JP2021141831A - Composition for production of alcohol food and drink - Google Patents

Abstract

To provide a method for improving yeast's ability to utilize proline.SOLUTION: A composition for the production of alcohol food and drink includes CAN1 mutant yeast or CAN1 inhibited yeast.SELECTED DRAWING: None

Description

The present disclosure relates to a composition for producing alcoholic foods and drinks, a method for producing alcoholic foods and drinks, and the like.

Grape contains about 40% proline as a nitrogen source. However, it is known that in winemaking (fermentation stage), proline is hardly consumed and remains in wine. Since proline is an amino acid that exhibits bitterness and sweetness, if the content of proline in wine increases, the bitterness increases and the acidity decreases, which affects the taste. Therefore, proline remaining in wine. Reducing the content has become an issue.
In addition, when the nitrogen source available to yeast such as ammonium is depleted during wine production (fermentation stage), it is necessary to add an artificial nitrogen source such as ammonium salt. For this reason as well, a method for utilizing the remaining proline that has not been assimilated as a nitrogen source has been developed (Patent Document 1).

JP 2006-006106

An object of the present disclosure is to improve the proline-utilizing ability of yeast.

The present inventors have found that the CAN1 gene is involved in the uptake of proline, and made further improvements.

The present disclosure includes, for example, the subjects described in the following sections.
Item 1.
A composition for producing alcoholic beverages, which contains CAN1 mutant yeast or CAN1-suppressed yeast.
Item 2.
The CAN1 mutant yeast encodes an amino acid sequence different from the amino acid sequence constituting the wild-type Can1 protein, in which at least one base is deleted, substituted, or added in the base sequence encoding the wild-type Can1 protein. Item 2. The composition for producing an alcoholic food or drink according to Item 1, which has a nucleic acid.
Item 3.
Item 2. The composition for producing alcoholic beverages according to Item 1 or 2, wherein the CAN1 mutant yeast or the CAN1 inhibitory yeast is a yeast having an improved proline uptake ability in the presence of arginine.
Item 4.
Item 4. The composition for producing alcoholic beverages according to any one of Items 1 to 3, wherein the yeast is a yeast belonging to the genus Saccharomyces.
Item 5.
A method for producing an alcoholic beverage, which comprises using a CAN1 mutant yeast or a CAN1 inhibitory yeast.
Item 6.
The CAN1 mutant yeast encodes an amino acid sequence different from the amino acid sequence constituting the wild-type Can1 protein, in which at least one base is deleted, substituted, or added in the base sequence encoding the wild-type Can1 protein. Item 5. The method for producing an alcoholic food or drink having a nucleic acid.
Item 7.
Item 5. The method for producing an alcoholic beverage according to Item 5 or 6, wherein the CAN1 mutant yeast or the CAN1 inhibitory yeast is a yeast having an improved proline uptake ability in the presence of arginine.
Item 8.
Item 8. The method for producing an alcoholic beverage according to any one of Items 5 to 7, wherein the yeast is a yeast belonging to the genus Saccharomyces.

Yeast with improved proline uptake in the presence of arginine is provided. A composition for producing an alcoholic food or drink containing the yeast is provided. Further, a method for producing an alcoholic food or drink using the yeast is provided.

The growth of wild-type strains and proline-requiring strains in arginine-added medium is shown. The growth of the proline-requiring strain and the CAN1 mutant in the arginine-added medium is shown. The amount of proline and the amount of arginine in the medium when the proline-requiring strain and the CAN1-disrupted strain were cultured in the arginine-added medium are shown. In the arginine-added medium, a strain in which an empty vector was introduced into a CAN1 disrupted strain (CAN1 disrupted strain), a strain in which wild-type CAN1 was expressed in a CAN1 disrupted strain (wild strain), and a mutant CAN1 (Can1 G434C) in a CAN1 disrupted strain. The growth of the strain expressing (CAN1 mutant strain) is shown.

Hereinafter, each embodiment included in the present disclosure will be described in more detail.

The present disclosure includes compositions containing CAN1 mutant yeast or CAN1-suppressed yeast. In the present specification, the composition may be referred to as "the composition of the present disclosure".

SEQ ID NO: 1 is an amino acid sequence constituting a wild-type Can1 protein derived from Saccharomyces cerevisiae. Further, SEQ ID NO: 2 is a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1. In other words, the amino acid sequence shown in SEQ ID NO: 1 is an example of the amino acid sequence constituting the wild-type Can1 protein. The base sequence shown in SEQ ID NO: 2 is an example of a base sequence encoding a wild-type Can1 protein.

In the present specification, yeast expressing the wild-type CAN1 gene or yeast expressing the wild-type Can1 protein may be referred to as "wild-type CAN1 yeast". Examples of the wild-type CAN1 yeast include wild-type strains. In addition, CAN1-suppressed yeast is excluded.

The wild-type CAN1 gene is a CAN1 gene that does not have a gene mutation (deletion, substitution, addition, etc.) due to artificial processing. Wild-type Can1 proteins are Can1 proteins that do not have amino acid changes (deletion, substitution, addition, etc.) due to artificial treatment. Examples of the wild-type CAN1 gene include the nucleotide sequence shown in SEQ ID NO: 2, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more. Nucleic acids consisting of base sequences having 97% or more, 98% or more, or 99% or more identity can be mentioned. It is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more. Examples of the wild-type Can1 protein include the amino acid sequence shown in SEQ ID NO: 1, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more. Examples thereof include polypeptides consisting of amino acid sequences having 97% or more, 98% or more, or 99% or more identity. It is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more.

The CAN1 mutant yeast used in the present disclosure may be a yeast expressing a mutant CAN1 gene or a yeast expressing a mutant Can1 protein.

CAN1 mutant yeast is a nucleic acid in which at least one base is deleted, substituted, or added in the base sequence encoding the wild-type Can1 protein, and the amino acid sequence is different from the amino acid sequence constituting the wild-type Can1 protein. It is preferable to have. In a base sequence in which at least one base is deleted, substituted, or added, the upper limit of the number of deleted, substituted, or added bases is, for example, 1770, 1500, 1000, 800, 600. 400 pieces, 200 pieces, 100 pieces, 50 pieces, 45 pieces, 40 pieces, 35 pieces, 30 pieces, 25 pieces, 20 pieces, 19 pieces, 18 pieces, 17 pieces, 16 pieces, 15 pieces, 14 pieces, It may be 13, 12, 11, 10, 9, 9, 8, 7, 6, 5, 4, 3, or 2. The CAN1 mutant yeast preferably has a nucleic acid encoding an amino acid sequence different from the amino acid sequence constituting the wild-type Can1 protein, and more specifically, at least one amino acid sequence constituting the wild-type Can1 protein. It is preferred that the amino acids have nucleic acids encoding different amino acid sequences. The upper limit of the number of amino acids different from the amino acid sequence constituting the wild-type Can1 protein is, for example, 590, 550, 500, 400, 300, 200, 100, 50, 45, 40, etc. 35, 30, 25, 20, 19, 19, 18, 17, 16, 15, 15, 14, 13, 12, 11, 10, 10, 9, 8, 7, 7 , 6, 5, 4, 3, or 2. Further, the CAN1 mutant yeast may have, for example, a nucleic acid encoding an amino acid sequence in which glycine at position 434 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with cysteine. Further, as shown in Examples described later, even if the gene encoding the Can1 protein is a yeast substituted with a canamycin resistance gene, the desired effect is exhibited. Therefore, the types of changes in bases and amino acids (for example, for example). The number of deleted, substituted, or added) and mutated bases and amino acids is not particularly limited as long as the ability to take up proline in the presence of arginine is improved. In short, the CAN1 mutant yeast used in the present disclosure may be a yeast in which all of the nucleotide sequences encoding the wild-type Can1 protein have been deleted.

Techniques for adding mutations such as deletions, substitutions, or additions of at least one base to a base sequence are known in the art and can be performed using any technique. For example, it can be performed by using restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., position-designated mutation introduction treatment, random mutation introduction treatment (for example, drug treatment, ultraviolet treatment, radiation treatment, etc.). Examples of the chemical treatment include ethyl methanesulfonate treatment and N-methyl-N-nitrosoguanidine treatment. Further, the CAN1 mutant yeast can be easily obtained by a known screening using an arginine analog such as canavanine.

The CAN1-suppressed yeast used in the present disclosure may be a yeast in which the expression of the wild-type CAN1 gene is suppressed, or a yeast in which the expression of the wild-type Can1 protein is suppressed.

Techniques for suppressing gene expression and protein expression are known in the art and can be carried out using any method. For example, CAN1-suppressed yeast can be produced by an RNAi method using siRNA, shRNA, microRNA, or the like, an antisense method, or the like.

CAN1 suppression can be confirmed, for example, by measuring the expression level of the wild-type CAN1 gene. Further, the measurement of the gene expression level can be confirmed by a method known in the art. A yeast in which the expression level of the wild-type CAN1 gene is reduced as compared with the wild-type CAN1 yeast can be determined to be a CAN1-suppressed yeast.

The CAN1 mutant yeast and the CAN1-suppressing yeast used in the present disclosure are preferably yeasts having improved proline uptake in the presence of arginine. The ability to take up proline in the presence of arginine can be confirmed by measuring the amount of proline contained in the medium when yeast is cultured in a medium containing proline and arginine. More specifically, using a medium containing 0.1% L-proline (4.6 mM) and 0.01% L-arginine hydrochloride (0.361 mM), the proline described in Examples described later will be used. It can be confirmed by the method of measuring the amount. Wild-type CAN1 yeast and CAN1 mutant yeast or CAN1-suppressed yeast are cultured under the same conditions, and CAN1 mutant yeast or CAN1-suppressed yeast is cultured by comparing the amount of proline contained in the medium in which wild-type CAN1 yeast is cultured. When the amount of proline contained in the yeast is small, it can be judged that the proline uptake ability is improved.

The ability of the CAN1 mutant yeast and the CAN1-suppressing yeast used in the present disclosure to take up arginine is preferably equal to or higher than that of the wild-type CAN1 yeast. The arginine uptake ability can be confirmed by measuring the amount of arginine contained in the medium in the same manner as the proline uptake ability.

The yeast is not particularly limited as long as it can be used for producing foods and drinks. For example, yeasts of the genus Saccharomyces, Pichia, Kluyberomyces and the like can be mentioned, and yeasts of the genus Saccharomyces are preferable. Further, as the yeast, for example, wine yeast, brewer's yeast, sake yeast and the like can be used.

Alcoholic food and drink means food and drink containing alcohol. Alcoholic foods and drinks include fruit liquor (eg wine, ceddle, etc.), beer, effervescent liquor, other effervescent liquors, brewed liquor such as sake; distilled liquor such as shochu, whiskey, brandy; wine lees, beer lees, Examples thereof include fermentation residues such as sake lees, shochu lees, and whiskey lees.

The content of the CAN1 mutant yeast or the CAN1 inhibitory yeast in the composition for producing alcoholic beverages can be appropriately set, and can be, for example, 1 to 100% by mass.

The composition for producing alcoholic beverages may contain CAN1 mutant yeast or CAN1-suppressed yeast, and may further contain other components.

Other components include, for example, solvents, dispersion media (eg, glycerol, DMSO, skim milk, etc.), preservatives, excipients, activators, preservatives, pH adjusters, stabilizers, medium components (eg, agar). , Glycerol, yeast extract, etc.), raw materials used in the production of alcoholic foods and drinks (for example, grapes, malt, hops, rice, wheat, extracts thereof, etc.); and the like. The raw material used in the production of alcoholic foods and drinks may contain proline. These can be used alone or in combination of two or more. In addition, the composition of the present disclosure may be prepared at the time of use by mixing CAN1 mutant yeast or CAN1 inhibitory yeast with other components.

The composition of the present disclosure may be in a solid form (for example, in a powder form, a granular form, a gel form, a paste form, etc.), a liquid form (for example, a solution form, a suspension form, etc.) and the like.

The composition of the present disclosure can be prepared by a conventional method in combination with a CAN1 mutant yeast or a CAN1 inhibitory yeast and, if necessary, other components.

The present disclosure also includes methods for producing alcoholic foods and drinks. In the present specification, the method may be referred to as "the method for producing an alcoholic beverage of the present disclosure".

The method for producing an alcoholic beverage of the present disclosure is characterized by using a CAN1 mutant yeast or a CAN1-suppressed yeast. In other words, the method for producing an alcoholic food or drink according to the present disclosure may use the composition of the present disclosure. Alcoholic foods and drinks can be produced by adopting conventional methods and arbitrary conditions depending on the type of alcoholic foods and drinks. For example, the method for producing an alcoholic food or drink of the present disclosure may include a fermentation step. The CAN1 mutant yeast or CAN1-suppressed yeast can be used at any stage in the production of alcoholic beverages. Further, when the method for producing an alcoholic food or drink of the present disclosure uses the composition of the present disclosure, the composition of the present disclosure can be used at any stage of the production of the alcoholic food or drink. Optional steps include, for example, a fermentation step, pre-fermentation, post-fermentation and the like.

The method for producing an alcoholic beverage of the present disclosure preferably includes a step of mixing a CAN1 mutant yeast or a CAN1 inhibitory yeast, or the composition of the present disclosure with a raw material used for producing an alcoholic beverage. The raw material used for producing the alcoholic food or drink can be appropriately selected depending on the type of the alcoholic food or drink, and may contain proline. The content of proline contained in the raw material used for producing alcoholic foods and drinks is not particularly limited, but may be, for example, 1% by mass to 99% by mass, and the upper or lower limit of the range is, for example, 10, 20, It may be 30, 40, 50, 60, 70, 80, or 90% by mass. More specifically, it may be, for example, 10% by mass to 90% by mass.

When yeast (for example, wine yeast, beer yeast, sake yeast, etc.) is used to produce alcohol in the method for producing alcoholic beverages of the present disclosure, the yeast and the CAN1 mutant yeast or CAN1 inhibitory yeast are referred to as each other. It may be the same or different.

According to the present disclosure, the ability of yeast to take up proline in the presence of arginine can be improved. Preferably, it is possible to obtain a yeast having an improved ability to take up proline in the presence of arginine as compared with the wild-type CAN1 yeast. Therefore, by using the composition of the present disclosure in the production of alcoholic foods and drinks, the content of proline in alcoholic foods and drinks can be reduced. Preferably, the proline content in alcoholic foods and drinks can be reduced as compared with the case of using wild-type CAN1 yeast. Therefore, the composition of the present disclosure can be suitably used for the production of alcoholic foods and drinks. In addition, the composition of the present disclosure can be suitably used for reducing the proline content in alcoholic foods and drinks. Further, according to the present disclosure, since the content of proline in alcoholic foods and drinks is reduced, alcoholic foods and drinks having excellent flavor are provided. Further, according to the present disclosure, since proline contained in the raw material can be effectively used, the amount of the artificial nitrogen source used in the production of alcoholic foods and drinks can be reduced. According to the present disclosure, it is possible to obtain a yeast having an arginine uptake ability comparable to that of wild-type CAN1 yeast. Therefore, according to the present disclosure, it is possible to maintain the same level of arginine content in alcoholic foods and drinks as when wild-type CAN1 yeast is used. Therefore, according to the present disclosure, since the content of arginine in alcoholic foods and drinks is low, alcoholic foods and drinks having excellent flavor are provided.

In addition, in this specification, "including" also includes "consisting essentially" and "consisting of" (The term "comprising" includes "consisting essentially of" and "consisting of."). The present disclosure also includes all combinations of the constituent requirements described herein.

In addition, the various properties (property, structure, function, etc.) described for each embodiment of the present disclosure described above may be combined in any way in specifying the subject matter included in the present disclosure. That is, the present disclosure includes all subjects consisting of any combination of each combinable property described herein.

The contents of the present disclosure will be specifically described with reference to the following experimental examples and examples. However, this disclosure is not limited to these. In the following, the experiments are carried out under atmospheric pressure and normal temperature conditions, unless otherwise specified. Also, unless otherwise specified, "%" means "mass%".

1. 1. Acquisition of Proline Requirement Strain Proline is known to be biosynthesized using glutamic acid or arginine as a starting material. In order to obtain a proline-requiring strain, glutamic acid-based biosynthetic pathway key enzyme γ-glutamyl kinase (Pro1) and arginine-based biosynthetic pathway key enzyme Ornithine aminotransferase (Car2), respectively, were used. A double-deficient strain (pro1Δcar2Δ strain) in which the encoding gene was disrupted was prepared. The specific production method is shown below.
First, using a plasmid containing a drug resistance gene (pFA6a-5FLAG-hphMX6: containing a hyglomycin resistance marker) as a template, a primer having a base sequence homologous to each of the upstream and downstream regions of the ORF of the PRO1 gene (SEQ ID NO: 3). And 4) were used to carry out PCR, and a nucleotide sequence homologous to the upstream and downstream of the PRO1 gene was ligated upstream and downstream of the drug resistance gene to prepare a fragment for disrupting the PRO1 gene. Saccharomyces cerevisiae X2180-1A strain cultured in YPD medium containing 2% glucose, 1% Bacto Yeast extract, and 2% Bacto Peptone _{until the logarithmic growth phase (OD 600} = 0.4-0.8) was collected and 0.1 M acetate. After suspending in lithium, the cells were incubated at 30 ° C. for 15 minutes. After removing the supernatant by centrifugation, 36 μL of 1 M lithium acetate, 25 μL of salmon sperm DNA, 50 μL of PRO1 gene disruption fragment, and 240 μL of 50% PEG4000 were added and mixed. After incubating this mixed solution at 30 ° C. for 30 minutes, 39 μL of DMSO was added, and the mixture was further incubated at 42 ° C. for 20 minutes. Then, the supernatant was removed, and recovery culture was carried out at 30 ° C. for 1 hour or longer. The cells after the recovery culture were inoculated into an appropriate selective medium to obtain transformants. Then, it was confirmed by the PCR method that the desired transformant (pro1Δ strain) was obtained.
Furthermore, using a plasmid containing the drug resistance gene (pFA6a-natMX6: containing the norseotricin resistance marker) as a template, primers having a base sequence homologous to each of the upstream and downstream regions of the ORF of the CAR2 gene (SEQ ID NO: 5 and PCR was performed using 6), and a nucleotide sequence homologous to the upstream and downstream of the CAR2 gene was ligated upstream and downstream of the drug resistance gene to prepare a fragment for disrupting the CAR2 gene. Regarding the obtained CAR2 gene disruption fragment, a proline-requiring strain (pro1Δcar2Δ strain) was obtained by the same method as above except that the pro1Δ strain was used instead of the Saccharomyces cerevisiae X2180-1A strain.

2. Growth test in solid medium (spot test) 1
Each strain cultured in proline medium (SD-N + Pro) _{was diluted and prepared so that OD 600} = 1.0, 0.1, 0.01, 0.001, 0.0001, 0.00001, and ammonium ionized medium (SD-N + Pro + AS) was added. ) Or arginine-added medium (SD-N + Pro + Arg) was spotted in 3 μL each. The composition of each medium was as follows, and powdered agar was added to a final concentration of 2% to prepare a solid medium.
Proline medium (SD-N + Pro): 2% glucose, 0.17% Yeast Nitrogen Base without amino acids and ammonium sulfate (Difco), and 0.1% L-proline (4.6 mM)
Ammonium ionized medium (SD-N + Pro + AS): 2% glucose, 0.17% Yeast Nitrogen Base without amino acids and ammonium sulfate (Difco), 0.1% L-proline (4.6 mM), And 0.5% ammonium sulfate (37.8 mM)
Arginine-added medium (SD-N + Pro + Arg): 2% glucose, 0.17% Yeast Nitrogen Base without Amino Acids and ammonium sulfate (Difco), 0.1% L-proline (4.6 mM), and 0.01% L-arginine hydrochloride (0.361 mM)
After incubating at 30 ° C. for 3 days, growth was observed. The results are shown in FIG.

As shown in FIG. 1, the growth of the proline-requiring strain was not confirmed in the arginine-added medium.

3. 3. Acquisition of "insensitive" strains that suppress proline assimilation and genome analysis To identify genes that control arginine-dependent suppression of proline assimilation, mutant strains that can assimilate proline in the presence of arginine (inhibition of assimilation "non-sensitive" strains) "Sensitive" strain) was acquired. The pro1Δcar2Δ strains cultured in proline medium (SD-N + Pro) was prepared so that the number 10 ⁷ bacteria were inoculated on the arginine supplemented media (SD-N + Pro + Arg ). Incubated at 30 ° C. for 5 days to isolate grown colonies. The isolated colonies were spotted on arginine-added medium to confirm phenotypic reproducibility. For the strains whose reproduction was confirmed, the genome was extracted using Gen Toru-kun (trademark) (for yeast) High Recovery (manufactured by Takara Bio Inc.). The whole genome sequence analysis of the obtained sample was outsourced to Biojet. The analysis results were compared with the genomic information of the X2180-1A strain on the database to identify gene mutations with amino acid substitutions.

As a result, it was found that in the obtained mutant (CAN1 mutant), the glycine at position 434 of the Can1 protein was replaced with cysteine.

4. Growth test in solid medium (spot test) 2
Growth tests of proline-requiring strains and CAN1 mutants were carried out by the same method as in the spot test in 2 above, except that a proline medium was used instead of the ammonium ion-added medium. The results are shown in FIG.

As shown in FIG. 2, the growth of the proline-requiring strain was not confirmed in the arginine-added medium, whereas the CAN1 mutant was found to be able to grow in the arginine-added medium.

5. Creation of CAN1 disrupted strain (triple-deficient strain in which the CAN1 gene of pro1Δcar2Δ strain was disrupted) Saccharomyces cerevisiae BY4741 strain can1:: kanMX4 (a strain in which the ORF of CAN1 in the genome is replaced with KanMX4: canamycin resistance gene) is used as a template. As a result, PCR was performed using the primers of SEQ ID NOs: 7 and 8 to obtain a PCR product. Regarding the obtained PCR product, a CAN1 disrupted strain (pro1Δcar2Δcan1Δ strain) was obtained by the same method as in 1 above except that the pro1Δcar2Δ strain was used instead of the Saccharomyces cerevisiae X2180-1A strain.

6. Measurement of Proline Amount and Arginine Amount The control (proline-requiring strain, pro1Δcar2Δ strain) cultured in proline medium (SD-N + Pro) and the CAN1 disrupted strain were arginine-added medium (SD) so that _{OD 600 = 1.5, respectively.} -N + Pro + Arg) was inoculated and cultured with shaking at 30 ° C. The culture supernatant was collected over time and used as a sample for amino acid measurement. The measurement sample was prepared by diluting with lithium acetate buffer (pH 2.0) and then filtering with a filter. After that, the amino acid concentration was measured using an amino acid analyzer (JLC-500 / V2 manufactured by JEOL Ltd.), and the consumption by cells was analyzed by comparing with the sample before culturing. A standard amino acid mixture (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the standard with a known concentration for amino acid quantification. The test was carried out at n = 2, and the average value is shown in FIG.

As shown in FIG. 3, no change was observed in the amount of proline in the culture supernatant in which the proline-requiring strain was cultured. On the other hand, since the amount of proline in the culture supernatant obtained by culturing the CAN1-disrupted strain decreased with time, it was found that the CAN1-disrupted strain can take up the proline contained in the medium (FIG. 3A). It was confirmed that the amount of arginine in the culture supernatant of both the proline-requiring strain and the CAN1-disrupted strain decreased with time (FIG. 3B).

7. Preparation of wild-type CAN1 or mutant CAN1 introduced strain PCR products were obtained by performing PCR using the primers of SEQ ID NOs: 9 and 10 using the genome of the wild-type CAN1 or CAN1 mutant as a template. The yeast expression plasmid pAUR123 (manufactured by Takara Bio Inc.) is linearized with restriction enzymes XbaI and SacI, mixed with the PCR product, and ligated by the infusion method (Takara Bio Inc.) to obtain wild-type CAN1 or mutant CAN1. A plasmid for (Can1 G434C) expression was constructed. CAN1 by the same method as in 1 above, except that the constructed plasmid and empty vector were used instead of the PCR product, and the CAN1 disrupted strain (pro1Δcar2Δcan1Δ strain) was used instead of the Saccharomyces cerevisiae X2180-1A strain. A strain expressing wild-type CAN1 in the disrupted strain (wild strain), a strain expressing mutant CAN1 (Can1 G434C) in the CAN1 disrupted strain (CAN1 mutant strain), and a strain in which an empty vector was introduced into the CAN1 disrupted strain (strain). CAN1 disrupted strain) was acquired. The obtained strain was seeded in a proline medium and an arginine-added medium, and the growth was examined. The results are shown in FIG.

On the arginine-free proline medium, each strain showed equivalent growth. On the other hand, in the arginine-added medium, the growth of the wild-type strain (proline-requiring strain) was completely suppressed, whereas the growth of the CAN1 disrupted strain and the CAN1 mutant strain was not suppressed at all. From these results, it was found that the CAN1 mutant strain having the Can1 G434C mutation was released from the suppression of proline assimilation as in the CAN1 disrupted strain.

Claims (8)

A composition for producing alcoholic beverages, which contains CAN1 mutant yeast or CAN1-suppressed yeast. The CAN1 mutant yeast encodes an amino acid sequence different from the amino acid sequence constituting the wild-type Can1 protein, in which at least one base is deleted, substituted, or added in the base sequence encoding the wild-type Can1 protein. The composition for producing an alcoholic food or drink according to claim 1, which comprises a nucleic acid. The composition for producing an alcoholic beverage according to claim 1 or 2, wherein the CAN1 mutant yeast or the CAN1 inhibitory yeast is a yeast having an improved proline uptake ability in the presence of arginine. The composition for producing alcoholic beverages according to any one of claims 1 to 3, wherein the yeast is a yeast belonging to the genus Saccharomyces. A method for producing an alcoholic beverage, which comprises using a CAN1 mutant yeast or a CAN1 inhibitory yeast. The CAN1 mutant yeast encodes an amino acid sequence different from the amino acid sequence constituting the wild-type Can1 protein, in which at least one base is deleted, substituted, or added in the base sequence encoding the wild-type Can1 protein. The method for producing an alcoholic food or drink according to claim 5, which has a nucleic acid. The method for producing an alcoholic beverage according to claim 5 or 6, wherein the CAN1 mutant yeast or the CAN1 inhibitory yeast is a yeast having an improved proline uptake ability in the presence of arginine. The method for producing an alcoholic beverage according to any one of claims 5 to 7, wherein the yeast is a yeast belonging to the genus Saccharomyces.

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