JPH0753102B2 - Yeast high in glutathione and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION This invention is, glutathione-rich support
The present invention relates to C. cerevisiae and a method for producing the same. More specifically, the present invention relates to Saccharomyces cerevisiae in which saccharomyces cerevisiae , which has been transduced with a gene and enhanced in glutathione-producing ability, is subjected to mutation treatment to produce and accumulate a large amount of glutathione in the cells , 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 controlling the yeast Saccharomyces cerevisiae to create an azaserine resistant strain, the rate-determining L
-The yeast Saccharomyces, which has a higher content of glutathione than conventional methods, by strengthening the intracellular synthesis system of cysteine.
The purpose is to obtain S. cerevisiae without adding an amino acid (L-cysteine).

[0006] Further, the yeast Saccharomyces containing highly glutathione by conjugating the azaserine-resistant strains obtained by the mutation treatment and further cell fusion treatment.
-For the purpose of obtaining cerevisiae .

[0007]

[Means for Solving the Problems] The present invention provides a azaserine-resistant strain by subjecting γ-glutamylcysteine synthase gene-introduced glutathione high-producing Saccharomyces cerevisiae to mutation treatment to obtain the azaserine-resistant strain. Saccharomycetes high in glutathione, characterized in that a large amount of glutathione is produced in the cells by aerobic culture, preferably by fed-batch culture.
It is a manufacturing method of Seth cerevisiae .

Further, preferably, the present invention mutates the glutathione high-producing Saccharomyces cerevisiae into which the γ-glutamylcysteine synthetase gene has been introduced to create an azaserine resistant strain, and the amount of the azaserine resistant strain obtained is 1 time. It was obtained by conjugating the body with a haploid of another glutathione high-producing strain to form a polyploid, sporulating the obtained strain, and screening a strain excellent in glutathione-producing and growing ability. A method for producing Saccharomyces cerevisiae having a high content of glutathione, which comprises producing a large amount of glutathione in the cells by aerobically culturing the strain, preferably by fed-batch culture.

More preferably, the present invention is a azaserine-resistant strain haploid obtained by subjecting γ-glutamylcysteine synthetase gene-introduced glutathione-producing Saccharomyces cerevisiae to mutation treatment to create an azaserine-resistant strain. A haploid strain having excellent glutathione-producing ability and growth ability, which was obtained by screening polyploidy by conjugating with a haploid of another glutathione high-producing strain, sporulation of the obtained strain, and screening Saccharomyces containing a high amount of glutathione, characterized in that a large amount of glutathione is produced in the cells by aerobically culturing the strain obtained by cell fusion of the two strains of A.
It is a method of producing S. cerevisiae .

The present invention will be described in detail below. Saccharoma highly producing transgenic glutathione used in the present invention
As Ices cerevisiae , Saccharomyces cerevisiae obtained by the method established by Otake et al. (Abstracts of the Japan Society for Agricultural Chemistry, 1990, p. 145, 1990) can be used. That is, to connect the γ- Guru Tamil cysteine synthetase gene is one of glutathione biosynthetic enzymes derived from yeast to strongly expressed promoter △ P8 of Saccharomyces Streptomyces cerevisiae Shi et chromosomes of Saccharomyces Streptomyces cerevisiae Shi et trp1 Saccharomyces cerevisiae that produces high levels of transduced glutathione can be created by introducing the gene into the site.

[0011] As such yeasts, those deposited by the International Depositary Authority stipulated in the Budapest Treaty with the deposit number of Micro Engineering Research 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, Saccharomyces cerevisiae having a higher glutathione content than in the conventional production method can be obtained without adding an amino acid (L-cysteine), and a mutation can be obtained. Saccharomyces containing highly glutathione by conjugating the azaserine-resistant strains obtained by the treatment and further by cell fusion treatment.
・ You can get cerevisiae .

[0016]

EXAMPLES The present invention will be described in detail below with reference to examples, but the invention is not limited thereto. (1) Saccharomyces which produces high levels of transgenic glutatin
Construction of S. cerevisiae In order to efficiently express the genes in the glutathione biosynthesis system, first, a strong transcription promoter was cloned from the Saccharomyces cerevisiae 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, it was created functional areas only of △ P8 (about 0.4kb).

Next, γ of Saccharomyces cerevisiae
-The glutamylcysteine synthetase gene was cloned. That is, S. cerevisiae YNN27 strain ( α tr
From p1 ura3 ), mutant strains lacking the above two enzymes were obtained. Next, a γ-glutamylcysteine synthetase deficient strain (YH
The γ-glutamylcysteine synthase gene was cloned using 1). When the YH1 strain was transformed with a gene library using YEp24 as a vector and screened using nitroprusside soda, one kind of plasmid (pYOG1001) that 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%.

[0019] (2) Construction YPD agar medium azaserine resistant strain (1% yeast extract, 1% peptone, 2% glucose, 1.5% agar) on at 30 ° C., Sa <br/> Kkaro My Seth cultured for 3 days cerevisiae the YHT178 strain (those without the agar from YPD agar) YPD liquid medium and loopful inoculated entered 500 ml volume shaking flask 100 ml, 30 ° C.,
Culture was performed overnight with shaking. 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 ℃, 20
5 ml of 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 nitrogen base W / O).
Amino acid 0.67%, glucose 2%, agar made by Difco
1.5%) and cultured 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 share and <br/> Saccharomyces Streptomyces cerevisiae of laboratory strains AH22 strain (AT
CC38626) was 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 similarly conjugated, spore formation and spore separation were performed, and about 100 strains 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 Saccharomyces cerevisiae highly producing glutathione obtained
The Sie 62D strain (ura3, his3) was further modified. First,
The plasmid YEp24 was completely digested with the restriction enzyme HindIII, and the plasmid YIo1 was completely digested with the restriction enzyme BamHI. DNA containing URA3 gene fragment subjected to agarose gel electrophoresis
The fragment and the remaining YIp1 plasmid fragment from which the HIS3 gene was removed were isolated. Maniatis (T.) et al.
{Molecular cloning,
Cold Spring Harbor Laboratory (Cold
Spring Harbor Laboratory), 1982}, and the URA3 gene was ligated to a YIp vector from which the HIS3 gene had been removed to prepare plasmid pYI-U3.

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.

Next, cell fusion of the above-prepared two strains was performed. A 500 ml shake flask containing 100 ml of YPD medium was inoculated with the above strain and cultured at 30 ° C. for about 16 hours. After collecting the bacteria by centrifugation and washing with sterile water, STE buffer (1 M sorbitol, 20 mM EDTA, 10 mM Tris-hydrochloric acid (pH 7.
4)) Suspended in 100 ml. 39 μl of β-mercaptoethanol was added thereto and treated at 30 ° C. for 10 minutes. Bacteria were collected by centrifugation, and 0.1 mg / ml concentration of Zymori Ace (Seikagaku Corporation, 10
Suspend in 10 ml of ST buffer (1M sorbitol, 10 mM Tris-hydrochloric acid (pH 7.4)) containing 10,000 units / g.
The reaction was allowed to proceed for minutes to give protoplasts. The cells were collected by centrifugation and washed once with ST buffer. Then 10 mM Ca
It was suspended in 10 ml of ST buffer (STC buffer) containing Cl ₂ . The number of cells in each suspension was counted by a hemocytometer, and 1 × 10 ⁷ cells 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), suspended in 10 mM CaCl ₂ ) and fused at 30 ° C. for 10 minutes. 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. About 100 strains of grown colonies were transferred to YPD medium and further cultured at 30 ° C. for 7 days.

Approximately 100 strains were cultivated in a test tube containing 5 ml of YPD medium at 30 ° C. for 3 days with shaking, and excellent strains were selected using the 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 3 shows each strain using YPD medium 3
The results of shaking culture at 0 ° C. for 3 days are shown.

[0029]

[Table 3] (5) Saccharomyces cerevisiae with high glutathione content
D. Cultivation, part 1 Glucose 2%, yeast extract (manufactured by Difco) 1%, Bactopeptone (manufactured by Difco) 1% was prepared as a medium and shake-cultured in a 500 ml shake flask at 100 ml for 24 hours. , As the inoculum solution. 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 the inoculation.

1 ml of the 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 absorption was increased at 412 nm. 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 4 shows a comparison with 178 strains. Table 4 shows 62D / 62D strain and Y
The comparison of HT178 strain is shown.

[0031]

[Table 4] (6) Saccharomyces cerevisiae with high glutathione content
D. Cultivation, Part 2 A medium consisting of 2% glucose, 1% yeast extract (manufactured by Difco) and 1% bactopeptone (manufactured by Difco) was 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.

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.

1 ml of a culture solution containing yeast cells was subjected to hot water extraction treatment, and after centrifugation, the cell extract obtained had a glutathione production amount 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 5 shows the amount of glutathione production, the glutathione content, and the dry cell weight after 48 hours of culture. Table 5 shows the glutathione production of the 62D / 62D strain.

[0034]

[Table 5]

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Claims (4)

[Claims] 1. A high-producing glutathione-producing Saccharomyces cerevisiae cell into which a γ-glutamylcysteine synthetase gene has been introduced.
A high abundance of glutathione, characterized by producing abundant amount of glutathione in cells by aerobically culturing the azaserine-resistant strains by mutating Leviche (Saccaromyces cerevisiae). Saccharomyces cerevisiae manufacturing method. 2. A saccharo with high production of transgenic glutathione.
Maisesu cerevisiae, Y HT178 strain (FERM BP first
13012), The method for producing Saccharomyces cerevisiae having a high glutathione content according to claim 1. 3. Saccharomyces containing high amounts of glutathione
Cerevisiae, containing glutathione height of claim 1, characterized in that 6 is a 2D strain (FERM BP-13010) Saccharopolyspora
Production method of Lomyces cerevisiae . 4. Saccharomyces containing high amounts of glutathione
Cerevisiae is a 62D / 62D strain (Mikoken Kenjoyori No. 13011)
The high content of glutathione according to claim 1, wherein
Saccharomyces cerevisiae manufacturing method.