JP7620382B2 - Glutathione stress resistant yeast - Google Patents

Description

The present invention relates to a method for breeding yeast that is resistant to glutathione stress.

Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine, and is a biologically active molecule that is widely conserved, for example, from Escherichia coli to humans.
Glutathione has attracted attention in the fields of medicine, food and cosmetics because it exhibits various functionalities such as antioxidant activity, immunostimulating activity, detoxification and liver function improving activity (Patent Document 1).
The functions of glutathione in cells are diverse, including the formation and maturation of iron-sulfur cluster proteins, elimination of reactive oxygen species, amino acid metabolism, and protein folding through homeostasis of redox balance (Non-Patent Document 1).

With the recent development of live cell imaging technology, it has become clear that the ratio of reduced glutathione to oxidized glutathione varies greatly from organelle to organelle, and that the spatial balance of glutathione is highly regulated. For example, the ratio of reduced glutathione to oxidized glutathione is 3,000:1 in the cytoplasm and mitochondrial inner membrane, but it has been reported that it is 3:1 to 1:1 in the endoplasmic reticulum (Non-Patent Document 2). The reason why the endoplasmic reticulum shifts to an oxidative environment is that secretory proteins and membrane proteins are properly folded by the action of protein disulfide isomerase and the like via glutathione (Non-Patent Document 1).

Currently, the mainstream industrial production method for glutathione is the fermentation method using yeast, and glutathione accumulates in yeast cells. It is known that the high physiological activity of glutathione as described above causes cytotoxicity (glutathione stress) when the degree of accumulation is excessive (Non-Patent Document 3).
Therefore, there is a report that, in order to reduce glutathione stress, glutathione was actively transported to the vacuole, thereby simultaneously improving glutathione accumulation and proliferation (Patent Document 2), and there is also a report that glutathione was excreted outside the cell (into the culture medium) to avoid cytotoxicity, thereby improving glutathione productivity (Non-Patent Document 4).
Regarding the cytotoxicity of glutathione in more detail, it has been reported that excessive accumulation of glutathione in yeast adversely affects protein folding in the endoplasmic reticulum and mitochondrial activity (Non-Patent Document 1).

However, factors that confer resistance to the cytotoxicity of glutathione have not been known in yeast or any other organisms. Therefore, attempts to improve glutathione productivity through the creation of glutathione stress-resistant yeast remain incomplete.

JP 2013-177188 A JP 2012-213376 A JP 2012-213361 A JP 2014-064472 A JP 2011-160739 A JP 2004-283125 A

Chitranshu Kumar et al. Glutathione revisited: a vital function in iron metabolism and ancillary role in thiol-redox control. The EMBO Journal, 30, p2044-2056 (2011). Michel B. Toledano et al. Functions and Cellular Compartmentation of the Thioredoxin and Glutathione Pathways in Yeast. ANTIOXIDANT and REDOX SIGNALING, 18 (13), p1699-1711 (2013) Srikanth CV, Vats P, Bourbouloux A et al. Multiple cis-regulatory elements and the yeast Sulfur regulatory network are required for the regulation of the yeast glutathione transporter, Hgt1p. Current Genetics, 47, p345-358 (2005) Kiriyama Kentaro, Kiyotaka Y. Hara, Akihiko Kondo, Extracellular glutathione fermentation using engineered Saccharomyces cerevisiae expressing a novel glutathione exporter. Appl Microbiol Biotechnol, 96(4), p1021-1027 (2012) Kassir Y. et al. The saccharomyces cerevisiae GSK-3 beta homologs. Current Drug Targets, 11, p1455-1465 (2006) Paula G. Bertram et al. The 14-3-3 proteins positively regulate rapamycin-sensitive signaling, Current Biology, 8, p1259-1267 (1998). Radcliffe P. et al. Deregulation of CLN1 and CLN2 in the Saccharomyces cerevisiae whi2 mutant, Yeast, 13(8), p707-715 (1997) Xiao, Y. and Mitchell.A.P., Shared roles of yeast glycogen synthase kinase 3 family members in nitrogen-responsive phosphorylation of meiotic regulator Ume6p. Molecular and Cellular Biology, 20, p5447-5453 (2000) Saul M. Honigberg and Kedar Purnapatre, Signal pathway integration in the switch from the meiotic cell cycle to meiosis in yeast. Journal of Cell Science, 116, p2137-2147 (2003) C. P. Hollenberg, P. Borst & E. F. J. Van Bruggen: Biochim. Biophys. Acta, 201(1), (1970) Tietze et al. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Analytical biochemistry, 27(3), p502-522, (1969) Aurelie Lardenois et al. The conserved histone deacetylase Rpd3 and its DNA binding subunit Ume6 control dynamic transcript architecture during mitotic growth and meiotic development.

In view of the above background, the present invention aims to: i) identify a factor that confers glutathione stress resistance to yeast; and ii) identify a biological process that confers glutathione stress resistance to yeast.
The objective of the present invention is to provide a method for producing a high-yield glutathione-producing yeast comprising the steps of:

In order to solve the above-mentioned problems, the inventors used a yeast chromosomal multicopy library to search for genes that suppress glutathione stress in multiple copies. As a result, they were able to find the gene RIM11 encoding the glycogen synthase kinase Rim11, the gene BMH1 encoding the 14-3-3 protein Bmh1, and the gene WHI2 encoding the protein Whi2, which down-regulates the G1 phase of cell division.
Furthermore, the inventors performed RNA sequencing and confirmed that the factor that endows yeast with glutathione stress resistance through increased expression of these genes is the change in the biological processes (a) and (b) below.
(a) Activation of the MAPK (Mitogen activated-protein kinase) pathway (b) Transition to the meiotic stage In addition, the present inventors focused on (a) glycogen synthase kinase Rim11, which functions downstream of the MAPK pathway. Using molecular genetic techniques, it was newly discovered that Ume6, known as a substrate phosphorylated by Rim11, and Rpd3 and Sin3, which cooperate with Ume6, function in conferring glutathione stress resistance to yeast. From this result, it was concluded that (a) the MAPK pathway, followed by the regulation of the epigenetic mechanism, is a factor in conferring glutathione stress resistance to yeast.

That is, the present invention provides:
(1) A yeast having improved glutathione stress resistance compared to a parent strain, which is obtained by transforming a parent strain with a gene belonging to the following biological process (a), (b) or (c):
(a) Mitogen activated-protein kinase (MAPK) pathway (b) meiosis (c) epigenetic mechanism

(2) A yeast having improved glutathione stress resistance compared to a parent strain, obtained by transforming a parent strain with DNA encoding any one of the following amino acid sequences (A) to (C):
(A) an amino acid sequence having 65% or more homology with the amino acid sequence of SEQ ID NO:1; (B) an amino acid sequence having 65% or more homology with the amino acid sequence of SEQ ID NO:2; (C) an amino acid sequence having 65% or more homology with the amino acid sequence of SEQ ID NO:3.

(3) A yeast according to (2) above, which is obtained by transforming a parent strain with DNA encoding an amino acid sequence having 65% or more homology to the amino acid sequence of SEQ ID NO:7, and which has improved glutathione stress resistance compared to the parent strain.

(4) (a) A yeast having improved glutathione stress resistance compared to a parent strain, obtained by transforming a parent strain with a gene belonging to the mechanism (I) below, which is a biological process that functions downstream of the MAPK pathway and transitions the cell division stage to somatic cell division, meiosis, or a pause in division (G1 arrest, G2 arrest) as appropriate in response to a change in the external growth environment.
(I) Epigenetic mechanisms

(5) A yeast having improved glutathione stress resistance compared to a parent strain, obtained by transforming a parent strain with DNA encoding any one of the following amino acid sequences (D) to (F):
(D) an amino acid sequence having 65% or more homology with the amino acid sequence of SEQ ID NO: 33; (E) an amino acid sequence having 65% or more homology with the amino acid sequence of SEQ ID NO: 34; (F) an amino acid sequence having 65% or more homology with the amino acid sequence of SEQ ID NO: 35

(6) A method for producing glutathione, comprising a step of culturing the yeast according to any one of (1) to (5).

This relates to:

According to the method of the present invention, it is possible to impart glutathione stress resistance to a host yeast against cytotoxicity induced by high accumulation of glutathione in yeast cells. By breeding the yeast that has acquired this trait, it is possible to produce a glutathione hyper-producing yeast.
The glutathione-rich yeast obtained according to the present invention has a proliferation rate equivalent to that of the wild type while containing a high amount of glutathione, and is therefore expected to contribute to improving the productivity in the industrial production of glutathione.

Schematic diagram of the mechanism by which glutathione stress resistance is conferred to yeast, as determined by transcriptome analysis. Plasmid map of the HGT1 gene strong expression cassette and schematic diagram of chromosomal homologous recombination The glutathione stress sensitivity was evaluated for the negative control (pRS426, library vector) and three mutants that showed glutathione stress resistance. The bacterial cell turbidity of the spots was _OD600 = 2.5, 0.25, 0.025, 0.0025, and 0.00025 from the left, and 10 μl of each was spotted. The samples were kept at 30°C for 3 days. Plasmid map of strong expression of the RIM11 gene and schematic diagram of chromosomal homologous recombination. Plasmid map of strong expression of the GSH1 gene and schematic diagram of chromosomal homologous recombination. Comparison of total glutathione content when each mutant was aerobically cultured in SD casamino acids medium until logarithmic growth phase or stationary phase (total glutathione content of BY4741 is set to 1). N number for each mutant = 4, two independent experiments. Comparison of growth ability of each mutant in SD casamino acid medium. N number of each yeast strain = 2, single test. The glutathione stress sensitivity was evaluated for the mutant Δume6, in which the gene encoding Ume6 was disrupted, and Δume6 HGT1. The bacterial cell turbidity of the spots was OD600 = 2.5, 0.25, 0.025, 0.0025, and 0.00025 (from the left), and 10 μl of each was spotted. The spots were kept at 30°C for 3 days. Comparison of growth ability of each mutant with and without glutathione stress. N number of each yeast strain = 7, 3 independent experiments. Error bars indicate standard deviation. The glutathione stress sensitivity was evaluated for the mutant Δsin3 HGT1 in which the gene encoding Sin3 of BY HGT1 was disrupted, and the same strain Δrpd3Δsin3 HGT1 in which the genes encoding Rpd3 and Sin3 were disrupted. The turbidity of the spots was OD600 = 2.5, 0.25, 0.025, 0.0025, and 0.00025 from the left, and 10 μl of each was spotted. Incubated at 30°C for 3 days.

The present invention will be described in detail below.
The present invention has newly discovered that genes encoding glycogen synthase kinase Rim11 (SEQ ID NO: 1), 14-3-3 protein Bmh1 (SEQ ID NO: 2), and somatic cell division G1 phase down-regulator Whi2 (SEQ ID NO: 3), specifically RIM11 (SEQ ID NO: 4), BMH1 (SEQ ID NO: 5), and WHI2 (SEQ ID NO: 6), confer glutathione stress resistance to yeast. According to the present invention, glutathione stress resistance can be imparted to yeast by upregulating the expression level of any of these genes and increasing the production of the protein. Particularly preferred is the improvement of expression of the gene encoding Rim11, and secondly preferred is the improvement of expression of the gene encoding Bmh1.

It has not been known so far that mass-produced glycogen synthase kinase, 14-3-3 protein, or somatic cell division G1 phase regulators confer glutathione stress resistance in any organism, including yeast.
The known functions of glycogen synthase kinase, such as the control of cell division and oxidative stress response (Non-Patent Document 7), the known functions of 14-3-3 proteins, such as the regulation of the MAPK pathway (Non-Patent Document 5), or the known functions of cell division control factors, such as the checkpoint in the G1 phase of somatic cell division (Non-Patent Document 6), are not directly linked in terms of cell physiology to the mechanism of action of glutathione stress reported in Non-Patent Document 1, i.e., a decrease in the activity of endogenous mitochondrial enzymes and induction of endoplasmic reticulum stress.

The present invention further relates to the mechanism of action of the glutathione stress resistance mechanism in yeast, which was discovered by transcriptome analysis.
The RIM11 gene, a member of the glycogen synthase kinase family, is known to be activated through the Tor pathway in response to nitrogen starvation and the subsequent MAPK pathway (mitogen-activated protein kinase) (Non-Patent Document 8). It has also been reported that Rim11 is involved in the control of the meiosis initiators Ime1 and Ime2 (IME) during early meiosis, and positively induces the transition of cells to the meiosis phase (Non-Patent Document 9). However, these functions of RIM11 are not easily linked to the mechanism of action of GSH stress.

Therefore, we performed RNA sequencing analysis of yeast with strong RIM11 expression, and unexpectedly, we confirmed that the biological process that confers glutathione stress resistance to yeast is the enhancement of the MAPK pathway and the transition from somatic cell division to the meiotic phase. Ume6 has been reported as a substrate of Rim11, and in addition, Rpd3 and Sin3 are proteins that cooperate with Ume6. Ume6, Rpd3, and Sin3 constitute a histone deacetylase complex (Conserved Histone Deacetylase; HDAC), and are known to be regulators of the epigenetic mechanism that controls the timing of gene expression in response to environmental changes (Non-Patent Document 12).
No relationship between epigenetic mechanisms and the physiological functions of glutathione (maintenance of redox balance, antioxidant activity, detoxification, and glutathione stress) can be found from existing information.
However, by using a molecular genetic technique in yeast, the inventors have discovered that there is a genetic interaction between the two mechanisms. This relationship is not something that can be easily predicted, and highly efficient production of glutathione based on this finding will have a great industrial impact.
From the above yeast chromosomal multicopy library screening, the 14-3-3 protein gene BMH1 was selected at the same time as RIM11, and interestingly, Bmh1 is also known to function in the MAPK pathway downstream of the Tor signal pathway (Non-Patent Document 6). The above results support the idea that the MAPK pathway confers glutathione stress resistance to yeast.

It is also known that the nitrogen starvation response causes the G1 phase of somatic cell division to stagnate, and the cyclin proteins Cln3 and Cln2 are suppressed. Cln2 is a negative regulator of Ime1, so when Cln2 function is reduced, the IME1 gene is upregulated, promoting the transition to the meiotic phase (Non-Patent Document 9). A gene encoding Whi2, WHI2, was found in a yeast chromosome multicopy library screening, and since this protein downregulates Cln1 and Cln2 (Non-Patent Document 7), the expression of the IME1 gene will be upregulated, promoting the transition to the meiotic phase. The above results suggest that the transition to the meiotic phase may be a factor that confers glutathione stress resistance to yeast.
The above biological processes are depicted in FIG.

The present invention also relates to a method for producing yeast having improved glutathione productivity.
The glutathione content in yeast can be increased by overexpressing a gene encoding glycogen synthase kinase Rim11 (SEQ ID NO: 1). In addition to this gene, a gene encoding a 14-3-3 protein Bmh1 (SEQ ID NO: 2) or a gene encoding a somatic cell division G1 phase regulator Whi2 (SEQ ID NO: 3) may also be used.

Furthermore, the gene may be a gene encoding Ume6 (sequence number 33), Rpd3 (sequence number 34), or Sin3 (sequence number 35), which are known to be regulators of the epigenetic mechanism.

In addition, in the present invention, an amino acid sequence having a homology of 65% or more, preferably 80% or more, and more preferably 90% or more with the amino acid sequences of SEQ ID NOs: 1 to 3 and 33 to 35 may be expressed.

Furthermore, when the Rim11, Bmh1 or Whi2 gene is overexpressed in a yeast in which the gene GSH1 (SEQ ID NO: 8) encoding glutathione synthetase-I (SEQ ID NO: 7) is strongly expressed, the glutathione content can be synergistically increased.
Yeast bred according to these methods has high intracellular glutathione content, yet its growth rate and cell yield (weight of yeast cells obtained per unit weight of sugar, biomass) are equivalent to those of the wild type, so the yeast of the invention is expected to improve glutathione productivity.

Furthermore, by applying previously reported methods for high glutathione production, such as adding cysteine and/or glycine to the medium in which yeast is cultured (Patent Document 3), a method of disrupting the glutathione reductase gene or enhancing the expression of the glutathione peroxidase gene (Patent Document 4), or a method of introducing a mutation into the MET30 gene (Patent Document 5), to the yeast of the present invention, it is expected that the glutathione content will be further increased.

<Types of yeast>
The yeast used in the present invention is not particularly limited, and examples thereof include the genus Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces rouxii, and Saccharomyces fragilis; the genus Candida, such as Candida utilis, Candida tropicalis, Candida glabrata, and Candida maltosa; the genus Zygosaccharomyces rouxii; rouxii) and the like.

<Method for overexpression of genes>
Methods for mass-expressing a gene encoding Rim11, such as RIM11, are well known to those skilled in the art, and can be carried out, for example, by using an expression plasmid in which DNA consisting of a base sequence encoding the gene is cloned into a multicopy vector (e.g., Non-Patent Document 10) that self-replicates in yeast, by integrating overlapping DNA fragments of the gene into the yeast genome to increase the copy number, or by placing the gene under the control of a promoter stronger than the promoter of the gene to increase the expression level, etc. Examples of strong promoters include upstream promoter sequences of known high expression promoters, such as PGK1, TDH3, TEF1, and CYC1 genes.

In addition, it is also possible to enhance the expression of the gene using standard mutagenesis techniques, such as treatment with chemical mutagens such as nitrosoguanidine or ethyl methanesulfonate, exposure to heavy particle beams, radiation, X-rays, UV light, or nitrous acid.

<Method for measuring total glutathione content>
The method is well known to those skilled in the art, and examples thereof include the enzyme-based rate assay by Tietze et al. (Non-Patent Document 11), a method using high performance liquid chromatography (HPLC) (Patent Document 6), and a method using a mass spectrometer (Non-Patent Document 1). Commercially available products such as GSSG/GSH Quantification Kit (manufactured by Dojindo Laboratories) can be used. The total glutathione content referred to here is the sum of the reduced glutathione (GSH) content and the oxidized glutathione (GSSG) content.

For example, in accordance with the method of Tietze et al., the modified yeast cells are collected by a centrifuge or the like, washed with water or the like, and then suspended in an aqueous solution of 5-sulfosalicylic acid. Glutathione can be extracted by known methods, i.e., a heat extraction method, an enzymatic decomposition method, or a physical disruption method using glass beads or a homogenizer. However, since it is known that the composition of the cell wall changes with the transition of the culture state of yeast, and that the reactivity of cell wall-dissolving enzymes decreases, particularly in the stationary phase, it is preferable to avoid the enzymatic decomposition method in this production method. The extract is clarified by subjecting it to a centrifuge or membrane filtration, and appropriately diluted with water or an aqueous solution of 5-sulfosalicylic acid to prepare a glutathione extract. However, since glutathione is more stable in a solution with a weak acidic pH, it is more preferable to use an aqueous solution of 5-sulfosalicylic acid.
The total glutathione content in the extract can be determined by tracking and measuring the change over time in the absorbance OD ₄₂₀ value of TNB (5-mercapto-2-nitrobenzoic acid) produced by reducing DTNB (5,5'-dithiobis-2-nitrobenzoic acid) with reduced glutathione.
On the other hand, a GSH standard solution is prepared by dissolving pure GSH in an aqueous solution of 5-sulfosalicylic acid, and then serially diluted with the same aqueous solution. A calibration curve is drawn from the relationship between the OD ₄₂₀ value obtained simultaneously with the above DTNB reaction and the dilution series of GSH concentrations. The total glutathione concentration and content of the glutathione extract can be calculated based on this calibration curve.

In the present invention, the glutathione reductase-DTNB method (hereinafter referred to as the "GR-DTNB method"), which is the method of Tietze et al., was used.
In the present invention, OD is a value measured using a spectrophotometer, specifically, a spectrophotometer (ratio beam spectrophotometer U-5100: manufactured by Hitachi High-Tech Science Corporation). pH is measured using a pH meter. Specifically, it was measured using a pH meter HM-30G (manufactured by DKK-TOA Corporation). In addition, in the analysis of total glutathione, it is preferable to measure the absorbance OD ₄₂₀ value using a plate reader, specifically, an ARVO X3 2030 Multilabel Reader (manufactured by PerkinElmer Co., Ltd.).

When evaluating glutathione productivity by yeast, the proliferation of yeast cells is examined, and the growth curve of the glutathione stress-resistant yeast is evaluated using a Compact Rocking Incubator TVS062CA (manufactured by Advantec).

The present invention will be described in detail below with reference to examples, but is not limited to these.
[Example 1]
(1) Construction of a cassette for strong expression of glutathione transporter HGT1 (OPT1) Using the genomic DNA of Saccharomyces cerevisiae BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) as a template, the LEU2 gene was PCR amplified using two types of primers (SEQ ID NOs: 9 and 10), and the amplified product was digested with restriction enzymes Spe I and Bam HI. This fragment was ligated to the Spe I and Bam HI digestion sites of the plasmid pBluescript SK-II(+) to obtain the LEU2 plasmid.
Using the genomic DNA as a template, two types of primers (SEQ ID NOs: 11 and 12) were used to amplify the promoter region of the TDH3 gene sequence, i.e., the nucleotide sequence from -680 nucleotides to -1 nucleotide upstream of the adenine of the start codon (ATG) as +1 nucleotide, and the amplified product was digested with Bam HI and Eco RI. This fragment was ligated to the Bam HI and Eco RI digestion sites of the LEU2 plasmid to obtain the LEU2-TDH3pr plasmid.
Furthermore, using the genomic DNA as a template, a part of the open reading frame (ORF) of the glutathione transporter HGT1 (OPT1) gene was PCR amplified with two types of primers (SEQ ID NOs: 13 and 14), and the amplified product was digested with Eco RI and Sal I. This was ligated to the Eco RI and Sal I digestion product of the LEU2-TDH3pr plasmid to obtain the LEU2-TDH3pr-HGT1 plasmid.
Finally, using the genomic DNA as a template, a part of the promoter region of the HGT1 gene sequence, i.e., the nucleotide sequence from -690 nucleotides to -197 nucleotides in the upstream region, was amplified with two types of primers (SEQ ID NOs: 15 and 16), and the amplified product was digested with Sac I and Spe I. This fragment was ligated to the Sac I and Spe I digestion sites of the LEU2-TDH3pr-HGT1 plasmid to construct the HGT1pr-LEU2-TDH3pr-HGT1 plasmid (FIG. 2).

SEQ ID NO:9
5'-ccggactagtaggagaacttctagtatatc-3'
SEQ ID NO:10
5'-ccggggatcctttctgacagagtaaaattc-3'
SEQ ID NO:11
5'-gccggatcccagttcgagtttatcattatc-3'
SEQ ID NO:12
5'-ggccgaattctttgtttgtttatgtgtgtt-3'
SEQ ID NO:13
5'-cggaattcatgagtaccatttatagggaga-3'
SEQ ID NO:14
5'-cggtcgactgattaccaccatttatcata-3'
SEQ ID NO:15
5'-cggagctcgagctgtgcaactcgagaca-3'
SEQ ID NO:16
5'-ggactagttctttcttcaacaacgattgct-3'

(2) Preparation of HGT1 Enhanced Strain The above HGT1pr-LEU2-TDH3pr-HGT1 plasmid was digested with the restriction enzyme Xho I to obtain a strong expression cassette of the HGT1 gene linked with the LEU2 gene.
Saccharomyces cerevisiae BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was transformed by double crossover chromosomal homologous recombination of the expression cassette using the lithium acetate method (Figure 2). The target chromosomal recombinant strain was selected by plating the LEU2 as a selection marker on a leucine-free selection medium (hereinafter referred to as SD-leu) and allowing it to form colonies.
The transformants were streaked on fresh SD-leu agar medium for single colony isolation, then inoculated into leucine-free SD liquid medium for growth, and spotted onto SD agar medium supplemented with reduced glutathione. The production of glutathione stress-sensitive yeast was confirmed by confirming that the transformants did not grow or that their growth was delayed.

(3) Screening of genes suppressing glutathione stress in multiple copies 3 g of a yeast chromosome library (2μ URA3) constructed from the chromosome of Saccharomyces cerevisiae was introduced into the glutathione stress-sensitive yeast (HGT1-enhanced strain) described in the previous section (2) by the lithium acetate method. Equal amounts of the library were spread on 60 SD-leu-ura agar plates containing GSH, and one SD-leu-ura agar plate not containing GSH. After incubation at 30° C. for 5 days, about 2,000 transformants were obtained on the SD-leu-ura agar plate not containing GSH. Thus, screening was performed on 60 SD-leu-ura agar plates containing GSH, which means that about 120,000 transformants were evaluated.
As a result, good growth was observed even in a glutathione stress environment for the three yeast mutants. For the three plasmids extracted from these yeast mutants, a part of the DNA base sequence derived from the Saccharomyces cerevisiae chromosome inserted into pRS426 was decoded using two primers (SEQ ID NOs: 17 and 18).

SEQ ID NO:17
5'-taatacgactcactataggg-3'
SEQ ID NO:18
5'-aattaaccctcactaaagg-3'

When the decoded sequence information was checked against the Saccharomyces Genome Database (SGD), it was found that chromosome fragments of chromosome number XIII (Chr III), chromosome number V (Chr V), and chromosome number XV (Chr XV) had been inserted. The genes located on each chromosome fragment are shown below.

Chromosome number XIII (Chr III): SIP5, RIM11, CIN4,
PSO2, GAT2
・Chromosome number V (Chr V): BMH1, PDA1, DMC1, ISC10
Chromosome number XV (Chr XV): HIR2, CKB2, GLO4, CUE5, WHI2

These genes were cloned singly into the pRS426 vector using the Saccharomyces cerevisiae BY4741 genome as a template.
Using the same procedure as for analyzing the DNA base sequence described above, it was confirmed that the target gene was carried alone in the pRS426 vector, and then the vector was introduced into an HGT1-enhanced strain and the presence or absence of glutathione stress resistance was evaluated. In each of the three types of chromosomal fragments obtained in this screening, the following three types of genes that suppress glutathione stress in multiple copies were identified (Figure 3).

Chromosome number XIII: RIM11
・Chromosome number V: BMH1
・Chromosome number XV: WHI2

FIG. 3 shows a comparison of glutathione stress sensitivity between the negative control (pRS426, library vector) and three mutants exhibiting glutathione stress resistance.
The turbidity of the spots was OD600 = 2.5, 0.25, 0.025, 0.0025, and 0.00025 from the left, and 10 μl of each was spotted and kept at 30° C. for 3 days.

[Example 2]
<Preparation of strains strongly expressing RIM11>
Using the genomic DNA of Saccharomyces cerevisiae BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) as a template, a region of 777 bases from the initiation codon of the RIM11 gene (SEQ ID NO: 2) was amplified by PCR using two types of primers (SEQ ID NO: 19 and 20). Meanwhile, TDH3pr was amplified by PCR using two types of primers (SEQ ID NO: 21 and 22). The primers of SEQ ID NO: 19 and 22 were synthesized so as to contain 15 base sequences complementary to each other.

The yeast-E. coli shuttle vector pRS303 carrying the HIS3 gene was digested with Sac I and Bam HI, mixed with the PCR amplified products of RIM11 and TDH3pr, and two-piece ligation was performed using the InFusion Cloning Kit manufactured by TaKaRa/Clontech. E. coli was transformed with the ligation solution, and the plasmid TDH3pr-RIM11-pRS303 was prepared from ampicillin-resistant colonies.

SEQ ID NO:19
5'-acataaacaaacaaaatgaatattcaaagcaataattctc-3'
SEQ ID NO:20
5'-gcagcccgggggatcctggagtacctaagattttaatg-3'
SEQ ID NO:21
5'-tatagggcgaattggagctccagttcgagtttatcattatc-3'
SEQ ID NO:22
5'-tttgtttgtttatgtgtgtttattc-3'

Next, using the same genomic DNA as a template, the promoter region of the RIM11 gene (from bases -961 to -48 upstream of the start codon, designated RIM11pr) was amplified by PCR using two types of primers (SEQ ID NOs: 23 and 24). TDH3pr-RIM11-pRS303 was cut with Sal I and Eco RI, and ligated to the PCR amplified product using the same kit as above to obtain RIM11pr-TDH3pr-RIM11-pRS303 (Figure 4).

SEQ ID NO:23
5'-ggccgaattcactaagtattatcaggaaac-3'
SEQ ID NO:24
5'-ccaagtcgactaatgctatgtcaagatctt-3'

<Preparation of a strain with strong GSH1 expression>
Using the genomic DNA of Saccharomyces cerevisiae BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) as a template, the region from the translation initiation codon to 1,118 bases of the glutathione synthetase-I gene (GSH1) (amino acid sequence: SEQ ID NO: 7, nucleotide sequence: SEQ ID NO: 8) was amplified by PCR using two types of primers (SEQ ID NOs: 25 and 26). Meanwhile, TDH3pr was PCR amplified using two types of primers (SEQ ID NOs: 27 and 28). The primers of SEQ ID NOs: 25 and 28 were synthesized so that their complementary 15-base sequences (underlined) overlap each other.

The yeast-E. coli shuttle vector pRS305 carrying the LEU2 gene was digested with Sac I and Bam HI, mixed with the PCR amplified products of GSH1 and TDH3pr, and two-piece ligation was performed using the InFusion Cloning Kit manufactured by TaKaRa/Clontech. E. coli was transformed with the ligation solution, and the plasmid TDH3pr-GSH1-pRS305 was prepared from ampicillin-resistant colonies.

SEQ ID NO:25
5'-acataaacaaacaaaatgggactcttagctttggg-3'
SEQ ID NO:26
5'-gcagcccgggggatccttcgacccacccaagaaaag-3'
SEQ ID NO:27
5'-tatagggcgaattggagctccagttcgagtttatcattatc-3'
SEQ ID NO:28
5'-tttgtttgtttatgtgtgtttattc-3'

Next, the promoter region of the GSH1 gene (from base -1,000 to base -441 upstream of the initiation codon, designated GSH1pr) was amplified by PCR using the same genomic DNA as a template and two types of primers (SEQ ID NOs: 29 and 30).
TDH3pr-GSH1-pRS305 was digested with Sma I and Xho I, and ligated to the PCR amplified product using the same kit as above to obtain GSH1pr-TDH3pr-GSH1-pRS305 (Figure 5). The PCR amplified sequence was analyzed for DNA base sequence to confirm the absence of errors such as base substitution, deletion, and insertion.

SEQ ID NO:29
5'-aaggatcccccgggctgcaggctcatcacggaactgtaac-3'
SEQ ID NO:30
5'-cgggccccccctcgagctccaactaccaaggttgt-3'

Using the lithium acetate method, Saccharomyces cerevisiae BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was transformed with the GSH1 overexpression cassette digested with Nco I and Hind III from the plasmid, and spread onto leucine-free selective medium SD-leu. A strain that formed colonies using LEU2 as a selection marker was selected to create a strain with strong GSH1 expression (designated BY GSH1).

Using the above-mentioned plasmid, RIM11pr-TDH3pr-RIM11-pRS303 (Figure 4), the RIM11 strong expression DNA fragment was introduced into Saccharomyces cerevisiae BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) or the above-mentioned BY GSH1 by chromosomal homologous recombination.

The former was spread on a histidine-free selective medium SD-his. A strain that formed colonies using HIS3 as a selective marker was selected to prepare a strain that strongly expresses RIM11 (designated BY RIM11).
The latter was spread on a selective medium SD-leu-his not containing leucine or histidine. A strain that formed colonies using LEU2 and HIS3 as selective markers was selected to prepare a strain that strongly expresses GSH1 and RIM11 (BY GSH1 RIM11).

<RNA sequence analysis of glutathione stress-resistant yeast (RIM11-enhanced strain)>
[Example 3]
(1) Production of glutathione stress resistant yeast, BY HGT1 RIM11 Using the plasmid described in Example 2, RIM11pr-TDH3pr-RIM11-pRS303 ( FIG. 4 ), a RIM11 strong expression DNA fragment was introduced into BY HGT1 described in Example 1(2) by chromosomal homologous recombination to produce a glutathione stress resistant yeast, BY HGT1 RIM11.

(2) Preparation of total RNA Total RNA was extracted from the BY HGT1 RIM11 strain by the hot phenol method. The procedure is as follows.
To 10 ml of SD Casamino Acid Medium (composition described in Example 4), BY HGT1 RIM11 preculture solution was added so that OD600 became 0.25. Two identical tubes were prepared. The culture was cultured with shaking at 30°C until OD600 became 1, and reduced glutathione (GSH) was aseptically added to one of the culture solutions to a final concentration of 500 μM, followed by shaking at 30°C.

Two hours after the addition of GSH, the supernatant was removed by centrifugation, and the yeast cells were collected and immediately flash frozen by immersion in liquid nitrogen. 500 μl of 65°C TES buffer (10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.5% SDS) was added, and 500 μl of 65°C water-saturated acidic phenol (for molecular biology, Wako Pure Chemical Industries, Ltd.) was immediately added, and the mixture was vigorously mixed with a vortex for 1 minute and stored at -20°C.

The -20°C stored samples (total of 2 samples) were incubated at 65°C for 30 minutes. After centrifugation, 200 μl of the supernatant was collected, and 750 μl of Trizol LS (Invitrogen) was added and mixed, and the mixture was left to stand at room temperature for 5 minutes. 200 μl of chloroform was added and mixed, and 300 μl of the supernatant was collected by centrifugation, after which 500 μl of isopropanol was added and mixed.

700 μl of this, including the precipitate, was subjected to RNeasy Mini Kit (QIAGEN), purified according to the standard procedure included with the kit, and dissolved in RNase-free water to obtain a total RNA solution.

(3) Library Preparation A library was prepared using a commercially available kit, TruSeq RNA Sample Prep Kit v2 (Illumina).

(4) Sequencing Using Illumina HiSeq 4000 (manufactured by Illumina), 100 bases each from both ends of the cDNA fragment were decoded (paired-end method).
The sequence was mapped to the Saccharomyces cerevisiae reference, GCF_000146054.2, using TopHat (Bowtie aligner).
The sample in which total RNA was extracted 2 hours after the addition of GSH to the culture medium is denoted as "GSH_posi_2hr", and the total RNA extraction sample in which GSH was not added, sampled at the same time as the above, is denoted as "GSH_nega_2hr".
Table 1 shows the total number of reads and the number of mapped reads in RNA sequencing, as well as their percentages.

(5) Data Analysis Using Cufflinks software, DEG (Differentially expressed genes) analysis was performed between the two groups, "GSH_posi_2hr" and "GSH_nega_2hr". From the KEGG pathway database (http://www.genome.jp/kegg/pathway.html), the MAPK pathway and meiosis were extracted as statistically significant biological processes.

<Glutathione production in strains with enhanced RIM11 gene expression>
[Example 4]
The BY RIM11 and BY GSH1 RIM11 strains were grown in SD casamino acid medium (containing 1.7 g/L yeast nitrogen base with amino acids and ammonium sulfate, 2 g/L vitamin assay casamino acids (both manufactured by Difco laboratories), 5 g/L ammonium sulfate, 20 g/L glucose (both manufactured by Wako Pure Chemical Industries, Ltd.), 20 mg/L adenine sulfate, 20 mg/L uracil (both manufactured by Sigma Aldrich), 20 mg/L tryptophan, and 20 mg/L urea sulfate ... The seed culture was carried out in 10 ml of 10 mg/L (manufactured by Wako Pure Chemical Industries, Ltd.) at 30° C. overnight with shaking.

Next, the seed culture liquid was inoculated into an L-shaped test tube containing 10 ml of fresh SD Casamino Acids medium with the same composition as above to give an OD600 of 0.25, and cultured for 7 hours at 30°C with stirring at 60 rpm (the culture stage at this time was in the logarithmic growth phase), and a portion of the culture liquid was recovered by centrifugation to give an OD·Units=5 [cm·ml]. The remainder was continued to be cultured under the same conditions, and 18 hours after the addition of the seed culture liquid (the culture stage at this time was in the stationary phase), the culture liquid was recovered by centrifugation to give an OD·Units=5 [cm·ml].

[Comparative Example 1]
For comparison, BY4741 and BY GSH1 were also cultured in the same manner as in Example 4 above, and the cells were collected.

[Example 5]
<Preparation of glutathione extracts of BY RIM11 and BY GSH1 RIM11>
The cells were washed twice with 0.1% ^w / _w 5-sulfosalicylic acid (5-SSA) and suspended in 80 μl of 1% ^w / _w 5-SSA. The cells were heat-treated at 95°C for 5 minutes to extract intracellular glutathione, cooled, centrifuged, and the collected supernatant was stored at -20°C. The extract was thawed and diluted 10-fold with water to prepare a 0.1% ^w / _w 5-SSA glutathione extract. The total glutathione concentration of the extract was analyzed by the glutathione reductase-DTNB method (GR-DTNB method).

[Comparative Example 2]
<Preparation of glutathione extracts of BY4741 and BY GSH1>
For comparison, glutathione extracts of BY4741 and BY GSH1 were prepared according to the same procedure as in Example 5 above.

[Example 6]
<Total glutathione analysis of BY RIM11 and BY GSH1 RIM11 extracts>
10 μl of the glutathione extract obtained in Example 5 was taken and diluted with 70 μl of 5-SSA aqueous solution. 120 μl of the reaction solution with the following composition was added to this, and the change in absorbance at OD 420 nm was measured three times every 5 minutes using a microplate reader. Each sample was measured in triplicate, and the measured value was the average value.
On the other hand, a 0.1% ^w / _w 5-SSA aqueous solution of reduced glutathione was prepared, and then diluted appropriately with the 0.1% ^w / _w 5-SSA aqueous solution to prepare glutathione standard solutions with different concentrations. The OD420 absorbance was measured simultaneously with the above analytical samples, three times every 5 minutes, in triplicate, and a calibration curve was drawn from the difference in the values and the glutathione concentration.
The total glutathione content in the BY RIM11 and BY GSH1 RIM11 extracts at OD·Units=5.0 [cm·ml] was calculated by multiplying the magnitude of the absorbance difference between each analysis sample by the above-mentioned calibration curve, the dilution ratio, and the extract volume of 80 μl.

[Comparative Example 3]
Total Glutathione Analysis of BY4741 and BY GSH1 Extracts
As a comparative control, the total glutathione content of BY4741 and BY GSH1 was calculated in the same manner as in Example 6. The results are shown in FIG.

Composition of reaction solution for GR-DTNB method:
(1) Preparation of potassium phosphate buffer (pH 7.5) a) 0.1 M Potassium Dihydrogen Phosphate
b) 0.1 M Dipotassium Hydrogen Phosphate
a) and b) were mixed and the pH was adjusted to 7.5.
c) 0.005 M EDTA・2Na・2H ₂ O (manufactured by Dojindo Laboratories)

(2) Preparation of reaction solution 2 mg of β-NADPH (manufactured by Oriental Yeast Co., Ltd.) and 8 mg of DTNB (manufactured by Dojindo Laboratories) were added to 12 ml of the potassium phosphate buffer (pH 7.5) and dissolved, and then 1.2 μl of glutathione reductase (manufactured by Oriental Yeast Co., Ltd.) was added. The reaction solution was prepared just before analysis.

[Example 7]
<RIM11 enhanced strain, other proliferation properties>
Regarding the three types of mutants used in Example 4 and Comparative Example 1 and BY4741,
The seed culture was inoculated into fresh SD casamino acid medium to give an OD600 of 0.1, and cultured with shaking at 30° C. and 70 rpm ⁻¹ . The results are shown in FIG.
7, BY GSH1 showed a slower rise in growth rate and a smaller number of cells (biomass) in the stationary phase than BY4741 (wild type). On the other hand, BY GSH1 RIM11 showed improvements in both growth rate and number of cells compared to BY GSH1.

From the results shown in Figure 6 of Example 6 and Figure 7 of Example 7, it was confirmed that glutathione productivity of yeast can be improved by imparting glutathione stress resistance.

[Example 8]
The RIM11 gene, IME1 gene, and UME6 gene disrupted strains (manufactured by OpenBiosystems) were each transformed with the cassette for strong expression of glutathione transporter HGT1 described in Example 1. LEU2 was used as a selection marker, and the strains were selected by plating on SD-leu and forming colonies. In this way, Δrim11 HGT1, Δime1 HGT1, and Δume6 HGT1 were obtained.
As shown in FIG. 8, Δume6 HGT1 became highly sensitive to glutathione stress, which revealed that Ume6 is involved in imparting glutathione stress resistance to yeast.

[Example 9]
Each of the mutants shown in FIG. 9 was inoculated into SD casamino acid liquid medium to a bacterial cell turbidity of OD600=0.1, and cultured at 30° C. and 70 rpm ⁻¹ with shaking.
9, the proliferation ability of Δrim11 HGT1 and Δume6 HGT1 was significantly decreased under glutathione stress-inducing conditions (with GSH). Together with the results of [Example 8] above, it was confirmed that Rim11 and Ume6 function in expressing glutathione stress resistance.

[Example 10]
The method described in Example 8 above was followed, except that the yeast strain of interest was a SIN3 gene disruptant (manufactured by OpenBiosystems). In this manner, Δsin3 HGT1 was obtained. Using two types of primers described in SEQ ID NOs: 31 and 32, the URA3 gene was PCR amplified using pRS306 (URA3) as a template. Δsin3 HGT1 was transformed with the amplified product and spread on SD-Ura agar medium. Δrpd3Δsin3 HGT1, in which the translation region of the RPD3 gene had been recombined with the PCR amplified product and gene function had been disrupted, was selected using the URA3 gene as a selection marker.
The spot assay in FIG. 10 was carried out in a similar manner to that described in Example 8.
As shown in FIG. 10, Δsin3 HGT1 had a higher glutathione sensitivity than BY HGT1, and Δrpd3Δsin3 HGT1 was even more sensitive.
As described above, the results of Examples 8 to 10 demonstrated that Ume6, RPD3, and SIN3, which function in coordination with each other in the epigenetic mechanism, are important in conferring glutathione stress resistance to yeast.

SEQ ID NO:31
5'-caattgcgccatacaaaacattcgtggctacaactcgatatccgtgcagcttaactatgcggcatcagag-3'
SEQ ID NO:32
5'-atgtaaataacacatataggcaattttcttcgaaacgtatgggacgcggtcctgatgcggtattttctcc-3'

Claims (3)

A method for producing glutathione, comprising: a gene belonging to the biological process (a) or (b) below is a DNA encoding any one of the amino acid sequences (A) to (C) below, the yeast being obtained by transforming a parent strain with the gene; and the yeast having improved glutathione stress resistance compared to the parent strain being cultured in the presence of glutathione to an extent that the parent strain of the yeast does not grow or grows slowly under glutathione stress.
(a) Mitogen-activated protein kinase (MAPK) pathway (b) Meiosis (A) An amino acid sequence having 90 % or more identity with the amino acid sequence of SEQ ID NO:1 (B) An amino acid sequence having 90 % or more identity with the amino acid sequence of SEQ ID NO:2 (C) An amino acid sequence having 90 % or more identity with the amino acid sequence of SEQ ID NO:3 A method for producing glutathione, comprising the step of culturing a yeast obtained by transforming a parent strain with a gene belonging to the biological process (a) or (b), the yeast being a DNA encoding any one of the amino acid sequences (A) to (C) above, the yeast being obtained by further transforming the parent strain with a DNA encoding a protein having glutathione synthesis ability and encoding an amino acid sequence having 90 % or more identity to the amino acid sequence of SEQ ID NO: 7, the yeast having improved glutathione stress resistance compared to the parent strain, in the presence of glutathione to an extent that the parent strain of the yeast does not grow or grows slowly due to glutathione stress. (a) A method for producing glutathione, comprising: culturing a yeast obtained by transforming a parent strain with DNA encoding any one of the amino acid sequences (D) to (F) below among genes belonging to the mechanism (I) below, which is a biological process that functions downstream of the MAPK pathway and which transitions the cell division stage to somatic cell division or meiosis, or to a temporary pause in division (G1 arrest, G2 arrest) as appropriate in response to changes in the external growth environment, and which has improved glutathione stress resistance compared to the parent strain, in the presence of glutathione to an extent that the parent strain of the yeast does not grow or grows slowly under glutathione stress.
(I) epigenetic mechanism (D) an amino acid sequence having 90 % or more identity with the amino acid sequence of SEQ ID NO: 33 (E) an amino acid sequence having 90 % or more identity with the amino acid sequence of SEQ ID NO: 34 (F) an amino acid sequence having 90 % or more identity with the amino acid sequence of SEQ ID NO: 35