JP4352877B2 - Method for producing γ-glutamylcysteine - Google Patents

Description

  The present invention relates to a yeast strain that produces γ-glutamylcysteine, a method for culturing the same, and a food and drink using the cells of the strain. A material containing γ-glutamylcysteine and a material containing cysteine produced therefrom are useful in the food field.

  Cysteine is used for the purpose of improving the flavor of food. Proteolytic methods and semi-synthetic methods are known as methods for producing cysteine, but the methods currently used mainly are proteolytic methods and semi-synthetic methods. For the purpose of using cysteine for improving the flavor of food, a natural food material having a high cysteine content is required. However, such a natural food material has hardly been known. On the other hand, it has been reported that if a yeast extract containing γ-glutamylcysteine is heated or enzymatically treated, a food material containing a high content of cysteine can be obtained (Patent Document 1).

  γ-glutamylcysteine is synthesized using cysteine and glutamic acid as substrates by the action of γ-glutamylcysteine synthetase. Glutathione is synthesized using γ-glutamylcysteine and glycine as substrates by the action of glutathione synthetase. It has been reported that yeast in which the glutathione synthetase gene is disrupted accumulates γ-glutamylcysteine (Non-patent Document 1).

  So far, yeasts with a high content of γ-glutamylcysteine have been reported in Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, and the like. However, these reports do not describe culture conditions for highly accumulating γ-glutamylcysteine using yeast in which the glutathione synthase gene has been disrupted or weakened.

  By the way, the technique regarding the culture | cultivation method which accumulates highly the glutathione which is a metabolite of (gamma) -glutamylcysteine in yeast is disclosed (patent document 2 etc.). This report describes that the amount of glutathione accumulated increases when cysteine, which is a constituent amino acid of glutathione, is added during the culture of yeast. Therefore, it is considered that γ-glutamylcysteine can be highly accumulated by adding cysteine during the cultivation of yeast in which the glutathione synthase gene has been disrupted or weakened. However, since a material containing γ-glutamylcysteine is useful for the purpose of producing a material containing cysteine, adding cysteine during the culture of yeast containing γ-glutamylcysteine for the purpose of obtaining a cysteine-containing material is This is impractical from a cost standpoint.

  Furthermore, Otake et al. Reported the content of γ-glutamylcysteine in yeast cells when 3 mM cysteine was added during the cultivation of the yeast YL1 strain in which the glutathione synthetase gene was disrupted (Non-patent Document 1). ). This report describes that the amount of γ-glutamylcysteine accumulated when cysteine was added to the YL1 strain and cultured was 0.533%, and the content of γ-glutamylcysteine when cultured without cysteine was 0.518%. ing. Also from this result, it is considered to be an impractical method to add cysteine during the cultivation of yeast in which the glutathione synthase gene has been disrupted or weakened and to culture it.

  It has also been reported that increasing the expression level of the MET25 gene in yeast increases the intracellular glutathione content. Furthermore, methods for increasing the expression level of the MET25 gene have been reported such as a method using a mutant MET4 gene (Non-patent Documents 4 and 3), a mutant MET30 gene (Non-patent Document 5), and the like.

  The expression of the MET25 gene is considered as follows (Non-Patent Document 6, Non-Patent Document 7). The MET4 product acts as a positive factor for expression in the MET25 gene. Normally, the MET4 product forms an SCFMET30 complex with the MET30 product and several other proteins, undergoes ubiquitination, and is degraded along with the MET30 product by the 26S proteasome proteolytic system, thus suppressing the expression of the MET25 gene Has been. On the other hand, when the function of the SCFMET30 complex decreases, the MET4 and MET30 products are not degraded and the MET25 gene is expressed.

  From the above findings, it is predicted that the γ-glutamylcysteine content is increased by increasing the expression level of the MET25 gene even in a yeast containing a high amount of γ-glutamylcysteine.

  In addition, studies on sake yeast have reported that when sake yeast is cultured in a deficient state of calcium pantothenate, hydrogen sulfide accumulates in the logarithmic growth phase. This report focuses on the generation of hydrogen sulfide from cysteine, and states that this phenomenon is promoted more in the pantothenic acid deficient state.

International Publication No. 00/30474 Pamphlet (WO 00/30474) JP-A-48-92579 Japanese Patent Laid-Open No. 10-33161 Otake et al., Agri. Biol. Chem., 54 (12), 3145-3150, 1990 Chris et al., Molecular Biology of the Cell., 8, 1699-1707, 1997 Inoue et al., Biochimica et Biophysica Acta, 1395 (1998) 315-320) Omura et al. FEBS Letters 387 (1996) 179-183 DOMINIQUE et al., MOLECULAR AND CELLUAR BIOLOGY, Dec 1995 p6526-6534 Patton et al., Genes Dev. 12: 692-705, 1998 Rouillon et al., EMBO Journal 19: 282-294, 2000

  The present invention provides a yeast suitable for γ-glutamylcysteine production, a method for producing γ-glutamylcysteine using the same, and a γ-glutamylcysteine-containing food / beverage product using the yeast strain based on the above technical background. The issue is to provide.

The inventors of the present invention have made extensive studies to solve the above problems, and it is not necessary that the amount of γ-glutamylcysteine accumulated in yeast is constant during culture. We thought that it should contain. And after cultivating yeast that has γ-glutamylcysteine-producing ability and that requires pantothenic acid with the minimum addition of pantothenic acid required by the yeast to obtain a sufficient cell content, By culturing in a state deficient in pantothenic acid, it was found that the amount of γ-glutamylcysteine accumulated increased, and the present invention was completed.
That is, the present invention is as follows.

(1) When cultivated in a medium having the ability to produce γ-glutamylcysteine, having pantothenic acid requirement and having a limited amount of pantothenic acid, the content of γ-glutamylcysteine per dry yeast cell Ascending yeast.
(2) The yeast according to (1), modified so that intracellular glutathione synthase activity is reduced or eliminated.
(3) The yeast according to (1) or (2), which is modified so that expression of the MET25 gene is derepressed.
(4) The expression of the MET25 gene is derepressed by retaining a mutant MET30 gene having a mutation in which the serine at position 569 of the encoded protein substitutes for an amino acid other than serine. yeast.
(5) The yeast according to (4), wherein the other amino acid is phenylalanine.
(6) The yeast according to any one of (1) to (5), which belongs to the genus Saccharomyces.
(7) A step of culturing the yeast according to any one of (1) to (6) in a medium containing a sufficient amount of pantothenic acid to proliferate the yeast, and culturing in a medium in which the amount of pantothenic acid is limited And a method for producing yeast having accumulated γ-glutamylcysteine, comprising a step of increasing the content of γ-glutamylcysteine in the microbial cells.
(8) A culture obtained by culturing the yeast according to any one of (1) to (6) under suitable conditions, a fraction of the culture containing γ-glutamylcysteine, or cysteine by heat treatment Food or drink containing these cultures or fractions produced.
(9) The food or drink according to (8), wherein the food or drink is an alcoholic beverage, bread food, or fermented food seasoning.
(10) A yeast extract produced using a culture obtained by culturing the yeast according to any one of (1) to (6) under suitable conditions.
(11) A culture obtained by culturing the yeast according to any one of (1) to (6) or a fraction thereof, or the heat-treated culture or fraction, A method for producing γ-glutamylcysteine or a cysteine-containing food or drink, which is mixed with a raw material and processed into a food or drink.
(12) A yeast in which expression of the MET25 gene is derepressed by retaining a mutant MET30 gene having a mutation in which serine at position 569 of the encoded protein is substituted with phenylalanine.

  Hereinafter, the present invention will be described in detail.

<1> Yeast of the Present Invention The yeast of the present invention is a yeast having the ability to produce γ-glutamylcysteine and having pantothenic acid requirement. Preferably, when the yeast of the present invention is cultured in a medium with a limited pantothenic acid content, the γ-glutamylcysteine content per dry yeast cell increases with time.

  In the present invention, “having the ability to produce γ-glutamylcysteine” means that a larger amount of γ-glutamylcysteine is accumulated in the microbial cells than the wild yeast strain, and preferably the yeast has a sufficient amount of pantothenic acid. It means that 1% or more of γ-glutamylcysteine is accumulated per dry yeast cell when cultured in a medium with a limited amount of pantothenic acid after culturing in a medium containing the same. More preferably, it means that the amount of glutathione accumulated per dry yeast cell is 0.1% or less.

  The accumulated amount of γ-glutamylcysteine or glutathione per dry yeast cell means, for example, the content (%) of γ-glutamylcysteine or glutathione with respect to the cell weight after heating at 105 ° C. for 4 hours.

  Examples of yeast having the ability to produce γ-glutamylcysteine include, for example, yeast in which intracellular glutathione synthetase activity has been reduced or eliminated, or yeast modified to enhance γ-glutamylcysteine synthetase activity, or Examples thereof include yeast modified so that intracellular glutathione synthetase activity is reduced or eliminated and γ-glutamylcysteine synthetase activity is enhanced.

  Yeast in which intracellular glutathione synthetase activity is reduced or eliminated is, for example, a gene that has been modified so that it does not produce a normally functioning enzyme by deleting a partial sequence of the gene encoding glutathione synthetase (GSH2), or It can be created by gene replacement using a DNA containing a gene having a mutation that reduces enzyme activity (hereinafter simply referred to as “mutant GSH2 gene”). Further, in the same manner as described in the examples, the yeast in which intracellular glutathione synthase activity is reduced or eliminated is treated with normal mutation treatment such as UV irradiation or N-methyl-N-nitrosoguanidine. (NTG), ethyl methanesulfonate (EMS), nitrous acid, acridine and other treatments with mutagens can also be obtained. It can be confirmed, for example, by the PCR method, that the obtained mutant has the target mutation.

  Examples of such a mutation that decreases glutathione synthetase activity include a mutation that changes the arginine residue at position 370 to a stop codon.

In addition, other mutations that reduce glutathione synthetase activity include the following mutations (see International Publication No. 03/046155 pamphlet).
(1) A mutation that replaces the threonine residue at position 47 with an isoleucine residue.
(2) A mutation that replaces the glycine residue at position 387 with an aspartic acid residue.
(3) A mutation in which the proline residue at position 54 is replaced with a leucine residue.
The mutations may be used alone or in any combination, but the combination of (1) and (3) and the combination of (2) and (3) are preferable.

  Introduction of the desired mutation into the glutathione synthetase gene can be performed by site-specific mutagenesis using a synthetic oligonucleotide.

  The gene replacement can be performed as follows. Yeast is transformed with a recombinant DNA containing the mutant GSH2 gene, and recombination occurs between the mutant GSH2 gene and the GSH2 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, and if the replication control region functioning in yeast is removed, a strain in which the recombinant DNA is incorporated into the chromosome can be efficiently obtained. be able to.

For transformation of yeast, methods usually used for transformation of yeast, such as protoplast method, KU method, KUR method, electroporation method, etc. can be employed.
A strain in which recombinant DNA is integrated into the chromosome as described above causes recombination between the mutant GSH2 gene and the GSH2 gene originally present on the chromosome, and a fusion gene of the wild-type GSH2 gene and the mutant GSH2 gene. Two of them are inserted into the chromosome with the other part of the recombinant DNA (vector part and marker gene) in between.

In order to leave only the mutant GSH2 gene out of the two fusion genes, one copy of the GSH2 gene is dropped from the chromosomal DNA together with the vector portion (including the marker gene) by recombination of the two GSH2 genes. At that time, the wild-type GSH2 gene is left on the chromosomal DNA and the mutant GSH2 gene is excised, whereas the mutant-type GSH2 gene is left on the chromosomal DNA and the wild-type GSH2 gene is excised. In any case, since the marker gene is lost, the occurrence of the second recombination can be confirmed by the phenotype corresponding to the marker gene. The target gene replacement strain can be selected by amplifying the GSH2 gene by PCR and examining its structure.

  The mutant GSH2 gene used for gene replacement may encode the full length glutathione synthetase protein, but may also be a gene fragment encoding a part of the protein as long as the deletion site is included.

The nucleotide sequence of Saccharomyces cerevisiae glutathione synthase gene (GSH2) has been reported (Inoue et al., Biochim. Biophys. Acta, 1395 (1998) 315-320, GenBank).
accession Y13804, SEQ ID NO: 1), can be obtained from Saccharomyces cerevisiae chromosomal DNA by PCR using an oligonucleotide prepared based on the base sequence as a primer. Moreover, as the gene to be introduced, a gene derived from a microorganism other than the genus Saccharomyces can be used.

  In addition to the mutations at positions 47, 387, and 54, the mutant GSH2 gene used in the present invention includes substitution or deletion of one or several amino acids at one or several positions in the amino acid sequence shown in SEQ ID NO: 2. It may encode a GSH2 product having an amino acid sequence that includes deletions, insertions or additions. Here, “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. Specifically, for example, the “several” is 2 to 10, preferably 2 to Six, more preferably 2-3. Alternatively, the mutant GSH2 gene may be DNA encoding a protein having a homology of 30 to 40% or more, preferably 55 to 65% or more with respect to the entire amino acid sequence of the GSH2 product.

  In addition, the above-mentioned base substitution, deletion, insertion, addition, or inversion is a naturally occurring mutation (mutant) based on individual differences of microorganisms holding the GSH2 gene, differences in species or genera, etc. Or variant).

  When the GSH2 gene is to be disrupted, the mutant GSH2 gene used for gene replacement does not necessarily need to contain the entire length, and may have a length necessary to cause gene disruption. The microorganism used for obtaining the GSH2 gene is not particularly limited as long as the gene has homology to the extent that homologous recombination occurs with the homologous gene of the microorganism used to create the gene disruption strain.

  Specifically, the DNA capable of undergoing homologous recombination with the GSH2 gene of Saccharomyces cerevisiae is, for example, 70% or more, preferably 80% or more, more preferably 90%, with DNA having the base sequence shown in SEQ ID NO: 1. Examples thereof include DNA having the above homology. More specifically, a DNA that hybridizes with a DNA having the base sequence shown in SEQ ID NO: 1 under stringent conditions can be mentioned. The stringent conditions include conditions under which washing is performed at a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS.

  In addition, the yeast in which glutathione synthetase activity is reduced or eliminated is treated with a normal mutation treatment such as UV irradiation or N-methyl-N-nitrosoguanidine (NTG), as described in the Examples. It can also be obtained by treatment with a mutagen such as ethyl methanesulfonate (EMS), nitrous acid or acridine.

  In addition, as a method for enhancing the γ-glutamylcysteine synthetase activity in yeast cells, yeast with a plasmid into which the same enzyme gene (for example, γ-glutamylcysteine synthetase gene of Saccharomyces cerevisiae: GenBank Accession No. D90220) is inserted is used. Transform and increase the intracellular copy number of the gene, or replace the promoter of the γ-glutamylcysteine synthase gene on the chromosome with a strong transcription promoter (Yasuyuki Otake et al., Bioscience and Industry, Vol. 50, Vol. No. 10, pages 989-994, 1992).

  The intracellular γ-glutamylcysteine synthetase activity and glutathione synthetase activity were determined by the method of Jackson (Jackson, RC, Biochem. J., 111, 309 (1969)) and the method of Gushima et al. (Gushima, T. et al., J. Appl. Biochem., 5, 210 (1983)).

  The yeast of the present invention has the ability to produce γ-glutamylcysteine as described above, and has pantothenic acid requirement. In the present invention, “pantothenic acid requirement” means that a higher concentration of pantothenic acid is required for growth than the concentration of pantothenic acid required for growth of an unmodified strain of yeast, for example, a wild strain.

  A yeast mutant having pantothenic acid requirement is a medium containing pantothenic acid, in which the mutant yeast is replicated on a medium containing pantothenic acid and a medium containing no pantothenic acid, and no colonies are formed on the medium containing no pantothenic acid. Can be obtained by selecting a strain that forms a colony. Also, pantothenic acid-requiring strains can be concentrated by culturing the mutated yeast in a medium that does not contain pantothenic acid and that has an additive that selectively acts on proliferating cells, such as nystatin. Can do.

  Specific examples of the medium not containing pantothenic acid include a medium having the following composition. Moreover, as a culture medium containing pantothenic acid, the culture medium which added the pantothenate to 0.1-10 mg / L, for example, 0.4 mg / L to the said culture medium is mentioned. An example of pantothenate is calcium pantothenate. In the case of a solid medium, it contains an appropriate amount of agar.

  The yeast of the present invention having the above properties can be grown in a medium containing a sufficient amount of pantothenic acid, and when cultured in a medium with a limited amount of pantothenic acid, The γ-glutamylcysteine content increases with time. By “sufficient pantothenic acid” is meant an amount that allows yeast in logarithmic growth to grow. This amount is usually 0.1 mg / L or more, preferably 0.4 mg / L or more. The upper limit of the pantothenic acid amount is not particularly limited, but is usually excessive when the amount is 10 mg / L or more. Therefore, the amount of pantothenic acid is usually 0.1 to 10 mg / L.

  On the other hand, “the amount of pantothenic acid is limited” means that even if yeast containing a sufficient amount of pantothenic acid is in the logarithmic growth phase, it cannot grow or the growth rate decreases in the medium. This means that the amount of pantothenic acid was limited. This amount is usually 0.1 mg / L or less, preferably 0.01 mg / L or less. The pantothenic acid amount may be 0 mg / L.

  In the present invention, “γ-glutamylcysteine content increases with time” means that the yeast of the present invention is grown in a medium containing a sufficient amount of pantothenic acid, and then the medium has a limited amount of pantothenic acid. When transferred and cultured, the maximum value of the γ-glutamylcysteine content after the medium exchange is preferably 1.5 times or more, more preferably, relative to the γ-glutamylcysteine content per dry yeast cell at the time of the medium exchange Means 1.8 times or more, particularly preferably 2 times or more.

  The yeast of the present invention may be modified so that the expression of the MET25 gene is derepressed. The expression of MET25 gene being derepressed means that the expression level of MET25 gene is not suppressed by methionine under the conditions described in the report of DOMINIQUE et al. (MOLLECULAR AND CELLUAR BIOLOGY Dec, 1995, p6526-6534).

As a method for derepressing the expression of the MET25 gene, specifically, for example, a mutant MET30 having a mutation in which serine at position 569 of the encoded protein is substituted with an amino acid other than serine
The method of making a gene hold | maintain in yeast is mentioned. Examples of the other amino acid include phenylalanine. Yeast having such properties can be obtained by mutation treatment of yeast as shown in the examples below, but since necessary mutations have been identified, yeast having the same mutations can be obtained by genetic engineering techniques. It can be acquired easily and reliably. For example, a yeast in which the expression of the MET25 gene is derepressed can be created by gene replacement using a mutant MET30 gene having the above mutation. Gene replacement can be performed in the same manner as the GSH2 gene described above. In addition, a yeast carrying a mutant MET30 gene can also be created by transforming the yeast with a plasmid into which the gene has been inserted and increasing the number of copies of the gene in the cell. Furthermore, the yeast carrying the mutant MET30 gene can be obtained by using wild-type yeast, as described in the Examples, by performing normal mutation treatment such as UV irradiation, N-methyl-N-nitrosoguanidine (NTG), ethyl It can also be obtained by treatment with a mutating agent such as methanesulfonate (EMS), nitrous acid, or acridine. It can be confirmed, for example, by the PCR method, that the obtained mutant has the target mutation. The yeast having the mutant MET30 gene having a mutation in which the serine at position 569 of the encoded protein is substituted with phenylalanine can also be used for the production of glutathione.

  In the present invention, the MET30 gene is a gene that forms a SCFMET30 complex together with the MET4 product and several other proteins and encodes a protein involved in the expression of the MET25 gene. For example, the nucleotide sequence shown in SEQ ID NO: 3 And the MET30 gene derived from Saccharomyces cerevisiae, and homologs thereof. Examples of the homolog include DNA that hybridizes with a DNA having the base sequence shown in SEQ ID NO: 3 under stringent conditions. The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify these conditions clearly, for example, DNAs with high homology, for example, DNAs having 50% or more homology hybridize and DNA with lower homology than that. A salt corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which does not hybridize with each other, or is a condition for washing of ordinary Southern hybridization Conditions for hybridizing at a concentration are mentioned.

  “Serine at position 569” means a serine residue located at position 569 in the amino acid sequence of the MET30 product shown in SEQ ID NO: 4. In addition, the position of a certain amino acid residue in the amino acid sequence can be changed by insertion or deletion of the amino acid residue in the sequence upstream of the position. In the present invention, “serine at position 569” corresponds to the serine residue at position 569 in the amino acid sequence shown in SEQ ID NO: 4 even when the absolute position in the amino acid sequence is changed as described above. Contains amino acid residues.

  The mutant MET30 gene used in the present invention is a conservative encoding a protein having a function equivalent to that of a protein having an amino acid sequence in which the serine at position 569 is substituted with another amino acid other than serine in the amino acid sequence shown in SEQ ID NO: 4. May encode a conservative variant, ie, a MET30 product having an amino acid sequence that includes substitution, deletion, insertion or addition of one or several amino acids at one or several positions other than position 569 . Here, “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. Specifically, for example, the “several” is 2 to 10, preferably 2 to Six, more preferably 2-3. Alternatively, the mutant MET30 gene may be a DNA encoding a protein having a homology of 30 to 40% or more, preferably 55 to 65% or more with respect to the entire amino acid sequence of the MET30 product.

  In addition, the above-mentioned base substitution, deletion, insertion, addition, or inversion is a naturally occurring mutation (mutant) based on individual differences in microorganisms holding the MET30 gene, differences in species or genera, etc. Or variant).

  The yeast of the present invention is not particularly limited as long as it can produce γ-glutamylcysteine, and specifically, Saccharomyces genus such as Saccharomyces cerevisiae, Candida genus such as Candida utilis, Shizo Examples include yeast belonging to the genus Schizosaccharomyces such as Saccharomyces pombe. In addition, the yeast strain of the present invention may be haploid, but preferably has diploidity or higher ploidy in terms of good growth.

  Yeast having double or higher ploidy is selected from a strain that mutates them to produce γ-glutamylcysteine, or a haploid yeast used for breeding a γ-glutamylcysteine producing strain, By joining a haploid of wild-type yeast and sporulating the resulting diploid yeast, a strain that weakens glutathione synthase activity and produces γ-glutamylcysteine is selected, and a different zygote is selected. It can be obtained by joining two γ-glutamylcysteine-producing haploid yeast strains. Similarly, a yeast having triploidy or higher ploidy can be obtained.

  The operations related to the breeding and modification of yeast as described above are as follows: “Chemistry and Biology Experiment 31 Yeast Experiment Techniques” First Edition Sasakawa Shoten; “Biomanual Series 10 Genetic Experiment Method Using Yeast” First Edition, Yodosha, “METHODS in YEAST GENETICS 2000 Edition "Cold Spring Harbor Laboratory Press etc.

<2> Utilization of Yeast of the Present Invention After the yeast of the present invention is cultured in a medium containing a sufficient amount of pantothenic acid to grow the yeast, the yeast is cultured in a medium in which the amount of pantothenic acid is limited. By increasing the content of γ-glutamylcysteine, yeast in which γ-glutamylcysteine is accumulated can be produced.

  Preferably, the “sufficient amount” of the pantothenic acid is necessary in order to experimentally measure the pantothenic acid requirement necessary for obtaining a certain amount of yeast cells in advance and obtain a target amount of cells. It can be determined by calculating the amount of pantothenic acid.

  In the step of growing yeast, pantothenic acid may be added to the initial culture medium in whole or in divided portions. The medium and culture conditions to be used are not particularly limited as long as the amount of pantothenic acid can be controlled, and the medium and conditions used for production of normal yeast extract and the like can be adopted.

  As a preferred embodiment of the yeast of the present invention, a yeast having reduced glutathione synthetase activity can grow well even in a medium not containing glutathione, and therefore, an industrially used medium can be used. If necessary, necessary nutrients are added to the medium according to the characteristics of the yeast to be used.

  When a sufficient amount of yeast is obtained, the cells are cultured in a medium with a limited amount of pantothenic acid. As a method for limiting the amount of pantothenic acid, a culture solution or yeast cells cultured in a medium containing a sufficient amount of pantothenic acid may contain a limited amount of pantothenic acid or may not contain pantothenic acid. The method of transferring to a culture medium is mentioned. In addition, the pantothenic acid amount can be limited by stopping the divided addition of pantothenic acid without replacing the medium. The period for limiting the amount of pantothenic acid is preferably performed in the logarithmic growth phase. For example, in baker's yeast, if the culture medium cultured until the logarithmic growth phase or the stationary phase is inoculated into a nutrient medium so as to be 2% and cultured at 30 ° C. for 8 to 16 hours with shaking, the bacteria in the logarithmic growth phase can be obtained. The body is obtained.

  During the cultivation in a medium with a limited pantothenic acid amount, the amount of γ-glutamylcysteine accumulated in the yeast cells increases with time. When the target accumulation amount is reached, the culture is terminated. Usually, under suitable conditions, the culture time is 10 to 30 hours, preferably 15 to 27 hours.

  The culture obtained as described above or a fraction thereof contains γ-glutamylcysteine. The culture may be a culture solution containing yeast cells, or may be a yeast cell, a crushed cell, or a cell extract (yeast extract) collected therefrom. A fraction containing γ-glutamylcysteine may be obtained from the crushed cell or yeast extract.

Cysteine can be released from γ-glutamylcysteine by heating the culture containing γ-glutamylcysteine or a fraction thereof.
Preparation of yeast extract and the like may be performed in the same manner as normal yeast extract preparation. The yeast extract may be one obtained by treating a yeast cell extracted with hot water, or one obtained by digesting a yeast cell.

  The culture containing γ-glutamylcysteine or cysteine or a fraction thereof can be used for the production of food and drink. Examples of the food and drink include alcoholic beverages, bread foods, and fermented food seasonings. Production of cysteine from γ-glutamylcysteine by heat treatment may be performed during or after the production of the food or drink.

  The said food / beverage products are manufactured by mixing the culture containing γ-glutamylcysteine or cysteine, or its fraction with the raw material for food / beverage products, and processing it into food / beverage products. 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 culture or fraction. 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.

  Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1]
<1> Acquisition of Yeast with Reduced Glutathione Synthase Activity A commercially available diploid Saccharomyces cerevisiae used for food applications was sporulated according to a conventional method. A haploid yeast strain YN0001 (MATα) was obtained from the formed spores by a random spore method. The YN0001 strain was mutated by EMS to obtain a mutant strain YN0002 (MATα) having a reduced glutathione content. A four-molecule analysis confirmed that the GSH2 gene of the YN0002 strain was mutated. Specifically, the 387th glycine residue of the encoded protein was replaced with an aspartic acid residue. In addition, a mutant YN0003 strain (MATa) having a reduced glutathione content was also obtained.

  The mutation treatment was performed under such conditions that the death rate was 90%. The YN0001 strain was cultured with shaking in 50 ml of YPD medium at 30 ° C. for 1 day to collect yeast cells. The yeast cells were washed 3 times with 0.2 M sodium phosphate buffer (pH 7.5). Suspend the yeast cells in a solution containing 9.2 ml of 0.2 M sodium phosphate buffer (pH 7.5), 0.5 ml of 40% D-glucose and 0.3 ml of EMS (Nacalai Tesque Code155-19), and shake at 90 ° C for 90 minutes. Cultured at last. To this suspension, 10 ml of 10% sodium thiosulfate (filter sterilized) was added and left at room temperature for 10 minutes to neutralize the mutagen. Yeast cells were collected and washed with 0.2 M sodium phosphate buffer (pH 7.5).

The YN0001 and YN0002 strains were each inoculated into a YPD medium and cultured at 30 ° C. with shaking. The culture product was inoculated 2% in SD medium and cultured with shaking at 30 ° C. The intracellular glutathione content in the logarithmic growth phase was measured. As a result, the glutathione content of the YN0001 strain was 0.52%. On the other hand, the glutathione content of the YN0002 strain was 0.006% or less.

<2> Acquisition of MET30 gene mutant strain The haploid yeast YN0001 strain (MATα) is mutated with EMS in the same manner as described above, and the mutant AJ14819 strain (MATα) in which the expression of the MET25 gene is not suppressed by methionine is obtained. I got it. This strain has been assigned a private number AJ14819. The National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (September 11, 1st East, 1-chome, Tsukuba, Ibaraki, 305-8566, Japan) ) And assigned the accession number FERM P-19007. Furthermore, this strain was transferred to an international deposit based on the Budapest Treaty on October 1, 2003, and assigned the accession number FERM BP-08502. Whether MET25 gene expression is suppressed by methionine can also be determined by the presence or absence of growth in a medium containing selenium (DOMINIQUE et al., MOLLECULAR AND CELLUAR BIOLOGY Dec, 1995, p6526-6534).

  Specifically, the mutant strain was selected as follows. The yeast subjected to the mutation treatment was spread on a YPD agar medium. The spread was performed so that the yeast growing on the agar medium was about 100 strains. The yeast grown on the YPD medium was replicated on a medium containing selenium and a medium containing no selenium (an agar medium described in DOMINIQUE et al.). A yeast strain that did not grow on a medium containing selenium but grew on a medium containing no selenium was selected.

It was confirmed as follows that the MET25 gene expression level of the selected yeast was increased. The selected yeast strain and YN0001 strain were cultured in SD medium and collected in the logarithmic growth phase. The RNA contained in the microbial cells was collected, and the amount of the MET25 gene transcript contained in the RNA was quantified using the ACT1 gene as an internal standard. Quantification was performed using PCR5700 (Applied Biosystems), which is quantitative PCR, and using the TaqMan One-Step RT-PCR kit (Applied Biosystems). TaqMan
Using ACT1-986T (SEQ ID NO: 5) and MET25-1077T (SEQ ID NO: 6) for Probe (Applied Biosystems), ACT1-963F (SEQ ID NO: 7) and ACT1-1039R (sequence) for amplification of ACT1 and MET25 genes No. 8), and MET251056F (SEQ ID NO: 9) and MET25-1134R (SEQ ID NO: 10) were used (Applied Biosystems). Thus, the yeast AJ14819 strain in which the expression level of the MET25 gene was increased more than twice that of the YN0001 strain was obtained.

  The mutant gene of the AJ14819 strain obtained in this way was identified by four-molecule analysis and the sequence of the gene was examined. As a result, the serine residue at position 569 of the protein encoded by the MET30 gene was mutated to a phenylalanine residue. . Thus, the yeast AJ14819 strain in which the expression of the MET25 gene was not suppressed by methionine was obtained.

<3> Acquisition of Yeast Having Calcium Pantothenate Requirement The haploid yeast strain YN0001 (MATα) was mutated with EMS as described above. In order to obtain a yeast that requires calcium pantothenate from the mutant-treated yeast, the yeast is cultured at 30 ° C. for 2 hours in a medium not containing calcium pantothenate and supplemented with nystatin (10 μg / ml). Was spread on YPD agar medium. The mutant yeast thus grown is replicated on an agar medium not containing calcium pantothenate and an agar medium containing calcium pantothenate (each having the composition shown in Table 1 above) and cannot grow on the former agar medium, but the latter agar medium Then, yeast that can grow was selected. In this way, a yeast strain Pa0001 (MATa) requiring calcium pantothenate was obtained.

<4> Acquisition of a yeast (GMP strain; Diploid gsh2 met30 pa-) having a mutant GSH2 gene and a mutant MET30 gene and having a requirement for calcium pantothenate According to a conventional method, AJ14819 strain and Pa0001 strain are joined. A diploid was obtained. The obtained diploid was sporulated, and a haploid yeast MP strain (MATa) having a mutant MET30 gene and showing calcium pantothenate requirement was obtained by the random spore method. Next, YN0002 strain and MP strain were joined to obtain a diploid. The obtained diploid is sporulated, and the haploid yeast GMP-1 (MATα), GMP-2 (having a mutant GSH2 gene and a mutant MET30 gene and exhibiting calcium pantothenate requirement by a random spore method MATa) stock was acquired. GMP-1 and GMP-2 strains were joined to obtain a diploid yeast GMP strain.

<5> Production of γ-glutamylcysteine using GMP strain
The GMP strain was inoculated into YPD medium (4 ml of test tube) and cultured with shaking at 30 ° C. for 1 day. The culture solution was inoculated into a medium containing 0.4 mg / dl of calcium pantothenate and cultured with shaking at 30 ° C. A medium is collected in the logarithmic growth phase, and a medium containing no calcium pantothenate and a medium containing 0.4 mg / L calcium pantothenate so that the bacterial cell concentration (weight of dry yeast) is 60 mg / dl (see Table 1 above). ), And cultured. The γ-glutamylcysteine content per dry yeast cell was measured over time. The results are shown in FIG. When cultured in a medium with a low calcium pantothenate concentration, the γ-glutamylcysteine content increased with time compared to when cultured in a medium with a high concentration.

  From the above results, it was shown that the γ-glutamylcysteine content of the GMP strain was increased due to the lack of calcium pantothenate.

[Comparative Example 1] Obtaining a yeast having a mutant GSH2 gene and a mutant MET30 gene According to a conventional method, the MET30 gene mutant AJ14819 is joined to a haploid yeast Pa0001 strain obtained from a commercially available yeast to double Acquired the body. This diploid was sporulated, and a haploid yeast strain M (MATa) having a mutant MET30 gene was obtained by the random spore method. Next, the M strain and the GSH2 gene mutant YN0002 were joined to obtain a diploid. The diploid was sporulated, and haploid yeast GM-1 strains (MATα) and GM-2 strains (MATa) having mutant GSH2 gene and mutant MET30 gene were obtained by random spore method. The GM-1 strain and the GM-2 strain were joined to obtain a diploid yeast GM strain.

[Example 2] Production of γ-glutamylcysteine by GMP strain and GM strain
The GM strain and GMP strain were each inoculated into YPD medium and cultured with shaking at 30 ° C. The culture solution was inoculated into a medium containing 0.4 mg / dl of calcium pantothenate and cultured with shaking at 30 ° C. The cells were collected in the logarithmic growth phase and inoculated into a medium containing no calcium pantothenate so that the cell concentration (dry yeast weight) was 60 mg / dl. After shaking culture at 30 ° C., the γ-glutamylcysteine content per dry yeast cell was measured over time. The results are shown in FIG.

  As a result, it was shown that in the GMP strain, the γ-glutamyl cisiten content per dry yeast cell was increased as the culture was continued in a state in which calbutium pantothenate was deficient.

[Example 3]
Next, the effect of further reducing the glutathione synthetase activity of the GMP strain was examined.

<6> Preparation of glutathione synthetase gene disruption cassette First, a glutathione synthetase gene disruption cassette was prepared as follows.
PCR was carried out using GSH2-AUR1-CF (SEQ ID NO: 11) and GSH2-AUR1-CR (SEQ ID NO: 12) as primers using the pAUR123 vector (Takara Shuzo code3602) cut with Kpn I as a template. Since the PCR product contains the N-terminal sequence of the ORF of the GSH2 gene and the C-terminal sequence of the ORF of the GSH2 gene at both ends of the AUR1-C gene, it is possible to destroy the glutathione synthase gene using this product. is there. PCR conditions are as follows.

1 μl of pAUR123 cut with Kpn I
10 × PCR Buffer 5 μl
dNTP 4μl
10 pmol/μl GSH2-AUR1-CF primer 1μl
10 pmol/μl GSH2-AUR1-CR primer 1μl
KOD Dash DNA polymerase 0.5μl
MilliQ 37.5μl
Total 50μl

94 ℃ 2min × 1cycle → 94 ℃ 40sec 、 54 ℃ 40sec 、 74 ℃ 1min × 30cycles → 4 ℃ ∞
(KOD Dash is codeLDP-101 made by TOYOBO)

<7> Acquisition of AJ14861 strain Using the glutathione synthetase gene disruption cassette prepared as described above, an attempt was made to disrupt the glutathione synthetase gene of the GMP strain as follows. That is, the GMP strain was cultured in a YPD medium and collected in the logarithmic growth phase. The cells washed twice with 1M sorbitol solution were suspended in a solution having the following composition and left at 5 ° C. for 1 hour.
Composition: 0.1 M LiCl
10 mM DTT
10 mM Tris-HCl (pH 7.5)
1 mM EDTA

Then, it was washed twice with 1M sorbitol solution. The thus prepared bacterial cells were mixed with the PCR product prepared as described above and electroporated. ("Bio Manual Series 10 Genetic Experiment Method Using Yeast", First Edition, Yodosha) Thereafter, the mixture was inoculated into YPD medium and cultured with shaking at 30 ° C for 16 hours. The culture product was spread on a YPD agar medium containing 0.2 μg / ml of aureobasidin A (Takara Shuzo code9000), which is a selection marker, and cultured at 30 ° C. for 3 days. (Minimum growth inhibitory concentration of GMP strain against aureobasidin A is 0.05μg / ml) The grown colonies were applied to YPD agar medium containing 0.2μg / ml aureobasidin A and resistant to aureobasidin A Selected colonies with. In this way, AJ14861 strain was obtained. This share has been assigned a private number AJ14861. As of November 19, 2003, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (305-8566)
It has been deposited internationally under the Budapest Treaty at Tsukuba City, Ibaraki Prefecture, Japan, 1-chome, 1-Chuo 6), and has been assigned the deposit number FERM BP-08553.

<8> Production of γ-glutamylcysteine using AJ14861 strain
AJ14861 strain was inoculated into YPD medium (4 ml of test tube) and cultured with shaking at 30 ° C. for 1 day. The culture solution was inoculated into a medium containing 0.4 mg / dl of calcium pantothenate and cultured with shaking at 30 ° C. A medium is collected in the logarithmic growth phase, and a medium containing no calcium pantothenate and a medium containing 0.4 mg / L calcium pantothenate so that the bacterial cell concentration (weight of dry yeast) is 60 mg / dl (see Table 1 above). ), And cultured. The γ-glutamylcysteine content per dry yeast cell was measured over time. The results are shown in Figure 3. When cultured in a medium with a low calcium pantothenate concentration, the γ-glutamylcysteine content increased with time compared to when cultured in a medium with a high concentration.

  From the above results, it was shown that the γ-glutamylcysteine content of the AJ14861 strain increases due to the lack of calcium pantothenate.

  According to the present invention, a yeast exhibiting pantothenic acid requirement is provided in which glutathione synthetase activity is reduced or eliminated, MET25 gene expression is derepressed. By culturing the yeast of the present invention under suitable conditions, a yeast culture solution containing a high amount of γ-glutamylcysteine is provided. The yeast and yeast culture solution of this invention can be utilized for manufacture of (gamma) -glutamylcysteine containing food / beverage products or cysteine containing food / beverage products.

The figure which shows the time-dependent change of γ-glutamylcysteine content in a cell when the yeast (GMP strain) of the present invention is cultured in a medium with or without calcium pantothenate (PaCa) added. The figure which shows the comparison of (gamma) -glutamylcysteine content of GM strain and GMP strain. The figure which shows a time-dependent change of γ-glutamylcysteine content in a cell when the yeast (AJ14861 strain) of the present invention is cultured in a medium with or without calcium pantothenate added.

Claims (8)

When cultivated in a medium that has the ability to produce γ-glutamylcysteine, has pantothenic acid requirement, and has a limited amount of pantothenic acid, the content of γ-glutamylcysteine per dry yeast cell increases over time The glutathione synthetase activity in the cell is modified so that it decreases or disappears by disruption or mutation of the gene encoding glutathione synthetase, and the expression of the MET25 gene is modified to be derepressed. Yeast . The yeast according to claim 1 , wherein expression of the MET25 gene is derepressed by retaining a mutant MET30 gene having a mutation in which serine at position 569 of the encoded protein substitutes phenylalanine . The yeast according to claim 1 or 2 , which belongs to the genus Saccharomyces. A step of culturing the yeast according to any one of claims 1 to 3 in a medium containing a sufficient amount of pantothenic acid to proliferate the yeast, and culturing the yeast in a medium with a limited amount of pantothenic acid, The manufacturing method of the yeast which accumulate | stored (gamma) -glutamylcysteine including the process of raising (gamma) -glutamylcysteine content in a body. A culture obtained by culturing the yeast according to any one of claims 1 to 3 under suitable conditions, a fraction of the culture containing γ-glutamylcysteine, or a cysteine produced by heat treatment Food or drink containing a culture or fraction of The food or drink according to claim 5 , wherein the food or drink is an alcoholic beverage, a bread food, or a fermented food seasoning. The yeast extract manufactured using the culture obtained by culture | cultivating the yeast as described in any one of Claims 1-3 on suitable conditions. A culture obtained by culturing the yeast according to any one of claims 1 to 3 under suitable conditions, or a fraction thereof, or the heat-treated culture or fraction is mixed with a food or drink raw material. And a process for producing γ-glutamylcysteine or a cysteine-containing food or drink, which is processed into a food or drink.

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