JP2004201677A - METHOD FOR PRODUCING gamma-GLUTAMYLCYCTEINE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a yeast suitable for producing γ-glutamylcysteine, and to provide a method for producing the γ-glutamylcysteine, by using the same. <P>SOLUTION: This yeast which has γ-glutamylcysteine producing ability and pantothenic acid requiring properties is produced through a process in which the yeast is proliferated by culturing the yeast in a culture medium containing an enough amount of the pantothenic acid and another process in which a content of the γ-glutamylcysteine in a body cell of the yeast is increased by culturing the yeast in another culture medium having a limited content of the pantothenic acid, so that the yeast in which the γ-glutamylcysteine is accumulated is produced. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  TECHNICAL FIELD The present invention relates to a yeast strain producing γ-glutamylcysteine, a method for culturing the yeast strain, and a food or drink using cells of the yeast strain. Materials containing γ-glutamylcysteine and materials containing cysteine produced therefrom are useful in the food field.

  Cysteine is used for the purpose of improving the flavor of foods and the like. As for the production method of cysteine, a proteolysis method and a semi-synthesis method are known, but currently mainly used methods are a proteolysis method and a semi-synthesis method. For the purpose of using cysteine to improve the flavor of foods, natural food materials having a high cysteine content have been demanded, but such natural food materials have hitherto hardly been known. On the other hand, it has been reported that a food material having a high cysteine content can be obtained by heating or enzymatically treating a yeast extract containing γ-glutamylcysteine (Patent Document 1).

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

  So far, yeasts containing 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, there is no description in these reports about culture conditions for highly accumulating γ-glutamylcysteine using yeast in which the glutathione synthase gene has been disrupted or weakened.

  By the way, a technique relating to a culture method for highly accumulating glutathione, a metabolite of γ-glutamylcysteine, in yeast is disclosed (Patent Document 2 and the like). This report states that addition of cysteine, a constituent amino acid of glutathione, during the culture of yeast increases glutathione accumulation. 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 destroyed or weakened. However, since a material containing γ-glutamylcysteine is useful for the purpose of producing a material containing cysteine, adding cysteine during the culturing of a yeast containing γ-glutamylcysteine for the purpose of obtaining a cysteine-containing material, This is impractical from a cost standpoint.

  Further, Otake et al. Report the γ-glutamylcysteine content in yeast cells when 3 mM cysteine was added during the culture of yeast YL1 strain in which the glutathione synthase gene was disrupted (Non-patent Document 1). ). In this report, the γ-glutamylcysteine accumulation when cysteine was added to the YL1 strain was 0.533%, and the γ-glutamylcysteine content when cultured without cysteine was 0.518%. ing. From these results, it is considered that it is an impractical method to add cysteine during the cultivation of the yeast in which the glutathione synthase gene has been destroyed or weakened, and culture the yeast.

  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 include a method using a mutant MET4 gene (Non-patent Document 4, Patent Document 3) and a method using a mutant MET30 gene (Non-patent Document 5).

  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 on 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 together with the MET30 product by the 26S proteasome proteolytic system, thus suppressing the expression of the MET25 gene. Have been. On the other hand, when the function of the SCFMET30 complex is reduced, the MET4 product and the MET30 product are not degraded, and the MET25 gene is expressed.

  From the above findings, it is predicted that, even in yeast having a high content of γ-glutamylcysteine, increasing the expression level of the MET25 gene will increase the γ-glutamylcysteine content.

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

WO 00/30474 pamphlet (WO 00/30474) JP-A-48-92579 JP-A-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 and a method for producing γ-glutamylcysteine using the same, and γ-glutamylcysteine-containing food and drink using the yeast strain, under the above technical background. The task is to provide.

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and the accumulated amount of γ-glutamylcysteine in yeast does not need to be constant during culture, and the required amount of γ-glutamylcysteine is obtained immediately before collection. We thought that it should be contained. And yeast having a γ-glutamylcysteine-producing ability, and requesting pantothenic acid, and culturing by adding the necessary minimum amount of pantothenic acid required by the yeast to obtain a sufficient amount of bacterial cells, It has been found that culturing in a state deficient in pantothenic acid results in an increase in the amount of accumulated γ-glutamylcysteine, thereby completing the present invention.
That is, the present invention is as follows.

(1) When cultured in a medium having a γ-glutamylcysteine-producing ability, having a requirement for pantothenic acid, and having a restricted amount of pantothenic acid, the γ-glutamylcysteine content per dry yeast cell increased over time. Yeast that rises.
(2) The yeast according to (1), wherein the yeast is modified so that intracellular glutathione synthetase activity is reduced or eliminated.
(3) The yeast according to (1) or (2), which has been modified so that expression of the MET25 gene is derepressed.
(4) The expression of the MET25 gene was derepressed by retaining a mutant MET30 gene having a mutation in which serine at position 569 of the encoded protein was substituted with another amino acid other than serine, thereby suppressing the expression of the MET25 gene. 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 grow the yeast, and culturing the yeast in a medium in which the amount of pantothenic acid is restricted; And a method for producing a yeast in which γ-glutamylcysteine is accumulated, comprising a step of increasing the content of γ-glutamylcysteine in the cells.
(8) A culture obtained by culturing the yeast according to any of (1) to (6) under suitable conditions, a fraction of the culture containing γ-glutamylcysteine, or cysteine by heat treatment. A food or drink containing the produced culture or fraction thereof.
(9) The food or drink according to (8), wherein the food or drink is an alcoholic beverage, a bread food, or a 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) under suitable conditions, or a fraction thereof, or the heat-treated culture or fraction, and A method for producing γ-glutamylcysteine or 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 desuppressed 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 a γ-glutamylcysteine-producing ability and having a requirement for pantothenic acid. Preferably, when the yeast of the present invention is cultured in a medium in which the amount of pantothenic acid is restricted, the γ-glutamylcysteine content per dry yeast cell increases with time.

  In the present invention, "having γ-glutamylcysteine-producing ability" refers to accumulating a larger amount of γ-glutamylcysteine in a bacterial cell than a yeast wild-type strain, and preferably, a yeast having a sufficient amount of pantothenic acid. This means that when cultured in a medium containing a limited amount of pantothenic acid after culturing in a culture medium containing 1% or more of γ-glutamylcysteine per dry yeast cell. More preferably, it means that the amount of accumulated glutathione per dry yeast cell is 0.1% or less.

  The accumulated amount of γ-glutamylcysteine or glutathione per dry yeast cell refers to, 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 γ-glutamylcysteine-producing ability include, for example, yeast in which intracellular glutathione synthetase activity has been reduced or disappeared, or yeast modified so that γ-glutamylcysteine synthetase activity has been enhanced, or A yeast modified so that intracellular glutathione synthetase activity decreases or disappears and γ-glutamylcysteine synthetase activity is enhanced is exemplified.

  A yeast in which the intracellular glutathione synthetase activity is reduced or eliminated is, for example, a gene modified so that the partial sequence of the gene encoding glutathione synthase (GSH2) is deleted and a normally functioning enzyme is not produced, or It can be created by gene replacement using DNA containing a gene having a mutation that reduces the enzyme activity (hereinafter, simply referred to as “mutant GSH2 gene”). In addition, yeast in which the intracellular glutathione synthetase activity has been reduced or eliminated, as described in the Examples, wild-type yeast can be subjected to ordinary mutation treatment, for example, UV irradiation, or N-methyl-N-nitrosoguanidine. (NTG), ethyl methanesulfonate (EMS), nitrite, acridine or the like, and can be obtained by treatment with a mutagen. Whether the obtained mutant has the desired mutation can be confirmed by, for example, PCR.

  Examples of the mutation that reduces glutathione synthase activity as described above include a mutation that changes an arginine residue at position 370 to a stop codon.

Other mutations that reduce glutathione synthase activity include the following mutations (see WO 03/046155).
(1) A mutation in which the threonine residue at position 47 is replaced with an isoleucine residue.
(2) A mutation in which the glycine residue at position 387 is replaced with an aspartic acid residue.
(3) A mutation in which the proline residue at position 54 is replaced with a leucine residue.
The above mutations may be used alone or in any combination, but a combination of (1) and (3) and a combination of (2) and (3) are preferred.

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

  The gene replacement can be performed as follows. Yeast is transformed with the recombinant DNA containing the mutant GSH2 gene to cause recombination between the mutant GSH2 gene and the GSH2 gene on the chromosome. At that time, if the recombinant DNA contains a marker gene according to the traits such as auxotrophy of the host, the operation is easy. In addition, the recombinant DNA is made linear by cutting with a restriction enzyme or the like, and further, if a replication control region that functions in yeast is removed, a strain in which the recombinant DNA is integrated into the chromosome can be efficiently obtained. be able to.

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

In order to leave only the mutant GSH2 gene among 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, there are cases where the wild-type GSH2 gene is left on the chromosomal DNA and the mutant GSH2 gene is cut out, and conversely, where the mutant GSH2 gene is left on the chromosomal DNA and the wild-type GSH2 gene is cut out. In any case, since the marker gene is dropped, 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 synthase protein, or may be a gene fragment encoding a part of the protein as long as it contains a deletion site.

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

  In the mutant GSH2 gene used in the present invention, in addition to the mutation at positions 47, 387, and 54, 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 containing a deletion, insertion or addition. Here, “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically, for example, the “several” is 2 to 10, preferably 2 to Six, more preferably two to three. Alternatively, the mutant GSH2 gene may be a DNA encoding a protein having 30 to 40% or more, preferably 55 to 65% or more homology to the entire amino acid sequence of the GSH2 product.

  In addition, substitution, deletion, insertion, addition, or inversion of the base as described above includes mutations that occur naturally (mutant) such as those based on individual differences, species, and genera of microorganisms that carry the GSH2 gene. Or variant) is also included.

  When the GSH2 gene is disrupted, the mutant GSH2 gene used for gene replacement does not necessarily need to include the full length, but 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 homologous recombination with a homologous gene of the microorganism used for creating a gene-disrupted strain.

  Specific examples of the DNA capable of causing homologous recombination with the GSH2 gene of Saccharomyces cerevisiae include a DNA having the nucleotide sequence shown in SEQ ID NO: 1, for example, 70% or more, preferably 80% or more, more preferably 90% or more. DNAs having the above homology are exemplified. More specifically, it includes a DNA having the nucleotide sequence shown in SEQ ID NO: 1 and a DNA that hybridizes under stringent conditions. Stringent conditions include conditions where washing is performed at a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS.

  Further, the yeast in which the glutathione synthetase activity has been reduced or eliminated can be obtained by subjecting a wild-type yeast to a normal mutation treatment, for example, UV irradiation, or N-methyl-N-nitrosoguanidine (NTG), as described in Examples. , Ethyl methanesulfonate (EMS), nitrous acid, acridine and the like.

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

  The intracellular γ-glutamylcysteine synthetase activity and glutathione synthase activity were measured by the method of Jackson (Jackson, RC, Biochem. J., 111, 309 (1969)) and the method of Gushima et al. 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 a requirement for pantothenic acid. In the present invention, “requiring pantothenic acid” means that a non-modified strain of yeast, for example, a wild strain, requires a higher concentration of pantothenic acid for growth than that required for growth.

  A yeast mutant strain having a pantothenic acid auxotrophy, the yeast subjected to the mutation treatment is replicated in a medium containing and not containing pantothenic acid, and does not form a colony in a medium containing no pantothenic acid, and contains a medium containing pantothenic acid. By selecting a strain that forms a colony in step (1). In addition, by enriching a pantothenic acid-requiring strain by culturing the mutated yeast in a medium containing no pantothenic acid and selectively adding an antibiotic that acts on growing cells, for example, nystatin. Can be.

  As the medium not containing pantothenic acid, a medium having the following composition is specifically mentioned. Examples of the medium containing pantothenic acid include a medium in which pantothenate is added to the above medium at 0.1 to 10 mg / L, for example, 0.4 mg / L. Pantothenates include 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-mentioned properties can be grown in a medium containing a sufficient amount of pantothenic acid, and when cultured in a medium in which the amount of pantothenic acid is restricted, per yeast yeast cells The γ-glutamylcysteine content increases over time. By "sufficient amount of pantothenic acid" is meant an amount that allows yeast in logarithmic growth to grow. This amount is usually at least 0.1 mg / L, preferably at least 0.4 mg / L. Although the upper limit of the amount of pantothenic acid is not particularly limited, it 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 restricted '' means that even if the yeast is in the logarithmic growth phase when it contains a sufficient amount of pantothenic acid, it cannot be grown or the growth rate is reduced in the medium. 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. Incidentally, the amount of pantothenic acid may be 0 mg / L.

  In the present invention, "the γ-glutamylcysteine content increases with time" means that after the yeast of the present invention is grown on a medium containing a sufficient amount of pantothenic acid, the amount of pantothenic acid in the medium is restricted. When transferred and cultured, the maximum value of the content of γ-glutamylcysteine after the medium replacement is preferably 1.5 times or more, more preferably, to the γ-glutamylcysteine content per dry yeast cells at the time of medium replacement. 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 the MET25 gene is derepressed means that the expression level of the 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 of desuppressing 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 another amino acid other than serine
There is a method in which a gene is retained in yeast. Examples of the other amino acids include phenylalanine. Yeast having such properties can be obtained by subjecting yeast to mutation treatment as shown in Examples below, but since the necessary mutation has been identified, yeast having the mutation can be obtained by genetic engineering techniques. It can be obtained easily and reliably. For example, yeast in which the expression of the MET25 gene has been 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 in the GSH2 gene described above. In addition, yeast having the mutant MET30 gene can also be created by transforming yeast with a plasmid having the gene inserted therein and increasing the intracellular copy number of the gene. Furthermore, as described in Examples, the yeast having the mutant MET30 gene can be prepared by subjecting a wild-type yeast to a conventional mutation treatment, for example, UV irradiation, N-methyl-N-nitrosoguanidine (NTG), ethyl acetate, or the like. It can also be obtained by treatment with a mutating agent such as methanesulfonate (EMS), nitrous acid, and acridine. Whether the obtained mutant has the desired mutation can be confirmed by, for example, PCR. The yeast having the mutant MET30 gene having a mutation in which serine at position 569 of the encoded protein is substituted with phenylalanine can be used for producing glutathione.

  In the present invention, the MET30 gene is a gene that forms a SCFMET30 complex 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 MET30 gene derived from Saccharomyces cerevisiae and a homolog thereof. Examples of the homolog include a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 3 under stringent conditions. The term "stringent conditions" used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, as an example, DNAs having high homology, for example, DNAs having homology of 50% or more hybridize to each other, and DNAs having lower homology A salt corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS at 60 ° C., which is a condition under which they do not hybridize with each other, or a washing condition for ordinary Southern hybridization. Conditions for hybridizing at a concentration are listed.

  “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 an 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. Contains amino acid residues.

  The constitutive MET30 gene used in the present invention is a conservative gene encoding a protein having a function equivalent to that of a protein having an amino acid sequence in which serine at position 569 is substituted with another amino acid other than serine in the amino acid sequence shown in SEQ ID NO: 4. It may encode a variant (conservative variant), that is, a MET30 product having an amino acid sequence containing 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, but specifically, for example, the “several” is 2 to 10, preferably 2 to Six, more preferably two to three. Alternatively, the mutant MET30 gene may be a DNA encoding a protein having 30 to 40% or more, preferably 55 to 65% or more homology to the entire amino acid sequence of the MET30 product.

  In addition, substitution, deletion, insertion, addition, or inversion of a base as described above includes mutations that occur naturally (mutant) such as those based on individual differences, species, and genera of microorganisms that carry the MET30 gene. Or variant) is also included.

  The yeast of the present invention is not particularly limited as long as it can produce γ-glutamylcysteine.Specifically, Saccharomyces genus such as Saccharomyces cerevisiae, Candida genus such as Candida utilis, and Shizo Examples include yeast belonging to the genus Schizosaccharomyces such as Saccharomyces pombe. Although the yeast strain of the present invention may be haploid, it is preferable that the yeast strain has a diploid or higher ploidy in terms of good growth.

  Yeast having a diploid or higher polyploidy, mutating them and selecting a strain that produces γ-glutamylcysteine, or a haploid yeast used for breeding a γ-glutamylcysteine producing strain, Glutathione synthetase activity is weakened by mating haploids of wild-type yeast and forming spores of the resulting diploid yeast, and a strain that produces γ-glutamylcysteine is selected. Γ-glutamylcysteine-producing haploid yeast can be obtained by conjugation. Similarly, a yeast having a triploid or higher ploidy can be obtained.

  The operations related to breeding and modification of yeast described above are described in “Chemistry and Biology Experiment Line 31 Yeast Experimental Techniques”, first edition, Hirokawa Shoten; “Bio Manual Series 10 Gene Experiments with Yeast”, first edition, Yodosha, “METHODS in YEAST GENETICS 2000 Edition "Cold Spring Harbor Laboratory Press.

<2> Utilization of the yeast of the present invention After culturing the yeast of the present invention in a medium containing a sufficient amount of pantothenic acid and growing the yeast, the yeast is cultured in a medium in which the amount of pantothenic acid is restricted. By increasing the content of γ-glutamylcysteine in the above, a yeast in which γ-glutamylcysteine has accumulated can be produced.

  Preferably, the `` sufficient amount '' of the pantothenic acid is determined in advance by experimentally measuring the required amount of pantothenic acid necessary to obtain a certain amount of yeast cells, and necessary to obtain the desired amount of cells. It can be determined by calculating the amount of pantothenic acid.

  In the step of growing the yeast, pantothenic acid may be added to the initial medium in its entirety or may be added in 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 the production of ordinary yeast extract and the like can be used.

  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 containing no glutathione, and therefore, a culture medium usually used industrially 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 cells is obtained, the cells are cultured in a medium in which the amount of pantothenic acid is restricted. As a method of limiting the amount of pantothenic acid, a culture solution or yeast cells obtained by culturing yeast in a medium containing a sufficient amount of pantothenic acid, containing a limited amount of pantothenic acid, or containing no pantothenic acid The method of transferring to a culture medium is mentioned. Also, the amount of pantothenic acid can be limited by stopping the divisional addition of pantothenic acid without depending on the medium exchange. It is preferable to limit the amount of pantothenic acid during the exponential growth phase. For example, in baker's yeast, a culture solution cultured to a logarithmic growth phase or a stationary phase is inoculated to a nutrient medium at 2% and shake-cultured at 30 ° C. for 8 to 16 hours. The body is obtained.

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

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

By heating the culture containing γ-glutamylcysteine or a fraction thereof, cysteine can be released from γ-glutamylcysteine.
The preparation of the yeast extract and the like may be performed in the same manner as the preparation of a normal yeast extract. The yeast extract may be obtained by treating a yeast cell that has been extracted with hot water, or a digested yeast cell that has been treated.

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

  The food or drink is produced by mixing a culture containing γ-glutamylcysteine or cysteine or a fraction thereof with a food or drink raw material and processing the mixture into a food or drink. The food and drink of the present invention can be produced by the same method using the same raw materials as ordinary food and drink except for using the culture or the fraction. Examples of such raw materials include rice, barley, corn starch and the like for alcoholic beverages, flour, sugar, salt, butter, yeast for fermentation and the like for bread foods, and soybean and wheat for fermented food seasonings.

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

[Example 1]
<1> Acquisition of yeast having reduced glutathione synthase activity A commercially available diploid Saccharomyces cerevisiae used for food applications was sporulated in a conventional manner. A haploid yeast strain YN0001 (MATα) was obtained from the formed spores by a random pore method. The YN0001 strain was subjected to mutation treatment by EMS to obtain a mutant YN0002 strain (MATα) having a reduced glutathione content. Four-molecule analysis confirmed that the GSH2 gene of the YN0002 strain was mutated. Specifically, the glycine residue at position 387 of the encoded protein was replaced with an aspartic acid residue. In addition, a mutant strain YN0003 (MATa) having a reduced glutathione content was also obtained.

  The mutation treatment was performed under such conditions that the mortality was 90%. The YN0001 strain was cultured with shaking at 30 ° C. for 1 day in 50 ml of YPD medium, and yeast cells were collected. The yeast cells were washed three times with a 0.2 M sodium phosphate buffer (pH 7.5). The yeast cells were suspended 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 shaken at 30 ° C for 90 minutes. Culture was continued. To this suspension was added 10 ml of 10% sodium thiosulfate (filter sterilized), and the mixture was left at room temperature for 10 minutes to neutralize the mutagen. The yeast cells were collected and washed with a 0.2 M sodium phosphate buffer (pH 7.5).

The YN0001 strain and the YN0002 strain were each inoculated in a YPD medium and cultured at 30 ° C. with shaking. The culture product was inoculated into SD medium at 2% and cultured with shaking at 30 ° C. The intracellular glutathione content during 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 The haploid yeast YN0001 strain (MATα) was mutated by EMS in the same manner as described above, and a mutant AJ14819 strain (MATα) in which the expression of the MET25 gene was not suppressed by methionine was obtained. I got it. This strain has been granted a private number AJ14819, and deposited on September 11, 2002, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (1-1-1, Higashi 1-1, Tsukuba, Ibaraki, Japan 305-8566, Japan) ) And assigned accession number FERM P-19007. Further, this strain was transferred to an international deposit based on the Budapest Treaty on October 1, 2003, and given the accession number FERM BP-08502. Whether or not the expression of the MET25 gene 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, mutants were selected as follows. The mutated yeast was spread on a YPD agar medium. Spreading was performed so that about 100 strains of yeast grew on the agar medium. The yeast grown on the YPD medium was replicated on a selenium-containing medium and a selenium-free medium (agar medium described in DOMINIQUE et al.). A yeast strain that does not grow on a medium containing selenium but grows on a medium not containing selenium was selected.

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

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

<3> Obtaining yeast having a requirement for calcium pantothenate The haploid yeast strain YN0001 (MATα) was subjected to mutation treatment by EMS in the same manner as described above. In order to obtain a calcium pantothenate-requiring yeast from the mutated yeast, the yeast was cultured at 30 ° C. for 2 hours in a medium not containing calcium pantothenate and to which nystatin (10 μg / ml) was added. Was spread on a YPD agar medium. The grown mutant yeast 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 be grown on the former agar medium, but on the latter agar medium. Then we selected yeasts that could grow. In this way, yeast Pa0001 strain (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, the AJ14819 strain and the Pa0001 strain are conjugated. A diploid was obtained. The obtained diploid was sporulated, and a haploid yeast MP strain (MATa) having a mutant MET30 gene and exhibiting a requirement for calcium pantothenate was obtained by a random pore method. Next, the YN0002 strain and the MP strain were conjugated to obtain a diploid. The obtained diploid is sporulated, and haploid yeasts GMP-1 (MATα), GMP-2 (GMP-1) having a mutant GSH2 gene and a mutant MET30 gene and exhibiting a requirement for calcium pantothenate are obtained by a random pore method. MATa) strain. The GMP-1 strain and the GMP-2 strain were conjugated to obtain a diploid yeast GMP strain.

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

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

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

[Example 2] Production of γ-glutamylcysteine by GMP strain and GM strain
Each of the GM strain and the GMP strain was inoculated in a YPD medium and cultured at 30 ° C. with shaking. The culture was inoculated into a medium containing 0.4 mg / dl of calcium pantothenate and cultured at 30 ° C. with shaking. The cells were collected during the logarithmic growth phase, and inoculated in 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 content of γ-glutamylcysteine per dried yeast cells was measured with time. FIG. 2 shows the results.

  As a result, it was shown that in the GMP strain, the γ-glutamylcysitene content per dry yeast cell increases as the culture is continued in a state in which the calcium pantothenate is deficient.

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

<6> Preparation of cassette for disrupting glutathione synthase gene First, a cassette for disrupting glutathione synthase gene was prepared as follows.
Using pAUR123 vector (Takara Shuzo code3602) cut with KpnI as a template, PCR was performed using GSH2-AUR1-CF (SEQ ID NO: 11) and GSH2-AUR1-CR (SEQ ID NO: 12) as primers. 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, the glutathione synthase gene can be disrupted using the same product. is there. The PCR conditions are as follows.

1 μl of pAUR123 cut with Kpn I
10 × PCR Buffer 5μl
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 synthase gene disruption cassette prepared as described above, the glutathione synthase gene of the GMP strain was disrupted as follows. That is, the GMP strain was cultured in the YPD medium, and collected during the logarithmic growth phase. The cells washed twice with a 1 M sorbitol solution were suspended in a solution having the following composition, and allowed to stand at 5 ° C for 1 hour.
Composition: 0.1 M LiCl
10 mM DTT
10 mM Tris-HCl (pH7.5)
1 mM EDTA

Then, it was washed twice with a 1 M sorbitol solution. The thus-prepared bacterial cells were mixed with the PCR product prepared as described above, and electroporation was performed. ("Bio Manual Series 10 Gene Experiment Using Yeast" First Edition, Yodosha) Thereafter, the mixture was inoculated into a 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) as a selection marker, and cultured at 30 ° C. for 3 days. (Minimum inhibitory concentration of GMP strain against Aureobasidin A is 0.05 μg / ml) The growing colonies are spread on YPD agar medium containing 0.2 μg / ml Aureobasidin A, and resistant to Aureobasidin A. The colonies that have been obtained were selected. Thus, AJ14861 strain was obtained. This strain has been granted a private number AJ14861 and, as of November 19, 2003, the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (〒305-8566
It has been deposited internationally under the Budapest Treaty at 1-1-1 Higashi, 1-chome, Tsukuba, Ibaraki, Japan, and is assigned the accession number FERM BP-08553.

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

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

  INDUSTRIAL APPLICABILITY The present invention provides a yeast showing reduced or eliminated glutathione synthetase activity, derepression of MET25 gene expression, and showing pantothenic acid requirement. By culturing the yeast of the present invention under suitable conditions, a yeast culture containing high content of γ-glutamylcysteine is provided. The yeast and the yeast culture solution of the present invention can be used for producing γ-glutamylcysteine-containing food or drink or cysteine-containing food or drink.

The figure which shows the time-dependent change of intracellular (gamma) -glutamylcysteine content when the yeast (GMP strain) of this invention is culture | cultivated in the medium to which calcium pantothenate (PaCa) is added or not. The figure which shows the comparison of (gamma) -glutamylcysteine content of GM strain and GMP strain. The figure which shows the time-dependent change of intracellular (gamma) -glutamylcysteine content when the yeast (AJ14861 strain) of this invention is cultured in the medium to which calcium pantothenate is added or not.

Claims (12)

Has a γ-glutamylcysteine-producing ability, has a requirement for pantothenic acid, and when cultured in a medium in which the amount of pantothenic acid is restricted, the γ-glutamylcysteine content per dry yeast cell increases with time. Yeast to do. The yeast according to claim 1, wherein the yeast is modified so that intracellular glutathione synthase activity is reduced or eliminated. The yeast according to claim 1 or 2, wherein the yeast is modified so that expression of the MET25 gene is derepressed. The yeast according to claim 3, wherein expression of the MET25 gene is desuppressed by retaining a mutant MET30 gene having a mutation in which serine at position 569 of the encoded protein is substituted with another amino acid other than serine. The yeast according to claim 4, wherein the other amino acid is phenylalanine. The yeast according to any one of claims 1 to 5, which belongs to the genus Saccharomyces. A step of culturing the yeast according to any one of claims 1 to 6 in a medium containing a sufficient amount of pantothenic acid to grow the yeast, and culturing the yeast in a medium in which the amount of pantothenic acid is restricted, A method for producing yeast having accumulated γ-glutamylcysteine, comprising a step of increasing the content of γ-glutamylcysteine in the body. A culture obtained by culturing the yeast according to any one of claims 1 to 6 under suitable conditions, or a fraction of the culture containing γ-glutamylcysteine, or a cysteine produced by heat treatment. A food or drink comprising a culture or a fraction of the above. The food or drink according to claim 8, wherein the food or drink is an alcoholic beverage, a bread food, or a fermented food seasoning. A yeast extract produced using a culture obtained by culturing the yeast according to any one of claims 1 to 6 under suitable conditions. A culture or a fraction thereof obtained by culturing the yeast according to any one of claims 1 to 6 under suitable conditions, or the culture or the fraction subjected to the heat treatment, and mixed with a food or drink raw material. And a process for producing γ-glutamylcysteine or a cysteine-containing food or beverage. A yeast in which expression of the MET25 gene is desuppressed by retaining a mutant MET30 gene having a mutation in which serine at position 569 of the encoded protein is substituted with phenylalanine.

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