JP2011103789A - Highly glutathione-producing yeast and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a variant yeast cell highly producing glutathione, and to provide a method for producing a yeast essence by using the yeast cell. <P>SOLUTION: The variant yeast cell has the characteristics of the following (a), (b) or both of the (a) and (b): (a) suppressing the function of a protein encoded by at least one kind of a gene concerning the inhibition of the production of the glutathione; and (b) enhancing the function of a protein encoded by at least one kind of a gene concerning the production of the glutathione. The method for producing the yeast essence by using the yeast cell is also provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to mutant yeast cells in which the production amount of glutathione, which is an antioxidant substance, in the cells is increased, and the use thereof.

  Glutathione is called γ-glutamyl-L-cysteinylglycine and is a tripeptide composed of three amino acids, glycine, L-glutamic acid, and L-cysteine, and is widely present in yeasts and animal livers. As an antidote for pharmaceuticals, it is used to prevent various addictions, chronic liver diseases, side effects of anticancer drugs and damage caused by radiation therapy. Further, it is used as an ophthalmic agent in the treatment of cataracts and corneal damage (Non-patent Document 1). Glutathione acts as a protective agent for cells against toxic substances such as cadmium, reactive oxygen derivatives such as active oxygen, and oxidative damage caused by radiation, and plays an important role in maintaining an intracellular redox balance.

  From the viewpoint of health orientation in recent years, health functional foods have attracted attention, but in the food field, foods with high glutathione content are attracting attention based on these physiological activities.

  Sulfur-containing amino acids such as cysteine and peptides such as γ-glutamyl-L-cysteine are used for the purpose of improving the flavor of foods. Various methods for producing cysteine are known, but the proteolytic method and the semisynthetic method are mainly used at present. In order to use cysteine for improving the flavor of foods, natural food materials having a high cysteine content are required. However, since cysteine exhibits cytotoxicity and it is difficult to increase the intracellular content of cysteine itself, an increase in the content of γ-glutamyl-L-cysteine has been reported. By heating or enzymatically treating a yeast extract containing γ-glutamyl-L-cysteine, a food material containing a high content of cysteine can be obtained (Patent Document 1).

  Similarly, it is considered that glutathione can be used for the purpose of improving the flavor of foods, and it has already been reported that glutathione imparts a rich taste (Non-patent Document 2). As an industrial production method of glutathione, a method of extracting from yeast is common, and for that purpose, acquisition of a glutathione-rich yeast is a necessary condition.

  Conventionally known methods for producing glutathione-rich yeast include a method of adding amino acids such as cysteine to the medium (Patent Document 2), a method of restricting zinc ions (Patent Document 3), and a culture temperature lower than usual. Examples thereof include a culture method (Patent Document 4). Examples of the method include obtaining an ethionine / sulfite resistant strain (Patent Document 5), a polyene antibiotic resistant strain (Patent Document 6), and the like by mutation treatment.

  Glutathione is biosynthesized by the following chain action of intracellular enzymes γ-glutamylcysteine synthase and glutathione synthase (see FIG. 1).

[Equation 1]
(1) L-glutamic acid + L-cysteine + ATP⇔γ-glutamyl-L-cysteine + ADP + Pi
(2) γ-Glutamyl-L-cysteine + glycine + ATP⇔glutathione + ADP + Pi

  The enzyme that catalyzes the reaction is synthesized by an enzyme coding gene. The enzyme coding gene for γ-glutamylcysteine synthase is GSH1, and the enzyme coding gene for glutathione synthase is GSH2. It has been reported that the production of glutathione is increased by about 30% by using the GSH1 gene in the cells of the yeast Saccharomyces cerevisiae (also referred to herein as baker's yeast) (Patent Document 7). . In addition, it has been reported that the amount of glutathione is increased by the use of GSH2 and the combined use of both GSH1 and GSH2 genes in E. coli and yeast Candida utilis (also referred to herein as Torula yeast). (Patent Documents 8 and 9). In addition to GSH1 and GSH2, it is reported that increasing the expression level of MET25 gene increases the glutathione content in the fungus, and the method for increasing the expression level of MET25 gene is a mutant MET4 gene (Non-patent Document 3, Patent literature 10), a method using a mutant MET30 gene (non-patent literature 4), and the like have been reported.

  In recent years, a gene-disrupted strain collection of commercially available Saccharomyces cerevisiae yeast has been used to screen genes that are resistant to immunosuppressive agents (Non-patent Document 5). Regarding secretion of glutathione to the outside of the cell, a mutant strain exhibiting high secretion and low secretion has been reported from analysis using a collection of yeast gene disruption strains (Non-patent Document 6). However, no increase or decrease in intracellular glutathione level has been reported.

International Publication No. 00/30474 Pamphlet (WO 00/30474) JP-A-53-94089 JP 2000-279164 A JP 60-156379 A JP 59-151894 JP 2003-284547 A JP-A-61-52299 JP 2005-73638 A JP-A 64-51098 Japanese Patent Laid-Open No. 10-33161

Nishikori et al., "Protein Nucleic Acid Enzyme", VOL.33, p.1625-1631, 1988 Y. Ueda et al., "Biosci. Biotech. Biochem.", VOL. 61, p. 1977-1980, 1997 F. Omura, et al., "FEBS Letters", VOL.387, p.179-183, 1996 D. Thomas et al., "Mol. Cell. Biol.", Vol. 15, p.6526-6534, 1995 C. Desmoucelles et al., `` J. Biol. Chem. '', Vol.277, p.27036-27044, 2002 G. Perrone et al., “Mol. Biol. Cell”, Vol. 16, p.218-230, 2005

  Therefore, the present invention, based on the above technical background, identifies a new gene related to glutathione content in yeast cells, and a yeast in which the gene is modified for high glutathione production in cells, and An object is to provide a glutathione-containing material using the yeast.

  In order to solve the above problems, the present inventors measured the intracellular glutathione content of all strains using a collection of non-essential gene disrupted strains of the budding yeast Saccharomyces cerevisiae, and determined the total intracellular glutathione (oxidized glutathione). And mutants whose total content of reduced glutathione) was changed by more than 10% compared to the wild type. As a result, it was found that the glutathione content in the specific gene-disrupted strain of Saccharomyces cerevisiae was significantly increased or decreased as compared with the wild-type strain.

  In addition, in the gene disruption strain with high glutathione production, when the GSH1 gene involved in high production of glutathione was overexpressed from the above findings, a synergistic increase in glutathione production was confirmed.

  Furthermore, in Candida utilis, the same results as Saccharomyces cerevisiae were obtained when high production of glutathione by overexpression of the same gene was examined. This suggests that these genes are involved in the production of glutathione in yeast in general.

  In the present invention, first, a mutant yeast cell having high glutathione productivity is provided. This mutant yeast cell body has (a) the function of a protein encoded by at least one gene involved in glutathione production inhibition is suppressed, and (b) at least one gene involved in glutathione production. The function of the encoded protein is enhanced or has both features (a) and (b).

  In the mutant yeast according to the present invention, the genes involved in glutathione production inhibition are not limited thereto, but include, for example, RTS1, PEP12, DDC1, UBP6, CST6, Y1H1, CHC1, MAL31, and PET123. Can be mentioned. Examples of genes involved in glutathione production include, but are not limited to, STR4 and GSH1.

  The yeast according to the present invention is preferably Saccharomyces cerevisiae or Candida utilis.

  The present invention also relates to a mutant yeast cell having high glutathione productivity, in which the function of a protein encoded by a gene involved in high production of glutathione is suppressed or enhanced.

Examples of the gene involved in high production of glutathione include, but are not limited to, DEF1.
The present invention further relates to a method for producing a yeast extract from the mutant yeast according to the present invention.

  According to the present invention, mutant yeast cells having a high intracellular glutathione content are provided. In addition, by using the mutant yeast according to the present invention, it is possible to produce a food material (for example, yeast extract) that is excellent in health functionality, good in flavor, and high in quality.

FIG. 1 is a view showing a sulfur-containing amino acid / glutathione metabolic pathway and genes encoding each enzyme in baker's yeast cells. Large squares represent yeast cells. FIG. 2 is a graph showing the intracellular glutathione content of a gene-disrupted strain derived from baker's yeast BY4742. The vertical axis indicates the relative amount of intracellular glutathione content per cell OD ₆₀₀ = 1 relative to the wild strain (Control), and the horizontal axis indicates the disrupted gene. The black bar indicates the result of the disrupted strain in which the glutathione concentration increased by 20% or more compared to the wild strain (Control). FIG. 3 is a graph showing the intracellular glutathione content of a gene-disrupted strain derived from baker's yeast BY4741. The vertical axis indicates the relative amount of intracellular glutathione content per cell OD ₆₀₀ = 1 relative to the wild strain (Control), and the horizontal axis indicates the disrupted gene. The black bar indicates the result of the disrupted strain in which the glutathione concentration increased by 20% or more compared to the wild strain (Control). FIG. 4 is a graph showing the intracellular glutathione content of a gene-disrupted strain derived from baker's yeast SYT001. The vertical axis indicates the relative amount of intracellular glutathione content per cell OD ₆₀₀ = 1 relative to the wild strain (Control), and the horizontal axis indicates the disrupted gene. The black bar indicates the result of the disrupted strain in which the glutathione concentration increased by 20% or more compared to the wild strain (Control). FIG. 5 is a graph showing the intracellular glutathione content when the GSH1 gene is overexpressed in baker's yeast DBY7286. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1. FIG. 6 is a graph showing the intracellular glutathione content when the GSH2 gene is overexpressed in baker's yeast DBY7286. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1. FIG. 7 is a graph showing the intracellular glutathione content when the STR1 gene is overexpressed in baker's yeast DBY7286. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1. FIG. 8 is a graph showing the intracellular glutathione content when the STR2 gene is overexpressed in baker's yeast DBY7286. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1. FIG. 9 is a graph showing the intracellular glutathione content when the STR3 gene is overexpressed in baker's yeast DBY7286. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1. FIG. 10 is a graph showing the intracellular glutathione content when the STR4 gene is overexpressed in baker's yeast DBY7286. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1. FIG. 11 is a graph showing the intracellular glutathione content when the GSH1 gene is overexpressed in the def1 and ubp6 gene disrupted strains derived from baker's yeast BY4742. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1, and the horizontal axis represents the type of transformant. FIG. 12 is a graph showing the intracellular glutathione content when the GSH1 gene is overexpressed in the rts1 gene-disrupted strain derived from baker's yeast BY4742. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1, and the horizontal axis represents the type of transformant. FIG. 13 is a graph showing the intracellular glutathione content when the GSH1 gene is overexpressed in the pep12 gene-disrupted strain derived from baker's yeast BY4742. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1, and the horizontal axis represents the type of transformant. FIG. 14 is a graph showing intracellular glutathione contents when DDC1, DEF1, PEP12, and UBP6 genes are overexpressed in baker's yeast DBY7286. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1, and the horizontal axis represents the overexpressed gene. FIG. 15 is a graph showing intracellular glutathione content when CuGSH1, CuDEF1, CuSTR4, CuPEP12, and CuUBP6 genes are overexpressed in Torula yeast NBRC0988 strain. The vertical axis represents the intracellular glutathione content per cell OD ₆₀₀ = 1, and the horizontal axis represents the overexpressed gene. FIG. 16 is a graph showing intracellular glutathione content when CuGSH1, CuDEF1, CuSTR4, CuPEP12, and CuUBP6 genes are overexpressed in Torula yeast NBRC0988 strain. The vertical axis represents the glutathione content per gram of dry yeast cells, and the horizontal axis represents the overexpressed gene.

  The present invention relates to a mutant yeast cell having high glutathione productivity (hereinafter simply referred to as the yeast cell of the present invention). In the yeast cell of the present invention, the function of a specific protein is suppressed or enhanced. Thereby, the yeast cell of the present invention is characterized in that glutathione is highly produced in the cell as compared with the wild-type yeast cell. Specifically, the yeast cell of the present invention is characterized in that when it is grown to the same extent under the same conditions as the wild type yeast cell, the glutathione content is significantly different from that of the wild type. For example, inoculate a test tube containing YPD medium (yeast extract 1%, peptone 2%, glucose 2%), and incubate at 30 ° C, 120rpm for 72 hours. Suspend and heat extract, dilute extract and measure total glutathione content (total of oxidized glutathione and reduced glutathione) using glutathione measurement kit (BIOXYTECH GSH / GSSH-412 manufactured by OxisResearch). This can be confirmed by comparing with the stock.

  In this specification, that the function of a protein is suppressed means that the gene encoding the protein is deleted from the genome, the expression of the gene encoding the protein is inhibited, and the activity of the protein is reduced. Including meaning.

  More specifically, the method for deleting a gene encoding a specific protein is not particularly limited, but a method of replacing a target gene with another gene by homologous recombination (see, for example, JP-A-2002-209574), A method for destroying a target gene using a transposon (see, for example, JP-A-2005-328769), a method for destroying a target gene using a selection marker that cancels auxotrophy (for example, see Table 01/014522), etc. Can be mentioned. Moreover, when deleting the said gene, the full length of the said gene may be deleted, and you may delete partially. Further, the function may be lost by introducing a point mutation into the enzyme active domain or the regulatory domain.

  In addition, the method for inhibiting the expression of a gene encoding a specific protein is not particularly limited, but a method for deleting a promoter that controls the expression of the gene, a promoter that controls the expression of the gene is expressed. A method of replacing with an inducible promoter, a method of introducing a mutation into a promoter controlling the expression of the gene, a method of degrading the transcription product of the gene using RNA interference, and using an antisense RNA The method of inhibiting the translation of the said gene can be mentioned.

  Furthermore, examples of a method for reducing the activity of a specific protein include a method in which a substance having a function of specifically binding to the protein and suppressing the activity of the protein is allowed to act. Examples of the substance include antibodies and inhibitors that can inhibit the function of the protein.

  In the present specification, enhancement of the function of a protein includes overexpression of a gene encoding the protein, stabilization of the protein by introduction of a point mutation, etc., and alteration of the intracellular localization of the protein. Meaning.

  Methods for overexpressing a protein-encoding gene are well known to those skilled in the art. For example, a large number of copies of the gene are incorporated into a plasmid and introduced into a yeast cell, or the gene is incorporated into a yeast genome and copied. It can be carried out by increasing the number, placing it under the control of a promoter stronger than that of the gene, or introducing a mutation into the promoter controlling the expression of the gene.

  In order to stabilize the protein by introducing a point mutation, etc., the protein may be introduced by introducing a mutation that makes the protein resistant to proteolytic enzymes, or by introducing a mutation that stabilizes the mRNA of the protein. it can.

  Changing the intracellular localization of a protein enhances the function of the protein and allows the protein to catalyze the reaction more efficiently. Altering the intracellular localization of a protein can be performed by introducing a point mutation and adding an organelle targeting sequence.

  The yeast cell of the present invention has (a) the function of a protein encoded by at least one gene involved in inhibition of glutathione production is suppressed, and (b) at least one gene involved in glutathione production. The function of the protein encoded by is enhanced or has both features (a) and (b).

  In the present invention, proteins capable of suppressing or enhancing the function for high production of glutathione are described in Tables 1 and 2 below.

  Alternatively, the yeast cell of the present invention suppresses the function of the protein described in Table 3 below, which is encoded by a gene involved in high production of glutathione, in addition to the suppression and / or enhancement of the function of the protein. Or have been strengthened.

  In the present invention, the protein whose function is suppressed and enhanced is not limited to a protein consisting of the amino acid sequence specified by the SEQ ID No. described in the above table, but also includes mutants thereof. As used herein, a “variant” is encoded by a mutant of a protein consisting of an amino acid sequence specified by a SEQ ID NO, and a homolog of each protein-coding gene derived from a yeast other than baker's yeast or torula yeast. Including proteins

  Specifically, the protein variant referred to in the present invention is a polypeptide comprising one or more amino acid substitutions, additions, deletions or insertions in the amino acid sequence of the protein of interest, and comprising the amino acid sequence. 70% or more identity, preferably 80% or more identity, more preferably 90% identity or most preferably 95% to the protein having the same function or the amino acid sequence of the target protein A protein having the above identity and having a function equivalent to that of a polypeptide comprising the amino acid sequence. Here, the amino acid to be substituted, deleted, added or inserted is, for example, 1 to 50, more preferably 1 to 25, more preferably 1 to 10, depending on the length of the amino acid sequence of the target protein. More preferably, it can be 1-5. The identity value means a value calculated by default setting using software (for example, FASTA, DNASYS, BLAST) that calculates identity between a plurality of amino acid sequences.

  In addition, the “equivalent function” used in the present specification means that the type of functional property of the mutant protein is substantially the same as that of the target protein, and the functional property of the mutant protein. The degree of characteristics is substantially the same. Whether or not the target protein has a function equivalent to that of the mutant protein can be determined by a method known to those skilled in the art according to the functional characteristics of the target protein. The functional characteristics possessed by each protein and the method for determining the functional characteristics are listed in Table 4 below.

  In the yeast cell of the present invention, a protein whose function is particularly preferably suppressed is a protein encoded by the DEF1 gene, the PEP12 gene or the UBP6 gene.

  In the yeast cell of the present invention, a protein whose function is particularly enhanced is a protein encoded by the DEF1 gene, GSH1 gene, or STR4 gene. DEF1 is thought to be involved in the regulation of intracellular homeostasis by glutathione because the glutathione content increases even if the transcription of the gene involved is increased or decreased. Therefore, the function of DEF1 may be enhanced or suppressed.

  Most preferably, the yeast of the present invention has the following characteristics (a) and (b): (a) the function of the protein encoded by the PEP12 gene is suppressed; ) The function of the protein encoded by the GSH1 gene is enhanced.

  The present invention can be applied to any yeast. That is, the present invention can be applied to any yeast as long as the wild-type yeast cell has the above-described protein. For example, but not limited to, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces rouxii, Saccharomyces carlsbergen, Pombe (Shizosaccharomyces pombe), Candida utilis, Candida tropicalis, Candida lipolytica, Candida flaveri, Candida boidinii ), Rhodotrura minuta, etc. The present invention can be applied. The present invention is particularly preferably applied to Saccharomyces cerevisiae or Candida utilis.

  The yeast cell of the present invention can be cultured according to a conventional method. For example, a medium containing a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by yeast and capable of efficiently cultivating transformants may be used as the medium. Specifically, YPD medium, YPG medium, YPDG medium, YPAD medium, minimum glucose synthesis medium (SD), minimum medium with iodine addition (DMM), Hartwell's complete medium (HC), GAL fermentation test medium, spore formation medium, etc. Can be used.

  As described above, since the yeast cell of the present invention has a higher glutathione content in the cell than the wild-type yeast, the yeast extract produced using the yeast strain of the present invention has excellent health functionality and flavor. But good quality. Therefore, the present invention further provides a method for producing a yeast extract having a high glutathione content using the yeast cells of the present invention.

  The yeast extract of the present invention may be produced using a method known to those skilled in the art. In general, glucose is used as a sugar source, a culture process in which culture is continuously performed while aerated in a fermentor, an extract extraction process in which bacteria are collected by centrifugation and heat-treated, followed by enzyme treatment, clarification, It consists of the steps of concentration and powdering. See, for example, JP-A-2004-229540 for the production method of yeast extract.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by a following example.

  Several genes, such as GSH1, GSH2, DUG1, DUG2, and DUG3, have already been reported as genes involved in the biosynthesis and degradation of glutathione in baker's yeast Saccharomyces cerevisiae. In addition to these genes, the following experiments were conducted with the aim of identifying new glutathione biosynthesis and degradation regulators by identifying new genes involved in glutathione synthesis and destroying or overexpressing them. It was.

[Example 1] Identification of genes involved in glutathione biosynthesis
From a glycerol-stocked plate of the laboratory yeast MATα haploid non-essential gene disruption strain collection (from BY4742) purchased from Open Biosystems, 150 μL of YPD medium (yeast extract 1%, Peptone 2%, glucose 2%) was inoculated using a 96-well sword mountain and cultured at 30 ° C for 1 day. Each culture solution was inoculated into 0.6 mL of YPD, and cultured at 30 ° C. for aerobic stirring for 3 days. After measuring the absorbance (OD ₆₀₀ ) at ₆₀₀ nm as the cell concentration of the culture solution, the culture solution was centrifuged (3000 rpm × 5 minutes) to obtain a cell body fraction, which was suspended in 0.5 mL of sterile water. Further, this suspension was centrifuged (3000 rpm × 5 minutes), the supernatant was discarded, and the bacterial cell fraction was suspended in 1 mL of sterilized water. These suspensions were heated at 70 ° C. for 10 minutes, the suspensions were centrifuged (3000 rpm × 5 minutes), and the glutathione in the cells was extracted by collecting the supernatant. Dilute the extract from 100-fold to 10000-fold, react according to the manual of glutathione measurement kit (Oxis Research BIOXYTECH GSH / GSSH-412), and absorb at 412 nm every minute with a microplate reader (OD ₄₁₂ ) The amount of change per minute (Δ412 / min) was calculated and divided by the bacterial cell concentration (OD ₆₀₀ ) to semi-quantify the glutathione content of the total amount of oxidized glutathione and reduced glutathione. Then, a gene-disrupted strain with more than 20% change compared to the wild strain was picked up as a strain containing a gene candidate involved in glutathione biosynthesis (the relative value of glutathione content when the wild strain is 1 is shown in FIG. 2). Show). Next, from the glycerol stock plate of the laboratory yeast MATa haploid non-essential gene disruption strain collection (BY4741) purchased from Invitrogen, the gene disruption strains listed as candidates are similarly inoculated and cultured, Glutathione concentration is measured, and gene disruption strains that change by more than 20% compared to wild strains are picked up (relative value of glutathione content when wild strain is 1 is shown in Fig. 3), glutathione biosynthesis and degradation We narrowed down the candidate genes involved in.

  For further refinement, a laboratory yeast haploid SYT001 without an auxotrophic marker (S. Yoshida et al., “Appl. Environ. Microbiol.” Vol. 74, p. 2787-2996, 2008) The genes listed as candidates in (1) are disrupted, and genes with more than 20% change compared to the parent strain SYT001 are candidates for genes involved in glutathione biosynthesis RTS1, PEP12, DDC1, DEF1, UBP6, CST6, YIH1 CHC1 was identified (the relative value of glutathione content when the wild strain is 1 is shown in FIG. 4).

[Example 2] Effect of gene overexpression on intracellular glutathione Based on the above findings, overexpressed strains of genes STR4, GSH1, and GSH2 in which the amount of intracellular glutathione decreased by 20% or more due to disruption were prepared. At that time, STR1, STR2, and STR3 in FIG. 1 were also evaluated.

By inserting the full length of these genes downstream of the GAP promoter in the plasmid vector pYES-G418GP (S. Yoshida et al., “Appl. Environ. Microbiol.” Vol. 74, p. 2787-2996, 2008) A plasmid for overexpression of these genes was constructed. Then, we decided to introduce these plasmids into DBY7286 strain with ura3 marker and investigate the effect on intracellular glutathione concentration. Transformants were selected using SD agar medium without uracil (Yeast Nitrogen Base without amino acids 0.67%, glucose 2%, agar 2%), and colonies that were grown for 3 days at 30 ° C. Was inoculated into a new SD agar medium. The cells were suspended in 6 mL of SD + casamino medium (Yeast Nitrogen Base without amino acids 0.67%, glucose 2%, casamino acids 2%) and cultured at 30 ° C. for 24 hours. After measuring the absorbance (OD ₆₀₀ ) at ₆₀₀ nm as the cell concentration of the culture solution, the culture solution was centrifuged (3000 rpm × 5 minutes) to obtain a cell fraction and suspended in 1 mL of sterilized water. These suspensions were heated at 70 ° C. for 10 minutes, the suspensions were centrifuged (3000 rpm × 5 minutes), and the glutathione in the cells was extracted by collecting the supernatant. Dilute the extract from 100-fold to 10000-fold, react according to the manual of glutathione measurement kit (Oxis Research BIOXYTECH GSH / GSSH-412), and absorb at 412 nm every minute with a microplate reader (OD ₄₁₂ ) The amount of change per minute (Δ412 / min) was calculated, and the total glutathione content was quantified by dividing by the cell concentration (OD ₆₀₀ ). As a result, it was found that only the two genes GSH1 and STR4 increase the amount of intracellular glutathione by overexpression, and the amount of intracellular glutathione decreases by overexpression of STR2 (results shown in FIGS. 5 to 10). To show).

[Example 3] Synergistic effect on glutathione production by gene disruption and gene overexpression Based on the knowledge of gene disruption strain and overexpression strain described above, in the synthesis of glutathione, in the yeast yeast, ubp6, pep12, def1, rts1 disruption strain It was considered that by overexpressing the GSH1 gene, yeast having an increased intracellular glutathione content compared to the control parent strain could be bred, and the following experiment including the hypothesis was conducted. That is, whether the intracellular glutathione content increases synergistically by overexpressing the gene obtained in [Example 2] in the gene-disrupted strain obtained in [Example 1]. A strain in which the GSH1 gene was overexpressed in the ubp6, pep12, def1, and rts1 disrupted strains of BY4742 obtained in [Example 1] was prepared. Transformants were selected by using SD agar medium containing amino acid without uracil (Yeast Nitrogen Base without amino acids 0.67%, glucose 2%, agar 2%, leucine 30 mg / L, histidine 20 mg / L, lysine 30 mg / L, The colonies that had grown after being cultured for 3 days at 30 ° C. were inoculated into a new amino acid-containing SD agar medium. 6 mL of amino acid-containing SD + casamino medium (Yeast Nitrogen Base without amino acids 0.67%, glucose 2%, casamino acid 2%, leucine 30 mg / L, histidine 20 mg / L, lysine 30 mg / L, tryptophan 20 mg / L) It was suspended and cultured at 30 ° C. for 24 hours. After measuring the absorbance (OD ₆₀₀ ) at ₆₀₀ nm as the cell concentration of the culture solution, the culture solution was centrifuged (3000 rpm × 5 minutes) to obtain a cell fraction and suspended in 1 mL of sterilized water. These suspensions were heated at 70 ° C. for 10 minutes, the suspensions were centrifuged (3000 rpm × 5 minutes), and the glutathione in the cells was extracted by collecting the supernatant. Dilute the extract from 100-fold to 10000-fold, react according to the manual of glutathione measurement kit (Oxis Research BIOXYTECH GSH / GSSH-412), and absorb at 412 nm every minute with a microplate reader (OD ₄₁₂ ) The amount of change per minute (Δ412 / min) was calculated, and the total glutathione content was quantified by dividing by the cell concentration (OD ₆₀₀ ). As a result, by overexpressing the GSH1 gene in the three gene-disrupted strains ubp6, pep12, and def1, the amount of intracellular glutathione was 1.8 times, 4.6 times, and 1.9, respectively, as compared to the strain in which the control parent strain had the vector. Each was successfully increased by a factor of 2 (results are shown in FIGS. 11 to 13). In addition, the result of overexpression of the GSH1 gene in the gene-disrupted strain rts1 also shows that the amount of intracellular glutathione is remarkably increased compared to its parent strain.

[Example 4] Effect of overexpression of genes involved in glutathione biosynthesis
The yeast overexpressing the PEP12, DDC1, DEF1, and UBP6 genes used in [Example 1] was used, and the effect of the overexpression of these genes on the amount of intracellular glutathione was investigated. That is, we decided to introduce these plasmids into DBY7286 strain with ura3 marker and investigate the effect on intracellular glutathione concentration. Transformants were selected using SD agar medium without uracil (Yeast Nitrogen Base without amino acids 0.67%, glucose 2%, agar 2%), and colonies that were grown for 3 days at 30 ° C. Was inoculated into a new SD agar medium. The cells were suspended in 6 mL of YPD + G418 medium (yeast extract 1%, peptone 2%, glucose 2%, G418 200 mg / L) and cultured at 30 ° C. for 24 hours. After measuring the absorbance (OD ₆₀₀ ) at ₆₀₀ nm as the cell concentration of the culture solution, the culture solution was centrifuged (3000 rpm × 5 minutes) to obtain a cell fraction and suspended in 1 mL of sterilized water. These suspensions were heated at 70 ° C. for 10 minutes, the suspensions were centrifuged (3000 rpm × 5 minutes), and the glutathione in the cells was extracted by collecting the supernatant. Dilute the extract from 100-fold to 10000-fold, react according to the manual of glutathione measurement kit (Oxis Research BIOXYTECH GSH / GSSH-412), and absorb at 412 nm every minute with a microplate reader (OD ₄₁₂ ) The amount of change per minute (Δ412 / min) was calculated, and the total glutathione content was quantified by dividing by the cell concentration (OD ₆₀₀ ). As a result, it was found that only one gene DEF1 increases the amount of intracellular glutathione by overexpression (the result is shown in FIG. 14).

  The above results revealed that the intracellular glutathione content of the DEF1 gene was increased by gene disruption and overexpression.

[Example 5] Effect of overexpression of a gene involved in glutathione biosynthesis in torula yeast Candida utilis What is described in the examples of the present invention is present in Candida utilis NBRC0988 strain. When using other strains of Candida utilis, such as NBRC0626 strain, NBRC0639 strain, NBRC1086 strain, etc., even if they are different from the gene sequence, those having the same function, that is, activity (other strain sequences) If it exists, it can be used as it is. The other strain sequence can be confirmed by those skilled in the art by a known method.

  As described above, it was shown that the glutathione content of the bacterial cells was increased by overexpressing the GSH1, DEF1, and STR4 genes in Saccharomyces cerevisiae. In addition, it was revealed that the glutathione content of the bacterial cells was increased by deleting the genes for DEF1, PEP12, and UBP6. Therefore, in the Torula yeast Candida utilis, which is recognized as a food yeast by the Food and Drug Administration (DA), the homology with the above five genes possessed by the yeast A high gene overexpression strain was constructed.

  Using DNA fragments obtained by amplifying ORF (Open Reading Frame) of GSH1, DEF1, STR4, UBP6, and PEP12 genes, each gene and each gene from a Candida utilis genomic DNA library constructed by a method known in the art We attempted to clone genes with high homology. As a result, the obtained new ORFs were named CuGSH1 gene (SEQ ID NO: 25), CuDEF1 gene (SEQ ID NO: 27), CuSTR4 gene (SEQ ID NO: 29), CuPEP12 gene (SEQ ID NO: 31) and CuUBP6 gene (SEQ ID NO: 33). did. The CuGSH1 gene was found to be identical to the nucleotide sequence already known in the document (see Patent Document 8 above).

  After linking these genes downstream of the GAP (Glyceraldehyde-3-phosphate dehydrogenase) gene promoter and upstream of the PGK (3-phosphoglycerate kinase) gene terminator, CuURA3 (Orotidine-5'-phosphate of Candida utilis NBRC0988 strain decarboxylase) (GenBank: E11619.1) gene locus (see JP-A-2003-144185). In addition, a strain incorporating a DNA fragment into which nothing was inserted between the GAP promoter and the PGK terminator was also constructed as a control for examining the effect of high expression of each gene (negative control strain). Since the plasmid also carries a hygromycin B resistance gene, positive clones were selected on a YPD agar medium supplemented with 600 mg / L hygromycin B.

The intracellular glutathione content of the obtained recombinant strain was examined. First, the bacterial cells cultured at 30 ° C. for 1 to 3 days on a YPD agar medium were cultured with shaking in aerobic conditions at 130 ° C. for 18 to 30 hours in 4 mL of YPD liquid medium (130 rpm). Absorbance (OD600) at 600 nm is 0.1-0.3 in 200 mL SD + casamino medium (Yeast Nitrogen Base without amino acids 0.67%, glucose 2%, casamino acid 2%) in a 500 mL Sakaguchi flask. After suspending in such a manner, the cells were cultured with aerobic shaking at 25 ° C for 46 hours (130 rpm). The glutathione content of the obtained bacterial cells was quantified by the method described above. Further, by measuring the dry cell weight, in addition to the glutathione content per OD ₆₀₀ (FIG. 15), the glutathione content per dry cell weight (307.33 g per 1 mole of glutathione) was also calculated (FIG. 16). Results were obtained from at least 3 independent trials.

  As a result, as expected, the overexpressed strains of CuGSH1 gene, CuDEF1 gene, and CuSTR4 gene had significantly higher glutathione content than the negative control strain. On the other hand, in baker's yeast, the CuUBP6 gene and CuPEP12 gene, which are homologs of genes known to bring about an effect of increasing glutathione content due to complete deficiency, did not show a significant change compared to the above three genes.

  According to the present invention, a yeast having a high intracellular glutathione content is provided. This makes it possible to produce a yeast extract with a high glutathione content at low cost. Therefore, the present invention is useful for the production of foods such as seasonings, pharmaceuticals, and others.

Claims (19)

  A mutant yeast cell having high glutathione productivity, wherein the function of a protein encoded by at least one gene selected from the group consisting of RTS1, PEP12, DDC1, DEF1, UBP6, CST6, and Y1H1 is suppressed. The mutant yeast cell according to claim 1, wherein the function of at least one protein described below is suppressed:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 2, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 2 and having a function equivalent to that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 4 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 6, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 6 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6;
A protein comprising the amino acid sequence shown in SEQ ID NO: 8, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 8 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 10, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 10 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 12, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 12 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12;
A protein comprising the amino acid sequence shown in SEQ ID NO: 14, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 14 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14. The mutant yeast cell according to claim 2, wherein the function of at least one protein described below is further suppressed:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 16, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 16 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 18, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 18 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 18;
A protein comprising the amino acid sequence shown in SEQ ID NO: 20, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 20 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20. The mutant yeast cell according to claim 1, wherein the function of at least one protein described below is suppressed:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 28, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 28 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 32, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 32 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32;
A protein comprising the amino acid sequence shown in SEQ ID NO: 34, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 34 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34.   A mutant yeast cell having high productivity of glutathione, wherein the function of a protein encoded by one gene selected from the group consisting of STR4 and DEF1 is enhanced. The mutant yeast cell according to claim 5, wherein the function of at least one protein described below is enhanced:
A protein comprising the amino acid sequence shown in SEQ ID NO: 8, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 8 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8;
A protein comprising the amino acid sequence shown in SEQ ID NO: 24, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 24 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24. The mutant yeast cell according to claim 5, wherein the function of at least one protein described below is enhanced:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 28, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 28 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28;
A protein comprising the amino acid sequence shown in SEQ ID NO: 30, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 30 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30.   A mutant yeast cell having high productivity of glutathione, wherein the function of the protein encoded by the DEF1 gene is suppressed or enhanced.   The protein has the same function as a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 8 and consisting of the amino acid sequence shown in SEQ ID NO: 8. 9. The mutant yeast cell according to claim 8, which is a protein having   The protein has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 28, or the polypeptide having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 28 and consisting of the amino acid sequence shown in SEQ ID NO: 28 9. The mutant yeast cell according to claim 8, which is a protein having   (a) the function of the protein encoded by at least one gene selected from the group consisting of RTS1, PEP12, DDC1, UBP6, CST6, and Y1H1 is suppressed, and (b) the group consisting of GSH1 and STR4 A mutant yeast cell having high glutathione productivity, wherein the function of at least one protein selected from the above is enhanced. 12. The mutant yeast cell according to claim 11, wherein the function of at least one protein described below is suppressed:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 2, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 2 and having a function equivalent to that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 4 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 6, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 6 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 10, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 10 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10;
A protein consisting of the amino acid sequence shown in SEQ ID NO: 12, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 12 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12;
A protein comprising the amino acid sequence shown in SEQ ID NO: 14, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 14 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14. 12. The mutant yeast cell according to claim 11, wherein the function of at least one protein described below is enhanced:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 22, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 22 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22;
A protein comprising the amino acid sequence shown in SEQ ID NO: 24, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 24 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24. 12. The mutant yeast cell according to claim 11, wherein the function of at least one protein described below is suppressed:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 32, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 32 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 32;
A protein comprising the amino acid sequence shown in SEQ ID NO: 34, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 34 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 34. 12. The mutant yeast cell according to claim 11, wherein the function of at least one protein described below is enhanced:
A protein consisting of the amino acid sequence shown in SEQ ID NO: 26, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 26 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26;
A protein comprising the amino acid sequence shown in SEQ ID NO: 30, or a protein having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 30 and having a function equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 30.   12. The mutant yeast cell according to claim 11, wherein the function of the protein encoded by the DEF1 gene is suppressed or enhanced.   The protein has the same function as a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 8 and consisting of the amino acid sequence shown in SEQ ID NO: 8. 17. The mutant yeast cell according to claim 16, which is a protein having   The protein has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 28, or the polypeptide having at least 90% amino acid identity with the amino acid sequence shown in SEQ ID NO: 28 and consisting of the amino acid sequence shown in SEQ ID NO: 28 17. The mutant yeast cell according to claim 16, which is a protein having   A method for producing a yeast extract having a high glutathione content, comprising extracting the yeast extract from the mutant yeast cells according to any one of claims 1 to 18.

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