JP3579716B2 - Breeding method of high alcohol producing yeast by mutant PDR3 gene - Google Patents

Description

【００２１】

【００２２】
【発明の効果】
本発明によって高アルコール生産酵母の育種が可能となり、本発明によって育種された酵母を使用することによってアルコール含量の高い醸造酒、アルコール発酵もろみ等を効率よく製造することが可能となった。
【００２３】
【配列表】

【図面の簡単な説明】
【図１】変異ＰＤＲ３遺伝子のＤＮＡシーケンスを示す。
【図２】同上続きを示す。
【図３】同上続きを示す。
【図４】同上続きを示す。
【図５】プライマーＡを示す。
【図６】プライマーＢを示す。
【図７】プラスミドｐＡＵＲ１０１−変異ＰＤＲ３の制限酵素地図を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to breeding of yeast, and more particularly, to breeding of high alcohol-producing yeast that produces high alcohol in mash of sake and other alcoholic beverage production processes. In addition, the present invention, in addition to the breeding of such yeasts, yeasts separated thereby and alcohols having a high alcohol content by using the yeasts (sake, grape wine, various brewed liquors, mash for distilled spirits production such as shochu, Including all mashes for alcohol production).
[0002]
[Prior art]
Although breeding examples in which high alcohol-producing yeasts that produce high amounts of alcohol have been isolated have been known, breeding of novel sake yeasts that use the mutant PDR3 gene according to the present inventors to produce high amounts of alcohol has been known. There have been no reports of successful cases.
[0003]
[Problems to be solved by the invention]
In brewing sake production, obtaining more alcohol and shortening the production period have been regarded as important issues to be solved.
[0004]
[Means for Solving the Problems]
The present invention has been made to solve the above problems, and has been made for the purpose of achieving a large amount of alcohol in a short period of time. Therefore, the present inventors aimed at increasing the yield of many alcohols in a shorter period of time, and set a new technical problem of breeding a production yeast with an increased fermentation rate in order to achieve the purpose.
[0005]
In order to achieve the above-mentioned object, as a result of various studies, the present inventors proceeded with research on a tricosecin-resistant sake yeast (Patent No. 3069679), which was first invented and has already been granted a patent, and renewed its multidrug drug. As a result of further intensive studies focusing on the resistance, it was found for the first time that high alcohol production was caused by the mutant PDR3 gene.
[0006]
The present invention has been completed as a result of further research based on the above-mentioned new and useful findings, and uses a specific gene to select from yeast belonging to the genus Cerevisiae of the genus Saccharomyces. The basic technical idea is to isolate and breed yeast that exhibits alcohol production. The specific gene includes a mutant PDR3 gene.
[0007]
That is, the present invention has further discovered that genes involved in multidrug resistance greatly contribute to high alcohol production, and based on these useful new findings, have been further studied and completed. It is also involved in the efficient selection of yeasts that have produced high alcohol by introduction of the PDR3 gene, and in the total system for producing alcoholic beverages, including the production of such yeasts.
[0008]
In the present invention, the desired high alcohol producing yeast may be separated from the yeast into which the mutant PDR3 gene has been introduced (positive selection), so that the separation operation is easy and simple. Is better.
[0009]
In practicing the present invention, a mutant PDR3 gene is introduced using a yeast belonging to the genus Saccharomyces cerevisiae, and a strain into which the mutant PDR3 gene has been introduced is selected and isolated. Thus, the desired high alcohol producing yeast can be obtained.
Furthermore, if desired, a small-batch test is performed using these yeasts to isolate yeasts having a high fermentative power, so that high-alcohol-producing practical yeasts having an excellent fermentative power can be selected and bred.
[0010]
The practice of the present invention may be performed according to a conventional method of genetic engineering. For example, a mutant PDR3 gene is amplified by a PCR method and the like, introduced into a vector, and the obtained expression plasmid is subjected to a lithium chloride method, a protoplast method, a PEG method. The cells may be introduced into a host cell such as yeast according to a conventional method such as electroporation, and the resulting transformant may be screened using a marker in a vector or a selection medium.
[0011]
The mutant PDR3 gene is a yeast which became trichothecin-resistant and became highly alcohol-producing from the association No. 10 yeast belonging to Saccharomyces cerevisiae (for example, K-10 Tri8 strain: National Institute of Advanced Industrial Science and Technology (currently, National Institute of Advanced Industrial Science and Technology)) The DNA may be amplified by the PCR method using the genomic DNA of FERM P-16040 (deposited with the Biotechnology Research Institute) as a template, or may be directly cut out from the genomic DNA of the above strain. The nucleotide sequence of the mutant PDR3 gene thus obtained is shown in SEQ ID NO: 1 (FIGS. 1, 2, 3, and 4) in the sequence listing. For the first time, it was found that the mutant PDR3 gene had mutations in codons encoding three amino acids of the PDR3 gene. That is, the codon TTG at positions 2233 to 2235 is mutated to TCG (amino acid leucine at position 553 → serine), the codon AAA at positions 2314 to 2316 is mutated to GAA (amino acid lysine at position 580 → glutamic acid), and The ド ン 3435 codon TTT is mutated to TTA (953 amino acid phenylalanine → leucine).
[0012]
In this way, the present inventors succeeded in breeding an excellent yeast with high alcohol production, named one strain as K10-PDR3, and instituted FERM with the National Institute of Advanced Industrial Science and Technology (AIST). Deposited as P-17940.
[0013]
In the present invention, any yeast can be used as the yeast as long as it belongs to the genus Saccharomyces (Saccharomyces) cerevisiae. For example, sake yeast (Kyoto No. 7 yeast, Kyokai No. 9 yeast, Kyokai No. 10) Practical yeasts such as yeast, Akari Ogawa yeast, etc.), wine yeast (Grape No. 1 yeast (Japan Brewing Association Wine No. 1 yeast), wine No. 3 yeast, wine No. 4 yeast, etc.), beer yeast, baker's yeast, etc. Any yeast can be advantageously used as long as it belongs to the genus Saccharomyces cerevisiae, including yeast commonly used for alcohol fermentation.
Hereinafter, examples of the present invention will be described.
[0014]
Embodiment 1
(Separation of mutant PDR3 transgenic yeast)
(1) Primer-A (SEQ ID NO: 2, FIG. 5) and primer B (SEQ ID NO: 3, SEQ ID NO: 3) were obtained from the DNA of a yeast that became tricosecin-resistant and became highly alcohol-producing from the association No. 10 yeast belonging to Saccharomyces cerevisiae. Using FIG. 6), the mutant PDR3 gene (SEQ ID NO: 1, FIGS. 1-4) was amplified by PCR, and then the plasmid vector pAUR101 (purchased from Takara Shuzo Co., Ltd.) and the amplified PDR3 gene were replaced with the restriction enzyme Sa1I. After cutting with XbaI and XbaI, ligation was carried out to select pAUR101 into which the mutant PDR3 gene was inserted, and a vector pAUR101-mutant PDR3 containing the PDR3 gene (restriction enzyme map is shown in FIG. 7). Was.
[0015]
(2) Saccharomyces a (Saccharomyces) genus cerevisiae (cerevisiae) belonging Kyokai No. 10 sake yeast (K-10), YPD medium (yeast extract 1%, peptone 2%, glucose 2%) was inoculated into, grown After that, the cells were aseptically collected and washed, and transformed using the vector pAUR101-mutant PDR3 (FIG. 7) containing the mutant PDR3 gene, and transferred to a YPD agar medium containing aureobasidin A (0.0005 mg / ml). The cells were spread, cultured at 30 ° C., and the grown colonies were further subcultured on a YPD agar medium containing aureobasidin A (0.0005 mg / ml).
[0016]
(3) That is, after the expression vector pAUR101-mutant PDR3 containing the mutant PDR3 gene obtained above was cut with the restriction enzyme XbaI, the K-10 was cloned according to the instructions for the Aureobasidin A resistant yeast transformation system of Takara Shuzo Co., Ltd. Transformation was performed by the lithium chloride method, and the transgenic yeast was selected on a plate containing aureobasidin A.
[0017]
Embodiment 2
In Example 1, the isolated aureobasidin A-resistant yeast strain was cultured in a YPD medium, applied to a YPD agar medium containing cycloheximide (0.0005 mg / ml), cultured at 30 ° C., and grown. did.
[0018]
Embodiment 3
(Further isolation of yeast having better fermentability than yeast expressing mutant PDR3 gene)
The yeast strain isolated in Example 2 was subjected to three-stage preparation using 70% polished white rice in a preparation mixture shown in Table 1 below. Yeast suspension was used for yeast (5 × 10 ⁶ per 1 ml of charged water). Addition was performed at 15 degrees, middle was performed at 12 degrees, and detention was performed at 9 degrees. The temperature of the product was increased, and thereafter fermentation was performed at a constant temperature of 15 degrees. The ethanol in the fermentation broth was measured using a vibrating dense clock (Kyoto Electronics Co., Ltd.), and the acidity was 0.1 N NaOH, the amino acid content was 0.1 N NaOH, and the National Tax Agency used a formalin solution (50% aqueous solution). The analysis was performed according to a predetermined analysis method. The components of the resulting sake (at the time of the upper tank) are shown in Table 2 below.
Thus, the high alcohol producing yeast was successfully bred. This strain was designated as K10-PDR3 and deposited with LIFEM as FERM P-17940.
[0019]
Although K-10 has been described as an example, mutant PDR3 transgenic yeast can be isolated from other yeasts in the same manner.
[0020]

[0021]

[0022]
【The invention's effect】
According to the present invention, it is possible to breed high-alcohol-producing yeast, and by using the yeast bred according to the present invention, it becomes possible to efficiently produce brewed liquor and alcohol-fermented moromi having a high alcohol content.
[0023]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the DNA sequence of a mutant PDR3 gene.
FIG. 2 shows a continuation of the above.
FIG. 3 shows a continuation of the above.
FIG. 4 shows a continuation of the above.
FIG. 5 shows primer A.
FIG. 6 shows primer B.
FIG. 7 shows a restriction map of plasmid pAUR101-mutated PDR3.

Claims (8)

A DNA represented by the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. A method for breeding a high alcohol producing yeast, which comprises introducing a DNA represented by the nucleotide sequence of SEQ ID NO: 1 into a yeast and expressing it. The breeding method according to claim 2, wherein the DNA includes DNA of a mutant PDR3 gene. A plasmid vector comprising the DNA represented by the nucleotide sequence of SEQ ID NO: 1. The plasmid vector according to claim 4, wherein the plasmid vector is pAUR101-mutated PDR3. A transformed high-alcohol-producing yeast obtained by transforming a yeast with the plasmid vector according to claim 4. Transformed high alcohol producing yeast, K10-PDR3 (FERM P-17940). A method for producing high alcohol liquors, comprising using the yeast according to claim 6 or 7 to produce high alcohol liquors.

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