JP2003144137A - Freeze-resistant baker's yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain baker's yeast which is provided with freeze resisting ability or has enhanced freeze resistance by elucidating a gene related to freeze resistance character of practical baker's yeast by a genetic method and to provide a new method for breeding freeze-resistant baker's yeast and a screening method. SOLUTION: This baker's yeast is provided with freeze resistance or has enhanced freeze resistance by raising the expression amount of YLR023C gene and/or YMR126C gene. The rise of the expression amount of YLR023C gene and/or YMR126C gene can be carried out by transformation using YLR023C gene and/or YMR126C gene, hybridization of baker's yeast having a large expression amount of YLR023C gene and/or YMR126C gene with another baker's yeast, etc. Freeze-resistant yeast can be selected by using the expression amount of YLR023C gene and/or YMR126C gene as an index.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a baker's yeast to which freeze resistance is imparted or enhanced, a method for selecting the baker's yeast, and a frozen dough and a frozen dough baking method using the baker's yeast.

[0002]

2. Description of the Related Art The frozen dough bread making method is a method in which the dough is temporarily frozen during the bread making process, thawed and baked if necessary. The frozen dough baking method is labor-saving at the time of baking, smoothes the manufacturing schedule, and can respond to the growing needs for small-lot, high-mix varieties represented by the recent "freshly baked bread" boom. It is a useful baking method and has been widely adopted in recent years. However, in the frozen dough baking method, baker's yeast is strongly damaged by freezing and storing the dough. Therefore, it is necessary to use baker's yeast having freezing tolerance in order to obtain bread having a product value. Therefore, in the yeast industry, in addition to the development of "freeze-tolerant baker's yeast" that can be used under various conditions, research on the mechanism of freezing-tolerance, etc. has been conducted in order to create higher quality frozen-tolerant baker's yeast. Various things are done.

As a conventional technique relating to freeze-tolerant baker's yeast, to date, yeast having enhanced intracellular trehalose accumulation by disrupting trehalase gene (NTH1, ATH1), which is a trehalose-degrading enzyme (Japanese Patent Laid-Open No. 10-1).
17771, JP-A-11-169180), plant-derived fatty acid desaturase gene (FAD)
2) into which the fluidity of the cell membrane is enhanced (Japanese Patent Application Laid-Open No. HEI-1)
1-75824), a yeast in which a specific amino acid such as arginine is highly accumulated by disruption of the arginine-degrading enzyme gene (CAR1) (JP 2001-238665 A).
Freeze-tolerant baker's yeast using a genetic engineering technique such as the above (Japanese Patent Publication) is known.

However, since the above-mentioned conventional freeze-tolerant baker's yeast is based on the knowledge obtained from laboratory yeasts, it often has characteristics different from those of the practical baker's yeast, and can be necessarily used as the practical baker's yeast. Not a thing. In fact, it has been pointed out that not all commercially available freeze-tolerant yeasts increase the amount of trehalose accumulated (Proceedings of the 42nd Annual Meeting of the Food Science and Technology Society of Japan, p136, title 2Gp4, 1995).

Therefore, the freezing tolerance trait of practical baker's yeast, which is expected to have an unknown mechanism, is being studied further by a genetic method, and fatty acid unsaturated as a gene complementing the freezing sensitive mutation. Oxygenase gene O
LE1 has been cloned (Japanese Patent Laid-Open No. 2000-37).
No. 185), there is a demand for the development of a technique that can be applied to breeding, such as further enhancing the freeze resistance of a commercially available freeze-tolerant baker's yeast and imparting the freeze-tolerance to a general-purpose strain having no freeze-tolerance.

[0006]

DISCLOSURE OF THE INVENTION The object of the present invention is to impart or enhance freezing tolerance by clarifying the genes involved in the freeze-tolerance trait of practical baker's yeast using a genetic method. To create baker's yeast. Further, it is an object of the present invention to provide a novel selection (screening) method and breeding method for freeze-tolerant yeast. And an object of the present invention is to provide a bread dough for freezing and a frozen dough baking method using the freeze-tolerant baker's yeast obtained as described above.

[0007]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies using genetic techniques to achieve the above-mentioned object, and as a result, have newly identified a gene that determines the freeze resistance of a practical freeze-resistant yeast. I was able to find it. Then, a new freeze-resistant strain can be created by using the gene, and a new method for selecting a freeze-resistant strain was successfully developed to complete the present invention.

[0008] That is, the present inventors have found that a strain in which a marker such as a gene is added to a haploid baker's yeast having freeze resistance used for imparting freeze resistance to a practical baker's yeast [among which, a uracil-requiring marker and a leucine-requiring marker The haploid baker's yeast UH41B with the addition of FERM P- at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary
18557 has been deposited with the accession number (hereinafter, those deposited at the center may be displayed only with the accession number)] as a strain to be analyzed for the freeze resistance gene,
Monoploid baker's yeast (UH41B strain) with the marker
To produce a freeze-sensitive mutant, and two freeze-sensitive mutants obtained thereby [dUS301B
Strain (accession number: FERM P-18586) and dUS4
Strain 04B (accession number: FERM P-18587)] was transformed by introducing a genome library of a freeze-tolerant strain (for example, the above-mentioned haploid baker's yeast), and the freeze-resistant strain was restored by the transformation. The gene was screened. As a result, among genes in baker's yeast, YLR023C gene and YMR126C
We obtained new findings that genes contribute to imparting or enhancing freezing tolerance. Therefore, for baker's yeast, YLR
When the treatment for increasing the expression level of 023C gene or YMR126C gene was performed, it was possible to impart or enhance freeze resistance. In particular, the above-mentioned haploid baker's yeast (UH41) to which a marker originally having freezing resistance is added.
BLR against YLR023C gene or YMR126
When the operation to increase the expression level of C gene was performed, Y
It was found that the strain having an increased expression level of the LR023C gene has an improved freezing tolerance of about 50%. Further, the present inventors can create a new baker's yeast having freeze resistance by using at least one of the YLR023C gene and the YMR126C gene described above, and further, the YLR023 in baker's yeast.
It was found that baker's yeast having freezing tolerance can be selected using the expression level of at least one of the C gene and YMR126C gene as an index, and the present invention was completed based on these various findings.

That is, the present invention provides (1) YLR02
It is a baker's yeast to which freezing resistance is imparted or enhanced, which is obtained by increasing the expression level of at least one of the 3C gene and the YMR126C gene.

The present invention provides (2) YLR023
The baker's yeast of (1) above, which has been transformed with at least one of the C gene and YMR126C gene; and (3) bread with a high expression level of at least one of the YLR023C gene and YMR126C gene. The baker's yeast of (1) above, which is a hybrid of yeast and other baker's yeast; (4) YLR0
Baker's yeast UH4 obtained by increasing the expression level of 23C gene
1BTF strain (accession number FERM P-18558); and (5) baker's yeast dUS301BMR strain (accession number FER) in which the expression level of the YMR126C gene is increased.
MP-18585);

Further, the present invention provides (6) YLR023.
A method of selecting a freeze-tolerant baker's yeast using the expression level of at least one of the C gene and YMR126C gene as an index; and (7) a freeze-tolerant baker's yeast selected by the method of (6) above. .

The present invention provides (8) above (1)-
A baker's yeast imparted with or enhanced freezing resistance, which is obtained by breeding with the baker's yeast according to any one of (5) and (7).

Further, the present invention is (9) above (1).
(5), (7) and (8) bread dough for freezing, which comprises using yeast; and (10) any of (1) to (5), (7) and (8) A method for producing bread by a frozen dough baking method using baker's yeast.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. Freezing-tolerant baker's yeast generally refers to baker's yeast that is not easily damaged by freezing, but "freezing-tolerance" in the present invention means that the degree of glucose consumption maintenance after freezing-thawing (freezing resistance) is a solution. It means to show 0.4 or more by the method. In the freeze-tolerant baker's yeast, the degree of maintaining the glucose consumption is preferably 0.5 or more,
It is more preferably 7 or more. The solution method is a method in which yeast is frozen in a state of being suspended in a solution and the ratio of glucose consumption before and after freezing is determined, as shown in Examples described later. This solution method has a different index from the conventional dough method for measuring the amount of carbon dioxide gas produced by making bread dough, but it is common in that the change in viable bacterial activity before and after freezing is measured. The freezing resistance (freezing resistance) evaluation result by the solution method has a good correlation with the evaluation result by the dough method. The solution method has the advantage that it can measure freezing tolerance with high sensitivity even when it is difficult to measure with the dough method, such as strains with auxotrophy, which can be easily processed in large quantities. Is very useful for.

The parent strain of the baker's yeast of the present invention (YLR0
As the baker's yeast for obtaining the 23C gene and / or the YMR126C gene, and the baker's yeast used as a base when increasing the expression level of the gene), any baker's yeast may be used, for example, Saccharomyces,
Examples include baker's yeasts of the genus Torluspora and the genus Cluveromyces. Among them, Saccharomyces
Baker's yeasts of the genus Saccharomyces such as S. cerevisiae, Saccharomyces ubalm, Saccharomyces chevalier, and Saccharomyces rosei are preferable, and those belonging to Saccharomyces cerevisiae are particularly preferable.

The sequence of the whole genomic DNA of the yeast Saccharomyces cerevisiae has been completely deciphered and can be found in databases such as "Saccharomyces Genome Database (htt
p: //genome-www.stanford.edu) ”etc. In the present invention, the above-mentioned “YLR023C gene” involved in imparting or enhancing freeze resistance in baker's yeast.
Is a Saccharomyces cerevisiae chromosome 12 right arm (about 186 kb from telomere) with a total length of about 1.6 kb
Is the gene. In addition, the above-mentioned "YMR126C gene"
Is a Saccharomyces cerevisiae chromosome 13 right arm (about 520 kb from the telomere) with a total length of about 1.0 kb
The gene is numbered in the above database. Therefore, YLR023C gene and YMR
The 126C gene can be easily obtained by cutting out from the yeast chromosomal gene by a conventional method, for example, using the nucleotide sequence information in the above database. Both genes were presumed to encode membrane proteins, but their functions have not been clarified so far, and the YLR023C gene and the YLR023C gene are involved in imparting or enhancing freeze resistance in baker's yeast. This is what the present inventors have found for the first time.

YLR023C gene and YMR126
The fact that the C gene is involved in imparting freeze resistance to baker's yeast and / or enhancing freeze resistance of baker's yeast can be confirmed as follows according to the experiments conducted by the present inventors. That is, genetic analysis can be performed by adding a gene marker such as ura3 mutation or leu2 mutation to a haploid freeze-tolerant strain of baker's yeast possessed by the present inventors, which is used for imparting freeze resistance to practical baker's yeast by a conventional method. Haploid freeze-resistant strain with a marker [eg UH41B (accession number:
FERM P-18557), etc.]. Then
Mutations are introduced into the haploid freeze-resistant strain with a marker (for example, UH41B) by a mutagenesis agent such as ethyl methanesulfonate, and a viable strain after the mutation treatment is changed to a freeze-sensitive strain (a strain having no freeze resistance). Select [In our experiments, the haploid freeze-sensitive strain dUS301B strain (accession number: FE
RM P-18586) and haploid freeze-sensitive strain dU.
S404B strain (trust number: FERM P-18587)
Two strains were obtained]. In general, when the mutation is recessive, it is known that the phenotype of the strain after mutation is restored to the wild type by transformation with the wild type gene, and thus this method was obtained above. It is applied to freeze-sensitive mutants (for example, dUS301B strain and dUS404B strain), and freeze-resistant strains (for example, haploid freeze-resistant strain of baker's yeast;
Each of the genomic libraries of the UH41B strain is a freeze-sensitive mutant strain (for example, dUS301B strain and dUS4 strain).
04B) individually, and by examining the plasmids of the strains reverted from freezing-sensitive mutant strains (eg, dUS301B strain and dUS404B strain) to freeze-resistant strains,
Genes involved in imparting or enhancing freeze resistance can be selected (screened).

In the experiments of the present inventors, the construction of the genomic library was carried out using YCp50 as a vector in Maniatis.
Et al. (Molecular Cloning, A Laboratory Man)
ual, Maniatis et al., Cold Spring Harbor Laborato
ry Press). Since YCp50 has the URA3 gene (uradylic acid synthase marker gene) as a selection marker, the uracil-free casamino acid agar medium (described in the Example section below) is used as the selection medium of the transformant. Using. As a result, in the freeze-sensitive mutant dUS404B strain, one strain was restored to freeze resistance,
In the freeze-sensitive mutant dUS301B strain, two strains in which the freeze resistance was reverted were obtained, and as a result of recovering the plasmid from each reverted strain and narrowing down the freeze resistance-related genes, the YLR023C gene in the freeze-sensitive mutant dUS404B strain was In the freeze-sensitive mutant dUS301B strain, YMR
It was revealed that the 126C gene is involved in the restoration of freeze resistance.

The baker's yeast of the present invention is the above YLR023C.
The expression level of at least one of the gene and the YMR126C gene is increased. YLR
In the baker's yeast of the present invention in which the expression level of the 023C gene and / or the YMR126C gene is increased, freeze resistance is imparted or freeze resistance is enhanced. In the baker's yeast of the present invention, whether only the expression level of the YLR023C gene is increased, the expression level of the YMR126C gene is increased, or the YLR023C gene and YMR12 are
The expression levels of both 6C genes may be increased.

Here, in the present invention, "YLR023C
The expression level of at least one of the genes and the YMR126C gene is increased "means that the expression level of at least one of the YLR023C gene and the YMR126C gene is YLR023C gene and / or YMR126C gene in baker's yeast that is not freeze-tolerant. Of the YLR023C gene and / or the YLR023C gene in the freeze-tolerant baker's yeast which is based on the expression level of the YLR023C gene and / or the YMR126C gene in the baker's yeast having the freeze-tolerance.
Alternatively, the YLR023C gene and / or in the baker's yeast in which the expression level of the YMR126C gene is further increased, or the YLR023C gene and / or the YMR126C gene expression level is decreased or is not expressed at all due to some factor YMR
It refers to the case where the expression level of the 126C gene is restored to the normal level before mutation.

Comparison of YLR023C gene and YMR126C gene amounts in baker's yeast, and YLR0
The presence or absence of a mutation in the 23C gene and the YMR126C gene can be determined, for example, by comparing and analyzing the transcribed mRNA or the translated protein. Northern blotting, dot blotting, nuclease protection, in situ hybridization, etc. are generally used for the specific detection of mRNA, which is one of the useful methods for comparing and quantifying differences in expression levels. , RT-PCR method. RT
-The PCR method is a method of sensitively detecting mRNA by combining reverse transcriptase and polymerase chain reaction,
Since quantitative information about its presence, length, and expression level can be obtained relatively easily, it is convenient even when a large amount of processing such as screening of a high expression strain is involved. Similarly, when comparing and quantifying the amounts of proteins, application of Western analysis may be considered, and an antibody against the expressed protein is obtained and RI
Alternatively, it is desirable to construct a system capable of comparative analysis of a large amount of fluorescent labels.

In the present invention, "imparting freeze resistance to baker's yeast" means imparting freeze resistance to baker's yeast showing freeze sensitivity (baker's yeast having no freeze resistance).
The freezing tolerance imparted by the present invention is, for example, a general-purpose baker's yeast (freezing-sensitive strain) having excellent traits but no freezing tolerance.
It is useful when you want to have freeze resistance. In addition, "enhancing the freeze resistance of baker's yeast" refers to further increasing the freeze resistance of baker's yeast exhibiting freeze resistance, or improving it to show freeze resistance under a wider range of conditions. For example, it is useful when making the freeze-tolerant yeast for medium sugar dough available in low sugar frozen dough.

YLR023C gene and / or YM
Examples of the method for obtaining the baker's yeast of the present invention in which the expression amount of the R126C gene is increased include (A) transformation, (B) mutation introduction, (C) crossing and the like. Among them, (A) transformation is a method of artificially introducing the YLR023C gene and / or the YMR126C gene into the cells of baker's yeast. YLR by transformation
Examples of the method for increasing the expression level of the 023C gene and / or the YMR126C gene include YLR02
Method of introducing the 3C gene and / or YMR126C gene into a baker's yeast cell with a vector, YLR023C gene and / or YMR126
Method for transforming baker's yeast with a multicopy vector containing the C gene to amplify the copy number of the gene, and to efficiently function the promoter of the YLR023C gene and / or the promoter sequence of the YMR126C gene on the plasmid or on the chromosome in the baker's yeast And a method of substituting the terminator of the YLR023C gene and / or the terminator of the YMR126C gene.

Various vectors have been developed as carriers of genes, and in the present invention, any of the vectors conventionally used for transformation of baker's yeast is used as the vector of YLR023C gene and / or YMR126C gene. it can. In the present invention, the YLR023C gene and / or YMR1 is transformed by transformation.
Examples of vectors that can be used to introduce the 26C gene into cells include YCp-type vectors capable of autonomous replication in yeast, YIp-type vectors capable of chromosome integration, etc. [Lundblad, V. et al. (1989) Yeast vector
s, "Carrent Protocols in Molecular Biology"
vol.2, New York, N.V. Y]. In addition, examples of the multi-copy type vector include YEp type vectors such as YEp13 and YEp24. In addition, promoters that function efficiently in baker's yeast include GAL7, TPI, PHO
There are 5 etc., GAL10, TP as terminator
I, GAPDH, etc. exist (yeast molecular genetics experimental method,
Yasuharu Oshima, Academic Publishing Center).

YLR023C gene and / or YM
For transformation of baker's yeast with a vector incorporating the R126C gene, the protoplast method [eg S. Harashima et al., Mol. Cell. Biol. , vol.4,7
71-778 (1984)], lithium acetate method [e.g. Ito et al. Bacteriol. , Vol.153,163
-168 (1983)], the electroporation method (for example, "Gene experiment method using yeast", Bio Manual series, edited by Masayuki Yamamoto, Yodosha) and the like. Also, ALKALI CATION YEA
ST TRANSFORMATION KIT (BIO
You may carry out using a commercial kit (made by 101 company).

For the selection of transformants, it is convenient to use a selection medium in which transformation is carried out using a vector having a marker gene and only a strain that has acquired the marker trait can grow. As the marker gene, for example, UR
A3, LEU2, TRP1, HIS3 (Methods in E
nzymology, vol.101, 202-211, R.N. Wu
Metabolic genes such as Academic Press), cerulenin resistance [N. Nakazawa et al. Ferment. Bioeng., Vo
76.60-63 (1993)], G418 resistance [T. D. Webster, Gene, vol.26, 243-2
52 (1983)] and the like. In addition, any marker can be used as long as it can confirm the transformation of the foreign plasmid.

(B) Mutagenesis refers to artificially mutating a gene, and there are two types: site-specific mutagenesis and random mutagenesis. Among the mutagenesis, examples of the site-specific mutagenesis method include a cassette replacement method for the mutation site and a method using a PCR reaction. The cassette replacement method of the mutation site can be performed by completely replacing the restriction enzyme fragment containing the site to be mutated with the synthetic DNA containing the mutation, and the mutation introduction using the PCR reaction is performed by the method of Ito et al. W. Ito et
al. Gene, vol.102, 67-70 (1991)].
And so on. These site-directed mutagenesis methods were applied to the YLR023C gene and / or YMR.
When used to obtain the baker's yeast of the present invention in which the expression level of the 126C gene is increased, it acts on an expression control sequence such as a promoter sequence of the gene or its expression control sequence in order to increase the transcription efficiency of the gene. The gene sequence of the transcription factor to be selected may be selected as a mutation introduction site, and in order to enhance the stability of mRNA transcribed from the gene,
The untranslated region sequence may be selected as the mutation introduction site.
Furthermore, a protein coding sequence may be selected as a mutagenesis site in order to enhance the specific activity of the translated protein.

Among the mutagenesis methods, the random mutagenesis method is a method of irradiating with ultraviolet light, or N-methyl-N'-nitro-N-nitrosoguanidine (NTG).
Generally, a method of treating with a mutagen such as or ethyl methanesulfonate (EMS) is used. Although not limited in any way, an example of the mutation method by EMS will be described. First, from the slant of the parent strain of baker's yeast, 1 platinum loop, YPD
Inoculate 5 ml of a medium (separately described), and incubate at 30 ° C. for 16 hours. The cultured cells are collected by centrifugation at 3000 rpm for 10 minutes, and washed once with 0.1 M pH 7.0 phosphate buffer. 5 ml of the same phosphate buffer
Suspension, and 0.15 ml of EMS was added, treated at 30 ° C. for 2 hours with occasional stirring, collected by centrifugation at 3000 rpm for 10 minutes, washed with phosphate buffer, and
Add 5 ml of 0% sodium thiosulfate, 30 ° C, 10
Let stand for minutes to inactivate EMS. 20% sodium thiosulfate treatment is performed twice, washed with 5 ml of physiological saline three times, and appropriately diluted on a YPD agar plate to apply the bacteria.
The baker's yeast of the present invention in which the expression level of the YLR023C gene and / or the YMR126C gene was increased was treated with this EM.
In order to obtain by the mutation method with S, a method of selecting, from among the mutant strains mutated with EMS, a strain in which the promoter activity of YLR023C gene and / or YMR126C gene is increased as compared with that before the mutation treatment, and the amount of transcribed mRNA is A method of selecting an increased strain, a method of selecting a strain having an increased amount of translated protein, or the like is adopted.

[0029] (C) Hybridization refers to the process of producing a hybrid strain by conjugation or cell fusion, preferably obtaining a spore strain to grow a strain having desired properties.
Mating is a haploid having a mating ability again due to a / α diploid vegetative cells generated through zygote formation by mixed culture between a and α both mating type haploid vegetative cells. It is a method for obtaining cells, and the collective conjugation method, direct conjugation method, conjugation by cross streak on a plate medium (experimental method of yeast molecular genetics, Yasuharu Oshima, Academic Publishing Center) and the like are widely used. Cell fusion is a method of directly fusing cells that have undergone protoplastization by removing the cell wall, and is used for breeding industrial yeasts for which mating-type strains cannot be obtained or for raising high-order polyploid strains. Has been done. YLR023C gene and / or YMR126
By crossing a freeze-tolerant baker's yeast having a high expression level of the C gene with a general-purpose yeast having the desired property, the freeze-tolerant baker's yeast of the present invention having both the freeze-tolerant property and the desired property can be constructed. Alternatively, a freeze-tolerant baker's yeast having desired properties and a YLR023C gene and / or YMR1
It is also possible to obtain the baker's yeast of the present invention having desired properties and further enhanced freezing tolerance by crossing yeasts with a high expression level of the 26C gene.

In addition, by adjusting the culture conditions,
The baker's yeast of the present invention in which the expression level of the YLR023C gene and / or the YMR126C gene is increased can be obtained. For example, utilization of environmental factors or metabolites that trigger high expression of YLR023C gene and / or YMR126C gene, such as stress load such as heat and salt, addition or depletion of specific medium components, may be effective.

YLR023C gene and / or YM
Among the baker's yeasts of the present invention in which the expression amount of R126C gene is increased, "UH41BTF strain" (accession number: FER
MP-18558) is a haploid freeze-tolerant baker's yeast UH41B strain (accession number: FERM) using YEp type plasmid p566 in which the YLR023C gene has been cloned.
P-18557) and transformed into YLR023.
It is baker's yeast into which multiple copies of the C gene have been introduced. "UH4
The degree of freezing resistance of the "1BTF strain" is improved to about 1.5 times that of the haploid freezing resistant baker's yeast UH41B strain before transformation, and the freezing resistance is further enhanced.

The "dUS301BMR strain" (accession number: FERM P-18585) is a haploid freezing-sensitive strain dUS301B (accession number: FERM P-) obtained by mutating the haploid freeze-tolerant baker's yeast UH41B strain.
18586) was transformed with the plasmid pC317 in which the YMR126C gene was cloned to introduce the YMR126C gene.
It is a baker's yeast in which the expression level of the C gene is increased. "DU
Freezing tolerance of "S301BMR strain" is freeze-sensitive strain dU
The freezing tolerance of S301B is about 2.2 times, and the freezing tolerance of the haploid freeze-resistant baker's yeast UH41B strain has been restored to about 0.88 times. It is a baker's yeast.

Further, in the case of the present invention, the baker's yeast obtained by transformation, mutation treatment, crossing or the like using the expression level of at least one of the YLR023C gene and the YMR126C gene in baker's yeast as an index, or Strains that have acquired freeze resistance can be selected (screened) from yeast groups collected from nature. In order to "use the expression level of YLR023C gene and / or YMR126C gene as an index", it is necessary to examine the amount of YLR023C gene and / or YMR126C gene in baker's yeast. In addition, comparative analysis of transcribed mRNA or translated protein can be performed. Northern blotting, dot blotting, nuclease protection, in situ hybridization, etc. are generally used for the specific detection of mRNA, which is one of the useful methods for comparing and quantifying differences in expression levels. , RT-PCR method. The content of the RT-PCR method is as described above, and it is also convenient when a large amount of processing such as screening of a highly expressing strain is involved. Similarly, when comparing and quantifying the amounts of proteins, application of Western analysis may be considered, and it is desirable to construct a system capable of obtaining an antibody against the expressed protein and conducting a comparative analysis of a large amount of RI or fluorescent label.

In the present invention, the YLR023C gene and / or YMR12 obtained by screening
For freeze-tolerant yeast with increased expression of 6C gene,
Further improvement by transformation, mutation treatment, crossing, etc.,
That is, by carrying out "breeding", baker's yeast having more excellent freezing resistance can be obtained, and the present invention includes such baker's yeast.

By producing bread dough using the baker's yeast obtained as described above, a quality close to that of a dough made by a normal bread-making method can be stably maintained even if a freeze-thaw treatment is put in between. Therefore, it becomes possible to manufacture breads with high product value. The dough of the present invention is
Except for using the freeze-tolerant baker's yeast of the present invention, it can be produced in the same manner as ordinary dough for freezing. The frozen bread dough in the present invention includes all bread dough for freezing, such as loaf of bread, confectionery bread, Danish pastry, Chinese manju, and yeast donut, which uses baker's yeast as an expanding agent. And using the bread dough of the present invention,
In the same manner as the conventional frozen dough baking method, various types of bread products of high quality as described above can be smoothly produced without causing freezing trouble.

[0036]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. In the following, “%” means mass% unless otherwise specified. The contents of the main media used in the following examples are as follows.・YPD medium : yeast extract 1%, bactopeptone 2
%, And 2% glucose in water.・Casamino acid medium : yeast nitrogen base 0.67
%, 0.4% casamino acid and 2% glucose in water. -Casamino acid agar medium : A medium obtained by adding agar to the above casamino acid medium at a ratio of 2%.・YNB 'medium : yeast nitrogen base 0.67
%, Casamino acid 0.4%, uracil 20 ppm and adenine 40 ppm in water.

The plasmid used in the present invention, DN
Conventional methods for handling A, various enzymes, Escherichia coli, yeast, etc. are described in detail in the following magazines and publications, and in the present invention, they were performed according to the descriptions in the following references. (1) "Protein / nucleic acid / enzyme" 26 (4), (198)
1) Extra edition "Genetic manipulation" (Kyoritsu Publishing); (2) "Protein / nucleic acid / enzyme" 35 (14), (19)
90) Extra edition "Genetic manipulation 1990" (Kyoritsu Shuppan); (3) "Bio Manual Series 10: Gene experimentation method by yeast" edited by Masayuki Yamamoto, Yodosha; (4) Method in Yeast Genetics, A Laboratory.
Course Manual, M.C. D. Rose et al, Cold Spring
Harbor Laboratory Press, Cold Spring Harbo
r, New York; (5) Molecular Cloning, A Laboratory Manua
l, Maniatis et al., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York; (6) Methods in Enzymology, L.S. Grossman et al., Volume 29, Academic Press; (7) Methods in Enzymology, R.M. Wu et al., 65
Volume Academic Press; (8) Methods in Enzymology, R.M. Wu et al., 10
Volume 1 Academic Press; (9) Methods in Enzymology, B.M. Kimmel et al.,
Volume 152 Academic Press; (10) Methods in Enzymology, edited by G. Fink et al., 19
Volume 4 Academic Press.

<< Freezing Tolerance Evaluation Method >> In the following examples, the freezing tolerance of baker's yeast was evaluated by the following solution method. (1) The above YPD medium 30 was placed on a microplate.
Dispense 0 μl, inoculate an appropriate amount of cells and incubate at 30 ° C for about 20
After static culture for a period of time, glucose and ethanol were added to the bacterial cell suspension from which 150 μl of the supernatant had been removed so that the final concentrations were 4% and 5%, respectively, and this was used for freezing and non-freezing. 80 μl was dispensed to each of two microplates. (2) To the non-freezing plate, 80 μl of 2 × YNB ′ medium (having twice the concentration of the YNB ′ medium described above) was added, and the mixture was shake-cultured at 38 ° C. for 2 hours, and then in the supernatant. Was measured for glucose concentration. In addition, the freezing plate,
Freeze in -20 ° C freezer (remove from expanded polystyrene container after slow cooling in expanded polystyrene container 2 hours-
Direct quenching in a freezer at 20 ° C for 1 hour), then immersing in a water bath for 8 minutes and thawing, and then adding 80 µl of 2xYNB 'medium, shaking culture at 38 ° C for 2 hours,
The glucose concentration in the supernatant was measured. "Glucose B Test Wako" (a kit manufactured by Wako Pure Chemical Industries, Ltd.) was used for the measurement of the glucose concentration, and the attached protocol was followed. (3) Freezing tolerance was evaluated by setting the glucose reduction amount on the frozen plate as A, the glucose reduction amount on the non-frozen plate as B, and the A / B value as "freezing tolerance".

<< Test Example 1 >> [Acquisition of Frozen Tolerance Gene] (1) Cloning Target Strain: As a target strain for cloning the freezing tolerance gene, a haploid freezing resistant strain UH41B strain (accession number: FERM P-18557) is used. I was there. UH4
The 1B strain is an SH2675 strain having auxotrophic markers of ura3, leu2, and trp1 against the haploid freeze-tolerant baker's yeast possessed by the present inventors, which is used to impart freeze resistance to practical baker's yeast. A spore strain was prepared from the hybrid diploid strain obtained by crossing, and a spore strain having auxotrophic markers for ura3 and leu2 and having freeze resistance equivalent to that of the original haploid freeze-tolerant baker's yeast was selected from the spore strains. The auxotrophic markers of ura3 and leu2 were added by selecting and backcrossing twice with haploid freeze-tolerant baker's yeast, and it was constructed as a strain capable of genetic analysis. (2) Acquisition of freezing-sensitive mutant: UH41B strain is subjected to mutation treatment with ethyl methanesulfonate, and a strain showing freezing sensitivity is obtained from about 4,000 colonies obtained under conditions showing a survival rate of about 10%. 6 strains were obtained. Single mutation, dominant / recessive test, and complementation test are performed by a conventional method, and dUS with different mutation phenotypes and mutations on different genes
301B strain (accession number: FERM P-18586) and dUS404B strain (accession number: FERMP-18587)
Got

(3) Screening of freeze-tolerant revertant strain and acquisition of freeze-tolerant gene: (i) Generally, when the mutation is recessive, the phenotype of the mutant parent strain becomes wild by transformation with the wild-type gene. It is known to return to the mold, and this was used as an index during screening. That is, in each of the two types of susceptible mutant strains dUS301B strain and dUS404B strain obtained above, a haploid freeze-resistant strain of the parent strain (UH41B strain)
The genome library was introduced and the strains whose resistance was restored were screened. At that time, the preparation of the genomic library was carried out using YCp50 as a vector according to the method of Maniatis et al. (Molecular Cloning, A Laboratory Manua).
l, Maniatis et al., Cold Spring Harbor Laboratory
Made according to Press). Since YCp50 has the URA3 gene as a selection marker, the above casamino acid agar medium containing no uracil was used as the selection medium for the transformant. Specifically, the chromosomal DNA prepared from UH41B was partially cleaved with the restriction enzyme Sau3AI, and then subjected to agarose gel electrophoresis to obtain 20 kbp from 20 kbp.
Fractional collection of the kbp DNA fragment was performed and the plasmid YCp5
0 BamHI site was inserted. In the obtained UH41B genomic library, two types of freeze-sensitive mutants dUS
Each of the 301B strain and the dUS404B strain was transformed to obtain about 60,000 dUS301B strains and dUS40 strains.
About 30,000 transformants were obtained for the 4B strain.

(Ii) dUS30 obtained in (i) above
As a result of investigating the presence or absence of freeze resistance of each of the 60,000 transformant strain 1B and 30,000 transformant strains of dUS404B using the solution method, it was found that freeze-sensitive dUS404
With the strains using the B strain, 1 strain was obtained in which the freeze resistance was restored, and in the strains using the freeze-sensitive dUS301B strain, 2 strains were restored with the freeze resistance. (Iii) When a plasmid was recovered from a freeze-resistant revertant obtained from a freeze-sensitive dUS404B strain, the nucleotide sequence of a chromosomal gene fragment inserted in the plasmid was determined and collated with a Saccharomyces cerevisiae genome database. It was found to contain the gene. Therefore, the chromosomal gene fragment inserted into the recovered plasmid was divided by various restriction enzymes and again incorporated into the plasmid YCp50, and the genes involved in freezing resistance were narrowed down by the method of examining the freezing resistance of the strain transformed with the dUS404B strain. Y for all plasmids that have returned to freezing resistance
Since the LR023C gene was included, YLR0
It was revealed that the 23C gene is a gene that restores freezing tolerance. (Iv) 2 obtained from freeze-sensitive dUS301B strain
When the plasmid was recovered from the freeze-resistant reverted strain of the strain, the nucleotide sequence of the chromosomal gene fragment inserted into the plasmid was determined and collated with the Saccharomyces cerevisiae genomic database, it was found that YMR126C gene was commonly contained. It was found that the gene is a gene that restores freezing tolerance.

Example 1 [Preparation of baker's yeast in which the expression level of YLR023C gene is increased] (1) From the ORF of YLR023C gene to about 2160
SphI site upstream of base and about 260 base
The region sandwiched by the downstream BamHI sites is the plasmid Y
It was introduced into Cp50 to prepare a plasmid pFT414 in which the YLR023C gene was cloned. (2) Using the plasmid pFT414 in which the YLR023C gene obtained in (1) above was cloned, freeze-sensitive strain dUS404B was transformed into the plasmid pFT414.
Then, the transformant dUS404BLR was obtained. The transformation in that case is ALKALI CATION
YAST TRANSFORMATION KIT
(Manufactured by BIO 101) was used and the attached protocol was followed. For the selection of transformants, the above-mentioned casamino acid agar medium was used and incubated at 37 ° C. for 2 days to form colonies. Obtained transformant dUS404BL
When the freeze resistance of R was examined by the method described above, the freeze resistance was 0.44. (3) In addition, the freeze resistance of the haploid freeze-resistant strain UH41B (parent strain) and the freeze-sensitive strain dUS404B was examined by the method described above. Table 1 below shows the freezing tolerance of each strain, and the ratio (relative value) of the frozen resistance of the freeze-sensitive strain dUS404B and the transformed strain dUS404BLR obtained in (2) above to the freeze resistance of the parent strain UH41B. .

[0043]

[Table 1]

As shown in Table 1 above, YLR02 was used for the strain that is not freeze-resistant (freeze-sensitive strain dUS404B).
The transformant dUS404BLR obtained in (2) above in which the expression level of the YLR023C gene was increased by carrying out transformation using a plasmid having the 3C gene.
Has a freezing resistance degree about twice that of the freezing sensitive strain dUS404B, and has freezing resistance close to that of the freezing resistant parent strain UH41B. From these results, it is understood that the YLR023C gene is involved in freezing resistance, and the baker's yeast obtained by increasing the expression amount of the YLR023C gene imparts or enhances freezing resistance.

Example 2 [Preparation of baker's yeast having an increased expression level of YMR126C gene] (1) Using plasmid pC317 (including YMR126C gene) recovered from a freeze-resistant revertant of dUS301B strain, freeze-sensitivity Strain dUS301B was transformed with the plasmid pC317 to obtain transformed strain dUS301.
BMR (accession number: FERM P-18585) was obtained. The transformation in that case is ALKALI CATION
YAST TRANSFORMATION KIT
(Manufactured by BIO 101) was used and the attached protocol was followed. For the selection of transformants, the above-mentioned casamino acid agar medium was used and incubated at 37 ° C. for 2 days to form colonies. Obtained transformant dUS301BM
When the freeze resistance of R was examined by the method described above, the freeze resistance was 0.46. (2) Further, the freeze resistance of the freeze-sensitive strain dUS301B was also examined by the method described above. Freezing resistance degree and parent strain UH41B of the above-described haploid freeze-tolerant strain UH41B (parent strain), freeze-sensitive strain dUS301B and transformant strain dUS301BMR obtained above.
Freezing-susceptible strain dUS301B to the degree of freezing tolerance of Escherichia coli and the transformant dUS301BMR obtained in (2) above
Table 2 below shows the ratio (relative value) of the freezing resistance of the above.

[0046]

[Table 2]

As shown in Table 2 above, YMR12 was added to the strain not freeze-resistant (freeze-sensitive strain dUS301B).
The transformant dUS301BMR obtained in the above (2) in which the expression level of the YMR126C gene was increased by carrying out transformation using a plasmid having the 6C gene.
Has a freezing tolerance of about 2.2 times that of the freeze-sensitive strain dUS301B, and has a freeze resistance close to that of the freeze-resistant parent strain UH41B. From these results, it is understood that the YMR126C gene is involved in freeze resistance, and that the baker's yeast obtained by increasing the expression level of the YMR126C gene imparts or enhances freeze resistance.

Example 3 [Preparation of baker's yeast into which multiple copies of YLR023C gene have been introduced] (1) About 1790 from ORF of YLR023C gene
SacI site upstream of base and about 260 base
The region sandwiched between the BamHI sites on the downstream side is represented by p566.
[PRS306 (Sikorski et al., Genetics 122, 1
9-27 (1989)) and 2 μDNA incorporated therein into a multicopy YEp type plasmid] to obtain plasmid pFT.
415 was produced. (2) The plasmid pFT415 obtained in (1) above
Freeze-susceptible strain dUS404B using ALKALI
CATION YEAS TRANSFORMATION
Transformed by ION KIT and transformed with the YLR023C gene in multiple copies dUS404BTF
Got At that time, the transformation and the selection of the transformant strain are
It carried out according to the method of Example 1. Obtained transformant d
When the freeze resistance of US404BTF was examined by the above method, it was 0.60. (3) Plasmid pFT415 obtained in (1) above
Was used to transform a haploid freeze-tolerant strain UH41B (parent strain) and a multi-copy YLR023C gene was introduced into the transformed strain UH41BTF (accession number: FERM P-1).
8558) was obtained. At that time, transformation and selection of transformants were performed according to the method of Example 1. When the freeze resistance of the obtained transformant UH41BTF was examined by the method described above, it was 0.76. (4) Parent strain UH41B, freeze-sensitive strain dUS404
B, transformant dUS404BTF and transformant U
Table 3 below shows the freeze resistance of H41BTF and the ratio (relative value) of the freeze resistance of each strain to the freeze resistance of the parent strain UH41B.

[0049]

[Table 3]

From the results shown in Table 3 above, by introducing multiple copies of the YLR023C gene, the freeze-sensitive strain dUS404.
It can be seen that the reversion degree of the freeze resistance of B can be highly enhanced, and further the freeze resistance of the freeze resistant strain UH41B can be further improved by about 50%.

[0051]

INDUSTRIAL APPLICABILITY The present invention clarifies a new genetic trait that determines the freeze resistance of baker's yeast, and provides a new practical freeze-tolerant baker's yeast and a new breeding method thereof. Further, in the case of the present invention, further improvement of the freeze resistance of the baker's yeast, such as further enhancement of the freeze resistance of the practical freeze-tolerant baker's yeast, or the addition of the freeze resistance to a general-purpose baker's yeast having no freeze-tolerance, can be expected. . The bread dough formed by using the baker's yeast of the present invention to which freezing resistance is imparted or enhanced, stably maintains a quality as close as possible to a dough made by a normal bread-making method, even if freeze-thaw treatment is performed. It becomes possible.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 15/09 C12N 15/00 A (72) Inventor Shun Harashima 3-29-2 Kamishodomuro, Takatsuki City, Osaka ( 72) Inventor Yoshinobu Kaneko 1-1-7-1204 F-term Kamihozumi, Ibaraki-shi, Osaka (reference) 4B024 AA05 CA01 DA12 EA04 GA11 HA08 4B032 DB01 DK58 4B065 AA72X AB01 AC03 BA02 CA41

Claims (10)

[Claims] 1. The YLR023C gene and YMR12
A baker's yeast imparted with or enhanced freeze resistance, which is obtained by increasing the expression level of at least one of the 6C genes. 2. The YLR023C gene and YMR12
The baker's yeast according to claim 1, which has been transformed with at least one of the 6C genes. 3. The YLR023C gene and YMR12
The baker's yeast according to claim 1, which is obtained by crossing baker's yeast having a high expression level of at least one of the 6C genes with another baker's yeast. 4. A baker's yeast UH41BTF strain (Accession No. FERM) in which the expression level of the YLR023C gene is increased.
P-18558). 5. A baker's yeast dUS301BMR strain (accession number FE) obtained by increasing the expression level of the YMR126C gene.
RM P-18585). 6. The YLR023C gene and YMR12
A method for selecting a freeze-tolerant baker's yeast using the expression level of at least one of the 6C genes as an index. 7. A freeze-tolerant baker's yeast selected by the method according to claim 6. 8. A baker's yeast imparted with or enhanced freeze resistance, which is bred using the baker's yeast according to any one of claims 1 to 5 and 7. 9. Any one of claims 1 to 5, 7 and 8
A frozen dough obtained by using the baker's yeast according to the item. 10. A method for producing bread by a frozen dough baking method using the baker's yeast according to any one of claims 1 to 5, 7 and 8.