a fragment of any of the polypeptides of (3-1A) to (3-1C) having glutamate-cysteine ligase activity can be.

[0049] In the above (3-1D), the fragment can preferably be a polypeptide having 200 or more, more preferably 300 or more, more preferably 400 or more, more preferably 450 or more, and more preferably 500 or more amino acids.

[0050] In the above (3-1B), "a plurality" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0051] In the above (3-1C), "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 56 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between the two.

[0052] The "gene encoding glutamate-cysteine ligase" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of glutamate-cysteine ligase.

[0053] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 56 of glutamate-cysteine ligase derived from Escherichia coli is shown in SEQ ID NO: 55. The base sequence of the nucleic acid encoding the amino acid sequence of glutamate-cysteine ligase may be codon-optimized according to the host.

[0054] That is, specific examples of the base sequence of the gene encoding the amino acid sequence of glutamate-cysteine ligase include: (3-1E) The base sequence shown in SEQ ID NO: 55; (3-1F) A base sequence in which 1 to a plurality of bases are added, deleted, or substituted in the base sequence shown in SEQ ID NO: 55 (particularly preferably, a total of 1 to a plurality of bases are substituted, deleted, and / or added at one or both of the 5' end and 3' end of the base sequence shown in SEQ ID NO: 55, preferably a base sequence with deletion and / or addition), and which encodes a polypeptide having glutamate-cysteine ligase activity; A base sequence having a sequence identity of 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 with respect to the base sequence shown in SEQ ID NO: 55, and encoding a polypeptide having glutamate-cysteine ligase activity; (3-1H) A partial base sequence encoding the amino acid sequence of a polypeptide having glutamate-cysteine ligase activity of any of the base sequences of (3-1E) to (3-1G); (3-1I) A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (3-1E) to (3-1H); (3-1J) A base sequence encoding the amino acid sequence of any of the polypeptides of (3-1A) to (3-1D); or, (3-1K) A base sequence having any of the base sequences of (3-1E) to (3-1J) as an exon sequence and having one or more intron sequences intervening therein may be mentioned.

[0055] In the above (3-1F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0056] In the above (3-1G), "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 55 when two base sequences are aligned and gaps are introduced as necessary so that the base match degree between the two is maximized.

[0057] <3-2. Glutathione Synthase> Glutathione synthetase (EC: 6.3.2.3) is an enzyme that catalyzes the reaction of recognizing γ-Glu-Cys as a substrate in the presence of ATP and binding it to glycine (Gly) to produce γ-Glu-Cys-Gly. As long as it has such activity, its origin, structure, etc. are not particularly limited. In the present invention, such activity is referred to as glutathione synthetase activity. 1 U of such activity means the activity of producing 1 μmol of glutathione per minute at 30°C, and it is measured under the following measurement conditions.

[0058] (Measurement conditions) Add the enzyme solution to a 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM ATP, 15 mM γ-glutamylcysteine, 15 mM glycine, and 10 mM magnesium sulfate, incubate at 30°C to carry out the reaction, and stop the reaction by adding 6N hydrochloric acid. Quantify glutathione in the reaction solution using high-performance liquid chromatography.

[0059] The conditions for high-performance liquid chromatography are the same as those described above for the activity measurement method of glutamate-cysteine ligase.

[0060] As the glutathione synthetase, it is preferably those having a glutathione synthetase activity (specific activity) of 0.5 U or more per 1 mg of protein.

[0061] The glutathione synthetase is not particularly limited, and those derived from microorganisms, animals, plants, etc. can be used. Glutathione synthetase derived from microorganisms is preferred, and in particular, glutathione synthetase derived from enterobacteria such as Escherichia coli, bacteria such as coryneform bacteria, eukaryotic microorganisms such as yeast, and microorganisms belonging to the family Hydrogenophilales is preferred.

[0062] The glutathione synthase derived from a microorganism belonging to the family Hydrogenophilales is preferably a glutathione synthase derived from a microorganism belonging to the genus Thiobacillus, more preferably a glutathione synthase derived from a microorganism belonging to Thiobacillus denitrificans. In particular, the glutathione synthase derived from Thiobacillus denitrificans ATCC25259 strain is preferred.

[0063] (Preferred embodiments of glutathione synthase derived from Escherichia coli or variants thereof) Specific examples of the nucleotide sequence of the glutathione synthase derived from Escherichia coli and the amino acid sequence encoded by the nucleotide sequence are shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively.

[0064] The glutathione synthase is not limited to the glutathione synthase consisting of the amino acid sequence shown in SEQ ID NO: 58, and other polypeptides having glutathione synthase activity, such as active variants and heterologous orthologs thereof, can also be used. Other polypeptides having glutathione synthase activity preferably exhibit an activity of 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 glutathione synthase consisting of the amino acid sequence shown in SEQ ID NO: 58 under the above activity measurement conditions.

[0065] Specific examples of the glutathione synthase derived from Escherichia coli or variants thereof include (3-2A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 58; (3-2B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 58 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 58), and having 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: 58, and having glutathione synthetase activity; or (3-2D) A fragment of any of the polypeptides of (3-2A) to (3-2C) having glutathione synthetase activity It can be.

[0066] In the above (3-2D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0067] In the above (3-2B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0068] In the above (3-2C), "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues in the amino acid sequence shown in SEQ ID NO: 58 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between them.

[0069] "Gene encoding glutathione synthetase" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of glutathione synthetase.

[0070] An example of DNA encoding the amino acid sequence shown in SEQ ID NO: 58 of glutathione synthase derived from Escherichia coli is shown in SEQ ID NO: 57. The base sequence of the nucleic acid encoding the amino acid sequence of glutathione synthase may be codon-optimized according to the host.

[0071] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of glutathione synthase derived from Escherichia coli or a variant thereof include: (3-2E) the base sequence shown in SEQ ID NO: 57; (3-2F) a base sequence in which one or more bases are added, deleted, or substituted in the base sequence shown in SEQ ID NO: 57 (particularly preferably, a total of one or more bases are substituted, deleted, and / or added at one or both of the 5' end and 3' end of the base sequence shown in SEQ ID NO: 57, preferably a base sequence with deletion and / or addition), and which encodes a polypeptide having glutathione synthase activity; (3-2G) a base sequence having a sequence identity of 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 with respect to the base sequence shown in SEQ ID NO: 57, and which encodes a polypeptide having glutathione synthase activity; (3-2H) a partial base sequence encoding the amino acid sequence of a polypeptide having glutathione synthase activity of any of the base sequences of (3-2E) to (3-2G); (3-2I) a base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced into any of the base sequences of (3-2E) to (3-2H); (3-2J) a base sequence encoding the amino acid sequence of any of the polypeptides of (3-2A) to (3-2D); or (3-2K) a base sequence having any of the base sequences of (3-2E) to (3-2J) as an exon sequence and having one or more intron sequences intervening therein may be mentioned.

[0072] In the above (3-2F), the term "plurality" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0073] In the above (3-2G), the term "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 57 when two base sequences are aligned and gaps are introduced as necessary to maximize the base match between the two.

[0074] (Preferred Embodiment of Glutathione Synthetase Derived from Thiobacillus denitrificans or Its Variant) Another preferred specific example of glutathione synthetase is a wild-type glutathione synthetase derived from Thiobacillus denitrificans ATCC 25259 strain or an active variant thereof. Specific examples of the base sequence of the wild-type glutathione synthetase of Thiobacillus denitrificans ATCC 25259 strain and the amino acid sequence encoded by the base sequence are shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. The active variant of the wild-type glutathione synthetase preferably exhibits an activity of 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, and still more preferably 90% or more when using the wild-type glutathione synthetase consisting of the amino acid sequence shown in SEQ ID NO: 50 under the above activity measurement conditions.

[0075] Specific examples of the glutathione synthetase of Thiobacillus denitrificans ATCC 25259 strain or its variant include: (3-3A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 50; (3-3B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 50 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 50), and having 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: 50, and having glutathione synthetase activity; or (3-3D) A fragment having glutathione synthetase activity of the polypeptide of any one of (3-3A) to (3-3C) It can be such.

[0076] In the above (3-3D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 250 or more amino acids, and still more preferably 300 or more amino acids.

[0077] In the above (3-3B), "a plurality" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0078] In the above (3-3C), "sequence identity" refers to the ratio (%) of the same amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 50 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between them.

[0079] An example of DNA encoding the amino acid sequence shown in SEQ ID NO: 50 of glutathione synthase of Thiobacillus denitrificans ATCC25259 strain is shown in SEQ ID NO: 49. The base sequence of the nucleic acid encoding the amino acid sequence of glutathione synthase may be codon-optimized according to the host.

[0080] That is, specific examples of the base sequence of the gene encoding the amino acid sequence of glutathione synthase of Thiobacillus denitrificans ATCC25259 strain or its variant include: (3-3E) The base sequence shown in SEQ ID NO: 49; (3-3F) In the base sequence shown in SEQ ID NO: 49, a base sequence in which one or more bases are added, deleted, or substituted (particularly preferably, a total of one or more bases are substituted, deleted, and / or added at one or both of the 5' end and 3' end of the base sequence shown in SEQ ID NO: 49, preferably a base sequence in which deletion and / or addition has occurred), and which encodes a polypeptide having glutathione synthase activity; (3-3G) A base sequence having a sequence identity of 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 with respect to the base sequence shown in SEQ ID NO: 49, and which encodes a polypeptide having glutathione synthase activity; (3-3H) A partial base sequence encoding the amino acid sequence of a polypeptide having glutathione synthase activity of any of the base sequences of (3-3E) to (3-3G); (3-3I) In any of the base sequences of (3-3E) to (3-3H), a base sequence into which a silent mutation (base substitution that does not change the encoded amino acid residue) has been introduced; (3-3J) A base sequence encoding the amino acid sequence of any of the polypeptides of (3-3A) to (3-3D); or, (3-3K) A base sequence in which any of the base sequences of (3-3E) to (3-3J) is used as an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0081] In the above (3-3F), "a plurality" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0082] In the above (3-3G), "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 49 when two base sequences are aligned and gaps are introduced as necessary to maximize the base match between the two.

[0083] (Preferred Embodiment of an Activity Variant of Glutathione Synthetase Derived from Thiobacillus denitrificans) Another preferred example of glutathione synthetase is an activity variant of the wild-type glutathione synthetase of Thiobacillus denitrificans ATCC 25259 strain containing the amino acid sequence shown in SEQ ID NO: 50, and the polypeptide described in International Publication WO2018 / 084165 is particularly preferred.

[0084] Specifically, the activity variant is (3-4A) A polypeptide consisting of an amino acid sequence in which one or more amino acids selected from the following group: 13th, 17th, 20th, 23rd, 39th, 70th, 78th, 101st, 113th, 125th, 126th, 136th, 138th, 149th, 152nd, 154th, 155th, 197th, 200th, 215th, 226th, 227th, 230th, 239th, 241st, 246th, 249th, 254th, 260th, 262nd, 263rd, 270th, 278th, 299th, 305th, 307th, and 310th positions of the amino acid sequence shown in SEQ ID NO: 50 are substituted, and which is composed of amino acid sequence 3-4A; (3-4B) A polypeptide consisting of an amino acid sequence in which one or more amino acids other than the amino acid sites in the amino acid sequence 3-4A are added, deleted, or substituted (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence 3-4A), and which has glutathione synthetase activity; (3 - 4C) A polypeptide consisting of an amino acid sequence that has the same amino acid sites as the amino acid sequence 3 - 4A and has a sequence identity of 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 in the portion other than the amino acid sites, and having glutathione synthetase activity; or (3 - 4D) A fragment having glutathione synthetase activity of any of the polypeptides of (3 - 4A) to (3 - 4C) It can be.

[0085] In the above (3 - 4D), as the fragment, 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 can be used.

[0086] In the above (3 - 4B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0087] In the above (3 - 4C), "sequence identity" refers to the ratio (%) of the same amino acid residues to the total number of amino acid residues of the amino acid sequence 3 - 4A when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between them.

[0088] The amino acid sequence 3 - 4A is more preferably in the following group in the amino acid sequence shown in SEQ ID NO: 50: 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 aspartic acid, the 226th is arginine, the 227th is serine, the 230th is proline, the 239th is serine, the 241st is histidine, the 246th is arginine, the 249th is glutamic acid, the 254th is aspartic acid, the 260th is alanine, cysteine, glycine, glutamine, threonine, the 262nd is cysteine, the 263rd is arginine, the 270th is isoleucine, the 278th is glycine, alanine, the 299th is alanine, the 305th is glycine, the 307th is valine and the 310th is threonine are substituted, which is an amino acid sequence into which one or more amino acid substitutions selected from

[0089] The amino acid sequence 3-4A is particularly preferably the following (1) to (35) among the amino acid sequences shown in SEQ ID NO: 50: (1) The 13th is serine, (2) The 17th is glutamic acid, the 113th is histidine, the 230th is proline, (3) The 20th is threonine, the 215th is aspartic acid, (4) The 20th is threonine, the 241st is histidine, (5) The 23rd is leucine, the 126th is asparagine, (6) The 39th is threonine, the 260th is alanine, (7) The 70th is serine, the 260th is alanine, (8) The 78th is leucine, the 278th is alanine, (9) The 101st is asparagine, (10) The 101st is glutamine, (11) The 101st is serine, (12) The 101st is serine and the 260th is alanine, (13) The 101st is threonine, (14) The 125th is valine and the 249th is glutamic acid, (15) The 125th is valine and the 152nd is glutamine, (16) The 136th is threonine, (17) The 138th is alanine, the 149th is glutamine, the 241st is histidine, and the 263rd is glutamine, (18) The 154th is asparagine and the 246th is arginine, (19) The 155th is leucine and the 239th is serine, (20) The 197th is glutamine, (21) The 200th is serine and the 260th is alanine, (22) The 226th is arginine and the 260th is alanine, (23) The 227th is serine and the 260th is alanine, (24) The 254th is aspartic acid and the 260th is alanine, (25) The 260th is alanine, (26) The 260th is alanine, the 278th is glycine, and the 307th is valine, (27) The 260th is alanine and the 299th is alanine, (28) The 260th is alanine and the 305th is glycine, (29) The 260th is alanine and the 310th is threonine, (30) The 260th is cysteine, (31) The 260th is glycine, (32) The 260th is glutamine, (33) The 260th is threonine, (34) The 262nd is cysteine, (35) The 270th is isoleucine, It is an amino acid sequence into which an amino acid substitution represented by any of the above is introduced.

[0090] The base sequence encoding the amino acid sequence of any one of the polypeptides (3-4A) to (3-4D) can be used as the "gene encoding glutathione synthetase".

[0091] In the amino acid sequence shown in SEQ ID NO: 50 of glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain, an example of the base sequence encoding the amino acid sequence of the active mutant in which valine at the 260th position is substituted with alanine (SEQ ID NO: 52) is shown in SEQ ID NO: 51. The base sequence of the nucleic acid encoding the amino acid sequence of the active mutant of glutathione synthetase of Thiobacillus denitrificans ATCC25259 strain may be codon-optimized according to the host. For example, the base sequence codon-optimized for expression in Escherichia coli encoding the amino acid sequence of SEQ ID NO: 52 is shown in SEQ ID NO: 51.

[0092] <4. Bifunctional Glutathione Synthetase> Bifunctional glutathione synthetase is an enzyme that has the activity of catalyzing the reaction of generating γ-Glu-Cys by recognizing L-Cys as a substrate in the presence of ATP and binding it to L-Glu, and the activity of catalyzing the reaction of generating γ-Glu-Cys-Gly by recognizing γ-Glu-Cys as a substrate in the presence of ATP and binding it to Gly. As long as it has the activity, its origin, structure, etc. are not particularly limited. In the present invention, the activity is referred to as bifunctional glutathione synthetase activity. 1 U of the activity means the activity of generating 1 μmol of γ-Glu-Cys-Gly (glutathione) per minute at 30°C, and it is measured under the following measurement conditions.

[0093] (Measurement Conditions) The reaction is carried out by adding the enzyme solution to a 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 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.

[0094] The conditions for high-performance liquid chromatography are the same as those described above for the method of measuring the activity of glutamate-cysteine ligase.

[0095] As the bifunctional glutathione synthase, it is preferable to use one with a bifunctional glutathione synthase activity (specific activity) of 0.5 U or more per 1 mg of protein.

[0096] The origin of the bifunctional glutathione synthase is not particularly limited, and those derived from microorganisms, animals, plants, etc. can be used. The bifunctional glutathione synthase derived from microorganisms is preferred. Particularly preferred is the bifunctional glutathione synthase derived from bacteria. Specifically, bacteria belonging to the genus Streptococcus such as Streptococcus agalactiae, Streptococcus mutans, Streptococcus suis, Streptococcus thermophilus; bacteria belonging to the genus Lactobacillus such as Lactobacillus plantarum; bacteria belonging to the genus Desulfotalea such as Desulfotalea psychrophila; bacteria belonging to the genus Clostridium such as Clostridium perfringens; bacteria belonging to the genus Listeria such as Listeria innocua, Listeria monocytogenes; bacteria belonging to the genus Enterococcus such as Enterococcus faecalis, Enterococcus faecium; bacteria belonging to the genus Pasteurella such as Pasteurella multocida; bacteria belonging to the genus Mannheimia such as Mannheimia succiniciprodecens; and at least one bifunctional glutathione synthase derived from the group consisting of bacteria belonging to the genus Haemophilus such as Haemophilus somnus is preferred.

[0097] The nucleotide sequence of the bifunctional glutathione synthase derived from Streptococcus agalactiae and a specific example of the amino acid sequence encoded by the nucleotide sequence are shown in SEQ ID NO: 53 and SEQ ID NO: 54, respectively. The nucleotide sequence of SEQ ID NO: 53 is a nucleotide sequence encoding the bifunctional glutathione synthase derived from Streptococcus agalactiae consisting of the amino acid sequence shown in SEQ ID NO: 54, and is a nucleotide sequence adapted to the codon usage frequency in Escherichia coli.

[0098] The bifunctional glutathione synthase is not limited to the bifunctional glutathione synthase consisting of the amino acid sequence shown in SEQ ID NO: 54, and other polypeptides having bifunctional glutathione synthase activity, such as active mutants and heterologous orthologs thereof, can also be used. Other polypeptides having bifunctional glutathione synthase activity preferably exhibit an activity of 10% or more, preferably 40% or more, more preferably 60% or more, more preferably 80% or more, still more preferably 90% or more of the bifunctional glutathione synthase consisting of the amino acid sequence shown in SEQ ID NO: 54 under the above activity measurement conditions.

[0099] Specific examples of the bifunctional glutathione synthase include (4A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 54; (4B) a polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 54 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 54) and having bifunctional glutathione synthase 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: 54 and having bifunctional glutathione synthase activity; or A fragment having bifunctional glutathione synthase activity of any one of the polypeptides of (4D), (4A) to (4C) can be.

[0100] In the above (4D), as the fragment, a polypeptide having 400 or more amino acids, more preferably 500 or more amino acids, more preferably 600 or more amino acids, more preferably 700 or more amino acids, and more preferably 730 or more amino acids can be used.

[0101] In the above (4B), "a plurality of" 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. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0102] In the above (4C), "sequence identity" refers to the ratio (%) of the same amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 54 when two amino acid sequences are aligned and gaps are introduced if necessary so that the amino acid identity between the two is maximized.

[0103] "Gene encoding bifunctional glutathione synthase" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of bifunctional glutathione synthase.

[0104] Specific examples of the nucleotide sequence of the gene encoding the amino acid sequence of bifunctional glutathione synthase include (4E) the nucleotide sequence shown in SEQ ID NO: 53; (4F) a nucleotide sequence in which 1 to a plurality of nucleotides are added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 53 (particularly preferably, a nucleotide sequence in which a total of 1 to a plurality of nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and 3' end of the nucleotide sequence shown in SEQ ID NO: 53), and which encodes a polypeptide having bifunctional glutathione synthase activity; A base sequence having a sequence identity of 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 with respect to the base sequence shown in SEQ ID NO: 53, and encoding a polypeptide having bifunctional glutathione synthase activity; (4H) A partial base sequence encoding the amino acid sequence of a polypeptide having bifunctional glutathione synthase activity of any of the base sequences of (4E) to (4G); (4I) A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (4E) to (4H); (4J) A base sequence encoding the amino acid sequence of any of the polypeptides of (4A) to (4D); or, (4K) A base sequence having any of the base sequences of (4E) to (4J) as an exon sequence and having one or more intron sequences intervening therein may be mentioned.

[0105] In the above (4F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0106] In the above (4G), "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 53 when two base sequences are aligned and gaps are introduced as necessary to maximize the base match between the two.

[0107] <5. Tryptophanase> Tryptophanase (EC: 4.1.99.1) is an enzyme protein having the activity of decomposing cysteine. TnaA can be exemplified as tryptophanase in microorganisms. The gene encoding the amino acid sequence of TnaA is tnaA. The "gene encoding tryptophanase" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of tryptophanase, and is contained in the genomic DNA on the chromosome of a wild-type microbial strain before deletion of the gene.

[0108] In the microbial strain according to one or more embodiments of the present invention described below, preferably, the tnaA gene is deleted.

[0109] Specific examples of the TnaA protein include (5A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 38; (5B) a polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 38 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 38), and having tryptophanase activity; (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: 38, and having tryptophanase activity; or (5D) a fragment having tryptophanase activity of any of the polypeptides of (5A) to (5C) can be.

[0110] In the above (5D), the number of amino acids of the fragment can preferably be 200 or more, more preferably 300 or more, more preferably 400 or more, and more preferably 450 or more.

[0111] In the above (5B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, substitution of amino acids is preferably a conservative amino acid substitution.

[0112] In (5C), the "sequence identity" refers to the percentage (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 38 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between them.

[0113] The "tnaA gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of TnaA, and is contained in the genomic DNA on the chromosome of the wild-type microbial strain before deletion of the gene.

[0114] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 38 of TnaA derived from Escherichia coli is shown in SEQ ID NO: 37. However, in the genomic DNA of the wild-type microbial strain, the nucleotide sequence of SEQ ID NO: 37 does not necessarily exist as it is. The nucleotide sequence of SEQ ID NO: 37 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0115] That is, specific examples of the gene encoding the amino acid sequence of TnaA or the nucleotide sequence of the tnaA gene include: (5E) The nucleotide sequence shown in SEQ ID NO: 37; (5F) A nucleotide sequence in which one or more bases are added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 37 (particularly preferably, a nucleotide sequence in which a total of one or more bases are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and 3' end of the nucleotide sequence shown in SEQ ID NO: 37), and which encodes a polypeptide having tryptophanase activity; (5G) A nucleotide sequence having a sequence identity of 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 to the nucleotide sequence shown in SEQ ID NO: 37, and which encodes a polypeptide having tryptophanase activity; (5H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having tryptophanase activity of any of the nucleotide sequences of (5E) to (5G); A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (5I), (5E) to (5H); A base sequence encoding the amino acid sequence of any of the polypeptides of (5J), (5A) to (5D); or, A base sequence in which any of the base sequences of (5K), (5E) to (5J) is an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0116] In the above (5F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0117] In the above (5G), "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 37 when two base sequences are aligned and gaps are introduced as necessary so that the base match degree between the two is the highest.

[0118] <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.

[0119] Specific examples of tripeptide peptidase include (6A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26; (6B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 26 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminal and C-terminal of the amino acid sequence shown in SEQ ID NO: 26), and having tripeptide peptidase activity; 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 tripeptide peptidase activity; or (6D) A fragment having tripeptide peptidase activity of the polypeptide according to any one of (6A) to (6C) It can be.

[0120] In the above (6D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 300 or more amino acids, and even more preferably 350 or more amino acids.

[0121] In the above (6B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0122] In the above (6C), "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 26 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between the two.

[0123] "A gene encoding tripeptide peptidase" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of tripeptide peptidase, and is contained in the genomic DNA on the chromosome of a wild-type microorganism before deletion of tripeptide peptidase.

[0124] An example of DNA encoding the amino acid sequence shown in SEQ ID NO: 26 of tripeptide peptidase derived from Escherichia coli is shown in SEQ ID NO: 25. However, in the genomic DNA of a wild-type microorganism, the nucleotide sequence of SEQ ID NO: 25 does not necessarily exist as it is, and the nucleotide sequence of SEQ ID NO: 25 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0125] That is, specific examples of the nucleotide sequence of the gene encoding the amino acid sequence of tripeptide peptidase include: (6E) the nucleotide sequence shown in SEQ ID NO: 25; (6F) a nucleotide sequence in which one or more nucleotides are added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 25 (particularly preferably, a nucleotide sequence in which a total of one or more nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5'-end and 3'-end of the nucleotide sequence shown in SEQ ID NO: 25), and which encodes a polypeptide having tripeptide peptidase activity; (6G) a nucleotide sequence having a sequence identity of 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 with the nucleotide sequence shown in SEQ ID NO: 25, and which encodes a polypeptide having tripeptide peptidase activity; (6H) a partial nucleotide sequence encoding the amino acid sequence of a polypeptide having tripeptide peptidase activity among the nucleotide sequences of any one of (6E) to (6G); (6I) a nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) is introduced into the nucleotide sequence of any one of (6E) to (6H); (6J) a nucleotide sequence encoding the amino acid sequence of any one of the polypeptides of (6A) to (6D); or, (6K) a nucleotide sequence having the nucleotide sequence of any one of (6E) to (6J) as an exon sequence and having one or more intron sequences intervening therein may be mentioned.

[0126] In the above (6F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0127] In the above (6G), "sequence identity" means the ratio (%) of the identical nucleotides to the total number of nucleotides of the nucleotide sequence shown in SEQ ID NO: 25 when two nucleotide sequences are aligned and gaps are introduced as necessary to maximize the nucleotide identity between the two.

[0128] <7. Protein Involved in Glutathione Uptake> A protein involved in glutathione uptake is a protein that has the function of taking up extracellular glutathione into the cell. 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. YliA, YliB, YliC, and YliD proteins may be collectively referred to as "YliABCD", and yliA, yliB, yliC, and yliD genes may be collectively referred to as "yliABCD". The "gene encoding a protein involved in glutathione uptake" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of a protein involved in glutathione uptake, and is contained in the genomic DNA on the chromosome of the wild-type microbial strain before deletion of the gene.

[0129] 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, all of the genes yliA, yliB, yliC, and yliD are deleted.

[0130] Specific examples of the YliA protein (glutathione transport system ATP-binding protein) include (7-1A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30; (7-1B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 30 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 30), and having the activity as YliA; (7-1C) 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: 30, and having the activity as YliA; or (7-1D) A fragment having the activity as YliA of any of the polypeptides of (7-1A) to (7-1C) It can be the above.

[0131] In the above (7-1B) to (7-1D), and in (7-1F) to (7-1H) described later, "having the activity as YliA" means having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30, particularly, the glutathione transport system ATP binding activity.

[0132] In the above (7-1D), the fragment can preferably be a polypeptide having 400 or more amino acids, more preferably 500 or more amino acids, and even more preferably 600 or more amino acids.

[0133] In the above (7-1B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0134] In the above (7-1C), "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 30 when two amino acid sequences are aligned and gaps are introduced if necessary to maximize the amino acid identity between them.

[0135] The "yliA gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of YliA, and is contained in the genomic DNA on the chromosome of a wild-type microbial strain before deletion of the gene.

[0136] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 30 of YliA derived from Escherichia coli is shown in SEQ ID NO: 29. However, in the genomic DNA of a wild-type microbial strain before deletion of the gene, the base sequence of SEQ ID NO: 29 does not necessarily exist as it is. The base sequence of SEQ ID NO: 29 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0137] That is, specific examples of the gene encoding the amino acid sequence of YliA or the base sequence of the yliA gene include: (7-1E) The base sequence shown in SEQ ID NO: 29; (7-1F) A base sequence in which one or more bases are added, deleted, or substituted in the base sequence shown in SEQ ID NO: 29 (particularly preferably, a total of one or more bases are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5'-end and 3'-end of the base sequence shown in SEQ ID NO: 29), and which encodes a polypeptide having the activity as YliA; (7-1G) A base sequence having a sequence identity of 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 with the base sequence shown in SEQ ID NO: 29, and which encodes a polypeptide having the activity as YliA; (7-1H) A partial base sequence encoding the amino acid sequence of a polypeptide having the activity as YliA of any of the base sequences of (7-1E) to (7-1G); (7-1I) A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (7-1E) to (7-1H); A base sequence encoding the amino acid sequence of any one of the polypeptides (7-1J), (7-1A) to (7-1D); or, A base sequence in which any one of the base sequences (7-1K), (7-1E) to (7-1J) is an exon sequence and one or more intron sequences are intervening in the middle may be mentioned.

[0138] In the above (7-1F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6 or 2 to 3.

[0139] In the above (7-1G), "sequence identity" refers to the ratio (%) of the same bases to the total number of bases of the base sequence shown in SEQ ID NO: 29 when two base sequences are aligned and gaps are introduced as necessary so that the base match degree between the two is the highest.

[0140] Specific examples of the YliB protein (glutathione transport system substrate-binding protein) include (7-2A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32; (7-2B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 32 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 32), and having the activity as YliB; (7-2C) 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: 32, and having the activity as YliB; or (7-2D) A fragment having the activity as YliB of any one of the polypeptides (7-2A) to (7-2C) can be.

[0141] In the above (7-2B) to (7-2D), and in the following (7-2F) to (7-2H) described later, "having the activity as YliB" refers to having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32, particularly, the glutathione transport system substrate binding activity.

[0142] In the above (7-2D), the fragment can be a polypeptide having preferably 300 or more amino acids, more preferably 400 or more amino acids, and even more preferably 500 or more amino acids.

[0143] In the above (7-2B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0144] In the above (7-2C), "sequence identity" refers to the ratio (%) of the same amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 32 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between the two.

[0145] The "yliB gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of YliB, and is contained in the genomic DNA on the chromosome of the wild-type microbial strain before deletion of the gene.

[0146] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 32 of YliB derived from Escherichia coli is shown in SEQ ID NO: 31. However, in the genomic DNA of the wild-type microbial strain before deletion of the gene, the nucleotide sequence of SEQ ID NO: 31 does not necessarily exist as it is, and the nucleotide sequence of SEQ ID NO: 31 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0147] That is, specific examples of the gene encoding the amino acid sequence of YliB or the nucleotide sequence of the yliB gene include: (7-2E) The nucleotide sequence shown in SEQ ID NO: 31; (7-2F) In the nucleotide sequence shown in SEQ ID NO: 31, a nucleotide sequence in which one or more nucleotides are added, deleted, or substituted (particularly preferably, a total of one or more nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 31), which encodes a polypeptide having the activity as YliB; (7-2G) A nucleotide sequence having a sequence identity of 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 with respect to the nucleotide sequence shown in SEQ ID NO: 31, which encodes a polypeptide having the activity as YliB; (7-2H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having the activity as YliB in any of the nucleotide sequences of (7-2E) to (7-2G); (7-2I) In any of the nucleotide sequences of (7-2E) to (7-2H), a nucleotide sequence into which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) is introduced; (7-2J) A nucleotide sequence encoding the amino acid sequence of any of the polypeptides of (7-2A) to (7-2D); or, (7-2K) A nucleotide sequence in which any of the nucleotide sequences of (7-2E) to (7-2J) is an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0148] In the above (7-2F), "a plurality" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0149] In the above (7-2G), "sequence identity" means the ratio (%) of the identical nucleotides to the total number of nucleotides in the nucleotide sequence shown in SEQ ID NO: 31 when two nucleotide sequences are aligned and gaps are introduced as necessary to maximize the nucleotide identity between them.

[0150] Specific examples of the YliC protein (glutathione transport system permease protein) include, A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34; (7-3B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 34 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 34), and having the activity as YliC; (7-3C) 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: 34, and having the activity as YliC; or (7-3D) A fragment having the activity as YliC of any of the polypeptides of (7-3A) to (7-3C) It can be any of the above.

[0151] In the above (7-3B) to (7-3D), and in (7-3F) to (7-3H) described below, "having the activity as YliC" means having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34, particularly, glutathione transport system permease activity.

[0152] In the above (7-3D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0153] In the above (7-3B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, amino acid substitution is preferably a conservative amino acid substitution.

[0154] In the above (7-3C), "sequence identity" refers to the ratio (%) of the number of identical amino acid residues to the total number of amino acid residues in the amino acid sequence shown in SEQ ID NO: 34 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid match between the two.

[0155] The "yliC gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of YliC, and is contained in the genomic DNA on the chromosome of the wild-type microbial strain before deletion of the gene.

[0156] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 34 of YliC derived from Escherichia coli is shown in SEQ ID NO: 33. However, in the genomic DNA of the wild-type microbial strain before deletion of the gene, the nucleotide sequence of SEQ ID NO: 33 does not necessarily exist as it is. The nucleotide sequence of SEQ ID NO: 33 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0157] That is, specific examples of the nucleotide sequence of the gene encoding the amino acid sequence of YliC or the yliC gene include: (7-3E) The nucleotide sequence shown in SEQ ID NO: 33; (7-3F) A nucleotide sequence in which one or more bases are added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 33 (particularly preferably, a nucleotide sequence in which a total of one or more bases are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and 3' end of the nucleotide sequence shown in SEQ ID NO: 33), and which encodes a polypeptide having the activity of YliC; (7-3G) A nucleotide sequence having a sequence identity of 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 with the nucleotide sequence shown in SEQ ID NO: 33, and which encodes a polypeptide having the activity of YliC; (7-3H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having the activity of YliC of any of the nucleotide sequences of (7-3E) to (7-3G); A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (7-3I), (7-3E) to (7-3H); A base sequence encoding the amino acid sequence of any of the polypeptides of (7-3J), (7-3A) to (7-3D); or, A base sequence in which any of the base sequences of (7-3K), (7-3E) to (7-3J) is an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0158] In the above (7-3F), "a plurality" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6 or 2 to 3.

[0159] In the above (7-3G), "sequence identity" means the ratio (%) of the same bases to the total number of bases of the base sequence shown in SEQ ID NO: 33 when two base sequences are aligned and gaps are introduced as necessary so that the base match degree between the two is the highest.

[0160] Specific examples of the YliD protein (glutathione transport system permease protein) include (7-4A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 36; (7-4B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 36 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 36), and having the activity as YliD; (7-4C) 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: 36, and having the activity as YliD; or A fragment of any of the polypeptides (7-4D), (7-4A) to (7-4C) having the activity as YliD can be such.

[0161] In the above (7-4B) to (7-4D) and (7-4F) to (7-4H) described later, "having the activity as YliD" means having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 36, particularly, glutathione transport system permease activity.

[0162] In the above (7-4D), the fragment can be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids, still more preferably 300 or more amino acids.

[0163] In the above (7-4B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0164] In the above (7-4C), "sequence identity" means the ratio (%) of the same amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 36 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between them.

[0165] The "yliD gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of YliD and is contained in the genomic DNA on the chromosome of the wild-type microbial strain before deletion of the gene.

[0166] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 36 of YliD derived from Escherichia coli is shown in SEQ ID NO: 35. However, in the genomic DNA of the wild-type microbial strain before deletion of the gene, the nucleotide sequence of SEQ ID NO: 35 does not necessarily exist as it is. The nucleotide sequence of SEQ ID NO: 35 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0167] That is, specific examples of the gene encoding the amino acid sequence of YliD or the nucleotide sequence of the yliD gene include: (7-4E) The nucleotide sequence shown in SEQ ID NO: 35; (7-4F) In the nucleotide sequence shown in SEQ ID NO: 35, a nucleotide sequence in which one or more nucleotides are added, deleted, or substituted (particularly preferably, a nucleotide sequence in which a total of one or more nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 35), and which encodes a polypeptide having the activity as YliD; (7-4G) A nucleotide sequence having a sequence identity of 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 with respect to the nucleotide sequence shown in SEQ ID NO: 35, and which encodes a polypeptide having the activity as YliD; (7-4H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having the activity as YliD of any of the nucleotide sequences of (7-4E) to (7-4G); (7-4I) In any of the nucleotide sequences of (7-4E) to (7-4H), a nucleotide sequence into which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) is introduced; (7-4J) A nucleotide sequence encoding the amino acid sequence of any of the polypeptides of (7-4A) to (7-4D); or, (7-4K) A nucleotide sequence in which any of the nucleotide sequences of (7-4E) to (7-4J) is used as an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0168] In the above (7-4F), the "one or more" 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.

[0169] In the above (7-4G), "sequence identity" refers to the ratio (%) of the number of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 35 when two base sequences are aligned and gaps are introduced as necessary to maximize the base match rate between the two.

[0170] <8. Glutathione Reductase> Glutathione reductase (EC: 1.8.1.7) is an enzyme that catalyzes the reaction of reducing oxidized glutathione (glutathione disulfide) to produce reduced glutathione in the presence of NADPH.

[0171] Specific examples of glutathione reductase include (8A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28; (8B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 28 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 28), and having glutathione reductase activity; (8C) 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, and having glutathione reductase activity; or (8D) A fragment of any of the polypeptides of (8A) to (8C) having glutathione reductase activity can be such.

[0172] In the above (8D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 300 or more amino acids, and even more preferably 400 or more amino acids.

[0173] In the above (8B), "a plurality" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, conservative amino acid substitutions are desirable for amino acid substitutions.

[0174] In the above (8C), "sequence identity" refers to the percentage (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 28 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between the two.

[0175] The "gene encoding glutathione reductase (EC: 1.8.1.7)" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of glutathione reductase, and is contained in the genomic DNA on the chromosome of a wild-type microorganism before deletion of glutathione reductase.

[0176] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 28 of glutathione reductase derived from Escherichia coli is shown in SEQ ID NO: 27. However, in the genomic DNA of a wild-type microorganism, the nucleotide sequence of SEQ ID NO: 27 does not necessarily exist as it is, and the nucleotide sequence of SEQ ID NO: 27 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0177] That is, specific examples of the nucleotide sequence of the gene encoding the amino acid sequence of glutathione reductase include (8E) the nucleotide sequence shown in SEQ ID NO: 27; (8F) a nucleotide sequence in which 1 to a plurality of bases are added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 27 (particularly preferably, a nucleotide sequence in which a total of 1 to a plurality of bases are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and 3' end of the nucleotide sequence shown in SEQ ID NO: 27), and which encodes a polypeptide having glutathione reductase activity; A base sequence having a sequence identity of 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 with respect to the base sequence shown in SEQ ID NO: 27, and encoding a polypeptide having glutathione reductase activity; (8H) A partial base sequence encoding the amino acid sequence of a polypeptide having glutathione reductase activity of any of the base sequences of (8E) to (8G); (8I) A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (8E) to (8H); (8J) A base sequence encoding the amino acid sequence of any of the polypeptides of (8A) to (8D); or, (8K) A base sequence in which any of the base sequences of (8E) to (8J) is an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0178] In the above (8F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0179] In the above (8G), "sequence identity" means the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 27 when two base sequences are aligned and gaps are introduced as necessary to maximize the base match degree between them.

[0180] <9. Protein Involved in Putrescine Excretion> Putrescine is a compound having the following structure and is biosynthesized in microbial cells.

Chemical Formula

[0181] Putrescine is known to have an effect of promoting 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-glutamylcysteine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione has not been conventionally studied. The inventors have found an unexpectedly advantageous effect that a microbial strain in which the expression of one or more of the genes encoding proteins involved in putrescine excretion is enhanced has significantly higher productivity of γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione than the host strain. This genetic modification is presumed to reduce the intracellular putrescine concentration. The protein involved in putrescine excretion is a protein having a function of excreting intracellular putrescine extracellularly. Examples of the protein involved in putrescine excretion in microorganisms include one or more proteins selected from a cationic peptide transport system substrate-binding protein, a cationic peptide transport system permease protein, and a cationic peptide transport system ATP-binding protein. As long as it is a protein involved in putrescine excretion, not limited to these, by enhancing the expression of one or more of the genes encoding it, the productivity of γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione by the microbial strain can be increased.

[0182] SapA can be exemplified as the cationic peptide transport system substrate-binding protein. SapA is a protein derived from Escherichia coli. The cationic peptide transport system substrate-binding protein is not limited to those having an amino acid sequence or three-dimensional structure similar to SapA, as long as it has a cationic peptide transport system substrate-binding activity and is a protein involved in putrescine excretion.

[0183] Examples of the 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 having an amino acid sequence or a three-dimensional structure similar to SapB or SapC, and may be any protein having a cationic peptide transport system permease activity and involved in putrescine excretion.

[0184] Examples of the 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 having an amino acid sequence or a three-dimensional structure similar to SapD or SapF, and may be any protein having a cationic peptide transport system ATP-binding activity and involved in putrescine excretion.

[0185] The protein involved in putrescine excretion in microorganisms is preferably one or more selected from SapA, SapB, SapC, SapD, and SapF. The 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 the 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". The "gene encoding a protein involved in putrescine excretion" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of a protein involved in putrescine excretion, and is contained in the genomic DNA on the chromosome of the microbial strain.

[0186] In the microbial strains 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 of sapA, sapB, sapC, sapD, and sapF is enhanced.

[0187] Specific examples of the SapA protein (cationic peptide transport system substrate-binding protein) include (9-1A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 40; (9-1B) a polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 40 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 40), and having the 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: 40, and having the activity as SapA; or (9-1D) a fragment having the activity as SapA of any of the polypeptides of (9-1A) to (9-1C) can be.

[0188] In the above (9-1B) to (9-1D) and (9-1F) to (9-1H) described below, "having the activity as SapA" means having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 40, particularly, the cationic peptide transport system substrate-binding activity.

[0189] In the above (9-1D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 300 or more amino acids, more preferably 400 or more amino acids, and more preferably 500 or more amino acids.

[0190] In the above (9-1B), "a plurality" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0191] In the above (9-1C), "sequence identity" refers to the ratio (%) of the same amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 40 when two amino acid sequences are aligned and gaps are introduced as necessary so that the amino acid identity between the two is maximized.

[0192] The "sapA gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of SapA and is contained in the genomic DNA on the chromosome of a microbial strain.

[0193] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 40 of SapA derived from Escherichia coli is shown in SEQ ID NO: 39. However, in the genomic DNA of a microbial strain, the base sequence of SEQ ID NO: 39 does not necessarily exist as it is. The base sequence of SEQ ID NO: 39 is an exon sequence, and one or more intron sequences may be intervening in the middle.

[0194] That is, specific examples of the gene encoding the amino acid sequence of SapA or the base sequence of the sapA gene include: (9-1E) The base sequence shown in SEQ ID NO: 39; (9-1F) A base sequence in which 1 to a plurality of bases are added, deleted, or substituted in the base sequence shown in SEQ ID NO: 39 (particularly preferably, a total of 1 to a plurality of bases are substituted, deleted, and / or added at one or both of the 5'-end and 3'-end of the base sequence shown in SEQ ID NO: 39, preferably a base sequence with deletion and / or addition), and which encodes a polypeptide having the activity as SapA; A base sequence having a sequence identity of 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 with respect to the base sequence shown in SEQ ID NO: 39, and encoding a polypeptide having activity as SapA; (9-1H) A partial base sequence encoding the amino acid sequence of a polypeptide having activity as SapA of any of the base sequences of (9-1E) to (9-1G); (9-1I) A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (9-1E) to (9-1H); (9-1J) A base sequence encoding the amino acid sequence of any of the polypeptides of (9-1A) to (9-1D); or, (9-1K) A base sequence having any of the base sequences of (9-1E) to (9-1J) as an exon sequence and having one or more intron sequences intervening therein may be mentioned.

[0195] In the above (9-1F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0196] In the above (9-1G), "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the base sequence shown in SEQ ID NO: 39 when two base sequences are aligned and gaps are introduced as necessary so that the base match degree between the two is maximized.

[0197] Specific examples of the SapB protein (cationic peptide transport system permease protein) include, (9-2A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 42; (9-2B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 42 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 42), and having the 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: 42, and having the activity as SapB; or (9-2D) A fragment having the activity as SapB of the polypeptide of any one of (9-2A) to (9-2C) It can be the above.

[0198] In the above (9-2B) to (9-2D), and (9-2F) to (9-2H) described below, "having the activity as SapB" means having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 42, particularly, the activity of a cationic peptide transport system permease.

[0199] In the above (9-2D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0200] In the above (9-2B), "one or more" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0201] In the above (9-2C), "sequence identity" refers to the percentage (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 42 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between the two.

[0202] The "sapB gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of SapB, which is contained in the genomic DNA on the chromosome of a microbial strain.

[0203] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 42 of SapB derived from Escherichia coli 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 it is. The nucleotide sequence of SEQ ID NO: 41 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0204] That is, specific examples of the gene encoding the amino acid sequence of SapB or the nucleotide sequence of the sapB gene include: (9-2E) The nucleotide sequence shown in SEQ ID NO: 41; (9-2F) A nucleotide sequence in which one or more nucleotides are added, deleted, or substituted in the nucleotide sequence shown in SEQ ID NO: 41 (particularly preferably, a nucleotide sequence in which a total of one or more nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and 3' end of the nucleotide sequence shown in SEQ ID NO: 41), and which encodes a polypeptide having the activity of SapB; (9-2G) A nucleotide sequence having a sequence identity of 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 with the nucleotide sequence shown in SEQ ID NO: 41, and which encodes a polypeptide having the activity of SapB; (9-2H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having the activity of SapB of any of the nucleotide sequences of (9-2E) to (9-2G); A base sequence in which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced in any of the base sequences of (9-2I), (9-2E) to (9-2H); A base sequence encoding the amino acid sequence of any of the polypeptides of (9-2J), (9-2A) to (9-2D); or, A base sequence having, as an exon sequence, any of the base sequences of (9-2K), (9-2E) to (9-2J), with one or more intron sequences intervening therein may be mentioned.

[0205] In the above (9-2F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0206] In the above (9-2G), "sequence identity" means the ratio (%) of the same bases to the total number of bases of the base sequence shown in SEQ ID NO: 41 when two base sequences are aligned and gaps are introduced as necessary to maximize the base match between them.

[0207] Specific examples of the SapC protein (cationic peptide transport system permease protein) include (9-3A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 44; (9-3B) A polypeptide consisting of an amino acid sequence in which 1 to a plurality of amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 44 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of 1 to a plurality of amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 44), and having the 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: 44, and having the activity as SapC; or A fragment of any of the polypeptides of (9-3D), (9-3A) to (9-3C) having activity as SapC can be.

[0208] In the above (9-3B) to (9-3D), and (9-3F) to (9-3H) described below, "having activity as SapC" refers to having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 44, particularly, cationic peptide transport system permease activity.

[0209] In the above (9-3D), the fragment can be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids.

[0210] In the above (9-3B), "a plurality of" 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. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0211] In the above (9-3C), "sequence identity" refers to the ratio (%) of identical amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 44 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between them.

[0212] The "sapC gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of SapC and is contained in the genomic DNA on the chromosome of a microbial strain.

[0213] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 44 of SapC derived from Escherichia coli 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 it is, and the nucleotide sequence of SEQ ID NO: 43 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0214] That is, specific examples of the gene encoding the amino acid sequence of SapC or the nucleotide sequence of the sapC gene include: (9-3E) the nucleotide sequence shown in SEQ ID NO: 43; (9-3F) In the nucleotide sequence shown in SEQ ID NO: 43, a nucleotide sequence in which one or more nucleotides are added, deleted, or substituted (particularly preferably, a total of one or more nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5'-end and 3'-end of the nucleotide sequence shown in SEQ ID NO: 43), and which encodes a polypeptide having the activity as SapC; (9-3G) A nucleotide sequence having a sequence identity of 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 with respect to the nucleotide sequence shown in SEQ ID NO: 43, and which encodes a polypeptide having the activity as SapC; (9-3H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having the activity as SapC of any of the nucleotide sequences of (9-3E) to (9-3G); (9-3I) In any of the nucleotide sequences of (9-3E) to (9-3H), a nucleotide sequence into which a silent mutation (base substitution that does not change the encoded amino acid residue) is introduced; (9-3J) A nucleotide sequence encoding the amino acid sequence of any of the polypeptides of (9-3A) to (9-3D); or, (9-3K) A nucleotide sequence having any of the nucleotide sequences of (9-3E) to (9-3J) as an exon sequence and having one or more intron sequences intervening therein may be mentioned.

[0215] In the above (9-3F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0216] In the above (9-3G), "sequence identity" means the ratio (%) of identical bases to the total number of bases of the nucleotide sequence shown in SEQ ID NO: 43 when two nucleotide sequences are aligned and gaps are introduced as necessary so that the base match degree between the two is the highest.

[0217] Specific examples of the SapD protein (cationic peptide transport system ATP-binding protein) include: (9-4A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 46; (9-4B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 46 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 46), and having the 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: 46, and having the activity as SapD; or (9-4D) A fragment having the activity as SapD of any of the polypeptides of (9-4A) to (9-4C) can be.

[0218] In the above (9-4B) to (9-4D), and (9-4F) to (9-4H) described below, "having the activity as SapD" means having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 46, particularly, the ATP-binding activity of the cationic peptide transport system.

[0219] In the above (9-4D), the fragment can preferably be a polypeptide having 200 or more amino acids, more preferably 250 or more amino acids, and even more preferably 300 or more amino acids.

[0220] In the above (9-4B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0221] In (9-4C), "sequence identity" refers to the ratio (%) of the number of identical amino acid residues to the total number of amino acid residues in the amino acid sequence shown in SEQ ID NO: 46 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between the two.

[0222] The "sapD gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of SapD, which is contained in the genomic DNA on the chromosome of a microbial strain.

[0223] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 46 of SapD derived from Escherichia coli is shown in SEQ ID NO: 45. However, in the genomic DNA of a microbial strain, the base sequence of SEQ ID NO: 45 does not necessarily exist as it is. The base sequence of SEQ ID NO: 45 may be an exon sequence, and one or more intron sequences may be interposed in the middle.

[0224] That is, specific examples of the gene encoding the amino acid sequence of SapD or the base sequence of the sapD gene include: (9-4E) The base sequence shown in SEQ ID NO: 45; (9-4F) A base sequence in which one or more bases are added, deleted, or substituted in the base sequence shown in SEQ ID NO: 45 (particularly preferably, a base sequence in which a total of one or more bases are substituted, deleted, and / or added at one or both of the 5' end and 3' end of the base sequence shown in SEQ ID NO: 45, preferably a base sequence in which deletion and / or addition occurs), and which encodes a polypeptide having the activity of SapD; (9-4G) A base sequence having a sequence identity of 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 with respect to the base sequence shown in SEQ ID NO: 45, and which encodes a polypeptide having the activity of SapD; (9-4H) A partial base sequence encoding the amino acid sequence of a polypeptide having the activity of SapD among the base sequences of any one of (9-4E) to (9-4G); A nucleotide sequence in which a silent mutation (a base substitution that does not change the encoded amino acid residue) is introduced in any of the nucleotide sequences of (9-4I), (9-4E) to (9-4H); A nucleotide sequence encoding the amino acid sequence of any of the polypeptides of (9-4J), (9-4A) to (9-4D); or, A nucleotide sequence in which any of the nucleotide sequences of (9-4K), (9-4E) to (9-4J) is an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0225] In the above (9-4F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6 or 2 to 3.

[0226] In the above (9-4G), "sequence identity" refers to the ratio (%) of identical bases to the total number of bases of the nucleotide sequence shown in SEQ ID NO: 45 when two nucleotide sequences are aligned and gaps are introduced as necessary so that the base match degree between the two is the highest.

[0227] Specific examples of the SapF protein (cationic peptide transport system ATP-binding protein) include (9-5A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 48; (9-5B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 48 (particularly preferably, a polypeptide consisting of an amino acid sequence in which a total of one or more amino acids are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the N-terminus and C-terminus of the amino acid sequence shown in SEQ ID NO: 48), and having the 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 shown in SEQ ID NO: 48, and having the activity as SapF; or A fragment of any of the polypeptides of (9-5D), (9-5A) to (9-5C) having activity as SapF It can be.

[0228] In the above (9-5B) to (9-5D) and (9-5F) to (9-5H) described later, "having activity as SapF" means having the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 48, particularly, cationic peptide transport system ATP binding activity.

[0229] In the above (9-5D), the fragment can be a polypeptide having preferably 200 or more amino acids, more preferably 250 or more amino acids.

[0230] In the above (9-5B), "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3. Also, the amino acid substitution is preferably a conservative amino acid substitution.

[0231] In the above (9-5C), "sequence identity" refers to the ratio (%) of the same amino acid residues to the total number of amino acid residues of the amino acid sequence shown in SEQ ID NO: 48 when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the amino acid identity between the two.

[0232] The "sapF gene" refers to a nucleic acid (DNA or RNA, preferably DNA) encoding the amino acid sequence of SapF and is contained in the genomic DNA on the chromosome of a microbial strain.

[0233] An example of the DNA encoding the amino acid sequence shown in SEQ ID NO: 48 of SapF derived from Escherichia coli is shown in SEQ ID NO: 47. However, in the genomic DNA of a microbial strain, the nucleotide sequence of SEQ ID NO: 47 does not necessarily exist as it is, and the nucleotide sequence of SEQ ID NO: 47 is an exon sequence, and one or more intron sequences may be interposed in the middle.

[0234] That is, specific examples of the gene encoding the amino acid sequence of SapF or the nucleotide sequence of the sapF gene include: (9-5E) the nucleotide sequence shown in SEQ ID NO: 47; (9-5F) In the nucleotide sequence shown in SEQ ID NO: 47, a nucleotide sequence in which one or more nucleotides are added, deleted, or substituted (particularly preferably, a total of one or more nucleotides are substituted, deleted, and / or added, preferably deleted and / or added, at one or both of the 5' end and the 3' end of the nucleotide sequence shown in SEQ ID NO: 47), and which encodes a polypeptide having the activity as SapF; (9-5G) A nucleotide sequence having a sequence identity of 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 with the nucleotide sequence shown in SEQ ID NO: 47, and which encodes a polypeptide having the activity as SapF; (9-5H) A partial nucleotide sequence encoding the amino acid sequence of a polypeptide having the activity as SapF among the nucleotide sequences of any one of (9-5E) to (9-5G); (9-5I) A nucleotide sequence in which a silent mutation (a nucleotide substitution that does not change the encoded amino acid residue) is introduced in the nucleotide sequence of any one of (9-5E) to (9-5H); (9-5J) A nucleotide sequence encoding the amino acid sequence of any one of the polypeptides of (9-5A) to (9-5D); or, (9-5K) A nucleotide sequence in which any one of the nucleotide sequences of (9-5E) to (9-5J) is used as an exon sequence and one or more intron sequences are interposed in the middle may be mentioned.

[0235] In the above (9-5F), "a plurality of" means, for example, 2 to 60, 2 to 45, 2 to 30, 2 to 21, 2 to 15, 2 to 6, or 2 to 3.

[0236] In the above (9-5G), "sequence identity" refers to the ratio (%) of identical nucleotides to the total number of nucleotides of the nucleotide sequence shown in SEQ ID NO: 47 when two nucleotide sequences are aligned and gaps are introduced as necessary to maximize the nucleotide identity between the two.

[0237] <Microbial strain according to the present invention> One or more embodiments of the present invention are [1] A microbial strain capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione, having the gene modification of [1]: [1] Enhancement of the expression of the gene encoding serine-O-acetyltransferase (EC: 2.3.1.30) relates to.

[0238] The microbial strain has a high ability to produce γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione by fermentation.

[0239] As used herein, the "microbial strain capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione" means a microbial strain having a higher ability to produce γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione than the host strain (wild strain or parental strain) before introducing a predetermined gene modification.

[0240] The microbial strain more preferably further has the gene modification of the following [2] and has one or more gene modifications selected from [3] and [4]. [2] Deletion of the gene encoding γ-glutamyltransferase (EC: 3.4.19.13); [3] Enhancement of the expression of the gene encoding glutamate-cysteine ligase (EC: 6.3.2.2) and / or the gene encoding glutathione synthetase (EC: 6.3.2.3); [4] Enhancement of the expression of the gene encoding bifunctional glutathione synthetase.

[0241] The microbial strain having the gene modification of [2] and one or more gene modifications selected from [3] and [4] can produce more γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcystine, reduced glutathione and / or oxidized glutathione than the host strain not having one or more of the gene modification of [2] and one or more gene modifications selected from [3] and [4]. Therefore, when combined with the gene modification of [1] above, the substance can be produced particularly efficiently.

[0242] When the microorganism is used for the production of γ-glutamylcysteine, bis-γ-glutamylcysteine and / or γ-glutamylcystine, it preferably has the gene modification of [3] among the gene modifications of [3] and [4]. In this case, the gene modification of [3] is preferably the enhancement of the expression of the gene encoding glutamate-cysteine ligase.

[0243] When the microorganism is used for the production of reduced glutathione and / or oxidized glutathione, it may have either of the gene modifications of [3] and [4], or both. In this case, the gene modification of [3] may be the enhancement of the expression of only one of the gene encoding glutamate-cysteine ligase and the gene encoding glutathione synthase, but more preferably the enhancement of the expression of both.

[0244] More preferably, the microbial strain further has one or more gene modifications selected from the following [5], [6], [7], [8] and [9]. [5] Deletion of the gene encoding tryptophanase (EC: 4.1.99.1); [6] Deletion of the gene encoding tripeptide peptidase (EC: 3.4.11.4); [7] Deletion of one or more of the genes encoding proteins involved in glutathione uptake; [8] Deletion of the gene encoding glutathione reductase (EC: 1.8.1.7); [9] Enhancement of the expression of one or more genes encoding proteins involved in putrescine excretion.

[0245] The microbial strain having one or more of the above genetic modifications selected from [5], [6], [7], [8] and [9] can produce more γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione compared to the host strain having none of the genetic modifications selected from [5], [6], [7], [8] and [9]. Therefore, when combined with the genetic modification of [1] above, more preferably, when combined with the genetic modification of [1] above, the genetic modification of [2], and one or more genetic modifications selected from [3] and [4], the substance can be produced particularly efficiently.

[0246] In a more preferred embodiment of the microbial strain, among [5], [6], [7], [8] and [9], it preferably has 2 or more, more preferably 3 or more, more preferably 4 or more, and more preferably all genetic modifications.

[0247] The microorganism serving as the host of the microbial strain according to one or more embodiments of the present invention is as described above.

[0248] Preferred examples of each gene 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.

[0249] The enhancement of the expression of a predetermined gene in the microbial strain according to one or more embodiments of the present invention will be described.

[0250] The gene to be enhanced in expression as defined in [1], [3], [4] and [9] may be referred to as the "expression-enhanced gene". The microbial strain having one or more gene modifications among [1], [3], [4] and [9] means that when the host strain (wild strain or parental strain) of the microbial strain originally expresses the expression-enhanced gene, the expression level of the expression-enhanced gene is increased as compared with the host strain, and when the host strain does not originally express the expression-enhanced gene, the ability to express the expression-enhanced gene is imparted to the host strain.

[0251] The increase in the expression level of the expression-enhanced gene can be achieved by replacing the promoter that controls the expression of the expression-enhanced gene on the genomic DNA of the microbial cell with a more powerful expression promoter, or by increasing the copy number of the expression-enhanced gene in the microbial cell. In the host strain originally having the expression-enhanced gene, when a genetically modified microbial strain into which a gene modification has been introduced to modify the expression promoter that controls the expression of the expression-enhanced gene into a second expression promoter different from the first expression promoter originally possessed by the host strain has the ability to express more of the expression-enhanced gene than the host strain, the second expression promoter is a "more powerful expression promoter".

[0252] When replacing the promoter of the expression-enhancing gene with a more potent expression promoter on the genomic DNA of the cells of the microorganism, preferred specific examples of the expression promoter include the tac promoter, trc promoter, ompF promoter, ompA promoter, cysK promoter, and lpp promoter. An example of the nucleotide sequence in which the tac promoter is linked to the SD sequence is shown in SEQ ID NO: 6. An example of the nucleotide sequence in which the trc promoter is linked to the SD sequence is shown in SEQ ID NO: 8. An example of the nucleotide sequence in which the ompF promoter is linked to the SD sequence is shown in SEQ ID NO: 10. An example of the nucleotide sequence in which the ompA promoter is linked to the SD sequence is shown in SEQ ID NO: 18. An example of the nucleotide sequence in which the cysK promoter is linked to the SD sequence is shown in SEQ ID NO: 19. An example of the nucleotide sequence in which the lpp promoter is linked to the SD sequence is shown in SEQ ID NO: 20.

[0253] An inducible promoter may be used as the expression promoter. Further, the above expression promoter may be functionally linked to an operator sequence to be an inducible promoter.

[0254] Examples of the inducible promoter include an isopropyl-β-thiogalactopyranoside (IPTG)-inducible promoter, a light-inducible promoter that induces 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 temperature), a promoter containing the tetO or lacO operator as the operator sequence, etc. can be exemplified, and the IPTG-inducible promoter, araBAD promoter, rhaBAD promoter, tet promoter, penP promoter, cspA promoter, or a promoter containing the tetO or lacO operator as the operator sequence is preferred.

[0255] Specific examples of IPTG-inducible promoters include the lacUV5 promoter, lac promoter, lacT5 promoter, lacT7 promoter, and T5 promoter, T7 promoter, tac promoter, etc. that are made IPTG-inducible by being functionally linked to an operator sequence. As the inducible promoter, an IPTG-inducible promoter is particularly preferred, and among the IPTG-inducible promoters, in particular, the T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter or tac promoter is preferred.

[0256] As the promoter, by using various reporter genes, a promoter obtained by modifying a native promoter into a highly active form can also be used. For example, by making the -35 and -10 regions in the promoter region closer to the consensus sequence, the activity of the promoter can be enhanced (International Publication WO00 / 18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574). Methods for evaluating the strength of promoters and examples of strong promoters are described in the paper by Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)) and the like.

[0257] In order to enhance the expression of the expression-enhancing gene, increasing the copy number of the expression-enhancing gene in the cells of the microbial strain can be achieved by (A) introducing an expression vector containing the expression-enhancing gene into the cells of the microbial strain, or (B) introducing the expression-enhancing gene into the genomic DNA of the cells of the microbial strain and can be achieved thereby.

[0258] As the expression vector used in the aspect of (A) above, a plasmid vector containing the expression enhancement gene can be used. The expression vector is preferably capable of autonomous replication in a microbial cell. The expression vector preferably contains DNA encoding a predetermined protein and a promoter functionally linked to a position where the DNA can be transcribed. The expression vector is preferably a recombinant DNA that is capable of autonomous replication in a microbial cell and contains a base sequence composed of a promoter, a ribosome binding sequence, a base sequence encoding the amino acid sequence of the above one or more enzymes, and a transcription termination sequence. The microbial strain according to one or more embodiments of the present invention preferably holds an expression vector containing a base sequence encoding the expression enhancement gene in a state where the expression enhancement gene can be expressed. Here, "the expression enhancement gene can be expressed" may mean that the constitutive expression of the expression enhancement gene is possible, or may mean that the inducible expression of the expression enhancement gene is possible.

[0259] Examples of 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 Co., Ltd.), pTipRT2 (available from Hokkaido System Science Co., Ltd.); pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16A, pNH18A 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 (can be prepared by modifying pUC18 (available from Takara Bio Inc.)), pSTV28 (available from Takara Bio Inc.), pUCNT (International Publication No. 94 / 03613), etc.

[0260] The expression vector preferably contains a promoter that controls the transcription of the expression-enhancing gene, more preferably an inducible promoter. Preferred examples of the promoter are as described above.

[0261] When introducing the expression vector containing the expression-enhancing gene 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, more preferably 20 or more.

[0262] When increasing the expression levels of two or more of the expression-enhancing genes in the cells of a microbial strain, two or more genes may be contained in one expression vector. In this case, two or more genes may be arranged under the control of one expression promoter. Also, two or more genes may be contained in separate expression vectors, respectively.

[0263] According to the aspect of (B) above, when introducing the expression-enhancing gene into the genomic DNA of the cells of a microbial strain, homologous recombination can be utilized.

[0264] In the microbial strain according to one or more embodiments of the present invention, the degree of enhancement (increase in expression level) of the expression of the expression-enhancing gene is not particularly limited. The expression level of the expression-enhancing gene can be represented as the amount of mRNA corresponding to the expression-enhancing gene extracted from the cells. The expression level based on this mRNA is preferably represented as a relative value with respect to the amount of mRNA encoding an appropriate internal standard protein. As the internal standard protein, a protein encoded by the hcaT gene known as a housekeeping gene can be exemplified. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, etc. (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). When the expression level in the host strain is set to 100%, the expression level based on the mRNA of the expression-enhancing gene is preferably 150% or more, 200% or more, 500% or more, 1000% or more, 1200%, 2000% or more, or 2500% or more. The upper limit of the expression level based on the mRNA of the expression-enhancing gene is not particularly limited, but when the expression level in the host strain is set to 100%, it can be, for example, 5000% or less, or 3000% or less.

[0265] The increase in the expression level of the expression-enhancing gene can also be represented as an increase in the activity of the protein encoded by the expression-enhancing gene extracted from the cell. The confirmation of the increase in the activity of the protein can be performed by measuring the amount or activity of the protein. The confirmation of the increase in the amount of the protein encoded by the expression-enhancing gene can be performed 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 expression-enhancing gene is preferably 150% or more, 200% or more, 500% or more, 1000% or more, 1200%, 2000% or more, or 2500% or more when the amount of the protein in the host strain is set to 100%. The upper limit of the expression level based on the protein of the expression-enhancing gene is not particularly limited, but can be, for example, 5000% or less, or 3000% or less when the expression level in the host strain is set to 100%.

[0266] Subsequently, the deletion of a predetermined gene in the microbial strain according to one or more embodiments of the present invention will be described.

[0267] In [2], [5], [6], [7] and [8] above, the gene to be deleted may be referred to as the "gene to be deleted". The "deletion" of the gene in [2], [5], [6], [7] and [8] means that the activity of the protein encoded by the gene to be deleted is decreased compared to the host strain, including the case where the activity has completely disappeared. The microbial strain according to one or more embodiments of the present invention is a microbial strain in a state where the function of the gene to be deleted is lost or the function is decreased. Specifically, it includes a state where the expression level of mRNA, which is the transcription product of the gene to be deleted, or protein, which is the translation product, is decreased, or a state where mRNA or protein, which is the transcription product or translation product of the gene to be deleted, does not function normally as mRNA or protein.

[0268] The deletion of the target gene can be achieved, for example, by artificially modifying the gene of the host strain. Such modification can be achieved, for example, by mutation treatment, gene recombination technology, gene expression suppression treatment using RNAi, etc.

[0269] Examples of the mutation treatment include ultraviolet irradiation or treatment with a mutagen commonly used in normal mutation treatments such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0270] As the gene recombination technology, known technologies (for example, FEMS Microbiology Letters 165 (1998) 335-340, JOURNAL OF BACTERIOLOGY, Dec. 1995, p7171-7177, Curr Genet 1986; 10(8):573-578, WO 98 / 14600, etc.) can be utilized.

[0271] The gene encoding the predetermined protein described in [2], [5], [6], [7] and [8] above refers to not only the coding region of the amino acid sequence of each protein, but also the expression regulatory sequence (such as promoter sequence), exon sequence, intron sequence, etc., without distinction. When modifying the expression regulatory sequence, the expression regulatory sequence is preferably modified by 1 base or more, more preferably 2 bases or more, and particularly preferably 3 bases or more.

[0272] The deletion of the target gene is more preferably the deletion of the target gene in the genomic DNA of the microbial strain. The deletion of the target gene may be a partial or complete deletion of the expression regulatory sequence, or a partial or complete deletion of the coding region of the amino acid sequence of each protein. Here, "deletion" means deletion or damage, and is preferably deletion.

[0273] In the genomic DNA of the host strain, the entire gene may be deleted, including the sequences before and after the target gene to be deleted. When a part or all of the coding region of the amino acid sequence of the protein encoded by the target gene to be deleted is deleted, as long as a decrease in protein activity can be achieved, the coding region of any region such as the N-terminal region, internal region, C-terminal region, etc. may be deleted. Usually, the longer the region to be deleted, the more surely the gene can be inactivated. Also, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. In a preferred embodiment, in the genomic DNA, at least a part of the coding region and / or expression regulatory sequence of the target gene to be deleted, for example, preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 100% of the total number of bases of the coding region and / or expression regulatory sequence, is a region deleted in the microbial strain. Particularly preferably, it is a microbial strain in which the region from the start codon to the stop codon of the target gene to be deleted is deleted in the genomic DNA.

[0274] Also, as other examples of deletion of the target gene to be deleted such that the activity of the protein decreases, introducing an amino acid substitution (missense mutation) into the amino acid sequence coding region of the target gene on the genomic DNA, introducing a stop codon (nonsense mutation), or introducing a frameshift mutation in which 1 to 2 bases are added or deleted, etc., can be exemplified as damage to the target gene to be deleted.

[0275] In addition, the deletion of the target gene that results in a decrease in protein activity can also be achieved, for example, by inserting another sequence into the expression regulatory sequence or the amino acid sequence coding region of the target gene on genomic DNA. The insertion site can be in any region of the gene, but a longer inserted sequence can more reliably inactivate the gene. Also, 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 can reduce or eliminate the function of the encoded protein. Examples thereof include a marker gene and a gene useful for the production of a target substance such as glutathione.

[0276] Deleting the target gene on genomic DNA as described above can be achieved, for example, by preparing an inactivated gene in which the target gene is modified so as not to produce a normally functioning protein, transforming a host strain with a recombinant DNA containing the inactivated gene, and causing homologous recombination between the inactivated gene and the gene on genomic DNA to replace the gene on genomic DNA with the inactivated gene. At that time, it is easier to operate if the recombinant DNA contains a marker gene according to the traits of the host such as auxotrophy. Also, if the recombinant DNA is linearized by cleavage with a restriction enzyme or the like, a strain in which the recombinant DNA is integrated into genomic DNA can be efficiently obtained. Even if the protein encoded by the inactivated gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost.

[0277] Also, for example, a linear DNA containing an arbitrary sequence, which is provided with sequences upstream and downstream of a site to be replaced on genomic DNA (typically, a part or all of the target gene to be deleted) at both ends of the arbitrary sequence, or a linear DNA in which the sequences upstream and downstream of the site to be replaced on genomic DNA are directly linked is used to transform a microorganism, and homologous recombination is caused to occur upstream and downstream of the site to be replaced on the genomic DNA of the host strain, respectively, so that the site to be replaced can be replaced with the sequence of the linear DNA in one step. The arbitrary sequence may contain, for example, a marker gene sequence. The marker gene may be removed later if necessary. When removing the marker gene, sequences for homologous recombination may be added to both ends of the marker gene so that the marker gene can be efficiently removed.

[0278] Confirmation that the target gene to be deleted is deleted in a microbial strain can be made by a decrease in the activity of the protein encoded by the target gene to be deleted. Confirmation that the activity of the protein has decreased can be carried out by measuring the amount or activity of the protein.

[0279] Confirmation that the transcription level of the target gene to be deleted has decreased can be carried out by comparing the amount of mRNA transcribed from the gene with that of the host strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, etc. (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of mRNA preferably has decreased, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared with the host strain.

[0280] Confirmation that the amount of the protein encoded by the target gene with a deficiency has decreased can be performed 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 target gene with a deficiency is preferably decreased to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the host strain.

[0281] <Method for producing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione according to the present invention> One or more further embodiments of the present invention are including culturing the microbial strain according to one or more embodiments of the present invention as described above, relate to a method for producing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione.

[0282] According to this method, it is possible to produce γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione at low cost. According to this method, the productivity of the target substance is high. In one aspect of this method, the yield of the target substance with respect to the sugar raw material supplied to the medium is high (high sugar yield). In another aspect, the target substance can be secreted at a high concentration into the medium.

[0283] When this method is a method for producing reduced glutathione and / or oxidized glutathione, the microbial strain to be used has the gene modifications of [1] and [2], and has one or more gene modifications selected from [3] and [4]. [3] is preferably the enhancement of the expression of both the gene encoding glutamate-cysteine ligase and the gene encoding glutathione synthase.

[0284] When this method is a method for producing γ-glutamylcysteine, bis-γ-glutamylcysteine, and / or γ-glutamylcysteine, the microbial strain to be used has the genetic modifications of [1] and [2], and has one or more genetic modifications selected from [3] and [4], and [3] is preferably the enhancement of the expression of the gene encoding glutamate-cysteine ligase.

[0285] The culture of the microbial strain according to one or more embodiments of the present invention can be carried out in a suitable medium. The medium can be either a synthetic medium or a natural medium as long as it contains nutrients necessary for the growth of the microorganisms used in the present invention, such as a carbon source, a nitrogen source, inorganic salts, vitamins, etc., and the biosynthesis of the target substance. Preferably, the M9 medium is used.

[0286] As the carbon source, any carbon source that can be assimilated by the microorganisms to be used may be used, and examples thereof include saccharides such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0287] 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 hydrolyzate. Examples of the inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0288] Examples of the vitamins include biotin and thiamine. Furthermore, substances required for the growth of the microbial strain according to one or more embodiments of the present invention (for example, the required amino acids in the case of an amino acid-requiring microbial strain) can be added as needed.

[0289] It is preferable to add at least one, preferably both, of a sulfur source and glycine to the medium. Examples of the addition concentration of glycine to the medium include 100 mM to 2000 mM, preferably 400 mM to 1200 mM. Examples of the addition concentration of the sulfur source to the medium include 100 mM to 2000 mM, preferably 400 mM to 1200 mM.

[0290] As the sulfur source, one or more inorganic sulfur compounds such as sulfuric acid, thiosulfuric acid, sulfurous acid, hyposulfurous acid, or sulfide or its salts can be added. Sulfuric acid, thiosulfuric acid, sulfurous acid, hyposulfurous acid, or sulfide may be in a free form, a salt form, or any mixture thereof. The salt is not particularly limited, and examples include sodium salts, calcium salts, ammonium salts, potassium salts, and the like.

[0291] Glycine may be in a free form, a salt form, or any mixture thereof. The salt is not particularly limited, and examples include sulfates and hydrochlorides.

[0292] 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 may be added to the medium all at once, or continuously or intermittently.

[0293] The sulfur source and / or glycine may be contained in the medium throughout the entire culture period, or may be contained in the medium only during a part of the culture period. For example, the addition amounts of the sulfur source and glycine do not necessarily need to be within the above ranges throughout the entire period of the stage of producing and accumulating the target substance. The sulfur source and / or glycine are contained in the medium such that the content is within the above ranges during the culture, and the sulfur source and / or glycine content may decrease as the culture time elapses. Also, the sulfur source and / or glycine may be added continuously or intermittently. In addition, the concentrations of medium components other than the sulfur source and / or glycine may vary during the culture period or may be additionally added.

[0294] The cultivation is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The cultivation temperature is 20 to 50 °C, preferably 20 to 42 °C, 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.

[0295] The target substance accumulated in the culture can be collected by ordinary purification methods. For example, after completion of the cultivation, the cells and solids in the culture are removed by centrifugation or the like, and then it can be collected by ion exchange, concentration, and fractional crystallization.

Examples

[0296] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0297] The genetic manipulations described below can be carried out with reference to the description in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Also, the enzymes, cloning hosts, etc. used in genetic manipulations can be purchased from market suppliers and used according to their instructions. The enzyme is not particularly limited as long as it can be used in genetic manipulations.

[0298] (Analysis of glutathione concentration in the culture solution) The glutathione concentration in the culture solution was measured using a high performance liquid chromatograph (HPLC, Shimadzu Corporation). The analysis conditions for HPLC 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, and adjust the pH to 3 with phosphoric acid. After adding 250 mL of methanol, adjust the pH to 3 with phosphoric acid again. Flow rate: 1 mL / min Detection: UV detector, λ = 210 nm Column temperature: 40 °C Injection volume: 10 μL

[0299] When analyzing the glutathione concentration contained in the culture solution, after removing the cells by centrifugation, 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.

[0300] (Production Example 1) Preparation of BW25113Δggt strain First, a plasmid vector for disrupting the ggt (γ-glutamyltransferase) gene (SEQ ID NO: 23) was prepared. By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 1) having the upstream sequence and the downstream sequence of the ggt gene on the chromosome was obtained. The obtained fragment was digested with XbaI and HindIII, and ligated with 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.

[0301] Next, the BW25113Δggt strain was prepared using pTH18cs1-ggt-UD. pTH18cs1-ggt-UD was introduced into Escherichia coli BW25113 strain by the electroporation method, spread on an LB agar plate containing 10 μg / mL of chloramphenicol, and cultured at 30 °C to obtain transformants. The obtained transformants were cultured overnight with shaking at 30 °C in an LB liquid medium containing 10 μg / mL of chloramphenicol, and the culture solution was spread on an LB agar plate containing 10 μg / mL of chloramphenicol and cultured at 42 °C to obtain transformants. The obtained transformants were cultured overnight at 42 °C in an LB liquid medium and then spread on an LB agar plate to obtain colonies. The obtained colonies were replicated onto an LB agar plate and an LB agar plate containing 10 μg / mL of chloramphenicol, respectively, and transformants showing chloramphenicol sensitivity were selected. From the selected transformants, one strain of bacteria with a deletion from the start codon to the stop codon of the ggt gene on the chromosome was isolated by analysis using PCR and a DNA sequencer. This gene disruption strain was named the BW25113Δggt strain.

[0302] The BW25113Δggt strain is a strain with a deletion from the start codon to the stop codon of the ggt gene on the chromosome, using Escherichia coli BW25113 strain as the host strain.

[0303] (Production Example 2) Preparation of BW25113ΔggtΔpepT strain First, a plasmid vector for disrupting the pepT (tripeptide peptidase) gene (SEQ ID NO: 25) was prepared. A DNA fragment (SEQ ID NO: 2) having the upstream sequence and downstream sequence of the pepT gene on the chromosome was obtained by PCR using synthetic oligonucleotide DNA. The obtained fragment was digested with XbaI and HindIII, ligated with a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver. 2, and the plasmid vector pTH18cs1-pepT-UD was obtained.

[0304] Next, using the BW25113Δggt strain prepared in Production Example 1 as the host strain, a strain in which the region from the start codon to the stop codon of the pepT gene on the chromosome was deleted was isolated in the same manner as in Production Example 1 using pTH18cs1-pepT-UD. This gene disruption strain was named the BW25113ΔggtΔpepT strain.

[0305] The BW25113ΔggtΔpepT strain is a strain in which the ggt gene and the region from the start codon to the stop codon of the pepT gene on the chromosome have been deleted, using the Escherichia coli BW25113 strain as the host strain.

[0306] (Production Example 3) Preparation of BW25113ΔggtΔpepTΔgor strain First, a plasmid vector for disrupting the gor (glutathione reductase) gene (SEQ ID NO: 27) was prepared. A DNA fragment (SEQ ID NO: 3) having the upstream and downstream sequences of the gor gene on the chromosome was obtained by PCR using synthetic oligonucleotide DNA. The obtained fragment was digested with XbaI and HindIII, ligated with a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver. 2, and the plasmid vector pTH18cs1-gor-UD was obtained.

[0307] Next, using the BW25113ΔggtΔpepT strain prepared in Production Example 2 as the host strain, a strain in which the region from the start codon to the stop codon of the gor gene on the chromosome was deleted was isolated in the same manner as in Production Example 1 using pTH18cs1-gor-UD. This gene disruption strain was named the BW25113ΔggtΔpepTΔgor strain.

[0308] (Production Example 4) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCD strain First, a plasmid vector was prepared to disrupt the yliABCD genes on the chromosome that form an operon consisting of the yliA (glutathione transport system ATP-binding protein) gene (SEQ ID NO: 29), yliB (glutathione transport system substrate-binding protein) gene (SEQ ID NO: 31), yliC (glutathione transport system permease protein) gene (SEQ ID NO: 33), and yliD (glutathione transport system permease protein) gene (SEQ ID NO: 35). By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 4) having the upstream sequence of the yliA gene and the downstream sequence of the yliD gene on the chromosome was obtained. The obtained fragment was digested with XbaI and HindIII, ligated with the fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver.2, and the plasmid vector pTH18cs1-yliABCD-UD was obtained.

[0309] Next, using the BW25113ΔggtΔpepTΔgor strain prepared in Production Example 3 as the host strain, a strain in which the yliABCD genes on the chromosome were deleted from the start codon to the stop codon was isolated in the same manner as in Production Example 1 using pTH18cs1-yliABCD-UD. This gene disruption strain was named BW25113ΔggtΔpepTΔgorΔyliABCD strain.

[0310] (Production Example 5) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA strain First, a plasmid vector was prepared to disrupt the tnaA (tryptophanase) gene (SEQ ID NO: 37). By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 5) having the upstream sequence and the downstream sequence of the tnaA gene on the chromosome was obtained. The obtained fragment was digested with XbaI and HindIII, ligated with the fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver.2, and the plasmid vector pTH18cs1-tnaA-UD was obtained.

[0311] Next, using the BW25113ΔggtΔpepTΔgorΔyliABCD strain prepared in Production Example 4 as a host strain, a strain in which the region from the start codon to the stop codon of the tnaA gene on the chromosome was deleted in the same manner as in Production Example 1 was isolated using pTH18cs1-tnaA-UD. This gene disruption strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA strain.

[0312] (Production Example 6) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF strain First, a plasmid vector for enhancing the expression of these genes was prepared by inserting a tac promoter and an SD sequence (SEQ ID NO: 6) upstream of the sapABCDF gene on the chromosome that forms an operon consisting of the sapA (cationic peptide transport system substrate-binding protein) gene (SEQ ID NO: 39), sapB (cationic peptide transport system permease protein) gene (SEQ ID NO: 41), sapC (cationic peptide transport system permease protein) gene (SEQ ID NO: 43), sapD (cationic peptide transport system ATP-binding protein) gene (SEQ ID NO: 45), and sapF (cationic peptide transport system ATP-binding protein) gene (SEQ ID NO: 47). By PCR using synthetic oligonucleotide DNA, a DNA fragment (SEQ ID NO: 7) having the upstream sequence of the sapA gene on the chromosome, the tac promoter and the SD sequence, and a sequence of 500 bp from the start codon of the sapA gene was obtained. The obtained fragment was digested with BamHI and HindIII, ligated with the fragment obtained by digesting pTH18cs1 with BamHI and HindIII using Ligation high Ver.2, and the plasmid vector pTH18cs1-Ptac-sapA-UD was obtained.

[0313] Next, using the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA strain prepared in Production Example 5 as the host strain, a strain in which a tac promoter and an SD sequence were inserted upstream of the sapA gene on the chromosome in the same manner as in Production Example 1 using pTH18cs1-Ptac-sapA-UD was isolated. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF strain.

[0314] (Production Example 7) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE strain First, a plasmid vector for enhancing the expression of these genes was prepared by inserting a trc promoter and an SD sequence (SEQ ID NO: 8) upstream of the cysE (serine-O-acetyltransferase) gene (SEQ ID NO: 21) on the chromosome. By PCR using synthetic oligonucleotide DNA, a DNA fragment (SEQ ID NO: 9) having an upstream sequence of the cysE gene on the chromosome, a trc promoter and an SD sequence, and a sequence of 500 bp from the start codon of the cysE gene was obtained. The obtained fragment was digested with XbaI and HindIII, ligated with the fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver.2, and a plasmid vector pTH18cs1-Ptrc-cysE-UD was obtained.

[0315] Next, using the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF strain prepared in Production Example 6 as the host strain, a strain in which a trc promoter and an SD sequence were inserted upstream of the cysE gene on the chromosome in the same manner as in Production Example 1 using pTH18cs1-Ptrc-cysE-UD was isolated. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE strain.

[0316] (Production Example 8) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF PompF-cysE strain First, an ompF promoter and an SD sequence (SEQ ID NO: 10) were inserted upstream of the cysE gene (SEQ ID NO: 21) on the chromosome to prepare a plasmid vector for enhancing the expression of these genes. By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 11) having an upstream sequence of the cysE gene on the chromosome, an ompF promoter and an SD sequence, and a sequence of 500 bp from the start codon of the cysE gene was obtained. The obtained fragment was digested with XbaI and HindIII, ligated with a fragment obtained by digesting pTH18cs1 with XbaI and HindIII using Ligation high Ver.2, and a plasmid vector pTH18cs1-PompF-cysE-UD was obtained.

[0317] Next, using the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF strain prepared in Production Example 6 as a host strain, a strain in which an ompF promoter and an SD sequence were inserted upstream of the cysE gene on the chromosome in the same manner as in Production Example 1 using pTH18cs1-PompF-cysE-UD was isolated. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF PompF-cysE strain.

[0318] (Production Example 9) Preparation of pQEK1-PT5-ABTd*-term First, to construct a vector for gene introduction into Escherichia coli, based on pQE-80L (QIAGEN), the drug resistance marker was changed to a tetracycline resistance gene, and a pQEK1 vector shown in SEQ ID NO: 12 was constructed. Further, a terminator sequence derived from lambda phage was inserted into the HindIII locus of pQEK1 to construct a pQEK1-term vector shown in SEQ ID NO: 13.

[0319] Next, a DNA fragment (SEQ ID NO: 14) consisting of a T5 promoter, an E. coli-derived gshA gene (SEQ ID NO: 55), and a Thiobacilus denitrificans-derived gshB gene (carrying the V260A mutation) (SEQ ID NO: 51) was obtained by PCR using synthetic oligo DNA. The obtained fragment was ligated with 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: 15.

[0320] (Production Example 10) Preparation of pQEK1-PT5-FSa-term A DNA fragment (SEQ ID NO: 16) consisting of a T5 promoter and a Streptococcus agalactiae-derived gshF gene (SEQ ID NO: 53) was obtained by PCR using synthetic oligo DNA. The obtained fragment was ligated with a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix to obtain pQEK1-PT5-FSa-term shown in SEQ ID NO: 17.

[0321] (Production Example 11) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-ABTd*-term Strain pQEK1-PT5-ABTd*-term prepared in Production Example 9 was introduced into the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF strain prepared in Production Example 6 by the electroporation method, and the transformants were selected by plating on an LB agar plate containing 20 μg / mL of tetracycline. One strain of the strain into which pQEK1-PT5-ABTd*-term was introduced was isolated by PCR analysis from the selected transformants. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-ABTd*-term strain.

[0322] (Production Example 12) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-ABTd*-term Strain Into the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE strain prepared in Production Example 7, pQEK1-PT5-ABTd*-term prepared in Production Example 9 was introduced using the electroporation method, and the transformants were selected by plating on an LB agar plate containing 20 μg / mL of tetracycline. One strain of the strain into which pQEK1-PT5-ABTd*-term was introduced was isolated by PCR analysis from the selected transformants. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-ABTd*-term strain.

[0323] (Production Example 13) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF PompF-cysE / pQEK1-PT5-ABTd*-term Strain Into the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF PompF-cysE strain prepared in Production Example 8, pQEK1-PT5-ABTd*-term prepared in Production Example 9 was introduced using the electroporation method, and the transformants were selected by plating on an LB agar plate containing 20 μg / mL of tetracycline. One strain of the strain into which pQEK1-PT5-ABTd*-term was introduced was isolated by PCR analysis from the selected transformants. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF PompF-cysE / pQEK1-PT5-ABTd*-term strain.

[0324] (Production Example 14) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-FSa-term Strain pQEK1-PT5-FSa-term prepared in Production Example 10 was introduced into the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF strain prepared in Production Example 6 using the electroporation method, and the transformants were selected by plating on an LB agar plate containing 20 μg / mL of tetracycline. One strain of the strain into which pQEK1-PT5-FSa-term was introduced was isolated by PCR analysis from the selected transformants. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-FSa-term strain.

[0325] (Production Example 15) Preparation of BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-FSa-term Strain pQEK1-PT5-FSa-term prepared in Production Example 11 was introduced into the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE strain prepared in Production Example 6 using the electroporation method, and the transformants were selected by plating on an LB agar plate containing 20 μg / mL of tetracycline. One strain of the strain into which pQEK1-PT5-FSa-term was introduced was isolated by PCR analysis from the selected transformants. This strain was named BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-FSa-term strain.

[0326] (Example 1) Fermentative Production of Glutathione by BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-ABTd*-term Strain The BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-ABTd*-term strain obtained in Production Example 12 was cultured under the following conditions to produce GSH and GSSG. The above-mentioned strain obtained in Production Example 12 was inoculated into 5 mL of LB medium (containing 20 μg / mL of tetracycline) and cultured with shaking at 300 rpm and 30 °C for 8 hours. 1 mL of this culture solution was inoculated into 100 mL of M9 medium (6 g / L of disodium hydrogen phosphate, 3 g / L of potassium dihydrogen phosphate, 0.5 g / L of sodium chloride, 1 g / L of ammonium chloride, 1 mM of magnesium sulfate, 0.001% of thiamine hydrochloride, 0.1 mM of calcium chloride, 2% of glucose) supplemented with 20 μg / mL of tetracycline, and cultured at 34 °C, pH 6.5, stirring at 1000 rpm, and aeration at 100 mL / min for 18 hours using a culture apparatus (Bio Jr.8 manufactured by Eppendorf). 20 mL of the culture solution after 18-hour culture was inoculated into 2 L of M9 medium supplemented with 20 μg / mL of tetracycline, and cultured at 34 °C, pH 6.7, stirring at 600 rpm, and aeration at 4 L / min using a culture apparatus (Bioneer-Neo manufactured by Marubishi BioEngineering Co., Ltd.). During the culture, a 50% (w / v) glucose solution was added as needed to adjust the glucose concentration in the system not to fall below 15 g / L. After 6-hour culture, 0.1 mM of isopropyl-β-thiogalactopyranoside was added, and at the same time, glycine and sodium sulfate were added to a final concentration of 100 mM. At the 30th hour of culture, an appropriate amount of the culture solution was sampled, and the cells and the supernatant were separated by centrifugation. The supernatant was appropriately diluted with distilled water, and GSH and GSSG were quantified by HPLC analysis. The quantification results are shown in Table 1.

[0327] (Example 2) Fermentative production of glutathione by the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF PompF-cysE / pQEK1-PT5-ABTd*-term strain The BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaAPtac-sapABCDFPompF-cysE / pQEK1-PT5-ABTd*-term strain obtained in Production Example 13 was cultured under the same conditions as in Example 1 to produce GSH and GSSG. The results are shown in Table 1.

[0328] (Comparative Example 1) Fermentative production of glutathione by the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-ABTd*-term strain The BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-ABTd*-term strain obtained in Production Example 11 was cultured under the same conditions as in Example 1 to produce GSH and GSSG. The results are shown in Table 1.

[0329] [Table 1]

[0330] [Discussion] Comparing the results of Examples 1 to 2 and Comparative Example 1 in Table 1, it can be seen that the productivity of glutathione (GSH + GSSG) increases significantly by enhancing the expression of the cysE (serine-O-acetyltransferase) gene. From this, it can be seen that enhancing the expression of the cysE gene is effective in the fermentative production of glutathione.

[0331] (Example 3) Fermentative production of glutathione by the BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-FSa-term strain The BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF Ptrc-cysE / pQEK1-PT5-FSa-term strain obtained in Production Example 15 was cultured under the same conditions as in Example 1 to produce GSH and GSSG. The results are shown in Table 2.

[0332] (Comparative Example 2) Fermentative production of glutathione by BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-FSa-term strain The BW25113ΔggtΔpepTΔgorΔyliABCDΔtnaA Ptac-sapABCDF / pQEK1-PT5-FSa-term strain obtained in Production Example 14 was cultured under the same conditions as in Example 1 to produce GSH and GSSG. The results are shown in Table 2.

[0333]

Table 2

[0334] <Discussion> Comparing the results of Example 3 and Comparative Example 2 in Table 2, it can be seen that the productivity of glutathione (GSH+GSSG) increases significantly by enhancing the expression of the cysE (serine-O-acetyltransferase) gene. From this, it can be seen that enhancing the expression of the cysE gene is effective in the fermentative production of glutathione. All publications, patents and patent applications cited herein are hereby incorporated by reference as they are.

Claims

**Claim 1** An Escherichia coli having the genetic modifications of [1] and [2], having one or more genetic modifications selected from [3] and [4], and having one or more genetic modifications selected from [5], [6], [7], [8] and [9], capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione: [1] Enhancement of the expression of the gene encoding serine-O-acetyltransferase (EC: 2.3.1.30); [2] Deletion of the gene encoding γ-glutamyltransferase (EC: 3.4.19.13); [3] Enhancement of the expression of the gene encoding glutamate-cysteine ligase (EC: 6.3.2.2) and / or the gene encoding glutathione synthetase (EC: 6.3.2.3); [4] Enhancement of the expression of the gene encoding bifunctional glutathione synthetase; [5] Deletion of the gene encoding tryptophanase (EC: 4.1.99.1); [6] Deletion of the gene encoding tripeptide peptidase (EC: 3.4.11.4); [7] Deletion of one or more of the genes encoding proteins involved in glutathione uptake; [8] Deletion of the gene encoding glutathione reductase (EC: 1.8.1.7); [9] Enhancement of the expression of one or more of the genes encoding proteins involved in putrescine excretion. **Claim 2** A method for producing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione, comprising culturing the Escherichia coli according to Claim 1.

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