JPH1066583A - New gene and new yeast containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene consisting of a gene regulating the fermentative power of yeast at low temperatures, capable of making hosts sensitive at low temperatures through transferrin into yeast so as to make suitably usable in refrigerating bread dough, thus useful for breeding low-temperature-sensitive bread yeast. SOLUTION: This new gene regulates the fermentative power of yeast at low temperatures, is related to energy-acquiring system or saccharide's membrane permeation system, lies on the left arm of the 11th chromosome of the yeast, and consists of about 5.2kb EcoRI-EcoRI fragment having a restriction enzyme map shown by the formula. This gene is capable of making hosts sensitive at low temperatures through transferring into bread yeast, therefore being suitably usable in refrigerating bread dough and useful for breeding low- temperature-sensitive yeast. This new gene is obtained, for example, by the following process: the parent strain of yeast is subjected to mutation treatment using e.g. ethyl methanesulfonate, a chromosome DNA is separated from the resultant variant followed by preparing a chromosome DNA library which, in turn, is put to screening with the reversion of low-temperature-sensitive hosts to wild type ones as indicator.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene that regulates the fermentative power of yeast at a low temperature, a plasmid containing the gene, a yeast having the plasmid, a method for breeding the yeast,
The present invention relates to bread dough containing the yeast and a method for producing the bread dough. More specifically, pastries such as baking, especially Danish and croissant, including the retarding process,
Furthermore, the present invention relates to a yeast suitable for refrigerated and frozen dough in all kinds of bread, a breeding method thereof, a dough containing the yeast, and a method of producing the dough.

[0002]

2. Description of the Related Art In the recent baking industry, the frozen dough method has been actively used in order to streamline the bread manufacturing process by labor saving and to meet various consumer needs. However, in the frozen dough method, not only is it costly to freeze, transport, store, and thaw the dough,
Further, there are problems such as acquisition of yeast that can withstand freezing and deterioration of dough. For example, after dough mixing, a slight fermentation (so-called pre-freezing fermentation) occurs in the freezing process, and ethyl alcohol is generated in the dough to reduce the freezing resistance of the yeast, or to open / close the freezer or to store storage conditions in the distribution process. Due to the temperature rise due to the defect, there was a problem that even the dough that had been frozen for a long time was partially thawed due to the rise in temperature and the quality was reduced.

[0003] As a means for solving this problem, in the production of frozen dough, the mixing temperature is extremely suppressed in order to suppress the fermentation before freezing, the mixing is carried out at a low temperature such as a refrigerator, and the spiral is used for freezing rapidly. Methods such as the use of a rapid refrigerator are employed. However, this method has disadvantages such as requiring a large amount of energy. In order to solve these problems, a so-called refrigerated dough method has been studied in which bread dough is stored in a refrigerated state without freezing and the dough is aged at the same time.

[0004] However, even if the dough is refrigerated at a low temperature using a conventionally used baker's yeast, the fermentation rate is slower than normal temperature, but the fermentation proceeds. There is a problem. For this reason, although some have been put to practical use, the fact is that they have not yet been widely applied.

Further, in the production of pastries such as Danish and croissant, a so-called "retard method" is employed in which dough is subjected to low-temperature treatment in order to fold roll-in margarine neatly into layers. However, due to some dough fermentation in this retarding process, roll-in margarine is not folded neatly, the resulting bread layer is not clean, and the dough cannot be refrigerated and stored, so that the remaining dough cannot be used. As a result, the yield of the baking process is reduced.

Therefore, in the cold storage or retarding step of bread dough, a low-temperature sensitive yeast having a low fermentation power in a low-temperature state is required. Of course, it is needless to say that the yeast has a fermenting power that can be used at ordinary fermentation temperatures as well as general yeast. Fermentation is suppressed at low temperatures, dormant or does not grow, but normally divides and grows at normal culture temperatures. Acta., Vo1.6
54 149-155 (1981), M. Moritz et al., Mol. Cell. Biol., Vo.
l.11 5681-5692 (1991)), a mutant related to the GTR1 gene (M. Bun-ya et al., Mol. Cell. Biol., vol. 122958-2966 (199)
2)) etc. are known. As a method for obtaining a cold-sensitive yeast, a method for obtaining a cold-sensitive strain using a tritium suicide method (BSLittlewood et al., Mutat. Res., Vol. 17 315-322 (1
973)) are known. However, these reported yeasts are not baker's yeasts that are used daily, and all have significantly lower fermentation power than baker's yeast, and furthermore, they are inferior in the unique flavor required for bread, and so yeast for baking. It is difficult to use as yeast.

Methods for obtaining or breeding baker's yeast whose fermentative power is suppressed at low temperatures are described in Japanese Patent Application Laid-Open Nos. 61-195637 and 4-23493.
9, JP-A-5-76348, JP-A-5-28896, JP-A-5-336872,
Alternatively, it is reported in Japanese Patent Publication No. 7-500006. However, any breeding method is complicated and time-consuming, and it is difficult to breed efficiently and effectively, or there is a problem in that the degree of suppression of fermentation at low temperatures is low and the practicability is poor.

[0008] From many disclosures about cold sensitive yeast,
It is clear that cold-sensitive yeasts exhibit their properties depending on the genetic background. M. Bun-ya et al., Mol. Cell. Bio
l., vol. 12, 2958-2966 (1992)) report that deletion of GTR1 renders yeast cold sensitive. In addition, Tokio Hei 5-500006
As disclosed in the gazette, lts1 to lts8, there are a plurality of genes relating to low-temperature sensitivity (hereinafter referred to as low-temperature sensitivity genes), and there is a difference in the low-temperature sensitivity level. Such facts indicate that selection of a cold-sensitive gene more practical for baker's yeast is one of the important issues.

As described above, the cause of making yeast sensitive to low temperature may be disruption (deletion) of a specific gene or a change in the properties of a gene product. Need to be identified. In the case where the properties of the gene product change, optimization becomes possible by evaluating the low-temperature sensitivity using, for example, the activity of an enzyme involved in the low-temperature sensitivity as an index. In addition, by introducing mutations into cold-sensitive genes in vitro and expressing them using yeast, E. coli, etc., and evaluating them using their enzyme activities as indicators, it is possible to determine whether the mutations in the cold-sensitive baker's yeast are good. Can be evaluated.

[0010] However, all the cold-sensitive baker's yeasts currently obtained or used merely breed the cold-sensitive strains obtained by mutation, and their most important property is the cold-sensitive strain. Is not optimized. Rather, it is a fact that a cold-sensitive strain is bred so as to have properties suitable for baker's yeast by improving properties other than cold sensitivity by a more complicated and complicated breeding method. In some cases, even such breeding is not performed, and the yeast is used as baker's yeast simply because it has a cold-sensitive gene (Japanese Patent Application Laid-Open No. 7-50000).
6) It is not always the optimal low-temperature sensitive yeast.

In order to breed an excellent low-temperature sensitive baker's yeast, it is important to improve the low-temperature sensitivity by combining the low-temperature-sensitive genes in addition to selecting the above-described optimal low-temperature-sensitive genes. For example, yeast has many metabolic systems, and it is considered that at least one of the genes involved in the metabolic system of yeast becomes cold-sensitive, thereby exhibiting a cold-sensitive trait. However, the effect of each metabolic system naturally differs. Combining multiple cold-sensitive genes related to the same metabolic system, or combining multiple cold-sensitive genes related to different metabolic systems is an excellent breeding method, but it can be said that crossing, sporulation, or The reality is that breeding techniques such as mutation are difficult. Thus, for the development of low-temperature sensitive baker's yeast, an efficient breeding method by establishing the evaluation of a gene product that imparts low-temperature sensitivity and the like is desired.

[0012]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems. An object of the present invention is to clone a gene that regulates the fermentative power of yeast at a low temperature and identify the gene. In addition, it has been very difficult with the conventional technique of breeding excellent cold-sensitive baker's yeast and breeding a strain having multiple cold-sensitive genes by evaluating the function of this gene product in vitro. A method is also provided that facilitates the breeding method.

[0013]

Means for Solving the Problems The present invention relates to a gene that regulates the fermentative power of yeast at a low temperature.

[0014] In a preferred embodiment, the gene is a gene related to an energy acquisition system or a sugar transmembrane system.

In a preferred embodiment, the gene is located on the left arm of chromosome 11 of yeast and has a restriction map of the following EcoRI-EcoRI fragment of about 5.2 kb:

[0016]

Embedded image

Exists within.

[0018] In a preferred embodiment, the gene corresponds to the SacI-HpaI in the approximately 5.2 kb fragment of EcoRI-EcoRI.
The following sequence present in the 1.3 kb fragment (SEQ ID NO: 1
1):

[0019]

Embedded image

[0020]

In a preferred embodiment, the gene has a sequence of about 1.1 kb (SEQ ID NO: 2: T2):

[0022]

Embedded image

Having the following.

In a preferred embodiment, the gene has a mutation in which one or more nucleotides in the gene have been substituted, added or deleted, and the regulation of fermentation power is equal to or improved from that before the mutation. Gene.

[0025] The present invention also relates to a yeast having the above-mentioned gene and capable of regulating fermentation power at a low temperature.

The present invention further relates to a yeast which exhibits complementation with respect to an existing yeast whose fermentation is regulated at a low temperature with respect to the ability to regulate fermentation at a low temperature. In which one or more nucleotides have a substitution, addition, or deletion, and thus have low temperature sensitivity.

[0027] The present invention also relates to a yeast which is complementary to an existing yeast whose fermentation is regulated at a low temperature with respect to the ability to regulate fermentation at a low temperature. The yeast showing the complementarity is a gene having a mutation in which one or more nucleotides have been substituted, deleted or added in the gene,
It has a gene that is complementary to the gene contained in the b fragment.

The term "yeast exhibiting complementarity with respect to the ability to regulate fermentation at low temperature to an existing yeast whose fermentation is regulated at low temperature" as used in the present invention refers to the yeast of the present invention, which is yeast or a spore thereof. When a strain has a conjugating ability and exhibits low-temperature sensitivity, it is a yeast which does not exhibit low-temperature sensitivity by conjugating with another low-temperature-sensitive yeast having the opposite mating type. Therefore, when the yeast of the present invention shows complementation to another cold-sensitive yeast, it means that the gene expressing cold sensitivity is different between the yeast of the present invention and the other cold-sensitive yeast.

The present invention also relates to a yeast having a gene capable of causing homologous recombination with a yeast gene whose fermentation is regulated at a low temperature. The gene capable of causing this homologous recombination is a sequence of about 1.1 kb (SEQ ID NO: 2: T2) present in the about 5.2 kb fragment of EcoRI-EcoRI or the about 1.3 kb fragment of SacI-HpaI contained therein. And a gene having a mutation in which one or more nucleotides have been substituted, deleted or added.

The present invention also relates to a yeast having a gene capable of causing homologous recombination with a yeast gene whose fermentation is regulated at a low temperature, a gene regulating the fermentation power of the yeast at a low temperature, an energy acquisition system or a sugar membrane. A yeast having a mutation in which one or more nucleotides have been substituted, deleted or added in a gene related to the permeation system.

The present invention further relates to a plasmid having the above-mentioned gene that regulates fermentation at a low temperature or a gene capable of causing homologous recombination with the gene.

The present invention also relates to a yeast containing the above-mentioned plasmid, the fermentation power of which is regulated at a low temperature.

In a preferred embodiment, the yeast is a yeast whose fermentative power is regulated at a low temperature, wherein the plasmid comprises a plasmid containing two or more different genes that regulate fermentative power.

Further, the present invention relates to bread dough containing a gene whose fermentative power is regulated at a low temperature or a plasmid containing this gene and containing a yeast whose fermentative power is regulated at a low temperature. In a preferred embodiment, the dough is refrigerated dough.

Further, the present invention relates to a method for producing bread dough containing the yeast.

Furthermore, the present invention provides a method for breeding yeast containing a plurality of genes that regulate the fermentative power of yeast at different low temperatures.

This method comprises the following steps: a step of isolating a plurality of genes that regulate the fermentative power of yeast at low temperatures; a spore strain having a gene that regulates fermentative power of yeast at low temperatures; Producing a diploid yeast by conjugating with a spore strain having a gene that regulates the fermentative power of yeast; a step of forming a spore in the diploid yeast to obtain a spore strain; Isolating a spore strain having a plurality of genes that regulate spores by hybridization using the isolated gene as a probe; and joining together spore strains having the plurality of genes.

In a preferred embodiment, there is provided a method for breeding yeast having two genes that regulate the fermentative power of yeast at a low temperature. This method comprises joining a spore strain having one gene that regulates the fermentative power of yeast at low temperature with a spore strain having one other gene that regulates the fermentative power of yeast at low temperature, and This is a method for breeding yeast by isolating a spore strain having two genes that regulate fermentation power.

In another preferred embodiment, a spore strain having two genes that regulate yeast fermentation at low temperatures and a spore strain having other genes that regulate yeast fermentation at low temperatures are mated. And breeding yeast having three or more improved genes.

The above objects are achieved by these inventions.

Hereinafter, the present invention will be described in detail.

As used herein, the term "gene product" refers to a polypeptide, tRNA, rRNA, etc., encoded by a gene. Further, "low temperature sensitivity" or "adjustment of yeast fermentation at low temperature" refers to the property of suppressing fermentation power (carbon dioxide generation) at 20 ° C or lower as compared with wild-type baker's yeast.

The gene of the present invention is present in an approximately 5.2 kb fragment of EcoRI-EcoRI present on the left arm of chromosome 11 of yeast.
Preferably, it has the restriction map shown in FIG. 1 or FIG. 2, and more specifically, it is present in a SacI-HpaI fragment of about 1.3 kb. This gene is Saccharomyces cerevisiae genom
e database This is a protein-encoding gene named YKL098w.

Hereinafter, a method for obtaining (cloning) the gene of the present invention using a mutant strain will be described.

Generally, the method of cloning a gene differs depending on whether the mutation of the gene is dominant or recessive. Whether a mutation is dominant or recessive can be determined genetically by genetic analysis of the hybrid. If the mutation is dominant, it may be used as an indicator that the wild-type host parent strain changes to low temperature sensitivity. When the mutation is recessive, it may be used as an indicator that the parent strain sensitive to low temperature returns to the wild type by transformation.

The mutation of the gene of the present invention that regulates the fermentative power of yeast at a low temperature (hereinafter sometimes referred to as a low-temperature sensitive gene) is considered recessive. Can be performed using the index as an index.

Accordingly, a target gene can be obtained by first obtaining a mutant strain of a yeast which is sensitive to low temperature and screening a gene for reverting to a wild type using the mutant strain as a host.

Examples of the gene that regulates fermentation include enzymes involved in the metabolism of glycolysis, or polypeptides that indirectly affect glycolysis, such as tRNA, rRNA, or a sugar transmembrane system, or Genes related to enzymes that indirectly affect sugar membrane permeation, such as enzymes in fatty acid synthesis, are considered. Genes can be identified by cloning the genes encoding these enzymes or polypeptides. DNA sequences encoding unidentified polypeptides can also be found. As a result of these analyses,
It can be clarified whether or not the gene of the present invention that regulates fermentation at low temperature and the yeast having the gene are novel.

First, the acquisition of a cold-sensitive yeast mutant and the creation of a suitable host for transforming the cold-sensitive yeast will be described, and then the chromosomal DNA (gene) 3.) A method for preparing a library and transforming a breeding host using the DNA library and cloning a target gene will be described.

(Acquisition of Cold Sensitive Yeast) The cold sensitive yeast for acquiring the gene of the present invention is obtained by screening from nature, mutation treatment such as nitrosoguanidine, ethyl methanesulfonate (hereinafter referred to as EMS), ultraviolet irradiation, etc. It is obtained by a breeding operation such as hybridization or cell fusion. Here, a method for obtaining a target yeast by mutation treatment is disclosed, but it goes without saying that the present invention is not limited to this method. When the single-stage mutation treatment is not sufficient, a method of repeating the mutation treatment, a method of obtaining spores for backcrossing, a cell fusion method, and the like can be used in combination.

(A) Mutation of yeast Here, a mutation method using EMS will be described. The medium to be used is as follows.

YPD Medium YPD Agar Plate Coolose 20 g Coolose 20 g Polypheton 20 g Polypheton 20 g Yeast Extract 10 g Yeast Extract 10 g Water 1000 ml Agar 20 g Water 1000 ml Incubate with shaking for 16 hours. After completion of culture, 3000 rp
Collect cells by centrifugation at m for 10 minutes, and wash once with a phosphate buffer of 0.1 M, pH 7.0. Bacteria cells in the same phosphate buffer 5
The suspension was treated with 0.15 ml of EMS at 30 ° C. for 2 hours with occasional stirring, collected by centrifugation at 3000 rpm for 10 minutes, washed with a phosphate buffer, and then washed with 20% Na ₂ S ₂ 5 ml of O ₃ is added and left at 30 ° C. for 10 minutes to inactivate the EMS. 20% Na ₂ S
₂ O ₃ treatment is performed twice, washed three times with 5 ml of physiological saline, and then suspended in 5 ml of physiological saline, diluted appropriately on a YPD agar plate having the above composition, and coated with bacteria.

(B) Screening of low-temperature sensitive yeast B-1. Primary screening: microplate method Bromcresol purple (BCP) is used, utilizing the properties of BCP that becomes blue-violet at high pH and yellow at low pH. This is a method for screening mutant strains.

The strain grown on the YPD agar plate is inoculated into a microplate containing one platinum loop and 200 μl of YPD medium, and cultured at 30 ° C. for 16 hours. 200 μl of BCP-containing YPD medium (30 mg of bromcresol purple (BCP) added to the above YPD medium) was injected into each well of the microplate, and the culture broth was added to each of two microplates.
Implant each at 0 μl and incubate at 10 ° C and 30 ° C respectively.
Incubate for static time. In the 30 ° C culture, the broth pH decreases and the color of the broth turns yellow for good fermentation, and the color of the broth turns yellow. Select strains whose pH does not decrease and the color of the broth does not turn yellow.

B-2. Secondary screening: Mycell method: 2
The next screening is a method for selecting a strain having a large weight difference before and after the mycell fermentation. The medium to be used is as follows.

Molasses medium Mycellar solution Molasses (as sugar) 30 g quorose 80 g Ammonium sulphate 5 g (NH ₄ ) ₂ HPO 5 g Phosphorus 3 g KH ₂ PO ₄ 5 g Urea 2 g Water 1000 ml Water 1000 ml The strain obtained by the primary screening is molasses medium 5 ml Then, inoculate one platinum loop and shake culture at 30 ° C for 24 hours. 5m of this culture
1 is transferred to 50 ml of molasses medium, and cultured with shaking at 30 ° C. for 24 hours. After culturing, combine the cultures from two flasks for a total of 8
Take 0 ml, collect by centrifugation at 3000 rpm for 10 minutes, and sterile water.
Wash once. Suspend in 100 ml of Mycellar solution, 50
The ml was incubated at 10 ° C. for 15 hours and at 30 ° C. for 5 hours. The difference between the weights before and after the mycel fermentation is defined as the mycell fermentative power, and a strain that ferments well at 30 ° C and has a low fermentative power at 10 ° C is selected.

From the viewpoint of obtaining a yeast whose fermentative power is suppressed at a low temperature, the mutated yeast is inoculated into a YPD medium containing an agent for killing the growing yeast, and is anaerobically cooled at a low temperature. It is more effective to add a step of culturing. This is because the cells that grow at a low temperature are killed, so that the screening efficiency is further improved. Examples of the agent that kills the growing yeast include nystatin, amphotericin B, cycloheximide, and the like, and any agent having a relatively low concentration and an effect of killing the proliferating cells may be used. The concentration of the drug in the medium is 10 μg / ml to 50 μg / ml for nystatin, 100 μg / ml to 500 μg / ml for amphotericin B, and for cycloheximide.
1000 μg / ml to 5000 μg / ml is suitable. As described above, a low-temperature sensitive yeast can be obtained.

(Breeding of Low Temperature Sensitive Yeast for Transformation) In general, practical yeasts often accept foreign plasmid DNA or the like, so-called transformation ability is extremely low, and is used as an index when introducing a foreign gene. There are usually no markers. In order to impart a high transformation ability and a gene marker to the low-temperature sensitive baker's yeast, breeding by crossing with a laboratory strain having the high transformation ability and the gene marker is effective. Of course, it is needless to say that breeding can be carried out by utilizing techniques such as mutation and cell fusion.

As the low-temperature sensitive baker's yeast, it is desirable to use a haploid strain for breeding by crossing. For diploid or more strains, haploids can be prepared and used. A laboratory strain exhibiting a high transformation ability is preferably, for example, one having a transformation ability of about 1,000 transformants / μg DNA or more in transformation with the plasmid YCp50 (8 kb). However, if the ability to receive foreign DNA is slight, it can basically be used in the present invention. Although the transformation efficiency is greatly affected by the transformation method, an example is ALKALI CATI0N manufactured by BIO 101.
YEAST TRANSF0RMATI0N KIT etc. can be used. However, it goes without saying that the transformation method is not limited to this method. Gene markers include, for example, ura3, leu2, tr
p1, his3 (Methods in Enzymology, vol. 101, 202-211
Auxotrophy such as R. Wu, Academic Press), cerulenin resistance (N. Nakazawa et al., J. Ferment. Bioeng. Vol. 76, 60-63).
(1993)), G-418 resistance (TDWebster, Gene, vo 1.26 242-2)
52 (1983)). In addition, any marker can be used as long as it can confirm the transformation of the foreign plasmid.

When cold-sensitive baker's yeast is used as a host,
A method for providing an appropriate marker will be described. A low-temperature-sensitive haploid baker's yeast was crossed with a haploid laboratory strain having a transforming ability of about 5,000 transformants / μg DNA and having gene markers such as ura3, leu2, and trp1, and Make it. Then, sporulation medium (Methods in Yeast Genet)
ics, Cold Spring Harbor Laboratory Press), and spores are separated from the spore sac by a manipulation method. The low temperature sensitivity of the obtained spore strain is examined by the method described above. Further, the auxotrophy is analyzed, and the transformability is examined using a plasmid having an auxotrophic marker such as YCp50 or a plasmid having a drug resistance marker such as pYI13G. In this way, it is cold sensitive and
A host of a cold-sensitive yeast having high transformation ability and having one or more markers such as ura3, leu2 and trp1 can be grown.

(Preparation of Chromosome DNA Library) The preparation of the yeast chromosome library is performed by preparing the yeast chromosome DNA and YCp50, YE
Using a plasmid such as p13, the method of Maniatis et al. (Mo1ecu
lar Cloning, T. Maniatis et al., Co1d Spring Harbor Labo
ratory Press). Yeast chromosomal DNA is H
ereford method (L. Hereford et al., Cell, vol. 18, 1261-1271 (197
9)) and potassium acetate method (CurrentProtocols in Molecula
r Biology, FM Ausubel, etc. John Wiley & Sons, Ne
w York (1989)), and the DNA-donating yeast can be any strain that does not contain the cold-sensitive gene. Also, commercially available gene libraries such as ATCC3741
5, ATCC 37323 can be used. ATCC37415 or ATCC3
Transformation is performed using the library DNA prepared from 7323, and a transformant is selected using a selection marker.
The transformed strain may be obtained in the order of 1,000 to 10,000 strains.
The above libraries are libraries of YCp type and YEp type plasmids, respectively, and it is known that these plasmids have different copy numbers in yeast.

(Transformation) Transformation into the above-mentioned host is performed by the protoplast method (S. Harashima et al., Mol. Cell. Biol., Vo1.4,
771-778 (1984)), lithium acetate method (H. Ito et al., J. Bact
eriol., vol. 153, 163-168 (1983)), electroporation method, electroinjection method (gene experiment method using yeast, biomanual series, edited by Masayuki Yamamoto, Yodosha) and the like. A made by BIO 101
LKALI CATI0N YEAST TRANSF0RMATI0N KIT or the like can be used.

When transformation is performed using ATCC 37415, any selective medium may be used as long as it is a minimal medium containing no uracil, and preferably a medium having the following composition: Yeast nitrogen base with amino acids 0.67% Glucose 2.0% Casamino acids 0.4% Agar 2.0% may be used.

When transformation is performed using ATCC 37323, any selective medium may be used as long as it is a minimal medium containing no leucine. Preferably, the medium has the following composition: Yeast nitrogen base without amino acids 0.67% Glucose 2.0% L-leucine drop-out mix * 0.2% Agar 2.0% * Drop-out mix refers to Methods in Yeast Genetics, A Labo
ratory Course Manual (MD Rose et al., Cold Spring
Harbor Laboratory Press) can be used.

(Cloning of a Gene that Regulates the Fermentation Ability of Yeast at Low Temperature) Screening of a target gene can be performed using an index indicating that a cold-sensitive host returns to a wild type. For example, the transformant is replicated in a YPGA medium, cultured at 30 ° C. for 2 to 3 days, and then overlaid with a dye agar medium kept at about 45 ° C., and immediately kept at 5 ° C. overnight. Low-temperature-sensitive yeast has a markedly reduced fermentation ability at low temperatures, so BC
The color tone of P remains bluish purple, but the color tone of BCP of the strain that has been desensitized to low temperature changes to yellow. In this way, many strains (positive clones) transformed with the plasmid containing the gene that releases the cold-sensitive trait can be selected.

Further, a selection method different from the above method may be used. A strain in which fermentation is significantly suppressed at low temperatures is a strain whose energy acquisition system is significantly suppressed at low temperatures, and the growth of such strains at low temperatures is significantly suppressed as compared with a wild strain. After culturing the transformed strain in YPD medium overnight,
Plate on agar medium. After culturing at 5 ° C. for about 2 months among the transformed strains, a number of strains that form colonies in the same manner as the wild strain can be isolated. In this way, a plurality of positive clones can be obtained.

(Recovery of Plasmid and Restriction Enzyme Map) Extraction of the plasmid from the transformant is performed by the Hereford method, the potassium acetate method, or the like. Thereafter, E. coli such as JM109 or HB101 is transformed with the extracted DNA according to a conventional method. By culturing the obtained transformant and extracting and purifying the plasmid, the plasmid can be recovered. Mapping of the gene of interest inserted into the plasmid (creation of restriction enzyme map) can be performed using various commercially available restriction enzymes. In this manner, the mapping of each plasmid of a plurality of positive clones results in the inserted DN
The intersection of the A fragments can be found. In this common part,
It is thought that there are genes that regulate fermentation at low temperatures.

(Determination of DNA base sequence) For purification of a gene fragment that regulates fermentation power at a low temperature, agarose gel electrophoresis or the like can be used. The obtained gene fragment is, for example, cut with an enzyme recognizing 4 bases such as restriction enzyme Sau3A or TthHB81, and subcloned into M13 phage having a multiple cloning site, for example, M13mp18 or M13mp19, to determine the nucleotide sequence of DNA. Do. Alternatively, the DNA base sequence can be determined using a plasmid vector such as pUC18 or pUC19, pUC118 or pTV119N instead of the M13 phage.

The DNA base sequence can be determined by the Sanger method or the Manger method.
axam-Gilbert method (Molecular Cloning, T. Maniatis et al., C
old Spring Harbor Laboratory Press). For example, Taq D of PerkinElmer Japan, Inc.
using the yeDeoxy ^TM Terminator cyclesequencing Kit, AB
This can be done with an I373A DNA sequencer. By comparing the obtained DNA sequence with various databases, genes in the DNA sequence can be identified. DDBJ (DNA Data Bank of Jap)
an) etc. can be used.

(Identification of Properties of Cold-Sensitive Genes) Among genes that regulate fermentation at low temperatures, genes whose activities can be measured relatively easily, for example, genes encoding glycolytic enzymes, have a degree of sensitivity. Can be measured as follows. First, a cell-free extract of the desired gene product was prepared (Masayuki Yamamoto, Bio Manual Series 10: Gene Experiment Using Yeast, Yodosha), and then the activity of each enzyme was measured in vitro. Cold sensitivity can be measured. In a cold-sensitive yeast having the same type of regulatory gene, a strain having high cold sensitivity can be selected by measuring the activity of the gene product at a low temperature. On the other hand, among cold-sensitive yeasts having different heterologous regulatory genes, strains having high cold sensitivity can be selected by comparing the rate of decrease in activity of the gene product at low temperature relative to the wild-type with each other.

(Analysis of Mutation Site) When a gene that regulates fermentation at low temperature has a mutation, the analysis of the mutation site includes the respective genes of wild-type yeast and mutant yeast that has become sensitive to low temperature. It is necessary to determine and compare the nucleotide sequences of

The parent strain of the cold-sensitive yeast, wild-type yeast
If you create a DNA library and use it to clone genes that regulate fermentation at low temperatures,
A mutation site can be determined by obtaining a gene fragment of a cold-sensitive yeast corresponding to the cloned gene and comparing the sequences.

On the other hand, when a DNA library of the parent strain is not used but a commercially available DNA library is used, the sequence of the gene fragment of the parent strain and its mutant, a cold-sensitive strain corresponding to the cloned gene fragment, is used. Need to be compared.

Whether or not a DNA (gene) fragment corresponding to the cloned DNA is present in the parent strain or the cold-sensitive yeast can be determined by Southern hybridization (Molecular Cloning, T. Maniatis et al., Cold Spri.
ng Harbor Laboratory Press).

The extraction of chromosomal DNA from yeast was performed by Hereford.
It can be performed using a method or a lithium acetate method. Extracted DN
A is cut with various restriction enzymes based on the above-mentioned restriction map, subjected to agarose gel electrophoresis, and blotted on a nylon filter or a nitrocellulose filter. A part or all of the above-mentioned cloned DNA fragment is used as a probe, isotope-labeled or fluorescent-labeled, and used for Southern hybridization. Fluorescein Gene Images ^™ random prime m manufactured by Amersham Japan Co., Ltd.
An odule or the like can be used. Also, instead of fluorescent labels
³² P labels can also be used.

From the results of Southern hybridization, both the parent strain and the cold-sensitive strain showed the cloned DNA.
It is determined whether the fragment has the same restriction map. Based on this information, the chromosomes of the parent strain and the cold-sensitive strain
The DNA is cleaved with an appropriate restriction enzyme, and the region where the target DNA fragment is present is purified by agarose electrophoresis or the like. The DNA thus obtained is inserted into an appropriate restriction enzyme site of a plasmid vector such as pUC18 or pUC19.
Instead of a plasmid vector, a phage vector such as λ phage can also be used. Using probes used for Southern hybridization, colony hybridization (Molecular Cloning, T. Maniatis et al., Cold
Spring Harbor Laboratory Press)
Clones having DNA fragments can be screened. In the case of a phage vector, a plaque hybridization method can be used.

By the above method, both wild-type and mutant DNA fragments of a gene that regulates fermentation power at a low temperature can be cloned. By determining and comparing the base sequences of both of these DNA fragments, the mutation site of the gene encoded by the DNA is revealed.

(Optimization of Mutation: Introduction of Mutation into Target Gene) By further optimizing the gene that regulates fermentation at low temperature according to the present invention, an excellent low-temperature sensitive strain can be bred.

To examine whether the gene that regulates fermentation at a low temperature is the best among the obtained mutations, an artificial mutation is introduced into the wild-type gene, and for example, an expression system using Escherichia coli is used. By using this, a large number of mutant gene products can be obtained. As an expression system, in addition to Escherichia coli, a system of yeast, Bacillus subtilis, animal cells and the like can be used. As a method for artificially introducing a mutation into a gene, there is a method for causing a mutation chemically or enzymatically. Methods for introducing mutations include a method for introducing mutations at random and a method for introducing mutations site-specifically.

As a method for introducing mutations at random, there is a method for chemically mutating DNA using a drug. Such agents include hydroxylamine hydrochloride, sodium nitrite, formic acid, hydrazine and the like. For example, in the mutation using hydroxylamine hydrochloride, first,
A DNA fragment of a gene that controls fermentation at low temperature is ligated to double-stranded DNA of M13 phage, for example, M13mp19. The ligated DNA is infected with E. coli JM109 or the like and cultured.
After the culture, phage DNA is prepared, and a mutation reaction is performed on the obtained phage particles. The mutagenesis reaction can be performed using hydroxylamidine hydrochloride. As hydroxylamine hydrochloride, 0.1 M to 2 M, preferably 0.25 M, pH 6.0 to 8.
A value of 0, preferably 6.0 can be used. Reaction is 37 ° C
And can be done for 1 to 24 hours. The phage particles that have undergone this mutation reaction are infected with E. coli JM109 or the like, and the double-stranded recombinant DNA into which the mutation has been introduced is recovered. The recovered mutant gene can be prepared by transferring it to a plasmid.

As a mutation method using sodium nitrite, formic acid, hydrazine or the like, basically, the method shown by Myers KW et al. (Science, vol. 229, 242-247 (1985)) can be used. This method can be performed by preparing a single-stranded DNA from a recombinant M13 phage into which a regulatory gene has been incorporated, and subjecting this to mutation treatment.

When sodium nitrite is used, the
The reaction is performed at a concentration of 2M, preferably 1M, and the pH is acidic, preferably pH 4.3, at a reaction temperature of 4 ° C to 37 ° C, preferably 25 ° C,
The reaction can be performed for 1 minute to 5 hours, preferably 30 minutes.

When formic acid is used, a concentration of 12 M and a temperature of 4 ° C.
The reaction can be performed at 37 ° C., preferably 15 ° C., for 2 to 10 minutes. When using hydrazine, at 25 ° C
This can be done by treating for 3 to 10 minutes.

The single-stranded DNA mutated in these reactions is
A mutated gene can be prepared by double-stranding using Klenow fragment of Escherichia coli DNA polymerase or SequenaseR and incorporating into a plasmid.

As a method of random mutation using an enzyme reaction, a method using a PCR reaction (polymerase chain reaction) (DW Leun et al., Journal of Methods in Cell a
nd Molecular Biology, vol. 1, 11-15 (1989)). In this method, Taq DNA polymerase is used to synthesize genes from synthetic DNAs (DNA primers) having sequences at both ends of the genes. The reaction conditions in this case, for example, using a reaction condition such as increasing the concentration of MgCl ₂ or the concentration of the substrate (dNTPs) from the normal reaction, or extremely decreasing the concentration of only one of the four substrates. , TaqDNA
By causing an error in gene synthesis by a polymerase, a gene having a mutation can be prepared.

Examples of a method for introducing a gene mutation site-specifically include an in vitro site-specific mutagenesis method using an oligonucleotide, a cassette replacement method for a mutation site, and a method using a PCR reaction. The method of introducing a site-specific mutation in vitro using an oligonucleotide is to prepare a single-stranded DNA from the M13 recombinant phage into which the gene has been inserted, and a synthetic DNA primer containing the mutated sequence of the portion to be mutated Is a method of double-stranding with an enzyme such as a DNA polymerase Klenow fragment. By introducing the double-stranded DNA into E. coli, a gene into which a mutation has been introduced can be prepared. This in vitro site-directed mutagenesis is performed using a commercially available kit, for example, Takara Shuzo Co., Ltd.
"Mutan ^TM -K" or "Mutan ^TM -G", Amersham
It can be easily carried out by using Japan's “site-specific in vitro mutant production system using oligonucleotide version 2.0”.

The cassette replacement method for the mutated site can be performed by completely replacing the restriction enzyme fragment containing the site to be mutated with the synthetic DNA containing the mutation.

Mutagenesis using the PCR reaction can be performed according to the method of Ito et al. (W. Ito et al., Gene, vol. 102, 67-70 (1991)). In this method, a PCR reaction is performed using a synthetic DNA primer containing the site to be mutated and a synthetic DNA at the end of the gene, and the synthesized DNA and the full-length DN of the gene are used.
After hybridizing with A, it is extended with an enzyme to synthesize the full length gene including the site to be mutated.
This is a method for performing the reaction again.

The gene into which the mutation has been introduced in this manner is
E. coli expression vectors, such as pKK233-2, pKK233-3
(Pharmacia Biotech) and introduce it into E. coli such as JM105. The desired polypeptide can be expressed by culturing these strains and adding a trait expression inducer such as IPTG. Extraction and purification can be performed according to the properties of the expressed polypeptide. Each of the purified gene products (polypeptides) is used at a high temperature (for example, 30
° C), low temperature (for example, 0 ° C to 20 ° C, preferably 5 ° C to 15 ° C)
The activity is measured by the above, and the difference in activity according to the temperature is determined. Among them, a gene product which exhibits a normal level or higher activity at a high temperature and a markedly lower activity than a wild type at a low temperature can be selected.

By comparing the temperature sensitivity of the product (polypeptide) of the gene into which the mutation was artificially introduced, it is determined whether or not the low-temperature-sensitive gene obtained by mutation or the like can further improve the function of suppressing fermentation at low temperatures. be able to. Therefore, if the improvement of the fermentation-suppressing function of this gene product at low temperatures can be expected, screening of more mutant strains should be performed to select yeasts containing genes exhibiting excellent cold sensitivity. Can be. Thus, artificial mutagenesis of the gene has provided useful information for screening of yeasts that are sensitive to low temperature, and cloning of the gene provides the only means to enable it.

As an application example of this technique, an optimal gene into which a mutation has been introduced can be selected, and the gene can be replaced with a wild-type baker's yeast gene to breed a superior strain. In general, in yeast, gene replacement using homologous recombination of genes in vivo can be performed relatively easily, and gene replacement can be similarly performed for two or more genes that regulate fermentation power at low temperatures. It is possible. As described above, the present invention also enables the breeding of low-temperature sensitive baker's yeast using the gene recombination technique.

(Plasmid) The gene obtained by the above method and capable of controlling fermentation power at a desired low temperature can be incorporated into a suitable plasmid, and yeast can be transformed. Any plasmid can be used as long as it is maintained in yeast. For example, yeast plasmids represented by YIp, YEp, or YCp can be used. Preferably, YIp1, YIp5, YCp5
0, a plasmid such as YEp13 can be used.

The thus obtained plasmid having a gene capable of controlling fermentation at a low temperature is introduced into baker's yeast, and a transformed yeast can be obtained.

The plasmid may contain two or more different genes that regulate fermentation power. Also,
A plurality of plasmids each having a different gene may be transformed into yeast.

(Breeding) A large number of yeasts containing genes that regulate fermentation power at low temperatures can be obtained by the method described above. Differences between yeast genes can be determined using a genetic method such as a complementation test. However, it is often difficult to clarify differences in mutations within genes.

Breeding a yeast containing a plurality of different regulatory genes from a yeast containing one gene that regulates fermentation at a low temperature by using breeding techniques such as hybridization provides an excellent cold-sensitive yeast. Is the way.

In the method using the genetic technique, it is necessary to carry out a detailed tetrad analysis, including crossing. In general, spore strains may show poor growth or strains that do not germinate at all, and this tendency is particularly strong in practical baker's yeast. For this reason, tetrad analysis becomes difficult, and in many cases, haploid strains containing a plurality of genes that regulate fermentation at different low temperatures cannot be easily selected. As described above, the method using the genetic technique actually requires a great deal of labor and time. In the present invention, a method for breeding a yeast which is difficult to apply the four-molecule analysis such as a practical yeast (for example, baker's yeast) is provided. It also provides a method for easily breeding yeast containing a plurality of genes that regulate fermentation power at low temperatures.

The gene of the present invention cannot be easily and accurately evaluated unlike distinct markers such as auxotrophy. Therefore, it is necessary to examine the degree of gene expression. Therefore, conventional methods such as tetrad analysis are not efficient because the fermentation power of all the formed spores must be examined.
Thus, once the target gene is isolated, a method for breeding a practical yeast using the target gene is provided.

Hereinafter, a method for breeding yeast containing a plurality of genes that regulate fermentation power at low temperature will be described.

First, a plurality of genes that regulate the fermentative power of yeast at a low temperature are isolated. Isolation of the gene can be performed in the manner described above. This isolated gene is used as a probe during breeding. Next, spores are formed in the cold-sensitive yeast, and a spore strain having a gene that regulates the fermentative power of the yeast at a low temperature is isolated. Next, spore strains having different cold-sensitive genes and having different mating types were mated,
Create diploid yeast. Spores are formed in this diploid yeast to obtain a spore strain. From among these, a spore strain having a plurality of genes that regulate the fermentative power of yeast at a low temperature is detected and isolated using the above gene as a probe. A spore strain having a plurality of genes selected in this manner and having different mating types and having a plurality of genes that regulate fermentation power can be conjugated to breed a desired yeast.

Preferably, yeast having two genes that regulate the fermentative power of yeast at low temperatures can be bred. This method comprises joining a spore strain having one gene that regulates the fermentative power of yeast at low temperature with a spore strain having one other gene that regulates the fermentative power of yeast at low temperature, and This is a method for breeding yeast by isolating a spore strain having two genes that regulate fermentation power.

Further, yeast having three or more genes that regulate the fermentative power of yeast can be bred. A spore strain having two genes that regulate yeast fermentation at low temperatures and a spore strain having other genes that regulate yeast fermentation at low temperatures, and three or more genes that regulate fermentation Can be bred.

In another preferred embodiment, a spore strain having two improved genes and a spore strain having other improved genes are mated to form a yeast having three or more improved genes. Is a method of breeding.

The case of introducing two genes will be described more specifically. To select strains having two different cold-sensitive genes from haploid spores, the method of MD Rose et al. (Methods in Yeast Geneti
cs, A Laboratory Course Manual, Cold Spring Harbor
Laboratory Press) is preferably performed. As the probe, for example, DNA encoding before and after the mutation site of the gene
According to the sequence, a 21-nucleotide probe consisting of 10 nucleotides each on the 5'-side and 3'-side centered on the mutant base can be suitably used. The sequence and length of the probe
Any type of wild type and mutant synthetic DNA can be used as long as there is a difference that can be detected as a dissociation condition in colony hybridization. γ- ³² P-ATP
Alternatively, if the end of DNA is labeled with a fluorescent reagent, it can be used as a probe. The two probes remain hybridized to the mutated DNA, but wild-type DNA
The washing conditions for dissociation from are determined.

First, haploid yeasts containing different cold-sensitive genes and having different mating types are conjugated to each other to prepare a diploid strain. Diploidization can be performed not only by hybridization but also by cell fusion or the like. The resulting diploid vegetative cells are applied to a spore-forming medium to form spores, and spores are separated by manipulation or random spore separation. Usually four haploid spores are isolated from a diploid strain. The resulting spore strain inherits the chromosomes of the parents in a mixed form, which may include chromosomes that have recombination between homologous chromosomes of the parents. From these spore strains, a spore strain having chromosomal DNA that remains hybridized to the mutant probes of the two genes by the above-described method but is dissociated from the wild-type probe is selected. This strain is a spore strain having two kinds of low-temperature-sensitive genes, and strains having different mating types among them can be joined together to prepare a diploid strain having two kinds of low-temperature-sensitive genes. Most of the strains bred in this way have improved cold sensitivity compared to the cold sensitivity exhibited by the parent strain.

In this way, the desired cold-sensitive yeast can be bred. The yeast thus obtained has a low fermentative power at a low temperature, and is therefore used for bread making by the refrigerated dough manufacturing method.

[0107]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the present invention.

[0108] The main culture media used in the present invention are as follows.

(YPDA medium) Bacto-yeast extract 1% Bacto-peptone 2% Glucose 2% Adenine Hydorochloride 0.04% YPDA agar medium is a medium obtained by adding 2% agar to this YPDA medium.

(Molasses medium) Molasses (as sugar) 3.0% Ammonium sulfate 0.5% Phosphorous acid 0.3% Urea 0.2% (YPGA medium) Bacto-yeast extract 1% Bact-polypeptone 2% Glycerol 3% Adenine Hydrochloride 0.04% Bacto-agar 2 % (Casamino acid medium) Yeast nitrogen base with amino acids 0.67% Glucose 2.0% Casamino acids 0.4% Bacto-agar 2.0% (Mycell solution) Glucose 3% Diammonium hydrogen phosphate 0.5% Potassium Dihydrogen phosphate 0.5% (Dye agar medium) Bacto- yeast extract 0.5% Bacto-polypeptone 1% Sucrose 10% 2% BCP / Et0H 1% Bacto-agar
0.8% (minimal medium) Yeast nitrogen base with amino ac
ids 0.67% Glucose
2.0% drop-out mix * 0.2% Bacto-agar 2.0% * Drop-out mix is for Methods in Yeast Genetics, a Laboratory Course Manual
(MDRose et al., Cold Spring Harbor Laboratory Press). (Leucine-free agar medium) Yeast nitrogen base without amino acids 0.67% Glucose 2.0% L-Leucine drop-out mix 0.2% Bacto-agar 2.0%

Further, various detailed procedures for handling plasmids, phages, DNAs, various enzymes, Escherichia coli, yeast, and the like used in the examples are described in the following magazines and books. 1. Protein, Nucleic Acid, Enzyme, 26 (4), (1981) Extra edition "Genetic Manipulation" (Kyoritsu Shuppan) 2. Proteins, nucleic acids and enzymes, 35 (14), (1990) Extra edition "Gene manipulation 1990" (Kyoritsu Shuppan). 3. Biomanual Series 10 "Gene Experiments with Yeast" Masayuki Yamamoto Editing Yodosha 4. Methods in Yeast Genetics, A Laboratory Course
Manua1, MDRose et al., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York Molecular Cloning, a Laboratory Manual, Maniati
s et al., Cold Spring Harbor Laboratory Press, Cold Sp.
ring Harbor, New York Methods in Enzymology, L. Grossman et al., Vol. 29, Aca
demic Press 7. Methods in Enzymology, edited by R. Wu et al., Volume 65, Academic
Press 8. Methods in Enzymology, edited by R. Wu et al., Volume 101, Academi
c Press 9. Methods in Enzymology, edited by B. Kimmel, 152 Academ
ic Press 10. Methods in Enzymology, edited by G. Fink, Vol. 194, Ac
ademic Press

Example 1 Using the spore strain 53R of Kanebuchi Chemical Co., Ltd. as a parent strain, the above-mentioned (A) was mutated by the EMS method, and the above-mentioned (B) was screened. As a result, three low-temperature-sensitive haploid yeasts designated as 53R-2-120, 53R-2-170, and 53R-5-89 were obtained. 53R-2-1
70 shares were transferred to the Saccha
It has been deposited as romyces cerevisiae 53R-2-170 (FERM P-15666). 53R-5-89 strain was sent to Saccharomyces cerevisiae
(FERM P-15667). For these strains, the low sugar dough fermentative power at 5 ° C and the low sugar dough fermentative power at 30 ° C were measured. The fermenting power of the low sugar dough is based on the low sugar dough fermenting power specified by the East Industry Association, and the following ingredients are blended: flour 100g yeast 2g sucrose 5g salt 2g water 62g, the dough temperature is 20 ± 1 ℃ And the dough was placed in a cylinder, and the amount of the dough at the start was measured in advance. The difference between the dough amount after fermentation at 5 ° C. for 24 hours and the dough amount at the start was defined as 5 ° C. (low temperature) low sugar dough fermentation power.

Further, the dough was mixed with the above raw materials so that the kneading temperature was 30 ± 1 ° C., and the dough was put into a cylinder, and the amount of the dough at the start was measured in advance. The difference between the amount of dough after fermentation at 30 ° C. for 80 minutes and the amount of dough at the start was defined as 30 ° C. low sugar dough fermentation power. Table 1 shows the results
Shown in

[0114]

[Table 1]

The mutant strain had a 5 ° C. (low temperature) low sugar dough fermentability that was much lower than that of the control strain 53R, Kaneka Red Yeast, and the 30 ° C. low sugar dough fermentability showed almost the same value as the control.

(Example 2) The transformation ability of the parent strain 53R and the strain 53R-2-120 obtained in Example 1 derived from this strain into cold-sensitive yeast was examined. LEU2, G-41 as plasmid
8 0.1 to 10 μg of pYE13G (ATCC37276) having a resistance marker
Used, BIO 101 company ALKALI CATI0N YEASTTRANSF0RMATI
Transformation was performed using 0N KIT. G-418 (250μg / m1)
After culturing for 3 days at 30 ° C. in a YPDA medium containing, the colonies that appeared were counted. As a control, SH2676 strain (zygous a: u
ra3-52, leu2-3, 112, trpl, his1-29, pho3-1, pho5-
1) was used. Table 2 shows the results.

[0117]

[Table 2]

(Example 3) In order to improve the transforming ability of the low-temperature sensitive strain 53R-2-120 and to provide a selection marker, it was crossed with a laboratory strain having a high transforming ability. The SH2676 strain was used as one parent of the cross. Using the method described in Example 2, pYE13G was used to transform strain SH2676, and SH2676 (pYE
13G) was prepared.

53R-2-120 (conjugated α) strain, SH2676 (pYE13G)
Each strain was streaked on YPDA agar medium and cultured at 30 ° C. for 2 days. 53R-2-120 strain and SH2676 (pYE1
The 3G) strain was replicated in a cross and cultured at 30 ° C. for 1 day. Thereafter, the cells were replicated on a minimal medium containing 250 μg / m1 of G-418, and further cultured at 30 ° C. for 3 days. The growing colonies were isolated and streaked in Fowell's sporulation medium to form spores at 30 ° C. A strain in which sporulation was observed was defined as a hybrid strain. Each hybrid strain was subcultured several times in a YPDA medium to remove the plasmid. Confirmation of the dropout of the plasmid was performed using the restoration of G-418 sensitivity as an index.

The spores of the hybrid strain 53R-2-120 / SH2676 were separated by manipulation. Among the low-temperature-sensitive and uracil-requiring strains, the growth was good, and the judgment by the low-temperature-sensitive dye plate was clear 53R-2-120 / SH27 (conjugated α; ura3-52, leu2-3, 112, trp1, his1-29, lts (low temperature sensitive mutation)) (hereinafter abbreviated as W27) were selected. The transformation efficiency of this cold-sensitive and uracil-requiring strain was determined by YCp50
It measured using. Table 3 shows the results.

[0121]

[Table 3]

(Example 4) A gene for restoring the low-temperature sensitivity of the W27 strain to the wild type was cloned. Created by M. Rose as a wild-type gene library, AT
ATCC No. 37415 (Name of Libr) distributed through CC
ary: CEN BANK) was used. ALKALI CATI0N manufactured by BIO 101
The W27 strain was transformed with CEN BANK DNA using YEAST TRANSF0RMATI0N KIT according to the protocol of the kit. Transformants were grown on casamino acid agar at 30 ° C, 2
It was obtained by culturing for days.

The transformant was replicated on a YPGA medium and a casamino acid agar medium as a master plate.
Cultured for days. After 10 ml of a dye agar medium was overlaid on the colonies grown on the YPGA medium and cultured overnight at 5 ° C, colonies that formed yellow halos were screened. The cold-sensitive strain did not change the bluish-violet hue of BCP.
As a result of screening about 8,000 transformed strains, four positive strains were obtained. These strains were named RTS301 to RTS304, respectively.

In order to remove the plasmid from the obtained four positive strains, an agar medium containing 5-fluoroorotic acid (Methods in Enzymology, edited by G. Fink, vol. 194, 302-319) was used.
(1991)). As a result of culturing at 30 ° C., strains from which the plasmid was eliminated were obtained. These strains were again replicated on YPGA medium, and the presence or absence of low-temperature sensitivity was examined on a dye agar medium.As a result, two out of four strains (R
TS301 and RTS302) showed cold sensitivity, and two strains (RTS303 and RTS304) did not show cold sensitivity. 2 positive strains
(RTS301, RTS302). Two
DNA was extracted and purified from the strain by the Hereford method, and E. coli HB10
1 and the plasmid was recovered. PRC each
Named A and pRCB. Transformation was performed again using each plasmid and the W27 strain as a host to obtain each transformed strain. Examination of each strain for the presence of low-temperature sensitivity on a dye agar medium revealed that both pRCA and pRCB returned the low-temperature sensitivity to the wild type.

From the above results, the plasmids pRCA and pRCB carried by the RTS301 and RTS302 strains respectively contained W2
It was found that a DNA fragment that restored the cold sensitivity of the seven strains to the wild type was cloned. FIG. 1 shows a restriction map of the above DNA fragment present in the plasmid pRCA.

Example 5 Next, an experiment was performed to identify a gene region that restores cold sensitivity to the wild type.
pRCA was cut with EcoRI, and after agarose electrophoresis, the cut insert DNA fragment was extracted using GENE CLEAN (manufactured by BIO 101). Using DNA Ligation Kit ver.2,
PRS316 (Robert S. Si) in which a DNA fragment was digested with EcoRI
Korski et al. Genetics, 122: 19-27 (1989)). E. coli HB101 was transformed, and a plasmid was extracted from the resulting transformant. Plasmid Ec
As a result of cleavage and analysis with various restriction enzymes such as oRI,
Plasmid pRCA6 containing a yeast chromosome DNA fragment that restores the cold sensitivity of the W27 strain to the wild type in the 5.2 kb EcoRI-EcoRI fragment
was gotten. Plasmid pRCA6 was introduced into E. coli HB101 and sent to E. coli HB1
Deposited as 01 (pRCA6) (FERM-15668).

This pRCA6 was digested with HindIII and DNA
Ligation was performed using Ligation kit ver.2. E. coli HB101 was transformed, and a plasmid was extracted from the resulting transformant. The plasmid was digested with various restriction enzymes such as HindIII and analyzed.
About 2.from the HindIII site in A to the HindIII site of pRS316.
3 kb deleted pRCA14 was obtained.

PRCA6 was digested with HpaI and SacI, and after agarose gel electrophoresis, a fragment of about 1.3 kb was extracted using GENE CLEAN (manufactured by BIO 101). This extracted fragment was
DNA Ligation Kit ver. To pRS316 cut with SacI.
Ligation was performed using 2. E. coli HB101 was transformed, and a plasmid was extracted from the resulting transformant. The plasmid was digested with various restriction enzymes such as SacI, and analyzed.As a result, SacI was removed from the HpaI site in the yeast chromosomal DNA.
PRCA16 with about 1.3 kb inserted to the site was obtained.

PRCA6 was digested with HindIII, and digested with the above method.
A 1.3 kb fragment was extracted and pRS316 cut with HindIII.
Was ligated. This was transformed, extracted with a plasmid, and analyzed as described above.
PRCA19 into which the HindIII fragment was inserted was obtained.

FIG. 2 shows a restriction map of each plasmid. PRCA6, pRCA14, pRC produced by the above method
W27 according to the method described above using A16 and pRCA19, respectively.
The strain was transformed, and the transformed strain was selected on a casamino acid medium. The low-temperature sensitivity of each transformant was examined using a dye agar medium. The results are shown in Table 4.

[0131]

[Table 4]

From the results, it was found that the EcoRI site of pRCA6
There is a gene that releases the cold sensitivity of the W27 strain on the approximately 5.2 kb DNA fragment up to the site, and furthermore, the cold sensitivity of the W27 strain is reduced on the approximately 1.3 kb DNA from the SacI site to the HpaI / SmaI site of pRCA16. It became clear that there is a high possibility that the gene to be released exists.

(Example 6) From the gene region that releases cold sensitivity determined in Example 5, ie, from the SacI site
The nucleotide sequence of about 1.3 kb up to the HpaI site was determined. p
RCA6 was digested with EcoRI, a DNA fragment of about 5.2 kb was separated by agarose electrophoresis, and the fragment was extracted and purified using GENE CLEAN (manufactured by BI0101). Similarly pRC
A6 was cut with XbaI, a DNA fragment of about 2.5 kb was separated by agarose electrophoresis, and the same fragment was extracted and purified. Furthermore, pRCA6 was cut with EcoRI and XbaI, and about 1.0 kb
Was separated by agarose electrophoresis, and the same fragment was extracted and purified. Next, about 5.2 kb, about 2.5 kb, about 1.
Each DNA fragment of 0 kb was subcloned into pUC118. After processing in accordance with a conventional method, Inc. PerkinElmer Japan Co., Ltd. of Taq DyeDeoxy ^TM Terminator Cycle Sequenc
Using ing Kit, the reaction was performed according to the protocol of the kit. The DNA base sequence was determined using AB1373A DNA sequencer manufactured by the company. PUC118 in the sequence listing
The nucleotide sequences of the subcloned DNA fragments are shown as T1 sequence (SEQ ID NO: 1), T3 sequence (SEQ ID NO: 3), T4 sequence (SEQ ID NO: 4), and T5 sequence (SEQ ID NO: 5). Based on this base sequence, homology search was performed using the database DDBJ. As a result, a DNA fragment of about 1.3 kb
It was revealed that it was included in the entry name SCHAPLAP (35757bp). Furthermore, it was revealed that the plasmid contained an open reading frame of about 1.1 kb: YKL098w. D
The NA fragment is located on the left arm of chromosome 11.

(Example 7) Cloning of a gene region of about 1.3 kb that regulates fermentation at low temperatures determined in Examples 5 and 6 was performed from wild-type yeast 53R and low-temperature sensitive yeast 53R-2-120. Was. Chromosomal DNA of 53R strain and 53R-2-120 strain was prepared by the Herford method, and each extracted DNA
Was digested with SacI and HpaI, and a fragment of about 1.3 kb containing the entire region of the cold-sensitive gene was recovered (see FIG. 2). This extracted fragment was ligated to the SacI-SmaI site of pUC118, and JM109 was transformed to prepare a library. pRCA6
Colony hybridization was carried out using about 1.3 kb of the SacI-HpaI fragment as a probe, and positive clones were obtained from the respective libraries. As a result of analyzing the plasmid recovered from each strain using various restriction enzymes, it was confirmed that the clone was the target clone. Using each plasmid as a template, the following primers:

[0135]

[Table 5]

The nucleotide sequence of the SacI-HpaI / SmaI fragment of about 1.3 kb was determined using the above method. The nucleotide sequence was determined according to Example 6. As a result, as shown in FIG. 3 to FIG.
In strain 2-120, it was revealed that cytosine (C) at position 289 of YKL098w had a base substitution for thymine (T), and glutamine (CAG) had been converted to a stop codon (TAG).

Example 8 The 1.3 kb SacI-HpaI / SmaI fragment whose base sequence was determined in Example 7 was examined for its ability to regulate fermentation power at low temperatures. According to Example 5, SacI-Sma of pRS316
At the I site, about 1.3 kb of the above DNA fragment SacI-HpaI was ligated, and from 53R strain and 53R-2-120 strain, pRCA28 and pR
CA29 was obtained. A cold-sensitive yeast obtained by cross-breeding the 53R-2-120 strain with the SH2676 strain; a transformed strain W07 (pRS3
16), W07 (pRCA28) and W07 (pRCA29) were examined for low-temperature sensitivity using a dye agar medium according to Example 4. In addition, low temperature sensitivity was measured by the amount of gas generated. Agar-free casamino acid liquid medium (5 ml)
And cultured overnight at 30 ° C. The whole amount was subcultured in 50 ml of the same medium and cultured overnight at 30 ° C. The an OD ₆₀₀ of the culture solution was measured, aligned cell mass was harvested by centrifugation. The measurement of the gas generation amount was performed as follows. As the fermented liquid, the following K10 fermented liquid capable of obtaining a good correlation with the fermentation pattern in the dough was used.

[0138]

[Table 6]

The cells were suspended in 2.5 ml of K10 fermentation broth, transferred to an aluminum-capped test tube (15 × 130 mm), and cultured with shaking at 30 ° C. or 5 ° C. at a speed of 220 rpm. It was measured. The dry weight of the cells was measured after drying at 105 ° C. for 5 hours. The results are shown in Table 7 and FIGS. At 30 ° C, all three strains showed the same amount of gas generation, but at 5 ° C, the W07 (pRCA28) strain showed a remarkable increase in gas generation,
Cold sensitivity was suppressed.

[0140]

[Table 7]

(Example 9) A complementation test on the low-temperature sensitivity of the three strains (zygous α) obtained in Example 1 and another low-temperature-sensitive yeast was performed. Plasmid pRBC1 (E. coli HB
It has been transformed into E. coli HB101 (pRBC1) (FERM P-14888) and deposited with the National Institute of Bioscience and Human Technology. ), A cold-sensitive yeast having a mutation in the same DNA region as the yeast DNA region; 9195 / SH38A (conjugation type a;
) And plasmid pRAB12 (transformed into E.coli HB101 and sent to E.coli
Deposited as HB101 (pRAB12) (FERM P-14889). ), A cold-sensitive yeast having a mutation in the same DNA region as the yeast DNA region; 9230 / SH42C (conjugation type a; hereinafter abbreviated as 42C); and a cold-sensitive yeast obtained by cross-breeding 53R-2-120 with SH2676; (Zygous type a), the resulting diploid was examined for low-temperature sensitivity using a dye agar medium, and the result of evaluation for complementation is shown in Table 8.

[0142]

[Table 8]

(Example 10) YKL0 for releasing low-temperature sensitivity
The 98w gene was disrupted. FIG. 9 shows an outline of plasmid preparation used for gene disruption. First, pBluescriptII
LE of YEp13 at SalI-XhoI site of KS + (Funakoshi Co., Ltd.)
P506 into which the U2 fragment was inserted was created. Next, about 2.3 of pRCA6
pRCA21 was prepared by inserting the EcoRI-HindIII fragment of kb into the EcoRI-HindIII site of pUC118. p506 was cut with SpeI and XhoI, pRCA21 was cut with SpeI and XbaI, and each was DNA bluntin
After blunt-ending with g kit (Takara Shuzo), pRCA-d2 in which the LEU2 fragment of p506 was inserted into pRCA21 was prepared according to Example 5. As a result, the SpeI-XbaI fragment of the yeast DNA gene in pRCA21 was deleted, and a yeast gene in which the YKL098w gene had been disrupted by insertion of the LEU2 gene was obtained.

As a host for gene disruption, strain SH2675 (conjugated α: ura3-52, leu2-3, 112, trp1, pho3-1, pho5-1)
Diploid strains SH2675 / SH2676 obtained by crossing
The strain was used. Transformation was carried out according to Example 2 using about 30 μg of pRCA-d2, and the transformant was obtained by culturing at 30 ° C. on a leucine-free agar medium. From the transformant,
Chromosomal DNA was prepared by the Hereford method, cut with HindIII, and Fluorescein manufactured by PerkinElmer Japan, Inc.
Fluorescent labeling was performed using the Gene Images ^™ random prime module, and analysis by Southern hybridization was performed using the same kit. Probe uses pRCA6 with EcoRI
Approximately 0.5 kb DNA fragment obtained by digestion with HindIII and pRCA6
An approximately 0.8 kb DNA fragment obtained by digesting with XbaI and HindIII and an approximately 2 kb LEU2 fragment obtained by digesting p506 with SpeI and XhoI were used. SH2675 / SH267 whose gene was disrupted as a result of analysis
It was confirmed that 6 (YKL098W :: LEU2) was obtained.

[0145] SH2675 / SH2676 (YKL098W :: LEU2) was cultured in Fowell sporulation medium to form spores and subjected to tetrad analysis. As a result, as shown in Table 9, for all the four molecules, the growth showed 2: 2 segregation, the growth of dispores was extremely poor, and the segregation of the LEU2 marker led to the gene disruption of this poorly grown spore strain. It was revealed that it was a strain. From this fact, it was found that the gene related to the low-temperature sensitivity is a gene strongly related to the growth.

[0146]

[Table 9]

[0147]

The mutant gene of the present invention that regulates the fermentative power of yeast at low temperatures, when introduced into baker's yeast, renders the host sensitive to low temperatures. Therefore, it can be suitably used for refrigerated bread dough. Furthermore, the gene and the method of breeding of the present invention can be used for breeding a cold-sensitive yeast which is more practical.

[0148]

[Sequence list]

[0149]

[SEQ ID NO: 1] Sequence length: 27 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: chromosomal DNA Source organism name: yeast Sequence: TTAATTGGTC TACATAGCGG CAAGATG 27

[0150]

[SEQ ID NO: 2] Sequence length: 1074 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: chromosomal DNA Origin organism: yeast Sequence: ATGGGCGATC ATAATCTGCC GGATTTTCAG ACATGTTTGA AGTTTTCTGT TACAGCTAAA 60 AAGAGCTTTC TATGTATGTA TAGAGACAGC GTCTCAAAGG AAAAATTAGC CTCTTCGATG 120 CCAAGTACTT GTGATATACA ACTGAAGAGG GCAATCAATG ACGCTTATCC GGGCGGAGGA 180 ATAAAGGTCA CGGTTCTGAA CTCAACAACT GCAAGCTTGG ACTCGTTAGC AACTACACAT 240 GTTAAGGAAT TTGAGATTGT TATCATTCCA GACATAAATT CTCTTTTACA GCCAGACCAA 300 GCGAAATTAG TAAAAATTAT GAGAGATTGC ACAGTGGCGA TTGAGAAGGC ACAGTCAACA 360 CGTATATTTA TAGGAGTGGT TCATTGGAAT AATCCCGTGC AGCCTTCAGG CGCCGCTAAG 420 GATGGGGATG AAGCAGGTAA ACCAGCTCCC AAAACTCGCA TCTTCCTGCC CACGTCTTTC 480 CGTATGGGTG CTTGGCTCAA ACACAAATTC TGGTTTGCAT GTGCACCCCC ATATTTGGAT 540 TTTGAAAGTT CAACTGAGTC TAGCATTAAT ACAAGAGCCA ATAATAGCAT TGGTATGGCT 600 GAGGAAGAGA AGCAGGAGCC AGAGAGCAAG AGGTCGATCA TATTAAATGA AGAGGCAAAT 660 CTGAACGATG TATTTGGGGTCCACTTA CGATAT CATGGTCCAT 720 TTGCGAACGC ATAGATTGAC GTATAACGCC AAAGCCGGCG GGGTATATAC AAATTCATTG 780 GACGACGTGG TGTTATTGTC TAGATTAATT GGTCTACATA GCGGCAAGAT GTTTGTGTCA 840 CCTTCACATG TCAAAGAGGC GTCCAGGTGG TACTTCCCCA TGCACTTGGA ATTGGTTCAG 900 CGTTCCTCCA TGGATAGTTC CTTATTGTAC GGTAGCGATC CCAACTTGGT GGACGAAATG 960 CTAGAGAAAC TTGCGAAGAT TAAGTGTGAG GAAGTTAATG AGTTTGAGAA TCCCCTTTTC 1020 CTGGAATCTC TCGTAGTGAA AAACGTACTA AGTAAAGTCG TGCCGCCCGT GTAA 1074

[0151]

[SEQ ID NO: 3] Sequence length: 139 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: chromosomal DNA Origin Organism name: Yeast Sequence: GTTCATCTTC TCTCGGGAAT CGACGAATAT TGTATGATTC CCACTGCTTT TGAACATCAA 60 CTGTTTCGCT CCTTTCTCCA GCTTTTTCAG CTCTATTTGC CTCAGCGTCC TCACATAATT 120 ACTATCTTGC GTTTTGAGT 139

[0152]

[SEQ ID NO: 4] Sequence length: 130 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: chromosomal DNA Origin Organism name: Yeast Sequence: ACTCAATGAA CTTTTGTCCT CACTACAAATT GCC
CCTCGGA TGGATTTGAA CTTACAAGAA 60 ATATCACGTTT GGCGCGCGCT AAAGTCGCTTT GCC
AATGCGC TATGAAATTTCATGACCTG 120 GCACATTTTTA
130

[0153]

[SEQ ID NO: 5] Sequence length: 60 Sequence type: nucleic acid Number of strands: double strand Topology: linear Sequence type: chromosomal DNA Origin Organism name: Yeast Sequence: TTCGGAATCG GCAATGACCA TCTTAGCCAA GGCGAATAAA GCGACAGACA AAGCAGTGGA 60

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing a restriction map of a pRCA-inserted yeast chromosomal DNA.

[Figure 2] Yeast chromosomal DNA of pRCA6, pRCA14, pRCA16, pRCA19
It is a figure which shows the restriction map of a part.

FIG. 3 is a diagram showing a control for comparing the DNA base sequences of the approximately 1.3 kb SacI-HpaI fragment of the 53R strain and the 53R-2-120 strain.

FIG. 4 is a continuation of FIG. 3;

FIG. 5 is a continuation of FIG. 4;

FIG. 6 is a continuation of FIG. 5;

FIG. 7 shows strains W07 (pRS316), W07 (pRCA28), and W07 (pR28).
It is a figure which shows the gas generation amount at 30 degreeC of CA29) strain.

FIG. 8: W07 (pRS316), W07 (pRCA28), and W07 (pR
FIG. 7 is a view showing the amount of gas generated at 5 ° C. of strain CA29).

FIG. 9 is a diagram showing a method for preparing a plasmid for gene disruption.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location // (C12N 1/19 C12R 1: 865)

Claims (18)

[Claims] 1. A gene that regulates the fermentative power of yeast at low temperatures. 2. The gene according to claim 1, wherein the gene is a gene related to an energy acquisition system or a sugar transmembrane system. 3. The gene is located on the left arm of chromosome 11 of yeast, and has the following restriction enzyme map: EcoRI-Ec
Approximately 5.2 kb fragment of oRI: The gene according to claim 1, which is present in 4. The gene according to claim 4, wherein said gene is about 5.2 of said EcoRI-EcoRI.
The following sequence present in an approximately 1.3 kb fragment of SacI-HpaI in the kb fragment (T1: SEQ ID NO: 1): The gene according to claim 3, having the following. 5. The gene has a sequence of about 1.1 kb (T2:
SEQ ID NO: 2): The gene according to claim 4, having the following. 6. The gene according to claim 1, wherein the gene is 1 or 2 of the gene.
The gene according to any one of claims 1 to 5, wherein the nucleotide has a substitution, addition or deletion mutation, and the regulation of fermentation power is equal to or improved from that before the mutation. 7. A plasmid having the gene according to any one of claims 1 to 6. 8. A yeast containing the plasmid according to claim 7, the fermentation power of which is regulated at a low temperature. 9. The plasmid, wherein the plasmid regulates fermentation power.
The yeast according to claim 8, which is a plasmid containing two or more different genes. 10. A yeast having a gene according to any one of claims 1 to 6, the fermentation power of which is regulated at a low temperature. 11. A yeast which is complementary to an existing yeast whose fermentation is regulated at a low temperature with respect to the ability to regulate fermentation at a low temperature, wherein the gene of the yeast exhibiting the complementarity is a yeast according to any one of claims 3 to 6. A yeast which is a gene that exhibits low-temperature sensitivity by having a mutation in which one or more nucleotides have been substituted, added or deleted in the gene described in any of the above items. 12. A yeast which exhibits complementation with respect to an existing yeast whose fermentation is regulated at a low temperature with respect to the ability to regulate fermentation at a low temperature, wherein the gene of the yeast exhibiting the complementarity is as defined in claim 1 or 2. A gene having a mutation in which one or more nucleotides have been substituted, deleted or added in the gene described above, and further exhibits complementarity to the gene according to any one of claims 3 to 5. Yeast with genes. A dough containing the yeast according to any one of claims 8 to 12. 14. The dough according to claim 13, wherein the dough is refrigerated dough. A method for producing a dough containing the yeast according to any one of claims 8 to 12. 16. A method for breeding yeast, each comprising a plurality of different genes that regulate the fermentative power of yeast at low temperatures, the method comprising the following steps: a plurality of genes that regulate fermentative power of yeast at low temperatures. Isolating a spore strain having a gene that regulates the fermentative power of yeast at a low temperature and a spore strain having a gene that regulates the fermentative power of yeast at another low temperature to produce a diploid yeast Forming a spore strain in the diploid yeast to obtain a spore strain; spore strain having a plurality of genes that regulate the fermentative power of yeast at a low temperature is simply ligated by hybridization using the isolated gene as a probe. Separating; and
Conjugating the spore strains having the plurality of genes with each other. 17. A spore strain having a gene that regulates the fermentative power of yeast at a low temperature and a spore strain having another gene that regulates the fermentative power of the yeast at a low temperature, each of which regulates the fermentative power of the yeast at a low temperature. 17. The yeast of claim 16, comprising the step of isolating a spore strain having two different genes that have one different gene and that regulates the fermentative power of the yeast at low temperatures by conjugating the two spore strains. Breeding method. 18. A spore strain having a gene that regulates the fermentative power of yeast at a low temperature and a spore strain having another gene that regulates the fermentative power of the yeast at a low temperature, each of which controls the fermentative power of the yeast at a low temperature. A spore strain having two genes that regulates yeast fermentation at a low temperature by conjugating the two spore strains with one different gene; 17. The method for breeding yeast according to claim 16, comprising a step of conjugating with a spore strain having another gene that regulates the fermentative power of yeast at a low temperature.