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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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