JP6284139B2 - Screening method for yeast having mating ability - Google Patents

Description

  The present invention relates to a method for breeding yeast strains for carrying out hybridization of yeast.

  Yeast has been closely related to human life since ancient times. In addition to the production of alcoholic beverages such as sake, wine and beer, and fermented foods such as bread, miso, and soy sauce, biotechnology has led to the development of pharmaceuticals in recent years. (Hereafter, “yeast that can be used in industrially useful applications such as the manufacture of products such as beverages, foods, and pharmaceuticals” will also be indicated as “practical yeast”).

  For effective use of yeast, it is essential to breed excellent yeast. So far, various breeding methods have been used to improve practical yeast strains. In particular, the cross breeding method has been widely used since ancient times. The cross breeding method is a method of artificially crossing two different strains (parent strains) and selecting a hybrid strain having a desired trait, which is effective for obtaining a strain having both superior traits of the parent strain. Is the method. Since mating is used for yeast crossing, the two strains used as the parental strains of the crossing have the property of mating with each other (hereinafter also referred to as “joining ability”), that is, each having a mating type of type a and α is necessary.

  In yeast, there are haploids having a basic number of chromosomes, diploids having twice the basic number of chromosomes, and N-ploids having N times the basic number of chromosomes (N represents a natural number). There are various cells with different ploidy. Here, the case where the locus for determining the mating type of yeast (hereinafter also referred to as “MAT locus”) is composed of only a, such as a / a / a / a, is a type, α / α / If we define only α as α and α and α / α as α / α / α / α, we will define any polyploidy The yeast mating type can also be classified into any of the above-mentioned a type, α type, or a / α type. Hereinafter, “a-type”, “α-type”, and “a / α-type” shall be expressed according to the above definition, unless the ploidy property is clearly indicated. In addition, the notation clearly indicating the a-type and α-type of the haploid is a-type (haploid) and α-type (haploid). When the a-type and α-type of a higher-order polyploid are specified, they are described in the same format.

  Most practical yeasts are higher-order polyploids such as diploids or tetraploids, and usually have a / α-type mating types that do not have mating ability. Thus, by forming spores from such yeast through meiosis, a-type or α-type meiotic isolates having mating ability are obtained. However, since there are many strains having a very low spore-forming ability among practical yeasts, a great deal of labor is required to produce a-type or α-type yeasts.

In recent years, yeast that does not have conjugation ability can be converted from yeast mating type to a type (diploid) or α type (diploid) yeast by expressing the mating type conversion gene (HO) in yeast cells. A technique (Patent Document 1) for imparting a bonding ability to a material has been developed. However, according to this method, since a-type (diploid) and α-type (diploid) yeast are produced at the same time, it is inevitable that self-doubling occurs due to the joining of both during the growth process. As a result, the frequency of appearance of hybrids between target yeasts having a desired mating type is reduced.
The present inventors recently focused on the yeast mating regulation mechanism, and in the a-type (diploid) cells transformed by the mating-type conversion gene (HO) expression, the α2 gene and the α-type (double-fold) are used. Suppressing self-doubling of a-type (diploid) yeast and α-type (diploid) yeast by forcibly expressing a1 gene in cells The yeast breeding method technology based on this is established and a patent application has been filed (Non-patent Document 1, Japanese Patent Application No. 2013-014863). With these technologies, even if the yeast has no sporulation, the yeast of type a (diploid) or α type (diploid) that inherited all the genetic backgrounds except the mating type locus of the parent strain can It can be provided stably without doubling, and the desired hybrid can be obtained by applying the hybridization method. However, the conjugating ability imparting step by HO gene expression is indispensable also by the inventors' self-doubling suppression method. In order for HO to act, a specific sequence is required at the mating type locus of the target yeast (hereinafter also referred to as “MAT locus”), and even when there is a mutation of only one base, HO acts. It is known that it cannot be performed (Non-Patent Document 2). That is, such a technique can be applied only to a certain limited range of yeast.

On the other hand, a technique (Non-patent Document 3) for producing yeast having a-type or α-type conjugation ability by utilizing loss of heterozygosity (hereinafter also referred to as “LOH”) is also developed. Has been. LOH is a state in which a specific locus is composed of different alleles (or sequences) by chromosomal rearrangement in diploid or higher polyploid cells (hereinafter also referred to as “heterozygous”) To a state having the same gene sequence (hereinafter also referred to as “homozygous type”). LOH is generally known to occur only at a low frequency of 1/10 ⁴ or less (Non-Patent Document 4). However, in the method of Non-Patent Document 3, by applying UV irradiation to yeast, LOH This indicates that it is possible to increase the frequency to about 1/10.
That is, if this technique is used, it is possible to generate high-frequency LOH at the MAT locus of yeast, and it is possible to easily produce a-type or α-type yeast from a / α-type yeast. Become.

  However, as pointed out in Non-Patent Document 5, UV irradiation used to increase the frequency of LOH has the risk of inducing undesired mutations at other loci at the same time. That is, despite the fact that a yeast having an excellent trait is a parent strain, such a trait is not always inherited by the produced a-type or α-type yeast. Therefore, it is essential to select a yeast strain having a desired trait from the produced hybrid population.

  A technique for homogenizing a specific locus in a chromosome without using UV irradiation (Patent Document 2) has also been developed. In this technique, homologous recombination is used to introduce a selectable marker gene into one of the loci of yeast homologous chromosomes, and then select the yeast from which the selectable marker gene has been dropped by LOH, thereby homogenizing the locus. Is achieved. If this technique is applied to the production of yeast having a-type (diploid) or α-type (diploid) mating ability, it is necessary to introduce a selection marker gene into the MAT locus that determines the mating type of yeast. is there.

  However, on the yeast chromosome, there are silent gene loci called HML and HMR loci that can also be called pseudogenes of the MAT locus, and these HML and HMR loci are the same upstream and over several hundred base pairs. It has a downstream sequence. Therefore, even if a selectable marker gene is introduced with the MAT locus as a target, there is a possibility that it will be introduced not at the MAT locus but at the HML locus and / or the HMR locus. Therefore, the efficiency of introduction into the MAT locus is expected to be significantly lower than when targeting other gene loci, and after the selection process, a complicated confirmation process of whether or not it has been correctly introduced into the MAT locus is also essential. become.

  As described above, tremendous efforts are still required to obtain a combination of a-type and α-type yeast inheriting each trait from different practical yeasts having a desired trait.

JP 2010-220481 A JP 2009-178104 A

Nobuo Fukuda, et. Al., 2013. ACS Synth Biol, in press, DOI: 10.1021 / sb400016j Bryan L. Ray, et.al., 1991, Mol Cell Biol., Vol. 11, 5372-5380. Shinji Hashimoto, et.al., 2006, Appl Microbiol Biotechnol., Vol. 69, 689-696. Mina Hiraoka, et.al., 2000, Genetics.Vol.156, 1531-1548. Ryosuke Yamada, et. Al., 2010, Appl Microbiol Biotechnol, Vol. 88, 849-857.

  An object of the present invention is to provide a method for efficiently and reliably producing an a-type or α-type yeast from a / α-type yeast having no mating type without using a mutagen.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have introduced a yeast cell growth control technology that depends on the mating type, so that a a naturally derived from a / α-type yeast by LOH. It was found that type or α-type yeast can be selectively obtained. After a selection marker gene corresponding to a desired selection pressure is inserted into a yeast a-type cell-specific or α-type cell-specific gene expression vector, the vector is introduced into yeast having an excellent trait in advance. Thus, the a-type or α-type yeast produced by low-frequency LOH was successfully selected, and the present invention was completed.

That is, the present invention includes the following.
[1] A method for producing yeast having mating ability from yeast that does not have a mating type, which is transformed with an expression vector in which a selection marker gene is inserted downstream of a promoter, and corresponds to the selection marker gene A method comprising a step of selecting a-type or α-type yeast under a selective pressure.
[2] The method according to [1], wherein the yeast is brewery yeast or baker's yeast.
[3] The method according to [1] or [2] above, wherein the selection marker gene is a URA3 gene derived from Saccharomyces cerevisiae genus yeast.
[4] The method according to any one of [1] to [3], wherein the expression vector is a YCp-type plasmid.
[5] The method according to any one of [1] to [4] above, wherein when the a-type yeast is a selection target, the promoter is a promoter of a gene that is specifically expressed by a-type.
[6] The method according to [5], wherein the promoter is P _STE2 .
[7] The method according to any one of [1] to [4], wherein when the α-type yeast is a selection target, the promoter is a promoter of a gene that is specifically expressed by α-type.
[8] The method according to [7], wherein the promoter is P _STE3 .
[9] Expression of α2 gene or a1 gene in a-type or α-type yeast cells using the same or different expression vector containing the selection marker gene to form an a1-α2 complex. The method according to any one of [1] to [8], wherein a step of suppressing self-doubling is provided.
[10] The method according to [9] above, wherein the promoter under control of the selection marker gene is a promoter of a gene that is expressed specifically in a haploid cell of yeast.
[11] The method according to [10] above, wherein the promoter is _PHO .
[12] A method for producing a hybrid strain of yeast, wherein at least one parent strain of the yeast having a conjugating ability produced by the method described in any one of [1] to [11].
[13] A yeast produced by the method according to any one of [1] to [12].

  According to the present invention, an a-type or α-type yeast can be produced simply, efficiently and reliably from an a / α-type yeast having excellent traits, avoiding the risk of unwanted mutations. . If cross breeding is performed using the produced yeast, it is considered that it is possible to efficiently breed a cross strain having both superior traits of the parent strain. That is, the production method of the present invention makes it possible to remarkably increase the efficiency of cross breeding of practical yeasts that could not be cross-bred or difficult by the conventional methods.

Schematic diagram of screening method for yeast having mating ability (A) Schematic diagram of method of screening a-type yeast (B) Schematic diagram of method of screening α-type yeast Behavior of yeast when using YEp type (multicopy type) vector with marker gene URA3 inserted (A) Genetic map of vector pHY-2U using a type specific promoter P _STE2 (B) Introduction of pHY-2U (C) Gene map of vector _pLY -3U using α-type specific promoter P _STE3 (D) Growth of yeast cells of each mating type introduced with pLY-3U Graph showing characteristics Yeast behavior when using YCp type (single copy type) vector inserted with marker gene URA3 (A) Gene map of vector pLS-2U using a type specific promoter P _STE2 (B) Introducing pLS-2U (C) Gene map of vector pHS-3U using α-type specific promoter P _STE3 (D) Growth of each conjugated yeast cell introduced with pHS-3U Graph showing characteristics Two α type specific URA3 gene expression technique share a vector, and URA3 gene expression vector pHS-HOU using yeast behavior (A) P _HO when using such techniques, and using the P _STE2 Gene map of α2 gene expression vector pL3G-2α (B) Graph showing the growth characteristics of each mating type yeast cell into which pHS-HoU and pL3G-2α have been introduced Results of screening of a-type yeast and α-type yeast (A) In the screening of a-type (diploid) yeast, the growth of colonies composed of a-type (diploid) yeast from the Ura-deficient agar plate is shown. Photo (Applying 1 ml of yeast suspension adjusted to OD ₆₀₀ = 1) (B) Graph showing fluorescence intensity emitted by yeast isolated as a-type (diploid) yeast (C) α-type (diploid) In yeast screening, a photograph showing the growth of colonies composed of α-type (diploid) yeast on a Ura-deficient agar plate (applied with 1 ml of yeast suspension adjusted to OD ₆₀₀ = 0.1) (D) α-type (Diploid) Graph showing fluorescence intensity emitted by yeast isolated as yeast (E) Photograph showing growth of hybrid yeast produced by conjugation with parent strains having different auxotrophy

1. Features of the present invention The present invention relates to a method for producing yeast having a-type or α-type conjugation ability from a / α-type yeast having no mating type. The production method of the present invention includes a step of selecting a type or α type yeast by introducing a selection marker gene linked to a promoter having expression characteristics depending on the mating type into a yeast cell using a vector. As long as this step is included, it is not excluded to include all technically applicable steps among those known to those skilled in the art. Further, the genotype or phenotype of the parent strain and the yeast strain obtained by the production method of the present invention is preferably invariant except for the portion related to the mating type, but the gene other than the portion related to the mating type. Does not exclude changes in type or phenotype.
In the production method of the present invention, the “step of selecting a-type or α-type yeast” includes a selection marker gene involved in yeast growth, a promoter controlled by the yeast mating type, or a haploid-specific expression characteristic. It is inserted downstream of a promoter having There is no particular limitation as long as it is a step for growing only a desired mating type yeast using the presence or absence of expression of a selection marker gene introduced using a vector in the presence of a specific selection pressure.
Examples of the selection marker gene include a blasticidin resistance gene (BSR gene), a G418 resistance gene (kanMX4 gene), a histidine synthesis gene (HIS3 gene), a leucine synthesis gene (LEU2 gene), a uracil synthesis gene ( URA3 gene) can be preferably exemplified. In the embodiment of the present invention, the URA3 gene derived from Saccharomyces cerevisiae yeast is used for illustration, but the present invention is not limited thereto.
A preferred example of the URA3 gene derived from Saccharomyces cerevisiae yeast is the URA3 gene having the base sequence shown in SEQ ID NO: 1. It should be noted that one or several bases are deleted, substituted, inserted or added in the sequence exemplified above as long as it is a gene capable of conferring uracil synthesis ability to such yeast by being expressed in yeast cells. It is also possible to use a variant having a different sequence or a variant. In the present invention, “one or several” means 1 to 50, preferably 1 to 20, more preferably 1 to 10, and 80% or more, preferably 85% or more of the full-length base sequence. More preferably, it indicates that there is a mutation within the range having 90% or more identity, and further preferably 95% or more, or that it has been modified.
Accordingly, a preferred URA3 gene in the present invention is a gene that can confer uracil synthesis ability to such yeast by, for example, expression in yeast cells, and from any of the following (1) to (2) It can be expressed as a gene having a selected base sequence.
(1) the base sequence represented by SEQ ID NO: 1,
(2) a base sequence in which one or several base residues of the base sequence shown in SEQ ID NO: 1 are deleted, substituted, inserted or added;

2. Regarding “yeast” to which the present invention can be applied As the above-mentioned “yeast”, yeast (practical yeast) that can be used for industrially useful applications such as production of products such as beverages, foods, and pharmaceuticals, specifically, Saccharomyces (Saccharomyces), Pichia (Pichia), yeast belonging to the genus Kluyveromyces (Kluyveromyces) can be preferably exemplified, yeast that can be used in the production of beverages and foods can be more preferably used, Yeasts that can be used for production and belong to the genus Saccharomyces can be particularly preferably used. Preferred examples of the “yeast that can be used in the production of beverages and foods” include brewing yeast such as bottom fermentation yeast, top fermentation yeast, wine yeast, sake yeast, shochu yeast, whiskey yeast, and bread yeast. be able to.

3. 3. Method for producing yeast having conjugation ability in the present invention (3-1) Outline of method When a / α-type yeast is cultured and grown repeatedly, most cells are a / α-type in the same manner as mother cells. However, by generating LOH at the MAT locus, a-type and α-type yeasts appear slightly. However, in addition to the low frequency of LOH, the a-type and α-type that appeared appeared to form a / α-type yeast again, so the proportion of a-type or α-type yeast in the cultured cell group is: It will remain at a very low level.
Therefore, in the present invention, when only the a-type yeast is to be selected from the a / α-type yeast, a selection marker gene (for example, URA3 gene) is connected under the control of the a-type specific promoter, and conversely the α-type When only yeast is to be selected, a vector in which a selection marker gene is linked under the control of an α-type specific promoter is introduced into the yeast, and the yeast is cultured for at least one night, followed by selection medium (for example, uracil-deficient medium). ), It is possible to obtain yeast colonies having the desired mating type (a type or α type). At that time, if a vector in which a marker gene (for example, GFP gene) is linked to an a-type or α-type specific promoter is also inserted at the same time, positive or false positive can be discriminated using the label intensity as an index.

(3-2) Promoter used in the present invention Although it is not particularly limited as long as it is a promoter capable of expressing a selectable marker gene (for example, URA3 gene) in yeast cells having a desired mating type, In the case of expression of a promoter (hereinafter also referred to as “a-type specific promoter”), the expression of α-type is specific. A promoter having expression characteristics (hereinafter also referred to as “α-type specific promoter”) is used.
_Preferred examples of the “a-type specific promoter” include P _STE2 (see Non-patent Document 1) and P _STE6 (see Patent Document 1), and the “α-type specific promoter” includes P _STE3 ( Non-patent document 1), _PMFA1 (see reference 1), _PMFA2 (see reference 1) and the like can be preferably exemplified.
Then, these vectors are introduced into yeast cells by a conventional method using a yeast vector (preferably a plasmid capable of autonomous replication in yeast) in which these promoters are linked upstream of the selection marker gene.

Furthermore, in the present invention, when an a1-α2 complex is artificially formed using the following “self-ploidy suppression technology (Non-patent Document 5)”, it has haploid-specific expression characteristics. A promoter (hereinafter also referred to as “haploid-specific promoter”) can be used. The "haploid-specific promoters", can be preferably exemplified, and more P _STE18 (see Resources 2) or P _HO (see reference 3).
In the case of expressing a selectable marker gene (for example, URA3 gene) in a-type yeast cells in which self-doubling is suppressed, the a-type specific promoter “P _STE2 (SEQ ID NO: 2)” is particularly preferred, When expressed in α-type yeast cells, the haploid-specific promoter “P _HO (SEQ ID NO: 3)” is particularly preferred.
In the case where a selectable marker gene (for example, URA3 gene) is expressed in an a-type yeast cell in which self-folding is not suppressed, the a-type specific promoter “P _STE2 ” is particularly preferable. In particular, the haploid-specific promoter “P _STE3 (SEQ ID NO: 4)” is particularly preferable.

As long as the selectable marker gene can be expressed, a modified promoter having a base sequence in which one or several bases are deleted, substituted, inserted or added in the promoter sequence exemplified above as preferred can also be used.
Therefore, a preferred promoter in the present invention can be expressed as having a base sequence selected from any of the following (1) to (4), for example.
(1) the base sequence shown in SEQ ID NO: 2,
(2) A base sequence in which one or several amino acid residues of the base sequence shown in SEQ ID NO: 2 have been deleted, substituted, inserted or added.
(3) the base sequence shown in SEQ ID NO: 3,
(4) A base sequence in which one or several amino acid residues of the base sequence shown in SEQ ID NO: 3 have been deleted, substituted, inserted or added.
(5) the base sequence shown in SEQ ID NO: 4,
(6) A base sequence in which one or several amino acid residues of the base sequence shown in SEQ ID NO: 4 have been deleted, substituted, inserted or added.

(3-3) Method for selecting a-type or α-type yeast As described above, a transformant obtained by introducing a vector for expressing a selectable marker gene (for example, URA gene) into yeast having a desired mating type And a selective pressure (for example, it is applied on an agar medium lacking uracil), a-type or α-type yeast can be isolated. In the present invention, whether or not an isolated yeast cell has a desired mating type can be easily confirmed by observing a label (for example, observing the presence or absence of GFP fluorescence).
Here, preferable examples of the marker gene include a fluorescent marker gene, a chromogenic marker gene, and a luminescent marker gene. Examples of fluorescently labeled genes include green fluorescent protein (GFP), red fluorescent protein (DsRed), and cyan fluorescent protein (CFP). Examples of the chromogenic marker gene include β-galactosidase (lacZ). Examples of the luminescent marker gene include luciferase (luc). In the embodiment of the present invention, the GFP gene (SEQ ID NO: 5) is described for illustration, but the present invention is not limited to this.
The marker gene is a vector that is incorporated under the control of an a-type specific promoter when isolating a-type yeast, and under the control of an α-type specific promoter when isolating α-type yeast. In addition, when self-ploidy is suppressed, a “haploid-specific promoter” can be used.
In this case, the vector may be incorporated into the same vector (preferably a plasmid capable of autonomous replication in yeast) as the vector for introducing the selection marker gene of the present invention.

(3-4) “Self-doubling suppression technology” In the present invention, in producing yeast having mating ability, a method of expressing a “gene that controls mating” in yeast cells, specifically, the present inventors. It is preferable to use together the “self-doubling suppression technology (Non-patent Document 1)” previously developed.
The a type yeast originally expresses the a1 gene, the α type yeast expresses the α2 gene, and the a / α type yeast expresses the a1 gene and the α2 gene. a1 and α2 co-expressed in a / α-type yeast cells form an a1-α2 complex and function as a repressor of a haploid-specific promoter (Reference 1). The self-doubling suppression technology developed by the present inventors (Non-patent Document 1) is a technology for artificially expressing an α2 gene and an a1 gene in a type yeast and α type yeast, respectively, to form an a1-α2 complex. It is. A gene group (hereinafter also referred to as “hsg”) that induces conjugation of yeast that is present downstream of a haploid-specific promoter and specifically expressed in a-type or α-type haploid cells, Since the expression is suppressed by the a1-α2 complex, it is possible to suppress the joining ability of the yeast regardless of the a-type yeast or the α-type yeast.

Here, as a typical a1 gene, the a1 gene encoding the amino acid sequence shown in SEQ ID NO: 6 (GenBank accession number: CAA24622) derived from Saccharomyces cerevisiae yeast is used, and the α2 gene is derived from Saccharomyces cerevisiae yeast. The α2 gene encoding the amino acid sequence shown in SEQ ID NO: 7 (GenBank accession number: DAA07518) can be shown. It should be noted that one or several amino acid residues are deleted, substituted, inserted or added in the sequences exemplified above as long as the mating ability of the paired yeast generated upon yeast mating type conversion can be deleted. Mutants having modified sequences or modifications can also be used. In the present invention, “one or several” means 1 to 50, preferably 1 to 20, more preferably 1 to 10, and 80% or more, preferably 85% or more of the full-length amino acid sequence. More preferably, it indicates that there is a mutation within the range having 90% or more identity, and further preferably 95% or more, or that it has been modified.
Therefore, a preferred gene that controls conjugation in the present invention is a gene that can lack the mating ability of a counter-yeast produced during such yeast mating type conversion by, for example, expression in yeast cells. And a gene encoding an amino acid sequence selected from any of the following (1) to (4).
(1) the amino acid sequence represented by SEQ ID NO: 6,
(2) an amino acid sequence in which one or several amino acid residues of the amino acid sequence shown in SEQ ID NO: 6 are deleted, substituted, inserted or added;
(3) the amino acid sequence shown in SEQ ID NO: 7,
(4) An amino acid sequence in which one or several amino acid residues of the amino acid sequence shown in SEQ ID NO: 7 are deleted, substituted, inserted or added.
Other "genes that control conjugation" include GPA1 (GenBank accession number: NP_011868), SST2 (GenBank accession number: NP_013557), DIG1 (GenBank accession number: NP_015276), etc. as α2 gene, a1 gene, etc. It may be used instead.

(3-5) Mechanism of appearance of a-type or α-type yeast from a / α-type yeast In the present invention, a-type or α-type yeast appears in the process of cell division of a / α-type yeast. The main mechanism includes the above-mentioned LOH, but is not limited thereto. For example, “chromosome loss” caused by uneven distribution of chromosomes during division of mother cells and daughter cells is also included as a mechanism for appearance of a-type or α-type yeast from a / α-type yeast. In this case, the appearing a-type or α-type yeast may have a ploidy different from the original a / α-type yeast. For example, a-type (haploid) or α-type (haploid) yeast may appear from a / α-type (diploid) yeast.
As described above, it is preferable that the genotype or phenotype (other than the portion related to the mating type) of the a / α-type yeast (parent strain) and the produced yeast strain is unchanged, so that the produced a-type or α The type yeast preferably has the same ploidy as the parent strain, but does not exclude having a different ploidy.

(3-6) Embodiment of the Present Invention Shown in FIG. 1 A specific embodiment of the present invention will be described with reference to FIG. (In FIG. 1, the "P _STE2" as a type-specific promoter, the "P _STE3" as α-type specific promoter, using a "P _HO" as haploid-specific promoters, as a selective marker gene URA3 This is a case where a gene is selected and labeled with a GFP gene, and the case where the “self-doubling suppression technology” of Non-Patent Document 1 is used is exemplified, but the present invention is not limited to this embodiment.

In the screening for a-type yeast in FIG. 1A, colonies of a-type yeast are isolated on an agar medium lacking uracil by expressing URA3 gene and GFP gene under the control of a-type specific promoter (P _STE2 ). It becomes possible. Moreover, since the isolated colony expresses GFP by the action of the a-type specific promoter, the high fluorescence intensity indicates that it is a positive cell group. On the other hand, in the α-type yeast screening shown in FIG. 1B, by expressing the URA3 and GFP genes under the control of the α-type specific promoter (P _STE3 ), the α-type yeast colonies were isolated on the agar medium lacking uracil. The yeast mating type can be confirmed using GFP fluorescence intensity as an index.

4). Transformation of Yeast (4-1) Method for Introducing Yeast Introducing the above construct into yeast involves incorporating the construct into a vector and introducing the vector into yeast. As a method for introducing the vector into yeast, all methods known to those skilled in the art, such as electroporation, metal treatment, and protoplast method, can be used.
In this case, a plasmid to be incorporated into genomic DNA is not suitable as a vector to be used, and a plasmid capable of autonomous replication in yeast cells is used.
Examples of the “plasmid capable of autonomous replication in yeast cells” used in the present invention include YCp type, YEp type, and YRp type plasmids.
YEp type and YRp type plasmids are multi-copy type plasmids and are capable of high gene expression. Therefore, expression of a1 or α2 gene for suppression of self-doubling, and GFP gene for evaluating mating type It is preferably used for the expression of.
On the other hand, the YCp type, which is a single copy plasmid, is limited in gene expression level, but at the same time it can reduce leaky gene expression, so it is particularly preferable to use it for the expression of a selectable marker gene (for example, URA3). . That is, by using the YCp type plasmid for the expression of the selection marker gene, the background when screening yeast having mating ability can be greatly reduced.

(4-2) Method for removing unnecessary plasmids The production method of the present invention is “after selecting yeast having a desired mating type” or “after allowing isolated yeast to join with other yeast”. In addition, an unnecessary plasmid, ie, “a plasmid incorporating a construct for expressing the URA3 gene”, “a plasmid incorporating a construct for expressing the GFP gene”, or “a1 gene or It is preferable to further include a step of dropping “a plasmid incorporating a construct for expressing the α2 gene”, and it is more preferable to further include a step of dropping all of these constructs. Thus, since the strain from which the plasmid has been dropped does not leave any trace of foreign DNA (the above-mentioned plasmid) in the chromosomal DNA of the yeast, such a strain is a recombinant as defined in the current legal regulations as described above. It is interpreted as not applicable to living things. Therefore, such yeast before joining can be suitably used as a mother of cross breeding to obtain a hybrid strain that can be used without limitation for beverages and foods. However, it is preferable in that it can be used without limitation.

  The method for dropping the plasmid from the yeast is not particularly limited, but after culturing the yeast under conditions that do not apply selective pressure in the direction of retaining the plasmid, the plasmid no longer has the selection marker gene contained therein. By selecting cells, a strain from which the plasmid has been removed can be easily obtained.

5. Method for producing the hybrid strain of the present invention As a method for producing the hybrid strain of the present invention, a yeast having a desired mating type produced by the production method of the present invention (hereinafter also referred to as “yeast according to the present invention”). Is crossed as at least one parent strain. Such a method for hybridizing yeast is not particularly limited, and a known method such as mixing and culturing both parent strains can be used. In addition, if a separate selection marker gene (preferably a drug resistance gene) is introduced into each of both parental strains, a hybrid strain can be easily isolated by selecting a strain having both of these selection marker genes. It becomes possible to do. Examples of the marker gene include a blasticidin resistance gene (BSR gene), a G418 resistance gene (kanMX4 gene), a histidine synthesis gene (HIS3 gene), a leucine synthesis gene (LEU2 gene), and a uracil synthesis gene (URA3). Gene) can be preferably exemplified. In the method for producing the above-mentioned hybrid strain of the present invention, the yeasts according to the present invention may be crossed as parent strains, and the non-yeast parent strain according to the present invention originally has a-type or α-type mating ability. You may use the stock which you have. In the examples described later, tetraploid yeast was prepared by introducing separate marker genes into the same parent strain and then joining the created a-type (diploid) and α-type (diploid) yeast. Any form that is technically applicable among forms known to those skilled in the art is not excluded.

  When using the method for producing the above-mentioned hybrid strain of the present invention, a yeast (for example, a practical yeast) that could not be practically cross-breeded by the conventional method or has a superior trait of the parent strain. It becomes possible to breed a hybrid strain (hereinafter also referred to as “cross hybrid according to the present invention”) extremely efficiently. Examples of the hybrid strain according to the present invention include, for example, a hybrid strain having two or more excellent traits as brewing yeast and a hybrid strain having two or more excellent traits as baker's yeast. Preferred examples of characteristics that are excellent as a brewing yeast include high fermentability, low hydrogen sulfide productivity, high aggregability, high sulfite productivity, and high alcohol tolerance.

(References)
Reference 1: Botstein D, et. Al., 2004, Genetics, Vol.166, 653-660.
Reference 2: Kentaro Furukawa, et. Al., 2011, PLoS One, Vol.6, e26584.
Reference 3: Brenda J. Andrews, et. Al., 1989, Cell. Vol. 57, 21-29.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent publication, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

(experimental method)
(1) Preparation of medium YPD medium containing 1% (w / v) yeast extract, 2% (w / v) peptone and 2% (w / v) glucose, 0.67% (w / v) amino acid-free Yeast was cultured in an SD medium containing yeast-containing nitrogen base (Becton Dickinson) and 2% (w / v) glucose. An amino acid and a nucleic acid corresponding to the selection marker gene were appropriately added to the SD medium. YPD and SD solid media were prepared by adding 2% (w / v) agar to these media.

(2) Preparation of yeast Saccharomyces cerevisiae methionine-requiring a-type (haploid) strain BY4741 (see reference 4), lysine-requiring α-type (haploid) strain BY4742 (reference 4) Table 1 shows information on yeast strains prepared in the following examples and plasmids introduced into the yeast strains.

(3) Fluorescence measurement As shown in FIG. 1, a GFP gene encoding a green fluorescent protein was used as a reporter gene for showing a yeast mating type. Yeast transformants were cultured in SD medium at 30 ° C. for 18 hours, and then collected, washed and resuspended in sterile water. The fluorescence intensity of 100 μL of the yeast suspension adjusted so that the bacterial cell concentration OD ₆₀₀ = 5.0 was measured using a microplate reader Infinite200 (manufactured by Tecan Japan). The excitation filter center wavelength was set to 485 nm (filter bandwidth 20 nm), the fluorescence filter center wavelength was set to 535 nm (filter bandwidth 25 nm), and the gain was fixed to 50 for measurement.

(4) Proliferation assay A- type, α-type, and a / α-type yeasts were prepared using uracil-containing medium (+ Ura) or uracil-deficient medium (-Ura) from the initial cell concentration OD ₆₀₀ = 0.03, respectively. And cultured at 30 ° C. The growth characteristics of each conjugated yeast were evaluated by measuring the OD ₆₀₀ of the yeast culture solution at each culture time.

(5) Conjugation assay In order to evaluate the conjugation ability of yeast, a proliferation assay using an agar medium prepared so that only the zygote can grow using the difference in auxotrophy of yeasts of type a and α. Carried out. The a-type and α-type yeast strains were seeded in 1 mL of YPD medium at an initial cell concentration of OD ₆₀₀ = 0.1, respectively, and co-cultured at 30 ° C. for 1.5 hours. The cells were collected and washed, and then resuspended in sterile distilled water. After preparing yeast suspensions with OD ₆₀₀ = 1.0, 0.1 and 0.001, spotting 10 μL each on an agar medium on which only zygotes can grow and culturing at 30 ° C. for 2 days. The presence or absence of colony formation was examined.

(Example 1) Expression example of URA3 gene using YEp-type plasmid (1-1) Construction of a-type cell-specific URA3 gene expression plasmid URA3 gene was primer 1 (SEQ ID NO: 8) and primer 2 (SEQ ID NO: 9) The plasmid pRS316 (provided by the Ministry of Education, Culture, Sports, Science and Technology NBRP “yeast”) was amplified as a template by PCR, inserted between NotI-BamHI sites of plasmid pHY-2GA (see Non-Patent Document 1), and plasmid pHY -2U.

(1-2) Construction of α-type cell-specific URA3 gene expression plasmid The URA3 gene was amplified by PCR using primer 3 (SEQ ID NO: 10) and primer 2 (SEQ ID NO: 9) using plasmid pRS316 as a template, and plasmid pLY It was inserted between NotI-BamHI sites of -3GC (see Non-Patent Document 1) to obtain plasmid pLY-3U.

(1-3) Proliferation assay of each conjugated yeast cell having YEp-type URA3 gene expression plasmid BY4741 is selected as a-type cells, BY4742 is selected as α-type cells, and BY4743 is selected as a / α-type cells, and pHY- A transformant in which 2U (FIG. 2A) or pLY-3U (FIG. 2C) was introduced by the lithium acetate method (see Reference 5) was created. The measurement results of OD ₆₀₀ after culturing each transformant for 18 hours are shown in FIGS. 2B and 2D, respectively. Error bars in the figure indicate standard deviations in the results of three independent experiments.

As a result, when the plasmid pHY-2U was introduced and cultured in SD-Ura medium, the a-type cells succeeded in expressing and propagating the URA3 gene, but the α-type and a / α-type cells were also clearly OD. It was confirmed that the value of ₆₀₀ increased (FIG. 2B). On the other hand, when plasmid pLY-3U was introduced and cultured in SD-Ura medium, it was confirmed that not only α-type cells but also α-type and a / α-type cells expressed and proliferated (FIG. 2D). ). As described above, it was shown that when the YEp-type URA3 expression plasmid was introduced into yeast cells, yeast cells having an undesired mating type also grew on the SD-Ura medium. Since the YEp-type plasmid is maintained at several tens of copies per cell, it is _surmised that the leakage of gene expression possessed by P _STE2 and P _STE3 was increased. Therefore, in order to avoid an increase in leaky gene expression, an attempt was made to use a YCp-type plasmid that maintains only one copy per cell, as shown below.

(Example 2) Example of expression of URA3 gene using YCp-type plasmid (2-1) Construction of a-type cell-specific URA3 gene expression plasmid A DNA fragment containing P _STE2 -URA3 was primer 4 (SEQ ID NO: 11) and primer 5 (SEQ ID NO: 12) was amplified by PCR using plasmid pHY-2U as a template, inserted between SacII-XhoI sites of plasmid pRS315 (provided by the Ministry of Education, Culture, Sports, Science and Technology NBRP "yeast"), and plasmid pLS- 2 U.

(2-2) Construction of α-type cell-specific URA3 gene expression plasmid A DNA fragment containing P _STE3 -URA3 was subjected to PCR using plasmid 4 (SEQ ID NO: 11) and primer 5 (SEQ ID NO: 12) to form plasmid pLY-3U. Was used as a template and inserted between the SacII-XhoI sites of plasmid pRS313 (provided by the Ministry of Education, Culture, Sports, Science and Technology NBRP “yeast”) to obtain plasmid pHS-3U.

(2-3) Growth assay of yeast cells of each mating type having YCp-type URA3 gene expression plasmid According to the lithium acetate method, pLS-2U (FIG. 3A) or pHS-3U (FIG. 3C) was obtained as BY4741, BY4742, or BY4743, respectively. The transformant introduced into was created. FIGS. 3B and 3D show the OD ₆₀₀ measurement results for each culture time when each transformant was cultured for up to 24 hours. Error bars in the figure indicate standard deviations in the results of three independent experiments.

As a result, when plasmid pLS-2U was introduced and cultured in SD-Ura medium, only a-type cells succeeded in expressing and propagating URA3 gene, and the OD of α-type and a / α-type cells. It was confirmed that the value of ₆₀₀ hardly changed until 24 hours (FIG. 3B). On the other hand, when plasmid pHS-3U is introduced and cultured in SD-Ura medium, α-type cells can proliferate by expressing the URA3 gene. It was confirmed that the proliferation of a / α type cells was suppressed (FIG. 3D). However, after 21 hours of culture, the OD ₆₀₀ values of α-type and a / α-type cells were shown to increase slightly. As described above, by introducing the YCp-type URA3 expression plasmid into yeast cells, the inventors succeeded in significantly reducing leaky gene expression.

However, although the leakage of P _STE3 is slightly remaining, it is expected that a false positive will occur in the screening of α-type yeast. The difference in leakage between P _STE2 and P _STE3 is presumed to be due to the difference in the expression control mode between the a-type specific promoter and the α-type specific promoter shown in Reference Document 1. P _STE2 is directly suppressed by repressor α2, but P _STE3 has no repressor, and downstream gene expression is regulated by the expression level of activator α1. The a1-α2 complex is known to function as an α1 repressor (see Reference 1). In FIG. 3D, it was shown that the a / α type yeast showed lower leakage of P _STE3 than the α type cells. This is because the a1-α2 complex in the a / α type cells expressed α1. It is presumed that the leakage of P _STE3 was indirectly reduced by suppressing the above. However, also in a / α-type cells, it is expected that it is difficult to completely inhibit the leakage of P _STE3 since the OD ₆₀₀ value tends to increase after 24 hours of culture. Therefore, as shown below, it attempted a technique used capable a1-[alpha] 2 complex acts as a direct repressor, a haploid-specific promoters P _HO as a substitute for P _STE3.

(2-4) by using the construction of the URA3 gene expression plasmid using P _HO P _HO Primer 6 (SEQ ID NO: 13) and primer 7 (SEQ ID NO: 14), the chromosomal DNA of BY4741 strain by PCR was amplified as a template Was inserted between the SacII-NotI sites of plasmid pHS-3U to obtain plasmid pHS-HoU.

(2-5) Proliferation assay of each mating type yeast cell having P _STE2 -α2 expression plasmid and P _HO -URA3 expression plasmid According to the lithium acetate method, pL3G-2α and pHS-HoU (FIG. 4A) were obtained as BY4741, A transformant introduced into BY4742 or BY4743 was created. FIG. 4B shows the OD ₆₀₀ measurement results for each culture time when each transformant was cultured for up to 24 hours. Error bars in the figure indicate standard deviations in the results of three independent experiments.

In a / α-type cells, the original a1-α2 complex suppresses the expression of the URA3 gene. In a-type cells, in addition to the original a1, the introduced α2 gene is expressed, and the a1-α2 complex is expressed. By forming, the expression of the URA3 gene is suppressed. On the other hand, the α-type cells by expressing the URA3 gene function is P _HO, is expected to be possible to grow in SD-Ura medium. As shown in FIG. 4B, among the three types of transformants, it was confirmed that only α-type cells were able to grow when cultured in SD-Ura medium. Accordingly, as shown in FIG. 1, in the screening of a type cells how to express the URA3 gene using P _STE2, in the screening of α type cell applying the method of expressing the URA3 gene using the P _HO It is effective.

(Example 3) Screening of a-type or α-type yeast Example (3-1) Screening of a-type yeast For BY4743 which is a / α-type yeast, pH2G-Pa1 and pLS-2U were obtained by the lithium acetate method. The introduced transformant was created. The obtained transformant was cultured in SD + Ura medium for 24 hours, washed with sterilized water, applied to SD-Ura agar medium, and cultured at 30 ° C. for 2 days to form colonies (FIG. 5A).

(3-2) Confirmation of junction type (a type)
FIG. 5B shows the results of fluorescence intensity measurement after culturing a colony (FIG. 5A) obtained in the screening for a-type yeast overnight in SD-URA medium. As shown in Table 1, the GFP gene encoded by pH2G-Pa1 is specifically expressed in a-type cells. As shown in FIG. 5B, it was confirmed that all the obtained colonies emitted green fluorescence. That is, a-type cells derived from L / OH from a / α-type BY4743 were successfully obtained selectively. Among these, a colony having the highest fluorescence intensity was selected and cultured overnight in a YPD medium, and then the yeast was collected, washed, and resuspended in distilled water. BY4743A strain selected from the colonies formed by applying the diluted yeast suspension on the YPD solid medium and culturing at 30 ° C. for 2 days, from which the plasmids pH2G-Pa1 and pLS-2U were dropped. (Table 1). Specifically, those that could not grow in either the medium not containing histidine or the medium not containing leucine were determined to be colonies from which both plasmids had dropped.

(3-3) Screening of α-type yeast A transformant in which pL3G-2α and pHS-HoU were introduced by a lithium acetate method into BY4743 which is an a / α-type yeast was created. The obtained transformant was cultured in SD + Ura medium for 24 hours, washed with sterilized water, applied to SD-Ura agar medium, and cultured at 30 ° C. for 2 days to form colonies (FIG. 5C).

(3-4) Confirmation of junction type (α type)
The colony obtained in the screening of α-type yeast (FIG. 5C) is cultured overnight in SD-URA medium, and then the fluorescence intensity is measured. The result is shown in FIG. 5D. As shown in Table 1, the GFP gene encoded by pL3G-2α is specifically expressed in a-type cells. As shown in FIG. 5D, it was confirmed that all the obtained colonies emitted green fluorescence. That is, α-type cells derived from L / OH from a / α-type BY4743 were successfully obtained selectively.
Among these, a colony having the highest fluorescence intensity was selected and cultured overnight in a YPD medium, and then the yeast was collected, washed, and resuspended in distilled water. By applying a diluted yeast suspension on a YPD solid medium and culturing at 30 ° C. for 2 days, a plasmid from which plasmids pL3G-2α and pHS-HoU have been removed is selected and strain BY4743AL (Table 1). Specifically, those that could not grow in either the medium not containing histidine or the medium not containing leucine were determined to be colonies from which both plasmids had dropped.

(3-5) Production of hybrids A transformant was created by introducing pH2G-Pa1 or pL3G-2α into the BY4743 by the lithium acetate method. Similarly, a transformant in which pH2G-Pa1 was introduced into BY4743A and a transformant in which pL3G-2α was introduced into BY4743AL were created. After culturing the obtained transformant in SD + Ura medium for 24 hours, a combination of two kinds of BY4743 transformants or a combination of BY4743A and BY4743AL transformants was mixed and cultured in YPD medium, and then contained histidine and leucine. First, a yeast suspension was applied to an SD solid medium for hybrid selection (SD-His, Leu plate) containing 20 mg / L uracil and 20 mg / L lysine, and cultured at 30 ° C. for 2 days. The observation results of colony growth on SD-His and Leu plates are shown in FIG. 5E. As a result, no hybrids were formed with BY4743 having no bonding ability, whereas BY4743A (a-type) and BY4743AL (α-type) produced using this technology were able to form hybrids. Was confirmed.

(References)
Reference 4: Brachmann CB, et. Al., 1998, Yeast. Vol 14: 115-132.
Reference 5: Gietz D, et. Al., 1992, Nucleic Acids Res. Vol20: 1425.

The present invention makes it possible to remarkably improve the efficiency of yeast cross breeding, which was difficult or difficult to perform practical cross breeding by conventional methods, such as production of products such as beverages and foods. It can be used very effectively in the field of hybrid breeding of yeast (practical yeast) that can be used for industrially useful applications.
[Sequence Listing Free Text]
SEQ ID NO: 1 URA3 gene (Saccharomyces cerevisiae)
Sequence number 2: P _STE2 (Saccharomyces cerevisiae)
Sequence number 3: _PHO (Saccharomyces cerevisiae)
Sequence number 4: P _STE3 (Saccharomyces cerevisiae)
SEQ ID NO: 5: GFP gene SEQ ID NO: 6: a1 protein (Saccharomyces cerevisiae)
SEQ ID NO: 7: α2 protein (Saccharomyces cerevisiae)
SEQ ID NO: 8: Primer 1 (F) URA3 gene amplification SEQ ID NO: 9: Primer 2 (R) URA3 gene amplification SEQ ID NO: 10: Primer 3 (F) URA3 gene amplification SEQ ID NO: 11: Primer 4 (F) in pRS series plasmid MCS amplification SEQ ID NO: 12: MCS amplification in primer 5 (R) pRS series plasmid SEQ ID NO: 13: Primer 6 (F) _PHO amplification SEQ ID NO: 14: Primer 7 (R) _PHO amplification

Claims (15)

  A method for producing yeast having mating ability from yeast having no mating ability, wherein a selection marker gene is inserted downstream of a promoter of a gene that specifically expresses a-type or a gene that expresses α-type specifically A method comprising a step of selecting an a-type or α-type yeast by transformation using an expression vector that is a YCp-type plasmid, and culturing under a selection pressure corresponding to a selection marker gene.   The method according to claim 1, wherein the promoter is a promoter of a gene that is specifically expressed in a-type, and the selection target is a-type yeast. The method according to claim 2, wherein the promoter is PSTE2.   The method according to claim 1, wherein the promoter is a promoter of a gene that is expressed specifically in α-type, and the selection target is α-type yeast.   The method of claim 4, wherein the promoter is PSTE3.   A method for producing yeast having mating ability from yeast having no mating ability, comprising a promoter of a gene specifically expressed by type a, a promoter of a gene specifically expressed by type α, or haploid-specifically An expression vector that is a YCp-type plasmid that expresses a selectable marker gene by a promoter of the gene to be expressed, and an expression vector that is the same or different from the expression vector, wherein the a1 gene or α2 gene is expressed in α-type or a-type yeast cells By transforming yeast that does not have conjugation ability using an expression vector that is expressed to form an a1-α2 complex and culturing while suppressing self-doubling while applying a selection pressure corresponding to the selection marker gene A method comprising a step of selecting a-type or α-type yeast.   The method according to claim 6, wherein the promoter under control of the selection marker gene is a promoter of a gene that is expressed specifically in a haploid cell of yeast.   8. The method of claim 7, wherein the promoter is PHO.   The method according to any one of claims 6 to 8, wherein the expression vector for forming the a1-α2 complex is a vector for expressing the a1 gene in α-type yeast cells, and the selection target is a-type yeast. .   The method according to any one of claims 6 to 8, wherein the expression vector for forming the a1-α2 complex is a vector for expressing the α2 gene in a-type yeast cells, and the selection target is α-type yeast. .   The promoter under control of the selection marker gene is a promoter of a gene that is specifically expressed in yeast a-type, and the expression vector that forms the a1-α2 complex is a vector that expresses the a1 gene in α-type yeast cells. The method according to claim 6, wherein the selection target is a-type yeast.   The promoter under the control of the selectable marker gene is a promoter of a gene that is expressed specifically in yeast α-type, and the expression vector that forms the a1-α2 complex is a vector that expresses the α2 gene in a-type yeast cells. The method according to claim 6, wherein the selection target is α-type yeast.   The method according to any one of claims 1 to 12, wherein the yeast is brewery yeast or baker's yeast.   The method according to any one of claims 1 to 13, wherein the selectable marker gene is a URA3 gene derived from a genus Saccharomyces cerevisiae.   A method for producing a hybrid strain of yeast, wherein the yeast having the ability to be conjugated produced by the method according to any one of claims 1 to 14 is used as at least one parent strain.

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