JPH0670752A - Highly glutathione-containing yeast and its production - Google Patents

Abstract

PURPOSE:To obtain the yeast massively producing and accumulating glutathione in its body by subjecting a glutathione production ability-increased yeast to a mutational treatment and subsequently aerobically culturing the produced azaserine-resistant strain, the glutathione production ability-increased yeast being produced by the introduction of a gene. CONSTITUTION:A highly glutathione-producing yeast [preferably Saccharomyces.cerevisiae YHT178 strain (FERM P-13012)] produced by introducing a gamma-glutamylcysteine-synthesizing enzyme gene into an yeast is first subjected to a mutational treatment, and the produced azaserine-resistant strain is aerobically cultured to provide the objective yeast producing glutathione in a remarkable amount in its body. When a yeast having a higher glutathione content is further desired, the azaserine-resistant strain is preferably subjected to a conjugation treatment or cell-fusion treatment. The objective yeast includes Saccharomyces.cerevisiae 62D strain (FERN P-13010) obtained from the YHT 178 strain.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a yeast having a high content of glutathione, and a method for producing the same. More specifically, the present invention relates to a yeast having a gene-introduced glutathione production-enhancing ability that is mutated to produce a large amount of glutathione in the cells and accumulate the yeast, and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Glutathione is a peptide composed of glutamic acid, cysteine and glycine, and is an important compound widely used as a therapeutic drug or a reagent for liver diseases. Conventionally, glutathione is produced by an organic synthesis method or a method of extracting from yeast. However, the organic synthesis method has a problem in that the reaction step is long and complicated, and that the extraction method from yeast is limited in the amount of glutathione extracted from the cells.

A number of improvements have been made so far in order to solve these drawbacks. For example, a method for producing glutathione by adding an amino acid related to glutathione production (Japanese Patent Publication No. 47-26314, Japanese Patent Publication No. 53-94089, Japanese Patent Publication No. 54-8989).
997, JP-A-48-44487 and JP-A-51-36357). However, these methods are not satisfactory in terms of glutathione production. Further, there is a method for producing glutathione by obtaining a mutant strain (Japanese Patent Publication No. 56-46826, Japanese Patent Publication No. 48-61689, Japanese Patent Publication No. 53-31956, Japanese Patent Publication No. 61-209583). Furthermore, a method for producing glutathione by microorganisms utilizing genetic engineering (Japanese Patent Laid-Open Nos. 58-20188 and 58-201
96, JP 61-52299, JP 62-275685, JP 64-5
1098).

Although these improve the glutathione synthesizing ability, the supply of L-cysteine, which is necessary for glutathione synthesis and is rate-limiting, has not been sufficiently improved, and addition of the same amino acid is necessary for high production of glutathione. it is conceivable that.

[0005]

Therefore, according to the present invention,
By mutating yeast to create an azaserine-resistant strain, the rate-determining intracellular synthesis system of L-cysteine is strengthened, and the yeast having a higher glutathione content than conventional methods is treated with amino acids (L-cysteine). The purpose is to obtain without adding.

Another object of the present invention is to obtain a yeast highly containing glutathione by conjugating the azaserine resistant strains obtained by the mutation treatment and further cell fusion treatment.

[0007]

MEANS FOR SOLVING THE PROBLEMS The present invention is to produce azaserine resistant strains by performing mutation treatment on a glutathione high-producing yeast into which a γ-glutamylcysteine synthetase gene has been introduced, and aerobically obtain the azaserine resistant strains. It is a method for producing a yeast having a high content of glutathione, characterized in that a large amount of glutathione is produced in the microbial cells by a static culture, preferably a fed-batch culture.

Further, according to the present invention, a glutathione-producing yeast into which a γ-glutamylcysteine synthetase gene has been introduced is preferably subjected to a mutation treatment to construct an azaserine resistant strain, and a haploid of the obtained azaserine resistant strain is obtained. By joining with a haploid of another glutathione high-producing strain, a polyploid is constructed,
The resulting strain is screened for excellent sporulation glutathione-producing ability and growth ability strain, by aerobically culturing the obtained strain, preferably by fed-batch culture, to obtain glutathione in the cells. It is a method for producing a yeast having a high glutathione content, which is characterized in that a large amount is produced.

More preferably, the present invention mutates a glutathione high-producing yeast into which a γ-glutamylcysteine synthetase gene has been introduced to construct an azaserine resistant strain, and obtain a haploid of the obtained azaserine resistant strain. Of a haploid strain having excellent glutathione-producing ability and growth ability, obtained by conjugating with a haploid of a glutathione high-producing strain of By aerobically culturing the strain obtained by cell fusion of the strain, preferably by fed-batch culture, a method for producing a glutathione-rich yeast characterized in that a large amount of glutathione is produced in the cells. Is.

The present invention will be described in detail below. As the gene-introduced glutathione high-producing yeast used in the present invention, yeast obtained by the method established by Ohtake et al. (1990 Annual Meeting of the Japanese Society of Agricultural Chemistry, p. 145, 1990) can be used. That is, γ-glutamylcysteine synthetase gene, which is one of the yeast glutathione biosynthetic enzymes, is connected to the Saccharomyces cerevisiae strong expression promoter ΔP8 and introduced into the chromosome trp1 site of Saccharomyces cerevisiae. A glutathione high-producing yeast can be created.

[0011] As such yeasts, those deposited with the International Depositary Authority under the Budapest Treaty under the accession number Microinvestigation Article No. 13012 are preferable. The mutagen for obtaining the azaserine-resistant strain may be any method generally used as a mutagen, and ultraviolet rays, ethyl methanesulfonic acid, nitrosoguanidine, X-rays and the like can be used.

The azaserine-resistant strain referred to in the present invention is a mutant strain in which cysteine biosynthesis is enhanced by enhancing the serine supply system required for cysteine biosynthesis. A strain that can grow in a medium supplemented with azaserine at a concentration of 25 μg / ml. The azaserine-resistant strain obtained in the present invention showed a characteristic that the amount of glutathione produced was increased as in the case of culturing by adding cysteine to the medium.

As a method for further improving the growth ability of the screened azaserine resistant strain, a conjugative haploid glutathione-producing excellent strain capable of conjugating with an azaserine resistant strain is obtained, and the haploid and azaserine are produced. Resistant strains can be mated to obtain polyploids. Furthermore, it is possible to sporulate polyploidy of the obtained azaserine resistant strains and to screen excellent strains producing glutathione. In order to obtain a good haploid strain capable of mating with an azaserine-resistant strain, the usual method of conjugating two haploid excellent strains, creating a polyploid strain, and then performing sporulation I can.

Further, a polyploid can be constructed in order to enhance the glutathione productivity of the obtained azaserine resistant strain. As a method of creating a polyploid body, joining,
Alternatively, it can be performed by cell fusion. The culture conditions of the strain of the present invention, as generally performed,
There is no particular limitation as long as it is aerobically cultivated at a culturing temperature of 20 to 34 ° C. and a culturing pH of 4.5 to 6.5, but it is particularly preferable to carry out fed-batch culturing in order to enhance glutathione productivity. Fed-batch culture was performed by inoculating a medium consisting of glucose, urea, yeast extract and inorganic salts using a jar fermenter, aeration of 1 vvm, temperature of 30 ° C and DO level of 1-5 ppm at the minimum. A sterilized mixture of glucose and yeast extract
Fed for 12 hours after inoculation at a rate of 0.05 l / h. Fed-batch time is approx.
Approximately 1/40 of the initial volume is added in 12 hours.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, a yeast having a higher glutathione content than the conventional production method can be obtained without adding an amino acid (L-cysteine), and azaserine obtained by a mutation treatment can be obtained. Yeast containing highly glutathione can be obtained by conjugating a resistant strain and further subjecting it to cell fusion.

[0016]

EXAMPLES The present invention will be described in detail below with reference to examples, but the invention is not limited thereto. (1) Construction of gene-introduced glutatin high-producing yeast In order to efficiently express genes in the glutathione biosynthesis system, a strong transcription promoter was first cloned from the yeast chromosome. The promoter detection vector pMC1585 was used for cloning using β-galactosidase activity as an index. As a result, a P8 promoter fragment (about 3.8 kb) was obtained as a highly active DNA fragment.
From this P8 promoter fragment, ΔP8 (approx.
0.4 kb) was created.

Next, the yeast γ-glutamylcysteine synthetase gene was cloned. That is, S. c
From the erevisiae YNN27 strain ( α trp1 ura3 ), a mutant strain lacking the above two enzymes was learned. Next, the γ-glutamylcysteine synthase gene was cloned using the γ-glutamylcysteine synthase deficient strain (YH1). YH
When one strain was transformed with a gene library using YEp24 as a vector and screened using nitroprusside soda, one kind of plasmid (pYOG1001) which was considered to be the target gene was obtained. When the resulting gene was subcloned, the target gene (GSH1) was present on a fragment of about 4.4 kb. Therefore, when the entire base sequence of this gene fragment was determined, this fragment consisted of 4.410 bases.

[0018] was prepared YHT178 strain inserted into trp1 on the chromosome of a transcriptional promoter GSH1 gene this cloning was replaced with △ P8, a strong expression of the △ P8-GSH1 fragment YNN27 strain. When the GSH-1 activity and glutathione content of the YHT178 strain were measured, the GSH-1 activity was 0.29 unit and the glutathione content was about 1.5%.

(2) Preparation of Azaserine-Resistant Strain Saccharomyces cerevisiae YHT178 strain cultured for 3 days on YPD agar medium (1% yeast extract, 1% peptone, 2% glucose, 1.5% agar) in YPD liquid medium (YPD agar medium from which agar had been removed) One platinum loop was inoculated into a 500 ml shake flask containing 100 ml, and cultured at 30 ° C. with shaking overnight. The cells were collected by centrifugation (3000 rpm, 10 minutes) and washed with 50 ml of distilled water. Then, it was suspended in 20 ml of a phosphate buffer (pH 7.5, 0.1 M), and 600 μl of ethyl methanesulfonic acid was added. After shaking for 1 hour at 30 ℃, 20ml
5% sodium thiosulfate solution was added and left for 5 minutes.
The cells were collected by centrifugation and resuspended in 20 ml of distilled water.
This suspension was added to an SD agar medium containing 25 μg / ml of azaserine (Difco yeast yeast base W / O amino acid 0.67%, glucose 2%, Difco agar 1.5.
%) And cultivated at 30 ° C. for 3 days. The growing colonies were selected, inoculated into a test tube containing 5 ml of YPD medium, and cultured by shaking at 30 ° C. for 30 hours. The cells were collected by centrifugation, washed with distilled water and suspended in 1 ml of distilled water. This suspension was heated at 100 ° C. for 5 minutes and then centrifuged to obtain a supernatant. The extracted glutathione in the supernatant was subjected to colorimetric determination.

To 2.5 ml of 0.36 M pH 7 phosphate buffer containing 0.7 mM of EDTA, 100 μl of 4 mg / ml solution of dithiobis (2-nitrobenzoic acid) and 4 mg / ml solution of NADPH 50
μl and 0.5 unit of glutathione reductase (manufactured by Oriental Yeast Co., Ltd.) were added, and the mixture was placed in a glass cuvette having an optical path length of 1 cm and heated to 25 ° C. Dilute the cuvette appropriately and measure glutathione.
l was added to start the reaction. Absorbance at 412 nm was continuously recorded for 1 minute using Hitachi Autograph 320 type spectrophotometer. Similarly, a standard solution of glutathione having a known concentration was reacted as a sample, the increase in absorbance was continuously recorded, a calibration curve was created using the slope of the increase curve, and the content of glutathione in the sample was calculated from the slope of the sample.

About 1000 strains of colonies were treated and 60 strains that produced a large amount of glutathione were selected. 100 ml of selected strain
Inoculate a 500 ml shake flask containing YPD medium of
The medium was shaken at 30 ° C. for 3 days. After the medium was finished, the amount of glutathione produced by each strain was compared in exactly the same manner as in the case of culturing using a test tube. As a result, ASR6-32 strain with the highest glutathione content per cell was selected. YH in Table 1
A comparison of T178 and ASR6-32 is shown.

[0022]

[Table 1] The values are the values obtained by culturing in a 500 ml Sakaguchi flask containing 100 ml YPD medium at 30 ° C. for 3 days.

(3) Improvement 1 of Azaserine resistant strain ASR6-32
Construction of polyploid strain Next, in order to improve the growth ability of this mutant strain, an attempt was made to form a polyploid strain by conjugation. First, YHT178 strain and Saccharomyces cerevisiae laboratory strain AH22 strain (ATCC
38626) were joined. Since the former is auxotrophic for tryptophan and uracil, and the latter is auxotrophic for histidine and leucine, the strain conjugated with both can grow in a minimal medium (SD medium), but the non-conjugated strain cannot grow. Was used to select the zygote strain. The obtained strain was inoculated into a sporulation medium (yeast extract 0.1%, glucose 0.05%, potassium acetate 1.0%, agar 2.0%) and cultured at 30 ° C. for about 10 days. Take one platinum loop of the spore-forming zygote,
0.1 mg / ml Zymo Ace (Seikagaku, 100,000 units)
/ g) Suspended in 1 ml of aqueous solution, and allowed to stand at 30 ° C for 15 minutes. This suspension is a micromanipulator manufactured by Narishige Co., Ltd.
Forming spores were isolated using MN-188 / MD-188 type. From the separated spores, a strain YHT182 having a mating type of a and a glucose productivity of YHT178 was selected. YHT182 and ASR6-32 were conjugated in the same manner, sporulation and spore separation were performed, and about 100 strains thus obtained were cultured in YPD medium to compare glutathione-producing ability and growth.
Then, they acquired the strain 62D, which satisfies both growth and glutathione production. Both strains were inoculated into a 500 ml flask containing 100 ml of YPD medium and cultured by shaking at 30 ° C. for 3 days, and the glutathione content, the amount of cells, etc. were compared in exactly the same manner as in Example 1. The results are shown in Table 2. Table 2 shows a comparison between the YHT178 strain and the 62D strain. The 62D strain was deposited by Mitsukoshi Kenjou No. 13010.

[0024]

[Table 2]

(4) Construction of polyploid strain of azaserine resistant strain 62D strain Glutathione high-producing yeast 62D strain (ura3, his
3) was further modified. First, use plasmid YEp24 with restriction enzyme HindIII and plasmid YIo1 with restriction enzyme Bam.
Each was completely digested with HI. Used for agarose gel electrophoresis
A DNA fragment containing the URA3 gene fragment and the remaining YIp1 plasmid fragment from which the HIS3 gene had been removed were separated. Maniatis (T.) et al. {Molecular cloning, Cold Spring Harbor Laboratory)
, 1982} and ligated the URA3 gene to the YIp vector from which the HIS3 gene had been deleted, and then plasmid pYI
-U3 was produced.

The 62D strain was inoculated into YPD medium, shake-cultured at 30 ° C. for 16 hours, and the cells were collected by centrifugation. After washing, the cells were suspended in ice-cooled 1M sorbitol so that the bacterial concentration was about 1 × 10 ¹⁰ cells / ml. To 40 μl of this suspension, 5 μl of a solution containing the previously prepared plasmid pYI-U3 in a concentration of about 20 μl / ml was added, and the mixture was ice-cooled. The suspension in which the bacteria and the plasmid were mixed was put into an ice-cooled cuvette for electroporation (165-2086 manufactured by Bio-Rad). This cuvette was set in Gene Pulser (manufactured by Bio-Rad). The device was set to 1 kV and 25 μF, and the pulse controller was set to 200Ω. Electroporation was performed with a time constant of 4 msec. Ice-cooled 1M
1 ml of the sorbitol solution was added to the cuvette and stirred well. Then, histidine was inoculated on SD agar medium containing 20 μg / ml and cultured at 30 ° C. for about 3 days. The grown colonies were transferred to YPD agar medium. Similarly, YIp1 was introduced into the 62D strain for transformation. Then 62D (h
is3) and 62D (ura3) were obtained.

Glucose 2% and urea were added to three 5 L jar fermenters (MDL500 type manufactured by Marubishi Bio Engineering Co., Ltd.).
0.6%, yeast extract (meast, manufactured by Asahi Breweries) 0.4
_{%, KH 2 PO 4 0.15%} , MgSO 4 · 7H 2 O 0.05%, Na 2 SO 4 0.
2%, CaCl ₂ · 2H ₂ O 0.03%, ZnSO ₄ · 7H ₂ O 1.32ppm, Fe
2400 ml of a medium containing 1.45 ppm of Cl ₃ .6H ₂ O and 0.2 ppm of CuSO ₄ 5H ₂ O was added and sterilized at 120 ° C. for 15 minutes. Inoculum solution
After centrifuging 1750 ml at 3000 rpm for 5 minutes, the supernatant was discarded, washed with 500 ml of sterilized water, and centrifuged again under the same conditions. Sterilized water was added to the obtained precipitate to make 875 ml, and this 2-fold concentrated seed culture solution was placed in three jar-famenters at 125 ml and 250 m, respectively.
l, 500 ml was inoculated. The number of viable bacteria at the time of inoculation is 2.75 × 10 ⁷ each
cells / ml, 6.25 × 10 ⁷ cells / ml, 9.00 × 10 ⁷ cells / ml
Met. The aeration rate was 1.0 vvm, the agitation number was 600 rpm, the temperature was controlled to pH 5.0 with 2% ammonia water at 30 ° C., and the mixture was cultured for 48 hours using a 1% silicone antifoaming agent (TSA737F, manufactured by Toshiba Silicone Co.). During the culture, 600 ml of a medium consisting of 15% glucose and 3% mist was fed at a rate of 0.05 l / h for 12 hours from 12 hours after inoculation.

The glutathione production of the bacterial cell extract obtained by subjecting 1 ml of the culture solution containing yeast cells to hot water extraction and centrifugation was determined according to the Titze method (Anal. Biochem., 27, 502 (1969)). It was measured by the increase in absorption at 412 nm. The dry cell weight was measured by freeze-drying, and the glutathione content was calculated from the glutathione production. Table 3 shows the amount of glutathione produced during 48 hours of culture, the glutathione content, and the dry cell weight. Table 3 shows the glutathione production of the 62D / 62D strain.

[0029]

[Table 3] Next, cell fusion of the two strains prepared above was performed. YPD medium
A 500 ml shake flask containing 100 ml was inoculated with the above strain and cultured at 30 ° C. for about 16 hours. The bacteria were collected by centrifugation, washed with sterile water, and then washed with STE buffer (1M sorbitol, 20
It was suspended in 100 ml of mMEDTA and 10 mM Tris-hydrochloric acid (pH 7.4). Add 39 μl of β-mercaptoethanol and add 30 μl.
It was treated at 0 ° C for 10 minutes. The cells were collected by centrifugation, and ST buffer (1M sorbitol, 10 mM Tris-containing 0.1 mg / ml concentration of Zymoriace (manufactured by Seikagaku Corporation, 100,000 units / g)) was collected.
It was suspended in 10 ml of hydrochloric acid (pH 7.4) and reacted at 30 ° C. for 20 minutes to give protoplasts. The cells were collected by centrifugation and washed once with ST buffer. Then, the cells were suspended in 10 ml of ST buffer (STC buffer) containing 10 mM CaCl ₂ . The number of cells in each suspension was counted by a hemocytometer, and 1 × 10 ⁷ cells were mixed. After collecting cells by centrifugation, ST containing 10 mM CaCl ₂
The cells were suspended in 1 ml of buffer and heated at 30 ° C for 15 minutes. Again, the cells were collected by centrifugation and 1 ml of PCT buffer (35%
PEG4000, 10 mM Tris-hydrochloric acid (pH 7.4), 10 mM CaCl ₂ )
And suspended at 30 ° C. for 10 minutes for fusion. The cells were collected, washed with ST buffer, and suspended in 1 ml of STC buffer. To SD agar medium containing 1M sorbitol, 100 μl of this suspension was inoculated and cultured at 30 ° C. for about 5 days. Transfer about 100 strains of grown colonies to YPD medium, and
The cells were cultured at 30 ° C for a day.

About 100 of the above strains were cultured in a test tube containing 5 ml of YPD medium with shaking at 30 ° C. for 3 days, and excellent strains were selected using glutathione content and growth as selection indexes. Then,
The 62D / 62D strain was selected. The 62D / 62D strain is the Micro Engineering Research Institute No. 13
Deposited at No. 011. Table 4 shows each strain using YPD medium.
The results of shaking culture at 0 ° C. for 3 days are shown.

[0031]

[Table 4]

(5) Cultivation of yeast containing a high amount of glutathione, No. 1 Glucose 2%, yeast extract (manufactured by Difco) 1%, bactopeptone (manufactured by Difco) 1% were prepared and a 500 ml shake flask was prepared. Incubated with 100 ml for 24 hours with shaking to give a seed culture. A 5L jar fermenter (Maruhishi Bio Engineering Co. MDL500 type), 2% glucose, 0.6% urea, yeast extract (Meast, Asahi _{Breweries) 0.4%, KH 2 PO 4} 0.15%, MgSO 4 · 7H 2 O 0.05
%, Na ₂ SO ₄ 0.2%, CaCl ₂・ 2H ₂ O 0.03%, ZnSO ₄・ 7H
₂ O 1.32ppm, FeCl ₃ / 6H ₂ O 1.45ppm, CuSO ₄ / 5H ₂ O
Add 2400 ml of medium consisting of 0.2 ppm, sterilize at 120 ℃ for 15 minutes, inoculate 100 ml of the above inoculum, aeration rate 1.0 vvm, stirring number 4
The pH was controlled at 5.0 rpm with 00 rpm, 30 ° C and 2% ammonia water, and 1% of silicone defoamer (TSA7 manufactured by Toshiba Silicone Co., Ltd.).
37F) was used and cultured for 48 hours. Glucose 15 in culture
% Of the medium and 3% of the mist, 600 ml of the medium was fed at a rate of 0.05 l / h for 12 hours from 8 hours after inoculation.

1 ml of a culture solution containing yeast cells was subjected to hot water extraction treatment, and after centrifugation, the amount of glutathione produced in the obtained cell extract was determined by the Titze method (Anal. Biochem., 27, 502 (1969)).
According to the method described above, the increase in absorption at 412 nm was measured. The dry cell weight was measured by freeze-drying, and the glutathione content was calculated from the glutathione production. The amount of glutathione produced after 35 hours of culturing was 700 mg / L, the glutathione content was 4.22%, and the dry cell weight was 16.6 mg / ml. YHT under similar conditions
Table 5 shows a comparison with 178 strains. Table 5 shows 62D / 62D strain and Y
The comparison of HT178 strain is shown.

[0034]

[Table 5] (6) Culture of yeast with high glutathione content, 2 Glucose 2%, yeast extract (manufactured by Difco) 1%, bactopeptone (manufactured by Difco) 1% were prepared, and 500 ml was poured into a 2000 ml shake flask. . After sterilizing at 120 ℃ for 15 minutes, inoculate 62D / 62D strain,
The mixture was cultivated with shaking at 24 ° C. for 24 hours to give a seed culture.

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[Procedure amendment]

[Submission date] July 9, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction content]

Next, the yeast γ-glutamylcysteine synthetase gene was cloned. That is, S. c
cerevisiae YNN27 strain from (α trp1 ura3), were acquired the defective mutants of the two enzymes. Next, the γ-glutamylcysteine synthase gene was cloned using the γ-glutamylcysteine synthase deficient strain (YH1). YH
When one strain was transformed with a gene library using YEp24 as a vector and screened using nitroprusside soda, one kind of plasmid (pYOG1001) which was considered to be the target gene was obtained. When the resulting gene was subcloned, the target gene (GSH1) was present on a fragment of about 4.4 kb. Therefore, when the entire base sequence of this gene fragment was determined, this fragment consisted of 4.410 bases.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

[0024]

[Table 2]

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

[Correction content]

Next, cell fusion of the above two strains prepared was carried out.
It was Place in a 500 ml shake flask containing 100 ml of YPD medium.
The strains described above were inoculated and cultured at 30 ° C for about 16 hours. Centrifuge bacteria
Collected by separation, washed with sterile water, and then washed with STE buffer (1M solution).
Rubitol, 20 mM EDTA, 10 mM Tris-HCl (pH 7.
4)) Suspended in 100 ml. There β-mercaptoethan
39 μl was added and the mixture was treated at 30 ° C. for 10 minutes. Collect cells by centrifugation
, 0.1 mg / ml concentration of Zymori Ace (Seikagaku Corporation, 10
ST buffer (1M sorbito) containing 10,000 units / g)
Suspension in 10 ml of 10 mM Tris-HCl (pH 7.4) at 20
The reaction was allowed to proceed for minutes to give protoplasts. Collect cells by centrifugation
And washed once with ST buffer. Then 10 mM Ca
To 10 ml of ST buffer (STC buffer) containing Cl ₂
Suspended. Count the number of cells in each suspension with a hemocytometer
Well, 1 × 10 ⁷ pieces were mixed. After collecting the cells by centrifugation,
Suspend in 1 ml of ST buffer containing mM CaCl ₂ and incubate at 30 ℃ for 15
Heated for minutes. Again collect the cells by centrifugation and collect 1 ml of PCT
Buffer (35% PEG4000, 10 mM Tris-HCl (pH 7.
4), suspend in 10 mM CaCl ₂ ) and allow fusion at 30 ° C for 10 minutes.
It was The cells were collected, washed with ST buffer, and washed with 1 ml of STC buffer.
Suspended in stuffer. SD agar containing 1M sorbitol
Inoculate 100 μl of this suspension into the medium and incubate at 30 ° C for about 5 days.
I nourished. About 100 grown colonies, YPD medium
And further cultured for 7 days at 30 ° C.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

Approximately 100 strains described above containing 5 ml of YPD medium
Culture in a test tube at 30 ℃ for 3 days with shaking to determine the glutathione content and
Good strains were selected using the growth and growth as selection indexes. Then,
The 62D / 62D strain was selected. The 62D / 62D strain is the Micro Engineering Research Institute No. 13
Deposited at No. 011. Table 3 shows each strain using YPD medium 3
The results of shaking culture at 0 ° C. for 3 days are shown.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

[0029]

[Table 3] (5) Culture of yeast with high glutathione content, 1 glucose 2%, yeast extract (Difco)
1%, Bactopeptone (Difco) 1%
Prepare the culture medium and add it to a 500 ml shake flask at 100 ml for 24 hours.
Culture was carried out with shaking for use as an inoculum solution. 5L jar famentor
(Maruryo Bio Engineering MDL500 type) with glucose
2%, urea 0.6%, yeast extract (meast, Asahi Bee
Le _{Co.) 0.4%, KH 2 PO 4} 0.15%, MgSO 4 · 7H 2 O 0.05
%, Na ₂ SO ₄ 0.2%, CaCl ₂・ 2H ₂ O 0.03%, ZnSO ₄・ 7H
₂ O 1.32ppm, FeCl ₃ / 6H ₂ O 1.45ppm, CuSO ₄ / 5H ₂ O
Add 2400 ml of 0.2 ppm medium and sterilize at 120 ° C for 15 minutes.
After that, inoculate 100 ml of the above inoculum, aeration rate 1.0 vvm, stirring number 4
Controlled to pH 5.0 with 00rpm, 30 ℃, 2% ammonia water
1% silicone defoamer (Toshiba Silicone, TSA7
37F) was used and cultured for 48 hours. Glucose 15 in culture
%, Mist 3% 600 ml of medium for 8 hours after inoculation
It was fed at a rate of 0.05 l / h for 12 hours.

[Procedure correction 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0030

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[Correction content]

1 ml of culture solution containing yeast cells was extracted with hot water
After centrifugation, the resulting bacterial cell extract was treated with glutathione.
Yield is Titze method (Anal. Biochem., 27, 502 (1969))
According to the method described above, the increase in absorption at 412 nm was measured. Dry by freeze-drying
Glutathione production was measured by measuring the dry cell weight.
The on content was calculated. Glutathione production in 35 hours of culture
The amount is 700mg / L, glutathione content is 4.22%, dry
The cell weight was 16.6 mg / ml. YHT under similar conditions
Table 4 shows a comparison with 178 strains. Table 4 shows 62D / 62D strain and Y
The comparison of HT178 strain is shown.

[Procedure Amendment 7]

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[Correction method] Change

[Correction content]

[0031]

[Table 4] (6) Glutathione-rich yeast culture, 2 glucose 2%, yeast extract (Difco)
1%, Bactopeptone (Difco) 1%
Prepare a culture medium and pour 500 ml into a 2000 ml shake flask.
It was After sterilizing at 120 ℃ for 15 minutes, inoculate 62D / 62D strain,
The mixture was cultivated with shaking at 24 ° C. for 24 hours to give a seed culture.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

Three 5L jar famenters
Oendi MDL500 type), glucose 2%, urea
0.6%, yeast extract (meast, manufactured by Asahi Breweries) 0.4
_{%, KH 2 PO 4 0.15%} , MgSO 4 · 7H 2 O 0.05%, Na 2 SO 4 0.
2%, CaCl ₂ · 2H ₂ O 0.03%, ZnSO ₄ · 7H ₂ O 1.32ppm, Fe
Cl ₃ · 6H ₂ O 1.45ppm, culture consisting of CuSO ₄ · 5H ₂ O 0.2ppm
2400 ml of ground was added and sterilized at 120 ° C for 15 minutes. Inoculum solution
Centrifuge 1750 ml at 3000 rpm for 5 minutes, discard the supernatant, and kill.
The cells were washed with 500 ml of bacterial water and centrifuged again under the same conditions. Got
Sterilized water was added to the precipitated precipitate to make 875 ml, and this double concentrated seed was used.
125 ml, 250 m of fungal fluid to 3 jars
l, 500 ml was inoculated. The number of viable bacteria at the time of inoculation is 2.75 × 10 ⁷ each
cells / ml, 6.25 × 10 ⁷ cells / ml, 9.00 × 10 ⁷ cells / ml
Met. Aeration rate 1.0vvm, stirring speed 600rpm, 30 ℃, 2%
The pH is adjusted to 5.0 with ammonia water and 1% of silicone is consumed.
Foaming agent (Toshiba Silicone TSA737F) is used and cultivated for 48 hours.
I nourished. Consisting of 15% glucose and 3% mist during culture
600 ml of culture medium from 12 hours after inoculation at a rate of 0.05 l / h
Fed-batch.

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1 ml of culture solution containing yeast cells was extracted with hot water
Glutathione production of the bacterial cell extract obtained after centrifugation
The amount was determined by the Titze method (Anal. Biochem., 27, 502 (1969)).
Therefore, it was measured by an increase in absorption at 412 nm. Dried by freeze-drying
Glutathione was measured from the amount of glutathione produced by measuring the cell weight.
The content rate was calculated. Glutathione production in 48 hours of culture
The amount, glutathione content and dry cell weight are shown in Table 5.
You Table 5 shows the glutathione production of the 62D / 62D strain.

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[0034]

[Table 5]

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Claims (4)

[Claims] 1. A glutathione-producing strain into which a γ-glutamylcysteine synthetase gene has been introduced is mutated to produce an azaserine-resistant strain, and the resulting azaserine-resistant strain is aerobically cultured to produce glutathione in the cells. A method for producing a yeast having a high content of glutathione, which comprises producing a large amount of 2. A gene-producing glutathione high-producing yeast,
Saccaromyces cerevisi
e) The method for producing a yeast having a high content of glutathione according to claim 1, which is YHT178 strain (Ministry of Industrial Technology Article No. 13012). 3. The method for producing a yeast having a high content of glutathione according to claim 1, wherein the yeast having a high content of glutathione is Saccharomyces cerevisiae 62D strain (Mikokukenjojo 13010). 4. A yeast having a high glutathione content is a Saccharomyces cerevisiae 62D / 62D strain (Ministry of Engineering Kenjojo 13011).
No.). The method for producing a glutathione-rich yeast according to claim 1.