JP5874286B2 - Method for producing yeast extract containing γ-glutamyl compound and yeast used in the method - Google Patents

Description

  The present invention relates to a method for producing a yeast extract containing a γ-glutamyl compound and a yeast used in the method. The yeast extract and yeast are useful in the food field.

  Glutathione (γ-Glu-Cys-Gly), which is one of γ-glutamyl compounds, is a non-protein thiol that is present in the largest amount in cells of all living organisms. Glutathione has various functions necessary for living body maintenance such as antioxidant action, immune support, and removal of cytotoxins in vivo, and is used as a raw material for pharmaceuticals and cosmetics. Glutathione has a rich taste and is also used as a food additive. Therefore, it is important to produce glutathione in large quantities at low cost. Glutathione can be efficiently produced, for example, by separating and using a budding yeast that produces glutathione at a high level (Non-Patent Document 1). However, glutathione having a highly reactive thiol group is important for maintaining the living body, but is harmful when it is excessively accumulated, and growth delay is often observed in cells that accumulate glutathione at a high concentration. Therefore, in order to efficiently produce glutathione, it is necessary to isolate budding yeast that accumulates glutathione in cells at a high concentration and does not delay growth.

  As an enzyme that degrades glutathione in yeast cells, Ecm38p encoded by the ECM38 gene is known (Non-patent Documents 2 and 3). In addition, recently, a DUG complex has been found as an enzyme that degrades glutathione in yeast cells (Non-Patent Documents 4 and 5). However, there is no report that intracellular glutathione concentration increased by weakening these glutathione degradation systems. Therefore, there was no idea to produce a yeast extract using yeast in which the activity of these glutathione degrading enzymes was reduced. The DUG complex consists of Dug1p, Dug2p, and Dug3p, but it is not clear how much each DUG factor contributes to the degradation of glutathione.

  Ycf1p encoded by the YCF1 gene is a transporter that transports glutathione chelated to metal to the vacuole (Non-patent Documents 3 and 6). Ycf1p is thought to transport only glutathione to the vacuole during nutrient starvation, but the effect of Ycf1p function on intracellular glutathione concentration is unknown.

Appl Microbiol Biotechnol. DOI 10.1007 / s00253-010-2968-6 (2010) Yeast. 2003 Jul 30; 20 (10): 857-63 FEBS Lett. 2009 May 6; 583 (9): 1489-92 J Biol Chem. 2009 May 22; 284 (21): 14493-502 Genetics. 2007 Mar; 175 (3): 1137-51 FEBS Lett. 2010 Feb 19; 584 (4): 726-32 Biometals. 2009 Apr; 22 (2): 243-9 J Biol Chem. 2005 Feb 11; 280 (6): 4851-4857 BioMetals (2006) 19: 593-599 J Biol Chem. 2003 Dec 12; 278 (50): 49920-8 Appl Microbiol Biotechnol DOI 10.1007 / s00253-010-2946-z (2010)

  The present invention relates to a yeast that accumulates industrially useful γ-glutamyl compounds such as glutathione at a high concentration, a yeast extract containing a γ-glutamyl compound using the yeast, a food and drink containing the yeast extract, and those It is an object to provide a manufacturing method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has further found that DUG, which is a glutathione degrading enzyme, is further enhanced in a yeast that has enhanced glutathione synthesizing ability by high expression of GSH1 gene encoding γ-glutamylcysteine synthetase. An unexpected finding has been found that by reducing the activity of the complex, the growth delay of yeast can be reduced despite the increase in intracellular glutathione accumulation. In addition, the present inventors enhanced the expression of the vacuolar glutathione transporter and reduced the activity of Ecm38p, a vacuolar localized glutathione degrading enzyme, thereby preventing growth delay due to excessive accumulation of glutathione and feedback inhibition by glutathione. And the amount of glutathione accumulated in the yeast cells was found to increase. Furthermore, the present inventor also improved the growth of yeast by reducing the activity of the DUG complex in yeast that accumulates γ-glutamylcysteine, and reduced the activity of Ecm38p and enhanced expression of the vacuolar glutathione transporter. The inventors have found that the amount of γ-glutamylcysteine accumulated in yeast cells increases. Based on the above, the present invention has been completed.

That is, the present invention can be exemplified as follows.
[1]
A method for producing a yeast extract, characterized in that a yeast extract is prepared using as a raw material a yeast that has been modified to increase the ability to synthesize γ-glutamyl compounds and to reduce the activity of glutathione degrading enzymes.
[2]
The method, wherein the glutathione degrading enzyme is one or more proteins selected from the following (A) to (D).
(A) Protein encoded by DUG1 gene (B) Protein encoded by DUG2 gene (C) Protein encoded by DUG3 gene (D) Protein encoded by ECM38 gene
[3]
The method as described above, wherein the yeast is modified so that at least the activity of a vacuolar localized glutathione degrading enzyme is decreased and the expression of a vacuolar glutathione transporter is increased.
[4]
The method as described above, wherein the activity of γ-glutamylcysteine synthetase is increased to increase the ability to synthesize γ-glutamyl compound.
[5]
The method, wherein the activity of synthesizing a glutathione synthetase is increased or decreased to increase the ability to synthesize γ-glutamyl compounds.
[6]
The method, wherein the γ-glutamyl compound is a compound selected from glutathione, γ-glutamylcysteine, and γ-glutamyl-α-aminobutyric acid.
[7]
The method described above, wherein the yeast is cultured in a medium to which a compound selected from amino acids, amino acid derivatives, and peptides constituting the γ-glutamyl compound is added. [8]
The method as described above, wherein the yeast belongs to the genus Saccharomyces.
[9]
The method, wherein the yeast is Saccharomyces cerevisiae.
[10]
Food / beverage products containing the yeast extract manufactured by the said method.
[11]
Yeast modified so that the activity of γ-glutamylcysteine synthetase is increased and the activity of glutathione degrading enzyme is decreased.
[12]
Furthermore, the yeast having one or more properties selected from the following (I) and (II).
(I) Increase or decrease activity of glutathione synthase (II) Increase expression of vacuolar glutathione transporter

  According to the present invention, a yeast that accumulates γ-glutamyl compounds such as glutathione in cells and has good growth is provided. By using these yeasts as raw materials, yeast extracts containing γ-glutamyl compounds can be efficiently produced, and foods and drinks can be produced using these yeast extracts.

The figure which shows the intracellular glutathione density | concentration of the GSH1 expression reinforcement | strengthening strain | stump | stock derived from Saccharomyces cerevisiae Y006 strain | stump | stock which is a uracil non-requiring strain | stump | stock, and Y006 strain | stump | stock, and a glutathione degrading enzyme gene destruction strain. Y003: Y003 strain, AG1: Y006 enhanced GSH1 expression strain, d1: Y006 DUG1 disruption strain, d2: Y006 DUG2 disruption strain, d3: Y006 DUG3 disruption strain, e: Y006 ECM38 disruption strain, ed: Y006 DUG2 and ECM38 double disruption strain. The figure which shows the intracellular glutathione density | concentration of the Y003 strain, AG1 strain, and the glutathione degrading enzyme gene destruction strain derived from AG1 strain. Y003: Y003 strain, AG1: YF006 enhanced GSH1 expression strain, AG1-d: AG1 strain DUG2 disruption strain, AG1-e: AG1 strain ECM38 disruption strain, AG1-ed: AG1 strain DUG2 and ECM38 double disruption strain . The figure which shows the growth curve of the mutant strain derived from the Y003 strain and the Y006 strain. Y003: Y003 strain, AG1: Y006 strain enhanced GSH1 expression strain, d2: Y006 DUG2 disruption strain, AG1-d: AG1 strain DUG2 disruption strain. The figure which shows the amount of mRNA of DUG1, DUG2, and DUG3 which codes a DUG complex constituent factor. Panel a: mRNA level of DUG factor at OD600 = 5 (mid logarithmic growth phase). Panel b: DUG factor mRNA level at OD600 = 10 (stationary phase). The figure which shows the intracellular glutathione density | concentration of the Y003 strain | stump | stock, the GSH1 expression enhanced strain derived from the Y006 strain, the glutathione degrading enzyme gene disruption strain, and the glutathione vacuolar transporter expression enhanced strain. Y003: Y003 strain, AG1: Y006 strain enhanced GSH1 expression strain, e: Y006 strain ECM38 disruption strain, d: Y006 strain DUG2 disruption strain, PY: Y006 strain YCF1 expression enhanced strain. The figure which shows the intracellular glutathione density | concentration of the Y003 strain | stump | stock, AG1 strain | stump | stock, the glutathione degrading enzyme gene-disrupted strain derived from AG1 strain | stump | stock, and a vacuolar glutathione transporter expression enhanced strain. Y003: Y003 strain, AG1: Y006 strain with enhanced GSH1 expression, AG1-d: AG1 strain with DUG2 disruption, AG1-e: AG1 strain with ECM38 disruption, AG1-PY: AG1 strain with enhanced YCF1 expression, AG1 -PY-e: ECM38 disruption strain of AG1-PY strain. The figure which shows the growth curve of the Y003 strain, AG1 strain, the glutathione degrading enzyme gene disruption strain derived from AG1 strain, and the vacuolar glutathione transporter expression enhanced strain. Y003: Y003 strain, AG1: Y006 strain with enhanced GSH1 expression, AG1-d: AG1 strain with DUG2 disruption, AG1-e: AG1 strain with ECM38 disruption, AG1-PY: AG1 strain with enhanced YCF1 expression, AG1 -PY-e: ECM38 disruption strain of AG1-PY strain. The figure which shows the growth curve of AG1-gsh2 strain | stump | stock and AG1-d-gsh2 strain | stump | stock which are (gamma) -glutamylcysteine accumulation yeast. AG1-gsh2: GSH2-disrupted strain of AG1 strain, AG1-d-gsh2: GSH2-disrupted strain of AG1-d strain. The figure which shows the intracellular (gamma) -glutamylcysteine density | concentration of the gsh2 strain | stump | stock, AG1-gsh2 strain | stump | stock, and AG1-d-gsh2 strain | stump | stock which are (gamma) -glutamylcysteine accumulation yeast. gsh2: GSH2 disruption strain of Y003 strain, AG1-gsh2: GSH2 disruption strain of AG1 strain, AG1-d-gsh2: GSH2 disruption strain of AG1-d strain. The figure which shows the intracellular (gamma) -glutamylcysteine density | concentration of AG1-d-gsh2 strain | stump | stock, AG1-d-PY-gsh2 strain | stump | stock, and AG1-d-PY-e-gsh2 strain | stump | stock which are (gamma) -glutamylcysteine accumulation yeast. AG1-d-gsh2: AG1-d strain GSH2-disrupted strain, AG1-d-PY-gsh2: AG1-d-PY strain GSH2-disrupted strain, AG1-d-PY-e-gsh2: AG1-d-PY- e-strain GSH2 disruption strain. The figure which shows the growth curve of the AG1 strain | stump | stock at the time of Abu addition culture | cultivation, and the glutathione degrading enzyme gene destruction strain derived from AG1 strain | stump | stock. AG1: AG1 strain, AG1-d: DUG2 disruption strain of AG1 strain.

Hereinafter, the present invention will be described in detail.
(1) Yeast of the Present Invention (1-1) Yeast of the Present Invention The yeast of the present invention has an increased ability to synthesize γ-glutamyl compounds such as glutathione and a decrease in intracellular glutathione degrading enzyme activity. A modified yeast that accumulates γ-glutamyl compounds such as glutathione in cells. The yeast of the present invention preferably has improved yeast growth as a result of a decrease in intracellular glutathione degrading enzyme activity, as compared to a yeast in which the ability to synthesize γ-glutamyl compounds is merely enhanced. Furthermore, the yeast of the present invention has a lower intracellular glutathione-degrading enzyme activity, so that the yeast γ-glutamyl compound has a higher ability to synthesize γ-glutamyl compounds than the yeast in which the ability to synthesize γ-glutamyl compounds is merely enhanced. It is preferred that the concentration be increased. The yeast of the present invention is preferably 0.5% by weight or more, more preferably 1.5% by weight or more, still more preferably 3% by weight or more, and particularly preferably 5% by weight or more γ- such as glutathione by dry weight. Glutamyl compounds can accumulate in cells.

  The yeast of the present invention is preferably modified so that the activity of glutathione degrading enzyme localized in the vacuole is reduced and the activity of the vacuolar glutathione transporter is increased. In this case, as the glutathione-degrading enzyme, only the activity of the vacuolar-localized glutathione-degrading enzyme may be reduced, or in addition to the vacuolar-localized glutathione-degrading enzyme, the activity of other glutathione-degrading enzymes may be reduced. Good. A high concentration of glutathione causes growth retardation and feedback inhibition of the glutathione synthesis system, but increases the activity of the vacuolar glutathione transporter and decreases the vacuolar localized glutathione degrading enzyme activity, thereby converting glutathione into the vacuole. It is considered that glutathione can be stably accumulated in the vacuole by transporting and reducing the glutathione concentration in the cytoplasm. Thereby, it is considered that growth delay due to glutathione and feedback inhibition of the glutathione synthesis system can be avoided, and the amount of accumulated glutathione can be increased.

  The yeast of the present invention can be obtained by modifying an appropriate strain of yeast, for example, a strain described later.

  The yeast of the present invention is not particularly limited as long as it can be modified as described above and accumulates γ-glutamyl compounds such as glutathione in cells, and may be budding yeast or fission yeast. As budding yeast, Saccharomyces cerevisiae (Saccharomyces cerevisiae) genus Saccharomyces, Candida utilis (Candida utilis) genus Pichia pastoris (Pichia pastoris (Pichia pastoris) Pichia genus, Hansenula polymorpha (Hansenula polymorpha) Examples include yeast belonging to the genus Hansenula. Examples of the fission yeast include yeast belonging to the genus Schizosaccharomyces pombe such as Schizosaccharomyces pombe. Of these, Saccharomyces cerevisiae and Candida utilis, which are often used for the production of yeast extract, are preferable. The yeast of the present invention may be haploid or may have diploidity or higher ploidy.

  The glutathione biosynthetic system is possessed by all living organisms, and the vacuolar glutathione transporter and the homologue of glutathione degrading enzyme used in the examples are known to exist in these various yeasts (non- Patent Documents 5 and 9).

  As Saccharomyces cerevisiae, specifically, Saccharomyces cerevisiae Y006 strain can be used. Saccharomyces cerevisiae Y006 strain was internationally deposited on August 18, 2010 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (address postal number 305-8656 Tsukuba City, Ibaraki Prefecture, 1-Chome, 1-Chuo 1-Chuo 6) Accession number FERM BP-11299 is given.

  As Saccharomyces cerevisiae, specifically, the BY4743 strain (ATCC201390) and the S288C strain (ATCC26108) can be used. As Candida utilis, specifically, Candida utilis ATCC22023 strain can be used. These can be obtained, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.

(1-2) Decrease in glutathione degrading enzyme activity The yeast of the present invention is modified so that the activity of glutathione degrading enzyme is reduced.

  “Enzyme activity decreases” means that the target enzyme activity is reduced as compared to a non-modified strain such as a wild strain or a parent strain, and includes a case where the activity is completely lost.

  The glutathione degrading enzyme activity is preferably 50% or less, more preferably 20% or less, still more preferably 10% or less, and particularly preferably 5% or less, as compared to the unmodified strain. Moreover, it is preferable that the glutathione degrading enzyme activity is substantially lost.

  Modifications that reduce enzyme activity can be achieved, for example, by mutation treatment or genetic recombination techniques.

  Mutation treatment includes UV irradiation or normal mutation treatment such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methylmethanesulfonate (MMS), etc. Treatment with the mutagen used in the above.

  Examples of gene recombination techniques include known techniques (FEMS Microbiology Letters 165 (1998) 335-340, JOURNAL OF BACTERIOLOGY, Dec. 1995, p7171-7177, Curr Genet 1986; 10 (8): 573-578, WO 98/14600 etc.) can be used.

  The modification that reduces the enzyme activity can be achieved, for example, by reducing the expression of a gene encoding the target enzyme. Reduction of gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter. In the case of modifying the expression control sequence, the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted.

Moreover, the modification that decreases the enzyme activity can be achieved, for example, by deleting a part or all of the coding region of the gene encoding the target enzyme on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted. As long as the reduction in enzyme activity can be achieved, the region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region. Usually, the longer region to be deleted can surely inactivate the gene. Moreover, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.

  Modifications that reduce enzyme activity include, for example, introducing an amino acid substitution (missense mutation) into the coding region of the gene encoding the target enzyme on the chromosome, introducing a stop codon (nonsense mutation), Alternatively, it can also be achieved by introducing a frameshift mutation that adds or deletes 1 to 2 bases.

  Moreover, the modification that decreases the enzyme activity can be achieved, for example, by inserting another sequence into the coding region of the gene encoding the target enzyme on the chromosome. The insertion site may be any region of the gene, but the longer the sequence to be inserted, the more reliably the gene can be inactivated. Moreover, it is preferable that the reading frames of the sequences before and after the insertion site do not match. The other sequence is not particularly limited as long as it reduces or eliminates the function of the encoded protein, and examples thereof include genes useful for production of γ-glutamyl compounds such as marker genes and glutathione.

  Modifying a gene on a chromosome as described above includes, for example, deleting a partial sequence of the gene and preparing a deleted gene modified so as not to produce a normally functioning protein. This can be achieved by replacing the gene on the chromosome with the deletion-type gene by transforming yeast with the recombinant DNA containing, and causing homologous recombination between the deletion-type gene and the gene on the chromosome. At this time, the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host. In addition, if the recombinant DNA is linearized by cutting with a restriction enzyme or the like, a strain in which the recombinant DNA is incorporated into the chromosome can be efficiently obtained. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost.

  Depending on the structure of the recombinant DNA used, as a result of homologous recombination, the wild-type gene and the deletion-type gene are inserted into the chromosome with other parts of the recombinant DNA (for example, the vector part and the marker gene) sandwiched between them. May be. Since the wild-type gene functions in this state, homologous recombination occurs again between the two genes, and one copy of the gene is dropped from the chromosomal DNA together with the vector portion and the marker gene, leaving the deleted gene. It is necessary to select the ones.

  In addition, for example, a linear DNA containing an arbitrary sequence, wherein yeast is transformed with a linear DNA having sequences upstream and downstream of the site to be replaced on both ends of the arbitrary sequence, and the target to be replaced By causing homologous recombination respectively upstream and downstream of the site, the site to be replaced can be replaced with the arbitrary sequence in one step. As the arbitrary sequence, for example, a sequence including a marker gene may be used. The marker gene may then be removed if necessary. When removing the marker gene, sequences for homologous recombination may be added to both ends of the marker gene so that the marker gene can be efficiently removed.

Confirmation that the target enzyme activity has decreased can be performed by measuring the activity of the enzyme. The activity of glutathione degrading enzyme is determined by a known method (Penninckx M. et al., 1980.
Eur J Biochem 104: 119-123, Ganguli D. et al., Genetics. 2007 Mar; 175 (3): 1137-51 etc.).

Confirmation that the transcription amount of the gene encoding the target enzyme has decreased can be confirmed by comparing the amount of mRNA transcribed from the gene with the unmodified strain. Examples of 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 unmodified strain.

  Confirmation that the amount of the target enzyme is reduced 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 unmodified strain.

  When the yeast of the present invention has a polyploidy of diploid or higher, the yeast of the present invention is a gene and wild type modified so that the enzyme activity is reduced as long as γ-glutamyl compounds such as glutathione can be accumulated. Although it may have a heterogeneous gene, it is usually preferable to be a homozygous gene modified so that the enzyme activity decreases.

  Yeast transformation methods include protoplast method, KU method (H. Ito et al., J. Bateriol., 153-163 (1983)), 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, a method usually used for yeast transformation can be employed. In addition, for the procedures such as yeast spore formation and haploid yeast separation, see “Chemistry and Biology Experiment Line 31 Yeast Experiment Technique”, first edition, Yodogawa Shoten; “Biomanual Series 10 Genetic Experiment Method Using Yeast” First Edition , Yodosha, etc.

  In the present invention, glutathione degrading enzyme refers to a protein having an activity of degrading glutathione.

Examples of glutathione degrading enzymes include Dug1p, Dug2p, and Dug3p encoded by the DUG1 gene, DUG2 gene, and DUG3 gene, respectively. Dug1p, Dug2p, and Dug3p are known to function as DUG complexes. Therefore, “having the activity of degrading glutathione” not only means that the protein encoded by the gene alone has the activity of degrading glutathione, but also the activity of the enzyme complex incorporating the protein degrading glutathione. Including the case where It is known that degradation of glutathione by the DUG complex requires all of Dug1p, Dug2p, and Dug3p (Ganguli D. et al., Genetics.
2007 Mar; 175 (3): 1137-51), reducing the activity of one or more proteins selected from Dug1p, Dug2p, and Dug3p can reduce glutathione degrading enzyme activity. Of these, it is preferable to reduce at least the activity of Dug2p.

  Saccharomyces cerevisiae DUG1 gene, DUG2 gene, and DUG3 gene nucleotide sequences are disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). The nucleotide sequence of Saccharomyces cerevisiae DUG1 gene obtained from the above database and the amino acid sequence encoded by the gene are shown in SEQ ID NOs: 1 and 2, respectively. Similarly, the nucleotide sequence of the DUG2 gene and the amino acid sequence encoded by the gene are shown in SEQ ID NOs: 3 and 4, respectively. Similarly, the nucleotide sequence of the DUG3 gene and the amino acid sequence encoded by the gene are shown in SEQ ID NOs: 5 and 6, respectively.

Since there may be differences in the base sequence of the gene encoding glutathione-degrading enzyme depending on the organism from which the enzyme is derived, the gene encoding glutathione-degrading enzyme is SEQ ID NO: It may be a variant of the base sequence shown in 1, 3, or 5. Each gene variant includes a homolog of each gene. Homologs of each gene can be easily obtained from public databases by BLAST search (http://blast.genome.jp/) or FASTA search using the nucleotide sequence shown in SEQ ID NO: 1, 3, or 5 as a query sequence. Can do. The homologue of each gene is a base of SEQ ID NO: 1, 3, or 5 using a chromosome of an arbitrary organism, for example, a eukaryotic microorganism such as yeast, or a bacterium such as an enteric bacterium or coryneform bacterium, as a template. Examples include genes that can be amplified by PCR using synthetic oligonucleotides prepared on the basis of sequences.

  In addition, as long as the gene encoding the enzyme used in the present invention encodes a protein having an activity of degrading glutathione, the amino acid sequence of the protein encoded by the above gene, for example, SEQ ID NOs: 2, 4, or 6 Or a gene encoding a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions. The “one or several” differs depending on the position of the protein in the three-dimensional structure of the amino acid residue and the type of the amino acid residue, but specifically preferably 1 to 20, more preferably 1 to 10, More preferably, it means 1-5. One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution means, for example, when a substitution site is an aromatic amino acid, between Phe, Trp, and Tyr, and when a substitution site is a hydrophobic amino acid, between Leu, Ile, and Val, a polar amino acid Is an amino acid having a hydroxyl group between Gln and Asn, in the case of a basic amino acid, between Lys, Arg, and His, and in the case of an acidic amino acid, between Asp and Glu. In some cases, it is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as cases based on individual differences in microorganisms from which genes are derived, or differences in species. Also included by

  Furthermore, the gene having a conservative mutation as described above is 80% or more of the entire amino acid sequence of the protein encoded by the above gene, for example, the entire amino acid sequence of SEQ ID NO: 2, 4, or 6. A gene encoding a protein having a homology of preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more and having an activity of degrading glutathione. Also good. In the present specification, “homology” may refer to “identity”.

The gene encoding the enzyme used in the present invention is a probe that can be prepared from a known gene sequence, for example, a complementary sequence to the whole or a part of the base sequence shown in SEQ ID NO: 1, 3, or 5 and stringent conditions. It may be DNA encoding a protein that hybridizes underneath and has an activity of degrading glutathione. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more DNAs having homology. Are hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C. Washing once, more preferably 2-3 times at a salt concentration and temperature corresponding to 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS The conditions to do are mentioned.

  As described above, the probe may be a part of the complementary sequence of the gene. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  In addition, the description regarding these variants shall apply mutatis mutandis also about the other gene and protein in this specification.

  Examples of glutathione degrading enzymes include vacuolar localized glutathione degrading enzymes. In the present invention, vacuole-localized glutathione degrading enzyme refers to a protein that is localized in the vacuole and has an activity of degrading glutathione. Examples of the vacuolar localized glutathione degrading enzyme include Ecm38p encoded by the ECM38 gene. Specifically, Ecm38p has γ-glutamyl transpeptidase (γ-GT) activity.

  The base sequence of the ECM38 gene of Saccharomyces cerevisiae is disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). The nucleotide sequence of Saccharomyces cerevisiae ECM38 gene obtained from the above database and the amino acid sequence encoded by the gene are shown in SEQ ID NOs: 7 and 8, respectively.

  Since the base sequence of the gene encoding the vacuolar localized glutathione degrading enzyme may vary depending on the organism from which the enzyme is derived, the gene encoding the vacuolar localized glutathione degrading enzyme is localized in the vacuole, As long as it encodes a protein having the activity of degrading glutathione, it may be a variant of the base sequence shown in SEQ ID NO: 7. The variant conforms to the description of the variant for the DUG gene group and the DUG complex described above.

  Moreover, it is considered that various peptidases such as Lap4p encoded by the LAP4 gene are involved in the degradation of glutathione, which is a tripeptide, in addition to the above-mentioned glutathione degrading enzyme (Non-patent Documents 3 and 7). Therefore, in the yeast of the present invention, it may be effective to reduce the activity of such peptidase.

(1-3) Enhancing the ability to synthesize γ-glutamyl compounds The yeast of the present invention has been modified to increase the ability to synthesize γ-glutamyl compounds. The γ-glutamyl compound is not particularly limited as long as it is a compound having a γ-glutamyl group, and examples thereof include γ-glutamyl peptides such as γ-glutamyl dipeptide and γ-glutamyl tripeptide. Specific examples of the γ-glutamyldipeptide include γ-glutamylcysteine (γ-Glu-Cys) and γ-glutamyl-α-aminobutyric acid (γ-Glu-Abu). Specific examples of the γ-glutamyl tripeptide include glutathione (γ-Glu-Cys-Gly) and γ-glutamyl-α-aminobutyric acid-glycine (γ-Glu-Abu-Gly). In the present invention, the amino acids and / or amino acid derivatives constituting the γ-glutamyl compound are all in the L form.

“The ability to synthesize γ-glutamyl compounds” means that the biosynthesis of γ-glutamyl compounds such as glutathione is improved as compared to unmodified strains such as wild strains and parental strains. γ
Modifications that increase the ability to synthesize glutamyl compounds can be achieved, for example, by increasing the activity of one or more enzymes involved in the biosynthesis of γ-glutamyl compounds. In vivo, γ-glutamylcysteine synthetase produces γ-glutamylcysteine (γ-Glu-Cys) from L-Glu and L-Cys, and L-Gly is further bound by glutathione synthetase to produce glutathione. . Therefore, the modification that increases the ability to synthesize glutathione can be achieved by, for example, increasing the activity of γ-glutamylcysteine synthetase and / or glutathione synthetase. Further, modification that increases the ability to synthesize γ-glutamylcysteine can be achieved by, for example, increasing the activity of γ-glutamylcysteine synthetase. Modification that increases the ability to synthesize γ-glutamylcysteine can also be achieved by reducing the activity of glutathione synthetase.

  In addition, γ-glutamylcysteine other than γ-glutamylcysteine and glutathione can be biosynthesized by a side reaction of γ-glutamylcysteine synthetase and / or glutathione synthetase. Therefore, a modification that increases the ability to synthesize γ-glutamyldipeptide can be achieved, for example, in the same manner as a modification that increases the ability to synthesize γ-glutamylcysteine. Moreover, the modification that increases the ability to synthesize γ-glutamyl tripeptide can be achieved, for example, in the same manner as the modification that increases the ability to synthesize glutathione.

  “Enzyme activity increases” means that the target enzyme activity is increased as compared to a non-modified strain such as a wild strain or a parent strain. In addition, “enzyme activity increases” not only increases the target enzyme activity in a strain originally having the target enzyme activity, but also imparts the target enzyme activity to a strain that does not originally have the target enzyme activity. Including doing. As a result, as long as the ability to synthesize glutathione is enhanced, a suitable glutathione biosynthesis system may be introduced after weakening the glutathione biosynthesis system inherent in yeast.

  When the γ-glutamylcysteine synthetase activity increases, the γ-glutamylcysteine synthetase activity is preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times that of the unmodified strain. It has improved more than twice.

  When glutathione synthetase activity increases, the glutathione synthetase activity is preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more compared to the unmodified strain. Yes.

  Modification that increases the enzyme activity can be achieved, for example, by enhancing the expression of a gene encoding the target enzyme.

  Enhancement of 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 that improves transcription of the gene over the native wild-type promoter. As a stronger promoter, a highly active type of a conventional promoter may be obtained by using various reporter genes. 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 replacement with a strong promoter can be used in combination with an increase in the copy number of a gene described later. As an example using a stronger promoter, a method for enhancing the γ-glutamylcysteine synthetase activity by replacing the promoter of the γ-glutamylcysteine synthetase gene on the chromosome with a strong transcription promoter is disclosed (Otake). Yasuyuki et al., Bioscience and Industry, Vol. 50, No. 10, pp. 989-994, 1992).

Further, enhancement of gene expression can be achieved, for example, by increasing the copy number of the gene.

  An increase in gene copy number can be achieved by introducing the gene of interest onto the chromosome. Introduction of a gene onto a chromosome can be performed using, for example, homologous recombination. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies in the chromosome as a target. Examples of the sequence having a large number of copies in a chromosome include an autonomously replicating sequence (ARS) consisting of a unique short repetitive sequence and an rDNA sequence having about 150 copies. An example of transformation of yeast using a plasmid containing ARS is described in International Publication No. 95/32289. Alternatively, a gene may be incorporated into a transposon and transferred to introduce multiple copies of the gene into the chromosome.

  An increase in gene copy number can also be achieved by introducing a vector containing the target gene into the host. As a vector, for example, a plasmid having a CEN4 replication origin and a multicopy plasmid having a 2 μm DNA replication origin can be preferably used. The target gene may be inserted into a vector in combination with a suitable promoter to express the target gene. In addition, when a vector containing a promoter suitable for expressing a gene is used, the target gene may be expressed using the promoter in the vector.

  Enhancement of the activity of γ-glutamylcysteine synthetase is disclosed in, for example, US Pat. No. 7553638, Yasuyuki Otake et al. (Bioscience and Industry, Vol. 50, No. 10, pages 989-994, 1992) and the like. . Other enzyme activities can be enhanced similarly to the activity of γ-glutamylcysteine synthetase.

  Modification that increases the enzyme activity can also be achieved, for example, by enhancing the specific activity of the target enzyme. Enzymes with enhanced specific activity can be obtained by searching for various organisms, for example. Alternatively, a highly active type may be obtained by introducing a mutation into a conventional enzyme. The enhancement of specific activity may be used alone or in any combination with the above-described method for enhancing gene expression.

  Confirmation that the target enzyme activity has increased can be performed by measuring the activity of the enzyme. The 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)).

  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 unmodified strain. Examples of 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 increases, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared with the unmodified strain.

  Confirmation that the amount of the target enzyme is increased 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 increased to 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain.

When the yeast of the present invention has a polyploidy of diploid or more and the enzyme activity is increased by modification of the chromosome, the yeast of the present invention has an increased enzyme activity as long as glutathione can be accumulated. Thus, the modified chromosome and the wild-type chromosome may be heterogeneous, or the homozygous chromosome modified so that the enzyme activity is increased.

  Further, when the glutathione synthetase activity decreases, the glutathione synthetase activity is preferably 50% or less, more preferably 20% or less, still more preferably 10% or less, particularly preferably, as compared to the unmodified strain. It has fallen below 5%. Moreover, when glutathione synthetase activity falls, it is preferable that glutathione degrading enzyme activity has substantially disappeared. The modification that reduces the glutathione synthetase activity can be achieved by the same method as described above for reducing the glutathione degrading enzyme activity.

  In the present invention, γ-glutamylcysteine synthetase is not particularly limited as long as it has an activity of recognizing L-Glu and L-Cys as substrates and catalyzing a reaction to produce γ-glutamylcysteine (γ-Glu-Cys). . Γ-glutamylcysteine synthetase recognizes any L-amino acid or amino acid derivative X as a substrate in addition to a reaction that generates γ-Glu-Cys, which is a positive reaction, and binds to L-Glu. -It may have an activity of catalyzing a reaction for producing Glu-X. Examples of γ-glutamylcysteine synthetase include Gsh1p encoded by the GSH1 gene.

  In the present invention, glutathione synthase is not particularly limited as long as it has an activity of recognizing γ-glutamylcysteine and Gly as substrates and catalyzing a reaction for producing glutathione (γ-Glu-Cys-Gly). An example of glutathione synthase is Gsh2p encoded by the GSH2 gene.

  The base sequences of Saccharomyces cerevisiae GSH1 and GSH2 genes are disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/), respectively. The nucleotide sequence of the GSH1 gene of Saccharomyces cerevisiae obtained from the above database and the amino acid sequence encoded by this gene are shown in SEQ ID NOs: 9 and 10, respectively. Further, the base sequence of Saccharomyces cerevisiae GSH2 gene obtained from the above database and the amino acid sequence encoded by the gene are shown in SEQ ID NOs: 11 and 12, respectively.

  Since there may be a difference in the base sequence of the gene encoding glutathione synthetase or γ-glutamylcysteine synthetase depending on the organism from which the enzyme is derived, the gene encoding them may be glutathione synthetase activity or γ-glutamylcysteine synthesis. As long as it encodes a protein having enzyme activity, it may be a variant of the base sequence shown in SEQ ID NO: 9 or 11. The variant conforms to the description of the variant for the DUG gene group and the DUG complex described above.

(1-4) Enhanced expression of vacuolar glutathione transporter In one aspect, the yeast of the present invention has been modified to increase the expression of the vacuolar glutathione transporter.

  “The expression of the vacuolar glutathione transporter is increased” means that the expression of the gene encoding the vacuolar glutathione transporter is increased as compared with a non-modified strain such as a wild strain or a parent strain.

  In the case of increasing the expression of the vacuolar glutathione transporter, the expression of the vacuolar glutathione transporter is preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times, compared to the unmodified strain. It has improved more than twice.

In addition, “the expression of the vacuolar glutathione transporter” is not only increased in the strain in which the vacuolar glutathione transporter is originally expressed, but the vacuolar glutathione transporter is not originally expressed. Expressing a vacuolar glutathione transporter in the strain. That is, for example, it includes introducing a gene encoding a vacuolar glutathione transporter into a strain that does not have a gene encoding a vacuolar glutathione transporter, and expressing the vacuolar glutathione transporter.

  Increasing the expression of the gene encoding the vacuolar glutathione transporter can be achieved by the same method as described above for increasing the expression of the gene encoding γ-glutamylcysteine synthetase and the gene encoding glutathione synthetase. it can.

  In the present invention, the vacuolar glutathione transporter means a protein having a function of transporting cytoplasmic glutathione to the vacuole, and is not particularly limited as long as it has the function. Examples of genes encoding the vacuolar glutathione transporter include the YCF1 gene and the BPT1 gene. Among them, it is preferable to increase the expression of the YCF1 gene. In addition, it is considered that various amino acid / peptide transporters such as Vba1p encoded by the VBA1 gene are involved in the transport of glutathione to the vacuole. In the present invention, the vacuolar glutathione transporter Various amino acid / peptide transporters are included.

  The base sequences of Saccharomyces cerevisiae YCF1 gene, BPT1 gene, and VBA1 gene are disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). Further, the base sequence of the Saccharomyces cerevisiae YCF1 gene obtained from the above database and the amino acid sequence encoded by the same gene are shown in SEQ ID NOs: 13 and 14, respectively.

  Since there may be a difference in the base sequence of the gene encoding the vacuolar glutathione transporter depending on the organism from which the enzyme is derived, as long as the gene encodes a protein having a function of transporting cytoplasmic glutathione to the vacuole, It may be a variant of the base sequence shown in SEQ ID NO: 13 or a variant of the base sequence of the gene encoding the vacuolar glutathione transporter disclosed in the Saccharomyces Genome Database. The variant conforms to the description of the variant for the DUG gene group and the DUG complex described above.

(1-5) Other Modifications The yeast of the present invention may be subjected to other arbitrary modifications in addition to the above modifications as long as γ-glutamyl compounds such as glutathione can be accumulated. Such modifications include modifications that increase the intracellular γ-glutamyl compound concentration. For example, it has been reported that the intracellular glutathione content increases when the expression level of the MET25 gene in yeast is increased. Methods for increasing the expression level of the MET25 gene include mutant MET4 gene (Omura et al., FEBS Letters 387 (1996) 179-183, Chris et. Al., Molecular Biology of the Cell., 8, 1699-1707, 1997). )), A method using a mutant MET30 gene (DOMINIQUE et. Al., MOLECULAR AND CELLUAR BIOLOGY, Dec 1995, p6526-6534, JP 2004-113155) has been reported. Moreover, the activity of aminotransferase may be increased. Examples of yeast aminotransferases include alanine: glyoxylate aminotransferase, branched-chain amino acid transaminase, aspartate aminotransferase, and γ-aminobutyric acid transaminase. (Gamma-aminobutyrate transaminase) and the like can be exemplified. In Saccharomyces cerevisiae, genes encoding these enzymes have already been identified, and AGX1 (systematic name: YFL030W), BAT1 (systematic name: YHR208W) and BAT2 (systematic name: YJR148W), AAT1 (systematic name: YKL106W), respectively. And AAT2 (systematic name: YLR027C), and UGA1 (systematic name: YGR019W). In particular, by increasing the activity of the branched-chain amino acid transaminase encoded by BAT1, it is possible to significantly promote intracellular α-aminobutyric acid synthesis, and intracellular γ-Glu-Abu (L-γ-glutamyl) -L-2-aminobutyric acid) and γ-Glu-Abu-Gly (L-γ-glutamyl-L-2-aminobutyric acid-glycine) accumulation can be increased. Increasing the activity of the aminotransferase can be achieved by the same method as described above for increasing the γ-glutamylcysteine synthetase activity or the glutathione synthetase activity. The base sequence of each gene is disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/).

  Modifications as described above, that is, increase in the ability to synthesize γ-glutamyl compounds, decrease in glutathione degrading enzyme activity, increase in expression of vacuolar glutathione transporter, and any other modification, any modification should be performed first. Also good.

(2) Yeast extract of this invention Next, the method to manufacture a yeast extract is demonstrated. The yeast extract of the present invention can be produced in the same manner as a normal yeast extract except that the yeast of the present invention is used as a raw material.

  First, the yeast of the present invention is cultured in a medium. The medium is not particularly limited as long as yeast can grow, and a medium usually used industrially can be used. For example, the YPD medium described in the examples can be suitably used.

  In addition, a compound that is a raw material for a γ-glutamyl compound such as glutathione (hereinafter also referred to as a raw material for a γ-glutamyl compound) may be added to the medium. The compound that is a raw material of the γ-glutamyl compound refers to, for example, an amino acid, an amino acid derivative, and / or a peptide that constitute the γ-glutamyl compound. When adding the raw material of the γ-glutamyl compound, the addition amount may be appropriately set according to various conditions such as the type of the γ-glutamyl compound and the biosynthetic ability of the raw material of the yeast of the present invention. The final concentration in the medium at the start of culture is preferably 1 ppm or more, more preferably 10 ppm or more, and even more preferably 100 ppm or more. The upper limit of the addition amount is not particularly limited, but for example, from the viewpoint of cost, the final concentration in the medium at the start of culture may be set to 100,000 ppm or less. The amount added may be, for example, preferably 10,000 ppm or less, more preferably 1,000 ppm or less, and even more preferably 500 ppm or less. In addition, when the raw material of the γ-glutamyl compound is added intermittently or continuously during the culture, the total amount added may be set to be equal to the final concentration in the medium at the start of the culture. Good. The raw material of the γ-glutamyl compound to be added may be one or more. The raw material of the γ-glutamyl compound to be added may be a purified product (pure product) or a composition containing the raw material. The composition containing the raw material is not particularly limited as long as it contains a necessary amount of the raw material.

  The culture conditions can be the same as those used in the production of normal yeast extract, and can be appropriately changed depending on the yeast used. Any method such as batch culture, fed-batch culture, or continuous culture can be used. In the case of Saccharomyces cerevisiae, it is preferable to cultivate aerobically by shaking culture or the like at 25 to 35 ° C, more preferably at 27 to 33 ° C, still more preferably at 28 to 32 ° C.

  When the yeast of the present invention is cultured as described above, a γ-glutamyl compound accumulates in the yeast cells.

Preparation of a yeast extract from the obtained yeast may be performed in the same manner as the preparation of a normal yeast extract. The yeast extract may be one obtained by treating a yeast cell extracted with hot water, or one obtained by digesting a yeast cell. Moreover, you may concentrate the yeast extract obtained as needed, and you may dry and make it into the form of a powder.

  As described above, a yeast extract having an increased content of the γ-glutamyl compound is obtained. In a preferred form, the yeast extract is 0.5% by weight or more, more preferably 1% by weight or more, more preferably 5% by weight or more, based on the total amount of solids in the yeast extract, based on the dry weight of the γ-glutamyl compound. The content is particularly preferably 10% by weight or more.

(3) Food / beverage products of this invention The yeast extract in which content of the (gamma) -glutamyl compound obtained as mentioned above was raised can be used for manufacture of food / beverage products. Examples of the food and drink include alcoholic beverages, bread foods, and fermented food seasonings.

  The food / beverage products of this invention are manufactured by adding the yeast extract of this invention to the raw material of food / beverage products, and processing it into food / beverage products. In addition, the addition of the yeast extract may be performed at any stage of the production process of the food or drink. The food / beverage products of this invention can be manufactured by the same method using the raw material similar to normal food / beverage products except using the said yeast extract. Examples of such raw materials include rice, barley and corn starch for alcoholic beverages, flour, sugar, salt, butter, yeast for fermentation, etc. for bread foods, and soybeans, wheat, etc. for fermented food seasonings. Yeast extract or a concentrate thereof, or a product obtained by drying them can be used as a fermented food seasoning by itself.

Example 1. Examination of the effect of lowering glutathione degrading enzyme activity In this example, the glutathione degrading enzyme activity of yeast was lowered and the influence on glutathione accumulating ability and growth rate was examined.

(1) Preparation of γ-glutamylcysteine synthetase gene high expression yeast (AG1 strain) It is known that intracellular glutathione concentration increases in a high expression strain of GSH1 gene encoding γ-glutamylcysteine synthetase ( Non-Patent Documents 10 and 11). Therefore, by replacing the promoter region of the GSH1 gene of Saccharomyces cerevisiae strain Y006 (FERM BP-11299) with the promoter region of the alcohol dehydrogenase gene (hereinafter referred to as ADH1), which is a constitutive expression promoter, the GSH1 high expression yeast (AG1 strain) ) Was produced. The procedure is shown below. The Y006 strain is a URA3 gene-deficient strain and exhibits uracil requirement.

(1-1) Preparation of template plasmid for ADH1 promoter replacement First, the URA3 gene was obtained by PCR using the primers of SEQ ID NOS: 15 and 16 and using the chromosomal DNA of Saccharomyces cerevisiae S288C strain distributed by NBRC as a template. Amplified. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 1 min), and 25 cycles. The obtained DNA fragment was purified by ethanol precipitation, digested with SphI and EcoRI, and inserted into the SphI-EcoRI site of plasmid pUC19 to obtain pUC19-URA3. Next, the ADH1 promoter region (pADH1) was amplified by PCR using the primers of SEQ ID NOs: 17 and 18 and the chromosomal DNA of the Y006 strain as a template. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 1 min), and 25 cycles. The obtained DNA fragment was digested with PstI and inserted into the PstI site of pUC19-URA3 digested with PstI and treated with CIAP to obtain pUC19-pADH1-URA3. Similarly, the ADH1 promoter region amplified using the primers of SEQ ID NOs: 19 and 20 was digested with AatII, inserted into the AatII site of pUC19-pADH1-URA3 digested with AatII and treated with CIAP, and pUC19-pADH1-URA3-ADH1p Got.

(1-2) Introduction of ADH1 promoter into GSH1 gene on chromosome
Using pUC19-pADH1-URA3-pADH1 as a template, using a primer of SEQ ID NO: 21 having an upstream sequence of GSH1 at the 5 ′ end and a primer of SEQ ID NO: 22 having a partial intra-ORF sequence starting from the start codon of the GSH1 gene PCR was performed to obtain a DNA fragment having URA3 sandwiched between ADH1 promoters. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (60 ° C., 10 sec), extension (72 ° C., 4 min), and 25 cycles. The Y006 strain was transformed with this DNA fragment and spread on an SD plate medium containing no uracil. From the grown transformant, a strain in which the GSH1 promoter was replaced with pADH1-URA3-pADH1 was obtained.

<SD medium composition>
Glucose 2%
Nitrogen Base 1x Concentration (10x Nitrogen Base is 1.7g Bacto Yeast Nitrogen Base w / o Amino Acids and
Ammonium Sulfate (Difco) mixed with 5 g of ammonium sulfate dissolved in 100 ml of sterilized water, pH adjusted to about 5.2, and filter sterilized by filtration)

(1-3) Removal of URA3 selection marker and promoter replacement of GSH1 gene
Since cells lacking the URA3 gene exhibit resistance to 5-fluoroorotic acid (5-FOA), a strain from which the URA3 selection marker has been removed can be selected using a medium containing 5-FOA. Therefore, the strain in which the GSH1 promoter was replaced with pADH1-URA3-pADH1 was cultured overnight in SD medium supplemented with uracil, and an appropriate amount was applied to a 5-FOA plate medium. A strain (AG1 ura3- strain) in which URA3 was removed and the GSH1 promoter was replaced with the ADH1 promoter was obtained from the grown colonies by homologous recombination between the two introduced ADH1 promoters.

(1-4) Insertion of URA3 Gene into ura3 Site Using the Saccharomyces cerevisiae S288C genome as a template, the primers shown in SEQ ID NOs: 23 and 24 were used to amplify the URA3 gene site. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 3 min), and 25 cycles. Next, the AG1 ura3- strain was transformed with this DNA fragment, and a strain in which the ura3 site was replaced with wild-type URA3 and became non-requiring uracil was obtained and designated AG1. Similarly, the Y006 strain was similarly transformed with this DNA fragment, and a strain that became non-uracil-requiring was obtained and designated as Y003 strain.

(2) Preparation of glutathione degrading enzyme gene disruption strain of Y006 strain In order to verify the effect of reducing glutathione degrading activity, mutant strains in which each glutathione degrading enzyme gene was disrupted were prepared based on Saccharomyces cerevisiae Y006 strain by the following procedure did.

(2-1) Preparation of a DUG1-disrupted strain of Y006 strain First, a primer of SEQ ID NO: 25 with 80 bases upstream from the start codon of DUG1 and a primer of SEQ ID NO: 26 with 80 bases downstream from the stop codon of DUG1 were used. The URA3 gene of Y006 strain was amplified. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. Y006 strain was transformed with the obtained DNA fragment and applied to SD medium containing no uracil. A dug1D strain (hereinafter referred to as d1 strain) of Y006 was obtained from the grown transformant.

(2-2) Preparation of a DUG2-disrupted strain of Y006 strain First, a primer of SEQ ID NO: 27 with 80 bases upstream from the start codon of DUG2 and a primer of SEQ ID NO: 28 with 80 bases downstream from the stop codon of DUG2 were used. The URA3 gene of Y006 strain was amplified. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. Y006 strain was transformed with the obtained DNA fragment and applied to SD medium containing no uracil. A dug2D strain of Y006 (hereinafter referred to as d2 strain) was obtained from the grown transformant.

(2-3) Preparation of a DUG3-disrupted strain of Y006 strain First, a primer of SEQ ID NO: 29 added with 80 bases upstream from the start codon of DUG3 and a primer of SEQ ID NO: 30 added with 80 bases downstream from the stop codon of DUG3 were used. The URA3 gene of Y006 strain was amplified. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. Y006 strain was transformed with the obtained DNA fragment and applied to SD medium containing no uracil. A dug3D strain of Y006 (hereinafter referred to as d3 strain) was obtained from the grown transformant.

(2-4) Preparation of ECM38-disrupted strain of Y006 strain First, the primer of SEQ ID NO: 31 with 80 bases upstream from the start codon of ECM38 and the primer of SEQ ID NO: 32 with 80 bases downstream from the stop codon of ECM38 were used. The URA3 gene of Y006 strain was amplified. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. Y006 strain was transformed with the obtained DNA fragment and applied to SD medium containing no uracil. An ecm38D strain of Y006 (hereinafter referred to as e strain) was obtained from the grown transformant.

(2-5) Production of ECM38-disrupted strain of dug2D strain First, using the ECM38-disrupted strain of Saccharomyces cerevisiae-disrupted strain set (OpenBiosystems) with BY4743 as a parent strain as a template, the primer of SEQ ID NO: 33 and the primer of SEQ ID NO: 34 were used. Used to amplify ECM38. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. The obtained DNA fragment was transformed into dug2Δ strain and applied to a YPD plate medium containing G418 having a final concentration of 50 mg / L. A dug2D ecm38D strain (hereinafter referred to as ed strain) was obtained from the grown transformant.

(3) Evaluation of glutathione accumulation ability of glutathione-degrading enzyme-disrupted strains The Saccharomyces cerevisiae gene mutant prepared as described above was cultured in a flask in a YPD medium, and the intracellular glutathione concentration was measured. The composition of the YPD medium is shown below.
<YPD medium>
D-glucose 20 g
Bacto Yeast Extract 10 g
Bacto Peptone 20 g
1 L water

  2.5 mL of SD medium overnight culture solution of Y003, AG1, d1, d2, d3, e, and ed strain was inoculated into 50 mL of YPD medium, respectively, and cultured in a flask for 18 hours. After completion of the culture, 5 mL of the culture solution used for dry cell weight measurement and 1 mL of the culture solution used for glutathione concentration analysis by HPLC were collected. The cells were collected from the culture solution, rinsed once with pure water, and stored as a sample at -80 ° C. Thereafter, the sample for measuring the dry cell weight was suspended in 100 mL of water, and then applied to a filter paper whose dry weight was measured in advance, and dried for 4 hours to calculate the dry cell weight per 5 mL of the culture solution. The sample used for glutathione concentration analysis was suspended in 10 mL of pure water and subjected to hot water extraction at 70 ° C. for 10 minutes. The hot water extract was mixed with a 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) reaction solution, the thiol group of glutathione was fluorescently labeled, and the amount of glutathione was calculated by HPLC analysis. The intracellular glutathione concentration was calculated as the amount of glutathione per dry cell weight.

The results are shown in FIG. The intracellular glutathione concentrations of the wild type strain Y003, the GSH1 high expression strain AG1, and the glutathione degrading enzyme gene disruption strains d1, d2, d3, e, and ed are Y003: 0.50%, respectively. AG1: 0.80%, d1: 0.47%, d2: 0.49%, d3: 0.54%, e: 0.53%, ed: 0.50%. The AG1 strain, which is a high expression strain of GSH1, has an improved glutathione concentration as compared with the wild-type strain Y003, as already reported (Non-Patent Documents 10 and 11). On the other hand, disruption of ECM38 or each DUG gene encoding glutathione degrading enzyme did not affect the intracellular glutathione concentration. In ECM38 or a single disruption strain of each DUG gene, it was expected that the intracellular glutathione concentration would not be affected because Ecm38p and DUG complex complement each other's functions. In the ed strain, which is a double disruption strain of the encoded gene, no increase in intracellular glutathione concentration was observed.

(4) Production of a glutathione degrading enzyme gene-disrupted strain derived from a strain having a high glutathione accumulation trait (AG1) Next, the effect of a decrease in glutathione degrading enzyme activity on the glutathione accumulating ability in a strain having a high glutathione accumulating trait is evaluated. Therefore, a glutathione degrading enzyme gene disruption strain was prepared based on the AG1 strain. The procedure is shown below.

(4-1) Production of AG1 strain DUG2 disruption strain
As in the preparation of the DUG2 disruption strain (d2 strain) of Y006 strain, the primer of SEQ ID NO: 27 with 80 bases upstream from the start codon of DUG2 and the primer of SEQ ID NO: 28 with 80 bases downstream from the stop codon of DUG2 were used. The URA3 gene of Y006 strain was amplified. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. AG1 ura3- strain was transformed with the obtained DNA fragment and applied to SD medium containing no uracil. AG1 dug2D strain (hereinafter referred to as AG1-d strain) was obtained from the grown transformant.

(4-2) Preparation of ECM38 disruption strain of AG1 strain
Similar to the preparation of the ECM38 disruption strain (e strain) of the Y006 strain, the primer of SEQ ID NO: 31 with 80 bases upstream from the start codon of ECM38 and the primer of SEQ ID NO: 32 with 80 bases downstream from the stop codon of ECM38 were used. The URA3 gene of Y006 strain was amplified. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. AG1 ura3- strain was transformed with the obtained DNA fragment and applied to SD medium containing no uracil. AG1 ecm38D strain (hereinafter referred to as AG1-e strain) was obtained from the grown transformant.

(5) Evaluation of glutathione accumulation ability of AG1 strain-derived glutathione-degrading enzyme gene-disrupted strain The Saccharomyces cerevisiae gene mutant prepared as described above was cultured in a flask in a YPD medium, and the intracellular glutathione concentration was measured. The composition of the YPD medium is shown below.
<YPD medium>
D-glucose 20 g
Bacto Yeast Extract 10 g
Bacto Peptone 20 g
1 L water

  2.5 mL of SD medium overnight culture solution of Y003, AG1, AG1-d, AG1-e, and AG1-ed strains was inoculated into 50 mL of YPD medium, respectively, and cultured in a flask for 18 hours. After completion of the culture, 5 mL of the culture solution used for dry cell weight measurement and 1 mL of the culture solution used for glutathione concentration analysis by HPLC were collected. The cells were collected from the culture solution, rinsed once with pure water, and stored as a sample at -80 ° C. Thereafter, the sample for measuring the dry cell weight was suspended in 100 mL of water, and then applied to a filter paper whose dry weight was measured in advance, and dried for 4 hours to calculate the dry cell weight per 5 mL of the culture solution. The sample used for glutathione concentration analysis was suspended in 10 mL of pure water and subjected to hot water extraction at 70 ° C. for 10 minutes. The hot water extract was mixed with a 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) reaction solution, the thiol group of glutathione was fluorescently labeled, and the amount of glutathione was calculated by HPLC analysis. The intracellular glutathione concentration was calculated as the amount of glutathione per dry cell weight.

  The results are shown in FIG. The intracellular glutathione concentrations of the Y003 strain, the AG1 strain, and the glutathione degrading enzyme-disrupted strain derived from the AG1 strain are Y003: 0.6%, AG1: 1.0%, AG1-d: 2.5%, AG1-e: 1.1%, AG1- ed: 2.4%.

  DUG complex deficiency, which did not affect intracellular glutathione concentration by single disruption, contributed to an increase in intracellular glutathione concentration in the high expression strain of GSH1. In addition, AG1 with high glutathione accumulation traits shows growth delay compared to Y003, but AG-d reduces growth delay in the logarithmic growth phase despite the higher intracellular glutathione concentration compared to AG1. (FIG. 3).

  From the above results, it was revealed that the deficiency of the DUG complex contributes to glutathione degradation in the GSH1 high expression strain. Therefore, it was suggested that high expression of GSH1 induces the activity of the DUG complex.

(6) Analysis of expression level of DUG complex component in GSH1 high expression strain
Whether the activity of the DUG complex was induced by high expression of GSH1 was analyzed by quantifying the amount of mRNA of each component of the DUG complex by RT-PCR.

  Y003 and 2.5 mL of the AG1 strain SD medium overnight culture solution were inoculated into 50 mL of YPD medium, respectively, and cultured in a flask. A 1 mL sample was taken at two points of OD600 = 5 and 10, rinsed twice with sterilized water, and then the pellet was frozen at −80 ° C. The next day, total RNA was extracted using an RNA extraction kit, RNeasy (Qiagen). 500 ng of the extracted RNA was reacted at 37 ° C. for 15 minutes using PrimeScript RT reagent Kit (TaKaRa) to prepare cDNA. At this time, in order to use as a negative control of RT-PCR, a sample was prepared in which the reaction was performed without adding an enzyme.

  Subsequently, the reaction solution was prepared with Power SYBR Green (Applied Biosystems). The primers shown in SEQ ID NOs: 35 and 36 were used for the DUG1 reaction. The primers shown in SEQ ID NOs: 37 and 38 were used for the DUG2 reaction. The primers shown in SEQ ID NOs: 39 and 40 were used for the DUG3 reaction. In addition, the ACT1 gene was amplified with the primers shown in SEQ ID NOs: 41 and 42 and used as an internal standard. As the cDNA solution, four different dilutions were used: a stock solution, a 10-fold diluted solution, a 100-fold diluted solution, and a 1000-fold diluted solution. For the RT-PCR reaction, 7500 Real Time PCR System (Applied Biosystems) was used.

  The results are shown in FIG. In the GSH1 high-expressing strain, DUG1 mRNA increased approximately 1.6-fold and DUG3 mRNA increased approximately 3-fold during the middle stage of culture (FIG. 4a). On the other hand, no effect due to high expression of GSH1 was observed in the stationary phase (FIG. 4b).

  From the above results, it was found that the activity of the DUG complex which is a glutathione degrading enzyme is increased by inducing the expression of the DUG component in the high expression strain of GSH1. In addition, as described above, from the comparison of the growth curves of the Y003, AG1, and AG1-d strains, growth delay was observed in the logarithmic growth phase in the AG1 strain, but this was reduced in the AG1-d strain (FIG. 3). . The expression level of DUG gene, which shows no difference between the two strains in the stationary phase, increased in the logarithmic growth phase in the high GSH1 expression strain, so the expression level of DUG gene in the logarithmic growth phase has an effect on growth. I understood.

As described above, by reducing the activity of the DUG complex in the GSH1 high-expressing strain, (1) the intracellular glutathione concentration is increased, (2) the intracellular glutathione concentration is increased,
It was clarified that two effects of reducing the growth delay can be obtained.

Example 2 Examination of the effect of enhancing the vacuolar glutathione transporter In this example, the expression of the yeast vacuolar glutathione transporter was enhanced, and the influence on the glutathione accumulation ability and growth rate was examined.

(7) Production of Yeast Glutathione Vacuolar Transporter Gene Highly Expressing Yeast (PY Strain) YCF1 gene is known as a gene encoding yeast glutathione vacuolar transporter. In order to verify the effect of enhancing glutathione vacuolar transporter, the YCF1 gene promoter region of Saccharomyces cerevisiae strain Y006 was replaced with the promoter region of the PGK1 gene, which is a high expression promoter of the YCF1 strain (PY strain). ) Was produced. The procedure is shown below.

(7-1) Preparation of template plasmid for PGK1 promoter substitution First, the URA3 gene was amplified by PCR using the primers of SEQ ID NOs: 15 and 16 and the chromosomal DNA of Saccharomyces cerevisiae S288C as a template. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 1 min), and 25 cycles. The obtained DNA fragment was purified by ethanol precipitation, digested with SphI and EcoRI, and inserted into the SphI-EcoRI site of plasmid pUC19 to obtain pUC19-URA3. Next, using the primers shown in SEQ ID NOs: 43 and 44, the PGK1 promoter region (pPGK1) was amplified by PCR using the chromosomal DNA of the Y006 strain as a template. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 1 min), and 25 cycles. The obtained DNA fragment was digested with PstI and inserted into the PstI site of pUC19-URA3 digested with PstI and treated with CIAP to obtain pUC19-pPGK1-URA3. Similarly, the PGK1 promoter region amplified using the primers shown in SEQ ID NOs: 45 and 46 was digested with AatII, inserted into the AatII site of pUC19-pPGK1-URA3 digested with AatII and treated with CIAP, and pUC19-pPGK1-URA3- pPGK1 was obtained.

(7-2) Introduction of PGK1 promoter into YCF1 gene on chromosome
Using pUC19-pPGK1-URA3-pPGK1 as a template, using a primer of SEQ ID NO: 47 having an upstream sequence of YCF1 at the 5 ′ end and a primer of SEQ ID NO: 48 having a partial intra-ORF sequence starting from the start codon of the YCF1 gene PCR was performed to obtain a DNA fragment having URA3 sandwiched between PGK1 promoters. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (60 ° C., 10 sec), extension (72 ° C., 4 min), and 25 cycles. The Y006 strain was transformed with this DNA fragment, and a strain in which the YCF1 promoter was replaced with pPGK1-URA3-pPGK1 was obtained from a transformant obtained by applying it to an SD plate medium containing no uracil.

(7-3) Removal of URA3 selectable marker and promoter replacement of YCF1 gene
The strain in which the YCF1 promoter was replaced with pPGK1-URA3-pPGK1 was cultured overnight in SD medium supplemented with uracil, and an appropriate amount was applied to a 5-FOA plate medium. From the grown colonies, a strain (PY ura3- strain) was obtained in which URA3 was removed by homologous recombination between the two introduced PGK1 promoters and the YCF1 promoter was replaced with the PGK1 promoter.
(7-4) Insertion of URA3 gene into ura3 site The URA3 gene site was amplified using the Saccharomyces cerevisiae S288C genome as a template and the primers shown in SEQ ID NOs: 23 and 24. Next, the PY ura3- strain was transformed with this DNA fragment to obtain a PY strain which was a strain in which the ura3 site was replaced with wild-type URA3.

(8) Evaluation of glutathione accumulation ability of the Y006 strain-derived glutathione vacuolar transporter-enhanced strain The Saccharomyces cerevisiae gene mutant prepared as described above was cultured in a flask in a YPD medium, and the glutathione concentration in the cells was measured. The composition of the YPD medium is shown below.
<YPD medium>
D-glucose 20 g
Bacto Yeast Extract 10 g
Bacto Peptone 20 g
1 L water

  Y003, AG1, e, d, and PY strain SD medium overnight culture solution 2.5 mL was inoculated into 50 mL of YPD medium, respectively, and cultured in a flask for 18 hours. After completion of the culture, 5 mL of the culture solution used for dry cell weight measurement and 1 mL of the culture solution used for glutathione concentration analysis by HPLC were collected. The cells were collected from the culture solution, rinsed once with pure water, and stored as a sample at -80 ° C. Thereafter, the sample for measuring the dry cell weight was suspended in 100 mL of water, and then applied to a filter paper whose dry weight was measured in advance, and dried for 4 hours to calculate the dry cell weight per 5 mL of the culture solution. The sample used for glutathione concentration analysis was suspended in 10 mL of pure water and subjected to hot water extraction at 70 ° C. for 10 minutes. The hot water extract was mixed with a 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) reaction solution, the thiol group of glutathione was fluorescently labeled, and the amount of glutathione was calculated by HPLC analysis. The intracellular glutathione concentration was calculated as the amount of glutathione per dry cell weight.

  The results are shown in FIG. The intracellular glutathione concentrations of Y003 and its single mutant AG1, e, d, and PY strains are Y003: 0.75%, AG1: 1.20%, e: 0.70%, d2: 0.75%, PY: 0.65%, respectively. Met. The enhanced expression of YCF1, a glutathione vacuolar transporter, did not affect intracellular glutathione concentration.

(9) Production of a glutathione vacuolar transporter-enhanced strain derived from a strain (AG1) having a high glutathione accumulating trait The enhancement of glutathione vacuolar transporter alone did not affect the intracellular glutathione concentration. Therefore, in order to evaluate the effect of enhancing glutathione vacuolar transporter in strains with high glutathione accumulation traits, the promoter region of AGCF strain YCF1 gene should be replaced with the promoter region of PGK1 gene, which is a high expression promoter. Thus, a YCF1 high expression yeast (AG1-PY strain) was prepared. Furthermore, in order to evaluate the combined effect of reduced vacuolar localized glutathione degrading enzyme activity and enhanced glutathione vacuolar transporter, ECM38 encoding vacuolar localized glutathione degrading enzyme was destroyed based on the AG1-PY strain. Strain (AG1-PY-e strain) was prepared. The procedure is shown below.

(9-1) Production of AG1 strain enhanced YCF1 expression

(9-1-1) Introduction of PGK1 promoter into YCF1 gene on chromosome of AG1 strain
Using pUC19-pPGK1-URA3-pPGK1 as a template, using a primer of SEQ ID NO: 47 having an upstream sequence of YCF1 at the 5 ′ end and a primer of SEQ ID NO: 48 having a partial intra-ORF sequence starting from the start codon of the YCF1 gene PCR was performed to obtain a DNA fragment having URA3 sandwiched between PGK1 promoters. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (60 ° C., 10 sec), extension (72 ° C., 4 min), and 25 cycles. The AG1 ura3- strain was transformed with this DNA fragment, and a strain in which the YCF1 promoter was replaced with pPGK1-URA3-pPGK1 was obtained from a transformant obtained by applying to an SD plate medium containing no uracil.

(9-1-2) Removal of URA3 selection marker and promoter replacement of YCF1 gene
The strain in which the YCF1 promoter was replaced with pPGK1-URA3-pPGK1 was cultured overnight in SD medium supplemented with uracil, and an appropriate amount was applied to a 5-FOA plate medium. From the grown colonies, a strain (AG1-PY ura3- strain) was obtained in which URA3 was removed and the YCF1 promoter was replaced with the PGK1 promoter by homologous recombination between the two introduced PGK1 promoters.

(9-1-3) Insertion of URA3 Gene into ura3 Site Using the Saccharomyces cerevisiae S288C genome as a template, the primers shown in SEQ ID NOs: 23 and 24 were used to amplify the URA3 gene site. Next, the AG1-PY ura3- strain was transformed with this DNA fragment, and the AG1-PY strain, which was a strain in which the ura3 site was replaced with wild-type URA3, was obtained.

(9-2) Preparation of ECM38-disrupted strain of AG1-PY strain First, the primer of SEQ ID NO: 31 added 80 bases upstream from the start codon of ECM38 and the primer of SEQ ID NO: 32 added 80 bases downstream from the stop codon of ECM38 Was used to amplify the URA3 gene of S288C strain. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. The obtained DNA fragment was transformed into AG1-PY ura3- strain and applied to SD medium not containing uracil. AG1-PY ecm38D strain (hereinafter referred to as AG1-PY-e strain) was obtained from the grown transformant.

(9-3) Preparation of AG1-d strain enhanced YCF1 expression strain In the same manner as the preparation of AG1 strain YCF1 expression-enhanced strain in (9-1) above, the AG1-d ura3- strain was obtained from the YCF1 promoter. AG1-d-PY ura3- strain in which PGK1 promoter was substituted was obtained, and further, the ura3 site was substituted with wild-type URA3 to obtain AG1-d-PY strain.

(9-4) Preparation of ECM38-disrupted strain of AG1-d-PY strain AG1-d-PY ura3- strain in the same manner as the ECM38-disrupted strain of AG1-PY strain was prepared in (9-2) above. Thus, an AG1-d-PY ecm38D strain (hereinafter referred to as AG1-d-PY-e strain) in which ECM38 was destroyed was obtained.

(10) Evaluation of glutathione accumulation ability and growth of various AG1-derived multiple mutant strains The Saccharomyces cerevisiae gene mutant strains produced as described above were cultured in a jar fermenter, and intracellular glutathione concentration and growth were measured. The composition of the medium used is as follows.

<Medium composition>
I) 5xSD medium
D-glucose 5g
Difco Yeast Nitrogen base w / o Amino Acids and Ammonium Sulfate 8.5 g
Ammonium sulfate 25 g
1 L water

II) 50% glucose
D-glucose 500 g
1 L water

  Inoculate 30 mL of SD medium overnight culture solution of Y003, AG1, AG1-d, AG1-de, AG1-PY, AG1-PY-e strains into 270 mL of 5xSD medium, respectively, and 50% glucose solution as required Cultivation was carried out for 36 hours with a jar fermenter. After completion of the culture, 5 mL of a culture solution used for dry cell weight measurement and 1 mL of a culture solution used for analysis of glutathione concentration by HPLC were collected. The cells were collected from the culture solution, rinsed once with pure water, and stored as a sample at -80 ° C. Thereafter, for the sample for measuring the weight of the dried cells, the cells were completely dried with a vacuum dryer at 42 ° C. for 3 days, and the weight of the dried cells per 5 mL of the culture solution was calculated. The sample used for glutathione concentration analysis was suspended in 10 mL of pure water and subjected to hot water extraction at 70 ° C. for 10 minutes. The hot water extract was mixed with the ABD-F reaction solution, the thiol group of glutathione was fluorescently labeled, and the amount of glutathione was calculated by HPLC analysis. The intracellular glutathione concentration was calculated as the amount of glutathione per dry cell weight.

  The intracellular glutathione concentration of each strain is shown in FIG. 6, and the growth curve is shown in FIG. The intracellular glutathione concentration of each strain is Y003: 0.7%, AG1: 1.1%, AG1-d: 2.5%, AG1-e: 1.2%, AG1-PY: 1.3%, AG1-PY-e: 4.3% there were. As it is known that intracellular high-concentration glutathione inhibits growth, the growth rate of AG1 strain decreased with the increase of intracellular glutathione.

  In the AG1-PY strain in which the expression of YCF1 encoding the glutathione vacuolar transporter was enhanced, the intracellular glutathione concentration was not increased, but the growth rate was improved to the wild type Y003 strain. This is presumably because a high concentration of intracellular glutathione, which is the cause of growth delay, was transported to the vacuole, and the glutathione concentration in the cytoplasm decreased.

  The AG1-PY-e strain, in which ECM38 encoding the vacuolar localized glutathione degrading enzyme was further disrupted in the AG1-PY strain, grew much faster than the wild type, and the intracellular glutathione concentration was significantly increased. . In the AG1-e strain, there was no increase in intracellular glutathione concentration, indicating that glutathione transported to the vacuole by Ycf1p was accumulated in the vacuole without being degraded by Ecm38p, and in the cytoplasm. It is thought that the feedback inhibition to the glutathione biosynthetic system was eliminated and the glutathione biosynthesis ability was improved by reducing the glutathione concentration.

  Based on the above results, high expression of YCF1, which encodes the glutathione vacuolar transporter, transports cytoplasmic glutathione to the vacuole, which can cause growth delay and feedback inhibition of the glutathione biosynthesis system. It was revealed that by further destroying ECM38 encoding glutathione-degrading enzyme and accumulating glutathione in vacuoles, it is possible to achieve further increase in intracellular glutathione concentration in strains with high glutathione content while avoiding growth delay. It was.

Example 3 Production of Yeast Extract In this example, a yeast extract was produced using yeast obtained by increasing the intracellular glutathione concentration obtained in the above example and improving growth as a raw material.

(11) Manufacture of yeast extract About 50 ml of culture broths of AG1-d strain and AG1-PY-e strain obtained in (10) above were collected, and the cells and culture supernatant were separated by centrifugation. The bacterial cells were washed by suspending the bacterial cells in 50 ml of pure water and separating the bacterial cells by centrifugation. This washing operation was repeated again and then suspended in pure water so that the bacterial cell concentration was about 10 g / dL. The bacterial cell concentration was estimated from the result of a preliminary experiment in which the correlation between the absorbance and the dry cell weight was examined. This suspension was heated at 70 ° C. for 10 minutes and then rapidly cooled on ice. The extract and the cell residue were separated by centrifugation. In this way, a yeast extract (extract) was produced. A yeast extract having a GSH content of about 8.2% per solid extract was obtained from the AG1-d strain, and a yeast extract having a GSH content of about 13.1% per solid extract was obtained from the AG1-PY-e strain.

Example 4 Application to production of γ-glutamyl peptides other than glutathione (1)
In this example, taking γ-glutamylcysteine as an example, in the accumulation of γ-glutamyl peptides other than glutathione, the decrease in the activity of the DUG complex contributes to the reduction of growth delay, and the enhancement of the glutathione vacuolar transporter The combination with reduced vacuolar localized glutathione degrading enzyme activity is shown to be effective. Yeast that accumulates γ-glutamylcysteine, for example, enhances the expression of GSH1, which encodes an enzyme that catalyzes a reaction that produces a dipeptide having a γ-glutamyl group, and synthesizes glutathione from γ-glutamylcysteine and glycine. It can be made by destroying GSH2, which encodes the enzyme to catalyze.

(12) Preparation of GSH2-disrupted strain First, GSH2 was amplified using the primer of SEQ ID NO: 49 and the primer of SEQ ID NO: 50 using the GSH2-disrupted strain of Saccharomyces cerevisiae-disrupted strain set (OpenBiosystems) with BY4743 as a parent strain as a template. did. PCR conditions were heat denaturation (94 ° C., 10 sec), annealing (50 ° C., 10 sec), extension (72 ° C., 2 min), and 25 cycles. The obtained DNA fragment is transformed into Y003 strain, AG1 strain, AG1-d strain, AG1-d-PY strain and AG1-d-PY-e strain to a YPD plate medium containing G418 at a final concentration of 50 mg / L. Applied. From the grown transformants, Y003 strain GSH2 disruption strain (hereinafter referred to as gsh2 strain), AG1 strain GSH2 disruption strain (hereinafter referred to as AG1-gsh2 strain), AG1-d strain GSH2 disruption strain (hereinafter referred to as AG1-d-gsh2), AG1 -D-PY GSH2 disruption strain (AG1-d-PY-gsh2 strain) and AG1-d-PY-e GSH2 disruption strain (AG1-d-PY-e-gsh2 strain) .

(13) Evaluation of γ-glutamylcysteine accumulation concentration and growth in various GSH2-disrupted strains The GSH2-disrupted strain of Saccharomyces cerevisiae prepared as described above was cultured with a jar fermenter, and the intracellular γ-glutamylcysteine concentration and growth Was measured. The composition of the medium used is as follows.

<Medium composition>
I) 5xSD medium
D-glucose 5g
Difco Yeast Nitrogen base w / o Amino Acids and Ammonium Sulfate 8.5 g
Ammonium sulfate 25 g
1 L water

II) 50% glucose
D-glucose 500 g
1 L water

  Inoculate 30 ml of SD medium overnight culture solution of gsh2 strain, AG1-gsh2 strain, and AG-d-gsh2 strain into 270 mL of 5xSD medium respectively, and add jar fermenter while adding 50% glucose solution dropwise as necessary. Was cultured. After completion of the culture, 5 mL of a culture solution used for dry cell weight measurement and 1 mL of a culture solution used for γ-glutamylcysteine concentration analysis by HPLC were collected. The cells were collected from the culture solution, rinsed once with pure water, and stored as a sample at -80 ° C. Thereafter, for the sample for measuring the weight of the dried cells, the cells were completely dried with a vacuum dryer at 42 ° C. for 3 days, and the weight of the dried cells per 5 mL of the culture solution was calculated. A sample used for γ-glutamylcysteine concentration analysis was suspended in 10 mL of pure water and subjected to hot water extraction at 70 ° C. for 10 minutes. The hot water extract was mixed with the ABD-F reaction solution, the thiol group of γ-glutamylcysteine was fluorescently labeled, and the amount of γ-glutamylcysteine was calculated by HPLC analysis. The intracellular γ-glutamylcysteine concentration was calculated as the amount of γ-glutamylcysteine per dry cell weight.

  The growth curve is shown in FIG. 8, and the intracellular γ-glutamylcysteine concentration is shown in FIG. An increase in intracellular γ-glutamylcysteine concentration was observed in the AG1-gsh2 strain, which is a GSH1 expression-enhanced strain of the gsh2 strain, compared to the gsh2 strain, which is a single disruption strain of GSH2. In the AG1-gsh2 strain, the AG1-d-gsh2 strain in which DUG2 encoding glutathione-degrading enzyme was further disrupted did not show an increase in intracellular γ-glutamylcysteine concentration compared to the AG1-gsh2 strain. This is probably because the DUG complex originally did not contribute to γ-glutamylcysteine degradation at all or hardly contributed. However, although the destruction of DUG2 did not contribute to the increase in intracellular γ-glutamylcysteine concentration (FIG. 9), the growth delay was greatly reduced in the AG1-d-gsh2 strain (FIG. 8).

  From the above results, in the yeast that accumulates γ-glutamylcysteine, which is a γ-glutamyl peptide other than glutathione, not only the growth delay of yeast that accumulates glutathione at a high concentration is reduced by the decreased activity of the DUG complex. It was also found that growth delay was reduced. Therefore, it is considered that the growth delay of yeast that accumulates various γ-glutamyl peptides produced by Gsh1p can be eliminated by reducing the activity of the DUG complex.

(14) Effect of accumulation of γ-glutamylcysteine in vacuoles
Inoculate 30 mL of the SD medium overnight culture solution of AG1-d-gsh2 strain, AG-d-PY-gsh2 strain, and AG-d-PY-e-gsh2 strain into 270 mL of 5xSD medium, respectively, as necessary. Then, culturing was performed with a jar fermenter while dropping a 50% glucose solution. After completion of the culture, 5 mL of a culture solution used for dry cell weight measurement and 1 mL of a culture solution used for γ-glutamylcysteine concentration analysis by HPLC were collected. The cells were collected from the culture solution, rinsed once with pure water, and stored as a sample at -80 ° C. Thereafter, for the sample for measuring the weight of the dried cells, the cells were completely dried with a vacuum dryer at 42 ° C. for 3 days, and the weight of the dried cells per 5 mL of the culture solution was calculated. A sample used for γ-glutamylcysteine concentration analysis was suspended in 10 mL of pure water and subjected to hot water extraction at 70 ° C. for 10 minutes. The hot water extract was mixed with the ABD-F reaction solution, the thiol group of γ-glutamylcysteine was fluorescently labeled, and the amount of γ-glutamylcysteine was calculated by HPLC analysis. The intracellular γ-glutamylcysteine concentration was calculated as the amount of γ-glutamylcysteine per dry cell weight.

  The results are shown in FIG. Similar to the glutathione-accumulating strain, the AG1-d-PY-e-gsh2 strain, which enhanced YCF1 expression and disrupted ECM38, had an intracellular γ-glutamylcysteine concentration compared to the AG1-d-gsh2 strain. Increased significantly. On the other hand, in the AG1-d-PY-gsh2 strain having wild type ECM38, the intracellular γ-glutamylcysteine concentration was not changed compared to the AG1-d-gsh2 strain. It is thought that it is degraded by Ecm38p localized in the vesicle. Therefore, in the AG1-d-PY-e-gsh2 strain, it is considered that γ-glutamylcysteine transferred to the vacuole due to enhanced expression of YCF1 is stably accumulated in the vacuole.

  From the above results, by enhancing the expression of YCF1 and decreasing the activity of ECM38, not only glutathione but also γ-glutamylcysteine such as γ-glutamylcysteine accumulates without being degraded in the vacuole, resulting in an increase in intracellular concentration. I found out that I could do it.

Example 5 FIG. Application to production of γ-glutamyl peptides other than glutathione (2)
By enhancing the expression of GSH1, which encodes an enzyme that catalyzes a reaction that generates a dipeptide having a γ-glutamyl group, various γ-glutamyl peptides are generated in addition to the production of γ-glutamylcysteine, which is a positive reaction. Therefore, in this example, as an example of γ-glutamyl peptides other than glutathione, the production of γ-glutamyl-α-aminobutyric acid (γ-Glu-Abu) and the effect of its accumulation on growth were examined.

(15) γ-Glu-Abu accumulation concentration and evaluation of growth First, the AG1 strain and the AG1-d strain were inoculated in an SD medium, and cultured with shaking at 30 ° C. Next, these overnight cultures were inoculated into SD medium containing 100 ppm of L-α-aminobutyric acid (Abu) and cultured with shaking at 30 ° C. During the logarithmic growth phase, a culture solution used for dry cell weight measurement and a culture solution used for γ-glutamyl peptide concentration analysis were collected. The cells were collected from the culture solution, rinsed once with pure water, and stored as a sample. Thereafter, the sample for measuring the dry cell weight was suspended in water so as to have an appropriate concentration, then applied to a filter paper whose dry weight was measured in advance, and dried for 4 hours to calculate the dry cell weight. On the other hand, the sample used for γ-Glu-Abu concentration analysis was suspended in water to an appropriate concentration, and then extracted with hot water at 70 ° C. for 10 minutes. In this step, the extract contained in the cells was extracted. Next, the extract and the cell residue were separated by centrifugation. Cell debris was removed from the extract using a 10 kDa centrifugal filtration membrane (MILLIPORE: Amicon Ultra-0.5 mL 10K (catalog number UFC501096)), and the obtained fraction was used as a yeast extract by the method described below. Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly contents in the extract were measured. The intracellular concentration of each compound was calculated as the amount per dry cell weight.

  Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly content were measured by derivatizing these compounds with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), LC-MS / It was performed by detecting by MS. Specifically, 2.5 μL of yeast extract diluted to an appropriate concentration, or 2.5 μL of MillQ water in 2.5 μL of a standard solution containing 1 μM of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly, 5 μM internal standard solution (internal standard is 3-methyl-His-d2 (Sigma) and Gly-d2 (Sigma), both are labeled with stable isotopes)) 5 μL, borate buffer ( 30 μL of AccQ-Fluor (registered trademark) reagent kit (manufactured by Japan Waters) was added. To these mixtures, 10 μL of AQC reagent solution (prepared by dissolving the above AccQ-Fluor reagent kit reagent powder in 1 mL of acetonitrile) was added. The obtained mixture was heated at 55 ° C. for 10 minutes, and then 100 μL of 0.1% formic acid aqueous solution was added to prepare an analytical sample.

  The analytical sample prepared as described above was then separated by reverse phase liquid chromatography. The separation conditions are as follows.

(1) HPLC: Agilent 1200 series (2) Separation column: Unison UK-Phenyl ID 2.0 mm, length 100 mm, particle size 3 μm (Imtakt)
(3) Column temperature: 40 ° C
(4) Mobile phase A: Aqueous solution of 25 mM formic acid aqueous solution adjusted to pH 6.0 with aqueous ammonia (5) Mobile phase B: Methanol (6) Flow rate: 0.25 mL / min
(7) Elution conditions: Elution was performed using a mixed solution of mobile phase A and mobile phase B. The ratio of mobile phase B to the mixture is as follows. 0 minutes (5%), 0 minutes to 17 minutes (5% to 40%), 17 minutes to 17.1 minutes (40% to 80%), 17.1 minutes to 19 minutes (80%), 19 minutes to 19.1 minutes (80 % -5%), 19.1-27 minutes (5%).

  Thereafter, derivatized products of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly eluted under the above-described separation conditions were introduced into a mass spectrometer and quantified by mass chromatogram. The analysis conditions are as follows.

(1) Mass spectrometer: AB Sciex API3200 QTRAP
(2) Detection mode: Selected Ion Monitoring (positive ion mode)
(3) Selected ions: Table 1

Quantification of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly derivatized products can be performed using the analysis software Analyst v.
er 1.4.2 (AB Sciex). As an internal standard substance for quantification, derivatized 3-methyl-His-d3 in the case of Abu derivatized, Gly- in the case of γ-Glu-Abu or γ-Glu-Abu-Gly derivatized Each derivatized product of d2 was used. When γ-Glu-Abu was quantified, if a rare peak was observed in the sample, it was quantified using 145.2 or 104.1 as the selected ion in the second mass analyzer. As a result, as shown in Table 2, the AG1-d strain accumulated a significant amount of γ-Glu-Abu as compared to the AG1 strain.

  Next, the effect of γ-Glu-Abu accumulation on growth was examined. AG1 strain and AG1-d strain were inoculated in SD medium and cultured at 30 ° C. with shaking. Next, these overnight cultures were inoculated into 4 ml of a small L-type culture vessel (ADVANTEC model TV100030) in an SD medium containing 100 ppm Abu so that the absorbance at 660 nm was 0.01, and a biophoto recorder (ADVANTEC) Growth was confirmed by measuring the absorbance at 660 nm every hour using a model TVS062CA). The culture temperature was set to 30 ° C., and the shaking number was set to 70 rpm. The growth curve is shown in FIG. As shown in the figure, the AG1-d strain that accumulated more γ-Glu-Abu showed almost the same growth as the AG1 strain.

  From the above results, the decrease in the activity of the DUG complex not only reduces the growth delay of yeast that accumulates glutathione at a high concentration, but also accumulates γ-Glu-Abu, which is a γ-glutamyl peptide other than glutathione. It became clear that growth delay was reduced in Therefore, it is considered that the growth delay of yeast that accumulates various γ-glutamyl peptides produced by Gsh1p can be eliminated by reducing the activity of the DUG complex.

  As a summary of Examples 1-5, γ-glutamyl peptide is accumulated at a high concentration by reducing growth delay by destroying DUG and enhancing expression of YCF1 and accumulation in vacuole by ECM38 disruption. It has been clarified that the yeast cells can be efficiently obtained and a high γ-glutamyl peptide yeast extract can be produced using the yeast cells.

  According to the present invention, a yeast that accumulates γ-glutamyl compounds such as glutathione in cells and has good growth is provided. By using these yeasts as raw materials, yeast extracts containing γ-glutamyl compounds can be efficiently produced, and foods and drinks can be produced using these yeast extracts.

Description of Sequence Listing SEQ ID NO: 1: SUGARMYCES cerevisiae DUG1 base sequence SEQ ID NO: 2: Saccharomyces cerevisiae Dug1p amino acid sequence SEQ ID NO: 3: Saccharomyces cerevisiae DUG2 base sequence SEQ ID NO: 4: Saccharomyces cerevisiae Dug2p Amino acid sequence SEQ ID NO: 5: nucleotide sequence of DUG3 of Saccharomyces cerevisiae SEQ ID NO: 6: amino acid sequence of Dug3p of Saccharomyces cerevisiae SEQ ID NO: 7: nucleotide sequence of ECM38 of Saccharomyces cerevisiae SEQ ID NO: 8: amino acid sequence of Ecm38p of Saccharomyces cerevisiae SEQ ID NO: 9 Saccharomyces cerevisiae GSH1 nucleotide sequence SEQ ID NO: 10 Saccharomyces cerevisiae Gsh1p amino acid sequence SEQ ID NO: 11 Saccharomyces cerevisiae GSH2 nucleotide sequence SEQ ID NO: 12 Saccharomyces cerevisiae Gsh2p Amino acid sequence SEQ ID NO: 13: Saccharomyces cerevisiae YCF1 nucleotide sequence SEQ ID NO: 14: Saccharomyces cerevisiae Ycf1p amino acid sequence SEQ ID NO: 15, 16: URA3 amplification primer SEQ ID NO: 17-20: ADH1 promoter region amplification primer sequence number 21, 22: Primer sequence number 23 for pADH1-URA3-pADH1 fragment amplification, 24: Primer sequence number 25 for URA3 amplification, 26: Primer sequence number 27 for DUG1 destruction, 28: Primer sequence number 29 for DUG2 destruction, 30: DUG3 Disruption primer SEQ ID NOs: 31, 32: ECM38 disruption primer SEQ ID NO: 33, 34: ECM38 disruption primer sequence number 35 using BY4743 gene disruption strain set as template, 36: DUG1 amplification primer SEQ ID NO: 37 used for RT-PCR, 38: DUG2 amplification primer used for RT-PCR SEQ ID NO: 39, 40: Used for RT-PCR DUG3 amplification primer SEQ ID NO: 41, 42: ACT1 amplification primer used for RT-PCR SEQ ID NO: 43-46: PGK1 promoter region amplification primer SEQ ID NO: 47, 48: pPGK1-URA3-pPGK1 fragment amplification primer SEQ ID NO: 49, 50: GSH2 disruption primer using BY4743 gene disruption strain set as template

Claims (9)

A method for producing a yeast extract, characterized in that a yeast extract is prepared using as a raw material a yeast that has been modified so that the ability to synthesize γ-glutamyl compound and the activity of glutathione-degrading enzyme decreases .
The yeast has been modified so that at least the activity of the vacuolar localized glutathione degrading enzyme is decreased, and the expression of the vacuolar glutathione transporter is increased,
A production method wherein the activity of γ-glutamylcysteine synthetase is increased to increase the ability to synthesize the γ-glutamyl compound . The method according to claim 1, wherein the glutathione degrading enzyme is one or more proteins selected from the following (A) to (D).
(A) Protein encoded by DUG1 gene (B) Protein encoded by DUG2 gene (C) Protein encoded by DUG3 gene (D) Protein encoded by ECM38 gene The method according to claim 1 or 2 , wherein the γ-glutamyl compound synthesis ability is increased by increasing or decreasing the activity of glutathione synthetase. The method according to any one of claims 1 to 3 , wherein the γ-glutamyl compound is γ-glutamyl dipeptide and / or γ-glutamyl tripeptide. The .gamma.-glutamyl compounds, glutathione, .gamma.-glutamyl -α- amino acid - glycine, .gamma. guru Tamil cysteine, and .gamma.-glutamyl -α- amino compound selected from butyric acid, claim 1-4 2. The method according to item 1. Amino acids constituting the γ- glutamyl compound, amino acid derivative, and is characterized by culturing the yeast in a medium a compound selected from the peptides were added, according to any one of claims 1 to 5 Method. The method according to any one of claims 1 to 6 , wherein the yeast belongs to the genus Saccharomyces. The method according to claim 7 , wherein the yeast is Saccharomyces cerevisiae. Yeast modified to increase the activity of γ-glutamylcysteine synthase and decrease the activity of glutathione degrading enzyme, and further have the following properties (I) and (II):
(I) Increase or decrease of glutathione synthase activity
(II) Increased expression of vacuolar glutathione transporter and decreased activity of vacuolar localized glutathione degrading enzyme

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