Method for producing useful material

Abstract

The present specification discloses a method for producing a peptide such as glutathione, and a microorganism that can be used by the method. One or more embodiments of a first aspect pertain to a method for producing a peptide such as glutathione, the method comprising culturing a prokaryotic microorganism strain in which the expression level of one or more genes selected from the group consisting of gshA genes, gshB genes, and gshF genes has been increased than that of a wild strain in a culture medium in which the total concentration of cysteine and cystine is 0.5 g / L or less. The second aspect relates to a microorganism in which the gamma-glutamyltransferase gene and the glutathione reductase gene are deleted and the expression of the gshA gene and the gshB, or gshF gene is enhanced.

Description

[0001] This application is a divisional application of Chinese invention application "Method for manufacturing useful substances" filed on November 20, 2020, with application number 202080080653.9. Technical Field

[0002] One or more embodiments of the first aspect relate to a method for manufacturing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione.

[0003] Another or more embodiments of the first aspect involve prokaryotic microbial strains capable of producing excessive amounts of γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione.

[0004] The second aspect relates to a microorganism that produces glutathione but lacks the glutathione reductase gene, and a method for producing glutathione using this microorganism. Background Technology

[0005] Glutathione exists in both reduced and oxidized forms. Reduced glutathione is a peptide composed of three amino acids: L-cysteine, L-glutamic acid, and glycine. Oxidized glutathione is a compound formed by the disulfide bonds of the thiol groups of two molecules of reduced glutathione. Glutathione is present not only in humans but also in many other organisms, including animals, plants, and microorganisms. It plays a crucial role in the elimination of reactive oxygen species, detoxification, and amino acid metabolism. Therefore, it has attracted considerable attention in the pharmaceutical, food, and cosmetic industries. Furthermore, recent studies have shown that oxidized glutathione promotes plant growth, raising expectations for its applications in various fields, including agriculture.

[0006] Glutathione exists in organisms in either reduced glutathione (hereinafter sometimes referred to as "GSH") or oxidized glutathione (hereinafter sometimes referred to as "GSSG"). Reduced glutathione is the SH form in which the thiol group of the L-cysteine ​​residue is reduced, and oxidized glutathione is the form in which the thiol group of the L-cysteine ​​residue is oxidized, forming a disulfide bond between two glutathione molecules.

[0007] As a method for manufacturing glutathione, there are known methods that use yeast or Escherichia coli to produce it through fermentation (Patent Document 1); and methods that use microorganisms to produce γ-glutamylcysteine ​​synthase and glutathione synthase, and then enzymatically link L-glutamic acid, L-cysteine ​​and glycine to manufacture it (Patent Documents 3, 4), etc.

[0008] For example, Patent Document 1 describes a method for manufacturing glutathione, characterized in that yeast with increased thiol oxidase activity compared to the parent strain is cultured in a culture medium to produce glutathione, and then the glutathione is recovered from the obtained culture medium.

[0009] Furthermore, Patent Document 2 discloses a method for manufacturing glutathione or γ-glutamylcysteine. This method includes: culturing microorganisms in a culture medium with proteins exhibiting higher activity than the parent strain, including proteins related to glutathione transport and biosynthesis of glutathione or γ-glutamylcysteine; thereby generating and accumulating glutathione or γ-glutamylcysteine ​​in the culture medium; and collecting glutathione or γ-glutamylcysteine ​​from the culture. Example 4 of Patent Document 2 describes culturing an Escherichia coli strain that overexpresses the gshA and gshB genes in a culture medium supplemented with amino acids. The result was a glutathione concentration of 160 mg / L in the culture medium. The gshA gene is a glutamate-cysteine ​​ligase gene derived from E. coli, and the gshB gene is a glutathione synthase gene.

[0010] Non-Patent Literature 1 describes a method comprising: culturing Escherichia coli in a medium supplemented with L-cysteine, L-glutamic acid and glycine, which are the constituent amino acids of glutathione, to produce glutathione, wherein the Escherichia coli is transformed by an expression vector containing a bifunctional glutathione synthase gshF gene configured to be controlled by a constitutive promoter.

[0011] Existing technical documents

[0012] Patent documents

[0013] Patent Document 1: International Publication WO2016 / 140349

[0014] Patent Document 2: International Publication WO2008 / 126784

[0015] Patent Document 3: Japanese Patent Application Publication No. 60-27396

[0016] Patent Document 4: Japanese Patent Application Publication No. 60-27397

[0017] Non-patent literature

[0018] Non-patent literature 1: Journal of Biotechnology (2018), https: / / doi.org / 10.1016 / j.jbiotec.2018.11.001 Summary of the Invention

[0019] The problem that the invention aims to solve

[0020] First, a new method is required for producing prokaryotic microbial strains capable of producing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione, and for manufacturing methods using the aforementioned peptides produced by the prokaryotic microbial strain.

[0021] Second, there is a need for new methods for producing glutathione using microorganisms and glutathione using these microorganisms.

[0022] Methods for solving problems

[0023] The first aspect of this specification includes the embodiments shown in (1) to (14) below.

[0024] (1) A method for manufacturing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione, the method comprising:

[0025] Prokaryotic microbial strains whose expression levels were increased compared to wild-type strains were selected from genes encoding glutamate-cysteine ​​ligase, genes encoding glutathione synthase, and genes encoding bifunctional glutathione synthase were cultured in a medium with a total cysteine ​​and cystine concentration of less than 0.5 g / L.

[0026] (2) According to the method of (1), wherein the prokaryotic microbial strain is capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione through the induced expression of the more than one gene.

[0027] (3) A method for manufacturing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione, the method comprising:

[0028] Prokaryotic microbial strains with increased expression levels of one or more genes selected from genes encoding glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase compared to wild-type strains were cultured in a culture medium.

[0029] This method does not involve adding cysteine ​​or cystine to the culture medium.

[0030] (4) The method according to (3), wherein the prokaryotic microbial strain is capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione through the induced expression of one or more genes.

[0031] (5) A prokaryotic microbial strain capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione, wherein it retains one or more genes expressibly linked to a promoter, selected from genes encoding glutamate-cysteine ​​ligase, genes encoding glutathione synthase, and genes encoding bifunctional glutathione synthase, wherein...

[0032] The promoter is a promoter that, when one or more genes are glutamate-cysteine ​​ligase genes, enables the transcription amount of the glutamate-cysteine ​​ligase gene of the prokaryotic microorganism strain to be more than 20 times that of the wild-type strain.

[0033] (6) The prokaryotic microbial strain according to (5), wherein the promoter is an inducible promoter.

[0034] (7) The prokaryotic microbial strain according to (6), wherein the inducible promoter is an IPTG inducible promoter, a photoinducible promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing a tetO or lacO operon as an operon sequence.

[0035] (8) The prokaryotic microbial strain according to (7), wherein the inducible promoter is a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter, a tac promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing a tetO or lacO operon as an operon sequence.

[0036] (9) The prokaryotic microbial strain according to (8), wherein the inducible promoter is a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter or a tac promoter.

[0037] (10) The prokaryotic microbial strain according to (9), wherein the inducible promoter is the T5 promoter.

[0038] (11) The prokaryotic microbial strain according to any one of (5) to (10) is a transformant of intestinal bacteria.

[0039] (12) The prokaryotic microbial strain according to any one of (5) to (10) is a transformant of Escherichia coli.

[0040] (13) The method according to (1) or (2), wherein the prokaryotic microbial strain is any one of (5) to (12).

[0041] (14) The method according to (3) or (4), wherein the prokaryotic microbial strain is any one of (5) to (12).

[0042] The second aspect of this specification includes the embodiments shown in (15) to (22) below.

[0043] (15) A microorganism that lacks the following [1] and [2] genes and enhances the expression of [3] or [4] genes:

[0044] [1] The gene encoding γ-glutamyltransferase (EC:2.3.2.2);

[0045] [2] The gene encoding glutathione reductase (EC: 1.8.1.7);

[0046] [3] Genes encoding glutamate-cysteine ​​ligase (EC:6.3.2.2) and glutathione synthase (EC:6.3.2.3);

[0047] [4] Gene encoding bifunctional glutathione synthase.

[0048] (16) The microorganism described in (15) lacks the following [5] gene:

[0049] [5] Gene encoding tripeptipeptidase (EC:3.4.11.4).

[0050] (17) The microorganism according to (15) or (16), wherein the microorganism is a transformant of bacteria.

[0051] (18) The microorganism according to (15) or (16), wherein the microorganism is a transformant of intestinal bacteria.

[0052] (19) The microorganism according to (15) or (16), wherein the microorganism is a transformant of a Gram-negative bacterium.

[0053] (20) The microorganism according to (15) or (16), wherein the microorganism is a transformant of Escherichia coli.

[0054] (21) A method for manufacturing glutathione, the method comprising: culturing the microorganisms described in any one of (15) to (20) in a culture medium.

[0055] This specification includes the disclosures of Japanese Patent Application No. 2019-211477 and Japanese Patent Application No. 2020-002363, which form the basis of the priority claim of this application.

[0056] The effects of the invention

[0057] According to the method of the first aspect of this specification, since the process of adding cysteine ​​or cystine is not required, it is possible to manufacture γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione at low cost.

[0058] The prokaryotic microbial strains of the first aspect of this specification are capable of efficiently producing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione.

[0059] The second aspect of this specification describes the high glutathione production capacity of microorganisms based on fermentation.

[0060] The method for manufacturing glutathione according to the second aspect of this specification enables efficient and effective production of glutathione. Detailed Implementation

[0061] The preferred embodiments of the first and second aspects of this specification will be described in detail below, but the scope of the first and second aspects of this specification is not limited to these embodiments.

[0062] <1. Enzymes>

[0063] <1.1. Gamma-glutamyltransferase>

[0064] γ-Glutamyltransferase (EC:2.3.2.2) is an enzyme that hydrolyzes γ-glutamyl peptides such as glutathione.

[0065] “Gamma-glutamyl transferase” is also known as “gamma-glutamyl transpeptidase” or “Ggt”. In this specification, “gamma-glutamyl transferase”, “gamma-glutamyl transpeptidase”, and “Ggt” are interchangeable.

[0066] Specific examples of γ-glutamyltransferases include:

[0067] (1A) A polypeptide consisting of the amino acid sequence shown in sequence number 22;

[0068] (1B) A polypeptide consisting of an amino acid sequence formed by the addition, deletion or substitution of one or more amino acids in the amino acid sequence shown in Serial No. 22 (particularly preferred is a polypeptide consisting of an amino acid sequence formed by the substitution, deletion and / or addition of one or more amino acids in total at the N-terminus and C-terminus of the amino acid sequence shown in Serial No. 22), and is a polypeptide having γ-glutamyl transferase activity.

[0069] (1C) A polypeptide consisting of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the amino acid sequence shown in sequence number 22, and being a polypeptide having γ-glutamyl transferase activity; or

[0070] Fragments of any polypeptide in (1D), (1A) to (1C) that have γ-glutamyl transferase activity.

[0071] In the above (1D), the fragment may be a polypeptide with an amino acid number of 200 or more, more preferably 300 or more, more preferably 400 or more, more preferably 500 or more, and more preferably 550 or more.

[0072] The aforementioned polypeptides can be chemically modified as appropriate.

[0073] In (1B) above, "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 amino acids. Furthermore, the substitution of amino acids is preferably a conservative amino acid substitution. "Conservative amino acid substitution" refers to the 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, as basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), non-polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acids (leucine, valine, isoleucine), and aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc.

[0074] In (1C) above, "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein shown in SEQ ID NO. 22, achieved by aligning (comparing) two amino acid sequences and introducing gaps as needed to maximize amino acid similarity. Sequence identity can be calculated using protein search systems based on BLAST or FASTA (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, SF et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Hereinafter, "sequence identity" of amino acid sequences will be used with the same meaning.

[0075] "The gene encoding γ-glutamyltransferase (EC:2.3.2.2)" refers to the gene (nucleic acid is DNA or RNA, preferably DNA) that encodes the amino acid sequence of γ-glutamyltransferase, which is contained in the genomic DNA of the chromosome of the wild-type microorganism before the deletion of γ-glutamyltransferase.

[0076] An example of DNA from *E. coli* encoding the amino acid sequence shown in sequence number 22, which is γ-glutamyltransferase, is shown in sequence number 21. However, in the genomic DNA of wild-type microorganisms, the base sequence of sequence number 21 is not limited to this state; the base sequence of sequence number 21 is an exon sequence, which may contain more than one intron sequence.

[0077] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of γ-glutamyltransferase can be given as follows:

[0078] (1E) The base sequence shown in Serial No. 21;

[0079] (1F) A base sequence formed by adding, deleting or substituting one or more bases in the base sequence shown in Serial No. 21 (particularly preferred is a base sequence formed by substituting, deleting and / or adding one or both of the 5' end and 3' end of the base sequence shown in Serial No. 21, preferably a base sequence formed by deleting and / or adding a total of one or more bases), and is a base sequence encoding a polypeptide having γ-glutamyltransferase activity;

[0080] (1G) A base 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 with respect to the base sequence shown in Serial No. 21, and being a base sequence encoding a polypeptide having γ-glutamyl transferase activity.

[0081] A partial base sequence of the amino acid sequence encoding a polypeptide with γ-glutamyl transferase activity from any base sequence in (1H) (1E) to (1G).

[0082] The base sequences in (1I) (1E)~(1H) contain silent mutations (base substitutions that do not change the amino acid residues being encoded);

[0083] (1J) The base sequence encoding the amino acid sequence of any polypeptide in (1A)~(1D); or

[0084] (1K) A base sequence that uses any base sequence from (1E) to (1J) as an exon sequence and contains one or more intron sequences.

[0085] In the above (1G), "sequence identity" refers to the percentage (%) of identical bases relative to all bases in Serial No. 21 when two base sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest sequence consistency. Sequence identity can be calculated using base sequence retrieval systems based on BLAST and FASTA (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, SF et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Hereinafter, "sequence identity" will be used with the same meaning in this specification.

[0086] <1.2. Glutathione reductase>

[0087] Glutathione reductase (EC: 1.8.1.7) is an enzyme that catalyzes the reduction of oxidized glutathione (glutathione disulfide) to reduced glutathione in the presence of NADPH.

[0088] Specific examples of glutathione reductase include:

[0089] (2A) A polypeptide consisting of the amino acid sequence shown in sequence number 26;

[0090] (2B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted or replaced in the amino acid sequence shown in Serial No. 26 (particularly preferred is a polypeptide consisting of an amino acid sequence in which one or both of the N-terminus and C-terminus of the amino acid sequence shown in Serial No. 26 are replaced, deleted and / or added, preferably deleted and / or added in total of one or more amino acids), and is a polypeptide having glutathione reductase activity.

[0091] (2C) A polypeptide consisting of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the amino acid sequence shown in sequence number 26, and being a polypeptide having glutathione reductase activity; or

[0092] Fragments of any polypeptide in (2D) (2A)~(2C) that have glutathione reductase activity.

[0093] In the above (2D), the fragment may be a polypeptide with an amino acid number of 200 or more, more preferably 300 or more, and more preferably 400 or more.

[0094] In (2B) above, "multiple" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" is as described in (1B) in the section <1.1. γ-glutamyltransferase>.

[0095] The "sequence identity" in (2C) above is as described in the explanation of (1C) in the section <1.1. γ-glutamyltransferase>. That is, in (2C) above, "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein shown in sequence number 26 when two amino acid sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest amino acid consistency between the two.

[0096] "The gene encoding glutathione reductase (EC: 1.8.1.7)" refers to the gene (nucleic acid is DNA or RNA, preferably DNA) that encodes the amino acid sequence of glutathione reductase, which is contained in the genomic DNA of the chromosome of the wild-type microorganism before the absence of glutathione reductase.

[0097] An example of DNA from *E. coli* encoding the amino acid sequence shown in sequence number 26 is shown in sequence number 25. However, in the genomic DNA of wild-type microorganisms, the base sequence of sequence number 25 is not limited to this state; the base sequence of sequence number 25 is an exon sequence, which may contain more than one intron sequence.

[0098] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of glutathione reductase can be given as follows:

[0099] (2E) The base sequence shown in sequence number 25;

[0100] (2F) A base sequence formed by adding, deleting or substituting one or more bases in the base sequence shown in Serial No. 25 (particularly preferred is a base sequence formed by substituting, deleting and / or adding one or both of the 5' end and 3' end of the base sequence shown in Serial No. 25, preferably a base sequence formed by deleting and / or adding a total of one or more bases), and is a base sequence encoding a polypeptide having glutathione reductase activity;

[0101] (2G) A base 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 with respect to the base sequence shown in Serial No. 25, and being a base sequence encoding a polypeptide having glutathione reductase activity.

[0102] A partial base sequence of the amino acid sequence encoding a polypeptide with glutathione reductase activity from any base sequence in (2H) (2E)~(2G).

[0103] The base sequences in (2I) (2E)~(2H) contain silent mutations (base substitutions that do not change the amino acid residues being encoded);

[0104] (2J) The base sequence encoding the amino acid sequence of any polypeptide in (2A)~(2D); or

[0105] (2K) A base sequence that uses any base sequence from (2E) to (2J) as an exon sequence and contains one or more intron sequences.

[0106] The "sequence identity" in (2G) above is as described in the section on (1G) in <1.1. γ-glutamyltransferase>. That is, in (2G) above, "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in sequence number 25 when two base sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest base identity.

[0107] <1.3. Tripeptidase>

[0108] Tripeptidase (EC:3.4.11.4) is an enzyme that catalyzes the reaction that releases terminal amino acid residues from a tripeptide.

[0109] "Tripeptidase" is also known as "peptidase T" or "PepT". In this specification, "tripeptidase", "peptidase T", and "PepT" are interchangeable.

[0110] Specific examples of tripeptidases include:

[0111] (5A) A polypeptide consisting of the amino acid sequence shown in sequence number 24;

[0112] (5B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted or replaced in the amino acid sequence shown in Serial No. 24 (particularly preferred is a polypeptide consisting of an amino acid sequence in which one or both of the N-terminus and C-terminus of the amino acid sequence shown in Serial No. 24 are replaced, deleted and / or added, preferably deleted and / or added in total of one or more amino acids), and is a polypeptide having tripeptipeptidase activity.

[0113] (5C) A polypeptide composed of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the amino acid sequence shown in sequence number 24, and being a polypeptide having tripeptipeptidase activity; or

[0114] Fragments of any polypeptide in (5D), (5A) to (5C) that have tripeptipeptidase activity.

[0115] In the above (5D), the fragment may be a polypeptide with an amino acid number of 200 or more, more preferably 300 or more, and more preferably 350 or more.

[0116] The aforementioned polypeptides can be chemically modified as appropriate.

[0117] In (5B) above, "multiple" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" is as described in (1B) in the section <1.1. γ-glutamyltransferase>.

[0118] The “sequence identity” in (5C) above is as described in the explanation of (1C) in the section <1.1. γ-glutamyltransferase>. That is, in (5C) above, “sequence identity” refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein shown in sequence number 24 when two amino acid sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest amino acid identity between the two.

[0119] "The gene encoding tripeptipeptidase (EC:3.4.11.4)" refers to the gene (nucleic acid is DNA or RNA, preferably DNA) that encodes the amino acid sequence of tripeptipeptidase, contained in the genomic DNA of the chromosome of the wild-type microorganism before tripeptipeptidase is lost.

[0120] An example of DNA encoding a tripeptipeptidase from *E. coli*, with the amino acid sequence shown in sequence number 24, is shown in sequence number 23. However, in the genomic DNA of wild-type microorganisms, the base sequence of sequence number 23 is not limited to this state; the base sequence of sequence number 23 is an exon sequence, which may contain more than one intron sequence.

[0121] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of a tripeptipeptidase can be given as follows:

[0122] (5E) The base sequence shown in sequence number 23;

[0123] (5F) A base sequence formed by adding, deleting or substituting one or more bases in the base sequence shown in Serial No. 23 (particularly preferred is a base sequence formed by substituting, deleting and / or adding one or more bases at the 5' end and 3' end of the base sequence shown in Serial No. 23, preferably a base sequence formed by deleting and / or adding a total of one or more bases), and is a base sequence encoding a polypeptide having tripeptipeptidase activity;

[0124] (5G) A base 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 with respect to the base sequence shown in Serial No. 23, and being a base sequence encoding a polypeptide having tripeptipeptidase activity.

[0125] A partial base sequence encoding the amino acid sequence of a polypeptide with tripeptipeptidase activity from any base sequence in (5H) (5E)~(5G).

[0126] The base sequences in (5I) (5E)~(5H) contain silent mutations (base substitutions that do not change the amino acid residues being encoded);

[0127] (5J) The base sequence encoding the amino acid sequence of any polypeptide in (5A)~(5D); or

[0128] (5K) A base sequence that uses any base sequence from (5E) to (5J) as an exon sequence and contains one or more intron sequences.

[0129] The "sequence identity" in (5G) above is as described in the section on (1G) in <1.1. γ-glutamyltransferase>. That is, in (5G) above, "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in sequence number 23 when two base sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest base identity.

[0130] <1.4. Glutamate-cysteine ​​ligase>

[0131] Glutamate-cysteine ​​ligase (EC: 6.3.2.2) is an enzyme that catalyzes the reaction in the presence of ATP, recognizing L-cysteine ​​as a substrate and binding it to L-glutamate to generate γ-glutamylcysteine. The source and structure of the enzyme are not particularly limited, as long as it possesses this activity. In this specification, this activity is referred to as glutamate-cysteine ​​ligase activity. 1 U of this activity refers to the activity of generating 1 μmol of γ-glutamylcysteine ​​in 1 minute at 30°C, and it was measured under the following conditions.

[0132] "Glutamate-cysteine ​​ligase" is also known as "glutamate-cysteine ​​ligase" or "GshA". In this specification, "glutamate-cysteine ​​ligase", "glutamate-cysteine ​​ligase", and "GshA" are interchangeable.

[0133] (Measurement conditions)

[0134] The enzyme solution was added to 50 mM Tris hydrochloride buffer (pH 8.0) containing 10 mM ATP, 15 mM L-glutamate, 15 mM L-cysteine, and 10 mM magnesium sulfate, and the reaction was carried out by incubation at 30 °C. The reaction was stopped by adding 6 N hydrochloric acid. γ-glutamylcysteine ​​in the reaction solution was quantified using high-performance liquid chromatography (HPLC).

[0135] 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) are dissolved in that order.

[0136] [HPLC conditions]

[0137] Chromatographic column: ODS-HG-3 (4.6mm φ×150mm, manufactured by Nomura Chemicals Co., Ltd.);

[0138] Eluent: Dissolve 12.2g of potassium dihydrogen phosphate and 3.6g of sodium heptanesulfonate in 1.8L of distilled water, adjust the pH of the solution to 2.8 with phosphoric acid, and add 186ml of methanol to dissolve the resulting liquid.

[0139] Flow rate: 1.0 ml / min;

[0140] Column temperature: 40℃;

[0141] Measurement wavelength: 210nm

[0142] As a glutamate-cysteine ​​ligase, it is preferred to use a glutamate-cysteine ​​ligase with a specific activity (specific activity) of 0.5 U or more per 1 mg of protein, more preferably 1 U or more, further preferably 5 U or more, and most preferably 10 U or more.

[0143] The source of glutamate-cysteine ​​ligase is not particularly limited, and enzymes from microorganisms, animals, plants, etc., can be used. Glutamate-cysteine ​​ligases of microbial origin are preferred, and those derived from intestinal bacteria such as Escherichia coli, bacilli such as rod-shaped bacteria, eukaryotic microorganisms such as yeast, etc., are particularly preferred.

[0144] Examples of the base sequence of glutamate-cysteine ​​ligase from Escherichia coli and the amino acid sequence encoded by the base sequence are shown in Serial No. 12 and Serial No. 13, respectively.

[0145] Furthermore, the glutamate-cysteine ​​ligase is not limited to the glutamate-cysteine ​​ligase consisting of the amino acid sequence shown in Serial No. 13; other polypeptides with glutamate-cysteine ​​ligase activity, such as its active mutants or orthologs of other species, can also be used. Preferably, the other polypeptide with glutamate-cysteine ​​ligase activity exhibits at least 10%, more preferably 40%, more preferably 60%, more preferably 80%, and even more preferably 90% or more of the activity shown when the glutamate-cysteine ​​ligase consisting of the amino acid sequence shown in Serial No. 13 was used under the activity assay conditions described above.

[0146] Specific examples of glutamate-cysteine ​​ligases include:

[0147] (3-1A) A polypeptide consisting of the amino acid sequence shown in sequence number 13;

[0148] (3-1B) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been added, deleted, or substituted in the amino acid sequence shown in Serial No. 13 (particularly preferred is a polypeptide consisting of an amino acid sequence in which one or both of the N-terminus and C-terminus of the amino acid sequence shown in Serial No. 13 have been substituted, deleted, and / or added, preferably deleted and / or added, in total one or more amino acids), and is a polypeptide having glutamate-cysteine ​​ligase activity;

[0149] (3-1C) A polypeptide consisting of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the amino acid sequence shown in sequence number 13, and being a polypeptide having glutamate-cysteine ​​ligase activity; or

[0150] Fragments of any polypeptide in (3-1D) (3-1A)~(3-1C) that have glutamate-cysteine ​​ligase activity.

[0151] In the above (3-1D), the fragment may be a polypeptide with an amino acid number of 200 or more, more preferably 300 or more, more preferably 400 or more, more preferably 450 or more, and more preferably 500 or more.

[0152] The aforementioned polypeptides can be chemically modified as appropriate.

[0153] In (3-1B) above, "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 amino acids. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" is as described in section (1B) of <1.1. γ-glutamyltransferase>.

[0154] The "sequence identity" in (3-1C) above is as described in the explanation of (1C) in the section <1.1. γ-glutamyltransferase>. That is, in (3-1C) above, "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein shown in sequence number 13, when two amino acid sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest amino acid consistency between the two.

[0155] "The gene encoding glutamate-cysteine ​​ligase (EC: 6.3.2.2)" refers to the nucleic acid (DNA or RNA, preferably DNA) that encodes the amino acid sequence of glutamate-cysteine ​​ligase.

[0156] An example of DNA from *E. coli* encoding the amino acid sequence shown in sequence number 13, which is an amino acid sequence of glutamate-cysteine ​​ligase, is shown in sequence number 12. The base sequence of the gene encoding the amino acid sequence of glutamate-cysteine ​​ligase can be codon-optimized for the host.

[0157] That is, specific examples of the base sequence of a gene encoding the amino acid sequence of a glutamate-cysteine ​​ligase can be given as follows:

[0158] (3-1E) The base sequence shown in sequence number 12;

[0159] (3-1F) A base sequence formed by adding, deleting or substituting one or more bases in the base sequence shown in Serial No. 12 (particularly preferred is a base sequence formed by substituting, deleting and / or adding one or both of the 5' end and 3' end of the base sequence shown in Serial No. 12, preferably a base sequence formed by deleting and / or adding a total of one or more bases), and is a base sequence encoding a polypeptide having glutamate-cysteine ​​ligase activity;

[0160] (3-1G) A base 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 with respect to the base sequence shown in Serial No. 12, and being a base sequence encoding a polypeptide having glutamate-cysteine ​​ligase activity.

[0161] A partial base sequence of the amino acid sequence encoding a polypeptide with glutamate-cysteine ​​ligase activity, from any base sequence in (3-1H) (3-1E) to (3-1G).

[0162] The base sequences in (3-1I) (3-1E)~(3-1H) contain silent mutations (base substitutions that do not change the amino acid residues being encoded);

[0163] (3-1J) The base sequence encoding the amino acid sequence of any polypeptide in (3-1A)~(3-1D); or

[0164] (3-1K) A base sequence that uses any base sequence from (3-1E) to (3-1J) as an exon sequence and contains one or more intron sequences.

[0165] The "sequence identity" in (3-1G) above is as described in the section on (1G) of <1.1. γ-glutamyltransferase>. That is, in (3-1G) above, "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in sequence number 12 when two base sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest base identity.

[0166] <1.5. Glutathione synthase>

[0167] Glutathione synthase (EC: 6.3.2.3) is an enzyme that catalyzes the reaction in the presence of ATP, whereby γ-glutamylcysteine ​​recognizes γ-glutamylcysteine ​​as a substrate and binds it to glycine to generate GSH. The source and structure of the enzyme are not particularly limited, as long as it possesses this activity. In this specification, this activity is referred to as glutathione synthase activity. 1 U of this activity refers to the activity of generating 1 μmol of glutathione per minute at 30°C, measured under the following conditions.

[0168] Glutathione synthase is also known as "GshB". In this instruction manual, "glutathione synthase" and "GshB" are used interchangeably.

[0169] (Measurement conditions)

[0170] The enzyme solution was added to 50 mM Tris hydrochloride buffer (pH 8.0) containing 10 mM ATP, 15 mM γ-glutamylcysteine, 15 mM glycine, and 10 mM magnesium sulfate, and the reaction was carried out by incubation at 30 °C. The reaction was stopped by adding 6 N hydrochloric acid. Glutathione in the reaction solution was quantified using high-performance liquid chromatography (HPLC).

[0171] The high-performance liquid chromatography (HPLC) conditions were the same as those described above for the determination of glutamate-cysteine ​​ligase activity.

[0172] As a glutathione synthase, it is preferred to use a glutathione synthase activity (specific activity) of 0.5 U or more per 1 mg of protein, more preferably 1 U or more, further preferably 5 U or more, and most preferably 10 U or more.

[0173] There are no particular limitations on the glutathione synthase used; enzymes from microorganisms, animals, plants, etc., can be used. Glutathione synthases from microorganisms are preferred, especially those derived from intestinal bacteria such as Escherichia coli, bacteria such as rod-shaped bacteria, eukaryotic microorganisms such as yeast, and microorganisms belonging to the Hydrogenophilales family.

[0174] The glutathione synthase derived from microorganisms belonging to the Hydrogenophilales family is preferably derived from microorganisms belonging to the Thiobacillus genus, more preferably from microorganisms belonging to the Thiobacillus denitrificans. Glutathione synthase derived from Thiobacillus denitrificans strain ATCC25259 is particularly preferred.

[0175] (Preferred embodiment of glutathione synthase or its mutant derived from Escherichia coli)

[0176] Examples of the base sequence of glutathione synthase from Escherichia coli and the amino acid sequence encoded by the base sequence are shown in Serial No. 14 and Serial No. 15, respectively.

[0177] Furthermore, the glutathione synthase is not limited to the glutathione synthase composed of the amino acid sequence shown in Serial No. 15; other polypeptides with glutathione synthase activity, such as its active mutants or orthologs of other species, may also be used. Preferably, the other polypeptide with glutathione synthase activity exhibits at least 10%, more preferably 40%, more preferably 60%, more preferably 80%, and even more preferably 90% or more of the activity shown when the glutathione synthase composed of the amino acid sequence shown in Serial No. 15 was used under the activity assay conditions described above.

[0178] Specific examples of glutathione synthase or its mutants derived from Escherichia coli include:

[0179] (3-2A) A polypeptide consisting of the amino acid sequence shown in sequence number 15;

[0180] (3-2B) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been added, deleted, or substituted in the amino acid sequence shown in Serial No. 15 (particularly preferred is a polypeptide consisting of an amino acid sequence in which one or both of the N-terminus and C-terminus of the amino acid sequence shown in Serial No. 15 have been substituted, deleted, and / or added, preferably deleted and / or added, in total one or more amino acids), and is a polypeptide having glutathione synthase activity.

[0181] (3-2C) A polypeptide composed of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the amino acid sequence shown in Serial No. 15, and being a polypeptide having glutathione synthase activity; or

[0182] Fragments of any polypeptide in (3-2D) (3-2A)~(3-2C) that have glutathione synthase activity.

[0183] In the above (3-2D), the fragment may be a polypeptide with an amino acid number of 200 or more, more preferably 250 or more, and more preferably 300 or more.

[0184] The aforementioned polypeptides can be chemically modified as appropriate.

[0185] In (3-2B) above, "multiple" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" is as described in section (1B) of <1.1. γ-glutamyltransferase>.

[0186] The "sequence identity" in (3-2C) above is as described in section (1C) of <1.1. γ-glutamyltransferase>. That is, in (3-2C) above, "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein shown in sequence number 15, when two amino acid sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest amino acid consistency between the two.

[0187] "The gene encoding glutathione synthase (EC: 6.3.2.3)" refers to the gene (the nucleic acid is DNA or RNA, preferably DNA) that encodes the amino acid sequence of glutathione synthase.

[0188] An example of DNA encoding glutathione synthase, as shown in sequence number 15, from *E. coli* is illustrated in sequence number 14. The base sequence of the gene encoding the amino acid sequence of glutathione synthase can be codon-optimized for the host.

[0189] Specifically, examples of the base sequence of a gene encoding the amino acid sequence of glutathione synthase or a mutant of Escherichia coli can be given as follows:

[0190] (3-2E) The base sequence shown in sequence number 14;

[0191] (3-2F) A base sequence formed by adding, deleting or substituting one or more bases in the base sequence shown in Serial No. 14 (particularly preferred is a base sequence formed by substituting, deleting and / or adding one or both of the 5' end and 3' end of the base sequence shown in Serial No. 14, preferably a base sequence formed by deleting and / or adding a total of one or more bases), and is a base sequence encoding a polypeptide having glutathione synthase activity;

[0192] (3-2G) A base 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 with respect to the base sequence shown in Serial No. 14, and being a base sequence encoding a polypeptide having glutathione synthase activity.

[0193] A partial base sequence of the amino acid sequence encoding a polypeptide with glutathione synthase activity from any base sequence in (3-2H) (3-2E)~(3-2G).

[0194] The base sequences in (3-2I) (3-2E)~(3-2H) contain silent mutations (base substitutions that do not change the amino acid residues being encoded);

[0195] (3-2J) The base sequence encoding the amino acid sequence of any polypeptide in (3-2A)~(3-2D); or

[0196] (3-2K) A base sequence that uses any base sequence from (3-2E) to (3-2J) as an exon sequence and contains one or more intron sequences.

[0197] The "sequence identity" in (3-2G) above is as described in the section on (1G) of <1.1. γ-glutamyltransferase>. That is, in (3-2G) above, "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in sequence number 14 when two base sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest base identity.

[0198] (Preferred embodiment of glutathione synthase or its mutant derived from Thiobacillus denitrification)

[0199] Other suitable specific examples of glutathione synthase are wild-type glutathione synthase or its active mutants derived from Thiobacillus denitrificans strain ATCC25259. Specific examples of the base sequence of wild-type glutathione synthase from Thiobacillus denitrificans strain ATCC25259 and the amino acid sequence encoded by that base sequence are shown in Serial Nos. 16 and 17, respectively. The active mutant of the aforementioned wild-type glutathione synthase preferably exhibits at least 10%, preferably at least 40%, more preferably at least 60%, more preferably at least 80%, and even more preferably at least 90% activity under the aforementioned activity assay conditions compared to the wild-type glutathione synthase composed of the amino acid sequence shown in Serial No. 17.

[0200] Specific examples of glutathione synthase or its mutants from Thiobacillus denitrification strain ATCC25259 can be cited as follows:

[0201] (3-3A) A polypeptide consisting of the amino acid sequence shown in sequence number 17;

[0202] (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 Serial No. 17 (particularly preferred is a polypeptide consisting of an amino acid sequence in which one or both of the N-terminus and C-terminus of the amino acid sequence shown in Serial No. 17 are substituted, deleted, and / or added, preferably deleted and / or added in total, a total of one or more amino acids), and is a polypeptide having glutathione synthase activity.

[0203] (3-3C) A polypeptide composed of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the amino acid sequence shown in sequence number 17, and being a polypeptide having glutathione synthase activity; or

[0204] Fragments of any polypeptide in (3-3D) (3-3A)~(3-3C) that have glutathione synthase activity.

[0205] In the above (3-3D), the fragment may be a polypeptide with an amino acid number of 200 or more, more preferably 250 or more, and more preferably 300 or more.

[0206] The aforementioned polypeptides can be chemically modified as appropriate.

[0207] In (3-3B) above, "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 amino acids. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" is as described in section (1B) of <1.1. γ-glutamyltransferase>.

[0208] The "sequence identity" in (3-3C) above is as described in section (1C) of <1.1. γ-glutamyltransferase>. That is, in (3-3C) above, "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein shown in sequence number 17 when two amino acid sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest amino acid consistency between the two.

[0209] An example of the DNA encoding the amino acid sequence shown in sequence number 17 of *Thiobacillus denitrificationus* strain ATCC25259 is shown in sequence number 16. The base sequence of the gene encoding the amino acid sequence of glutathione synthase can be codon-optimized for the host.

[0210] Specifically, examples of the base sequence of a gene encoding the amino acid sequence of glutathione synthase or a mutant of *Thiobacillus denitrifyingus* strain ATCC25259 can be given as follows:

[0211] (3-3E) The base sequence shown in sequence number 16;

[0212] (3-3F) A base sequence formed by adding, deleting or substituting one or more bases in the base sequence shown in Serial No. 16 (particularly preferred is a base sequence formed by substituting, deleting and / or adding one or both of the 5' end and 3' end of the base sequence shown in Serial No. 16, preferably a base sequence formed by deleting and / or adding a total of one or more bases), and is a base sequence encoding a polypeptide having glutathione synthase activity;

[0213] (3-3G) A base 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 with respect to the base sequence shown in Serial No. 16, and being a base sequence encoding a polypeptide having glutathione synthase activity.

[0214] A partial base sequence of the amino acid sequence encoding a polypeptide with glutathione synthase activity from any base sequence in (3-3H) (3-3E)~(3-3G).

[0215] The base sequences in (3-3I) (3-3E)~(3-3H) contain silent mutations (base substitutions that do not change the amino acid residues being encoded).

[0216] (3-3J) The base sequence encoding the amino acid sequence of any polypeptide in (3-3A)~(3-3D); or

[0217] (3-3K) A base sequence that uses any base sequence from (3-3E) to (3-3J) as an exon sequence and contains one or more intron sequences.

[0218] The "sequence identity" in (3-3G) above is as described in the section on (1G) of <1.1. γ-glutamyltransferase>. That is, in (3-3G) above, "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in sequence number 16 when two base sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest base identity.

[0219] (Preferred embodiment of an active mutant of glutathione synthase derived from Thiobacillus denitrification)

[0220] Other preferred examples of glutathione synthase are active mutants of wild-type glutathione synthase of Thiobacillus denitrification strain ATCC25259 containing the amino acid sequence shown in sequence number 17, and particularly preferred are the polypeptides described in international publication WO2018 / 084165.

[0221] The specific examples of the aforementioned active mutants include:

[0222] (3-4A) A polypeptide consisting of amino acid sequences 3-4A in which one or more amino acids selected from positions 13, 17, 20, 23, 39, 70, 78, 101, 113, 125, 126, 136, 138, 149, 152, 154, 155, 197, 200, 215, 226, 227, 230, 239, 241, 246, 249, 254, 260, 262, 263, 270, 278, 299, 305, 307 and 310 are substituted.

[0223] (3-4B) A polypeptide consisting of an amino acid sequence formed by the addition, deletion or substitution of one or more amino acids in the amino acid sequence 3-4A other than the amino acid sites described above (particularly preferred is a polypeptide consisting of an amino acid sequence formed by the substitution, deletion and / or addition of one or more amino acids in the N-terminus and C-terminus of the amino acid sequence 3-4A, preferably a polypeptide consisting of the deletion and / or addition of a total of one or more amino acids), and is a polypeptide having glutathione synthase activity;

[0224] (3-4C) A polypeptide composed of an amino acid sequence that is identical to the amino acid sites described above in the amino acid sequence 3-4A and has 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity in portions other than the amino acid sites, and is a polypeptide having glutathione synthase activity; or

[0225] Fragments of any polypeptide in (3-4D) (3-4A)~(3-4C) that have glutathione synthase activity.

[0226] In the above (3-4D), as a fragment, a polypeptide with 150 or more amino acids is preferably used, more preferably 200 or more, and more preferably 300 or more.

[0227] The aforementioned polypeptides can be chemically modified as appropriate.

[0228] In (3-4B) above, "multiple" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" is as described in section (1B) of <1.1. γ-glutamyltransferase>.

[0229] The "sequence identity" in (3-4C) above is as described in the explanation of (1C) in the <1.1. γ-glutamyltransferase> section. That is, in (3-4C) above, "sequence identity" refers to the percentage (%) of identical amino acid residues other than the aforementioned amino acid site in the above amino acid sequences 3-4A relative to the total number of amino acid residues other than the aforementioned amino acid site when two amino acid sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest amino acid consistency between the two sequences.

[0230] More preferably, the amino acid sequence 3-4A described above is an amino acid sequence in which one or more amino acid substitutions selected from the group consisting of the amino acid sequence shown in sequence number 17 are introduced:

[0231] The 13th position was replaced with serine, the 17th position with glutamic acid, the 20th position with threonine, the 23rd position with leucine, the 39th position with threonine, the 70th position with serine, the 78th position with leucine, the 101st position with asparagine, glutamine, serine, threonine, the 113th position with histidine, the 125th position with valine, the 126th position with asparagine, the 136th position with threonine, the 138th position with alanine, the 149th position with glutamine, the 152nd position with glutamine, the 154th position with asparagine, the 155th position with leucine, the 197th position with glutamine, and the 200th position with serine. Position 215 was replaced with aspartic acid, position 226 with arginine, position 227 with serine, position 230 with proline, position 239 with serine, position 241 with histidine, position 246 with arginine, position 249 with glutamic acid, position 254 with aspartic acid, position 260 with alanine, cysteine, glycine, glutamine, threonine, position 262 with cysteine, position 263 with arginine, position 270 with isoleucine, position 278 with glycine, alanine, position 299 with alanine, position 305 with glycine, position 307 with valine, and position 310 with threonine.

[0232] The above-mentioned amino acid sequence 3-4A is particularly preferred to be an amino acid sequence formed by introducing any one of the following (1) to (35) amino acid substitutions into the amino acid sequence shown in sequence number 17:

[0233] (1) The 13th position is serine,

[0234] (2) The 17th position is glutamic acid, the 113th position is histidine, and the 230th position is proline.

[0235] (3) The 20th position is threonine, and the 215th position is aspartic acid.

[0236] (4) The 20th position is threonine, and the 241st position is histidine.

[0237] (5) The 23rd position is leucine, and the 126th position is asparagine.

[0238] (6) The 39th position is threonine, and the 260th position is alanine.

[0239] (7) The 70th position is serine, and the 260th position is alanine.

[0240] (8) The 78th position is leucine, and the 278th position is alanine.

[0241] (9) The 101st position is asparagine.

[0242] (10) The 101st position is glutamine,

[0243] (11) The 101st position is serine,

[0244] (12) The 101st position is serine, and the 260th position is alanine.

[0245] (13) The 101st position is threonine.

[0246] (14) Position 125 is valine, position 249 is glutamic acid.

[0247] (15) Position 125 is valine, position 152 is glutamine.

[0248] (16) The 136th position is threonine.

[0249] (17) Alanine is at position 138, glutamine is at position 149, histidine is at position 241, and glutamine is at position 263.

[0250] (18) The 154th position is asparagine, and the 246th position is arginine.

[0251] (19) The 155th position is leucine, and the 239th position is serine.

[0252] (20) The 197th position is glutamine,

[0253] (21) The 200th position is serine, and the 260th position is alanine.

[0254] (22) Position 226 is arginine, position 260 is alanine.

[0255] (23) The 227th position is serine, and the 260th position is alanine.

[0256] (24) Position 254 is aspartic acid, position 260 is alanine.

[0257] (25) Position 260 is alanine.

[0258] (26) Position 260 is alanine, position 278 is glycine, and position 307 is valine.

[0259] (27) Alanine is at position 260 and position 299.

[0260] (28) Alanine is at position 260, and glycine is at position 305.

[0261] (29) Position 260 is alanine, position 310 is threonine.

[0262] (30) The 260th position is cysteine.

[0263] (31) The 260th position is glycine.

[0264] (32) The 260th position is glutamine,

[0265] (33) The 260th position is threonine.

[0266] (34) Position 262 is cysteine.

[0267] (35) The 270th position is isoleucine.

[0268] The base sequence of the amino acid sequence encoding any of the polypeptides in (3-4A) to (3-4D) above can be used as the "gene encoding glutathione synthase (EC: 6.3.2.3)".

[0269] An example of the base sequence encoding the amino acid sequence of an active mutant is shown in Serial No. 18, which is formed by replacing valine at position 260 with alanine in the amino acid sequence shown in Serial No. 17 of the glutathione synthase of *Thiobacillus denitrifyingus* strain ATCC25259. The base sequence of the gene encoding the amino acid sequence of the active mutant of glutathione synthase of *Thiobacillus denitrifyingus* strain ATCC25259 can be codon-optimized for the host.

[0270] <1.6. Bifunctional glutathione synthase>

[0271] Bifunctional glutathione synthase is an enzyme that simultaneously possesses the following activities: the activity of catalyzing the reaction in the presence of ATP to recognize L-cysteine ​​as a substrate and bind it to L-glutamate to generate γ-glutamylcysteine, and the activity of catalyzing the reaction in the presence of ATP to recognize γ-glutamylcysteine ​​as a substrate and bind it to glycine to generate GSH. The presence of this activity is sufficient, and its source and structure are not particularly limited. In this specification, this activity is referred to as bifunctional glutathione synthase activity. 1U of this activity refers to the activity of generating 1 μmol of GSH in 1 minute at 30°C, and it was measured under the following conditions.

[0272] "Bifunctional glutathione synthase" is also known as "GshF". In this instruction manual, "bifunctional glutathione synthase" and "GshF" are interchangeable.

[0273] (Measurement conditions)

[0274] The enzyme solution was added to 50 mM Tris hydrochloride buffer (pH 8.0) containing 10 mM ATP, 15 mM L-glutamate, 15 mM L-cysteine, 15 mM glycine, and 10 mM magnesium sulfate, and the reaction was carried out by incubation at 30 °C. The reaction was stopped by adding 6 N hydrochloric acid. Glutathione in the reaction solution was quantified using high-performance liquid chromatography (HPLC).

[0275] The high-performance liquid chromatography (HPLC) conditions were the same as those described above for the determination of glutamate-cysteine ​​ligase activity.

[0276] As a bifunctional glutathione synthase, it is preferred to use a bifunctional glutathione synthase with an activity (specific activity) of 0.5 U or more per 1 mg of protein, more preferably 1 U or more, further preferably 5 U or more, and most preferably 10 U or more.

[0277] The source of bifunctional glutathione synthase is not particularly limited; enzymes from microorganisms, animals, plants, etc., can be used. Bifunctional glutathione synthases from microorganisms are preferred. Particularly preferred are bifunctional glutathione synthases derived from bacteria. Specifically, bifunctional glutathione synthases derived from at least one of the following bacteria are preferred: Streptococcus spp. bacteria such as *Streptococcus agalactiae*, *Streptococcus mutans*, *Streptococcus suis*, and *Streptococcus thermophilus*; Lactobacillus spp. bacteria such as *Lactobacillus plantarum*; Desulfotalea spp. bacteria such as *Desulfotalea psychrophila*; Clostridium spp. bacteria such as *Clostridium perfringens*; and Listeria monocytogenes. Listeria species, including *Listeria innocua* and *Listeria monocytogenes*; Enterococcus species, including *Enterococcus faecalis* and *Enterococcus faecium*; Pasteurella species, including *Pasteurella multocida*; Mannheimia species, including *Mannheimia succiniciprodecens*; and Haemophilus species, including *Haemophilus somnus*.

[0278] The base sequence of a bifunctional glutathione synthase from *Streptococcus agalactiae*, and specific examples of the amino acid sequence encoded by that base sequence, are shown in Serial No. 19 and Serial No. 20, respectively. It should be noted that the base sequence of Serial No. 19 encodes the bifunctional glutathione synthase from *Streptococcus agalactiae* consisting of the amino acid sequence shown in Serial No. 20, and is a base sequence that conforms to the codon usage frequency in *Escherichia coli*.

[0279] Furthermore, the bifunctional glutathione synthase is not limited to the bifunctional glutathione synthase consisting of the amino acid sequence shown in Serial No. 20; other polypeptides with bifunctional glutathione synthase activity, such as its active mutants or orthologs of other species, may also be used. Preferably, the other polypeptide with bifunctional glutathione synthase activity exhibits at least 10%, more preferably 40%, more preferably 60%, more preferably 80%, and even more preferably 90% or more of the activity shown when the bifunctional glutathione synthase consisting of the amino acid sequence shown in Serial No. 20 was used under the activity assay conditions described above.

[0280] Specific examples of bifunctional glutathione synthases include:

[0281] (4A) A polypeptide consisting of the amino acid sequence shown in sequence number 20;

[0282] (4B) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been added, deleted, or substituted in the amino acid sequence shown in Serial No. 20 (particularly preferred is a polypeptide consisting of an amino acid sequence in which one or both of the N-terminus and C-terminus of the amino acid sequence shown in Serial No. 20 have been substituted, deleted, and / or added, preferably deleted and / or added, in total one or more amino acids), and is a polypeptide having bifunctional glutathione synthase activity.

[0283] (4C) A polypeptide consisting of an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the amino acid sequence shown in sequence number 20, and being a polypeptide having bifunctional glutathione synthase activity; or

[0284] Fragments of any polypeptide in (4D), (4A) to (4C) that have bifunctional glutathione synthase activity.

[0285] In the above (4D), the fragment may be a polypeptide with an amino acid number of 400 or more, more preferably 500 or more, more preferably 600 or more, more preferably 700 or more, and more preferably 730 or more.

[0286] The aforementioned polypeptides can be chemically modified as appropriate.

[0287] In (4B) above, "multiple" 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. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. "Conservative amino acid substitution" is as described in section (1B) of <1.1. γ-glutamyltransferase>.

[0288] The "sequence identity" in (4C) above is as described in the explanation of (1C) in the section <1.1. γ-glutamyltransferase>. That is, in (4C) above, "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total number of amino acid residues in the protein shown in sequence number 20 when two amino acid sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest amino acid consistency between the two.

[0289] "Gene encoding bifunctional glutathione synthase" refers to the gene (nucleic acid is DNA or RNA, preferably DNA) that encodes the amino acid sequence of bifunctional glutathione synthase.

[0290] Specific examples of the base sequence of a gene encoding the amino acid sequence of a bifunctional glutathione synthase can be given as follows:

[0291] (4E) The base sequence shown in sequence number 19;

[0292] (4F) A base sequence formed by adding, deleting or substituting one or more bases in the base sequence shown in Serial No. 19 (particularly preferred is a base sequence formed by substituting, deleting and / or adding one or both of the 5' end and 3' end of the base sequence shown in Serial No. 19, preferably a base sequence formed by deleting and / or adding a total of one or more bases), and is a base sequence encoding a polypeptide having bifunctional glutathione synthase activity;

[0293] (4G) A base 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 with respect to the base sequence shown in Serial No. 19, and being a base sequence encoding a polypeptide having bifunctional glutathione synthase activity.

[0294] A partial base sequence of the amino acid sequence encoding a polypeptide with bifunctional glutathione synthase activity, from any base sequence in (4H) (4E) to (4G).

[0295] The base sequences in (4I) (4E)~(4H) contain silent mutations (base substitutions that do not change the amino acid residues being encoded);

[0296] (4J) The base sequence encoding the amino acid sequence of any polypeptide in (4A)~(4D); or

[0297] (4K) A sequence of bases from (4E) to (4J) that is an exon sequence and contains one or more intron sequences.

[0298] The "sequence identity" in (4G) above is as described in the section on (1G) in <1.1. γ-glutamyltransferase>. That is, in (4G) above, "sequence identity" refers to the percentage (%) of identical bases relative to the total number of bases in sequence number 19 when two base sequences are aligned (compared) and vacancies are introduced as needed to achieve the highest base identity.

[0299] <2. First Aspect>

[0300] The first aspect of this specification describes the methods for manufacturing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione, as well as prokaryotic microbial strains.

[0301] <2.1. Prokaryotic microbial strains of the first aspect of this specification>

[0302] One or more embodiments of the first aspect relate to a prokaryotic microbial strain in which the expression level of one or more genes selected from genes encoding glutamate-cysteine ​​ligase, genes encoding glutathione synthase, and genes encoding bifunctional glutathione synthase is increased compared with that of a wild-type strain, enabling the overproduction of γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione.

[0303] The prokaryotic microbial strains of one or more embodiments of the first aspect can produce excess γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione by culturing in a culture medium, and accumulate them in the culture medium, thus allowing them to be used for the purpose of efficiently producing the aforementioned peptides. For the prokaryotic microorganisms of one or more embodiments of the first aspect, even when cultured in a culture medium with a total concentration of cysteine ​​and cystine of 0.5 g / L or less, or in a culture medium prepared without the addition of cysteine ​​or cystine, the aforementioned peptides can be produced, thus reducing the production cost of the aforementioned peptides.

[0304] The prokaryotic microbial strain in one or more embodiments of the first aspect is preferably a prokaryotic microbial strain capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione through the induced expression of one or more of the aforementioned genes. The prokaryotic microbial strain in one or more embodiments of the first aspect can overproduce γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione by culturing in a culture medium and inducing the expression of one or more of the aforementioned genes, thereby accumulating them in the culture medium, and can therefore be used for the purpose of efficiently producing the aforementioned peptides.

[0305] In the prokaryotic microbial strains of one or more embodiments of the first aspect, the enzymes whose gene expression levels are increased are selected from one or more enzymes chosen from glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase. Specific examples of each enzyme are as described above. When the above-mentioned prokaryotic microbial strains are used for the production of γ-glutamylcysteine, bis-γ-glutamylcysteine, and / or γ-glutamylcysteine, it is preferable that the gene expression levels of glutamate-cysteine ​​ligase and / or bifunctional glutathione synthase are increased compared to the wild-type strain. When the above-mentioned prokaryotic microbial strains are used for the production of reduced glutathione and / or oxidized glutathione, it is preferable that the gene expression levels of glutamate-cysteine ​​ligase and glutathione synthase are increased compared to the parental strain, or that the gene expression level of bifunctional glutathione synthase is increased compared to the wild-type strain.

[0306] In one or more embodiments of the first aspect, examples of prokaryotic microbial strains serving as hosts include bacteria, particularly cells of microorganisms belonging to the genera *Escherichia*, *Bacillus*, *Brevibacterium*, or *Corynebacterium*, with cells of microorganisms belonging to the genus *Escherichia* being particularly preferred, and cells of *Escherichia coli* being most preferred. Alternatively, the prokaryotic microbial strain serving as the host can be intestinal bacteria. The prokaryotic microbial strain in one or more embodiments of the first aspect can be a transformant of a prokaryotic microorganism that retains a given gene.

[0307] "Wild-type strain" refers to a host strain before the introduction of one or more genes selected from the genes encoding glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase; it can also be called "parental strain".

[0308] Here, "the expression level of one or more genes selected from the genes encoding glutamate-cysteine ​​ligase, the genes encoding glutathione synthase, and the genes encoding bifunctional glutathione synthase is increased compared with that of wild-type plants" includes the following two aspects: when the wild-type plants originally express one or more of the above genes, the expression level of the above-mentioned genes is increased compared with that of wild-type plants; and when the wild-type plants originally do not express one or more of the above genes, the wild-type plants are endowed with the ability to express one or more of the above genes.

[0309] The increase in the expression of one or more of the above genes can be achieved by: increasing the copy number of one or more of the above genes in the cells of prokaryotic microorganisms; or replacing the promoter controlling the expression of one or more of the above genes with a stronger expression promoter on the genomic DNA of the cells of prokaryotic microorganisms.

[0310] The increase in the copy number of one or more of the above-mentioned genes in the cells of prokaryotic microorganisms can be achieved in the following ways.

[0311] (1) Introduce an expression vector containing one or more of the above genes into the cells of prokaryotic microorganisms, or

[0312] (2) Introduce one or more of the above genes into the genomic DNA of prokaryotic microorganisms.

[0313] As the expression vector used in the manner described in (1) above, a plasmid vector containing one or more of the aforementioned genes can be used. The expression vector is preferably capable of autonomous replication within prokaryotic microbial cells. The expression vector preferably contains: DNA encoding one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase, and a promoter operatively linked to a position capable of transcribing the DNA. The expression vector is preferably a recombinant DNA capable of autonomous replication within prokaryotic microbial cells and containing a base sequence consisting of a promoter, a ribosome-binding sequence, an amino acid sequence encoding one or more of the aforementioned enzymes, and a transcription termination sequence.

[0314] Suitable plasmid vectors include: pQEK1, pCA24N (DNA RESEARCH, 12, 191-299 (2005)), pACYC177, pACYC184 (available from Nippon Gene), pQE30, pQE60, pQE70, pQE80, and pQE9 (available from Qiagen); pTipQC1 (available from Qiagen or Hokkaido System Science), and pTipRT2 (available from Hokkaido System Science). (Available from Science); 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), pUCN18 (prepared by modifying pUC18 (available from Takara Bio), pSTV28 (available from Takara Bio), pUCNT (International Publication No. 94 / 03613), etc.

[0315] The expression vectors described above preferably contain promoters that control the transcription of one or more of the genes mentioned above.

[0316] In one or more embodiments of the first aspect, the promoter is preferably an inducible promoter.

[0317] Examples of inducible promoters include: isopropyl-β-thiogalactopyranoside (IPTG) inducible promoters, light-inducible promoters that induce gene expression under light irradiation, araBAD promoters (arabinose inducible), rhaBAD promoters (rhamnose inducible), tet promoters (drug-inducible), penP promoters (drug-inducible), cspA promoters (temperature-inducible promoters that respond to low temperatures), and promoters containing tetO or lacO operons as operon sequences. Preferred promoters include IPTG inducible promoters, araBAD promoters, rhaBAD promoters, tet promoters, penP promoters, cspA promoters, or promoters containing tetO or lacO operons as operon sequences.

[0318] Specific examples of IPTG-inducible promoters include: T5 promoter, lacUV5 promoter, lac promoter, T7 promoter, lacT5 promoter, lacT7 promoter, tac promoter, etc. IPTG-inducible promoters are particularly preferred, and among IPTG-inducible promoters, T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, or tac promoter are particularly preferred.

[0319] As promoters, existing promoters can also be modified into highly active promoters using various reporter genes. For example, promoter activity can be improved by making the -35 and -10 regions within the promoter region closer to the common sequence (International Publication WO2000 / 018935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574). Methods for evaluating promoter strength and examples of strong promoters are described in the paper by Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0320] When an expression vector containing one or more of the above-mentioned genes is introduced into the cells of prokaryotic microorganisms, the copy number of the above-mentioned expression vector in the cells is preferably 2 or more, more preferably 3 or more, more preferably 5 or more, more preferably 10 or more, more preferably 15 or more, and more preferably 20 or more.

[0321] In prokaryotic microbial cells, to increase the expression of two or more of the aforementioned genes, these two or more genes can be contained within a single expression vector. In this case, the two or more genes can be configured to be controlled by a single expression promoter. Alternatively, the two or more genes can also be contained in separate expression vectors.

[0322] When one or more of the above genes are introduced into the genomic DNA of a prokaryotic microorganism cell in the manner described in (2) above, homologous recombination can be utilized.

[0323] When the promoters of one or more of the above-mentioned genes are replaced with stronger expression promoters on the genomic DNA of prokaryotic microorganisms, the same promoter as the expression vector can be used as the expression promoter, and more preferably an inducible promoter can be used. Specific examples of preferred promoters are described above.

[0324] In the prokaryotic microbial strains of one or more embodiments of the first aspect, the degree of increase in the expression level of the aforementioned one or more genes is not particularly limited. The expression level of the aforementioned one or more genes can be expressed as the amount of mRNA (i.e., mRNA encoding the amino acid sequence of one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase) extracted from the cell corresponding to the aforementioned one or more genes. The expression level based on this mRNA is preferably expressed as a relative value relative to the amount of mRNA encoding a suitable internal standard protein. In embodiments where the expression level of the glutamate-cysteine ​​ligase gene is increased, the expression level of glutamate-cysteine ​​ligase in the prokaryotic microbial strain (preferably a relative value obtained by dividing the amount of glutamate-cysteine ​​ligase mRNA in the prokaryotic microbial strain by the amount of mRNA encoding the internal standard protein in the same strain) is preferably 5 times or more, more preferably 10 times or more, and more preferably 20 times or more. In embodiments that increase the expression level of the glutathione synthase gene, the expression level of glutathione synthase in the prokaryotic microbial strain (preferably a relative value obtained by dividing the amount of glutathione synthase mRNA in the prokaryotic microbial strain by the amount of mRNA encoding the internal standard protein in the same strain) is preferably 5 times or more, more preferably 10 times or more, and more preferably 20 times or more, than the expression level of glutathione synthase in the wild-type strain (preferably a relative value obtained by dividing the amount of glutathione synthase mRNA in the wild-type strain by the amount of mRNA encoding the internal standard protein in the same strain). As an internal standard protein, the protein encoded by hcaT (sequence number 27), a known housekeeping gene, can be cited as an example.

[0325] In one or more embodiments of the first aspect, a more preferred prokaryotic microbial strain is one that possesses a gene for an enzyme with activity in breaking down cysteine, γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, or oxidized glutathione, or whose expression level of a glutathione uptake transporter gene is lower than that of a wild-type strain, or whose expression of the aforementioned genes is lost. When such a prokaryotic microbial strain is cultured, the aforementioned peptides tend to accumulate in the culture medium.

[0326] Examples of enzymes possessing activity in breaking down γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, or oxidized glutathione include γ-glutamyltransferase and tripeptipeptidase. Examples of genes possessing enzyme activity in breaking down cysteine ​​include the glutaminase gene tnaA. Examples of genes possessing glutathione uptake transporter activity include yliABCD.

[0327] Specific examples of γ-glutamyltransferase are as described above.

[0328] Specific examples of tripeptipeptidase are as described above.

[0329] Prokaryotic microbial strains possessing genes for enzymes that decompose cysteine, γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, or oxidized glutathione, or whose expression levels of glutathione uptake transporter genes are lower than those of wild-type strains, or whose expression of the aforementioned genes has been lost, can be manufactured by introducing base deletions, substitutions, or additions into the base sequences encoding the aforementioned enzymes on the genomic DNA of prokaryotic microbial strains. As an example of such a method, a method utilizing homologous recombination can be cited, specifically the method described in Japanese Patent Application Laid-Open No. 2004-344029.

[0330] A more preferred embodiment of the prokaryotic microbial strain of one or more embodiments of the first aspect described above is a prokaryotic microbial strain that retains one or more genes expressibly linked to an inducible promoter, selected from genes encoding glutamate-cysteine ​​ligase, genes encoding glutathione synthase, and genes encoding bifunctional glutathione synthase, wherein...

[0331] The aforementioned inducible promoters are inducible promoters that, when one or more of the above genes are genes encoding glutamate-cysteine ​​ligase, result in an expression level of glutamate-cysteine ​​ligase gene in the above prokaryotic microbial strains that is more than 20 times that of the wild-type strains.

[0332] Through the induced expression of one or more of the above genes, the prokaryotic microbial strain can overproduce γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione.

[0333] In the prokaryotic microbial strain of this preferred embodiment, the one or more genes expressibly linked to the inducible promoter can be maintained either as a part of the genomic DNA contained in the prokaryotic microbial strain or as an expression vector contained in the prokaryotic microbial strain. Specific examples of expression vectors are as described above.

[0334] As an inducible promoter, it is acceptable to achieve an expression level of the glutamate-cysteine ​​ligase gene in the prokaryotic microorganism strain that is at least 20 times higher than that in the wild-type strain, provided that one or more of the aforementioned genes are encoding glutamate-cysteine ​​ligase. There are no particular limitations. Here, the transcription level can be evaluated based on mRNA levels. Preferably, the gene encoding glutamate-cysteine ​​ligase used to confirm that the inducible promoter has a given expression capacity is of the same type as the gene encoding glutamate-cysteine ​​ligase present in the wild-type strain.

[0335] Such highly expressive inducible promoters can be selected from the examples of inducible promoters mentioned above. Preferred examples include IPTG-inducible promoters, photo-inducible promoters, araBAD promoters, rhaBAD promoters, tet promoters, penP promoters, cspA promoters, or promoters containing tetO or lacO operons as operon sequences. IPTG-inducible promoters are particularly preferred as highly expressive inducible promoters, and among IPTG-inducible promoters, T5 promoters, T7 promoters, lacT5 promoters, lacT7 promoters, or tac promoters are particularly preferred.

[0336] As a highly expressed inducible promoter, as mentioned above, highly active inducible promoters modified with various reporter genes can also be used.

[0337] <2.2. Method for manufacturing useful substances based on cell culture, which is part of the first aspect of this specification>

[0338] One or more embodiments of the first aspect relate to a method for manufacturing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione and / or oxidized glutathione (hereinafter referred to as the "target peptide"), the method comprising:

[0339] Prokaryotic strains whose expression levels were increased compared to wild-type strains were cultured in a medium with a total cysteine ​​and cystine concentration of less than 0.5 g / L.

[0340] The prokaryotic microbial strain used in this method is preferably a prokaryotic microbial strain that can overproduce the target peptide through the induction expression of one or more of the above genes.

[0341] One or more embodiments of the first aspect relate to a method for manufacturing the aforementioned target peptide, the method comprising:

[0342] Prokaryotic microbial strains with increased expression levels of one or more genes selected from genes encoding glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase compared to wild-type strains were cultured in a culture medium.

[0343] This method does not involve adding cysteine ​​or cystine to the above-mentioned culture medium.

[0344] The prokaryotic microbial strain used in this method is preferably a prokaryotic microbial strain that can overproduce the target peptide through the induction expression of one or more of the above genes.

[0345] This method is based on the unexpected discovery that prokaryotic microbial strains described in one or more embodiments of the first aspect above can accumulate the target peptide, containing cysteine ​​or cystine as a constituent amino acid, in the culture medium even when cultured in a low-concentration culture medium with a total cysteine ​​and cystine concentration of 0.5 g / L or less, or in a culture medium prepared without adding cysteine ​​or cystine. According to this method, the target peptide can be manufactured at low cost.

[0346] The culture medium used in the above method only needs to contain the nutrients necessary for the proliferation of microorganisms used in the first aspect and the biosynthesis of the target peptide, such as carbon source, nitrogen source, inorganic salts, and vitamins. It can be any kind of synthetic culture medium or natural culture medium.

[0347] As a carbon source, any carbon source that the microorganisms to be used can assimilate is acceptable. Examples include sugars such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0348] Examples of nitrogen sources include: ammonium salts such as ammonia and ammonium sulfate, nitrogen compounds such as amines, natural nitrogen sources such as peptone and soybean hydrolysate.

[0349] Examples of inorganic salts include: potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0350] Examples of vitamins include biotin and thiamine. Furthermore, substances required for the growth of the microorganisms mentioned in the first aspect can be added as needed (e.g., auxotrophic amino acids in the case of auxotrophic microorganisms).

[0351] It is preferable to add at least one of a sulfur source and glycine to the above-mentioned culture medium, and more preferably both. Examples of glycine concentrations added to the culture medium include 100 mM to 2000 mM, and more preferably 400 mM to 1200 mM. Examples of sulfur source concentrations added to the culture medium include 100 mM to 2000 mM, and more preferably 400 mM to 1200 mM.

[0352] As a sulfur source, one or more inorganic sulfur compounds such as sulfuric acid, thiosulfate, sulfurous acid, dithionite, sulfides, or their salts can be added. Sulfuric acid, thiosulfate, sulfurous acid, dithionite, or sulfides can be in free form, as salts, or any mixture thereof. There are no particular restrictions on the salts used; examples include sodium salts, calcium salts, ammonium salts, and potassium salts.

[0353] Glycine can be in its free form, as a salt, or any mixture thereof. As a salt, there are no particular limitations; examples include sulfates and hydrochlorides.

[0354] Sulfur sources and / or glycine can be added to the culture medium at the beginning of culture or during culture. Sulfur sources and / or glycine can be added to the culture medium all at once, or continuously or intermittently.

[0355] Sulfur source and / or glycine may be included in the culture medium throughout the entire culture period, or only for a portion of the culture. For example, the amount of sulfur source and glycine added does not need to be within the range described above throughout the entire production and accumulation phase of the target peptide. The culture medium can be maintained at the sulfur source and / or glycine levels within the range described above during the culture process, with the sulfur source and / or glycine content decreasing over time. Alternatively, sulfur source and / or glycine may be added continuously or intermittently. It should be noted that the concentrations of culture medium components other than sulfur source and / or glycine may vary during the culture period, and additional additions may also be made.

[0356] The culture is preferably carried out under aerobic conditions such as shaking culture and aerated stirring culture. The culture temperature is 20~50℃, preferably 20~42℃, more preferably 28~38℃. The pH during culture is 5~9, preferably 6~7.5. The culture time is 3 hours~5 days, preferably 5 hours~3 days.

[0357] The target peptides accumulated in the culture can be collected using conventional purification methods. For example, after the culture is completed, the culture can be collected by ion exchange, concentration, crystallization, or fractionation after removing the bacterial cells and solid matter by centrifugation.

[0358] <3. Second Aspect>

[0359] The second aspect of this specification describes the microorganisms and the method for manufacturing glutathione.

[0360] In this specification, "glutathione" can refer to reduced glutathione, oxidized glutathione, or a mixture of reduced and oxidized glutathione. In this specification, "glutathione" and "reduced glutathione and / or oxidized glutathione" are interchangeable.

[0361] <3.1. Host Microorganisms>

[0362] In one or more embodiments of the second aspect, the host (parental strain) of a microorganism that lacks the following [1] and [2] genes and enhances the expression of [3] or [4] genes is preferably a bacterium. The aforementioned bacteria may be intestinal bacteria. The aforementioned bacteria may be Gram-negative bacteria such as Escherichia bacteria and Pantoea bacteria, or Gram-positive bacteria such as Bacillus bacteria, Brevibacterium bacteria, and Corynebacterium bacteria, preferably Gram-negative bacteria, and particularly preferably Escherichia coli.

[0363] In one or more embodiments of the second aspect, the microorganism may be a transformant in which a given gene is deleted and the given gene is expressed and retained.

[0364] <3.2. Microorganisms, the second aspect of this specification>

[0365] One or more embodiments of the second aspect involve microorganisms that delete the following [1] and [2] genes and enhance the expression of [3] or [4] genes:

[0366] [1] The gene encoding γ-glutamyltransferase (EC:2.3.2.2);

[0367] [2] The gene encoding glutathione reductase (EC: 1.8.1.7);

[0368] [3] Genes encoding glutamate-cysteine ​​ligase (EC:6.3.2.2) and glutathione synthase (EC:6.3.2.3);

[0369] [4] Gene encoding bifunctional glutathione synthase.

[0370] The aforementioned microorganisms have a high fermentation-based glutathione production capacity, making them suitable for glutathione manufacturing. These microorganisms can produce glutathione through cultivation in a culture medium.

[0371] More preferably, the microorganisms mentioned above are microorganisms that further lack the following [5] genes.

[0372] [5] Gene encoding tripeptipeptidase (EC:3.4.11.4).

[0373] The microorganisms mentioned above that further lack the tripeptipeptidase gene have particularly high glutathione production capacity and are therefore preferred.

[0374] The microorganisms that serve as hosts for one or more embodiments of the second aspect are as described above.

[0375] The deletion of a given gene in a microorganism of one or more embodiments of the second aspect will be explained.

[0376] In one or more embodiments of the second aspect, the "deletion" of the gene encoding γ-glutamyltransferase and the gene encoding glutathione reductase, or further encoding tripeptipeptidase (hereinafter sometimes referred to as the "deleted target gene") in the microorganism means that the activity of the enzyme encoded by the aforementioned deleted target gene is reduced compared to the parent strain, including the case where the activity is completely lost. The microorganisms in one or more embodiments of the second aspect are microorganisms in a state of loss of function or reduced function of the aforementioned deleted target gene. Specifically, examples include: microorganisms in a state of reduced expression levels of mRNA as a transcription product of the aforementioned deleted target gene or protein as a translation product; and microorganisms in a state where the mRNA as a transcription product of the aforementioned deleted target gene or protein as a translation product cannot function normally as mRNA or protein.

[0377] The deletion of the aforementioned target genes can be achieved, for example, by artificially modifying the genes of the parent microbial strain. Such modifications can be achieved, for example, through mutation treatment, gene recombination technology, gene expression suppression treatment using RNAi, gene editing, etc.

[0378] Examples of mutation treatments include treatments using ultraviolet irradiation or mutagens commonly used in mutagenesis treatments, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0379] As a gene recombination technology, well-known techniques can be used, such as 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.).

[0380] In addition to the coding regions of the amino acid sequences of the proteins, genes encoding γ-glutamyltransferase, glutathione reductase, or tripeptipeptidase also indiscriminately represent their expression regulatory sequences (promoter sequences, etc.), exon sequences, intron sequences, etc. When the expression regulatory sequences are modified, it is preferred that at least one base is modified, more preferably at least two bases, and particularly preferably at least three bases.

[0381] The deletion of the target gene in the second aspect is more preferably a deletion of the target gene in the genomic DNA of a microorganism. The deletion of the target gene can 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 the enzyme. Here, "deletion" refers to a deletion or damage, preferably a deletion.

[0382] In the genomic DNA of the parent microbial strain, the entire gene, including the sequences preceding and following the aforementioned target gene, can be deleted. When deleting part or all of the coding region of the amino acid sequence of the enzyme encoded by the aforementioned target gene, any region, such as the N-terminal region, internal region, or C-terminal region, can be deleted, as long as a reduction in enzyme activity can be achieved. Generally, a longer deleted region can reliably inactivate the gene. Furthermore, the sequences preceding and following the deleted region are preferably not aligned with the reading frame. In a preferred embodiment, a microorganism has a deletion in its genomic DNA of at least a portion of the coding region and / or expression regulatory sequence of the aforementioned target gene, for example, a region consisting of at least 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and more preferably 100% of the total number of bases relative to the coding region and / or expression regulatory sequence. Particularly preferred are microorganisms in which the region from the start codon to the stop codon of the aforementioned target gene in the genomic DNA is deleted.

[0383] In addition, as other examples of the deletion of the above-mentioned target genes, such as the reduction of enzyme activity, examples include: the introduction of amino acid substitutions (missense mutations), the introduction of stop codons (speechless mutations), or the introduction of frameshift mutations that add or delete 1 to 2 bases into the amino acid sequence coding region of the above-mentioned target genes on the genomic DNA.

[0384] Furthermore, the deletion of the aforementioned target gene, which reduces enzyme activity, can also be achieved, for example, by inserting other sequences into the expression regulatory sequence or amino acid coding region of the target gene on the genomic DNA. The insertion site can be any region of the gene, provided the inserted sequence is long enough to reliably inactivate the gene. Additionally, the sequences before and after the insertion site are preferably not aligned with the reading frame. As for the other sequences, there are no particular restrictions as long as they can reduce or eliminate the function of the encoded protein; for example, they can be marker genes or genes useful for the production of γ-glutamyl compounds such as glutathione.

[0385] The deletion of the target gene on the genomic DNA, as described above, can be achieved, for example, by preparing an inactivated gene modified to prevent the production of a normally functional protein; transforming a microorganism with recombinant DNA containing the inactivated gene; and replacing the gene on the genomic DNA with the inactivated gene through homologous recombination between the inactivated gene and the gene on the genomic DNA. In this case, if a marker gene is pre-contained in the recombinant DNA, depending on the host's trophic dystrophic traits, the process is easier. Furthermore, if the recombinant DNA is pre-formed into a linear structure using restriction enzymes, strains with the recombinant DNA introduced into the genomic DNA can be obtained efficiently. Even if the protein encoded by the inactivated gene is generated, it will have a different stereostructure than the wild-type protein, and its function will be reduced or lost.

[0386] Alternatively, for example, transforming microorganisms with linear DNA induces homologous recombination upstream and downstream of the target site on the microbial genomic DNA. This allows the target site to be replaced with the aforementioned linear DNA sequence in a single step. The linear DNA can be either a linear DNA containing any sequence, with upstream and downstream sequences of the target site on the genomic DNA (representatively part or all of the deleted target gene) at both ends of that arbitrary sequence, or a linear DNA consisting of directly linked upstream and downstream sequences of the target site on the genomic DNA. The arbitrary sequence may, for example, contain a marker gene sequence. The marker gene can be subsequently removed as needed. In the case of marker gene removal, sequences for homologous recombination can be added to both ends of the marker gene to efficiently remove it.

[0387] The deletion of the aforementioned target gene in microorganisms can be confirmed by a decrease in the activity of the enzyme encoded by the deleted target gene. This decrease in enzyme activity can be confirmed by measuring the enzyme's activity. For example, glutathione reductase activity can be measured using known methods (such as the Glutathione Reductase Assay Kit, model 7510-100-K, manufactured by Cosmo Biotech).

[0388] The reduced transcription level of the aforementioned missing gene can be confirmed by comparing the amount of mRNA transcribed from that gene with that of the parental strain. Methods for evaluating the amount of mRNA include Northern blotting and RT-PCR (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). Compared to the parental strain, the amount of mRNA is preferably reduced to, for example, less than 50%, less than 20%, less than 10%, less than 5%, or 0%.

[0389] The reduction in the amount of enzyme encoded by the aforementioned deleted gene can be confirmed using antibody-based Western blotting (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). In the microorganisms of one or more embodiments of the second aspect, compared to the parent strain, the amount of enzyme encoded by the aforementioned deleted gene is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0%.

[0390] Next, the enhancement of the expression of a given gene in a microorganism in one or more embodiments of the second aspect will be described.

[0391] A microorganism with "enhanced expression" of a given expression-enhancing gene (a gene encoding glutamate-cysteine ​​ligase and a gene encoding glutathione synthase, or a gene encoding a bifunctional glutathione synthase) includes the following two aspects: when the parental strain (wild-type strain) of the microorganism originally expressed the aforementioned expression-enhancing gene, the expression level of the aforementioned expression-enhancing gene is increased compared to the parental strain; and when the parental strain originally did not express the aforementioned expression-enhancing gene, the parental strain was endowed with the ability to express the aforementioned expression-enhancing gene.

[0392] The increase in the expression level of the aforementioned expression-enhancing genes can be achieved by: increasing the copy number of the aforementioned expression-enhancing genes in the microbial cells, or replacing the promoter controlling the expression of the aforementioned expression-enhancing genes on the genomic DNA of the microbial cells with a stronger expression promoter.

[0393] The increase in the copy number of the aforementioned expression-enhancing genes within microbial cells can be achieved in the following ways.

[0394] (A) Introducing an expression vector containing the aforementioned expression-enhancing genes into the cells of microorganisms, or

[0395] (B) The above expression-enhancing gene was introduced into the genomic DNA of microbial cells.

[0396] As the expression vector used in the manner described in (A) above, a plasmid vector containing the aforementioned expression-enhancing gene can be used. The expression vector is preferably capable of autonomous replication within microbial cells. The expression vector preferably contains: DNA encoding one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase, and a promoter operatively linked to a position capable of transcribing the DNA. The expression vector is preferably recombinant DNA capable of autonomous replication within microbial cells and containing a base sequence consisting of a promoter, a ribosome-binding sequence, an amino acid sequence encoding one or more of the aforementioned enzymes, and a transcription termination sequence.

[0397] In one or more embodiments of the second aspect, the microorganism preferably possesses an expression vector containing a base sequence encoding the aforementioned expression-enhancing gene. This microorganism is capable of expressing the aforementioned expression-enhancing gene using the expression vector.

[0398] Suitable plasmid vectors include: pQEK1, pCA24N (DNA RESEARCH, 12, 191-299 (2005)), pACYC177, pACYC184 (available from Nippon Gene), pQE30, pQE60, pQE70, pQE80, and pQE9 (available from Qiagen); pTipQC1 (available from Qiagen or Hokkaido System Science), and pTipRT2 (available from Hokkaido System Science). (Available from Science); 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), pUCN18 (prepared by modifying pUC18 (available from Takara Bio), pSTV28 (available from Takara Bio), pUCNT (International Publication No. 94 / 03613), etc.

[0399] The expression vector preferably contains a promoter that controls the transcription of the expression-enhancing gene.

[0400] In one or more embodiments of the second aspect, the promoter is preferably an inductive promoter.

[0401] Examples of inducible promoters include: isopropyl-β-thiogalactopyranoside (IPTG) inducible promoters, light-inducible promoters that induce gene expression under light irradiation, araBAD promoters (arabinose inducible), rhaBAD promoters (rhamnose inducible), tet promoters (drug-inducible), penP promoters (drug-inducible), cspA promoters (temperature-inducible promoters that respond to low temperatures), and promoters containing tetO or lacO operons as operon sequences. Preferred promoters include IPTG inducible promoters, araBAD promoters, rhaBAD promoters, tet promoters, penP promoters, cspA promoters, or promoters containing tetO or lacO operons as operon sequences.

[0402] Specific examples of IPTG-inducible promoters include: T5 promoter, lacUV5 promoter, lac promoter, T7 promoter, lacT5 promoter, lacT7 promoter, tac promoter, etc. IPTG-inducible promoters are particularly preferred, and among IPTG-inducible promoters, T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, or tac promoter are particularly preferred.

[0403] As promoters, existing promoters can also be modified into highly active promoters using various reporter genes. For example, promoter activity can be improved by making the -35 and -10 regions within the promoter region closer to the common sequence (International Publication WO2000 / 018935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574). Methods for evaluating promoter strength and examples of strong promoters are described in the paper by Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0404] When an expression vector containing the above-mentioned expression-enhancing gene is introduced into a cell of a microorganism, the copy number of the expression vector in the cell is preferably 2 or more, more preferably 3 or more, more preferably 5 or more, more preferably 10 or more, more preferably 15 or more, and more preferably 20 or more.

[0405] In microbial cells, to increase the expression levels of two or more of the aforementioned expression-enhancing genes, two or more genes can be contained within a single expression vector. In this case, the two or more genes can be configured to be controlled by a single expression promoter. Alternatively, the two or more genes can also be contained in separate expression vectors.

[0406] When the above-mentioned expression-enhancing gene is introduced into the genomic DNA of a microbial cell in the manner described in (B) above, homologous recombination can be utilized.

[0407] When the promoter of the aforementioned expression-enhancing gene is replaced with a more potent expression promoter on the genomic DNA of a microbial cell, the same promoter as the expression vector can be used as the expression promoter, and an inducible promoter is more preferably used. Specific examples of preferred promoters are described above.

[0408] In the microorganisms of one or more embodiments of the second aspect, the degree of enhancement (increase in expression level) of the expression-enhancing gene is not particularly limited. The expression level of the expression-enhancing gene can be expressed as the amount of mRNA (i.e., mRNA encoding the amino acid sequence of one or more enzymes selected from glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase) corresponding to the expression-enhancing gene extracted from the cell. The expression level based on this mRNA is preferably expressed as a relative value to the amount of mRNA encoding a suitable internal standard protein. In one or more embodiments of a microorganism with enhanced expression of the gene encoding glutamate-cysteine ​​ligase, the expression level of the gene encoding glutamate-cysteine ​​ligase in the microorganism (preferably a relative value obtained by dividing the amount of glutamate-cysteine ​​ligase mRNA in the microorganism by the amount of mRNA encoding the internal standard protein in the same strain) is preferably 5 times or more, more preferably 10 times or more, and more preferably 20 times or more. In one or more embodiments of the microorganism with enhanced expression of the gene encoding glutathione synthase, the expression level of the gene encoding glutathione synthase in the microorganism (preferably a relative value obtained by dividing the amount of glutathione synthase mRNA in the microorganism by the amount of mRNA encoding the internal standard protein in the same strain) is preferably 5 times or more, more preferably 10 times or more, and more preferably 20 times or more than the expression level of glutathione synthase in the wild strain (preferably a relative value obtained by dividing the amount of glutathione synthase mRNA in the wild strain by the amount of mRNA encoding the internal standard protein in the same strain). As an internal standard protein, the protein encoded by the hcaT gene, which is known as a housekeeping gene, can be cited as an example.

[0409] <3.3. Method for manufacturing glutathione, the second aspect of this specification>

[0410] One or more embodiments of the second aspect relate to a method for manufacturing glutathione, the method comprising:

[0411] The microorganisms of one or more embodiments of the second aspect described above are cultured in a culture medium.

[0412] The method for producing glutathione according to this embodiment can efficiently produce glutathione.

[0413] The aforementioned culture medium only needs to contain the nutrients necessary for the proliferation of microorganisms and the biosynthesis of glutathione used in the second aspect, such as carbon source, nitrogen source, inorganic salts, and vitamins. It can be any type of synthetic culture medium or natural culture medium. M9 culture medium is preferred.

[0414] As a carbon source, any carbon source that the microorganisms to be used can assimilate is acceptable. Examples include sugars such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0415] Examples of nitrogen sources include: ammonium salts such as ammonia and ammonium sulfate, nitrogen compounds such as amines, natural nitrogen sources such as peptone and soybean hydrolysate.

[0416] Examples of inorganic salts include: potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0417] Examples of vitamins include biotin and thiamine. Furthermore, substances required for the growth of microorganisms according to one or more embodiments of the second aspect (e.g., auxotrophic amino acids in the case of amino acid auxotrophic microorganisms) may be added as needed.

[0418] It is preferable to add at least one of a sulfur source and glycine to the above-mentioned culture medium, and more preferably both. Examples of glycine concentrations added to the culture medium include 100 mM to 2000 mM, and more preferably 400 mM to 1200 mM. Examples of sulfur source concentrations added to the culture medium include 100 mM to 2000 mM, and more preferably 400 mM to 1200 mM.

[0419] As a sulfur source, one or more inorganic sulfur compounds such as sulfuric acid, thiosulfate, sulfurous acid, dithionite, sulfides, or their salts can be added. Sulfuric acid, thiosulfate, sulfurous acid, dithionite, or sulfides can be in free form, as salts, or any mixture thereof. There are no particular restrictions on the salts used; examples include sodium salts, calcium salts, ammonium salts, and potassium salts.

[0420] Glycine can be in its free form, as a salt, or any mixture thereof. As a salt, there are no particular limitations; examples include sulfates and hydrochlorides.

[0421] Sulfur sources and / or glycine can be added to the culture medium at the beginning of culture or during culture. Sulfur sources and / or glycine can be added to the culture medium all at once, or continuously or intermittently.

[0422] Sulfur sources and / or glycine may be included in the culture medium throughout the entire culture period, or only for a portion of the culture. For example, the amounts of sulfur sources and glycine added do not need to be within the aforementioned ranges throughout the entire glutathione accumulation phase; the culture medium can be maintained at levels within the aforementioned ranges during culture, decreasing as culture time progresses. Alternatively, sulfur sources and / or glycine may be added continuously or intermittently. It should be noted that the concentrations of culture medium components other than sulfur sources and / or glycine may vary during culture, and additional additions may be made.

[0423] The culture is preferably carried out under aerobic conditions such as shaking culture and aerated stirring culture. The culture temperature is 20~50℃, preferably 20~42℃, more preferably 28~38℃. The pH during culture is 5~9, preferably 6~7.5. The culture time is 3 hours~5 days, preferably 5 hours~3 days.

[0424] Glutathione accumulated in cultures can be collected using conventional purification methods. For example, after culturing, it can be collected by ion exchange, concentration, and crystallization after removing bacterial cells and solid matter from the culture through centrifugation.

[0425] Example

[0426] The first and second aspects of this specification will be described in more detail below with reference to examples, but the first and second aspects of this specification are not limited to these examples.

[0427] The gene manipulations described below can be performed with reference to the documentation in *Molecular Cloning* (Cold Spring Harbor Laboratory Press (1989)). Furthermore, the enzymes and cloning hosts used in gene manipulation can be purchased from commercial suppliers and used according to their instructions. It should be noted that any enzyme described above that can be used for gene manipulation is acceptable; there are no particular limitations.

[0428] <First Aspect>

[0429] The following are experimental results used to illustrate the first aspect of this specification.

[0430] [Example 1-1] Construction of glutathione synthesis gene expression vector (1)

[0431] The T5 promoter, the *E. coli*-derived *gshA* gene (Sequence No. 12), and the *E. coli*-derived *gshB* gene (Sequence No. 14) were inserted between the SmaI and HindIII sites of the plasmid vector pQEK1-term, as described in sequence No. 4. Primers were designed based on the instructions for the NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and the vector was constructed according to the described steps.

[0432] [Examples 1-2] Construction of glutathione synthesis gene expression vector (2)

[0433] The plasmid vector pQEK1-term, described in sequence number 4, incorporated an insertion between the SmaI and HindIII sites: a T5 promoter, the *E. coli*-derived *gshA* gene (sequence number 12), and the TDgshB(V260A) gene (sequence number 18) encoding a mutant enzyme (WO2018 / 084165) of glutathione synthase derived from *Thiobacillus denitrificans*, a sulfur-producing bacterium. Primers were designed based on the instructions for the NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and the vector was constructed according to the described steps. The constructed plasmid vector was named pQEK1-PT5-ABTd(V260A)-term.

[0434] [Examples 1-3] Construction of glutathione synthesis gene expression vectors (3)

[0435] The plasmid vector pQEK1-term, described in sequence number 4, incorporated a T5 promoter and the SAgshF gene (sequence number 19) encoding a bifunctional glutathione synthase derived from Streptococcus agalactiae between the SmaI and HindIII sites. Primers were designed based on the instructions for the NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and the vector was constructed according to the described steps. The constructed plasmid vector was named pQEK1-PT5-FSa-term.

[0436] [Examples 1-4] Construction of glutathione synthesis gene expression vectors (4)

[0437] The plasmid vector pQEK1-term, described in sequence number 4, incorporated an insert between the SmaI and HindIII sites: the lac promoter, the *E. coli*-derived *gshA* gene (sequence number 12), and the TDgshB(V260A) gene (sequence number 18) encoding a mutant enzyme (WO2018 / 084165) of glutathione synthase derived from *Thiobacillus denitrificans*, a sulfur-producing bacterium. Primers were designed based on the instructions for the NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and the vector was constructed according to the described steps. The constructed plasmid vector was named pQEK1-Plac-ABTd(V260A)-term.

[0438] [Examples 1-5] Construction of glutathione synthesis gene expression vectors (5)

[0439] The plasmid vector pQEK1-term, described in sequence number 4, incorporated the lacUV5 promoter, the *E. coli*-derived *gshA* gene (sequence number 12), and the TDgshB(V260A) gene (sequence number 18) encoding a mutant enzyme (WO2018 / 084165) of glutathione synthase derived from *Thiobacillus denitrificans*, a sulfur-producing bacterium. Primers were designed based on the instructions for the NEBuilder HiFi DNA Assembly Master Mix (manufactured by New England Biolabs), and the vector was constructed according to the described steps. The constructed plasmid vector was named pQEK1-PlacUV5-ABTd(V260A)-term.

[0440] [Examples 1-6] Preparation of host strains

[0441] Using the plasmid pTH18cs1 obtained from the National Institute of Genetics of the National University, strain BW25113 of Escherichia coli was prepared with the same method as the method disclosed in Japanese Patent Application Publication No. 2004-344029 for the preparation of a cytosine deaminase-deleted strain, strains with deletions of the γ-glutamyltransferase gene (serial number 21) and the tripeptipeptidase gene (serial number 23) were prepared.

[0442] [Examples 1-7] Preparation of glutathione synthesis gene-enhanced strains

[0443] Competent cells of the host strains prepared in Examples 1-6 were prepared using the usual method and transformed using the plasmid vectors prepared in Examples 1-1 to 5 to obtain the respective transformants.

[0444] [Examples 1-8] Evaluation of glutathione fermentation production

[0445] The host strains (without plasmids) prepared in Examples 1-6 or the glutathione synthesis gene-enhanced strains prepared in Examples 1-7 were inoculated into 5 mL of LB medium (containing 20 μg / mL tetracycline) and cultured with shaking at 30°C and 300 rpm for 8 hours. 1 mL of this culture was then inoculated into 100 mL of M9 medium supplemented with 20 μg / mL tetracycline (6 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 1 mM magnesium sulfate, 0.001% thiamine-hydrochloric acid, 0.1 mM calcium chloride, 2% glucose). The inoculated culture was cultured for 18 hours using a culture apparatus (Able Bio Jr. 8) at 34°C, pH 6.5, stirring at 1000 rpm, and aeration at 100 mL / min. 20 mL of the culture after 18 hours of culture was then inoculated into 2 L of M9 medium supplemented with 20 μg / mL tetracycline. The culture medium after the second inoculation was further cultured using a culture apparatus (Bioneer-Neo, Marubishi Bioengineering) at 34°C, pH 6.7, stirring at 600 rpm, and aeration at 4 L / min. During culture, 50 w / v% glucose solution was added periodically to adjust the glucose concentration to no less than 15 g / L. After 6 hours of culture, 0.1 mM isopropyl-β-thiopyranoside, along with 780 mM glycine and 780 mM sodium sulfate, were added. At 24 hours of culture, a sample of the culture medium was taken, and the cells were separated from the supernatant by centrifugation. The supernatant was appropriately diluted with distilled water, and the GSH and GSSG in the culture supernatant were quantified according to the method described in WO2016 / 002884 to determine the total concentration. The quantitative results of the total concentrations of GSH and GSSG in the culture supernatant are shown in Table 1. The culture medium used in the above experiments did not actually contain cysteine ​​and cystine; their total concentration was less than 0.5 g / L.

[0446]

[0447] [Examples 1-9] Transcriptional analysis of glutamate-cysteine ​​ligase gene and glutathione synthase gene

[0448] Expression levels of genes overexpressed using plasmids were analyzed by real-time PCR. In the cultures described in Examples 1-8, after culturing the culture medium for 6 hours following the second inoculation, 0.1 mM isopropyl-β-thiogalactopyranoside was added, and samples were taken 1 hour later. RNA was extracted using NucleoSpin RNA (Takara Bio) according to the accompanying instructions. Each RNA sample was diluted with water to a concentration of 50 ng / μL. cDNA was synthesized from the RNA using the PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio) according to the accompanying instructions. gshA, TDgshB (V260A), gshB, and SAgshF in each sample were quantified using a TB Green Premix Ex Taq II (TliRNaseH Plus) and a Thermo Fisher Scientific QuantStudio 3 real-time PCR system (Takara Bio). hcaT (sequence number 27), a known housekeeping gene, was used as an internal standard. For hcaT, gshA, and gshB, calibration curves were constructed using host *E. coli* genomic DNA and the sample simultaneously in a real-time PCR reaction. Based on these calibration curves, the amount of each gene contained in each cDNA was quantified. For TDgshB (V260A), and for plasmids pTDGSH2m15 and SAgshF described in WO2018 / 084165, calibration curves were constructed using plasmid pNGSHF described in WO2016 / 017631. In the same sample, the expression levels of each gene were normalized by dividing the quantitative value of each gene by the quantitative value of hcaT (used as an internal standard), and this normalized value was used as the expression level of each gene. The forward primer used for amplifying hcaT was primer number 28, and the reverse primer used was primer number 29. The forward primer used for amplifying gshA was primer number 30, and the reverse primer used was primer number 31. The forward primer used for amplifying TDgshB(V260A) was primer number 32, and the reverse primer used was primer number 33. The forward primer used for amplifying gshB was primer number 34, and the reverse primer used was primer number 35. The forward primer used for amplifying SAgshF was primer number 36, and the reverse primer used was primer number 37.

[0449] When the expression level of the *E. coli*-derived *gshA* gene from the untransformed host strains prepared in Examples 1-6 (the expression level obtained by normalizing the quantitative value of the *gshA* gene expression level in the host strain by the quantitative value of the *hcaT* expression level in the same host strain) was set to 1, the relative values ​​of pQEK1-PT5-ABTd(V260A)-term and pQEK1-Plac-ABTd(V260A)-term, which contain the *gshA* gene from *E. coli*, were calculated. The expression level of the *E. coli*-derived gshA gene in the transformants of pQEK1-PlacUV5-ABTd(V260A)-term or pQEK1-PT5-ABEc-term (the expression level obtained by normalizing the quantitative value of gshA gene expression in each transformant by the quantitative value of hcaT expression in the same transformant) was evaluated as follows: a relative value of 5 or more but less than 10 was rated as "+", a relative value of 10 or more but less than 20 was rated as "++", and a relative value of 20 or more was rated as "++++". Similarly, when the expression level of the *E. coli*-derived *gshB* gene in the untransformed host strain (the expression level obtained by normalizing the quantitative value of the *gshB* gene expression in the host strain by the quantitative value of the *hcaT* expression in the same host strain) was set to 1, the expression level of the *E. coli*-derived *gshB* gene in the transformant introduced into the pQEK1-PT5-ABEc-term plasmid vector containing the *gshB* gene from *E. coli* (the expression level obtained by normalizing the quantitative value of the *gshB* gene expression in each transformant by the quantitative value of the *hcaT* expression in the same transformant) was calculated as a relative value. A relative value of 5 or higher and less than 10 was rated as "+", 10 or higher and less than 20 was rated as "++", and 20 or higher was rated as "+++". The results are shown in Table 2.

[0450]

[0451] The expression levels of the TDgshB(V260A) or SAgshF genes in the transformants were standardized by dividing the quantitative values ​​of each gene expression by the quantitative value of the hcaT gene in the same sample. The transformants were obtained by introducing plasmid vectors pQEK1-PT5-ABTd(V260A)-term, pQEK1-Plac-ABTd(V260A)-term, pQEK1-PlacUV5-ABTd(V260A)-term, or pQEK1-PT5-FSa-term containing the TDgshB(V260A) or SAgshF genes, which are not originally present in the aforementioned host strains of *E. coli*. As a negative control, gene expression levels were also determined for the untransformed host strains (without plasmids). The results are shown in Table 3.

[0452] The relative value when the expression level of the hcaT gene in the same sample is set to 1.

[0453] <Second aspect>

[0454] Next, the experimental results used to illustrate the second aspect of this specification will be presented below.

[0455] (Analysis of glutathione concentration in culture medium)

[0456] The concentration of glutathione in the culture medium was determined using high performance liquid chromatography (HPLC, Shimadzu Corporation).

[0457] The analytical conditions for HPLC are as follows.

[0458] Column: Develosil ODS-HG-3 4.6mm x 250mm (Nomura Chemicals)

[0459] Mobile phase: Dissolve 30.5 g of potassium dihydrogen phosphate and 18 g of sodium heptanesulfonate in 4.5 L of distilled water, then adjust the pH to 3 with phosphoric acid. Add 250 mL of methanol, and then adjust the pH to 3 again with phosphoric acid.

[0460] Flow rate: 1 mL / min

[0461] Detection: UV detector, λ = 210nm

[0462] Column temperature: 40℃

[0463] Injection volume: 10 μL

[0464] When analyzing the glutathione concentration in the culture medium, the bacterial cells were removed by centrifugation, and the supernatant was passed through a syringe filter (Advantech, φ = 0.2 μm) to obtain the culture supernatant. The obtained culture supernatant was diluted 10-fold with distilled water for HPLC analysis.

[0465] (Manufacturing Example 2-1) Preparation of BW25113Δggt strain

[0466] First, a plasmid vector for disrupting the ggt (γ-glutamyl transferase) gene (Sequence No. 21) was prepared. DNA fragments containing the upstream and downstream sequences of the ggt gene (Sequence No. 1) were obtained by PCR using synthetic oligoDNA. The obtained fragments were digested with XbaI and HindIII. The temperature-sensitive plasmid pTH18cs1 (GenBank accession number AB019610) [Hashimoto-Gotoh, T., Gene, 241, 185-191 (2000)] was digested with XbaI and HindIII, and the resulting fragments were ligated using Ligation high Ver.2 (Toyobo Co., Ltd.) to obtain the plasmid vector pTH18cs1-ggt-UD.

[0467] Next, the BW25113Δggt strain was prepared using pTH18cs1-ggt-UD. pTH18cs1-ggt-UD was introduced into *E. coli* strain BW25113 via electroporation and plated onto LB agar plates containing 10 μg / mL chloramphenicol. Transformants were obtained by incubation at 30°C. The transformed strains were then cultured overnight at 30°C with shaking in LB liquid medium containing 10 μg / mL chloramphenicol. The culture was then plated onto LB agar plates containing 10 μg / mL chloramphenicol and incubated at 42°C to obtain transformants. After incubating the transformed strains overnight at 42°C on LB liquid medium, colonies were obtained by plated onto LB agar plates. The obtained colonies were replicated and plated onto LB agar plates and LB agar plates containing 10 μg / mL chloramphenicol, respectively, and transformants exhibiting chloramphenicol sensitivity were screened. PCR and DNA sequencing analysis were used to isolate a strain from the screened transformants that lacked both the start and stop codons of the ggt gene on the chromosome. This gene-destroyed strain was named BW25113Δggt.

[0468] BW25113Δggt strain is a strain of Escherichia coli BW25113 that uses this strain as a host and has the start codon to the stop codon of the ggt gene on the chromosome missing.

[0469] (Manufacturing Example 2-2) Preparation of BW25113ΔggtΔpepT strain

[0470] First, a plasmid vector for disrupting the pepT (tripeptipeptidase) gene (SEQ ID NO: 23) was prepared. DNA fragments containing the upstream and downstream sequences of the pepT gene (SEQ ID NO: 2) were obtained using PCR with synthetic oligoDNA. The obtained fragments were digested with XbaI and HindIII, and pTH18cs1 was digested with XbaI and HindIII. The resulting fragments were ligated using Ligation high Ver.2 to obtain the plasmid vector pTH18cs1-pepT-UD.

[0471] Next, using the BW25113Δggt strain prepared in Manufacturing Example 2-1 as the parent strain, a strain lacking the start codon to the stop codon of the pepT gene on the chromosome was isolated using pTH18cs1-pepT-UD through the same method as in Manufacturing Example 2-1. This gene-damaged strain was named BW25113ΔggtΔpepT strain.

[0472] The BW25113ΔggtΔpepT strain is a strain of *Escherichia coli* BW25113 that uses this strain as its host and has the start codon to the stop codon of the ggt gene and the pepT gene on its chromosome missing.

[0473] (Manufacturing Example 2-3) Preparation of pQEK1-PT5-ABTd(V260A)-term

[0474] First, to construct a vector for gene introduction into *E. coli*, the pQE-80L(QIAGEN) vector was constructed by changing the drug resistance marker to a tetracycline resistance gene, as shown in sequence number 3. Further, a terminator sequence derived from λ phage was inserted into the HindIII locus of pQEK1, constructing the pQEK1-term vector shown in sequence number 4.

[0475] Next, using PCR to synthesize oligomeric DNA, a DNA fragment (Sequence No. 5) was obtained, consisting of the T5 promoter, the *E. coli*-derived *gshA* gene, and the *Thiobacillus denitrificans*-derived *gshB* gene (with a V260A mutation). This fragment was then ligated to a fragment obtained by digesting the pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), yielding the pQEK1-PT5-ABTd(V260A)-term shown in Sequence No. 6.

[0476] (Manufacturing Example 2-4) Preparation of BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term strain

[0477] The pQEK1-PT5-ABTd(V260A)-term prepared in Manufacturing Example 2-3 was introduced into the BW25113ΔggtΔpepT strain prepared in Manufacturing Example 2-2 using electroporation. The transformed strains were plated on LB agar plates containing 20 μg / mL tetracycline and screened. One strain infused with pQEK1-PT5-ABTd(V260A)-term was isolated from the screened transformants by PCR-based analysis. This strain was named BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term.

[0478] (Manufacturing Example 2-5) Preparation of BW25113ΔggtΔpepTΔgor strain

[0479] First, a plasmid vector for disrupting the gor (glutathione reductase) gene was prepared. DNA fragments (accession number 7) containing the upstream and downstream sequences of the gor gene were obtained using PCR with synthetic oligoDNA. The obtained fragments were digested with XbaI and HindIII, and pTH18cs1 was digested with XbaI and HindIII. The resulting fragments were ligated using Ligationhigh Ver.2 to obtain the plasmid vector pTH18cs1-gor-UD.

[0480] Next, using the BW25113ΔggtΔpepT strain prepared in Manufacturing Example 2-2 as the parent strain, a strain lacking the start codon to the stop codon of the gor gene on the chromosome was isolated using pTH18cs1-gor-UD in the same manner as in Manufacturing Example 2-1. This gene-damaged strain was named BW25113ΔggtΔpepTΔgor strain.

[0481] Preparation of (Manufacturing Example 2-6) BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term strain

[0482] The pQEK1-PT5-ABTd(V260A)-term prepared in Manufacturing Examples 2-3 was introduced into the BW25113 ΔggtΔpepTΔgor strain prepared in Manufacturing Examples 2-5 using electroporation. The transformed strains were plated on LB agar plates containing 20 μg / mL tetracycline and screened. PCR-based analysis revealed one strain containing the pQEK1-PT5-ABTd(V260A)-term, which was named BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term.

[0483] (Manufacturing Example 2-7) Preparation of pQEK1-PT5-ABEc-term

[0484] A DNA fragment (Sequence No. 8) consisting of the T5 promoter, the *E. coli*-derived *gshA* gene, and the *E. coli*-derived *gshB* gene was obtained using PCR with synthetic oligoDNA. The obtained fragment was ligated to a fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNA AssemblyMaster Mix, yielding the pQEK1-PT5-ABEc-term shown in Sequence No. 9.

[0485] (Manufacturing Example 2-8) Preparation of BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term strain

[0486] The pQEK1-PT5-ABEc-term prepared in Manufacturing Example 2-7 was introduced into the BW25113ΔggtΔpepT strain prepared in Manufacturing Example 2-2 using electroporation. The transformed strains were plated on LB agar plates containing 20 μg / mL tetracycline and screened. PCR-based analysis identified one strain from the screened transformants that had been infused with pQEK1-PT5-ABEc-term. This strain was named BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term.

[0487] (Manufacturing Example 2-9) Preparation of BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term strain

[0488] The pQEK1-PT5-ABEc-term prepared in Manufacturing Examples 2-3 was introduced into the BW25113ΔggtΔpepTΔgor strain prepared in Manufacturing Examples 2-5 using electroporation. The transformed strains were plated on LB agar plates containing 20 μg / mL tetracycline and screened. PCR-based analysis identified one strain from the screened transformants that had been infused with pQEK1-PT5-ABEc-term. This strain was named BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term.

[0489] (Manufacturing Example 2-10) Preparation of pQEK1-PT5-FSa-term

[0490] A DNA fragment (Sequence No. 10) consisting of the T5 promoter and the gshF gene from Streptococcus agalactiae was obtained by PCR using synthetic oligoDNA. The obtained fragment was ligated to the fragment obtained by digesting pQEK1-term with SpeI and HindIII using NEBuilder HiFi DNAAssembly Master Mix, resulting in pQEK1-PT5-FSa-term as shown in Sequence No. 11.

[0491] (Manufacturing Example 2-11) Preparation of BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term strain

[0492] The pQEK1-PT5-FSa-term prepared in Manufacturing Example 2-10 was introduced into the BW25113ΔggtΔpepT strain prepared in Manufacturing Example 2-2 using electroporation. The transformed strains were plated on LB agar plates containing 20 μg / mL tetracycline and screened. PCR-based analysis identified one strain containing pQEK1-PT5-FSa-term from the screened transformants. This strain was named BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term.

[0493] (Manufacturing Example 2-12) Preparation of BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term strain

[0494] The pQEK1-PT5-FSa-term prepared in Manufacturing Example 2-10 was introduced into the BW25113ΔggtΔpepTΔgor strain prepared in Manufacturing Example 2-5 using electroporation. The transformed strains were plated on LB agar plates containing 20 μg / mL tetracycline and screened. PCR-based analysis identified one strain containing pQEK1-PT5-FSa-term from the screened transformants. This strain was named BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term.

[0495] (Example 2-1) Fermentation production of glutathione using BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term strain

[0496] GSH and GSSG were produced by culturing the BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABTd(V260A)-term strain obtained in Examples 2-6 under the following conditions. The culture was inoculated into 5 mL of LB medium (containing 20 μg / mL tetracycline) and cultured with shaking at 300 rpm and 30°C for 8 hours. 1 mL of this culture was then inoculated into 100 mL of M9 medium supplemented with 20 μg / mL tetracycline (6 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 1 mM magnesium sulfate, 0.001% thiamine-hydrochloric acid, 0.1 mM calcium chloride, 2% glucose). The medium was then cultured for 18 hours using an Able Bio Jr. 8 incubator at 34°C, pH 6.5, stirring at 1000 rpm, and aeration at 100 mL / min. After 18 hours of culture, 20 mL of the culture medium was inoculated into 2 L of M9 medium supplemented with 20 μg / mL tetracycline. The medium was cultured using a Bioneer-Neo culture apparatus (Marubishi Bioengineering) at 34°C, pH 6.7, stirring at 600 rpm, and aeration at 4 L / min. During culture, 50% (w / v) glucose solution was added periodically to maintain a glucose concentration of at least 15 g / L. After 6 hours of culture, 0.1 mM isopropyl-β-thiogalactopyranoside, along with glycine and sodium sulfate, was added to achieve a final concentration of 100 mM. At 30 hours of culture, a sample of the culture medium was taken, and the cells were separated from the supernatant by centrifugation. The supernatant was appropriately diluted with distilled water, and GSH and GSSG were quantified by HPLC analysis. The quantitative results are shown in Table 4.

[0497] (Comparative Example 2-1) Glutathione production by fermentation using strain BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term

[0498] The BW25113ΔggtΔpepT / pQEK1-PT5-ABTd(V260A)-term strain obtained in Manufacturing Examples 2-4 was cultured under the same conditions as in Example 2-1, and GSH and GSSG were produced. The results are shown in Table 4.

[0499]

[0500] <Inspection>

[0501] Comparing the results of Example 2-1 and Comparative Example 2-1 in Table 4 shows that disrupting the gor gene significantly increases glutathione production (GSH+GSSG). This demonstrates that disrupting the gor gene is effective in glutathione fermentation production.

[0502] (Example 2-2) Fermentation production of glutathione using strain BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term

[0503] The BW25113ΔggtΔpepTΔgor / pQEK1-PT5-ABEc-term strain obtained in Manufacturing Examples 2-9 was cultured under the same conditions as in Example 2-1, and GSH and GSSG were produced. The results are shown in Table 5.

[0504] (Comparative Example 2-2) Glutathione production by fermentation using strain BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term

[0505] The BW25113ΔggtΔpepT / pQEK1-PT5-ABEc-term strain obtained in Manufacturing Examples 2-8 was cultured under the same conditions as in Example 2-1, and GSH and GSSG were produced. The results are shown in Table 5.

[0506]

[0507] <Inspection>

[0508] Comparing the results of Example 2-2 and Comparative Example 2-2 in Table 5 shows that disrupting the gor gene significantly increases glutathione production (GSH+GSSG). This demonstrates that disrupting the gor gene is effective in glutathione fermentation production.

[0509] (Examples 2-3) Fermentation production of glutathione using BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term strain

[0510] The BW25113ΔggtΔpepTΔgor / pQEK1-PT5-FSa-term strain obtained in Manufacturing Example 2-12 was cultured under the same conditions as in Example 2-1, and GSH and GSSG were produced. The results are shown in Table 6.

[0511] (Comparative Examples 2-3) Glutathione production by fermentation using BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term strain

[0512] The BW25113ΔggtΔpepT / pQEK1-PT5-FSa-term strain obtained in Manufacturing Example 2-11 was cultured under the same conditions as in Example 2-1, and GSH and GSSG were produced. The results are shown in Table 6.

[0513]

[0514] <Inspection>

[0515] Comparing the results of Examples 2-3 and Comparative Examples 2-3 in Table 6, it can be seen that disrupting the gor gene significantly increases glutathione productivity (GSH+GSSG). Therefore, it can be concluded that disrupting the gor gene is effective in glutathione fermentation production.

[0516] All publications, patents and patent applications referenced in this specification are incorporated herein by direct reference.

Claims

1. A method for manufacturing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione, the method comprising: Gram-negative bacteria are cultured in a medium in which the expression levels of one or more genes selected from genes encoding glutamate-cysteine ​​ligase, glutathione synthase, and bifunctional glutathione synthase are increased compared to wild-type strains. These Gram-negative bacteria are capable of overproducing γ-glutamylcysteine, bis-γ-glutamylcysteine, γ-glutamylcysteine, reduced glutathione, and / or oxidized glutathione through the induction of expression of these one or more genes. The method does not include adding cysteine ​​or cystine to the culture medium, but includes adding glycine to the culture medium continuously or intermittently.

2. The method according to claim 1, wherein, The Gram-negative bacteria retain at least one gene selected from the following groups that are expressibly linked to an inducible promoter: a gene encoding glutamate-cysteine ​​ligase, a gene encoding glutathione synthase, and a gene encoding a bifunctional glutathione synthase. The inducible promoter is an inducible promoter that, when one or more genes are genes encoding glutamate-cysteine ​​ligase, causes the expression level of the gene encoding glutamate-cysteine ​​ligase in the Gram-negative bacteria to be more than 20 times that of the wild-type strain.

3. The method according to claim 2, wherein, The inducible promoter is an IPTG inducible promoter, a photoinducible promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing a tetO or lacO operon as an operon sequence.

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

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

6. The method according to claim 5, wherein, The inducible promoter is the T5 promoter.

7. The method according to any one of claims 1 to 6, wherein the Gram-negative bacterium is a transformant of intestinal bacteria.

8. The method according to any one of claims 1 to 6, wherein the Gram-negative bacterium is a transformant of Escherichia coli.

9. A microorganism that lacks the following [1] and [2] genes and enhances the expression of [3] or [4] genes: [1] The gene encoding γ-glutamyltransferase (EC:2.3.2.2); [2] The gene encoding glutathione reductase (EC: 1.8.1.7); [3] Genes encoding glutamate-cysteine ​​ligase (EC:6.3.2.2) and glutathione synthase (EC:6.3.2.3); [4] Gene encoding bifunctional glutathione synthase.

10. The microorganism according to claim 9, wherein the following [5] gene is missing: [5] Gene encoding tripeptipeptidase (EC:3.4.11.4).

11. The microorganism according to claim 9, wherein, The gene in [3] or the gene in [4] can be expressively linked to an inducible promoter. The inducible promoter is an IPTG inducible promoter, a photoinducible promoter, an araBAD promoter, a rhaBAD promoter, a tet promoter, a penP promoter, a cspA promoter, or a promoter containing a tetO or lacO operon as an operon sequence.

12. The organism according to claim 11, wherein, The inducible promoter is a T5 promoter, T7 promoter, lacT5 promoter, lacT7 promoter, tac promoter, araBAD promoter, rhaBAD promoter, tet promoter, penP promoter, cspA promoter, or a promoter containing tetO or lacO operons as an operon sequence.

13. The microorganism according to claim 12, wherein, The inducible promoter is a T5 promoter, a T7 promoter, a lacT5 promoter, a lacT7 promoter, or a tac promoter.

14. A method for manufacturing glutathione, the method comprising: Culturing the microorganisms according to any one of claims 9 to 13 in a culture medium, and Glycine is added to the culture medium continuously or intermittently.

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