WO2020050273A1 - Method for producing glutathione - Google Patents

Method for producing glutathione Download PDF

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Publication number
WO2020050273A1
WO2020050273A1 PCT/JP2019/034630 JP2019034630W WO2020050273A1 WO 2020050273 A1 WO2020050273 A1 WO 2020050273A1 JP 2019034630 W JP2019034630 W JP 2019034630W WO 2020050273 A1 WO2020050273 A1 WO 2020050273A1
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Prior art keywords
amino acid
glutathione
yeast
acid sequence
seq
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PCT/JP2019/034630
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French (fr)
Japanese (ja)
Inventor
裕一 岩本
晃 岩崎
祐章 加藤
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株式会社カネカ
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Priority to CN201980054912.8A priority Critical patent/CN112639117A/en
Priority to JP2020541239A priority patent/JPWO2020050273A1/en
Publication of WO2020050273A1 publication Critical patent/WO2020050273A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for producing glutathione by yeast, a method for promoting the production of glutathione by yeast, an agent for promoting the production of glutathione by yeast, and a medium composition suitable for the production of glutathione by yeast.
  • Patent Document 14 discloses, as a medium that imparts the same or higher growth ability to yeast as a YPD medium, a saccharide as a carbon source, an amino acid as a nitrogen source, a vitamin, inositol, and zinc.
  • a yeast medium containing ions, potassium ions and magnesium ions and having an inositol concentration of 50 to 100 mg / L is described.
  • the glutathione reductase has the following (1a) to (1e): (1a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, (1b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having a glutathione reductase activity; (1c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having glutathione reductase activity; (1d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 2, and having a glutathione reductas
  • the ⁇ -glutamylcysteine synthase has the following (2a) to (2e): (2a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3, (2b) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in which one or more amino acids have been deleted, substituted, inserted and / or added, and which has ⁇ -glutamylcysteine synthetase activity; (2c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and having ⁇ -glutamylcysteine synthetase activity; (2d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 4, and having a ⁇ -glutamylcysteine synthetase activity; and (2e) one or more bases in the base sequence shown in SEQ
  • the method of the present invention is characterized in that yeast is cultured in a medium in which the concentration of a basic amino acid such as lysine is 0.8 g / L or more.
  • a basic amino acid such as lysine
  • concentration of a basic amino acid such as lysine
  • the concentration of glutathione by the yeast can be promoted, and it is possible to obtain a glutathione-rich yeast (a yeast having a high glutathione content). It is not clear why the presence of a basic amino acid at a concentration of 0.8 g / L or more promotes the production of glutathione by yeast, and achieves a glutathione-rich effect (effect of increasing the glutathione content of yeast).
  • the yeast of the present invention has an oxidized glutathione (GSSG) weight of preferably 20 or more, more preferably 40 or more, more preferably 60 or more, and more preferably 100, when the reduced glutathione (GSH) weight is 100.
  • GSSG oxidized glutathione
  • yeasts containing GSH and GSSG that is, yeasts high in GSSG, are more preferably 120 or more.
  • the GSSG-rich yeast further preferably contains GSH and GSSG such that the GSSG weight is preferably 300 or less, more preferably 200 or less, more preferably 150 or less when the GSH weight is 100.
  • ⁇ ⁇ Increased gene expression can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter.
  • strong promoter is meant a promoter whose gene transcription is improved over the naturally occurring promoter of the wild type.
  • a highly active type of a conventional promoter may be obtained by using various reporter genes.
  • a known high expression promoter for example, a promoter of a gene such as PGK1, PDC1, TDH3, TEF1, HXT7, ADH1, etc. may be used.
  • the substitution with a strong promoter can be used in combination with an increase in the copy number of the gene described below.
  • the vector may further include a control element such as a promoter operably linked to a base sequence encoding the amino acid sequence of the target enzyme.
  • the regulatory element refers to a base sequence having a functional promoter, any relevant transcription elements (for example, enhancer, CCAAT box, TATA box, SPI site, etc.).
  • operably linked means that various regulatory elements such as a promoter and an enhancer that regulate gene expression and a nucleotide sequence encoding an amino acid sequence of a target enzyme are linked in a state where they can be operable in a host cell. To be done. It is well known to those skilled in the art that the type of the regulatory element may vary depending on the host. It is preferable that the vector further contains a base sequence of a selection marker gene.
  • the integration of the vector containing the DNA containing the nucleotide sequence encoding the amino acid sequence of the desired enzyme into the genomic DNA of yeast can be performed, for example, using homologous recombination.
  • multiple copies of a gene can be introduced into genomic DNA by performing homologous recombination on a sequence in which multiple copies are present in the genomic DNA of the yeast chromosome.
  • Sequences in which a large number of copies exist in genomic DNA include an autonomously replicating sequence (ARS) consisting of a unique short repetitive sequence, and an rDNA sequence having about 150 copies.
  • ARS autonomously replicating sequence
  • yeast was transformed using an ARS-containing plasmid is described in WO 95/32289.
  • the gene may be incorporated into a transposon and transferred to introduce multiple copies of the gene into genomic DNA.
  • the following method can be exemplified as an example of a method for obtaining a vector containing a DNA containing a base sequence encoding the amino acid sequence of the target enzyme and transforming the host yeast.
  • DNA-F2 having a base sequence obtained by adding a cleavage sequence of restriction enzyme A to a base sequence homologous to the upstream region of the base sequence encoding the amino acid sequence of the target enzyme in the genomic DNA sequence of the host yeast is synthesized.
  • DNA-R2 having a base sequence obtained by adding a cleavage sequence of restriction enzyme B to a base sequence homologous to a complementary base sequence in a downstream region of the base sequence is synthesized.
  • PCR amplification was performed using the genomic DNA of the host yeast as a template and DNA-F2 and DNA-R2 as primers, and the amplified DNA was cut with restriction enzymes A and B.
  • a recombinant vector is prepared by ligating to a vector having a selectable marker cleaved with restriction enzyme B, and the recombinant vector is introduced into host yeast to obtain a transformant.
  • primer design and experimental operations are performed based on manuals such as In-Fusion Cloning kit (manufactured by Takara Bio Inc.), Gibson Assembly System, and NEBuilder (manufactured by New England Biolabs) to encode the amino acid sequence of the target enzyme.
  • ⁇ Confirmation that the transcription amount of the gene encoding the target enzyme has increased can be performed by comparing the amount of mRNA transcribed from the gene with that of the parent strain.
  • Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). It is preferable that the amount of mRNA is increased, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to the parent strain.
  • the modification that reduces the enzyme activity can be achieved, for example, by reducing the expression of the gene encoding the target enzyme.
  • the gene encoding the enzyme of interest is typically contained in the genomic DNA of the host yeast.
  • the gene encoding the target enzyme refers to a gene containing a base sequence encoding the amino acid sequence of the target enzyme, and unless otherwise limited, not only the coding region of the amino acid sequence but also its expression control sequence (promoter sequence, etc.) , Exon sequence, intron sequence and the like are shown without distinction.
  • the expression control sequence is modified, the expression control sequence is preferably modified at one or more bases, more preferably at least two bases, particularly preferably at least three bases.
  • the entire gene may be deleted, including the sequence before and after the gene in the genomic DNA of the host yeast.
  • any region such as the N-terminal region, internal region, or C-terminal region can be deleted as long as the reduction in enzyme activity can be achieved. Good. Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. In addition, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame.
  • deletion of a gene encoding the amino acid sequence of the target enzyme such that the enzyme activity is reduced include amino acid substitution (missense mutation) in the coding region of the gene encoding the target enzyme on genomic DNA. , A stop codon (nonsense mutation), or a frameshift mutation that adds or deletes one or two bases.
  • Deletion of a gene on genomic DNA as described above can be performed, for example, by preparing an inactive gene in which the gene encoding the amino acid sequence of the target enzyme is modified so as not to produce a protein that functions normally, and Achieved by transforming yeast with a recombinant DNA containing an active gene and causing homologous recombination between the inactive gene and the gene on the genomic DNA, thereby replacing the gene on the genomic DNA with the inactive gene. it can.
  • the marker DNA is included in the recombinant DNA according to the trait such as auxotrophy of the host, the operation is easy.
  • the recombinant DNA is made linear by cutting with a restriction enzyme or the like, a strain in which the recombinant DNA has been incorporated into genomic DNA can be efficiently obtained.
  • the protein encoded by the inactive gene if produced, has a different steric structure than the wild-type protein and has reduced or lost function.
  • a linear DNA containing an arbitrary sequence and both ends of the arbitrary sequence may have a site to be replaced on genomic DNA (typically, a part or all of a gene encoding a target enzyme).
  • the yeast is transformed with the linear DNA having the upstream and downstream sequences, and homologous recombination is caused upstream and downstream of the site to be replaced, whereby the site to be replaced is replaced with an arbitrary sequence in one step. be able to.
  • a sequence containing a marker gene may be used as the arbitrary sequence.
  • the marker gene may then be removed if necessary. When the marker gene is removed, a sequence for homologous recombination may be added to both ends of the marker gene so that the marker gene can be removed efficiently.
  • ⁇ Confirmation of a decrease in the amount of transcription of the gene encoding the target enzyme can be performed by comparing the amount of mRNA transcribed from the gene with that of the parent strain.
  • Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)).
  • the amount of mRNA is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the parent strain.
  • the yeast of the present invention may be modified with a wild-type gene and a gene modified to reduce the enzyme activity as long as a ⁇ -glutamyl compound such as glutathione can be accumulated.
  • the gene may be heterozygous, but is usually preferably a homozygous gene modified so as to reduce the enzyme activity.
  • ⁇ Yeast in which glutathione reductase activity is suppressed has a low activity of converting GSSG to GSH, and thus easily accumulates GSSG.
  • Such a yeast in which glutathione reductase activity is suppressed is particularly preferably, when the GSH weight is 100, the GSSG weight is preferably 20 or more, more preferably 40 or more, more preferably 60 or more, and more preferably 100 or more.
  • the yeast in which glutathione reductase activity is suppressed is more preferably, when GSH weight is 100, GSSG weight is preferably 300 or less, more preferably 200 or less, and more preferably 150 or less. contains.
  • the sequence identity with the amino acid sequence shown in SEQ ID NO: 1 is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, still more preferably 85% or more, and 90% or more. Is more preferably 95% or more, still more preferably 97% or more, further preferably 98% or more, and most preferably 99% or more.
  • the amino acid sequence identity can be determined by comparing the amino acid sequence shown in SEQ ID NO: 1 or 2 with the amino acid sequence to be evaluated, dividing the number of positions where amino acids match in both sequences by the total number of amino acids to be compared, and It is represented by a value multiplied by 100.
  • sequence identity to the DNA shown in SEQ ID NO: 2 is 70% or more, preferably 74% or more, more preferably 79% or more, and still more preferably 85% or more. Even more preferably, 90% or more, even more preferably, 95% or more, even more preferably, 97% or more, even more preferably, 98% or more, and most preferably, 99% or more of DNA.
  • the nucleotide sequence modified by substitution, insertion, deletion and / or addition may include only one type of modification (for example, substitution) or two or more types of modification (for example, substitution and insertion). May be included.
  • the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has glutathione reductase activity, but for example, 1 to 150, preferably 1 to 100, Preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases. .
  • the proteins (1a) to (1e) are proteins having glutathione reductase activity
  • a transformant expressing the protein whose activity is to be confirmed using a DNA recombination method is prepared. After producing the protein using the transformant, the protein, oxidized glutathione, and NADPH are allowed to exist in an aqueous medium, and it is determined whether reduced glutathione or NADP is produced and accumulated in the aqueous medium.
  • a method of analyzing by HPLC or the like can be mentioned.
  • the glutathione reductase activity is reduced to preferably 50% or less, more preferably 20% or less, further preferably 10% or less, and particularly preferably 5% or less, as compared with the parent strain. It is particularly preferable that the glutathione reductase activity is substantially eliminated.
  • the ⁇ -glutamylcysteine synthase in the present invention is an enzyme having an activity of synthesizing ⁇ -glutamylcysteine by condensing glutamic acid and cysteine (ie, ⁇ -glutamylcysteine synthase activity).
  • sequence identity with the amino acid sequence shown in SEQ ID NO: 3 is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, further preferably 85% or more, and 90%
  • the above is still more preferable, 95% or more is more preferable, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is most preferable.
  • the sequence identity of the amino acid sequence can be calculated by the method described above in (1).
  • a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 4 and a DNA hybridizing under stringent conditions are a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 4 And DNA obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like under stringent conditions using the resulting DNA as a probe.
  • Hybridization conditions and the like are as described above in (1).
  • DNA in which one or more bases have been substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 4 can be prepared according to the method described above in (1). it can.
  • the nucleotide sequence modified by substitution, insertion, deletion and / or addition may include only one type of modification (for example, substitution) or two or more types of modification (for example, substitution and insertion). May be included.
  • the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has ⁇ -glutamylcysteine synthetase activity, and for example, 1 to 150, preferably 1 to 100 , More preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 1, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases, Means
  • Means for confirming that the proteins (2a) to (2e) are proteins having ⁇ -glutamylcysteine synthetase activity include, for example, a transformant expressing a protein whose activity is to be confirmed by DNA recombination. And producing the protein using the transformant. Then, the protein, L-glutamic acid and L-cysteine are allowed to exist in an aqueous medium, and ⁇ -glutamylcysteine is produced and accumulated in the aqueous medium. A method of analyzing whether or not to do so by HPLC or the like can be mentioned. It is preferable that ATP is further present in the aqueous medium as needed.
  • the protein of any of the above (3a) to (3e) is not limited to the form consisting of only the polypeptide chain consisting of the amino acid sequence defined in the above (3a) to (3e), May be in the form chemically modified with a sugar chain or the like, or may be in the form of a fusion protein in which the polypeptide chain is fused with another polypeptide chain.
  • a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, inserted, deleted and / or added is the protein described in (1) above. It can be prepared according to the method, and is included in the above proteins as long as it has glutathione synthase activity.
  • the “one or more” amino acids are, for example, 1 to 60, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, and still more preferably One to five, one to four, one to three, or one to two amino acids.
  • a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 6 is obtained from the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 6
  • DNA obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like under stringent conditions using the resulting DNA as a probe Hybridization conditions and the like are as described above in (1).
  • sequence identity to the DNA shown in SEQ ID NO: 6 is 70% or more, preferably 74% or more, more preferably 79% or more, and still more preferably 85% or more. Even more preferably, 90% or more, even more preferably, 95% or more, even more preferably, 97% or more, even more preferably, 98% or more, and most preferably, 99% or more of DNA.
  • DNA sequence identity (%) is as described above in (1).
  • the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has glutathione synthase activity, and for example, 1 to 150, preferably 1 to 100, Preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases. .
  • the proteins (3a) to (3e) are proteins having glutathione synthase activity
  • a transformant expressing the protein whose activity is to be confirmed using a DNA recombination method is prepared.
  • the protein and ⁇ -glutamylcysteine and glycine are allowed to exist in an aqueous medium, and whether or not glutathione is produced and accumulated in the aqueous medium is determined by HPLC or the like. Methods for analysis can be mentioned. It is preferable that ATP is further present in the aqueous medium as needed.
  • GSH2 having the amino acid sequence shown in SEQ ID NO: 5 is preferable as the glutathione synthetase in the present invention.
  • SEQ ID NO: 6 shows the nucleotide sequence of the GSH2 gene encoding the amino acid sequence of GSH2 shown in SEQ ID NO: 5.
  • the glutathione synthetase activity of the protein of (3b), (3c), (3d) or (3e) is preferably about the same or higher than the glutathione synthase activity of the protein of (3a), and more preferably. Is 50% or more, 80% or more, 90% or more, or 100% or more, more preferably 200% or less or 150% or less of the glutathione synthase activity of the protein of (3a).
  • the glutathione transfer enzyme means an enzyme having a function of transferring cytosolic glutathione to the vacuole (that is, glutathione transfer enzyme activity), and is not particularly limited as long as it has the function.
  • Glutathione transferases include the following (4a) to (4e): (4a) a protein consisting of the amino acid sequence of SEQ ID NO: 7, (4b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 7, and which has a glutathione transferase activity; (4c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and having a glutathione transferase activity; (4d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 8, and having a glutathione transferase activity; and (4e) a protein comprising an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO:
  • the protein of any of the above (4a) to (4e) is not limited to the form consisting of only the polypeptide chain consisting of the amino acid sequence defined in the above (4a) to (4e), May be in the form chemically modified with a sugar chain or the like, or may be in the form of a fusion protein in which the polypeptide chain is fused with another polypeptide chain.
  • a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, inserted, deleted and / or added is the protein described in (1) above. It can be prepared according to the method, and is included in the above proteins as long as it has glutathione transferase activity.
  • the amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one kind of modification (for example, substitution) or may contain two or more kinds of modification (for example, substitution and insertion). May be included.
  • the amino acid to be substituted is preferably an amino acid having similar properties to the amino acid before substitution (homologous amino acid).
  • the homologous amino acids are as described above in (1).
  • amino acids refer to, for example, 1 to 60, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, and still more preferably One to five, one to four, one to three, or one to two amino acids.
  • sequence identity with the amino acid sequence shown in SEQ ID NO: 7 is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, further preferably 85% or more, and 90%
  • the above is still more preferable, 95% or more is more preferable, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is most preferable.
  • the sequence identity of the amino acid sequence can be calculated by the method described above in (1).
  • a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8 and a DNA hybridizing under stringent conditions are a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8 And DNA obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like under stringent conditions using the resulting DNA as a probe. Hybridization conditions and the like are as described above in (1).
  • the DNA capable of hybridizing under the above conditions has a sequence identity with the DNA shown in SEQ ID NO: 8 of 70% or more, preferably 74% or more, more preferably 79% or more, and still more preferably 85% or more. Even more preferably, 90% or more, even more preferably, 95% or more, even more preferably, 97% or more, even more preferably, 98% or more, and most preferably, 99% or more of DNA.
  • DNA sequence identity (%) is as described above in (1).
  • the DNA in which one or more bases have been substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 8 can be prepared according to the method described above in (1). it can.
  • the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has glutathione transferase activity, but for example, 1 to 150, preferably 1 to 100, Preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases. .
  • YCF1 having the amino acid sequence shown in SEQ ID NO: 7 is preferable.
  • SEQ ID NO: 8 shows the base sequence of the YCF1 gene encoding the amino acid sequence of YCF1 shown in SEQ ID NO: 7.
  • the glutathione transferase activity of the protein of the above (4b), (4c), (4d) or (4e) is preferably equal to or more than the glutathione transferase activity of the protein of the above (4a), and more preferably. Is 50% or more, 80% or more, 90% or more, or 100% or more, more preferably 200% or less, or 150% or less of the glutathione transferase activity of the protein (4a).
  • yeast of the present invention is not particularly limited as long as it has a glutathione-producing ability.
  • Saccharomyces cerevisiae 24-51-78 (accession number: FERM BP-19072) can be suitably used as a parent strain. Saccharomyces cerevisiae 24-51-78 was deposited internationally on October 18, 2002 with the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Patent Organism Depositary Center (Room 2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba, Japan). (Indication by the depositor for identification: Saccharomyces cerevisiae 24-51-78, accession number: FERM BP-19072).
  • the “basic amino acid” is typically one or more selected from lysine, arginine and histidine, more preferably selected from lysine and arginine. And most preferably lysine.
  • the basic amino acid is preferably in the L-form.
  • the medium used in the present invention has a basic amino acid concentration of 0.8 g / L or more, preferably 1 g / L or more, more preferably 2 g / L or more, and particularly preferably 4 g / L or more.
  • the total concentration of the basic amino acids may be within the above range. More preferably, the concentration of each basic amino acid is at least 0.8 g / L, and more preferably Is 1 g / L or more, more preferably 2 g / L or more, and particularly preferably 4 g / L or more.
  • the basic amino acid may be present in the form of a salt.
  • the medium used in the present invention may be any medium containing a basic amino acid, and other components are not particularly limited. That is, any of a synthetic medium, a semi-synthetic medium, and a natural (composite) medium can be used as long as the medium appropriately contains a carbon source, a nitrogen source, inorganic substances, and other nutrients.
  • carbohydrate raw materials such as molasses, glucose, glycerol, fructose, sucrose, maltose, mannose, mannitol, xylose, galactose, starch, and starch hydrolyzate are used as the carbon source contained in the medium containing basic amino acids. It can.
  • Various organic acids such as pyruvic acid, acetic acid, and lactic acid, and various amino acids such as aspartic acid and alanine can also be used.
  • a particularly preferred embodiment of the medium containing basic amino acids comprises molasses as a carbon source.
  • Molasses is a viscous, black-brown liquid by-product containing sugar as a main component, which is generated when sugar is purified from raw materials such as sugarcane and sugar beet.
  • the medium containing a basic amino acid is more preferably a liquid medium containing molasses in an amount of 1% by weight or more, more preferably 2% by weight or more, more preferably 3% by weight or more as sugar contained in the molasses.
  • the amount of air supplied to the culture medium during aerobic culture is preferably 0.2 L / min or more, more preferably 0.5 L / min or more, and still more preferably 1 L / min or more, per 1 L of the culture medium.
  • the stirring speed is preferably 200 rpm or more, more preferably 300 rpm or more, and still more preferably 400 rpm or more.
  • the capacity of the jar fermenter is not particularly limited. For example, a jar fermenter having a total capacity of 2 L can be exemplified.
  • the temperature during the culturing is usually 20 to 45 ° C., preferably 25 to 35 ° C., and most preferably 28 to 32 ° C.
  • the culturing period is usually 16 hours to 72 hours, preferably 24 hours to 48 hours.
  • the initial cell concentration (prepared concentration) in the medium varies depending on the type of yeast, medium composition, etc., but the initial turbidity (OD600) is preferably 0.01 to 2.0, more preferably 0.02 to 1.0. And more preferably 0.1 to 0.4.
  • the carbon source such as glucose
  • any of a batch system in which culturing is carried out by batch charging at the beginning of the culture, and a semi-batch system in which the mixture is added little by little throughout the culture period can be applied, but according to the results of the study by the present inventors.
  • the semi-batch method is more preferable because the growth rate is improved, the final bacterial concentration is higher, and the activity of the glutathione synthesis-related enzyme in the bacterial body is also higher.
  • a carbon source such as glucose in a semi-batch system
  • the turbidity of the culture solution, the rate of oxygen consumption, the rate of carbon dioxide generation, the consumption of a neutralizing agent for pH adjustment, and the like can be used.
  • a culture mixture consisting of a yeast containing a high concentration of glutathione mainly in the cells and a medium can be obtained.
  • the method for producing glutathione of the present invention preferably further includes recovering glutathione from the obtained culture mixture.
  • yeast cells in the culture mixture are separated from the medium by filtration or centrifugation, and the resulting yeast cells are subjected to hot water extraction, alkali extraction, Glutathione can be extracted by appropriately combining the method, autolysis method, physical crushing operation, and the like as necessary.
  • the above-mentioned extraction operation may be directly performed on the culture mixture after the culture, or the enzymatic decomposition method, the autolysis method, and the physical crushing operation are performed to convert glutathione in the yeast cells into a medium.
  • glutathione may be extracted by performing the above extraction operation.
  • Glutathione is purified from the thus obtained glutathione by a general method to obtain a fraction containing glutathione at a high level or a glutathione powder.
  • the enhanced expression of GSH1 and GSH2 described in the non-patent document can be achieved by operably linking multiple copies of the GSH1 gene and the strong expression promoter operably linked to the strong expression promoter to genomic DNA of the host yeast, respectively. This is due to the incorporation of the GSH2 gene. Furthermore, PCR amplification was performed using 5′-AAAAGGATCCATGGCTGGTAATCTTGTTTCATGGGCC-3 ′ (SEQ ID NO: 11) and 5′-AAAACTCGAGTTAATTTTCATTGACCAAACCAGCCTCC-3 ′ (SEQ ID NO: 12) using genomic DNA of Saccharomyces cerevisiae strain YPH499 as a template and primers as primers.
  • cultivation was performed as a control with 50 ml of molasses medium (4% of molasses (as glucose in molasses), 0.3% of urea, 0.08% of ammonium sulfate, 2% of phosphoric acid and 0.04% of ammonium). .
  • Glutathione in the cells was eluted by washing the cells collected by centrifugation of 1 ml of the culture solution twice with sterilized water and heat-treating at 80 ° C. for 5 minutes. The eluate was centrifuged at 20,000 ⁇ g for 5 minutes at 25 ° C., and the supernatant was analyzed for glutathione concentration by HPLC to determine the amount of reduced glutathione (GSH) and the amount of oxidized glutathione (GSSG) per 1 ml of the culture solution. did. Similarly, 1 ml of the culture solution is washed twice with sterile water, and allowed to stand in a desiccator at 80 ° C.
  • the GSH content (%) and the GSSG content (%) were calculated by dividing the GSH content and the GSSG content by the dry cell weight, respectively.
  • the GSH content (%) in the control test using the molasses medium without adding lysine was set to 100%, and the GSSG content (%) in the control test using the molasses medium and the molasses lysine medium were used.
  • the relative values of the GSH content (%) and the GSSG content (%) in the test of Example 1 were determined. The results are shown in the following table.
  • the GSH content and GSSG content can be dramatically increased by increasing the lysine concentration in the medium.
  • Example 2 As the yeast, the GLR1 disruption + YCF1 enhancement + GSH1 enhancement + GSH2 enhancement strain described in Production Example 2 was prepared using a YPD medium (10 g / L yeast extract (manufactured by Difco laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g / Seed culture was carried out by shaking with 7 ml of L glucose (manufactured by Nacalai Tesque, Inc.) at 30 ° C. for 16 to 24 hours.
  • a YPD medium 10 g / L yeast extract (manufactured by Difco laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g / Seed culture was carried out by shaking with 7 ml of L glucose (manufactured by Nacalai Tesque, Inc.) at 30 ° C. for 16 to 24 hours.
  • molasses arginine medium 50 ml (molasses 4% (as glucose in molasses), urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%, arginine 0.1%)
  • the Sakaguchi flask was inoculated so as to have a seed culture solution of 3 ml, cultured at 30 ° C. under agitation at 130 rpm for 40 hours, and 1 ml of the culture solution was collected.
  • molasses medium molasses (as the amount of glucose in molasses) 4%, urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%
  • Glutathione in the cells was eluted by washing the cells collected by centrifugation of 1 ml of the culture solution twice with sterilized water and heat-treating at 80 ° C. for 5 minutes. The eluate was centrifuged at 20,000 ⁇ g for 5 minutes at 25 ° C., and the glutathione concentration of the supernatant was analyzed by HPLC to determine the amount of glutathione per 1 ml of the culture solution (the total amount of GSH and GSSG). Similarly, 1 ml of the culture solution is washed twice with sterile water, and allowed to stand in a desiccator at 80 ° C. overnight to obtain a dry cell weight per 1 ml of the culture solution.
  • Glutathione content was calculated by dividing the amount of glutathione by the dry cell weight.
  • the relative value of the glutathione content (%) in the test of Example 2 using the molasses arginine medium was determined, assuming that the glutathione content (%) in the comparative control test using the molasses medium without adding arginine was 100%. .
  • the results are shown in the following table.
  • Example 3 In Example 2, in place of the molasses arginine medium, molasses histidine medium (50 ml, molasses 4% (as the amount of glucose in molasses), urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%) Histidine 0.1%), except that GLR1 disruption + YCF1 + GSH1 + GSH2-enriched strain prepared in Production Example 2 was used in a molasses histidine medium and a molasses medium without histidine under the same conditions and procedure as in Example 2. And cultured, and the glutathione content (%) was measured.
  • Example 2 the glutathione content (%) in the comparative control test using the molasses medium without adding histidine was set to 100%, and the glutathione content (%) in the test of Example 3 using the molasses histidine medium was determined. ) was determined.
  • Saccharomyces cerevisiae 24-51-78 (accession number: FERM BP-19072) was used as a yeast in YPD medium (10 g / L yeast extract (manufactured by Difco laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g / Seed culture was carried out by shaking with 7 ml of L glucose (manufactured by Nacalai Tesque, Inc.) at 30 ° C. for 16 to 24 hours.
  • YPD medium 10 g / L yeast extract (manufactured by Difco laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g / Seed culture was carried out by shaking with 7 ml of L glucose (manufactured by Nacalai Tesque, Inc.) at 30 ° C. for 16 to 24 hours.
  • molasses 50 ml molasses 4% (as glucose in molasses), urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%, lysine 0.1%), molasses Arginine medium (having the same composition as molasses lysine medium except that it contains 0.1% arginine instead of 0.1% lysine), or molasses histidine medium (0.1% histidine instead of 0.1% lysine) Is inoculated in a Sakaguchi flask containing 3 mol of a seed mother culture solution, and cultured at 30 ° C.
  • molasses medium molasses (as the amount of glucose in molasses) 4%, urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%
  • Example 2 ⁇ ⁇ Glutathione content (%) of the culture in each medium was measured by the procedure described in Example 2. As in Example 2, the content of glutathione (%) in the comparative control test using a molasses medium without adding lysine, arginine and histidine was 100%, and a molasses lysine medium, a molasses arginine medium or a molasses histidine medium was used. The relative value of the glutathione content (%) in the test of Example 4 was determined.
  • Saccharomyces cerevisiae 24-51-78 accesiae 24-51-78 (accession number: FERM BP-19072) is cultured in a medium to which a basic amino acid has been added, the glutathione content of the yeast can be dramatically increased.

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Abstract

The purpose of the present invention is to provide a method for economically and efficiently producing glutathione using a yeast. Provided are a method for producing glutathione, said method being characterized by comprising culturing a yeast in a medium having a basic amino acid concentration of 0.8 g/L or higher, and a method for promoting the production of glutathione by a yeast.

Description

グルタチオンの製造方法Glutathione production method
　本発明は、酵母によるグルタチオンの製造方法、酵母によるグルタチオンの生産を促進する方法、酵母によるグルタチオンの生産の促進剤、および、酵母によるグルタチオンの生産に適した培地組成物に関する。 The present invention relates to a method for producing glutathione by yeast, a method for promoting the production of glutathione by yeast, an agent for promoting the production of glutathione by yeast, and a medium composition suitable for the production of glutathione by yeast.
　ビール酵母やパン酵母を始めとするサッカロマイセス（Ｓａｃｃｈａｒｏｍｙｃｅｓ）属菌に属する酵母は、天然のビタミンＢ群、アミノ酸、およびミネラル等をバランス良く含有しており、ビールやパンの製造に使われる以外にも有効活用されている。例えば、乾燥酵母は我が国において長年にわたって医薬品、食品原料、および調味料などとして使われており、栄養価と安全性の高い素材として認知されている。また、近年は酵母エキスの原料酵母としても広く用いられている。  Yeasts belonging to the genus Saccharomyces such as brewer's yeast and baker's yeast contain natural vitamins B, amino acids, and minerals in a well-balanced manner. It is being used effectively. For example, dried yeast has been used in Japan for many years as pharmaceuticals, food materials, seasonings, and the like, and is recognized as a material with high nutritional value and safety. In recent years, it has been widely used as a raw material yeast for yeast extract.
　酵母エキスとは、酵母の培養物から調製され、アミノ酸等を豊富に含むものであり、従来から、旨味やコクを付与するための調味料等のような食品添加剤として使用されている。特に昨今の天然志向の高まりから、調味料としての酵母エキスの需要は増加傾向にある。呈味成分を豊富に含む酵母から調製された酵母エキスは、より優れた調味料として使用し得ることが期待できるため、呈味成分をより多く含む酵母の開発が盛んに行われている。 Yeast extract is prepared from a culture of yeast and contains abundant amino acids and the like, and has conventionally been used as a food additive such as a seasoning for imparting umami or richness. In particular, the demand for yeast extract as a seasoning has been on an increasing trend due to the recent increase in natural consciousness. Since a yeast extract prepared from a yeast rich in taste components can be expected to be used as a better seasoning, yeasts containing more taste components are being actively developed.
　酵母菌体内の代表的な含硫化合物として、グルタチオンとＳ－アデノシルメチオニンがあげられる。一般にグルタチオンは酵母および動物の肝臓等に広く分布しており、生体内の酸化還元反応に深く関与しているトリペプチドであり、肝機能回復作用や解毒作用さらには活性酸素の消去による細胞の老化を防ぐ作用などの重要な役割を果たすものとして、極めて有用な物質である。グルタチオンにはその形態として還元型グルタチオン（以下、ＧＳＨと略称する。）と、２つの還元型グルタチオンがシステイン残基でジスルフィド結合した酸化型グルタチオン（以下、ＧＳＳＧと略称する。）が存在する。両者とも生体内で生合成されており、酸化還元反応において重要な働きをしている。ＧＳＳＧは生体内ではグルタチオン還元酵素により一旦ＧＳＨになり、リサイクルしながら働いている。従って、ＧＳＳＧを投与した場合はＧＳＨと同様の作用が見込まれるといわれている（例えば、ジャーナルオブ　ニュートリショナル　サイエンス　アンド　ビタミノロジー　４４巻、６１３ページ、１９９８年、等）。 Glutathione and S-adenosylmethionine are typical sulfur-containing compounds in yeast cells. In general, glutathione is widely distributed in yeast and animal liver, etc., and is a tripeptide that is deeply involved in the redox reaction in the living body. It is an extremely useful substance as it plays an important role in preventing odors. Glutathione includes reduced glutathione (hereinafter abbreviated as GSH) and oxidized glutathione (hereinafter abbreviated as GSSG) in which two reduced glutathiones are disulfide-bonded with cysteine residues. Both are biosynthesized in vivo and play important roles in redox reactions. GSSG is once converted into GSH in vivo by glutathione reductase and works while being recycled. Therefore, it is said that the same action as GSH is expected when GSSG is administered (for example, Journal of Nutritional Science and Vitaminology, Vol. 44, p. 613, 1998, etc.).
　含硫化合物は、通常、メチオニンやシステイン等の含硫アミノ酸を用いて、ＭＥＴ遺伝子（メチオニン合成遺伝子）群をはじめとする多くの遺伝子の転写および翻訳産物により合成される。そこで、より含硫化合物を高生産する酵母を得るために、酵母が有しているこれらの含硫化合物の合成に係る遺伝子に変異を生じさせ、含硫化合物高含有酵母変異株を製造することが広く行われている。例えば、グルタチオン高含有酵母を製造する方法として、グルタチオン生産に使用する酵母への突然変異処理（特許文献１～３）やグルタチオンの合成に関与する酵素を遺伝子組換えにより導入すること（特許文献４～８）、また、培地中にグルタチオンを構成する３種のアミノ酸であるＬ－グルタミン酸、Ｌ－システイン、グリシンを添加すること（特許文献９～１２）により、培養菌体内のグルタチオン含量を向上させ生産性を改善させる試みがなされてきた。 硫 Sulfur-containing compounds are usually synthesized from the transcription and translation products of many genes including the MET gene (methionine synthesis gene) using sulfur-containing amino acids such as methionine and cysteine. Therefore, in order to obtain a yeast that produces a sulfur-containing compound with higher production, it is necessary to produce a mutation in a yeast-containing gene relating to the synthesis of these sulfur-containing compounds to produce a mutant strain containing a sulfur-containing compound-rich yeast. Is widely practiced. For example, as a method for producing a glutathione-rich yeast, a yeast used for glutathione production is subjected to mutation treatment (Patent Documents 1 to 3) and an enzyme involved in glutathione synthesis is introduced by genetic recombination (Patent Document 4). 8) In addition, by adding three kinds of amino acids constituting glutathione, L-glutamic acid, L-cysteine, and glycine to the medium (Patent Documents 9 to 12), the glutathione content in the cultured cells can be improved. Attempts have been made to improve productivity.
　更に、特許文献１３には、キャンディダ属あるいはブレタノマイセス属に属するメチオニンとＬ－リジン要求性の微生物を、メチオニンとリジンを含有する培地で培養することを特徴とする、発酵法によるグルタチオンの製造方法が開示されている。特許文献１３では、メチオニンとリジンを含有する培地として、メチオニンを２００～５００μｇ／ｍＬ、および、Ｌ－リジンを３００～７５０μｇ／ｍＬ含有する培地が記載されている。 Furthermore, Patent Document 13 discloses a method for producing glutathione by a fermentation method, comprising culturing a methionine- and L-lysine-requiring microorganism belonging to the genus Candida or Brettanomyces in a medium containing methionine and lysine. Is disclosed. Patent Document 13 describes, as a medium containing methionine and lysine, a medium containing 200 to 500 μg / mL of methionine and 300 to 750 μg / mL of L-lysine.
　一方、特許文献１４には、ＹＰＤ培地と同程度又はそれ以上の増殖能力を酵母に付与する培地として、炭素源としての糖類と、窒素源としてのアミノ酸類と、ビタミン類と、イノシトールと、亜鉛イオンと、カリウムイオンと、マグネシウムイオンが含まれ、かつ、イノシトール濃度が５０～１００ｍｇ／Ｌである酵母用培地が記載されている。 On the other hand, Patent Document 14 discloses, as a medium that imparts the same or higher growth ability to yeast as a YPD medium, a saccharide as a carbon source, an amino acid as a nitrogen source, a vitamin, inositol, and zinc. A yeast medium containing ions, potassium ions and magnesium ions and having an inositol concentration of 50 to 100 mg / L is described.
特開昭５９－１５１８９４号公報JP-A-59-151894 特公平０３－１８８７２号公報Japanese Patent Publication No. 03-18882 特開平１０－１９１９６３号公報JP-A-10-191963 特開昭６１－５２２９９号公報JP-A-61-52299 特開昭６２－２７５６８５号公報JP-A-62-275885 特開昭６３－１２９９８５号公報JP-A-63-129985 特開昭６４－５１０９８号公報JP-A-64-51098 特開平４－１７９４８４号公報JP-A-4-179484 特開昭４７－１６９９０号公報JP-A-47-16990 特開昭４８－９２５７９号公報JP-A-48-92579 特開昭５１－１３９６８６号公報JP-A-51-139686 特開昭５３－９４０８９号公報JP-A-53-94089 特開昭４８－６１６８９号公報JP-A-48-61689 ＷＯ２０１４／０３０７７４WO2014 / 030774
　グルタチオン自体やグルタチオンを豊富に含む酵母エキス等を、より低コストで効率よく工業上量産するためには、グルタチオン含有量の高い酵母を利用することが重要である。特許文献１に記載の方法のように、変異処理等を行い遺伝的な改変が施された酵母の中から、よりグルタチオン含有量の高い酵母をスクリーニングする方法によっても、グルタチオン高含有酵母を得ることができるが、変異処理およびスクリーニングは手間と労力を要し、また、必ずしもグルタチオン高含有酵母を得ることができない場合も多い。 In order to efficiently and industrially mass-produce glutathione itself or a yeast extract rich in glutathione at lower cost, it is important to use a yeast having a high glutathione content. As in the method described in Patent Literature 1, a method for screening a yeast having a higher glutathione content from yeasts genetically modified by performing mutation treatment or the like to obtain a glutathione-rich yeast. However, mutagenesis and screening require labor and effort, and in many cases, a glutathione-rich yeast cannot always be obtained.
　本発明は、酵母によるグルタチオンの生産性を高めるための安価で効率的な手段を提供する。 The present invention provides an inexpensive and efficient means for increasing the productivity of glutathione by yeast.
　本発明者らは、上記課題を解決すべく鋭意研究した結果、サッカロマイセス属酵母等の酵母を培養する際に、培地に所定量以上のリジン等の塩基性アミノ酸を添加することにより、当該酵母のグルタチオン含有量が高められ、グルタチオンを効率よく生産できることを見出し、本発明を完成させた。 The present inventors have conducted intensive studies to solve the above problems, and as a result, when culturing yeast such as Saccharomyces yeast, by adding a predetermined amount or more of a basic amino acid such as lysine to the medium, the yeast The inventors have found that the content of glutathione is increased and that glutathione can be produced efficiently, thereby completing the present invention.
　すなわち、本発明は以下の発明を包含する。
［１］酵母を、塩基性アミノ酸濃度が０．８ｇ／Ｌ以上である培地中で培養することを特徴とする、グルタチオンの製造方法。
[２]前記酵母が、還元型グルタチオン重量を１００としたとき、酸化型グルタチオン重量が２０以上となるように、還元型グルタチオンおよび酸化型グルタチオンを含有する酵母である、［１］に記載の方法。
［３］前記酵母が、親株と比較してグルタチオンレダクターゼの活性が低下した酵母である、［１］又は［２］に記載の方法。
［４］前記酵母が、グルタチオンレダクターゼをコードする遺伝子が欠損した酵母である、［１］～［３］のいずれかに記載の方法。
［５］前記グルタチオンレダクターゼが下記（１ａ）～（１ｅ）：
（１ａ）配列番号１に示すアミノ酸配列からなるタンパク質、
（１ｂ）配列番号１に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
（１ｃ）配列番号１に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
（１ｄ）配列番号２と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、および、
（１ｅ）配列番号２に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
からなる群から選択される、［３］または［４］に記載の方法。
［６］前記酵母が、親株と比較してγ－グルタミルシステイン合成酵素および／またはグルタチオン合成酵素の活性が増大した酵母である、［１］～［５］のいずれかに記載の方法。
［７］前記酵母が、γ－グルタミルシステイン合成酵素をコードする塩基配列を含むＤＮＡ、および／またはグルタチオン合成酵素をコードする塩基配列を含むＤＮＡにより形質転換された酵母である、［１］～［６］のいずれかに記載の方法。
［８］前記γ－グルタミルシステイン合成酵素が下記（２ａ）～（２ｅ）：
（２ａ）配列番号３に示すアミノ酸配列からなるタンパク質、
（２ｂ）配列番号３に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
（２ｃ）配列番号３に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
（２ｄ）配列番号４と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、および、
（２ｅ）配列番号４に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
からなる群から選択される、［６］または［７］に記載の方法。
［９］前記グルタチオン合成酵素が下記（３ａ）～（３ｅ）：
（３ａ）配列番号５に示すアミノ酸配列からなるタンパク質、
（３ｂ）配列番号５に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
（３ｃ）配列番号５に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
（３ｄ）配列番号６と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、および、
（３ｅ）配列番号６に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
からなる群から選択される、［６］または［７］に記載の方法。
［１０］前記酵母が、親株と比較してグルタチオン輸送酵素の活性が増大した酵母である、［１］～［９］のいずれかに記載の方法。
［１１］前記酵母が、グルタチオン輸送酵素をコードする塩基配列を含むＤＮＡにより形質転換された酵母である、［１］～［１０］のいずれかに記載の方法。
［１２］前記グルタチオン輸送酵素が下記（４ａ）～（４ｅ）：
（４ａ）配列番号７に示すアミノ酸配列からなるタンパク質、
（４ｂ）配列番号７に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
（４ｃ）配列番号７に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
（４ｄ）配列番号８と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、および、
（４ｅ）配列番号８に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
からなる群から選択される、［１０］または［１１］に記載の方法。
［１３］前記酵母が、サッカロマイセス属、キャンディダ属、またはピキア属の酵母である、［１］～［１２］のいずれかに記載の方法。
［１４］前記酵母が、塩基性アミノ酸非要求性の酵母である、［１］～［１３］のいずれかに記載の方法。
［１５］前記培地が炭素源として糖蜜を含む、［１］～［１４］のいずれかに記載の方法。
［１６］培養開始時において、前記培地中の塩基性アミノ酸濃度が０．８ｇ／Ｌ以上である、［１］～［１５］のいずれかに記載の方法。
［１７］前記培地中の塩基性アミノ酸濃度が２ｇ／Ｌ以上である、［１］～［１６］のいずれかに記載の方法。
［１８］前記培地中の塩基性アミノ酸濃度が４ｇ／Ｌ以上である、［１］～［１６］のいずれかに記載の方法。
［１９］前記塩基性アミノ酸がリジンである、［１］～［１８］のいずれかに記載の方法。
［２０］酵母を、塩基性アミノ酸濃度が０．８ｇ／Ｌ以上である培地中で培養することを特徴とする、酵母によるグルタチオンの生産を促進する方法。
［２１］前記酵母が、［２］～［１４］のいずれかで規定する特徴を有する酵母である、［２０］に記載の方法。
［２２］前記培地が炭素源として糖蜜を含む、［２０］又は［２１］に記載の方法。
［２３］培養開始時において、前記培地中の塩基性アミノ酸濃度が０．８ｇ／Ｌ以上である、［２０］～［２２］のいずれかに記載の方法。
［２４］前記培地中の塩基性アミノ酸濃度が２ｇ／Ｌ以上である、［２０］～［２３］のいずれかに記載の方法。
［２５］前記培地中の塩基性アミノ酸濃度が４ｇ／Ｌ以上である、［２０］～［２３］のいずれかに記載の方法。
［２６］前記塩基性アミノ酸がリジンである、［２０］～［２５］のいずれかに記載の方法。
［２７］塩基性アミノ酸を含む、酵母によるグルタチオンの生産の促進剤。
［２８］前記酵母が、［２］～［１４］のいずれかで規定する特徴を有する酵母である、［２７］に記載の剤。
［２９］前記塩基性アミノ酸がリジンである、［２７］又は［２８］に記載の剤。
［３０］塩基性アミノ酸濃度が０．８ｇ／Ｌ以上であり、炭素源として糖蜜を含む、酵母用培地組成物。
［３１］［２］～［１４］のいずれかで規定する特徴を有する酵母の培養のための、［３０］に記載の酵母用培地組成物。
［３２］塩基性アミノ酸濃度が２ｇ／Ｌ以上である、［３０］又は［３１］に記載の酵母用培地組成物。
［３３］塩基性アミノ酸濃度が４ｇ／Ｌ以上である、［３０］又は［３１］に記載の酵母用培地組成物。
［３４］前記塩基性アミノ酸がリジンである、［３０］～［３３］のいずれかに記載の酵母用培地組成物。 That is, the present invention includes the following inventions.
[1] A method for producing glutathione, comprising culturing yeast in a medium having a basic amino acid concentration of 0.8 g / L or more.
[2] The method according to [1], wherein the yeast contains reduced glutathione and oxidized glutathione such that the weight of oxidized glutathione is 20 or more when the weight of reduced glutathione is 100. .
[3] The method according to [1] or [2], wherein the yeast is a yeast having reduced glutathione reductase activity as compared to a parent strain.
[4] The method according to any one of [1] to [3], wherein the yeast is a yeast deficient in a gene encoding glutathione reductase.
[5] The glutathione reductase has the following (1a) to (1e):
(1a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
(1b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having a glutathione reductase activity;
(1c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having glutathione reductase activity;
(1d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 2, and having a glutathione reductase activity; and
(1e) a protein comprising an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 2, and which has glutathione reductase activity;
The method according to [3] or [4], wherein the method is selected from the group consisting of:
[6] The method according to any one of [1] to [5], wherein the yeast has an increased activity of γ-glutamylcysteine synthetase and / or glutathione synthase as compared to the parent strain.
[7] The yeast according to [1] to [1], wherein the yeast is a yeast transformed with a DNA containing a nucleotide sequence encoding γ-glutamylcysteine synthetase and / or a DNA containing a nucleotide sequence encoding glutathione synthase. 6].
[8] The γ-glutamylcysteine synthase has the following (2a) to (2e):
(2a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3,
(2b) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in which one or more amino acids have been deleted, substituted, inserted and / or added, and which has γ-glutamylcysteine synthetase activity;
(2c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and having γ-glutamylcysteine synthetase activity;
(2d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 4, and having a γ-glutamylcysteine synthetase activity; and
(2e) one or more bases in the base sequence shown in SEQ ID NO: 4 consist of an amino acid sequence encoded by substitution, deletion, insertion and / or addition of DNA, and have γ-glutamylcysteine synthetase activity protein,
The method according to [6] or [7], wherein the method is selected from the group consisting of:
[9] The glutathione synthase has the following (3a) to (3e):
(3a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5,
(3b) a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 5, and having a glutathione synthase activity;
(3c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and having a glutathione synthase activity;
(3d) a protein comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 6, and having a glutathione synthase activity; and
(3e) a protein having an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 6, and which has glutathione synthase activity;
The method according to [6] or [7], wherein the method is selected from the group consisting of:
[10] The method according to any of [1] to [9], wherein the yeast is a yeast having an increased activity of glutathione transferase as compared to the parent strain.
[11] The method according to any one of [1] to [10], wherein the yeast is a yeast transformed with a DNA containing a nucleotide sequence encoding glutathione transferase.
[12] The glutathione transfer enzyme is as described below in (4a) to (4e):
(4a) a protein consisting of the amino acid sequence of SEQ ID NO: 7,
(4b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 7, and which has a glutathione transferase activity;
(4c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and having a glutathione transferase activity;
(4d) a protein comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 8, and having a glutathione transferase activity; and
(4e) a protein comprising an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 8, and which has a glutathione transferase activity;
The method according to [10] or [11], wherein the method is selected from the group consisting of:
[13] The method according to any one of [1] to [12], wherein the yeast is a yeast belonging to the genus Saccharomyces, Candida, or Pichia.
[14] The method according to any one of [1] to [13], wherein the yeast is a yeast that does not require basic amino acids.
[15] The method according to any one of [1] to [14], wherein the medium contains molasses as a carbon source.
[16] The method according to any one of [1] to [15], wherein at the start of the culture, the concentration of the basic amino acid in the medium is 0.8 g / L or more.
[17] The method according to any one of [1] to [16], wherein the concentration of the basic amino acid in the medium is 2 g / L or more.
[18] The method according to any one of [1] to [16], wherein the concentration of the basic amino acid in the medium is 4 g / L or more.
[19] The method according to any one of [1] to [18], wherein the basic amino acid is lysine.
[20] A method for promoting the production of glutathione by yeast, which comprises culturing yeast in a medium having a basic amino acid concentration of 0.8 g / L or more.
[21] The method according to [20], wherein the yeast is a yeast having the characteristics defined in any one of [2] to [14].
[22] The method according to [20] or [21], wherein the medium contains molasses as a carbon source.
[23] The method according to any one of [20] to [22], wherein at the start of the culture, the concentration of the basic amino acid in the medium is 0.8 g / L or more.
[24] The method according to any one of [20] to [23], wherein the concentration of the basic amino acid in the medium is 2 g / L or more.
[25] The method according to any one of [20] to [23], wherein the concentration of the basic amino acid in the medium is 4 g / L or more.
[26] The method according to any one of [20] to [25], wherein the basic amino acid is lysine.
[27] An accelerator for the production of glutathione by yeast, comprising a basic amino acid.
[28] The agent according to [27], wherein the yeast is a yeast having the characteristics defined in any of [2] to [14].
[29] The agent according to [27] or [28], wherein the basic amino acid is lysine.
[30] A yeast medium composition having a basic amino acid concentration of 0.8 g / L or more and containing molasses as a carbon source.
[31] The medium composition for yeast according to [30], for culturing yeast having the characteristics defined in any one of [2] to [14].
[32] The yeast medium composition according to [30] or [31], wherein the basic amino acid concentration is 2 g / L or more.
[33] The yeast medium composition according to [30] or [31], wherein the basic amino acid concentration is 4 g / L or more.
[34] The medium composition for yeast according to any of [30] to [33], wherein the basic amino acid is lysine.
　本明細書は本願の優先権の基礎となる日本国特許出願番号２０１８－１６７６５５号の開示内容を包含する。 明細 This description includes the disclosure of Japanese Patent Application No. 2018-167655, which is a priority document of the present application.
　本発明によれば、酵母を、十分量のリジン等の塩基性アミノ酸を含む培地中で培養するという簡単な工程によって、酵母によるグルタチオンの生産を促進し、グルタチオンを効率的に製造することができる。また、使用する酵母としては、還元型グルタチオン（ＧＳＨ）重量を１００としたとき、酸化型グルタチオン（ＧＳＳＧ）重量が好ましくは２０以上、より好ましくは４０以上、より好ましくは６０以上、より好ましくは１００以上、より好ましくは１２０以上となる菌体内含量比でＧＳＨ及びＧＳＳＧを含有する酵母であることが好ましい。 According to the present invention, by a simple process of culturing yeast in a medium containing a sufficient amount of a basic amino acid such as lysine, the production of glutathione by yeast can be promoted, and glutathione can be efficiently produced. . When the weight of reduced glutathione (GSH) is set to 100, the weight of oxidized glutathione (GSSG) is preferably 20 or more, more preferably 40 or more, more preferably 60 or more, and more preferably 100 or more. As described above, yeasts containing GSH and GSSG at a bacterial cell content ratio of more preferably 120 or more are preferable.
　以下、本発明の方法を詳細に説明する。
　本発明の方法は、酵母を、リジン等の塩基性アミノ酸の濃度が０．８ｇ／Ｌ以上である培地中で培養することを特徴とする。この条件で酵母を培養することにより、酵母によるグルタチオンの生産を促進することができ、グルタチオン高含有酵母（グルタチオン含有量が高い酵母）を得ることが可能になる。０．８ｇ／Ｌ以上の濃度の塩基性アミノ酸の存在により酵母によるグルタチオンの生産が促進され、グルタチオン高含有効果（酵母のグルタチオン含有量を高める効果）が達成される理由は明らかではない。 Hereinafter, the method of the present invention will be described in detail.
The method of the present invention is characterized in that yeast is cultured in a medium in which the concentration of a basic amino acid such as lysine is 0.8 g / L or more. By culturing the yeast under these conditions, the production of glutathione by the yeast can be promoted, and it is possible to obtain a glutathione-rich yeast (a yeast having a high glutathione content). It is not clear why the presence of a basic amino acid at a concentration of 0.8 g / L or more promotes the production of glutathione by yeast, and achieves a glutathione-rich effect (effect of increasing the glutathione content of yeast).
　本発明の方法で培養される酵母（本発明の酵母）は特に限定されず、野生株（天然の酵母）であってもよいし、変異処理により得られた変異株であってもよいが、好ましくは、変異処理により得られた変異株である。なお、本発明および本願明細書において、「野生株」とは、自然界に元々存在していた酵母、すなわち、遺伝子に対して人工的な変異処理を施していない酵母を意味する。これに対して、「変異株」とは、遺伝子に対して人工的な変異処理を施して得られた酵母を意味する。 The yeast (yeast of the present invention) cultured by the method of the present invention is not particularly limited, and may be a wild type (natural yeast) or a mutant obtained by mutagenesis. Preferably, it is a mutant obtained by the mutation treatment. In the present invention and the specification of the present application, the term "wild strain" refers to a yeast originally existing in nature, that is, a yeast in which a gene has not been subjected to artificial mutation. On the other hand, the “mutant strain” means a yeast obtained by artificially mutating a gene.
　なお、本発明において、変異処理とは、酵母等の生物が有する遺伝子の一部を変異させ得る処理であれば、特に限定されるものではなく、酵母等の微生物の変異株を作製する場合に通常用いられるいずれの手法を用いて行ってもよい。例えば、変異原として、紫外線、電離放射線、亜硝酸、ニトロソグアニジン、エチルメタンスルホネート（Ｅｔｈｙｌｍｅｔｈａｎｅ　ｓｕｌｕｆｏｎａｔｅ、以下ＥＭＳと略記する）等を用いて酵母を処理することにより、酵母に変異処理を行うことができる。 In the present invention, the mutation treatment is not particularly limited as long as it is a treatment capable of mutating a part of a gene possessed by an organism such as yeast, and is used for producing a mutant strain of a microorganism such as yeast. It may be carried out by using any commonly used technique. For example, the yeast can be subjected to mutation treatment by treating the yeast with a mutagen such as ultraviolet ray, ionizing radiation, nitrous acid, nitrosoguanidine, ethyl methanesulfonate (hereinafter, abbreviated as EMS) or the like. .
　また、本発明の酵母は、還元型グルタチオン（ＧＳＨ）重量を１００としたとき、酸化型グルタチオン（ＧＳＳＧ）重量が好ましくは２０以上、より好ましくは４０以上、より好ましくは６０以上、より好ましくは１００以上、より好ましくは１２０以上となるように、ＧＳＨおよびＧＳＳＧを含有する酵母、すなわちＧＳＳＧ高含有酵母であることが好ましい。ＧＳＳＧ高含有酵母は、更に好ましくは、ＧＳＨ重量を１００としたとき、ＧＳＳＧ重量が好ましくは３００以下、より好ましくは２００以下、より好ましくは１５０以下となるように、ＧＳＨおよびＧＳＳＧを含有する。 In addition, the yeast of the present invention has an oxidized glutathione (GSSG) weight of preferably 20 or more, more preferably 40 or more, more preferably 60 or more, and more preferably 100, when the reduced glutathione (GSH) weight is 100. As described above, yeasts containing GSH and GSSG, that is, yeasts high in GSSG, are more preferably 120 or more. The GSSG-rich yeast further preferably contains GSH and GSSG such that the GSSG weight is preferably 300 or less, more preferably 200 or less, more preferably 150 or less when the GSH weight is 100.
　また本発明の酵母は、特にグルタチオンレダクターゼ活性が低下および／またはγ－グルタミルシステイン合成酵素（ＧＳＨ１）および／またはグルタチオン合成酵素（ＧＳＨ２）および／またはグルタチオン輸送酵素（ＹＣＦ１）の活性が増大した酵母であってもよい。 The yeast of the present invention is particularly a yeast in which the activity of glutathione reductase is decreased and / or the activity of γ-glutamylcysteine synthetase (GSH1) and / or glutathione synthase (GSH2) and / or glutathione transportase (YCF1) is increased. There may be.
　本発明において「酵素活性が増大する」とは、目的の酵素（活性を増大させようとする酵素）の活性が野性株等の親株と比較して増大していることを意味する。また、「酵素活性が増大する」とは、もともと目的の酵素活性を有する酵母株において目的の酵素活性を増大させることだけでなく、もともと目的の酵素活性を有さない酵母株に目的の酵素活性を付与することを含む。本発明において活性を増大させようとする酵素としては、γ－グルタミルシステイン合成酵素、グルタチオン合成酵素、グルタチオン輸送酵素が例示できる。 に お い て In the present invention, “enzyme activity is increased” means that the activity of a target enzyme (enzyme whose activity is to be increased) is increased as compared with a parent strain such as a wild-type strain. In addition, "enzyme activity is increased" means that not only a yeast strain originally having a desired enzyme activity but also a desired yeast enzyme strain which does not have the desired enzyme activity. Including providing In the present invention, examples of the enzyme whose activity is to be increased include γ-glutamylcysteine synthetase, glutathione synthase, and glutathione transferase.
　酵素活性の増大は、例えば、酵母の遺伝子を人為的に改変することにより達成できる。そのような改変は、例えば、目的の酵素をコードする遺伝子の発現を増大させることにより達成できる。 増 大 Enzyme activity can be increased, for example, by artificially modifying yeast genes. Such modifications can be achieved, for example, by increasing the expression of the gene encoding the enzyme of interest.
　遺伝子の発現の増大は、例えば、染色体上の遺伝子のプロモーターをより強力なプロモーターに置換することにより達成できる。「より強力なプロモーター」とは、遺伝子の転写が、もともと存在している野生型のプロモーターよりも向上するプロモーターを意味する。より強力なプロモーターとしては、各種レポーター遺伝子を用いることにより、在来のプロモーターの高活性型のものを取得してもよい。また、より強力なプロモーターとしては、公知の高発現プロモーター、例えば、ＰＧＫ１、ＰＤＣ１、ＴＤＨ３、ＴＥＦ１、ＨＸＴ７、ＡＤＨ１等の遺伝子のプロモーターを用いてもよい。なお、強力なプロモーターへの置換は、後述する遺伝子のコピー数の増加と組み合わせて利用できる。より強力なプロモーターを利用した例としては、染色体ＤＮＡ上のγ－グルタミルシステイン合成酵素遺伝子のプロモーターを強転写プロモーターで置換することによりγ－グルタミルシステイン合成酵素活性を増大させる方法が開示されている（大竹康之ら、バイオサイエンスとインダストリー、第５０巻第１０号、第９８９～９９４頁、１９９２年）。 増 大 Increased gene expression can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter. By "stronger promoter" is meant a promoter whose gene transcription is improved over the naturally occurring promoter of the wild type. As a stronger promoter, a highly active type of a conventional promoter may be obtained by using various reporter genes. Further, as a stronger promoter, a known high expression promoter, for example, a promoter of a gene such as PGK1, PDC1, TDH3, TEF1, HXT7, ADH1, etc. may be used. The substitution with a strong promoter can be used in combination with an increase in the copy number of the gene described below. As an example using a stronger promoter, there is disclosed a method of increasing γ-glutamylcysteine synthetase activity by replacing the promoter of γ-glutamylcysteine synthase gene on chromosomal DNA with a strong transcription promoter ( Yasuyuki Otake et al., Bioscience and Industry, Vol. 50, No. 10, pp. 989-994, 1992).
　また、遺伝子の発現の増大は、例えば、遺伝子のコピー数を増加させることによっても達成できる。 増 大 Increase in gene expression can also be achieved, for example, by increasing the copy number of the gene.
　遺伝子のコピー数の増加は、活性を増大しようとする目的の酵素のアミノ酸配列をコードする塩基配列を含むＤＮＡにより酵母を形質転換することにより達成することができる。前記ＤＮＡによる酵母の形質転換は、前記ＤＮＡを含むベクターを用いて行うことができる。前記ＤＮＡを含むベクターは、酵母のゲノムＤＮＡに組み込まれるベクターであってもよいし、酵母細胞内で自律複製可能なベクターであってもよい。 増 加 The copy number of the gene can be increased by transforming yeast with a DNA containing a nucleotide sequence encoding the amino acid sequence of the target enzyme whose activity is to be increased. Transformation of yeast with the DNA can be performed using a vector containing the DNA. The vector containing the DNA may be a vector that is incorporated into genomic DNA of yeast, or may be a vector that can autonomously replicate in yeast cells.
　前記ベクターは、目的の酵素のアミノ酸配列をコードする塩基配列と作動可能に連結されたプロモーター等の制御因子を更に含んでもよい。ここで制御因子とは、機能的プロモーター、任意の関連する転写要素（例えばエンハンサー、ＣＣＡＡＴボックス、ＴＡＴＡボックス、ＳＰＩ部位など）等を有する塩基配列をいう。また、作動可能に連結とは、遺伝子の発現を調節するプロモーター、エンハンサー等の種々の調節エレメントと、目的の酵素のアミノ酸配列をコードする塩基配列とが、宿主細胞中で作動し得る状態で連結されることをいう。制御因子のタイプが、宿主に応じて変わり得ることは、当業者に周知の事項である。前記ベクターは、選択マーカー遺伝子の塩基配列を更に含むことが好ましい。 The vector may further include a control element such as a promoter operably linked to a base sequence encoding the amino acid sequence of the target enzyme. Here, the regulatory element refers to a base sequence having a functional promoter, any relevant transcription elements (for example, enhancer, CCAAT box, TATA box, SPI site, etc.). In addition, operably linked means that various regulatory elements such as a promoter and an enhancer that regulate gene expression and a nucleotide sequence encoding an amino acid sequence of a target enzyme are linked in a state where they can be operable in a host cell. To be done. It is well known to those skilled in the art that the type of the regulatory element may vary depending on the host. It is preferable that the vector further contains a base sequence of a selection marker gene.
　目的の酵素のアミノ酸配列をコードする塩基配列を含むＤＮＡを含むベクターの、酵母のゲノムＤＮＡへの組み込みは、例えば相同的組み換えを利用して行うことができる。例えば、酵母の染色体のゲノムＤＮＡ中に多数のコピーが存在する配列を標的として相同的組み換えを行うことで、ゲノムＤＮＡへ遺伝子の多数のコピーを導入することができる。ゲノムＤＮＡ中に多数のコピーが存在する配列としては、特有の短い繰り返し配列からなる自律複製配列（ＡＲＳ）や、約１５０コピー存在するｒＤＮＡ配列が挙げられる。ＡＲＳを含むプラスミドを用いて酵母の形質転換を行った例が、国際公開９５／３２２８９号パンフレットに記載されている。また、トランスポゾンに遺伝子を組み込み、それをゲノムＤＮＡへ遺伝子の多数のコピーを導入するよう転移させてもよい。 組 み 込 み The integration of the vector containing the DNA containing the nucleotide sequence encoding the amino acid sequence of the desired enzyme into the genomic DNA of yeast can be performed, for example, using homologous recombination. For example, multiple copies of a gene can be introduced into genomic DNA by performing homologous recombination on a sequence in which multiple copies are present in the genomic DNA of the yeast chromosome. Sequences in which a large number of copies exist in genomic DNA include an autonomously replicating sequence (ARS) consisting of a unique short repetitive sequence, and an rDNA sequence having about 150 copies. An example in which yeast was transformed using an ARS-containing plasmid is described in WO 95/32289. Alternatively, the gene may be incorporated into a transposon and transferred to introduce multiple copies of the gene into genomic DNA.
　また、遺伝子のコピー数の増加を、前記自律複製可能なベクターを酵母に導入することによって達成する場合に用いる、前記自律複製可能なベクターとしては、例えば、ＣＥＮ４の複製開始点を持つプラスミドや２μｍ　ＤＮＡの複製開始点を持つ多コピー型プラスミドを好適に用いることができる。目的遺伝子（目的の酵素のアミノ酸配列をコードする塩基配列を含むＤＮＡ）を好適なプロモーターと組み合わせて挿入したベクターを宿主酵母に導入し、目的遺伝子を発現させてもよい。また、目的遺伝子を発現させるのに好適なプロモーターを含むベクターを用いる場合には、ベクター中のプロモーターを利用して目的遺伝子を発現させてもよい。 The autonomously replicable vector used when the copy number of the gene is increased by introducing the autonomously replicable vector into yeast is, for example, a plasmid having a replication origin of CEN4 or 2 μm. A multicopy plasmid having a replication origin of DNA can be suitably used. A vector into which a target gene (a DNA containing a base sequence encoding the amino acid sequence of the target enzyme) is inserted in combination with a suitable promoter may be introduced into host yeast to express the target gene. When a vector containing a promoter suitable for expressing the target gene is used, the target gene may be expressed using the promoter in the vector.
　目的の酵素のアミノ酸配列をコードする塩基配列を含むＤＮＡを含むベクターを取得して宿主酵母を形質転換する方法の一例として次の方法が例示できる。宿主酵母のゲノムＤＮＡ配列のうち、目的の酵素のアミノ酸配列をコードする塩基配列の上流領域と相同な塩基配列に制限酵素Ａの切断配列を付加した塩基配列をもつＤＮＡ－Ｆ２を合成する。一方で、前記塩基配列の下流領域の相補塩基配列と相同な塩基配列に制限酵素Ｂの切断配列を付加した塩基配列をもつＤＮＡ－Ｒ２を合成する。次に、宿主酵母のゲノムＤＮＡをテンプレートとし、ＤＮＡ－Ｆ２およびＤＮＡ－Ｒ２をプライマーとして、ＰＣＲ増幅を行い、増幅されたＤＮＡを制限酵素Ａおよび制限酵素Ｂで切断し、同様に制限酵素Ａおよび制限酵素Ｂで切断した、選択マーカーをもつベクターに連結して組換えベクターを作製し、該組換えベクターを宿主酵母に導入し形質転換体を得る。また、Ｉｎ－Ｆｕｓｉｏｎ　ｃｌｏｎｉｎｇ　ｋｉｔ（タカラバイオ社製）、Ｇｉｂｓｏｎ　ａｓｓｅｍｂｌｙシステム、ＮＥＢｕｉｌｄｅｒ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社製）等の説明書に基づいてプライマー設計および実験操作を行い、目的の酵素のアミノ酸配列をコードする塩基配列を含むＤＮＡのＰＣＲ断片をベクターへシームレスに連結して組換えベクターを作製し、該組換えベクターを宿主酵母に導入して形質転換体を得ても良い。組み換えベクターの宿主酵母への導入の方法は特に限定されず、例えば、カルシウムイオンを用いる方法、エレクトロポレーション法、スフェロプラスト法、酢酸リチウム法、アグロバクテリウム感染法、パーティクルガン法、ポリエチレングリコール法、リン酸カルシウム法、リポソーム法、ＤＥＡＥデキストラン法、マイクロインジェクション法、カチオン性脂質媒介トランスフェクション、形質導入または感染等が使用できる。目的の酵素のアミノ酸配列をコードする塩基配列を含むＤＮＡにより形質転換された酵母株は、組換えベクターの中の選択マーカーを指標に選抜することが可能である。 The following method can be exemplified as an example of a method for obtaining a vector containing a DNA containing a base sequence encoding the amino acid sequence of the target enzyme and transforming the host yeast. DNA-F2 having a base sequence obtained by adding a cleavage sequence of restriction enzyme A to a base sequence homologous to the upstream region of the base sequence encoding the amino acid sequence of the target enzyme in the genomic DNA sequence of the host yeast is synthesized. On the other hand, DNA-R2 having a base sequence obtained by adding a cleavage sequence of restriction enzyme B to a base sequence homologous to a complementary base sequence in a downstream region of the base sequence is synthesized. Next, PCR amplification was performed using the genomic DNA of the host yeast as a template and DNA-F2 and DNA-R2 as primers, and the amplified DNA was cut with restriction enzymes A and B. A recombinant vector is prepared by ligating to a vector having a selectable marker cleaved with restriction enzyme B, and the recombinant vector is introduced into host yeast to obtain a transformant. In addition, primer design and experimental operations are performed based on manuals such as In-Fusion Cloning kit (manufactured by Takara Bio Inc.), Gibson Assembly System, and NEBuilder (manufactured by New England Biolabs) to encode the amino acid sequence of the target enzyme. A transformant may be obtained by preparing a recombinant vector by seamlessly ligating a PCR fragment of DNA containing a base sequence to a vector, and introducing the recombinant vector into host yeast. The method for introducing the recombinant vector into the host yeast is not particularly limited, and examples thereof include a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, an Agrobacterium infection method, a particle gun method, and polyethylene glycol. Methods, calcium phosphate method, liposome method, DEAE dextran method, microinjection method, cationic lipid-mediated transfection, transduction or infection and the like can be used. A yeast strain transformed with a DNA containing the nucleotide sequence encoding the amino acid sequence of the desired enzyme can be selected using the selection marker in the recombinant vector as an indicator.
　また、酵素活性が増大するような改変は、例えば、目的の酵素の比活性を増大させることによっても達成できる。比活性が増大した酵素は、例えば、種々の生物を探索し取得することができる。また、在来の酵素に変異を導入することで高比活性型のものを取得してもよい。比活性の増大は、単独で用いてもよく、上記のような遺伝子の発現を増大させる手法と任意に組み合わせて用いてもよい。 改 変 Also, the modification that increases the enzyme activity can be achieved, for example, by increasing the specific activity of the target enzyme. An enzyme having an increased specific activity can be obtained by, for example, searching for various organisms. Alternatively, a high specific activity type may be obtained by introducing a mutation into a conventional enzyme. The increase in specific activity may be used alone, or may be used in any combination with the above-described method for increasing gene expression.
　目的の酵素活性が増大したことの確認は、同酵素の活性を測定することによって行うことが出来る。γ－グルタミルシステイン合成酵素活性は、Jacksonの方法（Jackson, R. C., Biochem.J., 111, 309 (1969)）によって測定することができる。グルタチオン合成酵素活性は、Gushimaらの方法（Gushima, T. et al., J. Appl. Biochem., 5, 210 (1983)）によって測定することができる。グルタチオン輸送酵素活性はTHE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 50, Issue of December 11, pp. 33449-33454, 1998を参考にすることによって測定することができる。 確認 Confirmation that the activity of the target enzyme has increased can be performed by measuring the activity of the enzyme. γ-glutamylcysteine synthetase activity can be measured by the method of Jackson (Jackson, R. C., Biochem. J., 111, 309 (1969)). Glutathione synthase activity can be measured by the method of Gushima et al. (Gushima, T. et al., J. Appl. Biochem., 5, 210 (1983)). Glutathione transferase activity can be measured by referring to THE JOURNAL OF OF BIOLOGICAL CHEMISTRY Vol. 273, No. 50, Issue of Dec. 11, pp. 33449-33454, 1998.
　目的の酵素をコードする遺伝子の転写量が増大したことの確認は、同遺伝子から転写されるｍＲＮＡの量を親株と比較することによって行うことが出来る。ｍＲＮＡの量を評価する方法としては、ノーザンハイブリダイゼーション、ＲＴ－ＰＣＲ等が挙げられる（Molecular cloning（Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001））。ｍＲＮＡの量は、親株と比較して、例えば、１．５倍以上、２倍以上、または３倍以上に増大しているのが好ましい。 確認 Confirmation that the transcription amount of the gene encoding the target enzyme has increased can be performed by comparing the amount of mRNA transcribed from the gene with that of the parent strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). It is preferable that the amount of mRNA is increased, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to the parent strain.
　目的の酵素の量が増大したことの確認は、抗体を用いてウェスタンブロットによって行うことが出来る（Molecular cloning（Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001））。目的の酵素の量は、親株と比較して、例えば、１．５倍以上、２倍以上、または３倍以上に増大しているのが好ましい。 (4) The increase in the amount of the target enzyme can be confirmed by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). It is preferable that the amount of the target enzyme is, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared with the parent strain.
　本発明の酵母が２倍体以上の倍数性を有する場合であって、染色体の改変により酵素活性が増大している場合には、本発明の酵母は、グルタチオンを蓄積できる限り、酵素活性が増大するように改変された染色体と野生型染色体とをヘテロで有していてもよく、酵素活性が増大するように改変された染色体のホモ型であってもよい。 When the yeast of the present invention has a diploid or higher polyploidy and the enzyme activity is increased due to chromosome modification, the yeast of the present invention has an increased enzyme activity as long as glutathione can be accumulated. The chromosome modified so as to have a heterozygous chromosome and a wild-type chromosome, or may be a homologous chromosome modified so as to increase enzyme activity.
　本発明において「酵素活性が低下する」とは、目的の酵素活性が野性株等の親株と比較して低下していることを意味し、活性が完全に消失している場合を含む。酵素活性が低下した酵母は、目的の酵素（活性を低下させようとする酵素）をコードする遺伝子の機能が失われている状態、または、当該機能が減少している状態にある酵母であり、具体的には、前記遺伝子の転写産物であるｍＲＮＡや翻訳産物であるタンパク質の発現量が低下している状態や、前記ｍＲＮＡまたは前記タンパク質が、ｍＲＮＡまたはタンパク質として正常に機能しない状態にある酵母が挙げられる。本発明において活性を低下させようとする酵素としては、グルタチオンレダクターゼが例示できる。 に お い て In the present invention, “the enzyme activity is reduced” means that the target enzyme activity is lower than that of a parent strain such as a wild type strain, and includes the case where the activity is completely lost. A yeast with a reduced enzyme activity is a yeast in which the function of the gene encoding the target enzyme (the enzyme whose activity is to be reduced) is lost, or in which the function is reduced, Specifically, a yeast in which the expression level of the mRNA which is a transcription product of the gene or the protein which is a translation product is reduced or the yeast where the mRNA or the protein does not function normally as an mRNA or a protein is used. No. In the present invention, examples of the enzyme whose activity is to be reduced include glutathione reductase.
　酵素活性の低下は、例えば、酵母の遺伝子を人為的に改変することにより達成できる。そのような改変は、例えば、突然変異処理、遺伝子組換え技術、ＲＮＡｉを用いた遺伝子発現抑制処理等により達成できる。 低下 Reduction of enzyme activity can be achieved, for example, by artificially modifying yeast genes. Such a modification can be achieved by, for example, a mutation treatment, a gene recombination technique, a gene expression suppression treatment using RNAi, or the like.
　突然変異処理としては、紫外線照射、または、Ｎ－メチル－Ｎ'－ニトロ－Ｎ－ニトロソグアニジン（ＭＮＮＧ）、エチルメタンスルホネート（ＥＭＳ）、メチルメタンスルフォネート（ＭＭＳ）等の通常変異処理に用いられている変異剤による処理が挙げられる。 The mutation treatment may be performed by ultraviolet irradiation or normal mutation treatment of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), etc. Treatment with a known mutagen.
　遺伝子組換え技術としては、例えば、公知の技術（FEMS Microbiology Letters 165 (1998) 335-340、JOURNAL OF BACTERIOLOGY, Dec. 1995, p7171-7177、Curr Genet 1986; 10(8):573-578、WO 98/14600等）を利用できる。 As a gene recombination technique, for example, a known technique (FEMS Microbiology Letters 165 (1998) 335-340, JOURNAL OF BACTERIOLOGY, Dec. 1995, p7171-7177, Curr Genet 1986; 10 (8): 573-578, WO 98/14600).
　酵素活性が低下するような改変は、例えば、目的の酵素をコードする遺伝子の発現を低下させることにより達成できる。ここで目的の酵素をコードする遺伝子は、典型的には宿主酵母のゲノムＤＮＡに含まれる。目的の酵素をコードする遺伝子とは、目的の酵素のアミノ酸配列をコードする塩基配列を含む遺伝子を指し、特に限定しない限り、アミノ酸配列のコード領域だけでなく、その発現調節配列（プロモーター配列等）、エクソン配列、イントロン配列等を区別することなく示す。発現調節配列を改変する場合には、発現調節配列は、好ましくは１塩基以上、より好ましくは２塩基以上、特に好ましくは３塩基以上が改変される。 改 変 The modification that reduces the enzyme activity can be achieved, for example, by reducing the expression of the gene encoding the target enzyme. Here, the gene encoding the enzyme of interest is typically contained in the genomic DNA of the host yeast. The gene encoding the target enzyme refers to a gene containing a base sequence encoding the amino acid sequence of the target enzyme, and unless otherwise limited, not only the coding region of the amino acid sequence but also its expression control sequence (promoter sequence, etc.) , Exon sequence, intron sequence and the like are shown without distinction. When the expression control sequence is modified, the expression control sequence is preferably modified at one or more bases, more preferably at least two bases, particularly preferably at least three bases.
　酵素活性が低下するような改変は、より好ましくは、宿主酵母のゲノムＤＮＡにおける、目的の酵素をコードする遺伝子の欠損を含む。前記遺伝子の欠損としては、発現調節配列の一部または全部の欠損であってもよいし、目的の酵素のアミノ酸配列のコード領域の一部または全部の欠損であってもよい。ここで「欠損」とは、欠失または損傷を意味し、好ましくは欠失である。 改 変 The modification that reduces the enzyme activity more preferably includes a deletion of the gene encoding the enzyme of interest in the genomic DNA of the host yeast. The deletion of the gene may be a deletion of part or all of the expression control sequence, or a deletion of part or all of the coding region of the amino acid sequence of the target enzyme. Here, “deletion” means deletion or damage, and preferably deletion.
　宿主酵母のゲノムＤＮＡにおける前記遺伝子の前後の配列を含めて、遺伝子全体を欠失させてもよい。目的の酵素のアミノ酸配列のコード領域の一部または全部を欠失させる場合、酵素活性の低下が達成できる限り、Ｎ末端領域、内部領域、Ｃ末端領域等のいずれの領域を欠失させてもよい。通常、欠失させる領域は長い方が確実に遺伝子を不活化することができる。また、欠失させる領域の前後の配列は、リーディングフレームが一致しないことが好ましい。好ましい実施形態では、ゲノムＤＮＡにおいて、目的の酵素をコードする遺伝子のうちアミノ酸配列のコード領域および／または発現調節配列の少なくとも一部、例えば、コード領域および／または発現調節配列の全体の塩基数に対して好ましくは５０％以上、より好ましくは６０％以上、より好ましくは７０％以上、より好ましくは８０％以上、より好ましくは９０％以上、より好ましくは１００％の塩基数からなる領域、が欠失した酵母を用いる。 (4) The entire gene may be deleted, including the sequence before and after the gene in the genomic DNA of the host yeast. When part or all of the coding region of the amino acid sequence of the target enzyme is deleted, any region such as the N-terminal region, internal region, or C-terminal region can be deleted as long as the reduction in enzyme activity can be achieved. Good. Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. In addition, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. In a preferred embodiment, in the genomic DNA, at least a part of the coding region of the amino acid sequence and / or the expression control sequence of the gene encoding the enzyme of interest, for example, the total number of bases of the coding region and / or the expression control sequence On the other hand, a region having a base number of preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and more preferably 100% is missing. Use the lost yeast.
　また、酵素活性が低下するような、目的の酵素のアミノ酸配列をコードする遺伝子の欠損の他の例としては、ゲノムＤＮＡ上の目的の酵素をコードする遺伝子のコード領域にアミノ酸置換（ミスセンス変異）を導入すること、終止コドンを導入すること（ナンセンス変異）、あるいは１～２塩基を付加または欠失するフレームシフト変異を導入すること等の、前記遺伝子の損傷が例示できる。 Other examples of deletion of a gene encoding the amino acid sequence of the target enzyme such that the enzyme activity is reduced include amino acid substitution (missense mutation) in the coding region of the gene encoding the target enzyme on genomic DNA. , A stop codon (nonsense mutation), or a frameshift mutation that adds or deletes one or two bases.
　また、酵素活性が低下するような、目的の酵素のアミノ酸配列をコードする遺伝子の欠損は、例えば、ゲノムＤＮＡ上の目的の酵素をコードする遺伝子の発現調節配列またはコード領域に他の配列を挿入することによっても達成できる。挿入部位は、遺伝子のいずれの領域であってもよいが、挿入する配列は長い方が確実に遺伝子を不活化することができる。また、挿入部位の前後の配列は、リーディングフレームが一致しないことが好ましい。他の配列としては、コードされるタンパク質の機能を低下または消失させるものであれば特に制限されないが、例えば、マーカー遺伝子やグルタチオン等のγ－グルタミル化合物の生産に有用な遺伝子が挙げられる。 Deletion of the gene encoding the amino acid sequence of the target enzyme, such as a decrease in enzyme activity, may be caused, for example, by inserting another sequence into the expression control sequence or coding region of the gene encoding the target enzyme on the genomic DNA. Can also be achieved. The insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. Further, it is preferable that the sequences before and after the insertion site do not match in the reading frame. The other sequence is not particularly limited as long as it reduces or eliminates the function of the encoded protein. Examples thereof include a marker gene and a gene useful for the production of a γ-glutamyl compound such as glutathione.
　ゲノムＤＮＡ上の遺伝子を上記のように欠損させることは、例えば、目的の酵素のアミノ酸配列をコードする遺伝子を、正常に機能するタンパク質を産生しないように改変した不活性遺伝子を作製し、該不活性遺伝子を含む組換えＤＮＡで酵母を形質転換して、不活性遺伝子とゲノムＤＮＡ上の遺伝子とで相同組換えを起こさせることにより、ゲノムＤＮＡ上の遺伝子を不活性遺伝子に置換することによって達成できる。その際、組換えＤＮＡには、宿主の栄養要求性等の形質にしたがって、マーカー遺伝子を含ませておくと操作がしやすい。また、前記組換えＤＮＡは、制限酵素で切断する等により直鎖状にしておくと、ゲノムＤＮＡに組換えＤＮＡが組み込まれた株を効率よく取得することができる。不活性遺伝子によってコードされるタンパク質は、生成したとしても、野生型タンパク質とは異なる立体構造を有し、機能が低下または消失する。 Deletion of a gene on genomic DNA as described above can be performed, for example, by preparing an inactive gene in which the gene encoding the amino acid sequence of the target enzyme is modified so as not to produce a protein that functions normally, and Achieved by transforming yeast with a recombinant DNA containing an active gene and causing homologous recombination between the inactive gene and the gene on the genomic DNA, thereby replacing the gene on the genomic DNA with the inactive gene. it can. At this time, if the marker DNA is included in the recombinant DNA according to the trait such as auxotrophy of the host, the operation is easy. If the recombinant DNA is made linear by cutting with a restriction enzyme or the like, a strain in which the recombinant DNA has been incorporated into genomic DNA can be efficiently obtained. The protein encoded by the inactive gene, if produced, has a different steric structure than the wild-type protein and has reduced or lost function.
　用いる組換えＤＮＡの構造によっては、相同組換えの結果として、野生型遺伝子と不活性遺伝子とが組換えＤＮＡの他の部分（例えば、ベクター部分およびマーカー遺伝子）を挟んだ状態でゲノムＤＮＡに挿入される場合がある。この状態では野生型遺伝子が機能するため、当該２個の遺伝子間で再度相同組換えを起こさせ、１コピーの野生型遺伝子を、ベクター部分およびマーカー遺伝子とともにゲノムＤＮＡから脱落させ、不活性遺伝子が残ったものを選抜することを必要に応じて行う。 Depending on the structure of the recombinant DNA used, as a result of homologous recombination, the wild-type gene and the inactive gene are inserted into the genomic DNA with other parts of the recombinant DNA (eg, a vector part and a marker gene) interposed therebetween. May be done. In this state, since the wild-type gene functions, homologous recombination occurs again between the two genes, and one copy of the wild-type gene is dropped from the genomic DNA together with the vector portion and the marker gene, and the inactive gene is removed. The selection of the remaining ones is performed as needed.
　また、例えば、任意の配列を含む線状ＤＮＡであって、当該任意の配列の両端にゲノムＤＮＡ上の置換対象部位（典型的には、目的の酵素をコードする遺伝子の一部または全部）の上流および下流の配列を備える線状ＤＮＡで酵母を形質転換して、置換対象部位の上流および下流でそれぞれ相同組換えを起こさせることにより、１ステップで置換対象部位を当該任意の配列に置換することができる。当該任意の配列としては、例えば、マーカー遺伝子を含む配列を用いればよい。マーカー遺伝子は、その後、必要により除去してもよい。マーカー遺伝子を除去する場合には、マーカー遺伝子を効率的に除去できるよう、相同組み換え用の配列をマーカー遺伝子の両端に付加しておいてもよい。 In addition, for example, a linear DNA containing an arbitrary sequence, and both ends of the arbitrary sequence may have a site to be replaced on genomic DNA (typically, a part or all of a gene encoding a target enzyme). The yeast is transformed with the linear DNA having the upstream and downstream sequences, and homologous recombination is caused upstream and downstream of the site to be replaced, whereby the site to be replaced is replaced with an arbitrary sequence in one step. be able to. As the arbitrary sequence, for example, a sequence containing a marker gene may be used. The marker gene may then be removed if necessary. When the marker gene is removed, a sequence for homologous recombination may be added to both ends of the marker gene so that the marker gene can be removed efficiently.
　目的の酵素活性が低下したことの確認は、同酵素の活性を測定することによって行うことが出来る。例えば、グルタチオンレダクターゼの活性は、公知の手法（コスモバイオ社製Glutathione Reductase Assay Kit品番7510-100-K等）により測定することができる。 確認 Confirmation that the activity of the target enzyme has been reduced can be made by measuring the activity of the enzyme. For example, the activity of glutathione reductase can be measured by a known method (such as Glutathione Reductase Assay Kit No. 7510-100-K manufactured by Cosmo Bio Inc.).
　目的の酵素をコードする遺伝子の転写量が低下したことの確認は、同遺伝子から転写されるｍＲＮＡの量を親株と比較することによって行うことが出来る。ｍＲＮＡの量を評価する方法としては、ノーザンハイブリダイゼーション、ＲＴ－ＰＣＲ等が挙げられる（Molecular cloning（Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001））。ｍＲＮＡの量は、親株と比較して、例えば、５０％以下、２０％以下、１０％以下、５％以下、または０％に低下しているのが好ましい。 確認 Confirmation of a decrease in the amount of transcription of the gene encoding the target enzyme can be performed by comparing the amount of mRNA transcribed from the gene with that of the parent strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the parent strain.
　目的の酵素の量が低下したことの確認は、抗体を用いてウェスタンブロットによって行うことが出来る（Molecular cloning（Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001））。目的の酵素の量は、親株と比較して、例えば、５０％以下、２０％以下、１０％以下、５％以下、または０％に低下しているのが好ましい。 (4) The decrease in the amount of the target enzyme can be confirmed by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of the target enzyme is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the parent strain.
　酵母の形質転換法としては、プロトプラスト法、ＫＵ法（H.Ito et al., J. Bacteriol., 153,163-168 (1983)）、ＫＵＲ法（発酵と工業 vol.43, p.630-637 (1985)）、エレクトロポレーション法（Luis et al., FEMS Micro biology Letters 165 (1998) 335-340）、キャリアＤＮＡを用いる方法（Gietz R.D. and Schiestl R.H., Methods Mol.Cell. Biol. 5:255-269 (1995)）等、通常酵母の形質転換に用いられる方法を採用することができる。また、酵母の胞子形成、１倍体酵母の分離、等の操作については、「化学と生物 実験ライン３１ 酵母の実験技術」、初版、廣川書店；「バイオマニュアルシリーズ１０ 酵母による遺伝子実験法」初版、羊土社等に記載されている。 Examples of the yeast transformation method include a protoplast method, a KU method (H. Ito et al., J. Bacteriol., 153,163-168 (1983)), and a KUR method (fermentation and industry vol.43, p.630-637). 1985)), electroporation method (Luis et al., FEMS Micro Biology Letters 165 (1998) 335-340), method using carrier DNA (Gietz RD and Schiestl RH, Methods Mol. Cell. Biol. 5: 255- 269 (1995)) and the like, which are usually used for yeast transformation. For operations such as yeast spore formation and haploid yeast separation, see “Chemistry and Biology—Experimental Line 31—Yeast Experimental Techniques”, First Edition, Hirokawa Shoten; “Bio Manual Series 10—Gene Experiments with Yeast”, First Edition , Yodosha, etc.
　本発明の酵母が２倍体以上の倍数性を有する場合には、本発明の酵母は、グルタチオン等のγ－グルタミル化合物を蓄積できる限り、酵素活性が低下するように改変された遺伝子と野生型遺伝子とをヘテロで有していてもよいが、通常は、酵素活性が低下するように改変された遺伝子のホモ型であるのが好ましい。 When the yeast of the present invention has a diploid or higher polyploidy, the yeast of the present invention may be modified with a wild-type gene and a gene modified to reduce the enzyme activity as long as a γ-glutamyl compound such as glutathione can be accumulated. The gene may be heterozygous, but is usually preferably a homozygous gene modified so as to reduce the enzyme activity.
（１）グルタチオンレダクターゼ
　グルタチオンレダクターゼとは、下記式（２）に示すような酸化型グルタチオンを、ＮＡＤＰＨ（還元型ニコチンアミドジヌクレオチドリン酸）を利用して還元する活性を有する酵素である。 (1) Glutathione reductase Glutathione reductase is an enzyme having an activity of reducing oxidized glutathione represented by the following formula (2) using NADPH (reduced nicotinamide dinucleotide phosphate).
　グルタチオンレダクターゼ活性の抑制された酵母は、ＧＳＳＧをＧＳＨに変換する活性が低いため、ＧＳＳＧを蓄積し易い。このようなグルタチオンレダクターゼ活性の抑制された酵母は、特に好ましくは、ＧＳＨ重量を１００としたとき、ＧＳＳＧ重量が好ましくは２０以上、より好ましくは４０以上、より好ましくは６０以上、より好ましくは１００以上、より好ましくは１２０以上となるように、ＧＳＨおよびＧＳＳＧを含有するＧＳＳＧ高含有酵母である。グルタチオンレダクターゼ活性の抑制された酵母は、更に好ましくは、ＧＳＨ重量を１００としたとき、ＧＳＳＧ重量が好ましくは３００以下、より好ましくは２００以下、より好ましくは１５０以下となるように、ＧＳＨおよびＧＳＳＧを含有する。 酵母 Yeast in which glutathione reductase activity is suppressed has a low activity of converting GSSG to GSH, and thus easily accumulates GSSG. Such a yeast in which glutathione reductase activity is suppressed is particularly preferably, when the GSH weight is 100, the GSSG weight is preferably 20 or more, more preferably 40 or more, more preferably 60 or more, and more preferably 100 or more. GSSG-rich yeast containing GSH and GSSG so as to be 120 or more. The yeast in which glutathione reductase activity is suppressed is more preferably, when GSH weight is 100, GSSG weight is preferably 300 or less, more preferably 200 or less, and more preferably 150 or less. contains.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　本発明におけるグルタチオンレダクターゼは、ＮＡＤＰＨもしくはＮＡＤＨを利用してジスルフィド結合を還元する活性（即ち、グルタチオンレダクターゼ活性）を有する酵素であれば良く、特に限定されないが、例えば、以下の（１ａ）～（１ｅ）：
（１ａ）配列番号１に示すアミノ酸配列からなるタンパク質、
（１ｂ）配列番号１に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
（１ｃ）配列番号１に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
（１ｄ）配列番号２と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、ならびに、
（１ｅ）配列番号２に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
のいずれかであることが好ましい。 Glutathione reductase in the present invention may be any enzyme having an activity of reducing a disulfide bond using NADPH or NADH (that is, glutathione reductase activity), and is not particularly limited. Examples thereof include the following (1a) to (1e) ):
(1a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
(1b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having a glutathione reductase activity;
(1c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having glutathione reductase activity;
(1d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 2, and having a glutathione reductase activity; and
(1e) a protein comprising an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 2, and which has glutathione reductase activity;
It is preferable that it is either.
　ここで、上記（１ａ）～（１ｅ）のいずれかのタンパク質は、上記（１ａ）～（１ｅ）で規定するアミノ酸配列からなるポリペプチド鎖のみからなる形態には限定されず、該ポリペプチド鎖が糖鎖等で化学修飾された形態であってもよいし、該ポリペプチド鎖が他のポリペプチド鎖と融合された融合タンパク質の形態であってもよい。 Here, the protein of any of the above (1a) to (1e) is not limited to the form consisting of only the polypeptide chain consisting of the amino acid sequence defined in the above (1a) to (1e), May be in the form chemically modified with a sugar chain or the like, or may be in the form of a fusion protein in which the polypeptide chain is fused with another polypeptide chain.
　上記（１ｂ）に記載の、配列番号１に示すアミノ酸配列において、１もしくは複数個のアミノ酸が置換、挿入、欠失および／または付加されたアミノ酸配列からなるタンパク質は、「Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（Ｊｏｈｎ　Ｗｉｌｅｙ　ａｎｄ　Ｓｏｎｓ，　Ｉｎｃ．，　１９８９）」等に記載の公知の方法に準じて調製することができ、グルタチオンレダクターゼ活性を有する限り上記タンパクに包含される。 In the amino acid sequence represented by SEQ ID NO: 1 described in (1b) above, a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, inserted, deleted and / or added is referred to as “Current \ Protocols in \ Molecular \ Biology ( John Wiley and Sons, Inc., 1989), etc., and are included in the above proteins as long as they have glutathione reductase activity.
　置換、挿入、欠失および／または付加により改変されたアミノ酸配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種以上の改変（例えば、置換と挿入）を含んでいても良い。また、置換の場合には、置換するアミノ酸は、置換前のアミノ酸と類似の性質を有するアミノ酸（同族アミノ酸）であることが好ましい。ここでは、以下に挙げる各群の同一群内のアミノ酸を同族アミノ酸とする。
（第１群：中性非極性アミノ酸）Ｇｌｙ，Ａｌａ，Ｖａｌ，Ｌｅｕ，Ｉｌｅ，Ｍｅｔ，Ｃｙｓ，Ｐｒｏ，Ｐｈｅ
（第２群：中性極性アミノ酸）Ｓｅｒ，Ｔｈｒ，Ｇｌｎ，Ａｓｎ，Ｔｒｐ，Ｔｙｒ
（第３群：酸性アミノ酸）Ｇｌｕ，Ａｓｐ
（第４群：塩基性アミノ酸）Ｈｉｓ，Ｌｙｓ，Ａｒｇ The amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one kind of modification (for example, substitution) or may contain two or more kinds of modification (for example, substitution and insertion). May be included. In the case of substitution, the amino acid to be substituted is preferably an amino acid having similar properties to the amino acid before substitution (homologous amino acid). Here, the amino acids in the same group of each group described below are homologous amino acids.
(Group 1: neutral non-polar amino acids) Gly, Ala, Val, Leu, Ile, Met, Cys, Pro, Phe
(Group 2: neutral polar amino acids) Ser, Thr, Gln, Asn, Trp, Tyr
(Group 3: acidic amino acids) Glu, Asp
(Group 4: Basic amino acids) His, Lys, Arg
　上記（１ｂ）において、「１もしくは複数個」のアミノ酸とは、例えば、１～６０個、好ましくは１～２０個、より好ましくは１～１５個、さらに好ましくは１～１０個、さらに好ましくは１～５個、１～４個、１～３個、または１～２個のアミノ酸を意味する。 In the above (1b), “one or more” amino acids include, for example, 1 to 60, preferably 1 to 20, more preferably 1 to 15, further preferably 1 to 10, and further preferably One to five, one to four, one to three, or one to two amino acids.
　上記（１ｃ）において、配列番号１に示すアミノ酸配列との配列同一性は、６０％以上が好ましく、７０％以上がより好ましく、８０％以上がさらに好ましく、８５％以上がさらに好ましく、９０％以上がさらに好ましく、９５％以上がさらに好ましく、９７％以上がさらに好ましく、９８％以上がさらに好ましく、９９％以上が最も好ましい。アミノ酸配列の配列同一性は、配列番号１または２に示すアミノ酸配列と、評価したいアミノ酸配列とを比較し、両方の配列でアミノ酸が一致した位置の数を比較総アミノ酸数で除して、さらに１００を乗じた値で表される。 In the above (1c), the sequence identity with the amino acid sequence shown in SEQ ID NO: 1 is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, still more preferably 85% or more, and 90% or more. Is more preferably 95% or more, still more preferably 97% or more, further preferably 98% or more, and most preferably 99% or more. The amino acid sequence identity can be determined by comparing the amino acid sequence shown in SEQ ID NO: 1 or 2 with the amino acid sequence to be evaluated, dividing the number of positions where amino acids match in both sequences by the total number of amino acids to be compared, and It is represented by a value multiplied by 100.
　上記（１ｄ）において、配列番号２に示す塩基配列と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡとは、配列番号２に示す塩基配列と相補的な塩基配列からなるＤＮＡをプローブとして、ストリンジェントな条件下にコロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法、あるいはサザンハイブリダイゼーション法等を用いることにより得られるＤＮＡを意味する。 In the above (1d), a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 is a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 And DNA obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like under stringent conditions using the resulting DNA as a probe.
　ハイブリダイゼーションは、「Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，　Ａ　ｌａｂｏｒａｔｏｒｙ　ｍａｎｕａｌ，　ｓｅｃｏｎｄ　ｅｄｉｔｉｏｎ（Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ，　１９８９）」等に記載されている方法に準じて行うことができる。ここで、ストリンジェントな条件でハイブリダイズするＤＮＡとは、例えば、コロニーあるいはプラーク由来のＤＮＡを固定化したフィルターを用いて、０．７～１．０ＭのＮａＣｌ存在下、６５℃でハイブリダイゼーションを行った後、２倍濃度のＳＳＣ溶液（１倍濃度のＳＳＣ溶液の組成は、１５０ｍＭ塩化ナトリウム、１５ｍＭクエン酸ナトリウムよりなる）を用い、６５℃条件下でフィルターを洗浄することにより取得できるＤＮＡを挙げることができる。好ましくは６５℃で１倍濃度のＳＳＣ溶液で洗浄、より好ましくは６５℃で０．５倍濃度のＳＳＣ溶液で洗浄、さらに好ましくは６５℃で０．２倍濃度のＳＳＣ溶液で洗浄、最も好ましくは６５℃で０．１倍濃度のＳＳＣ溶液で洗浄することにより取得できるＤＮＡである。 The hybridization can be performed according to a method described in “Molecular Cloning, A laboratory manual, second edition (Cold Spring Harbor Laboratory Press, 1989)” or the like. Here, DNA that hybridizes under stringent conditions refers to, for example, hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques is immobilized. After that, DNA which can be obtained by washing the filter at 65 ° C. using a 2 × concentration SSC solution (the composition of the 1 × concentration SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate) is used. Can be mentioned. Washing is preferably carried out at 65 ° C. with a 1-fold concentration SSC solution, more preferably at 65 ° C. with a 0.5-fold concentration SSC solution, even more preferably at 65 ° C. with a 0.2-fold concentration SSC solution, most preferably. Is a DNA that can be obtained by washing with a 0.1-fold concentration SSC solution at 65 ° C.
　以上のようにハイブリダイゼーション条件を記載したが、ハイブリダイゼーション条件はこれらの条件に特に制限されない。ハイブリダイゼーションのストリンジェンシーに影響する要素としては温度や塩濃度など複数の要素が考えられ、当業者であればこれら要素を適宜選択することで最適なストリンジェンシーを実現することが可能である。 ハ イ Although the hybridization conditions have been described above, the hybridization conditions are not particularly limited to these conditions. A plurality of factors such as temperature and salt concentration can be considered as factors that influence the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.
　上記の条件にてハイブリダイズ可能なＤＮＡとしては、配列番号２に示されるＤＮＡとの配列同一性が７０％以上、好ましくは７４％以上、より好ましくは７９％以上、さらに好ましくは８５％以上、さらにより好ましくは９０％以上、さらにより好ましくは９５％以上、さらにより好ましくは９７％以上、さらにより好ましくは９８％以上、最も好ましくは９９％以上のＤＮＡを挙げることができる。 As DNA hybridizable under the above conditions, the sequence identity to the DNA shown in SEQ ID NO: 2 is 70% or more, preferably 74% or more, more preferably 79% or more, and still more preferably 85% or more. Even more preferably, 90% or more, even more preferably, 95% or more, even more preferably, 97% or more, even more preferably, 98% or more, and most preferably, 99% or more of DNA.
　ＤＮＡの配列同一性（％）とは、対比される２つのＤＮＡを最適に整列させ、核酸塩基(例えば、Ａ、Ｔ、Ｃ、Ｇ、Ｕ、またはＩ)が両方の配列で一致した位置の数を比較塩基総数で除し、そして、この結果に１００を乗じた数値で表される。 DNA sequence identity (%) refers to the optimal alignment of two contrasted DNAs and the position of a nucleobase (eg, A, T, C, G, U, or I) that is identical in both sequences. The number is divided by the total number of comparison bases, and the result is multiplied by 100.
　ＤＮＡの配列同一性は、例えば、以下の配列分析用ツールを用いて算出し得る: ＧＣＧ　Ｗｉｓｃｏｎｓｉｎ　Ｐａｃｋａｇｅ（Ｐｒｏｇｒａｍ　Ｍａｎｕａｌ　ｆｏｒ　Ｔｈｅ　Ｗｉｓｃｏｎｓｉｎ　Ｐａｃｋａｇｅ，　Ｖｅｒｓｉｏｎ８，　１９９４年９月，　Ｇｅｎｅｔｉｃｓ　Ｃｏｍｐｕｔｅｒ　Ｇｒｏｕｐ，　５７５　Ｓｃｉｅｎｃｅ　Ｄｒｉｖｅ　Ｍｅｄｉｓｏｎ，　Ｗｉｓｃｏｎｓｉｎ，　ＵＳＡ　５３７１１；　Ｒｉｃｅ，　Ｐ．　（１９９６）　Ｐｒｏｇｒａｍ　Ｍａｎｕａｌ　ｆｏｒ　ＥＧＣＧ　Ｐａｃｋａｇｅ，　Ｐｅｔｅｒ　Ｒｉｃｅ，　Ｔｈｅ　Ｓａｎｇｅｒ　Ｃｅｎｔｒｅ，　Ｈｉｎｘｔｏｎ　Ｈａｌｌ，　Ｃａｍｂｒｉｄｇｅ，　ＣＢ１０　１ＲＱ，　Ｅｎｇｌａｎｄ）、および、ｔｈｅ．ｅ．ｘＰＡＳｙ　Ｗｏｒｌｄ　Ｗｉｄｅ　Ｗｅｂ分子生物学用サーバー（Ｇｅｎｅｖａ　Ｕｎｉｖｅｒｓｉｔｙ　Ｈｏｓｐｉｔａｌ　ａｎｄ　Ｕｎｉｖｅｒｓｉｔｙ　ｏｆ　Ｇｅｎｅｖａ，　Ｇｅｎｅｖａ，　Ｓｗｉｔｚｅｒｌａｎｄ）。 The sequence identity of DNA can be calculated, for example, using the following sequence analysis tool: GCG Wisconsin Package (Program Manual Manual for The Wisconsin Package, Version 8, September 1994, Genetics Computer Corporation, Germany, Germany, Germany). Rice, P. 53 (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Center, Hinxton Hall, Cambridge, Q.E.B., and CB10R. e. xPASy World Wide Web Server for Molecular Biology (Geneva University Hospital International University of Geneva, Geneva, Switzerland).
　上記（１ｅ）において、配列番号２に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡは、「Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（Ｊｏｈｎ　Ｗｉｌｅｙ　ａｎｄ　Ｓｏｎｓ，　Ｉｎｃ．，　１９８９）」等に記載の公知の方法に準じて調製することができる。 In the above (1e), DNA in which one or more bases have been substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 2 is referred to as “Current Protocols in Molecular Biology (John Wiley and Sons, Inc.). , {1989)] ”and the like.
　置換、挿入、欠失および／または付加により改変された塩基配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種類以上の改変（例えば、置換と挿入）を含んでいても良い。 The nucleotide sequence modified by substitution, insertion, deletion and / or addition may include only one type of modification (for example, substitution) or two or more types of modification (for example, substitution and insertion). May be included.
　上記（１ｅ）において、「１もしくは複数個」の塩基とは、該ＤＮＡによりコードされるタンパク質がグルタチオンレダクターゼ活性を有する限り特に限定されないが、例えば１～１５０個、好ましくは１～１００個、より好ましくは１～５０個、さらに好ましくは１～２０個、さらにより好ましくは１～１０個、１～５個、１～４個、１～３個、または１～２個の塩基、を意味する。 In the above (1e), the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has glutathione reductase activity, but for example, 1 to 150, preferably 1 to 100, Preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases. .
　上記（１ａ）～（１ｅ）のタンパク質がグルタチオンレダクターゼ活性を有するタンパク質であることを確認する手段としては、例えばＤＮＡ組換え法を用いて活性を確認したいタンパク質を発現する形質転換体を作製し、該形質転換体を用いて該タンパク質を製造した後、該タンパク質、ならびに酸化型グルタチオン、ＮＡＤＰＨを水性媒体中に存在せしめ、該水性媒体中に還元型グルタチオンまたはＮＡＤＰが生成、蓄積するか否かをＨＰＬＣ等により分析する方法を挙げることができる。 As a means for confirming that the proteins (1a) to (1e) are proteins having glutathione reductase activity, for example, a transformant expressing the protein whose activity is to be confirmed using a DNA recombination method is prepared. After producing the protein using the transformant, the protein, oxidized glutathione, and NADPH are allowed to exist in an aqueous medium, and it is determined whether reduced glutathione or NADP is produced and accumulated in the aqueous medium. A method of analyzing by HPLC or the like can be mentioned.
　本発明において、グルタチオンレダクターゼ活性は、親株と比較して、好ましくは５０％以下、より好ましくは２０％以下、さらに好ましくは１０％以下、特に好ましくは５％以下に低下している。また、グルタチオンレダクターゼ活性は、実質的に消失していることが特に好ましい。 In the present invention, the glutathione reductase activity is reduced to preferably 50% or less, more preferably 20% or less, further preferably 10% or less, and particularly preferably 5% or less, as compared with the parent strain. It is particularly preferable that the glutathione reductase activity is substantially eliminated.
　本発明におけるグルタチオンレダクターゼとしては、上記タンパク質のうち、配列番号１に示すアミノ酸配列からなるＧＬＲ１が好ましい。配列番号２に、配列番号１に示すＧＬＲ１のアミノ酸配列をコードするＧＬＲ１遺伝子の塩基配列を示す。 Glutathione reductase in the present invention is preferably GLR1 having the amino acid sequence shown in SEQ ID NO: 1 among the above proteins. SEQ ID NO: 2 shows the nucleotide sequence of the GLR1 gene encoding the amino acid sequence of GLR1 shown in SEQ ID NO: 1.
　上記（１ｂ）、（１ｃ）、（１ｄ）または（１ｅ）のタンパク質のグルタチオンレダクターゼ活性は、好ましくは、上記（１ａ）のタンパク質のグルタチオンレダクターゼ活性と同程度又はそれ以上であり、より好ましくは、上記（１ａ）のタンパク質のグルタチオンレダクターゼ活性の５０％以上、８０％以上、９０％以上または１００％以上であり、より好ましくは、２００％以下又は１５０％以下である。 The glutathione reductase activity of the protein of (1b), (1c), (1d) or (1e) is preferably about the same or higher than the glutathione reductase activity of the protein of (1a), and more preferably It is 50% or more, 80% or more, 90% or more or 100% or more, more preferably 200% or less or 150% or less of the glutathione reductase activity of the protein (1a).
（２）γ－グルタミルシステイン合成酵素
　本発明におけるγ－グルタミルシステイン合成酵素は、グルタミン酸とシステインとを縮合させγ－グルタミルシステインを合成する活性（即ち、γ－グルタミルシステイン合成酵素活性）を有する酵素であれば良く、特に限定されないが、例えば、以下の（２ａ）～（２ｅ）：
（２ａ）配列番号３に示すアミノ酸配列からなるタンパク質、
（２ｂ）配列番号３に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
（２ｃ）配列番号３に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
（２ｄ）配列番号４と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、ならびに、
（２ｅ）配列番号４に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
のいずれかであることが好ましい。 (2) γ-glutamylcysteine synthase The γ-glutamylcysteine synthase in the present invention is an enzyme having an activity of synthesizing γ-glutamylcysteine by condensing glutamic acid and cysteine (ie, γ-glutamylcysteine synthase activity). There is no particular limitation, but for example, the following (2a) to (2e):
(2a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3,
(2b) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in which one or more amino acids have been deleted, substituted, inserted and / or added, and which has γ-glutamylcysteine synthetase activity;
(2c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and having γ-glutamylcysteine synthetase activity;
(2d) a protein comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 4, and having a γ-glutamylcysteine synthetase activity; and
(2e) one or more bases in the base sequence shown in SEQ ID NO: 4 consist of an amino acid sequence encoded by substitution, deletion, insertion and / or addition of DNA, and have γ-glutamylcysteine synthetase activity protein,
It is preferable that it is either.
　ここで、上記（２ａ）～（２ｅ）のいずれかのタンパク質は、上記（２ａ）～（２ｅ）で規定するアミノ酸配列からなるポリペプチド鎖のみからなる形態には限定されず、該ポリペプチド鎖が糖鎖等で化学修飾された形態であってもよいし、該ポリペプチド鎖が他のポリペプチド鎖と融合された融合タンパク質の形態であってもよい。 Here, the protein of any of the above (2a) to (2e) is not limited to the form consisting of only the polypeptide chain consisting of the amino acid sequence defined in the above (2a) to (2e), May be in the form chemically modified with a sugar chain or the like, or may be in the form of a fusion protein in which the polypeptide chain is fused with another polypeptide chain.
　上記（２ｂ）に記載の、配列番号３に示すアミノ酸配列において、１もしくは複数個のアミノ酸が置換、挿入、欠失および／または付加されたアミノ酸配列からなるタンパク質は、上記（１）に記載の方法に準じて調製することができ、γ－グルタミルシステイン合成酵素活性を有する限り上記タンパクに包含される。 In the amino acid sequence of SEQ ID NO: 3 described in (2b), a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, inserted, deleted and / or added is the protein described in (1). It can be prepared according to the method and is included in the above proteins as long as it has γ-glutamylcysteine synthase activity.
　置換、挿入、欠失および／または付加により改変されたアミノ酸配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種以上の改変（例えば、置換と挿入）を含んでいても良い。また、置換の場合には、置換するアミノ酸は、置換前のアミノ酸と類似の性質を有するアミノ酸（同族アミノ酸）であることが好ましい。同族アミノ酸については（１）において上述した通りである。 The amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one kind of modification (for example, substitution) or may contain two or more kinds of modification (for example, substitution and insertion). May be included. In the case of substitution, the amino acid to be substituted is preferably an amino acid having similar properties to the amino acid before substitution (homologous amino acid). The homologous amino acids are as described above in (1).
　上記（２ｂ）において、「１もしくは複数個」のアミノ酸とは、例えば、１～６０個、好ましくは１～２０個、より好ましくは１～１５個、さらに好ましくは１～１０個、さらに好ましくは１～５個、１～４個、１～３個、または１～２個のアミノ酸を意味する。 In the above (2b), the “one or more” amino acids are, for example, 1 to 60, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, and still more preferably One to five, one to four, one to three, or one to two amino acids.
　上記（２ｃ）において、配列番号３に示すアミノ酸配列との配列同一性は、６０％以上が好ましいが、７０％以上がより好ましく、８０％以上がさらに好ましく、８５％以上がさらに好ましく、９０％以上がさらに好ましく、９５％以上がさらに好ましく、９７％以上がさらに好ましく、９８％以上がさらに好ましく、９９％以上が最も好ましい。アミノ酸配列の配列同一性は、（１）において上述した方法により算出できる。 In the above (2c), the sequence identity with the amino acid sequence shown in SEQ ID NO: 3 is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, further preferably 85% or more, and 90% The above is still more preferable, 95% or more is more preferable, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is most preferable. The sequence identity of the amino acid sequence can be calculated by the method described above in (1).
　上記（２ｄ）において、配列番号４に示す塩基配列と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡとは、配列番号４に示す塩基配列と相補的な塩基配列からなるＤＮＡをプローブとして、ストリンジェントな条件下にコロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法、あるいはサザンハイブリダイゼーション法等を用いることにより得られるＤＮＡを意味する。ハイブリダイゼーションの条件等は（１）において上述した通りである。 In the above (2d), a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 4 and a DNA hybridizing under stringent conditions are a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 4 And DNA obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like under stringent conditions using the resulting DNA as a probe. Hybridization conditions and the like are as described above in (1).
　上記の条件にてハイブリダイズ可能なＤＮＡとしては、配列番号４に示されるＤＮＡとの配列同一性が７０％以上、好ましくは７４％以上、より好ましくは７９％以上、さらに好ましくは８５％以上、さらにより好ましくは９０％以上、さらにより好ましくは９５％以上、さらにより好ましくは９７％以上、さらにより好ましくは９８％以上、最も好ましくは９９％以上のＤＮＡを挙げることができる。ＤＮＡの配列同一性（％）は（１）において上述した通りである。 The DNA hybridizable under the above conditions has a sequence identity of 70% or more, preferably 74% or more, more preferably 79% or more, further preferably 85% or more, with the DNA shown in SEQ ID NO: 4. Even more preferably, 90% or more, even more preferably, 95% or more, even more preferably, 97% or more, even more preferably, 98% or more, and most preferably, 99% or more of DNA. DNA sequence identity (%) is as described above in (1).
　上記（２ｅ）において、配列番号４に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および/または付加されたＤＮＡは、（１）において上述した方法に準じて調製することができる。 In the above (2e), DNA in which one or more bases have been substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 4 can be prepared according to the method described above in (1). it can.
　置換、挿入、欠失および／または付加により改変された塩基配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種類以上の改変（例えば、置換と挿入）を含んでいても良い。 The nucleotide sequence modified by substitution, insertion, deletion and / or addition may include only one type of modification (for example, substitution) or two or more types of modification (for example, substitution and insertion). May be included.
　上記（２ｅ）において、「１もしくは複数個」の塩基とは、該ＤＮＡがコードするタンパク質がγ－グルタミルシステイン合成酵素活性を有する限り特に限定されないが、例えば１～１５０個、好ましくは１～１００個、より好ましくは１～５０個、さらに好ましくは１～２０個、さらにより好ましくは１～１０個、１～５個、１～４個、１～３個、または１～２個の塩基、を意味する。 In the above (2e), the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has γ-glutamylcysteine synthetase activity, and for example, 1 to 150, preferably 1 to 100 , More preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 1, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases, Means
　上記（２ａ）～（２ｅ）のタンパク質がγ－グルタミルシステイン合成酵素活性を有するタンパク質であることを確認する手段としては、例えばＤＮＡ組換え法を用いて活性を確認したいタンパク質を発現する形質転換体を作製し、該形質転換体を用いて該タンパク質を製造した後、該タンパク質、ならびにＬ－グルタミン酸およびＬ－システインを水性媒体中に存在せしめ、該水性媒体中にγ－グルタミルシステインが生成、蓄積するか否かをＨＰＬＣ等により分析する方法を挙げることができる。前記水性媒体中には必要に応じ更にＡＴＰを存在させることが好ましい。 Means for confirming that the proteins (2a) to (2e) are proteins having γ-glutamylcysteine synthetase activity include, for example, a transformant expressing a protein whose activity is to be confirmed by DNA recombination. And producing the protein using the transformant. Then, the protein, L-glutamic acid and L-cysteine are allowed to exist in an aqueous medium, and γ-glutamylcysteine is produced and accumulated in the aqueous medium. A method of analyzing whether or not to do so by HPLC or the like can be mentioned. It is preferable that ATP is further present in the aqueous medium as needed.
　本発明におけるγ－グルタミルシステイン合成酵素としては、上記タンパク質のうち、配列番号３に示すアミノ酸配列からなるＧＳＨ１が好ましい。配列番号４に、配列番号３に示すＧＳＨ１のアミノ酸配列をコードするＧＳＨ１遺伝子の塩基配列を示す。 Γ As the γ-glutamylcysteine synthetase in the present invention, GSH1 having the amino acid sequence shown in SEQ ID NO: 3 is preferable among the above proteins. SEQ ID NO: 4 shows the nucleotide sequence of the GSH1 gene encoding the amino acid sequence of GSH1 shown in SEQ ID NO: 3.
　上記（２ｂ）、（２ｃ）、（２ｄ）または（２ｅ）のタンパク質のγ－グルタミルシステイン合成酵素活性は、好ましくは、上記（２ａ）のタンパク質のγ－グルタミルシステイン合成酵素活性と同程度又はそれ以上であり、より好ましくは、上記（２ａ）のタンパク質のγ－グルタミルシステイン合成酵素活性の５０％以上、８０％以上、９０％以上または１００％以上であり、より好ましくは、２００％以下又は１５０％以下である。 The γ-glutamylcysteine synthetase activity of the protein of the above (2b), (2c), (2d) or (2e) is preferably similar to or higher than that of the protein of the above (2a). And more preferably at least 50%, at least 80%, at least 90% or at least 100% of the γ-glutamylcysteine synthetase activity of the protein of (2a), more preferably at most 200% or at least 150%. % Or less.
（３）グルタチオン合成酵素
　本発明におけるグルタチオン合成酵素は、γ－グルタミルシステインとグリシンを縮合させグルタチオンを合成する活性（即ち、グルタチオン合成酵素活性）を有する酵素であれば良く、特に限定されないが、例えば、以下の（３ａ）～（３ｅ）：
（３ａ）配列番号５に示すアミノ酸配列からなるタンパク質、
（３ｂ）配列番号５に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
（３ｃ）配列番号５に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
（３ｄ）配列番号６と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、ならびに、
（３ｅ）配列番号６に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
のいずれかであることが好ましい。 (3) Glutathione synthase The glutathione synthase in the present invention may be any enzyme having an activity of synthesizing glutathione by condensing γ-glutamylcysteine and glycine (ie, glutathione synthase activity), and is not particularly limited. The following (3a) to (3e):
(3a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5,
(3b) a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 5, and having a glutathione synthase activity;
(3c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and having a glutathione synthase activity;
(3d) a protein comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 6, and having a glutathione synthase activity; and
(3e) a protein having an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 6, and which has glutathione synthase activity;
It is preferable that it is either.
　ここで、上記（３ａ）～（３ｅ）のいずれかのタンパク質は、上記（３ａ）～（３ｅ）で規定するアミノ酸配列からなるポリペプチド鎖のみからなる形態には限定されず、該ポリペプチド鎖が糖鎖等で化学修飾された形態であってもよいし、該ポリペプチド鎖が他のポリペプチド鎖と融合された融合タンパク質の形態であってもよい。 Here, the protein of any of the above (3a) to (3e) is not limited to the form consisting of only the polypeptide chain consisting of the amino acid sequence defined in the above (3a) to (3e), May be in the form chemically modified with a sugar chain or the like, or may be in the form of a fusion protein in which the polypeptide chain is fused with another polypeptide chain.
　上記（３ｂ）に記載の、配列番号５に示すアミノ酸配列において、１もしくは複数個のアミノ酸が置換、挿入、欠失および／または付加されたアミノ酸配列からなるタンパク質は、上記（１）に記載の方法に準じて調製することができ、グルタチオン合成酵素活性を有する限り上記タンパクに包含される。 In the amino acid sequence of SEQ ID NO: 5 described in (3b) above, a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, inserted, deleted and / or added is the protein described in (1) above. It can be prepared according to the method, and is included in the above proteins as long as it has glutathione synthase activity.
　置換、挿入、欠失および／または付加により改変されたアミノ酸配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種以上の改変（例えば、置換と挿入）を含んでいても良い。また、置換の場合には、置換するアミノ酸は、置換前のアミノ酸と類似の性質を有するアミノ酸（同族アミノ酸）であることが好ましい。同族アミノ酸については（１）において上述した通りである。 The amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one kind of modification (for example, substitution) or may contain two or more kinds of modification (for example, substitution and insertion). May be included. In the case of substitution, the amino acid to be substituted is preferably an amino acid having similar properties to the amino acid before substitution (homologous amino acid). The homologous amino acids are as described above in (1).
　上記（３ｂ）において、「１もしくは複数個」のアミノ酸とは、例えば、１～６０個、好ましくは１～２０個、より好ましくは１～１５個、さらに好ましくは１～１０個、さらに好ましくは１～５個、１～４個、１～３個、または１～２個のアミノ酸を意味する。 In the above (3b), the “one or more” amino acids are, for example, 1 to 60, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, and still more preferably One to five, one to four, one to three, or one to two amino acids.
　上記（３ｃ）において、配列番号５に示すアミノ酸配列との配列同一性は、６０％以上が好ましいが、７０％以上がより好ましく、８０％以上がさらに好ましく、８５％以上がさらに好ましく、９０％以上がさらに好ましく、９５％以上がさらに好ましく、９７％以上がさらに好ましく、９８％以上がさらに好ましく、９９％以上が最も好ましい。アミノ酸配列の配列同一性は、（１）において上述した方法により算出できる。 In the above (3c), the sequence identity with the amino acid sequence shown in SEQ ID NO: 5 is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, further preferably 85% or more, and 90% The above is still more preferable, 95% or more is more preferable, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is most preferable. The sequence identity of the amino acid sequence can be calculated by the method described above in (1).
　上記（３ｄ）において、配列番号６に示す塩基配列と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡとは、配列番号６に示す塩基配列と相補的な塩基配列からなるＤＮＡをプローブとして、ストリンジェントな条件下にコロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法、あるいはサザンハイブリダイゼーション法等を用いることにより得られるＤＮＡを意味する。ハイブリダイゼーションの条件等は（１）において上述した通りである。 In the above (3d), a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 6 is obtained from the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 6 And DNA obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like under stringent conditions using the resulting DNA as a probe. Hybridization conditions and the like are as described above in (1).
　上記の条件にてハイブリダイズ可能なＤＮＡとしては、配列番号６に示されるＤＮＡとの配列同一性が７０％以上、好ましくは７４％以上、より好ましくは７９％以上、さらに好ましくは８５％以上、さらにより好ましくは９０％以上、さらにより好ましくは９５％以上、さらにより好ましくは９７％以上、さらにより好ましくは９８％以上、最も好ましくは９９％以上のＤＮＡを挙げることができる。ＤＮＡの配列同一性（％）は（１）において上述した通りである。 As DNA hybridizable under the above conditions, the sequence identity to the DNA shown in SEQ ID NO: 6 is 70% or more, preferably 74% or more, more preferably 79% or more, and still more preferably 85% or more. Even more preferably, 90% or more, even more preferably, 95% or more, even more preferably, 97% or more, even more preferably, 98% or more, and most preferably, 99% or more of DNA. DNA sequence identity (%) is as described above in (1).
　上記（３ｅ）において、配列番号６に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および/または付加されたＤＮＡは、（１）において上述した方法に準じて調製することができる。 In the above (3e), DNA in which one or more bases have been substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 6 can be prepared according to the method described above in (1). it can.
　置換、挿入、欠失および/または付加により改変された塩基配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種類以上の改変（例えば、置換と挿入） を含んでいても良い。 The nucleotide sequence modified by substitution, insertion, deletion and / or addition may include only one type of modification (eg, substitution) or may include two or more types of modification (eg, substitution and insertion) May be included.
　上記（３ｅ）において、「１もしくは複数個」の塩基とは、該ＤＮＡがコードするタンパク質がグルタチオン合成酵素活性を有する限り特に限定されないが、例えば１～１５０個、好ましくは１～１００個、より好ましくは１～５０個、さらに好ましくは１～２０個、さらにより好ましくは１～１０個、１～５個、１～４個、１～３個、または１～２個の塩基、を意味する。 In the above (3e), the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has glutathione synthase activity, and for example, 1 to 150, preferably 1 to 100, Preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases. .
　上記（３ａ）～（３ｅ）のタンパク質がグルタチオン合成酵素活性を有するタンパク質であることを確認する手段としては、例えばＤＮＡ組換え法を用いて活性を確認したいタンパク質を発現する形質転換体を作製し、該形質転換体を用いて該タンパク質を製造した後、該タンパク質ならびにγ－グルタミルシステインおよびグリシンを水性媒体中に存在せしめ、該水性媒体中にグルタチオンが生成、蓄積するか否かをＨＰＬＣ等により分析する方法を挙げることができる。前記水性媒体中には必要に応じ更にＡＴＰを存在させることが好ましい。 As means for confirming that the proteins (3a) to (3e) are proteins having glutathione synthase activity, for example, a transformant expressing the protein whose activity is to be confirmed using a DNA recombination method is prepared. After producing the protein using the transformant, the protein and γ-glutamylcysteine and glycine are allowed to exist in an aqueous medium, and whether or not glutathione is produced and accumulated in the aqueous medium is determined by HPLC or the like. Methods for analysis can be mentioned. It is preferable that ATP is further present in the aqueous medium as needed.
　本発明におけるグルタチオン合成酵素としては、配列番号５に示すアミノ酸配列からなるＧＳＨ２が好ましい。配列番号６に、配列番号５に示すＧＳＨ２のアミノ酸配列をコードするＧＳＨ２遺伝子の塩基配列を示す。 GSH2 having the amino acid sequence shown in SEQ ID NO: 5 is preferable as the glutathione synthetase in the present invention. SEQ ID NO: 6 shows the nucleotide sequence of the GSH2 gene encoding the amino acid sequence of GSH2 shown in SEQ ID NO: 5.
　上記（３ｂ）、（３ｃ）、（３ｄ）または（３ｅ）のタンパク質のグルタチオン合成酵素活性は、好ましくは、上記（３ａ）のタンパク質のグルタチオン合成酵素活性と同程度又はそれ以上であり、より好ましくは、上記（３ａ）のタンパク質のグルタチオン合成酵素活性の５０％以上、８０％以上、９０％以上または１００％以上であり、より好ましくは、２００％以下又は１５０％以下である。 The glutathione synthetase activity of the protein of (3b), (3c), (3d) or (3e) is preferably about the same or higher than the glutathione synthase activity of the protein of (3a), and more preferably. Is 50% or more, 80% or more, 90% or more, or 100% or more, more preferably 200% or less or 150% or less of the glutathione synthase activity of the protein of (3a).
（４）グルタチオン輸送酵素
　本発明において、グルタチオン輸送酵素とは、細胞質のグルタチオンを液胞に輸送する機能（即ち、グルタチオン輸送酵素活性）を有する酵素を意味し、当該機能を有する限り特に限定されない。
　グルタチオン輸送酵素は、以下の（４ａ）～（４ｅ）：
（４ａ）配列番号７に示すアミノ酸配列からなるタンパク質、
（４ｂ）配列番号７に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
（４ｃ）配列番号７に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
（４ｄ）配列番号８と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、ならびに、
（４ｅ）配列番号８に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
のいずれかであることが好ましい。 (4) Glutathione transfer enzyme In the present invention, the glutathione transfer enzyme means an enzyme having a function of transferring cytosolic glutathione to the vacuole (that is, glutathione transfer enzyme activity), and is not particularly limited as long as it has the function.
Glutathione transferases include the following (4a) to (4e):
(4a) a protein consisting of the amino acid sequence of SEQ ID NO: 7,
(4b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 7, and which has a glutathione transferase activity;
(4c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and having a glutathione transferase activity;
(4d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 8, and having a glutathione transferase activity; and
(4e) a protein comprising an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 8, and which has a glutathione transferase activity;
It is preferable that it is either.
　ここで、上記（４ａ）～（４ｅ）のいずれかのタンパク質は、上記（４ａ）～（４ｅ）で規定するアミノ酸配列からなるポリペプチド鎖のみからなる形態には限定されず、該ポリペプチド鎖が糖鎖等で化学修飾された形態であってもよいし、該ポリペプチド鎖が他のポリペプチド鎖と融合された融合タンパク質の形態であってもよい。 Here, the protein of any of the above (4a) to (4e) is not limited to the form consisting of only the polypeptide chain consisting of the amino acid sequence defined in the above (4a) to (4e), May be in the form chemically modified with a sugar chain or the like, or may be in the form of a fusion protein in which the polypeptide chain is fused with another polypeptide chain.
　上記（４ｂ）に記載の、配列番号７に示すアミノ酸配列において、１もしくは複数個のアミノ酸が置換、挿入、欠失および／または付加されたアミノ酸配列からなるタンパク質は、上記（１）に記載の方法に準じて調製することができ、グルタチオン輸送酵素活性を有する限り上記タンパクに包含される。 In the amino acid sequence shown in SEQ ID NO: 7 described in (4b) above, a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, inserted, deleted and / or added is the protein described in (1) above. It can be prepared according to the method, and is included in the above proteins as long as it has glutathione transferase activity.
　置換、挿入、欠失および／または付加により改変されたアミノ酸配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種以上の改変（例えば、置換と挿入）を含んでいても良い。また、置換の場合には、置換するアミノ酸は、置換前のアミノ酸と類似の性質を有するアミノ酸（同族アミノ酸）であることが好ましい。同族アミノ酸については（１）において上述した通りである。 The amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one kind of modification (for example, substitution) or may contain two or more kinds of modification (for example, substitution and insertion). May be included. In the case of substitution, the amino acid to be substituted is preferably an amino acid having similar properties to the amino acid before substitution (homologous amino acid). The homologous amino acids are as described above in (1).
　上記（４ｂ）において、「１もしくは複数個」のアミノ酸とは、例えば、１～６０個、好ましくは１～２０個、より好ましくは１～１５個、さらに好ましくは１～１０個、さらに好ましくは１～５個、１～４個、１～３個、または１～２個のアミノ酸を意味する。 In the above (4b), “one or more” amino acids refer to, for example, 1 to 60, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, and still more preferably One to five, one to four, one to three, or one to two amino acids.
　上記（４ｃ）において、配列番号７に示すアミノ酸配列との配列同一性は、６０％以上が好ましいが、７０％以上がより好ましく、８０％以上がさらに好ましく、８５％以上がさらに好ましく、９０％以上がさらに好ましく、９５％以上がさらに好ましく、９７％以上がさらに好ましく、９８％以上がさらに好ましく、９９％以上が最も好ましい。アミノ酸配列の配列同一性は、（１）において上述した方法により算出できる。 In the above (4c), the sequence identity with the amino acid sequence shown in SEQ ID NO: 7 is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, further preferably 85% or more, and 90% The above is still more preferable, 95% or more is more preferable, 97% or more is more preferable, 98% or more is more preferable, and 99% or more is most preferable. The sequence identity of the amino acid sequence can be calculated by the method described above in (1).
　上記（４ｄ）において、配列番号８に示す塩基配列と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡとは、配列番号８に示す塩基配列と相補的な塩基配列からなるＤＮＡをプローブとして、ストリンジェントな条件下にコロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法、あるいはサザンハイブリダイゼーション法等を用いることにより得られるＤＮＡを意味する。ハイブリダイゼーションの条件等は（１）において上述した通りである。 In the above (4d), a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8 and a DNA hybridizing under stringent conditions are a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8 And DNA obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like under stringent conditions using the resulting DNA as a probe. Hybridization conditions and the like are as described above in (1).
　上記の条件にてハイブリダイズ可能なＤＮＡとしては、配列番号８に示されるＤＮＡとの配列同一性が７０％以上、好ましくは７４％以上、より好ましくは７９％以上、さらに好ましくは８５％以上、さらにより好ましくは９０％以上、さらにより好ましくは９５％以上、さらにより好ましくは９７％以上、さらにより好ましくは９８％以上、最も好ましくは９９％以上のＤＮＡを挙げることができる。ＤＮＡの配列同一性（％）は（１）において上述した通りである。 The DNA capable of hybridizing under the above conditions has a sequence identity with the DNA shown in SEQ ID NO: 8 of 70% or more, preferably 74% or more, more preferably 79% or more, and still more preferably 85% or more. Even more preferably, 90% or more, even more preferably, 95% or more, even more preferably, 97% or more, even more preferably, 98% or more, and most preferably, 99% or more of DNA. DNA sequence identity (%) is as described above in (1).
　上記（４ｅ）において、配列番号８に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および/または付加されたＤＮＡは、（１）において上述した方法に準じて調製することができる。 In the above (4e), the DNA in which one or more bases have been substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 8 can be prepared according to the method described above in (1). it can.
　置換、挿入、欠失および/または付加により改変された塩基配列としては、１種類の改変（例えば置換）のみを含むものであっても良いし、２種類以上の改変（例えば、置換と挿入）を含んでいても良い。 The nucleotide sequence modified by substitution, insertion, deletion and / or addition may include only one type of modification (eg, substitution) or may include two or more types of modification (eg, substitution and insertion) May be included.
　上記（４ｅ）において、「１もしくは複数個」の塩基とは、該ＤＮＡがコードするタンパク質がグルタチオン輸送酵素活性を有する限り特に限定されないが、例えば１～１５０個、好ましくは１～１００個、より好ましくは１～５０個、さらに好ましくは１～２０個、さらにより好ましくは１～１０個、１～５個、１～４個、１～３個、または１～２個の塩基、を意味する。 In the above (4e), the “one or more” bases are not particularly limited as long as the protein encoded by the DNA has glutathione transferase activity, but for example, 1 to 150, preferably 1 to 100, Preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases. .
　本発明におけるグルタチオン輸送酵素としては、配列番号７に示すアミノ酸配列からなるＹＣＦ１が好ましい。配列番号８に、配列番号７に示すＹＣＦ１のアミノ酸配列をコードするＹＣＦ１遺伝子の塩基配列を示す。 グ ル As the glutathione transferase in the present invention, YCF1 having the amino acid sequence shown in SEQ ID NO: 7 is preferable. SEQ ID NO: 8 shows the base sequence of the YCF1 gene encoding the amino acid sequence of YCF1 shown in SEQ ID NO: 7.
　上記（４ｂ）、（４ｃ）、（４ｄ）または（４ｅ）のタンパク質のグルタチオン輸送酵素活性は、好ましくは、上記（４ａ）のタンパク質のグルタチオン輸送酵素活性と同程度又はそれ以上であり、より好ましくは、上記（４ａ）のタンパク質のグルタチオン輸送酵素活性の５０％以上、８０％以上、９０％以上または１００％以上であり、より好ましくは、２００％以下又は１５０％以下である。 The glutathione transferase activity of the protein of the above (4b), (4c), (4d) or (4e) is preferably equal to or more than the glutathione transferase activity of the protein of the above (4a), and more preferably. Is 50% or more, 80% or more, 90% or more, or 100% or more, more preferably 200% or less, or 150% or less of the glutathione transferase activity of the protein (4a).
（５）本発明の酵母
　本発明の酵母は、グルタチオン生産能を有する酵母であれば、特に制限はなく、例えば、サッカロマイセス・セレビシエ(Saccharomyces cerevisiae)、サッカロマイセス・カールスベルゲンシス(Saccharomyces carlsbergensis)、サッカロマイセス・フラギリス(Saccharomyces fragilis)、サッカロマイセス・ルーキシー(Saccharomyces rouxii)などのサッカロマイセス属、キャンディダ・ユーティリス(Candida utilis)、キャンディダ・トロピカリス(Candida tropicalis)などのキャンディダ属、シゾサッカロマイセス・ポンべ(Schizosaccharomyces pombe)などのシゾサッカロミセス属、トルロプシス・バーサティリス(Torulopsis versatilis)、トルロプシス・ペトロフィラム(Torulopsis petrophilum)などのトルロプシス属、ピキア（Pichia）属、ブレタノマイセス(Brettanomyces)属、マイコトルラ(Mycotorula)属、ロードトルラ(Rhodotorula)属、ハンゼニュラ(Hansenula)属、エンドマイセス(Endomyces)属などの酵母が挙げられる。中でも、サッカロマイセス属、キャンディダ属、またはピキア属の酵母が好ましく、更にはサッカロマイセス属のサッカロマイセス・セレビシエ、キャンディダ属のキャンディダ・ユーティリスが好ましい。例えば、親株として、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　２４－５１－７８（受託番号：ＦＥＲＭ　ＢＰ－１９０７２）を好適に使用することができる。Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　２４－５１－７８は、２００２年１０月１８日付で独立行政法人　製品評価技術基盤機構　特許生物寄託センター（日本国千葉県木更津市かずさ鎌足２－５－８　１２０号室）に国際寄託されている（寄託者が付した識別のための表示：Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　２４－５１－７８、受託番号：ＦＥＲＭ　ＢＰ－１９０７２）。 (5) Yeast of the present invention The yeast of the present invention is not particularly limited as long as it has a glutathione-producing ability. For example, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Saccharomyces Fragilis (Saccharomyces fragilis), Saccharomyces rouxii (Saccharomyces rouxii) and other Saccharomyces genus, Candida utilis (Candida utilis), Candida genus such as Candida tropicalis (Candida tropicalis), Schizosaccharomyces pombe ( Schizosaccharomyces pombe and the like, Torulopsis versatilis (Torulopsis versatilis), Torulopsis petrophilum (Torulopsis petrophilum) and the like genus Torropsis, Pichia (genus Pichia), genus Brettanomyces (Brettanomyces) Yeasts such as Mycotorula, Rhodotorula, Hansenula, and Endomyces are exemplified. Among them, yeast of the genus Saccharomyces, Candida or Pichia is preferable, and Saccharomyces cerevisiae of the genus Saccharomyces and Candida utilis of the genus Candida are more preferable. For example, Saccharomyces cerevisiae 24-51-78 (accession number: FERM BP-19072) can be suitably used as a parent strain. Saccharomyces cerevisiae 24-51-78 was deposited internationally on October 18, 2002 with the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Patent Organism Depositary Center (Room 2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba, Japan). (Indication by the depositor for identification: Saccharomyces cerevisiae 24-51-78, accession number: FERM BP-19072).
（６）塩基性アミノ酸を含有する培地
　本発明において「塩基性アミノ酸」とは、典型的には、リジン、アルギニンおよびヒスチジンから選択される１以上であり、より好ましくは、リジンおよびアルギニンから選択される１以上であり、最も好ましくはリジンである。塩基性アミノ酸は好ましくはＬ体である。 (6) Medium containing basic amino acid In the present invention, the “basic amino acid” is typically one or more selected from lysine, arginine and histidine, more preferably selected from lysine and arginine. And most preferably lysine. The basic amino acid is preferably in the L-form.
　本発明で用いる培地は、塩基性アミノ酸の濃度が０．８ｇ／Ｌ以上であり、好ましくは１ｇ／Ｌ以上であり、より好ましくは２ｇ／Ｌ以上であり、特に好ましくは４ｇ／Ｌ以上である。２種以上の塩基性アミノ酸が含まれる場合、塩基性アミノ酸の合計濃度が上記の範囲であればよいが、より好ましくは、各塩基性アミノ酸の濃度がそれぞれ０．８ｇ／Ｌ以上であり、好ましくは１ｇ／Ｌ以上であり、より好ましくは２ｇ／Ｌ以上であり、特に好ましくは４ｇ／Ｌ以上である。 The medium used in the present invention has a basic amino acid concentration of 0.8 g / L or more, preferably 1 g / L or more, more preferably 2 g / L or more, and particularly preferably 4 g / L or more. . When two or more basic amino acids are contained, the total concentration of the basic amino acids may be within the above range. More preferably, the concentration of each basic amino acid is at least 0.8 g / L, and more preferably Is 1 g / L or more, more preferably 2 g / L or more, and particularly preferably 4 g / L or more.
　本発明で用いる培地の塩基性アミノ酸の濃度の上限は特に限定されないが、通常は１００ｇ／Ｌ以下であり、好ましくは５０ｇ／Ｌ以下であり、より好ましくは２０ｇ／Ｌ以下である。２種以上の塩基性アミノ酸が含まれる場合、各塩基性アミノ酸の濃度が、それぞれ、通常は１００ｇ／Ｌ以下であり、好ましくは５０ｇ／Ｌ以下であり、より好ましくは２０ｇ／Ｌ以下である。 上限 The upper limit of the concentration of the basic amino acid in the medium used in the present invention is not particularly limited, but is usually 100 g / L or less, preferably 50 g / L or less, and more preferably 20 g / L or less. When two or more basic amino acids are contained, the concentration of each basic amino acid is usually 100 g / L or less, preferably 50 g / L or less, and more preferably 20 g / L or less.
　塩基性アミノ酸は、塩の形態で存在してもよい。 The basic amino acid may be present in the form of a salt.
　本発明で用いる培地は、酵母の培養開始時において、塩基性アミノ酸を上記の濃度範囲となるように含有することが好ましい。本発明者らは、驚くべきことに、培養開始時に塩基性アミノ酸を上記の濃度範囲で含む培地中で酵母を培養する場合に、グルタチオン高含有効果が特に高いことを見出した。この場合、酵母の培養中に塩基性アミノ酸の培地中の濃度が０．８ｇ／Ｌを下回ることも本実施形態の範囲内である。 培 地 The medium used in the present invention preferably contains a basic amino acid in the above-mentioned concentration range at the start of culturing yeast. The present inventors have surprisingly found that when a yeast is cultured in a medium containing a basic amino acid in the above concentration range at the start of the culture, the effect of high glutathione content is particularly high. In this case, it is also within the scope of the present embodiment that the concentration of the basic amino acid in the medium during culture of the yeast is lower than 0.8 g / L.
　本発明で用いる培地は、塩基性アミノ酸を含有する培地であればよく、その他の成分は特に限定されない。すなわち炭素源、窒素源、無機物その他の栄養等を適宜含有していれば、合成培地、半合成培地、天然（複合）培地のいずれも使用可能である。 培 地 The medium used in the present invention may be any medium containing a basic amino acid, and other components are not particularly limited. That is, any of a synthetic medium, a semi-synthetic medium, and a natural (composite) medium can be used as long as the medium appropriately contains a carbon source, a nitrogen source, inorganic substances, and other nutrients.
　本発明で用いる培地は液体培地であってもよいし、固形培地であってもよいが、液体培地であることが特に好ましい。 培 地 The medium used in the present invention may be a liquid medium or a solid medium, but a liquid medium is particularly preferred.
　塩基性アミノ酸を含有する培地に含まれる炭素源としては糖蜜、グルコース、グリセロール、フラクトース、シュクロース、マルトース、マンノース、マニトール、キシロース、ガラクトース、デンプン、デンプン加水分解物液などの種々の炭水化物原料が使用できる。またピルビン酸、酢酸、乳酸などの各種有機酸、アスパラギン酸、アラニンなどの各種アミノ酸類も使用可能である。 Various carbohydrate raw materials such as molasses, glucose, glycerol, fructose, sucrose, maltose, mannose, mannitol, xylose, galactose, starch, and starch hydrolyzate are used as the carbon source contained in the medium containing basic amino acids. it can. Various organic acids such as pyruvic acid, acetic acid, and lactic acid, and various amino acids such as aspartic acid and alanine can also be used.
　塩基性アミノ酸を含有する培地の特に好ましい実施形態は、炭素源として糖蜜を含む。糖蜜とは、サトウキビ、テンサイ等の原料から砂糖を精製する際に生じる、糖を主成分とする粘状で黒褐色の液体副産物である。塩基性アミノ酸を含有する培地は、より好ましくは、糖蜜中に含まれる糖として１重量％以上、より好ましくは２重量％以上、より好ましくは３重量％以上となる量の糖蜜を含む液体培地であり、糖蜜の含有量の上限は特に限定されないが、例えば、糖蜜中に含まれる糖として１０重量％以下、５重量％以下、４重量％以下となる量の糖蜜を含む液体培地である。塩基性アミノ酸を含有する培地は、糖蜜以外の炭素源を更に含んでもよいが、より好ましくは、糖蜜を炭素源全体の少なくとも１０重量％以上とする培地（糖蜜培地）である。糖蜜中に含まれる糖は酸加水分解後ベルトラン法によって測定することができる。 A particularly preferred embodiment of the medium containing basic amino acids comprises molasses as a carbon source. Molasses is a viscous, black-brown liquid by-product containing sugar as a main component, which is generated when sugar is purified from raw materials such as sugarcane and sugar beet. The medium containing a basic amino acid is more preferably a liquid medium containing molasses in an amount of 1% by weight or more, more preferably 2% by weight or more, more preferably 3% by weight or more as sugar contained in the molasses. Yes, the upper limit of the molasses content is not particularly limited, and for example, a liquid medium containing molasses in an amount of 10% by weight or less, 5% by weight or less, and 4% by weight or less as sugar contained in the molasses. The medium containing a basic amino acid may further contain a carbon source other than molasses, but is more preferably a medium containing molasses at least 10% by weight or more of the total carbon source (molasses medium). The sugar contained in molasses can be measured by the Bertrand method after acid hydrolysis.
　塩基性アミノ酸を含有する培地に含まれる窒素源としてはアンモニアあるいは塩化アンモニウム、リン酸アンモニウム、硫酸アンモニウム、硝酸アンモニウム、炭酸アンモニウム、酢酸アンモニウムなどの各種の無機または有機アンモニウム塩類、あるいは尿素その他の窒素含有化合物、あるいはペプトン、ＮＺアミン、肉エキス、酵母エキス、コーンスティープリカー、カゼイン加水分解物、フィシュミールあるいはその消化物、脱脂大豆あるいはその消化物や加水分解物などの窒素性有機物質、あるいはアスパラギン酸、グルタミン酸、スレオニンなどの各種アミノ酸が使用可能である。 As a nitrogen source contained in a medium containing a basic amino acid, ammonia or ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, ammonium carbonate, various inorganic or organic ammonium salts such as ammonium acetate, or urea and other nitrogen-containing compounds, Alternatively, nitrogenous organic substances such as peptone, NZ amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fishmeal or its digest, defatted soybean or its digest or hydrolyzate, or aspartic acid or glutamic acid And various amino acids such as threonine can be used.
　塩基性アミノ酸を含有する培地に含まれる無機物としてはリン酸第一水素カリウム、リン酸第二水素カリウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、炭酸カルシウムなどが使用できる。 無機 As inorganic substances contained in the medium containing a basic amino acid, potassium hydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate and the like can be used.
（７）酵母によるグルタチオンの生産
　本発明の方法は、酵母を所定濃度の塩基性アミノ酸を含む培地中で培養することを含む。
　培養は振とう培養、通気攪拌深部培養などの好気的培養条件下で実施する。培養時ｐＨは、好ましくは３．０～８．０、より好ましくは４．０～６．０、最も好ましくは５．０に制御される。ｐＨ制御の中和剤としてはアンモニア水、水酸化ナトリウム、炭酸アンモニウムなどが使用可能である。好気的培養時の培地への空気供給量は、培地１Ｌに対し、好ましくは０．２Ｌ／分以上、より好ましくは０．５Ｌ／分以上、さらに好ましくは１Ｌ／分以上である。また、ジャーファーメンターを使用して培養する場合、攪拌数は好ましくは２００ｒｐｍ以上、より好ましくは３００ｒｐｍ以上、さらに好ましくは４００ｒｐｍ以上である。ジャーファーメンターの容量は特に限定されないが例えば総容量２Ｌのジャーファーメンターが例示できる。また、培養時の温度は、通常２０～４５℃、好ましくは２５～３５℃、最も好ましくは２８～３２℃である。培養期間は、通常１６時間～７２時間、好ましくは２４時間～４８時間である。培地中への初期細胞濃度（仕込み濃度）は酵母の種類、培地組成などにより異なるが、初発濁度（ＯＤ６００）は好ましくは０．０１～２．０、より好ましくは０．０２～１．０、さらに好ましくは０．１～０．４である。グルコースなどの上記炭素源に関しては培養初期に一括に仕込んで培養する回分方式、培養期間を通じて少しずつ添加する半回分方式のいずれの方式も適用可能であるが、本発明者らの検討結果によれば、使用する微生物にもよるが、半回分方式によれば、増殖速度が向上し、最終菌濃度もより高位となり、菌体内のグルタチオン合成関連酵素の活性も高まるのでより好ましい。半回分方式でグルコースなどの炭素源の流加速度を決定する指標としては、培養液の濁度、酸素消費速度、二酸化炭素発生速度、ｐＨ調整用中和剤の消費量などが利用でき、使用する酵母に合わせて適切な指標を選び、その変化に対応させて炭素源の流加速度を変化させることにより、酵母培養の最適化が図れ、最終菌濃度およびグルタチオン合成関連酵素の活性向上、その結果グルタチオンの生産性向上が実現できる。ただし上記の通り塩基性アミノ酸に関しては、培養初期に一括に回分方式により培地に添加することが好ましい。 (7) Production of glutathione by yeast The method of the present invention comprises culturing yeast in a medium containing a predetermined concentration of a basic amino acid.
The cultivation is performed under aerobic culturing conditions such as shaking culturing and aeration stirring submerged culturing. During the culture, the pH is controlled preferably at 3.0 to 8.0, more preferably at 4.0 to 6.0, and most preferably at 5.0. Ammonia water, sodium hydroxide, ammonium carbonate, and the like can be used as the pH control neutralizing agent. The amount of air supplied to the culture medium during aerobic culture is preferably 0.2 L / min or more, more preferably 0.5 L / min or more, and still more preferably 1 L / min or more, per 1 L of the culture medium. When culturing using a jar fermenter, the stirring speed is preferably 200 rpm or more, more preferably 300 rpm or more, and still more preferably 400 rpm or more. The capacity of the jar fermenter is not particularly limited. For example, a jar fermenter having a total capacity of 2 L can be exemplified. The temperature during the culturing is usually 20 to 45 ° C., preferably 25 to 35 ° C., and most preferably 28 to 32 ° C. The culturing period is usually 16 hours to 72 hours, preferably 24 hours to 48 hours. The initial cell concentration (prepared concentration) in the medium varies depending on the type of yeast, medium composition, etc., but the initial turbidity (OD600) is preferably 0.01 to 2.0, more preferably 0.02 to 1.0. And more preferably 0.1 to 0.4. Regarding the carbon source such as glucose, any of a batch system in which culturing is carried out by batch charging at the beginning of the culture, and a semi-batch system in which the mixture is added little by little throughout the culture period can be applied, but according to the results of the study by the present inventors. For example, although depending on the microorganism used, the semi-batch method is more preferable because the growth rate is improved, the final bacterial concentration is higher, and the activity of the glutathione synthesis-related enzyme in the bacterial body is also higher. As an index for determining the flow acceleration of a carbon source such as glucose in a semi-batch system, the turbidity of the culture solution, the rate of oxygen consumption, the rate of carbon dioxide generation, the consumption of a neutralizing agent for pH adjustment, and the like can be used. By selecting the appropriate index according to the yeast and changing the flow rate of the carbon source in response to the change, the yeast culture can be optimized, the final bacterial concentration and the activity of glutathione synthesis-related enzymes improved, and as a result glutathione Productivity can be improved. However, as described above, it is preferable that the basic amino acid be added to the medium in a batch system at the beginning of the culture.
　本発明の酵母を培地中で培養することで、高濃度のグルタチオンを主として菌体内に含有する酵母と培地とからなる培養混合物が得られる。本発明のグルタチオンの製造方法は、好ましくは、得られた培養混合物から、グルタチオンを回収することを更に含む。 培養 By culturing the yeast of the present invention in a medium, a culture mixture consisting of a yeast containing a high concentration of glutathione mainly in the cells and a medium can be obtained. The method for producing glutathione of the present invention preferably further includes recovering glutathione from the obtained culture mixture.
　培養混合物からグルタチオンを回収するためには、例えば、培養混合物中の酵母菌体を、濾過や遠心分離操作により培地と分離し、得られた酵母菌体から熱水抽出、アルカリ抽出法、酵素分解法、自己消化法、物理的な破砕操作などを必要に応じて適宜組み合わせてグルタチオンを抽出することができる。また、培養後の培養混合物に対し、直接に上記の抽出操作を実施してもよく、あるいは、酵素分解法、自己消化法、物理的な破砕操作を行って、酵母菌体中のグルタチオンを培地中に溶出させた後に、上記抽出操作を行って、グルタチオンを抽出しても良い。このようにして得られるグルタチオンから、一般的な方法によりグルタチオンを精製することによりグルタチオンを高度に含有する分画、またはグルタチオン粉末を得ることが出来る。 To recover glutathione from the culture mixture, for example, yeast cells in the culture mixture are separated from the medium by filtration or centrifugation, and the resulting yeast cells are subjected to hot water extraction, alkali extraction, Glutathione can be extracted by appropriately combining the method, autolysis method, physical crushing operation, and the like as necessary. In addition, the above-mentioned extraction operation may be directly performed on the culture mixture after the culture, or the enzymatic decomposition method, the autolysis method, and the physical crushing operation are performed to convert glutathione in the yeast cells into a medium. After being eluted in, glutathione may be extracted by performing the above extraction operation. Glutathione is purified from the thus obtained glutathione by a general method to obtain a fraction containing glutathione at a high level or a glutathione powder.
　また、上記の手順で得られたグルタチオンに含まれる酸化型グルタチオンを還元することにより、還元型の割合が高められたグルタチオンを得ることもできる。還元する方法としては特に限定しないが、好ましくはグルタチオンレダクターゼによる還元が挙げられる Further, by reducing oxidized glutathione contained in glutathione obtained by the above procedure, glutathione having an increased ratio of reduced form can also be obtained. The method for reduction is not particularly limited, but preferably includes reduction with glutathione reductase.
　以下、実施例などを挙げて本発明をより詳細に説明するが、本発明は以下の実施例などにより限定されない。なお、以下の実施例において、「乾燥菌体重量当たりの還元型グルタチオン含有量（％（ｗ／ｗ））」は、「ＧＳＨ含有率（％）」と言い換え、「乾燥菌体重量当たりの酸化型グルタチオン含有量（％（ｗ／ｗ））」は、「ＧＳＳＧ含有率（％）」と言い換えることがある。「％」は特に明示の無い限り「％（ｗ／ｗ）」を意味する。 Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited to the following Examples and the like. In the following examples, the “content of reduced glutathione per dry cell weight (% (w / w))” is referred to as “GSH content (%)”, which means “oxidation per dry cell weight”. “Glutathione content (% (w / w))” may be referred to as “GSSG content (%)”. “%” Means “% (w / w)” unless otherwise specified.
［製造例１］ＧＬＲ１遺伝子を破壊した酵母（ＧＬＲ１破壊株）の作製
　Ｓ．ｃｅｒｅｖｉｃｉａｅ用ｖｅｃｔｏｒ　ｐＤ１５１１（ＡＴＵＭ社）のｇＲＮＡ部分の配列を5′-GATTACCTCGTCATCGGGGG-3′（配列番号９）としたプラスミドを調製し、更にＤＮＡ配列5′-ACACTTCTGGTTTTTCTCATGCGCTTCTCACTCTCAGTATATTTTGCTGCTTTCCTTCATATGTATATATATCTATTTACATATTAGTTTACAGAACTTTAGCAACGAAACTAGACGTCCAATCTGCTGTTGCTATACTGGACTTTTGTACTCTTGTAAACAATCTTATATAGCATCCTGAAATACGTAGTAATTTTGTC-3′（配列番号１０）およびその相補ＤＮＡからなる２本鎖ＤＮＡ断片を用意し、これら両者を用いてＳａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　２４－５１－７８（受託番号：ＦＥＲＭ　ＢＰ－１９０７２）を宿主として形質転換を行い、配列番号１に示すアミノ酸配列をコードするグルタチオンレダクターゼ（ＧＬＲ１）遺伝子を破壊した、ＧＬＲ１破壊株を得た。このＧＬＲ１破壊株では、宿主酵母のゲノムＤＮＡにおいて、配列番号１に示すアミノ酸配列をコードするＧＬＲ１遺伝子のコード領域の一部または全体が破壊されている。 [Production Example 1] Production of yeast in which GLR1 gene was disrupted (GLR1-disrupted strain) cereviciae for vector pD1511 'prepared (SEQ ID NO: 9) and plasmid further DNA sequence 5'-ACACTTCTGGTTTTTCTCATGCGCTTCTCACTCTCAGTATATTTTGCTGCTTTCCTTCATATGTATATATATCTATTTACATATTAGTTTACAGAACTTTAGCAACGAAACTAGACGTCCAATCTGCTGTTGCTATACTGGACTTTTGTACTCTTGTAAACAATCTTATATAGCATCCTGAAATACGTAGTAATTTTGTC-3' The sequence of the gRNA portion 5'-GATTACCTCGTCATCGGGGG-3 of (ATUM Inc.) (SEQ ID NO: 10) and its A double-stranded DNA fragment comprising a complementary DNA is prepared, and both of them are used to transform Saccharomyces cerevisiae 24-51-78 (Accession number: FERM BP-19072) as a host, and the amino acid sequence shown in SEQ ID NO: 1 is obtained. A GLR1-disrupted strain in which the encoding glutathione reductase (GLR1) gene was disrupted was obtained. In this GLR1-disrupted strain, part or all of the coding region of the GLR1 gene encoding the amino acid sequence shown in SEQ ID NO: 1 is disrupted in the genomic DNA of the host yeast.
［製造例２］ＧＬＲ１遺伝子を破壊し、さらにＹＣＦ１、ＧＳＨ１、ＧＳＨ２の発現を強化した酵母（ＧＬＲ１破壊＋ＹＣＦ１強化＋ＧＳＨ１強化＋ＧＳＨ２強化株）の作製
　製造例１で作製したＧＬＲ１破壊株を宿主とし、非特許文献（Hara KY, Kiriyama K, Inagaki A, Nakayama H, Kondo A. (2012) Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 426(2):129-33.）に記載の方法で、配列番号３に示すアミノ酸配列からなるγ－グルタミルシステイン合成酵素（ＧＳＨ１）および配列番号５に示すアミノ酸配列からなるグルタチオン合成酵素（ＧＳＨ２）の発現を強化して、ＧＬＲ１破壊＋ＧＳＨ１強化＋ＧＳＨ２強化株を得た。前記非特許文献に記載のＧＳＨ１およびＧＳＨ２の発現の強化は、宿主酵母のゲノムＤＮＡに、それぞれ複数コピーの、強発現プロモーターと制御可能に連結されたＧＳＨ１遺伝子および強発現プロモーターと制御可能に連結されたＧＳＨ２遺伝子を組み込むことによるものである。更にＳａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　ＹＰＨ４９９株のゲノムＤＮＡをテンプレートとし、プライマーとして、5'- AAAAGGATCCATGGCTGGTAATCTTGTTTCATGGGCC-3'（配列番号１１）および5'- AAAACTCGAGTTAATTTTCATTGACCAAACCAGCCTCC-3'(配列番号１２)を用いてＰＣＲ増幅を行い、増幅物をＢａｍＨＩおよびＸｈｏＩで消化して、配列番号７に示すアミノ酸配列をコードする、配列番号８に示す塩基配列を含むグルタチオン輸送酵素（ＹＣＦ１）遺伝子ＢａｍＨＩ－ＸｈｏＩ断片を得た。この断片をｐ４２７ＴＥＦ（コスモバイオ社製）のＢａｍＨＩ－ＸｈｏＩ消化部位に連結し、ＹＣＦ１発現プラスミドｐ４２７－ＹＣＦ１を得た。このプラスミドを用いて、上記で得たＧＬＲ１破壊＋ＧＳＨ１強化＋ＧＳＨ２強化株を、更に形質転換した（ＧＬＲ１破壊＋ＹＣＦ１強化＋ＧＳＨ１強化＋ＧＳＨ２強化株）。 [Production Example 2] Production of yeast (GLR1 disruption + YCF1 enhancement + GSH1 enhancement + GSH2 enhanced strain) in which the GLR1 gene was disrupted and the expression of YCF1, GSH1, and GSH2 was further enhanced, using the GLR1 disruption strain produced in Production Example 1 as a host, Patent Document (Hara KY, Kiriyama K, Inagaki A, Nakayama H, Kondo A. (2012) Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of Saccharomyces cerevisiae.Appl Microbiol Biotechnol, 426 (2): 129-33.) , The expression of γ-glutamylcysteine synthetase (GSH1) consisting of the amino acid sequence shown in SEQ ID NO: 3 and glutathione synthase (GSH2) consisting of the amino acid sequence shown in SEQ ID NO: 5 is enhanced, and GLR1 disruption + GSH1 Enhanced + GSH2 enhanced strain was obtained. The enhanced expression of GSH1 and GSH2 described in the non-patent document can be achieved by operably linking multiple copies of the GSH1 gene and the strong expression promoter operably linked to the strong expression promoter to genomic DNA of the host yeast, respectively. This is due to the incorporation of the GSH2 gene. Furthermore, PCR amplification was performed using 5′-AAAAGGATCCATGGCTGGTAATCTTGTTTCATGGGCC-3 ′ (SEQ ID NO: 11) and 5′-AAAACTCGAGTTAATTTTCATTGACCAAACCAGCCTCC-3 ′ (SEQ ID NO: 12) using genomic DNA of Saccharomyces cerevisiae strain YPH499 as a template and primers as primers. Was digested with BamHI and XhoI to obtain a glutathione transferase (YCF1) gene BamHI-XhoI fragment encoding the amino acid sequence shown in SEQ ID NO: 7 and containing the nucleotide sequence shown in SEQ ID NO: 8. This fragment was ligated to the BamHI-XhoI digestion site of p427TEF (manufactured by Cosmo Bio) to obtain a YCF1-expression plasmid p427-YCF1. Using this plasmid, the GLR1 disruption + GSH1 enhanced + GSH2 enhanced strain obtained above was further transformed (GLR1 disrupted + YCF1 enhanced + GSH1 enhanced + GSH2 enhanced strain).
［実施例１］
　酵母として、製造例２に記載のＧＬＲ１破壊＋ＹＣＦ１強化＋ＧＳＨ１強化＋ＧＳＨ２強化株をＹＰＤ培地（１０ｇ／Ｌ酵母エキストラクト（Ｄｉｆｃｏ　ｌａｂｏｒａｔｏｒｉｅｓ製）、２０ｇ／Ｌポリペプトン（和光純薬（株）製）、２０ｇ／Ｌグルコース（ナカライテスク（株）製））７ｍｌで３０℃、１６～２４時間振盪することにより種母培養を行った。 [Example 1]
As the yeast, the GLR1 disruption + YCF1 enhancement + GSH1 enhancement + GSH2 enhancement strain described in Production Example 2 was prepared using a YPD medium (10 g / L yeast extract (manufactured by Difco laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g / Seed culture was carried out by shaking with 7 ml of L glucose (manufactured by Nacalai Tesque, Inc.) at 30 ° C. for 16 to 24 hours.
　次に糖蜜リジン培地５０ｍｌ（糖蜜４％（糖蜜中のグルコース量として）、尿素０．３％、硫酸アンモニウム０．０８％、リン酸２％、アンモニウム０．０４％、リジン０．１％、０．２％または０．４％）を含む坂口フラスコに種母培養液３ｍｌとなるよう植菌し、３０℃、攪拌１３０ｒｐｍの条件で４０時間培養を行い、培養液を１ｍｌ回収した。また、同時に糖蜜培地５０ｍｌ（糖蜜４％（糖蜜中のグルコース量として）、尿素０．３％、硫酸アンモニウム０．０８％、リン酸２％、アンモニウム０．０４％）でも比較対照として培養を行った。 Next, 50 ml of molasses lysine medium (4% molasses (as glucose in molasses), 0.3% urea, 0.08% ammonium sulfate, 2% phosphoric acid, 0.04% ammonium, 0.1% lysine, 0.1% lysine). (2% or 0.4%) was inoculated into a Sakaguchi flask so as to obtain 3 ml of a seed culture solution, and cultured at 30 ° C. under agitation at 130 rpm for 40 hours to collect 1 ml of the culture solution. At the same time, cultivation was performed as a control with 50 ml of molasses medium (4% of molasses (as glucose in molasses), 0.3% of urea, 0.08% of ammonium sulfate, 2% of phosphoric acid and 0.04% of ammonium). .
　培養液１ｍｌを遠心分離して集めた菌体を滅菌水にて２度洗い、８０℃で５分間熱処理することで、菌体内のグルタチオンを溶出した。この溶出液を、２５℃、２００００ｘｇにて５分間遠心した上清のグルタチオン濃度をＨＰＬＣ法にて分析し、培養液１ｍｌあたりの還元型グルタチオン（ＧＳＨ）量および酸化型グルタチオン（ＧＳＳＧ）量を決定した。同様に培養液１ｍｌを滅菌水にて２度洗い、８０℃デシケーター内で一晩静置することで、培養液１ｍｌあたりの乾燥菌体重量を得る。ＧＳＨ量およびＧＳＳＧ量をそれぞれ乾燥菌体重量で除することで、ＧＳＨ含有率（％）およびＧＳＳＧ含有率（％）を算出した。リジンを添加しない糖蜜培地を用いた比較対照試験でのＧＳＨ含有率（％）を１００％とし、糖蜜培地を用いた比較対照試験でのＧＳＳＧ含有率（％）、ならびに、糖蜜リジン培地を用いた実施例１の試験でのＧＳＨ含有率（％）およびＧＳＳＧ含有率（％）の相対値を求めた。結果を次表に示す。 Glutathione in the cells was eluted by washing the cells collected by centrifugation of 1 ml of the culture solution twice with sterilized water and heat-treating at 80 ° C. for 5 minutes. The eluate was centrifuged at 20,000 × g for 5 minutes at 25 ° C., and the supernatant was analyzed for glutathione concentration by HPLC to determine the amount of reduced glutathione (GSH) and the amount of oxidized glutathione (GSSG) per 1 ml of the culture solution. did. Similarly, 1 ml of the culture solution is washed twice with sterile water, and allowed to stand in a desiccator at 80 ° C. overnight to obtain a dry cell weight per 1 ml of the culture solution. The GSH content (%) and the GSSG content (%) were calculated by dividing the GSH content and the GSSG content by the dry cell weight, respectively. The GSH content (%) in the control test using the molasses medium without adding lysine was set to 100%, and the GSSG content (%) in the control test using the molasses medium and the molasses lysine medium were used. The relative values of the GSH content (%) and the GSSG content (%) in the test of Example 1 were determined. The results are shown in the following table.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表から分かるように、培地中のリジン濃度を高めることで、ＧＳＨ含有率およびＧＳＳＧ含有率を飛躍的に高めることができる。 分 か る As can be seen from the table, the GSH content and GSSG content can be dramatically increased by increasing the lysine concentration in the medium.
［実施例２］
　酵母として、製造例２に記載のＧＬＲ１破壊＋ＹＣＦ１強化＋ＧＳＨ１強化＋ＧＳＨ２強化株をＹＰＤ培地（１０ｇ／Ｌ酵母エキストラクト（Ｄｉｆｃｏ　ｌａｂｏｒａｔｏｒｉｅｓ製）、２０ｇ／Ｌポリペプトン（和光純薬（株）製）、２０ｇ／Ｌグルコース（ナカライテスク（株）製））７ｍｌで３０℃、１６～２４時間振盪することにより種母培養を行った。 [Example 2]
As the yeast, the GLR1 disruption + YCF1 enhancement + GSH1 enhancement + GSH2 enhancement strain described in Production Example 2 was prepared using a YPD medium (10 g / L yeast extract (manufactured by Difco laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g / Seed culture was carried out by shaking with 7 ml of L glucose (manufactured by Nacalai Tesque, Inc.) at 30 ° C. for 16 to 24 hours.
　次に糖蜜アルギニン培地５０ｍｌ（糖蜜４％（糖蜜中のグルコース量として）、尿素０．３％、硫酸アンモニウム０．０８％、リン酸２％、アンモニウム０．０４％、アルギニン０．１％）を含む坂口フラスコに種母培養液３ｍｌとなるよう植菌し、３０℃、攪拌１３０ｒｐｍの条件で４０時間培養を行い、培養液を１ｍｌ回収した。また、同時に糖蜜培地５０ｍｌ（糖蜜（糖蜜中のグルコース量として）４％、尿素０．３％、硫酸アンモニウム０．０８％、リン酸２％、アンモニウム０．０４％）でも比較対照として培養を行った。 Next, molasses arginine medium 50 ml (molasses 4% (as glucose in molasses), urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%, arginine 0.1%) The Sakaguchi flask was inoculated so as to have a seed culture solution of 3 ml, cultured at 30 ° C. under agitation at 130 rpm for 40 hours, and 1 ml of the culture solution was collected. Simultaneously, 50 ml of molasses medium (molasses (as the amount of glucose in molasses) 4%, urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%) was cultured as a control. .
　培養液１ｍｌを遠心分離して集めた菌体を滅菌水にて２度洗い、８０℃で５分間熱処理することで、菌体内のグルタチオンを溶出した。この溶出液を、２５℃、２００００ｘｇにて５分間遠心した上清のグルタチオン濃度をＨＰＬＣ法にて分析し、培養液１ｍｌあたりのグルタチオン量（ＧＳＨ量とＧＳＳＧ量の合計）を決定した。同様に培養液１ｍｌを滅菌水にて２度洗い、８０℃デシケーター内で一晩静置することで、培養液１ｍｌあたりの乾燥菌体重量を得る。グルタチオン量を乾燥菌体重量で除することで、グルタチオン含有率（％）を算出した。アルギニンを添加しない糖蜜培地を用いた比較対照試験でのグルタチオン含有率（％）を１００％とし、糖蜜アルギニン培地を用いた実施例２の試験でのグルタチオン含有率（％）の相対値を求めた。結果を次表に示す。 Glutathione in the cells was eluted by washing the cells collected by centrifugation of 1 ml of the culture solution twice with sterilized water and heat-treating at 80 ° C. for 5 minutes. The eluate was centrifuged at 20,000 × g for 5 minutes at 25 ° C., and the glutathione concentration of the supernatant was analyzed by HPLC to determine the amount of glutathione per 1 ml of the culture solution (the total amount of GSH and GSSG). Similarly, 1 ml of the culture solution is washed twice with sterile water, and allowed to stand in a desiccator at 80 ° C. overnight to obtain a dry cell weight per 1 ml of the culture solution. Glutathione content (%) was calculated by dividing the amount of glutathione by the dry cell weight. The relative value of the glutathione content (%) in the test of Example 2 using the molasses arginine medium was determined, assuming that the glutathione content (%) in the comparative control test using the molasses medium without adding arginine was 100%. . The results are shown in the following table.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表から分かるように、糖蜜培地にアルギニンを添加することで、酵母のグルタチオン含有率を飛躍的に高めることができる。 分 か る As can be seen from the table, the addition of arginine to the molasses medium can dramatically increase the glutathione content of the yeast.
［実施例３］
　実施例２において、糖蜜アルギニン培地の代わりに糖蜜ヒスチジン培地５０ｍｌ（糖蜜４％（糖蜜中のグルコース量として）、尿素０．３％、硫酸アンモニウム０．０８％、リン酸２％、アンモニウム０．０４％、ヒスチジン０．１％）を用いた以外は、実施例２と同じ条件及び手順で、製造例２で調製したＧＬＲ１破壊＋ＹＣＦ１強化＋ＧＳＨ１強化＋ＧＳＨ２強化株を糖蜜ヒスチジン培地およびヒスチジンを添加しない糖蜜培地にて培養し、グルタチオン含有率（％）を測定した。実施例２と同様に、ヒスチジンを添加しない糖蜜培地を用いた比較対照試験でのグルタチオン含有率（％）を１００％とし、糖蜜ヒスチジン培地を用いた実施例３の試験でのグルタチオン含有率（％）の相対値を求めた。 [Example 3]
In Example 2, in place of the molasses arginine medium, molasses histidine medium (50 ml, molasses 4% (as the amount of glucose in molasses), urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%) Histidine 0.1%), except that GLR1 disruption + YCF1 + GSH1 + GSH2-enriched strain prepared in Production Example 2 was used in a molasses histidine medium and a molasses medium without histidine under the same conditions and procedure as in Example 2. And cultured, and the glutathione content (%) was measured. Similarly to Example 2, the glutathione content (%) in the comparative control test using the molasses medium without adding histidine was set to 100%, and the glutathione content (%) in the test of Example 3 using the molasses histidine medium was determined. ) Was determined.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　アルギニン及びリジンと同様に、ヒスチジンを添加した糖蜜培地中での培養により、酵母のグルタチオン含有率を飛躍的に高めることができる。 Similar to arginine and lysine, cultivation in a molasses medium supplemented with histidine can dramatically increase the glutathione content of yeast.
［実施例４］
　酵母としてＳａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　２４－５１－７８（受託番号：ＦＥＲＭ　ＢＰ－１９０７２）をＹＰＤ培地（１０ｇ／Ｌ酵母エキストラクト（Ｄｉｆｃｏ　ｌａｂｏｒａｔｏｒｉｅｓ製）、２０ｇ／Ｌポリペプトン（和光純薬（株）製）、２０ｇ／Ｌグルコース（ナカライテスク（株）製））７ｍｌで３０℃、１６～２４時間振盪することにより種母培養を行った。 [Example 4]
Saccharomyces cerevisiae 24-51-78 (accession number: FERM BP-19072) was used as a yeast in YPD medium (10 g / L yeast extract (manufactured by Difco laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g / Seed culture was carried out by shaking with 7 ml of L glucose (manufactured by Nacalai Tesque, Inc.) at 30 ° C. for 16 to 24 hours.
　次に糖蜜リジン培地５０ｍｌ（糖蜜４％（糖蜜中のグルコース量として）、尿素０．３％、硫酸アンモニウム０．０８％、リン酸２％、アンモニウム０．０４％、リジン０．１％）、糖蜜アルギニン培地（リジン０．１％に代えてアルギニン０．１％を含有する以外は糖蜜リジン培地と同じ組成を有する）、又は、糖蜜ヒスチジン培地（リジン０．１％に代えてヒスチジン０．１％を含有する以外は糖蜜リジン培地と同じ組成を有する）を含む坂口フラスコに種母培養液３ｍｌとなるよう植菌し、３０℃、攪拌１３０ｒｐｍの条件で４０時間培養を行い、培養液を１ｍｌ回収した。また、同時に糖蜜培地５０ｍｌ（糖蜜（糖蜜中のグルコース量として）４％、尿素０．３％、硫酸アンモニウム０．０８％、リン酸２％、アンモニウム０．０４％）でも比較対照として培養を行った。 Next, molasses 50 ml (molasses 4% (as glucose in molasses), urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%, lysine 0.1%), molasses Arginine medium (having the same composition as molasses lysine medium except that it contains 0.1% arginine instead of 0.1% lysine), or molasses histidine medium (0.1% histidine instead of 0.1% lysine) Is inoculated in a Sakaguchi flask containing 3 mol of a seed mother culture solution, and cultured at 30 ° C. under agitation at 130 rpm for 40 hours to recover 1 ml of the culture solution. did. Simultaneously, 50 ml of molasses medium (molasses (as the amount of glucose in molasses) 4%, urea 0.3%, ammonium sulfate 0.08%, phosphoric acid 2%, ammonium 0.04%) was cultured as a control. .
　実施例２に記載の手順で各培地での培養物のグルタチオン含有率（％）を測定した。実施例２と同様に、リジン、アルギニン及びヒスチジンを添加しない糖蜜培地を用いた比較対照試験でのグルタチオン含有率（％）を１００％とし、糖蜜リジン培地、糖蜜アルギニン培地又は糖蜜ヒスチジン培地を用いた実施例４の試験でのグルタチオン含有率（％）の相対値を求めた。 グ ル Glutathione content (%) of the culture in each medium was measured by the procedure described in Example 2. As in Example 2, the content of glutathione (%) in the comparative control test using a molasses medium without adding lysine, arginine and histidine was 100%, and a molasses lysine medium, a molasses arginine medium or a molasses histidine medium was used. The relative value of the glutathione content (%) in the test of Example 4 was determined.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　酵母としてＳａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　２４－５１－７８（受託番号：ＦＥＲＭ　ＢＰ－１９０７２）を、塩基性アミノ酸を添加した培地中で培養した場合でも、酵母のグルタチオン含有率を飛躍的に高めることができる。 Even when Saccharomyces cerevisiae 24-51-78 (accession number: FERM BP-19072) is cultured in a medium to which a basic amino acid has been added, the glutathione content of the yeast can be dramatically increased.
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (22)

  1. 　酵母を、塩基性アミノ酸濃度が０．８ｇ／Ｌ以上である培地中で培養することを特徴とする、グルタチオンの製造方法。 (4) A method for producing glutathione, comprising culturing yeast in a medium having a basic amino acid concentration of 0.8 g / L or more.
  2. 　前記酵母が、還元型グルタチオン重量を１００としたとき、酸化型グルタチオン重量が２０以上となるように、還元型グルタチオンおよび酸化型グルタチオンを含有する酵母である、請求項１に記載の方法。 方法 The method according to claim 1, wherein the yeast contains reduced glutathione and oxidized glutathione such that the weight of oxidized glutathione is 20 or more when the weight of reduced glutathione is 100.
  3. 　前記酵母が、親株と比較してグルタチオンレダクターゼの活性が低下した酵母である、請求項１又は２に記載の方法。 方法 The method according to claim 1 or 2, wherein the yeast is a yeast having reduced glutathione reductase activity compared to the parent strain.
  4. 　前記酵母が、グルタチオンレダクターゼをコードする遺伝子が欠損した酵母である、請求項１～３のいずれか１項に記載の方法。 (4) The method according to any one of (1) to (3), wherein the yeast is a yeast deficient in a gene encoding glutathione reductase.
  5. 　前記グルタチオンレダクターゼが下記（１ａ）～（１ｅ）：
    （１ａ）配列番号１に示すアミノ酸配列からなるタンパク質、
    （１ｂ）配列番号１に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
    （１ｃ）配列番号１に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
    （１ｄ）配列番号２と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、および、
    （１ｅ）配列番号２に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオンレダクターゼ活性を有するタンパク質、
    からなる群から選択される、請求項３または４に記載の方法。     The glutathione reductase has the following (1a) to (1e):
    (1a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
    (1b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having a glutathione reductase activity;
    (1c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having glutathione reductase activity;
    (1d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 2, and having a glutathione reductase activity; and
    (1e) a protein comprising an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 2, and which has glutathione reductase activity;
    The method according to claim 3 or 4, wherein the method is selected from the group consisting of:
  6. 　前記酵母が、親株と比較してγ－グルタミルシステイン合成酵素および／またはグルタチオン合成酵素の活性が増大した酵母である、請求項１～５のいずれか１項に記載の方法。 (6) The method according to any one of (1) to (5), wherein the yeast has an increased activity of γ-glutamylcysteine synthetase and / or glutathione synthase as compared with the parent strain.
  7. 　前記酵母が、γ－グルタミルシステイン合成酵素をコードする塩基配列を含むＤＮＡ、および／またはグルタチオン合成酵素をコードする塩基配列を含むＤＮＡにより形質転換された酵母である、請求項１～６のいずれか１項に記載の方法。 The yeast according to any one of claims 1 to 6, wherein the yeast is a yeast transformed with a DNA containing a nucleotide sequence encoding γ-glutamylcysteine synthetase and / or a DNA containing a nucleotide sequence encoding glutathione synthase. 2. The method according to item 1.
  8. 　前記γ－グルタミルシステイン合成酵素が下記（２ａ）～（２ｅ）：
    （２ａ）配列番号３に示すアミノ酸配列からなるタンパク質、
    （２ｂ）配列番号３に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
    （２ｃ）配列番号３に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
    （２ｄ）配列番号４と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、および、
    （２ｅ）配列番号４に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつγ－グルタミルシステイン合成酵素活性を有するタンパク質、
    からなる群から選択される、請求項６または７に記載の方法。     The γ-glutamylcysteine synthase is represented by the following (2a) to (2e):
    (2a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3,
    (2b) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in which one or more amino acids have been deleted, substituted, inserted and / or added, and which has γ-glutamylcysteine synthetase activity;
    (2c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and having γ-glutamylcysteine synthetase activity;
    (2d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 4, and having a γ-glutamylcysteine synthetase activity; and
    (2e) one or more bases in the base sequence shown in SEQ ID NO: 4 consist of an amino acid sequence encoded by substitution, deletion, insertion and / or addition of DNA, and have γ-glutamylcysteine synthetase activity protein,
    The method according to claim 6 or 7, wherein the method is selected from the group consisting of:
  9. 　前記グルタチオン合成酵素が下記（３ａ）～（３ｅ）：
    （３ａ）配列番号５に示すアミノ酸配列からなるタンパク質、
    （３ｂ）配列番号５に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
    （３ｃ）配列番号５に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
    （３ｄ）配列番号６と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、および、
    （３ｅ）配列番号６に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン合成酵素活性を有するタンパク質、
    からなる群から選択される、請求項６または７に記載の方法。     The glutathione synthase has the following (3a) to (3e):
    (3a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5,
    (3b) a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 5, and having a glutathione synthase activity;
    (3c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and having a glutathione synthase activity;
    (3d) a protein comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 6, and having a glutathione synthase activity; and
    (3e) a protein having an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 6, and which has glutathione synthase activity;
    The method according to claim 6 or 7, wherein the method is selected from the group consisting of:
  10. 　前記酵母が、親株と比較してグルタチオン輸送酵素の活性が増大した酵母である、請求項１～９のいずれか１項に記載の方法。 (10) The method according to any one of (1) to (9), wherein the yeast has an increased activity of glutathione transferase as compared with the parent strain.
  11. 　前記酵母が、グルタチオン輸送酵素をコードする塩基配列を含むＤＮＡにより形質転換された酵母である、請求項１～１０のいずれか１項に記載の方法。 The method according to any one of claims 1 to 10, wherein the yeast is a yeast transformed with a DNA containing a nucleotide sequence encoding glutathione transferase.
  12. 　前記グルタチオン輸送酵素が下記（４ａ）～（４ｅ）：
    （４ａ）配列番号７に示すアミノ酸配列からなるタンパク質、
    （４ｂ）配列番号７に示すアミノ酸配列において、１もしくは複数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
    （４ｃ）配列番号７に示すアミノ酸配列と６０％以上の配列同一性を有するアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
    （４ｄ）配列番号８と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、および、
    （４ｅ）配列番号８に示す塩基配列において１もしくは複数個の塩基が置換、欠矢、挿入および／または付加されたＤＮＡによりコードされるアミノ酸配列からなり、かつグルタチオン輸送酵素活性を有するタンパク質、
    からなる群から選択される、請求項１０または１１に記載の方法。     The glutathione transferase is represented by the following (4a) to (4e):
    (4a) a protein consisting of the amino acid sequence of SEQ ID NO: 7,
    (4b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 7, and which has a glutathione transferase activity;
    (4c) a protein consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and having a glutathione transferase activity;
    (4d) a protein comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to SEQ ID NO: 8, and having a glutathione transferase activity; and
    (4e) a protein comprising an amino acid sequence encoded by DNA in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 8, and which has a glutathione transferase activity;
    The method according to claim 10 or 11, wherein the method is selected from the group consisting of:
  13. 　前記酵母が、サッカロマイセス属、キャンディダ属、またはピキア属の酵母である、請求項１～１２のいずれか１項に記載の方法。 The method according to any one of claims 1 to 12, wherein the yeast is a yeast belonging to the genus Saccharomyces, Candida, or Pichia.
  14. 　前記酵母が、塩基性アミノ酸非要求性の酵母である、請求項１～１３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 13, wherein the yeast is a yeast that does not require basic amino acids.
  15. 　前記培地が炭素源として糖蜜を含む、請求項１～１４のいずれか１項に記載の方法。 The method according to any one of claims 1 to 14, wherein the medium contains molasses as a carbon source.
  16. 　培養開始時において、前記培地中の塩基性アミノ酸濃度が０．８ｇ／Ｌ以上である、請求項１～１５のいずれか１項に記載の方法。 (16) The method according to any one of (1) to (15), wherein at the start of the culture, the concentration of the basic amino acid in the medium is 0.8 g / L or more.
  17. 　前記培地中の塩基性アミノ酸濃度が２ｇ／Ｌ以上である、請求項１～１６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 16, wherein the concentration of the basic amino acid in the medium is 2 g / L or more.
  18. 　前記培地中の塩基性アミノ酸濃度が４ｇ／Ｌ以上である、請求項１～１６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 16, wherein the concentration of the basic amino acid in the medium is 4 g / L or more.
  19. 　前記塩基性アミノ酸がリジンである、請求項１～１８のいずれか１項に記載の方法。 方法 The method according to any one of claims 1 to 18, wherein the basic amino acid is lysine.
  20. 　酵母を、塩基性アミノ酸濃度が０．８ｇ／Ｌ以上である培地中で培養することを特徴とする、酵母によるグルタチオンの生産を促進する方法。 (4) A method for promoting the production of glutathione by yeast, which comprises culturing yeast in a medium having a basic amino acid concentration of 0.8 g / L or more.
  21. 　塩基性アミノ酸を含む、酵母によるグルタチオンの生産の促進剤。 (4) An accelerator for glutathione production by yeast, containing a basic amino acid.
  22. 　塩基性アミノ酸濃度が０．８ｇ／Ｌ以上であり、炭素源として糖蜜を含む、酵母用培地組成物。 (4) A yeast medium composition having a basic amino acid concentration of 0.8 g / L or more and containing molasses as a carbon source.
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