JP3966887B2 - New yeast gene - Google Patents

Description

  The present invention relates to a refrigerated dough baking method and an ethanol manufacturing method.

  In the bakery industry, refrigerated dough bread making methods have become widespread for the purpose of saving labor in the manufacturing process and meeting the needs of various consumers. The refrigerated dough baking method is a method in which partially fermented dough is put in a refrigerator or the like and stored at low temperature, and then bread is produced through fermentation, proofing and baking. In the refrigerated dough making method, fermentation is suppressed while being stored at low temperature in the dough, so-called “refrigerated resistant yeast”, which has the ability to normally ferment and expand the dough in the temperature range of fermentation and proofing. Is used.

As a method for breeding refrigeration-resistant yeast, there are known methods in which a wild-type yeast is subjected to a mutation treatment to give the yeast a mutation that exhibits low-temperature sensitivity (for example, Patent Documents 1 to 3 and non-patent documents). Reference 1). Yeast to which a fermentative ability is imparted with a mutation exhibiting low-temperature sensitivity is used as refrigeration-resistant yeast or as a parent strain for breeding refrigeration-resistant yeast.
However, since mutations are caused randomly in the mutation treatment, not only low-temperature sensitive mutations but also mutations related to basic characteristics of fermentation such as bread dough expansion ability are given to yeast. There is a possibility that.

On the other hand, a method for imparting useful properties such as agglutination (see Non-Patent Document 2) and aroma generation (see Non-Patent Document 3) to baker's yeast or brewing yeast is known. Yes.
However, there are no known methods for breeding refrigeration-resistant yeast using genes involved in the low-temperature sensitivity of fermentability and gene manipulation techniques.

  Ethanol is produced by a fermentation method from a sugar raw material such as molasses or a starch raw material such as corn or potato as a carbon source. Fermentation is usually carried out at 30 to 43 ° C., but an increase in fermentation temperature causes death of yeast, poor growth or a decrease in fermentation power, so cooling is usually carried out to a temperature of 30 to 35 ° C. However, cooling is insufficient when the temperature rises, such as in summer, and the culture temperature may rise to about 35 to 38 ° C. during alcohol fermentation. For this reason, in alcohol fermentation, cooling is usually performed in order to suppress a temperature rise due to fermentation heat. In order to reduce the cooling cost, yeast having temperature resistance is required.

As a method for breeding yeast having temperature tolerance, a method for introducing mitochondria involved in temperature tolerance (see Non-Patent Document 4), a method for highly expressing heat shock protein HSP104 (see Non-Patent Document 5), and the like have been reported. However, application to alcoholic fermentation has not been studied. In addition, it is known that heat resistance is improved when the yeast is heat-treated at a non-lethal temperature (see Non-Patent Document 6), but this effect is transient and difficult to apply to alcohol fermentation. is there.
Japanese Patent Publication No. 7-71474 JP-A-7-213277 JP-A-7-79767 Applied and Enbiolomental Microbiology (Appl. Environ. Microbiol.), 61, 639-642 (1995) 23rd European Brewery Convention Proceeding (23rd Europran Brewry Conv., Proc.), 297-304 (1991) Current Genetics (Curr. Genet.), 20, 453-456 (1991) Curr. Genet., 13, 461-469 (1988) Proc. Natl. Acad. Sci. USA, 93, 5301-5306 (1996) J. Bacteriol., 156, 1363 (1983)

  An object of the present invention is to provide a refrigerated dough baking method and an ethanol manufacturing method.

The present invention is a protein comprising the amino acid sequence shown in SEQ ID NO: 2 , or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 2 , and has a low temperature sensitivity of fermentation ability. A protein that complements the indicated mutation, a gene encoding the protein, and a DNA consisting of the base sequence shown in SEQ ID NO: 1, or one or more DNAs added, deleted or substituted in the base sequence shown in SEQ ID NO: 1 In addition, the present invention relates to a gene comprising DNA that complements a mutation whose fermentability is low-temperature sensitive. Further, the present invention is a yeast belonging to the genus Saccharomyces ( Saccharomyces ), characterized in that the above-mentioned gene on the chromosome is inactivated, fermenting ability is low temperature sensitive yeast, dough containing the yeast, A method for producing bread characterized by adding yeast to the dough, a method for producing ethanol characterized by culturing the yeast in a medium, accumulating ethanol in the culture, and collecting ethanol from the culture About.

  According to the present invention, it is possible to provide a protein or gene that complements a mutation whose fermentation ability exhibits low-temperature sensitivity, a refrigeration-resistant yeast obtained by inactivating the gene, and a method for producing bread and ethanol using the yeast. it can.

  In the present invention, the fermentative ability exhibits low temperature sensitivity means that it has substantially no fermentative ability in the temperature range of low temperature storage and has normal fermentative ability in the temperature range of fermentation or proofing after low temperature storage. It means to show. For example, baker's yeast has substantially no dough swelling ability at 5 ° C. and normal dough swelling ability at 20-40 ° C. after refrigerated storage at 5 ° C. for 1-7 days. That is, in the case of alcoholic yeast, it does not substantially show alcoholic fermentation at 5 ° C, and after normal refrigerated storage at 5 ° C for 1 to 7 days, it has normal alcoholic fermentation ability at 20 to 40 ° C. Means.

For the basic procedures related to genetic engineering or biotechnology used to isolate a gene whose fermentative ability exhibits a low temperature sensitivity, isolate the gene, determine the gene sequence, and inactivate the gene, a commercially available experiment document For example, Gene Manual Kodansha, Yasutaka Takagi, Gene manipulation experiment method Kodansha, Molecular Cloning, Cold Spring Harbor Laboratory (1982), Molecular Cloning, Second Edition (Molecular Cloning, 2nd ed.) Cold Spring Harbor Laboratory (1989), Methods in Enzymology, 194 (1991), Experimental Medicine Separate Volume, Genetic Experiments with Yeast (1994) or the like.

  A gene that complements a mutation that exhibits low temperature sensitivity of the fermentation ability of the present invention (hereinafter referred to as a gene that complements low temperature sensitivity) is, for example, Saccharomyces cerevisiae RZT-3 (FERM BP-3871 described in JP-A-5-336872). ) Strain (hereinafter referred to as RZT-3 strain) can be isolated as a gene that complements the low temperature sensitivity of the fermentation ability. In other words, the RZT-3 strain is transformed with a yeast DNA library having a gene that complements low-temperature sensitivity, and the low-temperature sensitivity is complemented by obtaining DNA from a strain supplemented with a mutation that exhibits low-temperature sensitivity of fermentation ability. The gene can be isolated.

  A yeast DNA library having a gene complementary to low temperature sensitivity is obtained by cleaving a chromosomal DNA of a yeast having a wild type gene, for example, Saccharomyces cerevisiae strain X2180-1B (hereinafter referred to as X2180-1B strain) with a restriction enzyme. The obtained DNA fragment can be produced by ligating with a vector capable of being retained in yeast. Any restriction enzyme can be used as long as it can cleave chromosomal DNA, but a restriction enzyme that generates a DNA fragment of 20 Kbp or less is preferably used. In addition, the chromosomal DNA may be completely cut or partially cut by a restriction enzyme.

Examples of vectors that can be maintained in yeast include YCp type vectors, YEp type vectors, YRp type vectors, YIp type vectors, and YAC (yeast artificial chromosome) vectors.
Transformation of the DNA library into the RZT-3 strain is carried out by the spheroplast method [for example, Proc. Natl. Acad. Sci. USA, 75, 1929-1933. (1978)], lithium acetate methods [eg, Journal of Bacteriol., 153 , 163-168 (1983)], electroporation methods [eg, Methods in Enzymology, 194 , 182-187 (1991)] and the like, and can be carried out according to a method commonly used in the field of genetic engineering or biotechnology.

Complementation of a mutation that shows low-temperature sensitivity in fermentation ability can be confirmed by examining the transformed yeast for growth at low temperature or fermentation ability at low temperature [Applied and Enviromental Microbio Rosie, 61 , 639-642 (1995)]. In order to investigate the fermentability at low temperature, for example, the following dye agar overlay method can be used. The strain to be evaluated is cultured on YPG agar medium (1% yeast extract, 2% peptone, 3% glycerol, 2% agar) at 30 ° C. to form colonies, and then pigment agar (0.5% yeast extract) is formed on the medium. , 1% peptone, 10% sucrose, 0.02% bromcresol purple, 1% agar, pH 7.5) and keep at a low temperature, for example 5 ° C. Bromcresol purple is a pH indicator and shows a purple color immediately after overlaying. However, when yeast is fermented, the pH of the medium around the colony decreases, and the color tone around the colony changes to yellow. Therefore, a strain in which the color tone around the colony is changed to yellow while the stacked plates are kept at a low temperature can be selected as a strain exhibiting fermentability at a low temperature.

  As a method for recovering a plasmid from yeast and a method for transforming the recovered plasmid into E. coli, a method commonly used in the field of genetic engineering can be used. Examples of a method for recovering a plasmid from yeast include, for example, an experimental medicine separate volume, a genetic experiment method using yeast, and a method described in Yodosha (1994). As a method for transforming the recovered plasmid into Escherichia coli, for example, molecular -The method described in Cloning 2nd edition, Cold Spring Harbor Laboratory (1989) is mentioned.

The base sequence of a gene complementary to low temperature sensitivity can be determined by a method commonly used in the field of genetic engineering, such as the Maxam-Gilbert method or the dideoxy method.
A polypeptide encoded by a gene complementary to low temperature sensitivity can be easily obtained using current molecular genetic knowledge. If necessary, analysis using various computers is also possible [for example, cell engineering, 14 , 577-588 (1995)]. A polypeptide encoded by a gene that complements low-temperature sensitivity can be used, for example, as a substance that suppresses low-temperature sensitivity of fermentation ability in yeast that exhibits low-temperature sensitivity.

In the present invention, the nucleotide sequence of a gene that complements low-temperature sensitivity and the amino acid sequence of the polypeptide encoded by the gene have been clarified. For example, expression control of gene to be altered or modification of expression level, expression of various genes using promoters of genes complementary to low temperature sensitivity, preparation of genes and genes that fuse low temperature sensitivity with genes and fusion polypeptides, etc. It can be carried out using the method described in Methods in Enzymology, 194 , 594-597 (1991).

A method for inactivating a gene complementary to low temperature sensitivity in yeast will be described below.
In the present invention, gene inactivation means disruption of a gene [eg, Methods in Enzymology, 194 , 281-301 (1991)], introduction of a transposable element into a gene [eg, Methods in Enzyme] Imology, 194 , 342-361 (1991)], introduction and expression of antisense genes [for example, Japanese Patent Publication No. 7-40943, 23rd European Brewery Conv. Proc. , 297-304 (1991)], introduction of DNA involved in silencing in the vicinity of a gene [eg Cell, 75 , 531-541 (1993)], treatment of an antibody against a polypeptide encoded by the gene [for example, European journal of Biochemistry (European J. Biochem.), 231 , 329-336 (1995)] or the like, using a genetic engineering technique or biotechnological techniques, genes or co of the gene Sul polypeptide means reducing or inactivating the functions inherent.

Yeasts used for inactivation of genes complementary to low temperature sensitivity include yeasts belonging to the genus Saccharomyces, preferably yeasts belonging to Saccharomyces cerevisiae , baker's yeast, sake yeast, wine yeast, beer yeast , Miso / soy sauce yeast, ethanol-producing yeast, etc. may be used.
Destruction of a gene that complements low-temperature sensitivity is a gene that has a homologous base sequence to a gene that complements low-temperature sensitivity, but can cause mutations such as additions, deletions, and substitutions to serve as a gene that complements mutations that exhibit low-temperature sensitivity. This refers to introducing non-DNA into a yeast cell to cause homologous recombination and incorporating this mutation into a gene on the genome.

  As a method for producing DNA used for gene disruption, for example, a gene that complements low-temperature sensitivity is cleaved with a restriction enzyme or the like, and DNA is added, deleted, substituted, or the like. The method of mutating with (in vitro mutagenesis) is used. As a method of adding or replacing DNA, a method of inserting a marker gene or the like may be used.

To destroy a gene that complements low-temperature sensitivity, you can destroy any part of the gene that complements low-temperature sensitivity, such as the promoter part, open reading frame part, or terminator part. Also good. A gene can also be destroyed by deleting the entire gene that complements low-temperature sensitivity.
In order to destroy a gene that complements low-temperature sensitivity, for example, a plasmid or plasmid fragment for disrupting a gene that complements low-temperature sensitivity of yeast is transformed into yeast, and the plasmid or plasmid fragment contained in the transformed plasmid is included. The DNA fragment obtained can undergo homologous recombination with a gene on the yeast genome. As a DNA fragment that causes homologous recombination, a plasmid for disrupting a gene that complements cold sensitivity or a fragment thereof and a gene that complements cold sensitivity on the yeast genome have homology to the extent that homologous recombination can occur. Just do it. Whether the DNA fragment undergoes homologous recombination depends on whether or not a strain that has undergone homologous recombination can be isolated by introducing the DNA fragment into yeast, that is, whether or not a strain whose fermentation ability exhibits low-temperature sensitivity can be isolated. You can investigate.

As a vector used for preparing a plasmid for disrupting a gene complementary to low temperature sensitivity, in addition to a vector that can be maintained in yeast, a vector that can be maintained in E. coli, such as pUC19, pBR322, Bluscript II, etc. Any of SK ^{+ and the} like may be used.
As the marker gene, if it is a marker gene that can be used in yeast, for example, a gene that complements an auxotrophic mutation such as URA3, TRP1, LEU2, and HIS3, a chemical substance such as G418, hygromycin B, cerulenin, and parafluorophenylalanine Genes resistant to [eg, J. Ferment. Bioeng., 76 , 60-63 (1993), Enzyme and Microb. Technol .), 15 , 874-876 (1993)], etc. may be used.

The disruption of the gene complementary to low temperature sensitivity on the yeast genome can be carried out by transforming the yeast with a plasmid for disrupting the gene complementary to low temperature sensitivity.
Transformation of yeast can be performed by methods commonly used in the field of genetic engineering or biotechnology, for example, the spheroplast method, the lithium acetate method, the electroporation method and the like described above.

  By introducing a marker gene into a plasmid for disrupting a gene that complements low-temperature sensitivity, transformants can be easily isolated using the marker as an index. In addition, transformants can also be isolated by using as an index that fermentation ability shows low temperature sensitivity when a gene complementary to low temperature sensitivity on the yeast genome is destroyed. Confirmation of low temperature sensitivity of a strain in which a gene complementary to low temperature sensitivity is disrupted can be performed by examining the growth or fermentation ability of the yeast at low temperature.

As a yeast having a fermentative ability exhibiting low temperature sensitivity, characterized in that the gene complementary to low temperature sensitivity produced by the above method is inactivated, for example, Saccharomyces cerevisiae YHK1243 strain (hereinafter referred to as YHK1243 strain) can give. This strain was deposited as FERM BP-5327 at the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry (No. 1-3, Higashi 1-chome, Tsukuba, Ibaraki) on December 7, 1995, based on the Budapest Treaty. Yes.
A test example showing that the low temperature sensitivity of fermentation ability of YHK1243 strain is decreased is shown below.

Test Example 1 Low temperature sensitivity test for fermentability
One platinum ear of YHK1243 strain was inoculated into a test tube containing 5 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose) and cultured at 30 ° C. for 16 hours. After culturing, 1 ml of the culture was transferred to a 300 ml Erlenmeyer flask containing 50 ml of YPD medium and cultured at 30 ° C. for 24 hours. After completion of the culture, the cells were collected by centrifugation, washed twice with deionized water, and 0.61 g of the obtained wet cells were put into a 50 ml fermentation test medium [East Nitrogen Base W / O Amino Acid (Difco) ) Suspended in a test tube with an inner diameter of 22 mm and a height of 200 mm containing 0.67%, sucrose 2%, sodium succinate 1% (adjusted to pH 4.5 with concentrated hydrochloric acid)], and a silicon tube was attached to the mouth of the test tube After sealing with a silicon stopper, the cells were cultured at 5 ° C. for 24 hours. The gas generated during the culture period was collected in a saturated saline solution through a silicon tube, the volume of the gas was measured, and the amount of carbon dioxide generated per gram of cells was calculated. For the YOY655 strain, the amount of carbon dioxide generated per gram of cells was calculated using the same method as for the YHK1243 strain.

  The results are shown in Table 1.

YHK1243 株の5℃での炭酸ガス発生量は、YOY655株の炭酸ガス発生量の約1/9であった。

The amount of carbon dioxide generated by the YHK1243 strain at 5 ° C was about 1/9 that of the YOY655 strain.

Test Example 2 Low temperature sensitivity test of fermentability (2)
One platinum loop of YHK1243 strain was inoculated into a 300 ml Erlenmeyer flask containing 30 ml of YPD medium and cultured at 30 ° C. for 24 hours. After culturing, the entire culture solution was transplanted into a 2 liter baffled Erlenmeyer flask containing 270 ml of molasses medium (3% molasses, 0.193% urea, 0.046% potassium dihydrogen phosphate, 2 drops of antifoaming agent), 30 The cells were cultured at 24 ° C. for 24 hours. After completion of the culture, the cells were collected by centrifugation, washed twice with deionized water, and then water was removed on an unglazed absorbent plate to obtain bacterial cells. For the YOY655 strain, the cells were obtained using the same method as the YHK1243 strain.

Using these cells, doughs containing YOY655 strain and YHK1243 strain were prepared by the following dough composition and process.

Fabric composition: (Weight: g)
Powerful powder 100
Sand sugar 5
Food salt 2
Yeast cells (YHK1243 or YOY655) 3
Water 62
Process:
Mixing (2 minutes at 100 rpm using a National Complete Mixer)
↓
Split (1/5 each of the total fabric weight; 34.4g)
↓
Refrigerated storage (7 days in 5 ° C refrigerator)
↓
Defrosting (30 ° C, relative humidity 85% for 30 minutes)
↓
Measure the amount of carbon dioxide generated at 30 ° C for 2 hours using a farm graph (manufactured by Ato) After storing the obtained dough refrigerated, measure the amount of carbon dioxide generated at 30 ° C and evaluate the refrigeration resistance of the dough Went.

  The results are shown in Table 2.

The dough containing YHK1243 strain had higher carbon dioxide generation at 30 ° C after refrigerated storage than that of YOY655 strain. Further, during the refrigerated storage period, swelling was observed in the dough containing the YOY655 strain, but substantially no swelling was observed in the dough containing the YHK1243 strain.
A dough containing a yeast (hereinafter referred to as the yeast of the present invention) having a fermentative ability and low-temperature sensitivity, characterized by belonging to the genus Saccharomyces and having inactivated a gene complementary to low-temperature sensitivity, is described below. .

The dough containing the yeast of the present invention includes wheat, rye flour, etc., the yeast of the present invention, salt, water, and if necessary, oils, sugar, shortening, butter, skimmed milk powder, yeast food, eggs and other auxiliary materials Adds and kneads.
The condition for refrigeration of the dough containing the yeast of the present invention is -5 to 10 ° C, preferably 0 to 5 ° C for 1 to 10 days, preferably 1 to 7 days.

A method for producing the dough containing the yeast of the present invention and a method for producing bread characterized by adding the yeast of the present invention to the dough will be described below.
The yeast of the present invention is cultured in a normal medium containing a carbon source, nitrogen source, inorganic substance, amino acid, vitamin, etc., under aerobic conditions while adjusting the temperature to 27 to 32 ° C., and the cells are collected and washed. By performing, a yeast cell suitable for bread production can be obtained.

As the carbon source in the medium, glucose, sucrose, starch hydrolyzate, molasses and the like can be used, and particularly, molasses is preferably used.
As the nitrogen source, ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, urea, yeast extract, corn steep liquor and the like are used.
As inorganic substances, magnesium phosphate, potassium phosphate and the like are used, glutamic acid and the like are used as amino acids, and pantothenic acid and thiamine and the like are used as vitamins.

As the culture, fed-batch culture is appropriate.
After completion of the culture, the yeast cells of the present invention are recovered by centrifugation or the like, and this is combined with salt, water, and if necessary, flour together with fats and oils, sugar, shortening, butter, skimmed milk powder, yeast food, eggs, etc. The dough containing the yeast of the present invention is obtained by adding to and mixing with rye flour.

Bread can be manufactured by using a normal bread-making method using the obtained dough. There are two types of bread making methods for typical breads and confectionery breads: the straight rice method and the medium seed method.The former is a method in which all ingredients are mixed together from the beginning. This is a method in which water is added to form a medium seed and the remaining ingredients are combined after fermentation.
In the straight rice process, all raw materials are mixed, then fermented at 25-30 ° C, divided, benched, molded, and packed. After proofing (35-42 ° C), firing (200-240 ° C). In the middle seed method, about 70% of all used flour, yeast, yeast food, etc. are mixed with water and fermented at 25-35 ° C. for 3-5 hours, then the remaining ingredients (flour, salt, water, etc.) ), Chaos (main wall), split, bench, molding, mold filling. After passing through a proof (35-42 ° C), firing (200-240 ° C) is performed.

When manufacturing a Danish pastry, a croissant, etc., it carries out as follows, for example.
Add flour, salt, yeast of the present invention, sugar, shortening, egg, nonfat dry milk, and water and knead to make dough. Next, the fats and oils such as butter and margarine are wrapped in the dough, and rolling and folding are repeated to make a multilayer of the dough and the fats and oils. The process of folding oils and fats when preparing the dough is called roll-in. As the roll-in, the dough temperature is lowered to about 15 ° C., the method is performed without cooling to the desired number of layers, and the method of performing cooling several times in the refrigerator or freezer during the folding operation, There are two methods of so-called retard production.

The obtained dough is rolled, divided, molded and filled. After passing through a proof (30-39 ° C), firing (190-210 ° C) is performed.
A method for producing ethanol, which comprises culturing the yeast of the present invention in a medium, accumulating ethanol in the culture, and collecting ethanol from the culture, is described below.
The production of ethanol by the yeast of the present invention is carried out by a method used for normal yeast culture. At this time, the microorganism used in the present invention may be immobilized on a gel-like carrier such as agar, sodium alginate, polyacrylamide or carrageenan.

The medium for ethanol production in the present invention may be either a synthetic medium or a natural medium as long as it contains a suitable amount of carbon source, nitrogen source, inorganic substance, and other nutrients as required.
As the carbon source, a fermentation raw material containing at least sucrose is used, but any other carbon source that can be assimilated by the microorganism used in the present invention, for example, sugars such as glucose, fructose, galactose, or maltose can be used. The fermentation raw material containing sucrose may be either a synthetic raw material or a natural raw material as long as it contains sucrose. For example, sugar cane juice, sugar beet juice (beet) juice, or a sugar production process using these juices Waste molasses obtained after crystallizing sucrose (sugar) is used.

As the nitrogen source, organic or inorganic nitrogen sources such as urea, ammonia, ammonium sulfate and ammonium nitrate, and natural nitrogen sources such as corn steep liquor, peptone, meat extract and yeast extract are used.
As the inorganic salt, potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, potassium chloride, sodium chloride and the like are used.

As other nutrients, vitamins such as thiamine hydrochloride, p-aminobenzoic acid, folic acid, riboflavin, and inositol are used.
The culture is usually performed under aerobic conditions such as shaking culture or aeration and agitation culture. The temperature is maintained at 25 to 50 ° C., preferably 30 to 43 ° C., the pH of the culture is maintained at 3 to 7, preferably 4 to 6, and the culture is usually completed in 1 to 10 days.

  After completion of the culture, ethanol can be recovered from the culture solution by using a usual method such as distillation.

Cloning of genes complementary to low temperature sensitivity Addition of ura3 mutation to RZT-3 strain As a marker for introducing a plasmid into RZT-3 strain, a yeast whose fermentation ability is low-temperature sensitive, the method of Boeke et al. [Molecular and General Gene The ura3 mutation was added according to Tix (Mol. Gen. Genet.), 197 , 345-346 (1984)]. That is, RZT-3 strain was inoculated with one platinum ear in YPD medium and cultured overnight at 30 ° C. with shaking. 100 μl of the obtained culture solution was applied to a FOA plate [0.67% yeast nitrogen-based W / O amino acid (Difco), 0.1% 5-fluoroorotic acid, 0.005% uracil, 2% glucose, 2% agar] The cells were cultured at 30 ° C for 3 days. After completion of the culture, one of the resulting colonies, which is uracil-requiring, is selected for one strain that is complemented with uracil-requiring and transforms with low-temperature fermentability when transformed with plasmid YCp50 having URA3 as a marker. Saccharomyces cerevisiae RZT-3u strain (hereinafter referred to as RZT-3u strain).

2. Cloning
A DNA fragment obtained by partially decomposing chromosomal DNA of the X2180-1B strain (obtained from Yeast Genetic Stock Center) with Sau3AI was inserted into the BamHI site of plasmid YCp50 to prepare a gene library. Using the gene library, the RZT-3u strain was transformed, and transformants were selected without requiring uracil. The obtained transformant was cultured at 30 ° C. on a YPG agar medium to form colonies, and then overlayed with dye agar and cultured at 5 ° C. for 1 to 3 days. A strain in which the color tone around the colony changed to yellow during the incubation period at 5 ° C, that is, a strain in which fermentation was observed at 5 ° C, was isolated as a strain complemented with a mutation showing a low temperature sensitivity of fermentation ability. The recombinant plasmid pHK162 was extracted from the strain.

Plasmid pHK162 was introduced into Escherichia coli JM109 strain to prepare Escherichia coli EHK162 strain. This strain was deposited as FERM BP-5328 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry on December 7, 1995, based on the Budapest Treaty.

3. Complementarity test In the plasmid pHK162, a DNA fragment of about 12 Kbp from Sau3AI / BamHI to BamHI was inserted. Plasmid pHK162 was cleaved with various restriction enzymes, and the resulting DNA fragments were separated by electrophoresis, the molecular weight was measured, and a restriction enzyme map was prepared as shown in FIG. Based on this restriction enzyme map, a recombinant plasmid was prepared by inserting a DNA fragment obtained by cutting about 12 Kbp of Sau3AI / BamHI to BamHI with SphI, BamHI, MluI and ClaI into plasmid YCp50. RZT-3u strain was transformed with each recombinant plasmid.

Whether or not the obtained transformant complements a mutation showing low temperature sensitivity of fermentation ability was examined. As a result, as shown in FIG. 1, when the RZT-3u strain was transformed with the plasmid pHK162, a mutation showing low temperature sensitivity of the fermentation ability was complemented. When A was transformed, a mutation showing low temperature sensitivity of the fermentation ability was not complemented.
Based on the above, in order to complement the low temperature sensitivity mutation of the RZT-3u strain, BamHI (A) (1291 to 1296 of the nucleotide sequence of SEQ ID NO: 1) to SphI (B ) (7767th to 7680th positions of the base sequence of SEQ ID NO: 1) About 6.5 Kbp DNA fragment Shows that a DNA fragment having a longer sequence is required in the upstream region at the 5 ′ end and the downstream region at the 3 ′ end. It was done.

4). Determination of base sequence The base sequence of a 12 Kbp DNA fragment inserted in plasmid pHK162 was determined using the dideoxy method and a DNA sequencer (Pharmacia LKB, ALF DNA Sequencer II). As a result, a gene containing an approximately 6.5 Kbp region cleaved by BamHI (A) and SphI (B) in FIG. 1 in an open reading frame was found. This gene was named CSF1 gene. As shown in the amino acid sequence of SEQ ID NO: 2 , the polypeptide encoded by the CSF1 gene predicted from the determined base sequence was composed of 2958 amino acid residues and a molecular weight of 338 kDa. As a result of examining the homology of DNA with other genes, the upstream side of the CSF1 gene containing about 140 amino acids on the N-terminal side of the open reading frame of the CSF1 gene is the Saccharomyces cerevisiae GAA1 gene [Hamburger et al. .) Upstream of the sequence reported as the base sequence of J. Cell Biol., 129 , 629-639 (1995)] (outside the region encoded by the GAA1 gene) Matched. However, the report by Hamburger et al. Relates to the GAA1 gene, and there is no description regarding the presence of another gene (CSF1 gene) upstream of the GAA1 gene. In addition, the base sequence reported by them has a single base T inserted between the 198th T and the 199th G of the base sequence of SEQ ID NO: 1, and the polypeptide encoded by the CSF1 gene is their It cannot be deduced from the reported sequence.

Preparation of yeast showing low temperature sensitivity of fermentation ability Preparation of plasmid for gene disruption Dissolve about 5 μg of plasmid pHK162 in 20 μl of H buffer (50 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 1 mM dithiothreitol, 100 mM sodium chloride) and limit to 10 units Enzyme BamHI was added and reacted at 30 ° C. for 3 hours. The reaction products were separated by 0.8% agarose gel electrophoresis, and an approximately 8 kb DNA fragment of BamHI (A) to BamHI (C) in Fig. 1 was excised and extracted and purified using GENECLEAN II kit (Bio 101). did. A DNA fragment of about 2.8 kb was extracted and purified using the same method except that about 5 μg of plasmid pHK162 was replaced with about 5 μg of plasmid pUC19. A DNA fragment 0.1μg of approximately 2.8kb of about 8kb DNA fragment derived from 1μg plasmid pUC19 of from plasmid pHK162 was overnight ligation reaction at 16 ° C. using a Ligation Pack (Nippon Gene).

  After completion of the reaction, 2 μl of the reaction solution was used to transform competent high E. coli JM109 strain (Toyobo Co., Ltd.). The obtained transformant was applied to 5-bromo-4-chloro-3-indolyl-β-D-galactoside (hereinafter referred to as X-gal) ampicillin LB agar medium and cultured at 37 ° C. for 20 hours. X-gal ampicillin LB agar was prepared by adding 50 μl of LB agar medium (1% bactotryptone (Difco), 0.5% yeast extract, 1% sodium chloride, 1.5% agar) containing 50 μg / ml of ampicillin. % X-gal and 25 μl of isopropyl-1-thio-β-D-galactoside were added dropwise, spread with a spreader, and lightly dried. After completion of the culture, the resulting white colony was isolated, and the plasmid DNA obtained by culturing this was extracted and purified to prepare plasmid pHK179.

  About 5 μg of plasmid pHK179 was dissolved in 20 μl of H buffer, 10 units of restriction enzyme MluI and 10 units of restriction enzyme SpeI were added and reacted at 37 ° C. for 3 hours. The reaction product was subjected to blunt end treatment using DNA Blunting Kit (Takara Shuzo). After separation of the treated product by 0.8% agarose gel electrophoresis, about 0.6 kb of MluI (position 4388 to 4393 of the nucleotide sequence of SEQ ID NO: 1) to SpeI (position 5027 to 5032 of the nucleotide sequence of SEQ ID NO: 1) in FIG. An about 10 Kbp fragment excluding the DNA fragment was excised and extracted and purified using GENECLEAN II kit. In addition, plasmid YEp24, which is a vector containing the marker gene URA3 complementary to uracil-requiring mutation in HindIII to HindIII, was added to 20 μl of M buffer [10 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 1 mM dithiothreitol. , 50 mM sodium chloride] was dissolved in about 5 μg, 10 units of restriction enzyme HindIII was added, and the mixture was reacted at 37 ° C. for 3 hours. The reaction product was subjected to blunt end treatment using DNA Blunting Kit (Takara Shuzo). After the treated product was separated by 0.8% agarose gel electrophoresis, an about 1.1 kb fragment containing URA3 was excised and extracted and purified using the GENECLEAN II kit.

A ligation reaction was carried out overnight at 16 ° C. using Ligation Pack between 0.5 μg of about 10 kb DNA fragment derived from plasmid pHK179 and 0.5 μg of about 1.1 kb DNA fragment derived from plasmid YEp24. After completion of the reaction, 2 μl of the reaction solution was used to transform competent high E. coli JM109 strain. The obtained transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. for 20 hours. After completion of the culture, the resulting colonies were isolated, and the plasmid DNA obtained by culturing the colonies was extracted and purified to prepare plasmid pHK188 for disrupting the CSF1 gene. The plasmid pHK188 was confirmed to be the target plasmid by separating the plasmids before and after digestion with the restriction enzyme BamHI by 0.8% agarose gel electrophoresis and measuring the molecular weight.
In addition, FIG. 2 shows an outline of a process for preparing a plasmid for disrupting the CSF1 gene.

2. Disruption of CSF1 gene The plasmid pHK188 was used to disrupt the CSF1 gene of the YOY655u strain, which is a haploid strain of Saccharomyces cerevisiae. The YOY655u strain is a strain obtained by introducing a uracil auxotrophic (ura3) mutation into the YOY655 strain, which is a haploid strain of Saccharomyces cerevisiae, and has the same properties as the YOY655 strain. YOY655u strain was inoculated into an Erlenmeyer flask containing 100 ml of YPD medium, and cultured with shaking at 30 ° C. until 2 to 4 × 10 ⁷ . After completion of the culture, the cells were collected by centrifugation (2500 rpm, 5 minutes), and the obtained cells were brought into contact with plasmid pHK188 by the lithium acetate method. In order to promote homologous recombination between the CSF1 gene and the plasmid pHK188, the plasmid pHK188 was completely cut with BamHI and linearized before use before transformation. YOY655u strain contacted with plasmid pHK188 was inoculated on SGlu agar medium (0.67% yeast nitrogen base W / O amino acid, 2% glucose, 2% agar) and cultured at 30 ° C. for 2 to 5 days. After completion of the culture, the YHK1243 strain was obtained from one of the resulting colonies as a transformant in which the uracil requirement of the YOY655u strain was complemented.

  Inoculate YHK1243 strain, YOY655u strain and RZT-3 strain on YPG agar medium and incubate at 30 ° C for 1-2 days to form colonies, then overlay dye agar on the medium, and then at 5 ° C for 3 days Cultured. The color tone around the colonies of the YHK1243 and RZT-3 strains did not change throughout the culture period, but the color tone around the colonies of the YOY655u strain changed to yellow on the first day of culture.

How to make refrigerated dough 1. Baker's yeast culture
One platinum ear of YOY655 strain and YHK1243 strain was each inoculated into a 300 ml Erlenmeyer flask containing 30 ml of YPD medium and cultured at 30 ° C. for 24 hours. After culturing, the entire culture solution was transplanted into a 2 liter baffled Erlenmeyer flask containing 270 ml of molasses medium (3% molasses, 0.193% urea, 0.046% potassium dihydrogen phosphate, 2 drops of antifoaming agent), 30 The cells were cultured at 24 ° C. for 24 hours. After completion of the culture, the cells were collected by centrifugation, washed twice with deionized water, and then water was removed on an unglazed absorbent plate to obtain bacterial cells. The obtained microbial cells were used for bread making.

2. Bread making Bread was obtained by the dough composition and process shown below.

Fabric composition: (Weight: g)
Powerful powder 100
Sand sugar 5
Food salt 2
Yeast cells 2
Water 62
Process:
Mixing (100rpm, 2 minutes)
Split (34.4g)
Storage (5 ℃, 7 days)
Proofer (40 ℃, 90% RH, 75 minutes)
Annealing (220 ℃, 25 minutes)

As a result, the bread obtained using the YHK1243 strain as yeast cells had a larger volume than the bread obtained using the YOY655 strain.

Alcohol fermentation Yeast culture and alcohol fermentation
One platinum loop of each of YOY655 strain and YHK1243 strain was inoculated into a test tube containing 5 ml of YPD medium and cultured at 30 ° C. for 24 hours. After completion of the culture, 2 ml of the culture solution was transplanted into a large test tube containing 20 ml of molasses medium (25% molasses, 0.2% ammonium sulfate) and cultured at 37 ° C. After 16 hours and 40 hours from the start of the culture, 0.5 ml of the culture solution was sampled, and the ethanol concentration in the culture solution was measured.

  The results are shown in Table 3.

  As shown in Table 3, when the YHK1243 strain was used, ethanol production at 37 ° C. was higher than when the YOY655 strain was used.

  According to the present invention, it is possible to provide a protein or gene that complements a mutation whose fermentation ability exhibits low-temperature sensitivity, a refrigeration-resistant yeast obtained by inactivating the gene, and a method for producing bread and ethanol using the yeast. it can.

FIG. 1 is a diagram showing a restriction enzyme map of a DNA fragment containing the CSF1 gene and the results of subcloning and complementation tests performed for limiting the functional region of the CSF1 gene. FIG. 2 is a diagram showing a production process of a plasmid for disrupting the CSF1 gene.

Claims (8)

In yeast belonging to the Saccharomyces genus, genes comprising DNA consisting contained in chromosomes (1) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2, or (2) the nucleotide sequence shown in SEQ ID NO: 1
A method for producing a yeast, wherein the fermentability exhibits low-temperature sensitivity, wherein the fermentation is inactivated. Belonging to the genus Saccharomyces, comprising DNA consisting Included (1) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2, or (2) the nucleotide sequence shown in SEQ ID NO: 1 in the chromosomal gene
A yeast exhibiting low-temperature sensitivity in fermentability, characterized in that is inactivated. The yeast according to claim 2, belonging to Saccharomyces cerevisiae. The yeast according to claim 2 or 3, wherein genes 4388 to 5032 of the base sequence shown in SEQ ID NO: 1 are disrupted. Saccharomyces cerevisiae YHK1243 (FERM BP-5327) strain. The dough containing the yeast according to any one of claims 2 to 5. A method for producing bread, comprising adding the yeast according to any one of claims 2 to 5 to the dough. A method for producing ethanol, wherein the yeast according to any one of claims 2 to 5 is cultured in a medium, ethanol is accumulated in the culture, and ethanol is collected from the culture.

Images