JP5315600B2 - Y-glutamylcysteine-producing yeast and use thereof - Google Patents

Description

  The present invention relates to a yeast that produces γ-glutamylcysteine and a method for producing a yeast that accumulates γ-glutamylcysteine. γ-Glutamylcysteine is useful in foods and drinks, pharmaceuticals, chemical products, particularly in the seasoning field.

  At present, sulfur-containing compounds are used in a wide range of fields such as foods, pharmaceuticals, and chemical products because of their high efficacy. For example, glutathione (GSH), which is a tripeptide consisting of glutamic acid, cysteine, and glycine, is known to have a medicinal effect as a pharmaceutical product. Currently, GSH preparations as pharmaceuticals are positioned as antidotes and ophthalmic agents, and are used to prevent various intoxications, chronic liver diseases, side effects of anticancer drugs and damage caused by radiation therapy, skin diseases, cataracts and corneal damage. It is used (Non-patent Document 1). Moreover, it is known that glutathione is a substance that imparts a rich taste to food as a food application (Non-patent Document 2), and a yeast extract containing a high amount of glutathione is used as a seasoning.

  Recently, the usefulness of γ-glutamylcysteine, which is a precursor of glutathione and is a dipeptide consisting of glutamic acid and cysteine, is being reported. For example, it has been reported that a good flavor composition can be obtained by adding a saccharide to γ-glutamylcysteine and subjecting it to a heat treatment (Patent Document 1). Furthermore, it is reported that cysteine widely used as a flavor improving material is produced by heating or enzymatic treatment of γ-glutamylcysteine (Patent Document 2). In addition to this, a roast meat flavor-like seasoning can be obtained by adding saccharides to yeast extract containing 2 to 20% by weight of a sulfur-containing compound such as glutathione, cysteine, and glutamylcysteine, followed by heat treatment in the absence of fatty acids. (Patent Document 3).

  However, the usefulness of γ-glutamylcysteine has only begun to become clear compared to glutathione, and means for efficiently producing it have not been studied much. The prior knowledge to date is as follows.

  For example, the present inventors have reported that yeast having a high content of γ-glutamylcysteine was obtained by destroying or weakening glutathione synthetase that biosynthesizes glutathione using γ-glutamylcysteine and glycine as substrates. (Patent Document 2, Patent Documents 4 to 7). These techniques aim to suppress the metabolism of γ-glutamylcysteine to glutathione and to make yeast contain a high amount of γ-glutamylcysteine as a precursor, but the content of γ-glutamylcysteine is 1-2. It is weight percent and there is room for further efficiency.

  In addition, the present inventors cultured γ-glutamylcysteine-producing yeast having pantothenic acid requirement in a pantothenic acid-containing medium and then in a pantothenic acid-free medium, whereby γ- A technique for increasing glutamylcysteine is disclosed (Patent Document 8). In this document, yeast containing less than 4% of γ-glutamylcysteine is shown, which is considered to be a means for efficiently producing γ-glutamylcysteine. However, it is necessary to control the amount of pantothenic acid during the culturing in order for the yeast to stably contain γ-glutamylcysteine at a high content.

Furthermore, the present inventors retain the MET30 gene product in which the serine residue at position 569 of the protein encoded by the gene is mutated to another amino acid residue, thereby increasing the amount of γ-glutamylcysteine accumulated and the growth of yeast. It has been reported that both speeds can be satisfied (Patent Document 9).

On the other hand, studies on cerulenin resistance in yeast have been conducted, and it has been reported that at least the YAP1 gene (also known as PDR4 gene) (non-patent document 3) and the FAS2 gene (non-patent document 4) are involved in cerulenin resistance. . The YAP1 gene has been reported to be a transcription factor for the GSH1 gene (γ-glutamylcysteine synthase gene) and the GSH2 gene (glutathione synthase gene) (Non-patent Document 5). It has not been confirmed how the content of GSH or γ-glutamylcysteine in the body changes. Moreover, although the FAS2 gene is a fatty acid synthase gene, cerulenin resistance is not necessarily linked to a change in the content of γ-glutamylcysteine, because cerulenin resistance is imparted to yeast by mutation of the gene. In addition, there is a report (Patent Document 10) that cerrenin-resistant yeast is used for sake production, but there is no report that cerulenin resistance is imparted to yeast in which glutathione synthetase activity is reduced or eliminated.
Japanese Patent No. 2830415 WO 00/30474 Patent No. 2903659 WO01 / 090310 JP2003-159048 JP2003-159049 WO2003 / 080832 JP 2004-201677 A JP 2004-113155 A JP-A-8-23594 Protein Nucleic Acid Enzyme 1988-7 VOL.33 NO.9 ISSN 003909450 Special Issue “Epoc of Glutathione Research” p1626 Y. Ueda et al., Biosci. Biotech. Biochem., 61, 1977-1980 (1997) Nobusige Nagazawa et al., Journal of Fermentation and Bioengineering Vol.76, No 1, 60-63, 1993 Kazuo Aritomi et al., Biosci. Biotechnol. Bochem., 68 (1), 206-214, 2004 Sugiyama et al., Journal of Biological Chemistry (2000), 275 (20), 15535-15540

  An object of the present invention is to provide a yeast that efficiently produces γ-glutamylcysteine, and to provide a method for producing a yeast that accumulates γ-glutamylcysteine.

  As a result of intensive studies, the present inventors have found that yeast having γ-glutamylcysteine production ability and cerulenin resistance can stably and efficiently produce γ-glutamylcysteine. It came to be completed.

That is, the present invention is as follows.
(1) Yeast having γ-glutamylcysteine production ability and cerulenin resistance.
(2) The yeast having resistance to 10 μM or more of cerulenin.
(3) The yeast modified so that intracellular glutathione synthase activity is reduced or eliminated.
(4) The yeast belonging to the genus Saccharomyces.
(5) The yeast as described above, which is Saccharomyces cerevisiae.
(6) A method for producing a yeast in which γ-glutamylcysteine is accumulated, wherein the yeast is cultured in a medium.
(7) A culture obtained by culturing the yeast in a medium or a fraction thereof, or the heat-treated culture or fraction, mixed with a raw material for food and drink, and processed into a food or drink. A method for producing γ-glutamylcysteine or a cysteine-containing food or drink.

  The yeast of the present invention can produce γ-glutamylcysteine efficiently. In particular, in a preferred form, when cultivated at a high density, the increase in the amount of γ-glutamylcysteine accumulated over time is better than that of yeast that does not have cerulenin resistance.

<1> Yeast of the Present Invention The yeast of the present invention is a yeast having the ability to produce γ-glutamylcysteine and having cerulenin resistance.
The yeast of the present invention can be obtained by imparting cerulenin resistance to a yeast having the ability to produce γ-glutamylcysteine. The yeast of the present invention can also be obtained by imparting γ-glutamylcysteine-producing ability to yeast having cerulenin resistance, or increasing the γ-glutamylcysteine-producing ability of yeast having cerulenin resistance.

  In the present invention, “having the ability to produce γ-glutamylcysteine” refers to having the ability to produce γ-glutamylcysteine and accumulate it in the cells, and preferably a larger amount of γ-glutamyl than the yeast wild strain. Accumulation of cysteine in the fungus body. More preferably, when the yeast of the present invention is cultured in a minimal medium, 1% by weight or more, more preferably 1.4% by weight or more of γ-glutamylcysteine is accumulated per dry yeast cell in the logarithmic growth phase. Say. Moreover, it is preferable that the glutathione accumulation amount per dry yeast cell is 0.1% by weight or less.

  The yeast of the present invention is not particularly limited as long as it has the ability to produce γ-glutamylcysteine. Examples include the genus Saccharomyces such as Saccharomyces cerevisiae, the genus Candida such as Candida utilis, the genus Pipia such as Pipia pastoris, and the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Among the yeasts described above, techniques for improving γ-glutamylcysteine production ability, for example, methods for destroying or weakening glutathione synthase, and those in which the γ-glutamylcysteine synthase gene has been cloned, It is suitable for constructing the yeast of the present invention. Examples of such yeast include Saccharomyces cerevisiae and Candida utilis.

  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.
As long as the mutation includes at least (1) or (2), it may be used alone or in any combination, but the combination of (1) and (3) and the combination of (2) and (3) are preferred.

  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 is preferably a protein having homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more with respect to the entire amino acid sequence of the GSH2 product. It may be DNA that encodes.

  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 Saccharomyces cerevisiae GSH2 gene is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, with the DNA having the base sequence shown in SEQ ID NO: 1. Particularly preferred is DNA having a homology of 95% or more. 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.

  Moreover, as a method for enhancing γ-glutamylcysteine synthetase activity in yeast cells, a plasmid into which the same enzyme gene (GSH1) (for example, γ-glutamylcysteine synthetase gene of Saccharomyces cerevisiae: GenBank Accession No. D90220) is inserted. In yeast to increase the number of copies of the gene in the cell, or to replace the promoter of the γ-glutamylcysteine synthase gene on the chromosome with a strong transcription promoter (Yasuyuki Ohtake et al., Bioscience and Industry, 50, No. 10, 989-994, 1992). As a γ-glutamylcysteine synthetase gene, a gene encoding glutathione synthetase of Candida utilis is also known (Japanese Patent Application Laid-Open No. 2005-073638) and can be used in the present invention.

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)).
Also, conferring MNNG resistance (Japanese Patent Laid-Open No. 2003-159048), retaining a mutant MET30 gene (Japanese Patent Laid-Open No. 2004-113155), derepressing MET25 gene expression (Japanese Patent Laid-Open No. 2004-201677), pantothenic acid requirement By imparting γ (JP-A 2004-201677), it is possible to improve the ability of yeast to produce γ-glutamylcysteine, or to impart favorable properties to yeast.

  The yeast of the present invention is a yeast strain that has the above-mentioned ability to produce γ-glutamylcysteine and has been modified to have cerulenin resistance. In the present invention, “having cerulenin resistance” means that a non-modified yeast strain, for example, a wild strain, or a parent strain used for obtaining the yeast of the present invention can grow on a medium containing cerulenin at a concentration that cannot grow. In addition, yeast that grows faster than non-modified strains in a medium containing cerulenin at a concentration that allows growth of non-modified strains of yeast, such as wild-type or parent strains, has cerulenin resistance. Specifically, for example, when a yeast suspension is spread on an agar medium such as a YPD plate containing cerulenin and cultured at a suitable temperature, for example, 30 ° C., the unmodified strain does not form a colony, and the modified strain does not form a colony. The modified strain has cerulenin resistance. Here, the “colony” refers to a colony that can be confirmed with the naked eye.

[YPD medium composition]
Glucose 2%
Peptone 2%
Yeast extract 1%
(pH 5.0)
(In the case of agar medium, agar 2%)

  When the yeast is Saccharomyces cerevisiae, more specifically, the yeast is inoculated in a YPD medium, and a culture solution obtained by culturing at a temperature optimum for growth, for example, 30 ° C. for 3 days, is placed on a YPD plate containing a certain concentration of cerulenin. If the colonies form within 5 days when spread and cultured at the growth indication temperature, the yeast is resistant to that concentration of cerulenin. Saccharomyces cerevisiae that has not been modified to confer cerulenin resistance, such as the AJ14800 strain described in the Examples, hardly forms colonies within 3 days on YPD plates containing at least 7 μM cerulenin. The yeast of the present invention preferably has resistance to 10 μM or more, more preferably 15 μM or more, particularly preferably 20 μM or more.

  The yeast having cerulenin resistance is, for example, a strain that forms a colony by mutating a wild strain of yeast or a yeast having the ability to produce γ-glutamylcysteine, inoculating agar medium containing cerulenin at a concentration at which the wild strain cannot grow. It can be acquired by selecting. Further, the operation of spreading the culture solution of the strain thus obtained on an agar medium containing cerulenin again and selecting a strain having colony forming ability may be repeated twice or more. Further, the obtained yeast having cerulenin resistance may be subjected to mutation treatment again and the selection operation may be performed. Mutation treatment is the same as described for yeast in which glutathione synthetase activity has been reduced or eliminated. For example, UV irradiation, or N-methyl-N-nitrosoguanidine (NTG), ethylmethanesulfonate (EMS ), Treatment with mutagen such as nitrous acid and acridine.

  Moreover, the yeast having cerulenin resistance can be obtained as a natural mutant strain by culturing in a medium containing cerulenin in the same manner as described above from a wild strain without performing a mutation treatment.

The yeast 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 by selecting a strain that produces a γ-glutamylcysteine by mutating the yeast having ploidy, or by using the same for breeding a γ-glutamylcysteine producing strain. Production of γ-glutamylcysteine having different mating types by selecting a strain that produces γ-glutamylcysteine by sporulating the diploid yeast obtained by joining somatic yeast and wild type yeast haploid It can be obtained by joining two 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.

  As described above, yeast having cerulenin resistance efficiently produces γ-glutamylcysteine. Therefore, a strain that efficiently produces γ-glutamylcysteine can be screened or bred using cerulenin resistance as an index.

<2> Utilization of Yeast of the Present Invention By culturing the yeast of the present invention in a medium, a yeast in which γ-glutamylcysteine is accumulated in the microbial cells and a culture thereof can be produced.

  The medium used for culturing the yeast of the present invention is not particularly limited as long as the yeast of the present invention grows well and efficiently produces γ-glutamylcysteine, and is usually a medium that is industrially used. Can be used. If necessary, necessary nutrients are added to the medium according to the characteristics of the yeast to be used.

  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 24 hours with shaking, the bacteria in the logarithmic growth phase The body is obtained.

  When the target γ-glutamylcysteine 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.

In the present invention, high-density culture can be employed as a culture form. In the present invention, “high density culture” means a cell density in liquid culture of 1 × 10 ⁸ cells / ml or more, preferably 2.5 × 10 ⁸ cells / ml or more, more preferably 4 × 10 ⁸ cells. It means more than cells / ml. Alternatively, the case where the dry cell weight in liquid culture is 1 g / 100 ml or more, preferably 2.5 g / 100 ml or more, more preferably 4 g / 100 ml or more is also included in the high-density culture. It is not necessary for the whole culture period to be high-density culture, as long as the cell density is reached at least in part of the period. For example, the cell density is preferably reached at 1% or more, preferably 5% or more, more preferably 10% or more of the culture period.

  In the yeast of the present invention, the γ-glutamylcysteine content per cell increases with time in high-density culture. Although some conventional γ-glutamylcysteine yeasts that do not have cerulenin resistance also increase in γ-glutamylcysteine content during high-density culture, the increase in γ-glutamylcysteine content has ceased over the course of the culture period. Tend to be. In contrast, the yeast of the present invention, in a preferred form, has a significant increase in γ-glutamylcysteine content compared to conventional yeast.

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 beverages and foods such as 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. The present invention is not limited to the following examples.

[Reference Example 1] Molecular breeding of high γ-GC yeast by high expression of YAP1 gene A strain that highly expresses YAP1 gene was bred from γ-glutamylcysteine-producing yeast strain AJ14800.
Strain AJ14800 was obtained by joining strain AJ14809, which had weakened glutathione synthetase activity derived from wild-type Saccharomyces cerevisiae, and Saccharomyces cerevisiae wild strain AJ14810 (MATa), which had weakened glutathione synthetase activity and γ-glutamylcysteine. It is a diploid yeast (gsh2 / gsh2) obtained by selecting a strain that produces A (Japanese Patent Laid-Open No. 2003-159048, US Patent Publication 2004-0214308A1).

  On October 1, 2001, AJ14809 shares were transferred to FERM P-18546 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba City, Ibaraki 305-5466, Japan). And was transferred to an international deposit under the Budapest Treaty on November 1, 2002, and was given the deposit number FERM BP-8228. On October 1, 2001, AJ14810 shares were registered with FERM P on the patent biological deposit center of the National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1-chome, Tsukuba City, Ibaraki 305-5466, Japan). It was deposited with a deposit number of -18547, transferred to an international deposit under the Budapest Treaty on November 1, 2002, and assigned the deposit number FERM BP-8229.

First, based on a conventional method, a vector (YAP1 / pAUR123) that highly expresses the YAP1 gene was prepared as follows. The AJ14800 strain was cultured in YPD medium, and chromosomal DNA was recovered during the logarithmic growth phase. Using the collected chromosomal DNA as a template, the ORF region of the YAP1 gene was amplified using Pyrobest DNA polymerase (Takara Bio Inc.), primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4). Since the SmaI site or XbaI site is designed to be added to the end of the primer, the SmaI site is added to the N-terminal side of the PCR product and the XbaI site is added to the C-terminal side. This PCR product is purified, and the purified product and yeast protein expression shuttle vector pAUR123
(Takara Bio Inc.) were cleaved with restriction enzymes SmaI and XbaI, respectively, and ligated using TaKaRa DNA ligation kit ver. 2 (Takara Bio Inc.). Escherichia coli JM109 was transformed with the ligated product to obtain a transformant. A transformant carrying the target expression vector was selected, and a large amount of YAP1 / pAUR123 vector was prepared from the obtained strain.

  The AJ14800 strain was transformed with the YAP1 / pAUR123 vector and pAUR123 prepared as described above. Specifically, the following operation was performed. AJ14800 strain was cultured in 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

  Thereafter, the cells were washed twice with 1M sorbitol solution. The thus prepared bacterial cells were mixed with the PCR product prepared as described above, and electroporation was performed (“Biomanual Series 10 Gene 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 was spread on a YPD agar medium containing 0.2 μg / ml of aureobasidin A (Takara Shuzo code9000), which is a selection marker for the pAUR123 vector, and cultured at 30 ° C. for 3 days. The minimum inhibitory concentration of AJ14800 strain against aureobasidin A is 0.05 μg / ml. The emerged colonies were applied to a YPD agar medium containing 0.2 μg / ml aureobasidin A, and colonies having resistance to aureobasidin A were selected. From these colonies, transformants carrying the target vector were selected to obtain AJ14800 / YAP1 / pAUR123 strain and AJ14800 / pAUR123 strain, respectively.

The above two strains were inoculated into YPD medium (4 ml, test tube) containing 0.2 μg / ml aureobasidin A and cultured at 30 ° C. with shaking. The preculture was seeded 2% in SD medium (50 ml, Sakaguchi flask) containing 0.2 μg / ml aureobasidin A and cultured at 30 ° C. with shaking. Bacteria were collected in the logarithmic growth phase (16 hours after seeding), and the γ-glutamylcysteine content per dry cell was measured. The AJ14800 / YAP1 / pAUR123 strain was 1.7% by weight, and the AJ14800 / pAUR123 strain was 1.4% by weight. there were.
From the above, it was confirmed that by increasing the expression level of the YAP1 gene, the content of γ-glutamylcysteine in the microbial cells increased.

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

  In addition, YPD agar medium containing various concentrations of cerulenin was prepared, and the minimum growth inhibitory concentration of cerulenin for the aforementioned two strains was measured. Specifically, each strain is cultured with shaking in a YPD liquid medium at 30 ° C., 3 μl of a culture solution that has reached a stationary phase is spotted on an agar medium containing cerulenin, and cultured at 30 ° C. for 3 days to form a colony. The growth of bacterial cells was evaluated based on the presence or absence of. As a result, the minimum growth inhibitory concentration was 9 μM for the AJ14800 / YAP1 / pAUR123 strain and 7 μM for the AJ14800 / pAUR123 strain. From this, it was confirmed that cerulenin resistance was increased by improving the expression level of the YAP1 gene.

[Example 1]
(Acquisition of cerulenin resistant strain)
In the same manner as in Reference Example 1, the minimum growth inhibitory concentration of AJ14800 strain against cerulenin was determined to be 7 μM.

  Next, AJ14800 strain was inoculated into YPD medium and cultured at 30 ° C. for 3 days. The culture was spread on a YPD plate, a YPD plate containing 7 μM cerulenin, and a YPD plate containing 10 μM cerulenin to obtain a cerulenin resistant strain. The incidence of resistant strains was assessed by comparing the number of colonies formed on the YPD plate and on the YPD plate containing cerulenin, one in 100 strains on the 7 μM plate and 2000 on the 10 μM plate. There was one share per share.

[Example 2] Classification of cerulenin-resistant strains The cerulenin-resistant strains obtained in Example 1 were examined for the degree of cerulenin resistance (growth inhibitory concentration).

Serulenin resistance 7 μM or more and less than 10 μM: 420 strains Cellulinin resistance 10 μM or more: 613 strains

  The γ-glutamylcysteine content of these strains was measured, and the strains with higher γ-glutamylcysteine content and better growth than the parent strain were selected. The following number of strains were selected for each group: It was done.

Serlenin resistance 7 μM or more and less than 10 μM: 8 strains Cellulenin resistance 10 μM or more: 18 strains

  As the best strains from each group, 7-46 strains (selected from a group having a cerrenin resistance of 7 μM or more and less than 10 μM) and 10-29 strains (selected from a group having a cerulenin resistance of 10 μM or more) were selected. When these strains were cultured in SD medium and the γ-glutamylcysteine content in the logarithmic growth phase was measured, AJ14800 strain was 1.50%, 7-46 strain was 1.68% (1.1 times the parent strain), 10-29 strain Was 1.80% (1.2 times the parent strain).

  When the nucleotide sequences of YAP1 gene of AJ14800 strain and 10-29 strain were determined, no difference was found. From this, it was speculated that the cause of the increase in the γ-glutamylcysteine content of the 10-29 strain was other than the mutation of the YAP1 gene. The 10-29 strain could grow on a plate of cerulenin 25 μM.

[Example 3] γ-glutamylcysteine content during high-density culture
The AJ14800 strain, 7-46 strain, and 10-29 strain were cultured at high density using a minijar, and the γ-glutamylcysteine content was measured.

  One seed of each strain was inoculated into 50 ml of YPD medium (500 ml Sakaguchi flask), and pre-cultured by shaking at 30 ° C. and 120 rpm. Next, 5 ml of the pre-seed culture was inoculated into 500 ml of YPD medium (conical flask with 2 L baffle) and cultured by shaking at 30 ° C. and 120 rpm for 48 hours. 200 ml of the seed culture was inoculated into the main medium and cultured under the following conditions using a minijar.

  Zones A, B and C were each separately autoclaved (120 ° C for 20 minutes) and then mixed. Yeast Extract was added so that the total amount of nitrogen was 0.23 g / dl. Culturing was performed at a culture temperature of 30 ° C., an aeration rate of 1/1 VVM, and a stirring rate of 800 rpm. A 46.7% (W / V) glucose solution was used as a sugar feed solution, and the feed was started when the pH of the culture broth reached 6.0. The pH during the cultivation was controlled to pH 6.0 using ammonia gas after the sugar feed was started. The absorbance (OD660nm) of the culture solution was measured every 3 hours after the sugar feed, and glucose was added so as to be 0.2 μg-glucose / dry cell weight (DCW) g / hr. As a conversion formula, DCW = 0.22 × OD660 nm was used.

  The results of measuring the γ-glutamylcysteine content at regular intervals are shown in FIG. The results of measuring the dry cell weight (DCW) and γ-glutamylcysteine content (γ-GC) after 48 hours of culture are shown below.

AJ14800 strain: γ-GC 2.10% DCW3.65g / dl
7-46 stock: γ-GC 2.66% DCW3.23g / dl
10-29 strain: γ-GC 3.48% DCW 3.16g / dl

  In the 10-29 strain, the γ-glutamylcysteine content was remarkably increased over time. In addition, the γ-glutamylcysteine content of other strains selected from the group having a degree of cerulenin resistance of 10 μM or more markedly increased with time under high-density culture.

[Example 4] Further reduction study of glutathione synthetase activity of strain 10-29 Next, the effect of further reducing glutathione synthetase activity by disrupting the glutathione synthetase gene of strain 10-29 was examined. investigated. The glutathione synthase gene of 10-29 strain was disrupted by the same method as described in Example 3 of JP-A-2004-201677. The obtained strain is assigned a private number AJ14892, and has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of August 25, 2006, with a deposit number of FERM P-21007.

The AJ14892 strain obtained as described above was subjected to high-density culture in the same manner as in Example 3. A 60% glucose solution was used as the sugar feed solution, and the sugar feed was started when the pH of the broth reached 6.0. The sugar feed flow rate was set to 20 μl / min until 0.5 hours after the start of sugar feed, 75 μl / min until 5.9 hours, and 135 μl / min thereafter.
The results of measuring the γ-glutamylcysteine content at regular intervals are shown in FIG. Further, the dry cell weight (DCW) and γ-glutamylcysteine content (γ-GC) at 48 hours after culture were as follows.

γ-GC: 4.12%
DCW: 5.02g / dl

  Similar to the strain 10-29, the γ-glutamylcysteine content of this strain increased with time in the high-density culture. From this result, it was found that the content of γ-glutamylcysteine increased with time even when the glutathione synthetase activity was further decreased.

The figure which shows the relationship between the culture | cultivation time of yeast, and (gamma) -glutamylcysteine content. The figure which shows the relationship between the culture | cultivation time of yeast, and (gamma) -glutamylcysteine content.

Claims (6)

Under the high density culture, which has the ability to produce γ-glutamylcysteine and accumulate it in the microbial cells, and has a resistance to cerulenin of 10 μM or more, and the cultivated cell weight in liquid culture is 2.5 g / 100 ml or more. Yeast with increased γ-glutamylcysteine content. The yeast according to claim 1 , which has been modified so that intracellular glutathione synthetase activity is reduced or eliminated. The yeast according to claim 1 or 2 , which belongs to the genus Saccharomyces. The yeast according to claim 3 , which is Saccharomyces cerevisiae. A method for producing a yeast in which γ-glutamylcysteine is accumulated, wherein the yeast according to any one of claims 1 to 4 is cultured in a medium. A step of culturing the yeast according to any one of claims 1 to 4 in a medium to obtain a culture, and the obtained culture or a fraction thereof, or the heat-treated culture or fraction, The manufacturing method of (gamma) -glutamylcysteine or a cysteine containing food / beverage product which has the process mixed with food / beverage product raw materials and processed into food / beverage products.

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