JPH07114689B2 - Auxotrophic brewer's yeast - Google Patents

Description

TECHNICAL FIELD The present invention relates to an auxotrophic mutant yeast by gene disruption.

More specifically, the present invention relates to a URA3 gene of a yeast used for brewing an actual food or drink (called a practical brewing yeast) or
The present invention relates to a practical brewer's yeast that has become auxotrophic for uracil or uracil and tryptophan by disrupting the URA3 gene and TRP1 gene.

The auxotrophic practical brewer's yeast which is the transformant of the present invention is URA3 gene or URA3 gene and TRP1 gene deletion, so it cannot grow without the presence of uracil or uracil and tryptophan in the medium. .

In order to express a heterologous gene in a laboratory yeast strain from the outside, many plasmid vectors that are usually used have a URA3 gene or TRP1 gene as a selectable marker. That is, when a heterologous gene is introduced into a practical brewer's yeast, by using the transformant of the present invention, the target gene can be obtained by a plasmid vector having the same URA3 gene or TRP1 gene as that performed in the laboratory strain. Can be expressed in a practical brewer's yeast. Therefore, the present invention facilitates breeding of a practical brewing yeast having unprecedented properties, and greatly contributes to the production of alcoholic beverages, alcohols, and other brewed foods.

(Background of the Invention and Prior Art and Problems Thereof) In general, laboratory strain yeast is haploid, so that it is easily uracil auxotrophic strain (ura3) or tryptophan auxotrophic strain (trp
1) can be obtained, and therefore, in the laboratory strain yeast, using these strains as hosts, the URA3 gene and TRP1 can be obtained.
A transformation system using a plasmid vector having a gene has been established.

However, since the practical brewer's yeast (Saccharomyces cerevisiae) normally used for the production of sake, shochu, fruit wine, beer, whiskey, etc. is diploid (or higher polyploid), It was not possible to obtain such an auxotrophy which is a recessive mutation without impairing its brewing properties.

(Means for Solving Problems) In view of its outstanding usefulness, the inventors of the present invention dared to create an auxotrophic strain of practically brewing yeast, which was conventionally impossible to produce. Did research from the direction.

As a result, the present inventors succeeded in creating a new URA3 gene disruption plasmid pURA36 having a deletion within the translation region of the URA3 gene. Furthermore, the above-mentioned deletion mutation in which resistance to sulfoformulon methyl (SM) was taken as a selectable marker for practical yeast and the resistance gene (SMR1) was linked.
We also succeeded in constructing a new URA3 disruption plasmid pURA38, which also contains the URA3 gene.

As a result of further research to create an auxotrophic yeast using the novel plasmid thus created, it was discovered that the yeast can be created by the following method.

That is, first, transformation is performed using the plasmid pURA38, and then the plasmid pURA36 is introduced into the obtained transformant, and the uracil auxotrophic strain is selected by the FOA medium in which only the uracil auxotrophic strain grows. By this method, it was found that a uracil-requiring mutant strain in which the URA3 gene on two chromosomes of a practically brewing yeast is destroyed can be efficiently obtained. The auxotrophic yeast thus obtained has not been known so far and is novel.

Next, from the novel uracil auxotrophic strain thus obtained, a plasmid which was also successfully newly created
It was found that a tryptophan-requiring mutant strain can be obtained in the same manner using pTRP14. The auxotrophic yeast thus obtained has not been known so far and is novel.

The present invention is not only successful in creating a new auxotrophic yeast in this way, as a result of the growth test and the sake brewing test using the novel practical brewing yeast thus obtained, a heterologous gene. It was also confirmed that it is suitable as a host for transducing saccharomyces, comprehensively studied these, confirmed the usefulness, and finally completed the present invention.

Hereinafter, the present invention will be described in detail.

(1) Acquisition of uracil-requiring mutant strain by gene disruption Plasmid pURA36 was constructed by ligating the PstI-SmaI fragment containing the URA3 gene of plasmid YIp5 to plasmid pUC18.
URA30 is created, followed by deletion of the EcoRV-StuI site and Hinc II-Hinc II site within the URA3 gene. Further, pURA38 is constructed by ligating pURA36 with the SmaI-KpnI fragment containing the SMR1 gene of pWX500 (Fig. 1).

In transformation, yeast for practical brewing is diploid (or higher polyploid), so it is necessary to destroy the gene of interest on two chromosomes.

To do so, first, cut the plasmid pURA38 at the StuI site, transform it into a linear DNA and transform it into a practical brewer's yeast. select.

Next, the transformant was retransformed using a linear DNA of about 0.8 kbp obtained by cutting the plasmid pURA36 with PstI and SmaI sites, and the gene on the remaining chromosome was destroyed, resulting in uracil-requiring Select transformants that have become sex. It can be easily selected by using an FOA medium in which only such a uracil-auxotrophic strain can grow.

Whether or not the URA3 gene was destroyed depends on the chromosomal DNA.
Is prepared and confirmed by Southern blotting.

(2) Acquisition of tryptophan-requiring mutant strain by gene disruption The plasmid pTRP14 for gene disruption is prepared as follows.

First, a plasmid pTRP11 incorporating a 1 kbp EcoRI fragment containing the TRP1 gene is prepared, and subsequently, an EcoRV-NcoI site inside the TRP1 gene is deleted to prepare pTRP12. In addition, pTRP14 was constructed in the manner described above.
It is prepared by ligating PstI and SmaI fragments containing the URA3 gene to (Fig. 2).

In carrying out the transformation, the parent strain of the transformation,
Using the uracil-requiring mutant strain obtained by the method of (1), linearization is performed at the StuI site of pTRP14, and then transformation is performed. Transformants are selected in a minimal medium and then cultured in a nutrient medium to obtain a gene replacement-induced uracil-requiring strain on a FOA plate. From this, strains in which the TRP1 gene has a deletion inside are selected by Southern blotting.

Since the obtained strain was one in which one of the two chromosomes had the TRP1 gene disrupted, pT was transformed into this transformant strain.
Transform with RP14. By performing the same operation,
A target transformant is obtained. By this method,
Uracil- and tryptophan-requiring strains without E. coli-derived DNA can be obtained.

Next, examples and application examples of the present invention will be shown.

Example 1 Construction of plasmid for URA3 gene disruption Method for constructing plasmids pURA36 and pURA38: About 1 kbp SmaI and PstI DNA fragment containing URA3 gene was ligated to pUC18 using T4 ligase to prepare pURA30. Next, in order to delete the EcoRV-ScaI site and the HincII-HincII site inside the URA3 gene, the URA3 gene was treated with each restriction enzyme and ligated with T4 ligase to prepare. SM of pWX509 to this pURA36
The SmaI-KpnI fragment containing the R1 gene is ligated to prepare (FIG. 1).

Construction of plasmid pTRP14: 1.4 kbp E containing TRP1 gene
A plasmid pTRP11 incorporating the coRI fragment is created, followed by deletion of the EcoRV-StuI site within the TRP1 gene to create pTRP12. In addition, pTRP14 is created by pTRP12
It is constructed by ligating PstI and KpnI fragments containing the URA3 gene to (Fig. 2).

Example 2 Method for obtaining uracil auxotrophic strain (1) Sake yeast association No. 701 (diploid (commercially available) and sake yeast association No. 901 (diploid) using pura38 and pURA36 for URA3 gene disruption) (Commercially available) was transformed by Ito et al. (J.
Bacteriol., Vol 153, 163 (1983)).

That is, sake yeast (association 701, association 901) of 100 ml YPD
The culture was carried out with shaking in a medium (1% yeast extract, 2% polypeptone, 2% glucose, pH 5.3), and the cells were collected by centrifugation in the logarithmic growth phase. 2x10 in the same buffer after washing with TE buffer
Suspension was performed at a concentration of ⁸ cells / ml. Transfer 0.5 ml of this to a small test tube and add an equal volume of 0.2 M lithium acetate solution for 1 hour.
Shake at 30 ° C. From this, 0.1 ml was transferred to a 1.5 ml Eppendorf tube and the plasmid pURA38 was digested with the restriction enzyme St
10 μl of linearized DNA solution treated with uI (2 μg / μl)
Was added and static culture was carried out at 30 ° C. for 30 minutes. Then sterilized 70
% PEG4000 (150 μl) was added and mixed well. After standing at 30 ° C for 1 hour, the Eppendorf tube was left standing in a constant temperature water bath at 42 ° C for 5 minutes, then the cells were immediately cooled to room temperature and washed with sterilized water, and the SM-containing medium (east nitrogen base ( W / O amino acid) 0.67%, glucose 2%, agar 2%,
Transformed strains of 40 strains that grew at 100 ppm of sulfometuron methyl (added after autoclave) were obtained.

Next, for four of these strains, plasmid pURA36 was treated with restriction enzymes PstI and SmaI to form a linear DNA solution (0.8 μg / μm).
l) 10 μl was added, and transformation was performed by the same method as above. The target uracil-requiring strain is FOA plate (east nitrogen base (W / O amino acid) 0.67
%, 5-fluoroorotic acid 0.1%, uracil 20 ppm, glucose 2%, agar 2%), and about 36 strains were selected from colonies appearing after 1 week of culture at 30 ° C.

It was confirmed by Southern blotting that three types A, B, and C of URA3 gene-disrupted strains were obtained from the uracil-requiring strains thus obtained (3rd
Figure). The U-4-12 strain derived from Association No. 701 is FERM P
-11078 has been deposited with the Institute of Microtechnology.

Example 3 Method for obtaining uracil auxotrophic strain (2) Using the plasmid pURA36 for disrupting URA3 gene, yeast strain for sake 901 (diploid) (commercially available) was transformed by the above method.

That is, Sake Yeast Association No. 901 is 100 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% glucose, pH 5.3).
The cells were cultivated with shaking in the medium, and the cells were collected by centrifugation in the logarithmic growth phase. After washing with TE buffer, the cells were suspended in the same buffer at a concentration of 2 × 10 ⁸ cells / ml. 0.5 ml of this was transferred to a small test tube, an equal volume of 0.2 M lithium acetate solution was added, and the mixture was shaken at 30 ° C. for 1 hour. From this, 0.1 ml was transferred to a 1.5 ml Eppendorf tube, and 10 μl of a linearized DNA solution (1.6 μg / μl) obtained by treating plasmid pURA36 with restriction enzymes PstI and SmaI was added, followed by transformation in the same manner as above. I went. The target uracil-requiring strain is FOA plate (yeast nitrogen base (W / O amino acid) 0.67%, 5-fluoroorotic acid 0.1%, uracil 20ppm, glucose 2%, agar 2%) at 30 ° C for 1 week. 9 from colonies that appear in culture
Acquired stock.

From the uracil-requiring strains thus obtained, it was confirmed by Southern blotting that a URA3 gene-disrupted strain of type D (U-9-11) different from the above 3 types was obtained (Fig. 3). .

U-9-11 has been deposited with the Institute of Mechanical Engineering as FERM P-11079.

Example 4 Method for Obtaining Uracil- and Tryptophan-auxotrophic Strain Transformation of uracil-auxotrophic strain U-4-12 derived from Sake Yeast Society No. 701 (diploid) (commercially available) using TRP1 gene disruption pramid pTRP14 Was performed by the above method.

That is, the U-4-12 strain was shake-cultured in 100 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% glucose, pH 5.3), and the cells were collected by centrifugation in the logarithmic growth phase. TE
After washing with a buffer solution, the cells were suspended in the same buffer solution at a concentration of 2 × 10 ⁸ cells / ml. Transfer 0.5 ml of this to a small test tube,
Lithium acetate solution was added and shaken for 1 hour at 30 ° C.
From this, 0.1 ml was transferred to a 1.5 ml Eppendorf tube, and 10 μl of a linear DNA solution (1.4 μg / μl) obtained by treating plasmid pTRP14 with the restriction enzyme StuI was added, and transformation was performed by the same method as above. It was Fifty transformants were obtained which grew on SD plates (east nitrogen base (W / O amino acid) 0.67%, glucose 2%, agar 2%).

Next, by culturing 4 strains of these in YPD medium at 30 ° C for 24 hours, gene replacement was caused in pTRP14 integrated on the chromosome, and strains that grew on FOA plate were selected. Selected. In this way, with respect to 24 strains that became uracil auxotrophic again, a transformant strain (U-4-12-4 strain) in which one of the TRP1 genes on the chromosome was deleted was obtained by Southern blotting.

U-4-12-4 strain was cultivated in YPD medium at 30 ° C for 2 days with shaking to select a gene-disrupted strain in which deletion of the TRP1 gene of one chromosome was transferred to the other complete TRP1 gene by gene conversion. To do. As a selection method, first, tryptophan-deficient plate (yeast nitrogen base (W / O amino acid) 0.67%, uracil 30 ppm, tryptophan 1 ppm, casamino acid 0.1%, glucose 2%, agar 2%) was used to obtain about 20000%.
Colonies, and about 100 strains that form small colonies are selected, and tryptophan-added plates (yeast nitrogen base (W / O amino acid) 0.67
%, Uracil 30ppm, tryptophan 30ppm, glucose 2%, agar 2%) and tryptophan-free plate (yeast nitrogen base (W / O amino acid) 0.67)
%, Uracil 30 ppm, glucose 2%, agar 2%) were isolated as the target gene-disrupted strains (UT-1, UT-2).

It was confirmed by Southern blotting that these strains were gene-disrupted strains in which the EcoRV-StuI site inside the TRP1 gene was deleted.

In addition, UT-1 has been deposited with MIC as FERM P-11077.

Reference Example Method for Obtaining Tryptophan-auxotrophic Strain YIp5 having URA3 gene was used to transform uracil and tryptophan-auxotrophic strain U-4-12 derived from Sake Yeast Society No. 701 (diploid) (commercially available product) as described above. It was performed by the method of.

That is, the U-4-12 strain was shake-cultured in 100 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% glucose, pH 5.3), and the cells were collected by centrifugation in the logarithmic growth phase. TE
After washing with a buffer solution, the cells were suspended in the same buffer solution at a concentration of 2 × 10 ⁸ cells / ml. Transfer 0.5 ml of this to a small test tube,
Lithium acetate solution was added and shaken for 1 hour at 30 ° C.
From this, 0.1 ml was transferred to a 1.5 ml Eppendorf tube and the plasmid YIp5 was treated with restriction enzymes PstI and SmaI to form a linear DNA solution containing the URA3 gene (0.8 μg / μl) 10 μm
1 was added and transformation was performed by the same method as above.

SD plate with tryptophan (yeast nitrogen base (W / O amino acid) 0.67%, tryptophan 30ppm,
40 transformants were obtained which grew on glucose 2% and agar 2%).

It was confirmed by Southern blotting that one of the URA3 genes on the chromosome of this transformant was restored to a complete URA3 gene by Southern blotting.

Application Example 1 Production of Sake by Transformant Transformant U-4-12 (FERM P-11078), parent strain (Association No. 701) and U-4-12 strain transformed with YIp5 (p.
-4-12) was shake-cultured in 10 ml of YPD medium at 30 ° C. overnight,
Bacteria-collected and washed, and the total rice is prepared according to the formulation and manufacturing conditions shown in Table 1.
200g of sake was prepared.

The progress of alcohol fermentation was measured by the decrease in weight due to the generation of carbon dioxide, and is shown in FIG. Table 2 shows the primary components and organic acid composition of the produced sake.

As a result, the uracil auxotrophic strain U-4-12 had a slower alcohol fermentation rate than the parent strain, and the produced liquor was a type characterized by a large amount of acid, which was generally not desirable. However, in the p-4-12 strain into which the URA3 gene was introduced by one transformation, there was no difference in the fermentation rate and the quality of the produced sake from the parent strain. It is suggested that the method returns to and, and is useful as a host when expressing a heterologous gene product.

Thus, this gene-disrupted strain having the property of increasing organic acids such as malic acid and succinic acid has been shown to be effective for the production of sake, which is rich in acid, in terms of product differentiation.

Application Example 2 Expression of Heterologous Gene Product by Transformant α-Amylase Gene of Aspergillus oryzae
pYcT-1F in which the cDNA was ligated to the yeast expression vector pYcDE was used as a transformant UT-1 (FERM P-11077, a / α trpl / trpl
u ra3 / ura3) and laboratory strain NA87-11 (α trpl leu2
his3) to Ito et al. (J. Bacteriol., Vol 153,163 (198
Transduction was performed according to the method of 3)). The obtained transformant formed a halo indicating that amylase was secreted extracellularly in the medium containing starch. Also, in YPD medium, 30
The UT-1 derived from sake yeast was 1.
7 mg / l, laboratory strain NA87-11 produced 0.4 mg / l α-amylase protein, indicating that sake yeast is a promising host for heterologous protein production.

[Brief description of drawings]

Fig. 1 is an explanatory diagram of a plasmid for URA3 gene disruption, Fig. 2 is an explanatory diagram of a plasmid for TRP1 gene disruption, Fig. 3 is a Southern blotting analysis diagram of a URA3 gene disruption strain, and Fig. 4 is It is a progress chart of alcohol fermentation of a gene disruption strain.

Continuation of the front page (72) Inventor Kojiro Takahashi 10-chome, Odori Nishi, Chuo-ku, Sapporo, Hokkaido Sapporo Taxation Bureau Appraisal Office (56) Reference JP-A-61-56077 (JP, A) European Patent 286303 (EP, B) )

Claims (2)

[Claims] 1. A uracil-requiring practical brewing yeast obtained by disrupting the URA3 gene using the plasmids pURA36 and / or pURA38 derived from Saccharomyces yeast for disrupting the URA3 gene shown below. 2. A uracil-requiring brewer's yeast obtained by disrupting the URA3 gene using the plasmids pURA36 and / or pURA38 derived from Saccharomyces yeasts for disrupting the URA3 gene shown below: Uracil- and tryptophan-requiring practical brewing yeast obtained by disrupting the TRP1 gene using the plasmid pTRP14 derived from the yeast of the genus Saccharomyces for disrupting the TRP1 gene shown below.