JPH10243783A - Creation of stress-resistant yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject yeast that is useful in the production of food and beverage (brewage, baking) by fermentation by increasing the trehalose content in the cell bodies of the yeast. SOLUTION: The content of (neutral) trehalose in the cell bodies of a yeast such as Saccharomyces cerevisiae is increased by breaking the NTH1 gene coding neutral trehalose by partial defection (using a mismatch primer of NTHI gene) or by self-cloning (YEp24 is used as a vector and URA3 gene is employed) to prepare the objective yeast that maintains resistance to stress (for example, resistances to alcohol, osmotic pressure and heat, and durability) and is genetically stable. When this yeast is used, the conventional restriction as conditions for food production is improved and beverage and food products can efficiently be produced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a stress-resistant yeast, and more particularly to a method for producing a yeast having improved stress resistance by increasing the trehalose content in yeast cells.

[0002]

2. Description of the Related Art Conventionally, yeasts having improved stress resistance,
For example, yeast having alcohol resistance, osmotic resistance, durability, heat resistance and the like has been obtained by mutating yeast. There are two main methods for this mutation. The first is natural mutation, such as the acclimatization method that gradually increases the tolerance of yeast to each stress, and the selection method using natural yeast mutation. It is a method of utilizing. The second method is a method in which the yeast is irradiated with X-rays, radiation, ultraviolet rays, or the like, or a mutagen such as a chemical substance is allowed to act on the yeast, so that the mutation treatment is actively performed to induce the mutation.

[0003] On the other hand, although the mechanism of highly stress-resistant yeast has not been completely elucidated, since the trehalose content in the cells is higher than that of stress-sensitive yeasts, the yeast cells in the yeast cells have a higher trehalose content. There is increasing interest in the physiological effects of trehalose. Trehalose is a non-reducing disaccharide in which two molecules of D-glucose are linked by α-1,1 and is contained in microorganisms such as yeast, algae, lichens, and many insects. This trehalose has a biological protection effect such as retaining water in the living body. In particular, insects not only exist as the main blood sugar in the lymph of the blood but also have an effect as an antifreeze, and their concentration is adjusted according to the season to obtain cold resistance. well known.

[0004]

By the way, in the above-mentioned spontaneous mutation by the conventional acclimatization method and the selection method, the improvement of the stress resistance of the yeast is slight, and few of them can withstand practical use at present. . In addition, in breeding by the mutation method, a reversion mutation in which the wild-type phenotype is restored may occur, and it is difficult to stably obtain a mutant strain maintaining characteristics such as stress resistance. Therefore, there was a practical problem. Accordingly, an object of the present invention is to provide a method for producing a genetically stable yeast which can maintain excellent stress resistance and is genetically stable by a safe and simple method.

The present inventors believe that if trehalose, which is present in yeast cells and has a bioprotective effect, can be accumulated in the cells, a yeast with improved stress resistance can be produced. Was examined repeatedly. As a result, NTH1 encoding neutral trehalase in yeast
It has been found that it is effective to destroy a gene by using a genetic engineering technique, and that it is possible to accumulate trehalose in a yeast cell, and have reached the present invention.

[0006]

Means for Solving the Problems The present invention according to claim 1 is a method for producing a stress-tolerant yeast, characterized by increasing the trehalose content in a yeast cell. The present invention according to claim 2 provides the method for producing yeast according to claim 1, wherein the NTH1 gene of the yeast encoding neutral trehalase is disrupted by genetic engineering to increase the trehalose content in the yeast cells. is there. The present invention as set forth in claim 3 provides the yeast N-encoding neutral trehalase.
The method for producing yeast according to claim 2, wherein the TH1 gene is disrupted by partial deletion or self-cloning of the gene. According to a fourth aspect of the present invention, there is provided a method for producing a food or drink, wherein a stress-resistant yeast produced by the method according to the first aspect is used in producing a food or drink by a fermentation method.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The yeast (parent strain) that can be used in the present invention is not limited, but yeast used for producing food and drink by a fermentation method is preferable. For example, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces rosevi (S. rosei), Saccharomyces exiguus (S. exiguus), Zygosaccharomyces roxii (Zygos)
accharomyces rouxii), Candida Versatiris
(Candida versatilis), Candida Etchersi
(C. etchellsii) and the like, and particularly, Saccharomyces cerevisiae used as baker's yeast, sake yeast and the like are preferable.

[0008] As a method for accumulating trehalose in yeast cells, the present invention employs a method in which NTH1, which is a gene encoding neutral trehalase in yeast, is destroyed using genetic engineering techniques. That is, N, which encodes a neutral trehalase that has the action of hydrolyzing trehalose,
The purpose is to prevent the normal transcription and translation of the enzyme by mutating the base sequence of the TH1 gene. Specific genetic engineering techniques for disrupting the NTH1 gene include a method of partially deleting the NTH1 gene and the use of other genes in the same type of yeast as the NTH1 gene.
A method of homologous recombination with a DNA fragment inserted into the TH1 gene, that is, a method of self-cloning may be mentioned.

The method for partially deleting the yeast NTH1 gene is not particularly limited as long as it is a commonly used method, but a method using a mismatch primer of the NTH1 gene [Kunkel et al., Methods i
n Enzymology, 154, 367-382 (1987)]. The method of partially deleting the NTH1 gene using this mismatch primer is based on the base to be converted from the NTH1 gene, with 8 to 10 bases before and after, and 15 to 15 bases in total.
Synthesize an oligonucleotide of about 20 bases and use Mutan-K
A method using a kit (manufactured by Takara Shuzo Co., Ltd.) is preferable.

Next, self-cloning will be described. In this self-cloning, as a gene to be inserted into the NTH1 gene, in addition to the URA3 gene using YEp24 as a vector, for example, in the case of Saccharomyces cerevisiae, a TRP1 gene using YRp7 as a vector [Eco RI fragment of 1.5 kb, Strufl et al. ., Proceedingof
National Academic Science, 76, 1035 (1979)], pD
LYS2 gene using A6200 as a vector [4.6 kb Ec
o RI-Hind III fragment, Barnes et al., Molecular Cell B
iology, 6, 2828 (1986)], LEU2 gene using YEp13 as a vector [Sal I-Xho I fragment of 2.2 kb, Branch et al.
l., Gene, 8, 121 (1979)], ARG4 gene using pCL1 as a vector [3.0 kb Hind III-Hind III fragment, Hsiao
et al., Gene, 15, 157 (1981)].

The specific operation of self-cloning will be described in detail below. (1) Preparation of DNA vector fragment containing NTH1 gene Plasmid pNTH0.1 containing yeast NTH1 gene
~ 20 μg, preferably 10-15 μg of the restriction enzyme Eco.
RI 5 to 50 units, preferably 10 to 20 units,
1-5 μl of 10 × EcoRI buffer, preferably 1-2
Sterile water is added to μl to make a total of 10 to 50 μl, preferably 10 to 20 μl, and incubated at 30 to 37 ° C., preferably 37 ° C. for 30 to 180 minutes, preferably 60 to 120 minutes. After incubation, dye staining solution (BPB-XC solution; 0.25% bromophemol blue, 0.25%
Xylene cyanol, 25% ficoll 400) is added in an amount of 1 to 5 μl, preferably 2 to 3 μl, loaded into a sample slot of an agarose gel, and subjected to electrophoresis. The electrophoresis is carried out on an agarose gel of 0.8 to 1.5%, preferably 0.8 to 1.0%.
% To 100-200 in 1 × TAE buffer
After the electrophoresis, the gel is stained with 0.00005% EtBr-1 × TAE staining solution for 30 to 60 minutes, preferably 30 minutes. Further, the gel is irradiated with a long-wavelength UV lamp, and a band around 1.5 kb is cut out with a sterilized scalpel and attached to a TE buffer. Thereafter, the dialysis tube was immersed in TE buffer and autoclaved.
1), put the excised gel, and perform electrophoresis.
Electrophoresis is performed at 150 V for 30 minutes. After the electrophoresis, the TE buffer is extracted from the dialysis tube, and an equal volume of phenol-chloroform (1: 1) is added and stirred. After stirring, 10,000 to 15000 rpm, preferably 13
Centrifuge at 000 rpm for 10 to 20 minutes, preferably 15 minutes, and collect the aqueous layer. Further, an equal amount of chloroform is added to the aqueous layer, the mixture is stirred again, and then centrifuged in the same manner to collect the aqueous layer.
Two volumes of cold ethyl alcohol was added to the aqueous layer, and -30 was added.
15 minutes or more at -80 ° C, preferably -50 to -80
Cool at 30 ° C. for 30-60 minutes and centrifuge similarly. After discarding the supernatant, 1 ml of cold 70% ethyl alcohol was added to the precipitate.
In addition, after stirring, the same centrifugation is performed, and the mixture is air-dried at room temperature, and then dissolved in a TE buffer at a DNA concentration of 1 to 20 μg / μl, preferably 5 to 10 μg / μl. Use as a DNA fragment solution.

(2) Preparation of Escherichia coli Holding a Plasmid Modified with a Restriction Enzyme Cleavage Site According to the type of DNA fragment to be inserted into the NTH1 gene, a plasmid having a restriction enzyme cleavage site modified if necessary is prepared. Prepare the retained E. coli. Vector plasmid, preferably pUC19 or pUC11
9 Sterilize to 1 to 20 μg, preferably 5 to 10 μg, 5 to 50 units of restriction enzyme that cuts the site to be modified, preferably 10 to 20 units, 1 to 5 μl of 10-fold concentration restriction enzyme buffer, preferably 1 to 2 μl. Add water,
A total of 10 to 50 μl, preferably 10 to 15 μl, is incubated at 30 to 60 ° C., preferably 37 ° C. for 30 to 60 minutes, preferably 30 minutes. After the incubation, if necessary, add 0.1 to 2 units of Klenow enzyme, preferably 0.2 to 0.5 unit, and add 30 units.
Incubate at ３７37 ° C., preferably at 37 ° C. for 30-60 minutes, preferably 30 minutes, to blunt-end the restriction enzyme cut surface. Furthermore, T4 DNA ligase 0.1 ~
Add 1 unit, preferably 0.1 unit, at 16 ° C
For 12 to 24 hours, preferably 16 to 20 hours. E. coli, preferably HB
200 competent cells (concentration OD60
0 = 10) and cool in water for 30 minutes. continue,
Incubate at 42 ° C for 2 minutes, and immediately cool on ice for 5 minutes. This solution was applied to an LB-Amp plate (the composition of the medium is shown below), and the solution was applied at 37 ° C. for 12 to 24 hours.
The culture is performed for a time, preferably 16 to 20 hours. The appearing colony is defined as Escherichia coli holding a plasmid having a modified restriction enzyme cleavage site.

Composition of LB-Amp plate Bacto trypton (Difco) 1.0% Yeast extract (Difco) 0.5% NaCl 1.0% Ampicillin 0.005% Agar 2.0%

(3) Preparation of Plasmid with Modified Restriction Enzyme Cleavage Escherichia coli harboring a plasmid with a restriction enzyme cleavage site modified was inoculated into an LB-Amp medium at 37 ° C. and 140 rpm.
And culture overnight. After the culture, the cells are centrifuged at 5000 rpm for 5 minutes, and a plasmid having a restriction enzyme cleavage site modified from the cells using a QIAGEN-midi kit (Funakoshi) is prepared.

(4) Preparation of Plasmid Containing NTH1 Gene Subsequently, Escherichia coli carrying a plasmid containing the NTH1 gene is prepared. The operation of the plasmid prepared by the method of (3) is carried out under the conditions of (1). Then, a restriction enzyme EcoRI is allowed to act to prepare a plasmid containing the gene by the methods (2) and (3) described above.

(5) Preparation of another gene to be inserted Next, a gene to be inserted into the NTH1 gene is prepared from another plasmid, and the operation is performed in the same manner as in the above (1) preparation of a DNA vector fragment containing the NTH1 gene. . However,
The restriction enzymes used are Hind III or Eco RI, Bam HI, Sal I, Xh
o Let it be I. Thus, a DNA fragment containing another gene of 1.1 to 4.6 kb can be obtained.

(6) Preparation of a plasmid having another gene inserted into the NTH1 gene Plasmids 5 to 10 containing the NTH1 gene obtained in (4)
μg of the restriction enzyme Hind III or
EcoRI, BamHI, SalI, XhoI, and a fragment 1: 1 to 1:10, preferably 1: 1, containing the other gene of 1.1 to 4.6 kb obtained in (5). The mixture is mixed at a ratio of 2-1: 5, and ligation is performed by the method (2). By this operation, a plasmid having another gene inserted into the NTH1 gene is prepared.

(7) Preparation of a DNA vector fragment containing the NTH1 gene into which another gene has been inserted The DNA fragment obtained in the above (6) is prepared using the above (2), (3)
A plasmid containing the NTH1 gene into which another gene was inserted was prepared in the same manner as described above.
g, preferably 5 to 10 μg, of the restriction enzyme EcoRI is allowed to act by the above-mentioned method (1). By this operation, NTH
A DNA fragment having one gene inserted with another gene is prepared.

(8) Transformation Next, transformation is performed by homologously recombining the DNA fragment obtained in (7) with the NTH1 gene on the yeast chromosome. Transformation is not particularly limited as long as it is a commonly used method, but the lithium acetate method is particularly preferable. The parent strain (ura3) for which a transformant is to be obtained is designated Y
25 to 30 in a PD medium (medium composition is shown below)
The cells are cultured at 5 ° C. for 5 to 10 hours, preferably at 30 ° C. for 6 to 8 hours, and centrifuged at 3000 rpm for 5 minutes to collect the cells. The collected bacteria are suspended in 2 to 10 ml, preferably 4 to 6 ml of TE buffer, in which 0.4 to 1 ml, preferably 0.1 to 1 ml, is suspended.
Add 4 to 0.6 ml of 1M lithium acetate solution and add 25 to
30 to 90 minutes at 30 ° C, preferably 6 to 28 to 30 ° C.
Shake for 0-90 minutes. After shaking, 1 to 10 μg, preferably 2 to 5 μg of the DNA fragment prepared in the above (7) is added, and the mixture is added at 25 to 30 ° C. for 20 to 90 minutes, preferably 28 to
Incubate at 30 ° C. for 30-50 minutes. Next, 5
Add 500-1000 μl, preferably 600-800 μl, of 500% PEG 4000 and add 30-30 ° C. at 25-30 ° C.
Incubate for 90 minutes, preferably at 28-30 ° C for 50-70 minutes. Further, 400 to 1000 μl, preferably 400 to 600 μl of 1M sorbitol is added, and the mixture is centrifuged at 5000 rpm for 10 minutes. The transformant thus obtained was used in a uracil-deficient restriction medium (0.
67% Yeast nitrogen base w / o amino acid, Difco) at 25-30 ° C for 1-4 days, preferably 28-3
By culturing at 0 ° C. for 2 to 3 days, it can be obtained as a colony grown on the medium. Since this transformant has the URA3 gene inserted into the NTH1 gene, it can grow in a uracil-deficient restriction medium, and the NTH1 gene encoding trehalase is disrupted by other genes. It has the property that trehalase cannot be transcribed and translated normally.

Composition of YPD medium Pepton 1.0% Yeast extract 0.5% Glucose 3.0%

[0021] The yeast obtained by the above-described method accumulates trehalose in the cells three or more times in the logarithmic growth phase and 1.5 to 5 times in the stationary phase as compared with the parent strain, and has a low stress resistance. Since it is improved, it has excellent properties such as alcohol resistance, osmotic pressure resistance, durability, and heat resistance. For this reason, by using the yeast, in the industrial alcohol production, brewing liquor production, distilled liquor production, bread production, miso / soy sauce production, yeast production, and the like, the production conditions and the like are more restricted than in the case where conventional yeast is used. Is improved, and the target product can be manufactured effectively.

[0022]

The present invention will be described in detail below, but the present invention is not limited by these. Example 1 Saccharomyces cerevisiae (ura3, α strain) [Boeke
et al., Mol. Gen. Gent., 197, 345 (1984)], and a transformant in which the NTH1 gene was disrupted from the parent strain by self-cloning was prepared by the following method. The gene to be inserted into the NTH1 gene is plasmid YEp24 [Botstein et al., Gene, 8, 17-24 (1979)].
The 1.1 kb URA3 gene prepared above was used.
All reagents were prepared from commercially available special grade products, and all reagents and instruments used were autoclaved. Then
The trehalose content contained in the obtained transformant was measured.

(1) Preparation of DNA Vector Fragment Containing NTH1 Gene A plasmid pNTH containing the NTH1 gene of Saccharomyces cerevisiae [Kopp et al., J. Biol. Chem., 268,
4766-4774 (1993)] Restriction enzyme Eco RI (Takara Shuzo Co., Ltd.) 15 units to 10 μg, 10 × Eco RI buffer 2
Sterile water was added to the mixture to make a total of 20 μl.
For 120 minutes. After incubation, dye staining solution (BPB-XC solution; 0.25% bromophemol blu
e, 0.25% xylene cyanol, 25% ficoll 400)
Was added to a sample slot of an agarose gel, and electrophoresis was performed. The electrophoresis was performed at a concentration of 1.0% agarose S (manufactured by Nippon Gene Co., Ltd.) in 1 × TA.
The gel was stained with 0.00005% EtBr-1 × TAE staining solution for 30 minutes after electrophoresis at 100 V for 120 minutes in an E buffer. Further irradiate the gel with a long wavelength UV lamp,
Cut out a band around 1.5 kb with a sterilized scalpel,
Dipped in E buffer. Then, immersed in TE buffer, added 500 μl of TE buffer to the autoclaved dialysis tube, put the cut gel,
Electrophoresis was performed. Electrophoresis was performed at 150 V for 30 minutes. After the electrophoresis, remove the TE buffer from the dialysis tube, and use an equal volume of phenol-chloroform (1: 1).
Was added and stirred. After stirring, the mixture was centrifuged at 13000 rpm for 15 minutes to collect an aqueous layer. Further, an equal amount of chloroform was added to the aqueous layer, and the mixture was stirred again, followed by centrifugation in the same manner to collect the aqueous layer. To this aqueous layer was added two volumes of cold ethyl alcohol,
After cooling at 80 ° C. for 60 minutes, centrifugation was performed in the same manner. After discarding the supernatant, 1 ml of cold 70% ethyl alcohol was added to the precipitate.
In addition, the mixture was stirred, centrifuged in the same manner, air-dried at room temperature, and dissolved in a TE buffer to a DNA concentration of 10 μg / μl to obtain a 1.5 kb DNA fragment containing the gene.

(2) Preparation of Escherichia coli carrying a plasmid from which the Hind III site has been deleted Vector plasmid pUC19 (Takara Shuzo) 5
Twenty units of HindIII (manufactured by Takara Shuzo Co., Ltd.) were added to μg, and sterile water was added to 1.5 μl of 10 × HindIII buffer to make a total of 15 μl and incubated at 37 ° C. for 30 minutes. After the incubation, 0.5 unit of Klenow enzyme (Takara Shuzo Co., Ltd.) was added, and Hind III was added.
The mixture was incubated under the same conditions as described above, the cut surface of the restriction enzyme was blunt-ended, 0.1 unit of T4 DNA ligase (Takara Shuzo) was added, and the mixture was incubated at 16 ° C. for 16 hours. To this DNA solution, 200 µl of competent cells of Escherichia coli HB101 (manufactured by Takara Shuzo Co., Ltd.) (concentration OD 600 = 10) was added, followed by cooling in water for 30 minutes. Subsequently, the mixture was incubated at 42 ° C. for 2 minutes, and immediately after completion of the incubation, the mixture was ice-cooled for 5 minutes. This solution is LB-Am
It was applied to a p-plate and cultured at 37 ° C. for 16 hours. The appeared colonies were defined as Escherichia coli carrying the plasmid from which the Hind III site had been deleted.

(3) Preparation of plasmid from which Hind III site was deleted Escherichia coli harboring the plasmid from which Hind III site was deleted was inoculated into LB-Amp medium and cultured overnight at 37 ° C. and 140 rpm. After the culture, the cells were centrifuged at 5000 rpm for 5 minutes, and a plasmid from which HindIII site was deleted was prepared from the cells using a QIAGEN-midi kit (Funakoshi).

(4) Preparation of Plasmid Containing NTH1 Gene Subsequently, Escherichia coli carrying a plasmid containing the NTH1 gene is prepared, and the plasmid prepared by the above method (3) is prepared under the conditions of the above (1). The restriction enzyme EcoRI was allowed to act, and a plasmid containing the gene was prepared by the methods (2) and (3) described above.

(5) Preparation of URA3 Gene to be Inserted Next, a gene to be inserted into a 1.5 kb DNA vector fragment containing the NTH1 gene was prepared from the plasmid YEp24. The preparation was carried out by the above (4) DN containing the NTH1 gene.
The procedure was the same as that for preparing the A vector fragment. However, Hind III (manufactured by Takara Shuzo Co., Ltd.) was used as a restriction enzyme. As a result, a DNA fragment containing the 1.1 kb URA3 gene could be obtained.

(6) Preparation of a plasmid having the URA3 gene inserted into the NTH1 gene 5 μg of the plasmid containing the NTH1 gene obtained in (4) was treated with the restriction enzyme Hind III in the same manner as in (5) above.
Then, the DNA fragment containing the URA3 gene of 1.1 kb obtained in (5) was mixed at a ratio of 1: 5, and then mixed with the above (2).
Ligation was performed in the same manner. By this operation, N
A plasmid having the URA3 gene inserted in the TH1 gene was prepared.

(7) NTH1 into which URA3 gene is inserted
Preparation of DNA Vector Fragment Containing Gene The DNA fragment obtained in the above (6) is converted into the above (2), (3)
NTH1 into which the URA3 gene was inserted by the same method as described above.
A plasmid containing the gene was prepared, and the restriction enzyme EcoRI was allowed to act on 5 μg of the plasmid by the method described in (1) above. By this operation, a 2.6 kb DNA fragment in which the URA3 gene was inserted into the NTH1 gene was prepared.

(8) Transformation Next, the DNA fragment obtained in (7) is homologously recombined with the NTH1 gene on the chromosome of Saccharomyces cerevisiae (ura3, α strain). Made by. First, a parent strain (ura3) from which a transformant was to be obtained was cultured in a YPD medium at 30 ° C. for 8 hours, centrifuged at 3000 rpm for 5 minutes, and collected. The collected bacteria were suspended in 5 ml of TE buffer,
ml of 1M lithium acetate solution was added and shaken at 30 ° C. for 60 minutes. After shaking, 2 μg of the 2.6 kb DNA fragment prepared in the above (7) was added, and the mixture was incubated at 30 ° C. for 40 minutes. Next, 700 μl of 500% PEG 4000
(Manufactured by Wako Pure Chemical Industries, Ltd.) and incubated at 30 ° C. for 60 minutes. Further, 500 μl of 1M sorbitol was added, and the mixture was centrifuged at 5000 rpm for 10 minutes. The transformant thus obtained was used in a uracil-deficient restriction medium (0.67% yeast nitrogen base w / o amino acid,
By culturing the cells at 30 ° C. for 2 days in the same culture, colonies grown on the medium could be obtained.
The trehalose content of the thus obtained transformant and parent strain was extracted with 15% TCA and then measured by high performance liquid chromatography.

As a result, the trehalose content was 0% in the logarithmic growth phase and 0.56% in the transformed strain per log dry cell weight. In the stationary phase, the parent strain was 2.4%, while the transformed strain was 12.0%, indicating that the trehalose content was increased five-fold.

Example 2 Using the transformed strain of Saccharomyces cerevisiae (ura3, α strain) prepared in Example 1 and its parent strain in the logarithmic growth phase, the alcohol resistance of these yeasts was examined. The evaluation of alcohol resistance was performed based on the survival rate when the ethyl alcohol concentration in the medium was changed and kept at -4.5 ° C for a certain period. In addition, the measurement of the survival rate
The measurement was performed by the plate measurement method.

When maintained at an ethyl alcohol concentration of 14% for 2 weeks, the survival rate of the parent strain was 56%, whereas the survival rate of the transformed strain was 100%. Further, when the same period was maintained at an ethyl alcohol concentration of 20%, the survival rate of the parent strain was remarkably reduced to 0.29%, whereas the survival rate of the transformed strain was maintained at a high level of 76%. . Furthermore, the survival rate of the parent strain when it was maintained for 1 month at an ethyl alcohol concentration of 20% was further reduced to 0.16%, while the survival rate of the transformed strain was 70%.
It was almost the same level as when it was kept for two weeks. This indicated that the transformed strain had better alcohol tolerance than the parent strain due to the increased trehalose content in the cells.

Example 3 Using the transformed strain of Saccharomyces cerevisiae (ura3, α strain) prepared in Example 1 and its parent strain in the logarithmic growth phase, the osmotic resistance of these yeasts was examined. The osmotic pressure resistance was evaluated by changing the sodium chloride concentration in the medium and maintaining the survival rate for a certain period of time using a plate measurement method.

When the parent strain was kept at -4.5 ° C. for 1 week at a sodium chloride concentration of 29%, the survival rate of the parent strain was 16%, whereas the transformed strain was 71%. Further, when the cells were kept at a sodium chloride concentration of 28.5% at 4 ° C. for one month, the number of transformed strains was 4.0 compared to 0.16% of the parent strain.
% And a 25-fold higher viability, indicating excellent osmotic pressure resistance.

Example 4 The durability of these yeasts was examined using the transformed strain of Saccharomyces cerevisiae (ura3, α strain) prepared in Example 1 and its parent strain in the logarithmic growth phase.
The durability was examined based on the survival rate when kept at 15 ° C. for one month, and the survival rate was measured by a plate measurement method. As a result, the survival rate of the parent strain was 2.2%, while that of the transformed strain was 89%, which was 40 times or more higher than that of the parent strain. This revealed that the transformed strain had better durability than the parent strain.

Example 5 Using the transformed strain of Saccharomyces cerevisiae (ura3, α strain) prepared in Example 1 and its parent strain in the logarithmic growth phase, the heat resistance of these yeasts was examined.
The heat resistance was evaluated by the survival rate after heating the yeast together with the medium at 52 ° C. for 10 minutes, and the survival rate was measured by a plate measurement method. As a result, while the survival rate of the parent strain was 0.4%, the transformed strain was 47%, showing a survival rate 118 times higher than that of the parent strain, and the transformed strain had excellent heat resistance. It became clear that there was.

[0038]

According to the present invention, it is possible to produce a genetically stable yeast which is excellent in alcohol resistance, osmotic pressure resistance, durability and heat resistance and is safe and simple. Therefore, industrial alcohol production, brewing,
In the production of distilled liquor, bread, miso / soy sauce, yeast, etc., by improving the stress resistance of the yeast used in these methods by the method of the present invention, the desired product can be produced under simpler conditions than conventional methods. Can be manufactured well.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12G 3/02 119 C12G 3/02 119G C12N 1/19 C12N 1/19 // (C12N 15/09 C12R 1: 865) (C12N 1/19 C12R 1: 865)

Claims (4)

[Claims] 1. A method for producing a stress-resistant yeast, comprising increasing the trehalose content in a yeast cell. 2. The yeast N encoding a neutral trehalase
The method for producing yeast according to claim 1, wherein the trehalose content in the yeast is increased by disrupting the TH1 gene by a genetic engineering technique. 3. The yeast N encoding neutral trehalase
The method for producing yeast according to claim 2, wherein the TH1 gene is disrupted by partial deletion or self-cloning of the gene. 4. A method for producing a food or drink, comprising using the stress-resistant yeast produced by the method according to claim 1 in producing the food or drink by a fermentation method.