JPH08270A - Yeast aggregation gene FLO8 - Google Patents

Abstract

(57) [Summary] (Modified) [Objective] To provide a yeast agglutination gene and a method of using the same. [Structure] FLO having activity of imparting flocculation property to yeast
8 protein, FLO8 gene DN encoding FLO8 protein
A, a vector containing the DNA, the FLO8 gene D
Using NA, a method for producing a yeast having aggregating property or enhanced, a method for producing a yeast having a lacking or decreasing aggregating property, and suppressing the expression of the FLO8 gene, thereby aggregating yeast A method of deleting or reducing sex.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a yeast agglutinating gene and its use, and more particularly to a FLO8 protein having an activity of imparting aggregating property to yeast, a FLO8 gene DNA encoding the FLO8 protein, and the above DNA. Vector, method for producing yeast having aggregating property or enhanced using said FLO8 gene DNA, method for producing yeast having aggregating property deleted or reduced, and suppressing expression of said FLO8 gene According to the present invention relates to a method of deleting or reducing the agglutinability of yeast.

[0002]

2. Description of the Related Art It is well known that, in liquors such as beer and wine, the cohesiveness of yeast used for brewing is important not only for the flavor of the product but also for the workability of the brewing process. It is a fact. Therefore, many studies on yeast aggregation have been conducted so far. Besides, in the field of wastewater treatment, research on yeast flocculation is actively conducted.

[0003] Despite the enormous research on the agglutinability of yeast, which is an important property in industry, the mechanism of yeast agglutination has not yet been clarified, and the agglutinability can be successfully controlled by improving the yeast itself. Not not. From many years of research on the gene level of yeast, as genes involved in yeast agglutination, FLO1, flo3, FLO5, FLO8, sfl
1, fsu1, fsu2, tup1, cyc8, cka
2, genes such as FMC1 and mitochondrial DNA
The presence of the oli1 and oxi2 genes in the protein has been confirmed so far.

As a molecular-level study of these yeast genes involved in agglutinability, the FLO1 gene has been isolated and analyzed (YEAST, 9, 423 (1993) and
YEAST, 10, 211 (1994)). Also, the isolation and analysis of the FLO5 gene has been reported, in which F
Although the LO5 gene differs from the previously reported FLO1 gene in the location, it has been shown that the restriction map and the DNA base sequence are almost the same (J. Inst.
Brew., 85, 95 (1979) and Curr. Genet., 25, 196
(1994)).

However, the analysis of these genes at the molecular level is not sufficient, and it has not been clarified by what mechanism these genes are involved in yeast aggregation. Regarding genes other than FLO1 and FLO5 genes involved in yeast agglutination,
Neither isolation nor its structural analysis has been performed, nor have any proteins encoded by these genes been reported.

[0006] As an attempt to improve the aggregating ability of yeast by utilizing the genes involved in the aggregating ability of yeast as described above,
F, an aggregating gene of Saccharomyces cerevisiae
It has been reported that the non-aggregating yeast is imparted with aggregating property by introducing the LO1 gene. The aggregating ability of the transformant obtained here is expressed from the early stage of fermentation, and the fermentation of the transformed brewer's yeast is It has been reported to be later than the parent strain (Journal of the Brewing Society 88 p665 (1993)). Therefore, it cannot be said that the imparting of agglutinability to yeast by this FLO1 gene is controlled in a preferable manner, and further improvement was required for practical use.

As an attempt to utilize a gene other than the FLO1 gene, an attempt was made to impart a genetic trait to yeast by the FLO5 gene using the cell fusion method, and the usefulness of the genetic trait impartation was shown (J. Inst. Brew., 98, 315 (199
2)). However, since the gene transduction method is a cell fusion method, it is not only difficult to obtain a yeast having a target trait, but also the obtained cell fusion strain contains a gene other than the target aggregation-related gene. The DNA sequence is also introduced, and for example, the POF1 gene of experimental yeast that adds a phenol odor to beer is also introduced (Proc. Eu.
r. Brew. Conv. 497 (1981)).

[0008] As described above, the improvement in yeast aggregating ability using the gene involved in yeast agglutination, which has been attempted so far, has not been practical.

[0009]

Therefore, an object of the present invention is to provide a FLO8 protein having an activity of imparting aggregating property to yeast. Another object of the present invention is to provide a FLO8 gene DNA involved in yeast aggregability.

Furthermore, the present invention aims to provide a vector containing the above FLO8 gene DNA. Still another object of the present invention is to provide a method for producing yeast having imparted or enhanced cohesiveness. Another object of the present invention is to provide a method for producing yeast lacking agglutination property.

Further, the present invention aims to provide a method for deleting or reducing the flocculating property of yeast.

[0012]

The inventors of the present invention have found that FLO8
Isolate the gene to reveal its structure, then
It was found that when the FLO8 protein was produced in a non-aggregating yeast by introducing the LO8 gene, yeast aggregation was caused. Furthermore, the inventors
By introducing a disrupted FLO8 gene into an aggregating yeast, the yeast was successfully converted into a non-aggregating yeast, thus completing the present invention. That is, the present invention provides a FLO8 protein having substantially the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Further, the present invention is a FL containing a nucleotide sequence encoding a FLO8 protein having substantially the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
Provide O8 gene DNA. Furthermore, the present invention provides a DNA containing a nucleotide sequence encoding a FLO8 protein having an activity of imparting aggregating property to yeast and selected from the group consisting of (a) and (b) below.

(A) A DNA containing a sequence from the 267th base to the 2453th base in the base sequence shown in SEQ ID NO: 2 of the sequence listing or its complementary strand, and (b) (a) DNA
Or a DNA that hybridizes with the fragment

Furthermore, the present invention provides the plasmid pF415-
1, p7H12, pNEOF8, pYESF8 or pLF8 incorporated,
The present invention provides a DNA containing a nucleotide sequence encoding a FLO8 protein having an activity of imparting aggregating property to yeast.
The present invention also provides a vector containing the above DNA. Furthermore, the present invention provides a method for producing a yeast having an imparted or enhanced aggregating property, which comprises introducing the above-mentioned DNA. Furthermore, the present invention provides a method for producing a yeast having a deleted or reduced agglutinating property, which comprises introducing a DNA having the ability to express FLO8 protein deleted or reduced by destroying the DNA. I will provide a. The present invention also provides a method for deleting or reducing the agglutinability of yeast by suppressing the expression of the above DNA.

It is known that agglutination between yeast cells is caused by sexual agglutination between a-type cells and α-type cells, unseparation of budding daughter cells from mother cells, non-sexual agglutination, etc. The present invention is aimed at controlling non-sexual aggregation of these. Hereinafter, the present invention will be described in detail. In this specification,
The terms "DNA,""gene," and "gene DNA" are used interchangeably.

<FLO8 protein> The present invention includes FLO8 protein. The FLO8 protein can be derived from yeast, particularly Saccharomyces cerevisiae, and has the property of imparting aggregating property to yeast. The Saccharomyces cerevisiae referred to here is The yeast, a taxonomic study 3rd.
Edition (ed. By. NJW Kreger-van Rij. Elsevier
Science Publisher BV, Amsterdam (1984), p379) and Saccharomyces cerevisiae and its synonyms or mutants. F
LO8 protein has substantially the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. “Substantially the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing” means that, in addition to the “amino acid sequence shown in SEQ ID NO: 1 of the sequence listing”, the amino acid sequence has the activity of imparting aggregating property to yeast. An amino acid sequence obtained by modifying the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, that is, an amino acid sequence in which amino acids are added, inserted, deleted, deleted or substituted in a part of the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing. It includes.

Although not wishing to be bound by theory, the FLO8 protein, which is the product of the FLO8 gene of the present invention, is FLO1.
It is thought that yeast is aggregated by activating a gene and increasing its transcription amount. Where F
The FLO1 gene that can be activated by LO8 protein to impart yeast aggregating ability or enhance yeast aggregating ability is not limited to the yeast-derived FLO1 gene showing a strong aggregating ability reported so far, but a strong aggregating ability. Showing no sex,
F derived from yeast, which has been reported to be non-aggregating so far
It may be the LO1 gene.

The FLO8 protein of the present invention can be produced by introducing the FLO8 gene described below into a suitable host, particularly yeast, and expressing it.

<FLO8 Gene> The present invention includes FLO8 gene DNA. Here, "FLO8 gene DN
"A" means FLO8 having an activity of imparting cohesiveness to yeast
It means a DNA containing a nucleotide sequence encoding a protein.

Specifically, the present invention includes a FLO8 gene DNA containing a nucleotide sequence that substantially encodes a protein having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
The “base sequence encoding a protein having an amino acid sequence” as used herein means all base sequences in a degenerate relationship. Such FLO8 gene DNA is
For example, it can be acquired as follows. First,
An agglutinating yeast gene library is prepared using an Escherichia coli yeast shuttle vector, non-aggregating yeast is transformed with this library, and then the agglutinating gene is cloned from the clone that has become agglutinative.

Once the FLO8 gene DNA is obtained, a part of this DNA is used as a probe,
Other gene DNAs which are the same as or homologous thereto can be obtained by means such as plaque hybridization, colony hybridization, polymerase chain reaction (PCR) and the like. All of these methods are well known to those skilled in the art and can be easily implemented.

As a useful gene source for obtaining the FLO8 gene DNA of the present invention, yeasts of the genus Saccharomyces, yeasts closely related to the genus Saccharomyces, and the like can be used.

From another point of view, the present invention comprises a nucleotide sequence encoding a FLO8 protein having an activity of imparting aggregating property to yeast, which is selected from the group consisting of (a) and (b) below: Includes. (a) A DNA containing the sequence from the 267th base to the 2453th base of the base sequence shown in SEQ ID NO: 2 of the Sequence Listing or its complementary strand. (b) A DNA which hybridizes with the DNA of (a) or a fragment thereof.

The DNA of (a) includes, in addition to the sequence from the 267th base to the 2453th base of the base sequence shown in SEQ ID NO: 2 of the sequence listing, other bases or base sequences, for example, in the sequence listing. The base sequence shown in SEQ ID NO: 2 may contain a base other than the sequence from the 267th base to the 2453th base, or a base sequence, or the complementary chain thereof.

The DNA of (b) does not show its base sequence in particular, but it is compared among the base sequences shown in SEQ ID NO: 2 in the sequence listing from the 267th base to the 2453th base. It can be easily obtained from a yeast chromosomal gene library by using a method such as plaque hybridization, colony hybridization, PCR, etc., by selecting a sequence having a small number of bases and using the DNA containing this sequence as a probe. In the hybridization, in order to detect a nucleic acid sequence which is not completely complementary, the contact condition with the probe may be relaxed.
A typical hybridization method is 6 ×
SSC (0.9M sodium chloride, 0.09M sodium citrate), 5 x Denhardt's solution (0.1% polyvinylpyrrolidone, 0.1% ficoll, 0.1% bovine serum albumin), 0.5
% SDS, 10 μg / ml single-stranded salmon sperm DNA was contacted with the probe overnight at 60 ° C., then 2 × SS
An example of the method is to remove the probe not bound at 60 ° C. by using an aqueous solution containing C (0.3 M sodium chloride, 0.03 M sodium citrate) and 0.1% SDS. The detection of nucleic acids having lower complementarity is performed by increasing the concentration of SSC at the time of contact with the probe, lowering the temperature, or removing S that has not been bound to the SSC.
This can be performed by increasing the SC concentration or decreasing the temperature. The chain length of DNA as a probe is
If it is too short, sufficient hybridization will not occur, so it is preferably at least 20 bases, more preferably 100 bases or more. The gene library to which this probe is to be applied is preferably 5 × 10 ³ bases to 30 × 10 ³ bases obtained by cutting the yeast chromosome by chemical or physical means such as restriction enzyme or ultrasonic wave.
It is composed of DNA in which a DNA having chain lengths having almost the same range of bases is incorporated into a vector. Homology DN by a probe from a DNA library with such a vector
Recovery of A and D with the desired vector recovered
Cloning of NA, for example, cloning by yeast cells, can be performed according to a method already well known in genetic engineering. For more information, for example, Mo
lecular Cloning, Cold Spring Harbor Laboratory (198
See 9). As a specific example of such a method, a method (JP-A-6-62849) used for obtaining a homologous gene of a brewer's yeast alcohol acetyltransferase gene as a probe can be mentioned. The homologous gene obtained by this method had alcohol acetyltransferase activity.

[0026] From another point of view, the present invention relates to FL which is incorporated into plasmids pF415-1, p7H12, pNEOF8, pYESF8 or pLF8 and has the activity of imparting aggregating property to yeast.
It also includes a DNA containing a nucleotide sequence encoding the O8 protein. Escherichia coli JM109 into which the plasmid pF415-1 was introduced and Escherichia coli DH5 α into which the plasmid pLF8 was introduced were respectively transferred to the Institute of Biotechnology, Institute of Industrial Science, May 1994.
Deposited on the 10th, their microbial accession number is FERM B
P-4664 and FERM BP-4665. One example of such DNA is a DNA fragment of about 11.2 kb which is incorporated into the plasmid pF415-1 and has the restriction map shown in FIG.

The DNAs of the present invention described above will be generically referred to as "FLO8 gene DNA" hereinafter. The FLO8 gene DNA of the present invention may be derived from a natural product, totally synthesized, or synthesized using a part of the natural product, that is, a semi-synthetic one.

<Transformation> FLO8 gene DN of the present invention
By introducing A, it is possible to obtain a yeast to which cohesiveness is imparted or enhanced.

As a method for introducing the FLO8 gene DNA, a method commonly used in the field of genetic engineering may be used, and the method used is the ANALYTICAL BIOCHEMIS
TRY163.391 (1987) etc.). Specifically, a method of incorporating a desired DNA into a vector and introducing it into yeast, a method of directly introducing into a yeast without incorporating into a vector, and the like can be mentioned.

In the method of incorporating the above DNA into a vector and introducing it into yeast, examples of usable vectors include YRp system (a yeast multicopy vector having an ARS sequence of the yeast chromosome as an origin of replication), YE.
p system (a yeast multicopy vector having a 2 μm yeast origin of replication), YCp system (a yeast single copy vector having a yeast chromosome ARS sequence as a replication origin, and a yeast chromosome centromere DNA sequence), YIp All known yeasts such as a system (a vector for integrating a yeast chromosome having no origin of replication of yeast) can be used. These vectors have been described in the literature (Medical Publishing Center, "New Biotechnology of Yeast", p284) and can be easily prepared.

DNA directly into yeast without being incorporated into a vector
As a typical method for introducing the gene, there can be mentioned a cotransformation method in which yeast is simultaneously transformed with a plasmid having a marker gene such as a drug resistance gene and the gene DNA to be introduced (Japanese Patent Publication No. 5-60918). issue).

In order to express the introduced gene DNA in yeast or to increase or decrease the expression in the above method, the promoter which is a unit for controlling transcription and translation is used as the FLO8 gene DNA of the present invention. 5'- upstream of the terminator 3'-
It should be incorporated in each downstream area. The promoter and terminator are derived from the FLO8 gene itself, as well as an alcohol dehydrogenase gene (J. Biol. Chem., 257, 3018 (1982)) and a phosphoglycerate kinase gene (Nucleic Acids Res., 10, 77).
91 (1982)), glycerol aldehyde-3-phosphate dehydrogenase gene (J. Biol. Chem., 254, 9839 (197).
9)) or other known gene-derived ones or artificially improved ones can be used. More specifically, ADH (alias ADC), GAPDH (alias GPD),
Promoters and terminators such as PHO, GAL, PGK, ENO, TRP and HIP can be used.

Further, the FLO8 gene DNA of the present invention can be controlled and expressed in yeast by selecting an appropriate promoter. For example, if the promoter of the galactokinase gene is used, the expression level can be increased by changing the sugar source of the medium from, for example, glucose to galactose.

Further, by disrupting the FLO8 gene DNA of the present invention to introduce a DNA whose ability to express the FLO8 protein is deleted or reduced, it is possible to obtain a yeast in which agglutination is deleted or reduced. it can. F
The disruption of the LO8 gene DNA is due to the FLO8 gene FLO
It can be carried out by adding or deleting a single or a plurality of bases inside the region involved in expression of 8 proteins, for example, the promoter region or the coding region, or deleting the entire region. By thus destroying the FLO8 gene DNA, the DNA whose ability to express the FLO8 protein is deleted or reduced can be introduced into yeast by the same method as the above-mentioned DNA introduction method. By the introduction, homologous recombination occurs between the FLO8 gene in the chromosomal DNA of the host yeast and the introduced DNA, the FLO8 gene of the host yeast is disrupted, and FL
It is believed that the ability to express the O8 protein is deleted or diminished, resulting in a deletion or diminution of the host yeast aggregability.

The host yeast usable in the present invention is
Although it may be any that can be taxonomically included in the category of yeast, yeasts for producing alcoholic beverages belonging to Saccharomyces cerevisiae, specifically, beer yeasts, wine yeasts, etc., or yeasts used for alcohol production are preferable.

The present invention also includes a method for eliminating or reducing the agglutinability of yeast by suppressing the expression of the above FLO8 gene DNA. Examples of such a method include a method of introducing DNA in which the ability to express the FLO8 protein is deleted or reduced by destroying the FLO8 gene DNA, an antisense RNA method, and the like.

<Other FLO8 Proteins> According to the present invention, a part of DNA containing a sequence from the 267th base to the 2453rd base of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing is used as a probe. When a DNA that hybridizes with this was cloned from a yeast chromosomal DNA library and introduced into a non-aggregating yeast, this yeast was converted to aggregating. Therefore, the protein encoded by the DNA that hybridizes with the DNA containing the sequence from the 267th base to the 2453rd base in the base sequence shown in SEQ ID NO: 2 of the Sequence Listing is also equivalent to the FLO8 protein of the present invention. It is easily inferred that it has various biological activities, that is, the activity of adding aggregating ability to yeast.

[0038]

INDUSTRIAL APPLICABILITY The present invention provides a FLO8 protein having an activity of imparting aggregating property to yeast. In addition, the present invention provides FLO8 gene DNA. It is possible to transform yeast by introducing the DNA of the present invention as a foreign gene into yeast by a genetic engineering technique.
That is, by introducing this DNA into a yeast cell as an extranuclear and / or intranuclear gene, it is possible to impart agglutinability to yeast or enhance agglutinability of yeast. On the contrary, by introducing a disrupted version of this DNA into yeast cells or suppressing the expression of this DNA, the aggregating yeast can be converted into a non-aggregating yeast.

[0039]

【Example】

[Example 1] Yeast ATCC of flocculating gene FLO8
Cloning from strain 60715 (i) Preparation of yeast gene library Extraction of total yeast DNA was performed by the method of Hereford et al. (Cell, 18,
1261-1271, (1979)) was modified by the following method.

Agglutinative yeast ATCC60715 strain (a, leu2-3, leu2
-112, his4, STA1, FLO8) was inoculated into 200 ml of YPD medium (2% bactopeptone (Difco), 1% yeast extract (Difco), 2% glucose) and shaken at 30 ° C for 48 hours. After culturing and collecting the cells, the cells were washed successively with 100 mM EDTA and sterile water.
This was suspended in 12.5 ml of 0.2 M Tris solution, and 0.625 ml of β
-Add mercaptoethanol, leave at room temperature for 30 minutes,
The cells were collected by centrifugation at 12000 rpm for 5 seconds. 12.5 ml of zymolyce solution (1M sorbitol, 40 mM sodium phosphate buffer (pH 6.8), 0.125 mg / ml zymolyce 100T
(Seikagaku Co., Ltd.) and placed at 30 ° C. for 2 hours to completely transform the yeast into protoplasts. 10 this at 12000 rpm
Collect the cells by centrifuging for 2 seconds and collect 12.5 ml of lysis buffer (50 mM Tris-HCl buffer (pH 8.0), 0.2 M sodium chloride, 0.1 M ED).
The cells were suspended in TA, 5% SDS, 1 mg / ml Pronase E (Sigma) and placed at 60 ° C for 30 minutes to lyse the cells. An equal amount of phenol: chloroform: isoamyl alcohol (25: 2
The operation of adding 4: 1) and gently stirring and then collecting the aqueous layer was repeated twice. 25 ml of ethanol was added to the obtained aqueous layer, the precipitated filamentous nucleic acid was entangled with a Pasteur pipette and collected, and after washing with 70% ethanol,
Dried. This was dissolved in 3 ml of TE buffer (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA), put in a dialysis tube, and dialyzed overnight in 1 liter of TE buffer.
Collect the liquid in the dialysis tube and remove a part of it with ribonuclease.
After treatment with A (Sigma), the DNA concentration was quantified. The remaining solution was adjusted to 1 μg / μl and used as a DNA solution in the subsequent experiments.

1000 μg of DNA was partially digested with the restriction enzyme Sau3AI (Takara Shuzo), and 32 ml of 10% to 40% sucrose density gradient solution (1 M sodium chloride, 20 mM Tris-HCl buffer) prepared in Hitachi ultracentrifuge tube 40PA was prepared. Liquid (pH8.0), 5mM EDTA)
Then, using a Hitachi ultracentrifuge SCP70H2 and the same rotor SRP28SA1, it was centrifuged for 21.5 hours at 20 ° C. and 26000 rpm. After the centrifugation was completed, a hole was made in the bottom of the tube with an injection needle, and the falling sample was collected into about 1.5 ml each in a 1.5 ml tube. The molecular weight of the DNA was confirmed by agarose electrophoresis using a part of each fraction, and the DNA was recovered by ethanol precipitation from the fraction containing the DNA having a size of about 10 kb.

As a cloning vector, YIp5
At the EcoRI site of (ATCC 37061), there is a gene sequence data bank, Antrey (National Center for
CE obtained from Biotechnology Information)
A 1.2 kb fragment containing CEN3 obtained by PCR based on the nucleotide sequence of N3 and the entire nucleotide sequence of yeast chromosome 3 and a fragment containing the ARS sequence obtained as a YRP7-derived EcoRI-HindIII fragment were introduced. pYT37 was used. The unique BamHI site in this plasmid was cleaved, dephosphorylated with alkaline phosphatase (Takara Shuzo), and 2.1 μg of it and the above-mentioned approximately 10 kb
The ligation kit (Takara Shuzo) was used to ligate 11.6 μg of the yeast DNA fragment having the length of 1) according to the protocol, and a part thereof was introduced into Escherichia coli competent cell strain DH5 (Toyobo). As a result, a total of about 22000 transformants were plated on a total of 50 LB plates (1% bactotryptone (Difco), 0.5% yeast extract, 1% NaCl, 50 μg / ml).
Ampicillin, 1.5% agar, pH 7.0). The transformants were collected from the plate using sterilized water, and the cells were collected in a total of 10 tubes every 5 plates.
After washing once, a plasmid was prepared by the alkaline method. 1 μg of DNA was collected from each tube containing the plasmid solution, and a plasmid solution containing a total of 10 μg of DNA (concentration 480 ng / μl) was used for yeast transformation as a yeast gene library.

(Ii) Measurement of agglutination property The agglutination property was measured by the method of Watari et al. (Agric. Biol. Che.
m., 53, 901-903, (1989)). Flat bottom
300 μl of the liquid medium was dispensed into a 96-well microtiter plate, and 1 platinum loop of the yeast grown on the solid medium was inoculated into the 96-well microtiter plate, and this was statically cultured at 30 ° C. for 72 hours. The circumference of the microtiter plate was covered with vinyl tape to prevent evaporation of the medium. A silicon plate to prevent the scattering of the culture medium is sandwiched between the main body of the microtiter plate and the lid, shaken vigorously for 1 minute using the Iwaki shaker CST-100 (Iwaki glass), and then allowed to stand for 1 minute, The cohesiveness was visually observed. At this point, the non-aggregating yeast was still dispersed in the medium, whereas the aggregating yeast rapidly aggregated and settled, so that the two could be visually distinguished.

(Iii) Selection of Yeast Host for Obtaining Aggregation Gene In yeast, even if an agglutination gene is present, there is a case in which an agglutination inhibition gene having a function of suppressing it is present. Known to be. Even if the yeast gene library was introduced into a non-aggregating yeast and an attempt was made to obtain an agglutinating gene using the conversion from non-aggregating to aggregating as an index, the non-aggregating yeast serving as a host would have such an aggregating property. If the suppressor gene was present, acquisition of the aggregating gene would not be desired. Therefore, when selecting the yeast host for obtaining the agglutinin gene, it was confirmed that the yeast host did not have the agglutinin suppressor gene. That is, if the non-aggregating yeast does not have an agglutinating suppressor gene, it is allowed to aggregate with the aggregating yeast having the aggregating gene FLO8, then spores are formed, and its tetrad analysis is performed. sex:
Non-aggregating should separate 2: 2. Therefore, the flocculating yeast ATCC60715 strain having the FLO8 gene and the non-flocculating yeast YPH500
Strains (a, ura3-52, lys2-801 amber, ade2-101ochre, trp
1Δ63, his3Δ200, leu2Δ1, Stratagene) and 10 four molecules thereof were conjugated by the method described by Ono (Protein and Nucleic Acid Enzymes special issue, vol.39, No.4, 760-769).
(1994)) Each 4 spores were isolated and each 4
When the agglutinability of the strain in YPD medium was measured,
Aggregating: non-aggregating 2: 2 in all 0 tetrads
Separated. From this, it is expected that the YPH500 strain does not have an agglutination suppressor gene for FLO8, so this strain was selected as a yeast host for obtaining the agglutination gene FLO8.

(Iv) Transformation of yeast Yeast was transformed by the protoplast method as follows (Jikken Igaku, 8, 85-91, (1990)). yeast
The YPH500 strain was cultured in 100 ml of YPD medium at 30 ° C for 24 hours with shaking, and then centrifuged at 500 xg for 4 minutes to collect the cells.
It was washed once with M sorbitol. 20 ml of collected bacterial cells
SCE (1M sorbitol, 0.1M sodium citrate (p
H5.8), 10 mM EDTA), 0.2 ml of 1M DTT, and
134 μl of Zymolyase 100T solution (2 mg / ml SCE) was added, and the mixture was gently shaken at 30 ° C. for 1 hour to form protoplasts. The cells were collected by centrifugation at 300 g for 4 minutes, washed twice with STC (1 M sorbitol, 10 mM Tris-HCl buffer (pH 8.0), 10 mM calcium chloride), and then suspended in 4 ml of STC. 2 μ
l of the aforementioned yeast gene library and 20 μl of 20
After adding 1 ml of the protoplast suspension to mg / ml calf thymus DNA (Sigma) and allowing it to stand at room temperature for 10 minutes, 10 ml of PEG solution (20% (w / v) PEG8000, 10 mM Tris-HCl buffer (pH7)) was added. .
5), 10 mM calcium chloride) is gently added,
Let stand at room temperature for minutes. This was centrifuged at 300 xg for 4 minutes, the bacterial cells were suspended in 2 ml of SOC (1M sorbitol, 7 mM calcium chloride, 25% YPD medium), and left at 30 ° C for 30 minutes.
5 ml of SOB upper layer agar (1M sorbitol, 0.67% yeast nitrogen base (without amino acids, Difco)), 0.06% amino acid mix (without uracil) was dissolved in 40 μl of this solution and kept at 50 ° C. , 2% glucose, 2.5% agar) and gently mix with SOB
It was layered on a lower layer agar medium (the composition was the same as SOB upper layer agar except for the agar concentration of 2%) and cultured at 30 ° C. In this way, about 12000 transformants were obtained.

(V) Cloning of agglutinating gene Regarding 11130 clones among the obtained transformants, SD (uracil-free) medium (0.67% yeast nitrogen base (amino acid) Was not added), 0.06% amino acid mix (without uracil), 2% glucose, pH 5.8), and cultured, and then the aggregating property was measured. As a result, 4 clones with extremely strong aggregation were found.

From these clones, the introduced plasmid was recovered by the following method. After washing the clone with TE buffer, 30 μl of STES buffer (0.5 M sodium chloride, 0.2 M Tris-HCl buffer (pH 7.6), 0.01 M EDT) was added.
A, 1% SDS). Add 0.3 g of glass beads and shake it vigorously for 5 minutes to disrupt the cells, then add 30 ml.
Phenol: chloroform: isoamyl alcohol (25: 24: 1) was added, and the mixture was vigorously extracted by shaking for 2 minutes, and 5 μl of the aqueous layer obtained by centrifugation was added to Escherichia coli competent cell DH.
Introduced to 5 stocks (Toyobo).

A plasmid was prepared from the obtained transformant by the alkaline method. The resulting 4 plasmids were digested with HindIII (Boehringer) and electrophoresed on a 1% agarose gel to analyze the length of the resulting restriction enzyme fragment. kb, 1.
Since 4 types of common HindIII fragments of 6 kb and 0.9 kb appeared, it was presumed that they were all cloned genes of the same locus in yeast. A plasmid named p7H12 was selected from these plasmids, digested with XhoI (Boehringer), and the resulting XhoI fragment of about 11.2 kb was cloned into the XhoI site of plasmid pRS415 (Stratagene) to obtain pF415. It was named -1.

These two plasmids p7H12 and pF415
When -1 was introduced into the non-aggregating YPH500 strain, all the transformants were converted into agglutinin.
It was determined that the aggregating gene derived from the CC60715 strain could be obtained. A restriction map of pF415-1 is shown in Figure 1.

(Vi) Confirmation that the obtained aggregating gene is FLO8 In order to confirm that the aggregating gene obtained as described above is FLO8, as shown in FIG. 2, integration mapping was performed. Was carried out. That is, p7H12 is replaced with EcoRI
The plasmid p7H12ΔE from which part of the sequence derived from the ATCC60715 strain and CEN3 and ARS was removed by self-ligation after digestion with (Boehringer) (cleavage points are indicated by arrows 1) and 2) in FIG. 2) was performed. Then, the XhoI site uniquely present only in the sequence derived from the ATCC60715 strain in this plasmid was cleaved (the cleaved site in FIG.
3)), followed by the method of Chen et al. (Curr. Gene
t., 21, 83-84, (1992)) agglutinating yeast strain YF191 (a, ura
3-52, ade2-101 ocher, leu2Δ1, FLO8).
Since the obtained uracil-non-auxotrophic transformant showed aggregating property, it was judged that the FLO8 gene was not disrupted.

This transformant was named YF191URA, and the state of insertion of the vector sequence containing the URA3 gene was analyzed by Southern analysis. That is, the DNA of YF191URA was prepared by the method described above, digested with NruI (Takara Shuzo), electrophoresed on a 1% agarose gel, and blotted on a nylon membrane Hybond N + (Amersham) according to the protocol. 4.0 kb derived from the ATCC 60715 strain gene in p7H12
HindIII fragment as a probe, labeled with [α- ³² P] dCTP (Amersham), and hybridized with the membrane at 65 ° C. for 16 hours according to the Hybond N + protocol.
Finally washed with 0.1x SSPE, 0.1% SDS at 65 ° C,
When the signal from the hybrid band was exposed to X-ray film for autoradiography, YF19
1URA chromosomal DNA has A cloned in p7H12
It was confirmed that the vector sequence containing the URA3 gene was inserted into the gene sequence derived from the TCC60715 strain (see FIG. 2).

The YF191URA strain was used as a non-aggregating yeast strain TD1 (a, his4
-38, ura3-52, trp1-289, cans, MAK (K)) were allowed to undergo tetrad formation, and 15 tetrad analyzes for aggregation and uracil requirement were performed. As a result, 4 strains derived from one tetrad have agglutinability and non-aggregability of 2: 2,
In addition, uracil-requiring and non-requiring were separated at 2: 2, and all the aggregating strains were non-uracil-requiring (Table 1).

[0054]

[Table 1] Table 1. Tetramolecular analysis of FLO8 gene YF191URA (a, leu2, FLO8, URA3) × TD1 (α, LEU2, ura3, flo8) ──────────────────────── ─────────── Parent type 2 Non-parent type 2 Tetra type ─────────────────────────────── ──── Leu ⁺ -Ura ⁺ 1 4 10 Flo ⁺ -Ura ⁺ 150 0 ────────────────────────────── From these facts, it was proved that in the YF191URA strain, the URA3 gene was inserted in the immediate vicinity of the FLO8 gene by homologous recombination. Therefore, it is concluded that the aggregating gene derived from the ATCC60715 strain cloned in p7H12 is the FLO8 gene.

Example 2 Determination of Nucleotide Sequence of FLO8 Gene Three Hins of 2.6 kb, 1.6 kb, and 0.9 kb in the insert fragment of pF415-1 were used.
dIII fragment, pBluescriptII KS + (Stratagene)
Was subcloned into the HindIII site of K., and the deletion series was prepared according to the protocol using the deletion kit for kilosequencing (Takara Shuzo), and the nucleotide sequence was determined by the dideoxy method. Connection between each subclone is 5'GAGGGCTGAATTAATGTGTT3 '(SEQ ID NO: 3), 5'GGAAAAATTATTAACGATAG3' (SEQ ID NO: 4), 5 '
AAGTAGTAAAGGCAATCGTA3 '(SEQ ID NO: 5), 5'TTTAATACCA
The nucleotide sequence was determined using four kinds of synthetic DNA of GTTCTTCCAG3 '(SEQ ID NO: 6) as primers. The obtained base sequence is shown in SEQ ID NO: 2 in the sequence listing.

[Example 3] Confirmation of function of open reading frame of FLO8 gene The nucleotide sequence of FLO8 gene suggested the existence of an open reading frame of 2187 bp in total length. 5'TTTGAGCTCAGCAGCAGCAGTG before and after this part to confirm that this expected open reading frame works.
GATAAATCAACT 3 '(SEQ ID NO: 7) and 5'GCTCTAGAATTATG
Two synthetic DNAs of TTTCCTGTCATTAAGAGTT3 '(SEQ ID NO: 8)
Was used as a primer and pF415-1 was used as a template for PCR synthesis. The resulting PCR product was digested with SacI and XbaI (both Boehringer), and then pYES2 (Invitrogen) GAL1.
The plasmid pYESF8 obtained by cloning at the XbaI-SacI site downstream of the promoter was
It was introduced into the yeast YPH500 strain by the method of En et al. (Curr. Genet., 21, 83-84, (1992)).

The resulting transformant showed strong agglutination when SD (uracil-free) medium had 2% galactose as the sugar source. From this, it was determined that the open reading frame predicted from the nucleotide sequence of the FLO8 gene encodes a protein that imparts aggregating property.

[Example 4] Search for FLO8 homologous gene in yeasts having other genotypes In order to confirm whether or not the FLO8 gene is present in yeasts having other genotypes, three agglutinability experimental yeast strains ( ABXL-1D, AB
XR11A, ATCC60715), two non-aggregating experimental yeast strains (S288C,
X2180-1A), 2 strains of cohesive top-fermenting yeast (NCYC1007, NCYC
1195), three cohesive bottom fermenting yeast strains (KBY001, KBY002, TU
M59), two non-aggregating bottom fermenting yeast strains (KBY003, AJL2155
12) were subjected to Southern analysis by the above-mentioned method. However, the total DNA of the strain was digested with HindIII, and a 1574 bp HindIII fragment of the insertion fragment of pF415-1 was used as a probe (see FIG. 1). As a result, one signal was detected at around 1.6 kb in all the used strains. From this, it was judged that the FLO8 gene is widely present in yeast regardless of the presence or absence of aggregating property.

Example 5 Preparation and Evaluation of FLO8 Gene Disrupted Strain The FLO8 gene disrupted strain was prepared as shown in FIG. SacI plasmid pRS405 (Stratagene)
After removing the SacII site simultaneously digested with SmaI and SmaI, the ends were blunted using Klenow DNA polymerase (Takara Shuzo) and self-ligated using a ligation kit (Takara Shuzo). The plasmid pR thus obtained
A plasmid p containing the FLO8 gene at the HindIII site of S405Δ
Within the coding region of the FLO8 gene obtained from F415-1 15
The 74 bp HindIII fragment (see Figure 1) was cloned.
The 1574 bp HindIII fragment was obtained by deleting 284 bp at the carpoxyl terminal and bp at the amino terminal of the FLO8 gene. After digesting this plasmid with SacII, which is uniquely present in the 1574 bp HindIII fragment, the aggregating yeast Y was prepared by the method of Chen et al. (Curr. Genet., 21, 83-84, (1992)).
Introduced into the F191 strain, leucine non-auxotrophic transformant YE191L
Got the EU.

Southern analysis was carried out on the leucine non-auxotrophic transformant YF191LEU by the above-mentioned method to find the FLO8 locus.
The vector sequence containing the LEU2 gene has been inserted,
That is, it was confirmed that the FLO8 gene was disrupted. However, the total DNA of the strain was digested with HindIII, and the probe was the 1574 bp HindIII fragment of pF415-1. Therefore, YF
In 191LEU, the sequence derived from pRS405
As a result of being inserted into the FLO8 gene by homologous recombination, 2 copies of FL of the FLO8 gene lacking the carboxy terminus of 284 bp and the FLO8 gene lacking the amino terminus of 329 bp
The O8 gene is presumed to be aligned with the sequence derived from pRS405 in between (see FIG. 3). FLO8 thus obtained
When the aggregating property of the gene-disrupted strain YF191LEU was evaluated, it was found to be non-aggregating.

[Example 6] Confirmation of mechanism of aggregating expression by FLO8 gene Two strains of aggregating yeast YF191 strain and non-aggregating yeast YF191LEU strain, which is a FLO8 gene disrupted strain of YF191 strain, had an OD600 of 0.1.
The cells were inoculated in SD medium (the one containing no leucine was used for culturing the transformant) so as to be 5, and shake-cultured at 30 ° C. for 15 hours. After culturing, collect an amount of cells corresponding to 100 x OD600, and adjust to a final concentration of 50 μg / ml.
After adding μg / ml cycloheximide, the mixture was cooled in ice for 5 minutes, and the cells were collected by centrifugation. This was washed once with 8 ml of ice-cooled 50 mg / ml aqueous solution of cycloheximide. From the collected cells, the method of Elion and Warner (Cell, 39, 6
63-673, (1984)), and total RNA was prepared. 10 of the obtained RNA
16 μl of glyoxal / DMSO solution (1 M glyoxal, 50% DMSO, 10 mM sodium phosphate buffer (pH
7.0)) for 1 hour at 50 ° C. to perform glyoxalization, and then 2 μl of the applying buffer (50% (w / w
v) Glycerol, 10 mM phosphate buffer (pH 7.0), 0.4%
(w / v) bromophenyl blue) and 1 μl of 1 mg / ml ethidium bromide solution were added, and electrophoresis was performed in a gel containing 10 mM sodium phosphate buffer (pH 7.0) and 1% agarose. During electrophoresis, a peristaltic pump was used to constantly circulate the buffer solution in the electrophoretic layer to prevent a pH gradient from occurring. Bromphenyl blue is 70% of the length of the gel
When it reached a certain level, electrophoresis was stopped, and RNA in the gel stained with ethidium bromide was observed using an ultraviolet transilluminator, and it was confirmed that the RNA was not decomposed using ribosomal RNA as an index. Then in the gel
RNA was blotted on a nylon membrane Genescreen-Plus (DuPont) according to the attached protocol, and the RNA-blotted membrane was subjected to incubation treatment at 80 ° C. for 2 hours. This membrane was used for Northern analysis according to the protocol attached to Genescreen-Plus. The following two types were used as probes. As a part of the FLO8 gene fragment, a 1574 bp HindIII fragment derived from pF415-1 was used (see FIG. 1). Some fragments of the FLO1 gene include Te
Report by unisen, AWRH et al. (Yeast, 9, 423-427, 199
3)), the aggregative ABXL-1D strain (a, FLO1, Yeast Gen
5'GATGAA using the total DNA of the etic stock center as a template
ACTGTCATTGTTGTCAAA3 '(SEQ ID NO: 9) and 5'TCGTTTCAGCAG
PCR was performed using two synthetic primers of CTAAAGTAT3 '(SEQ ID NO: 10), the entire PCR product was electrophoresed in a 1% agarose gel, and the amplified 1045 bp DNA fragment was excised from the gel, and Prep-A- A DNA fragment recovered using Gene (Bio-Rad) was used. These two fragments are each [α-
^It was labeled with ³² P] dCTP (Amersham) and used for Northern analysis. Bio image analyzer BAS2000 (Fuji Photo Film Co., Ltd.) was used to measure the transcripts of these genes.
Co., Ltd.) was used according to the protocol. The results are shown in Table 2.

[0062]

[Table 2] Table 2. Transcription levels of FLO8 and FLO1 in the FLO8 gene-disrupted strain ─────────────────────────────────── YF191LEU strain YF191 strain ─────────────────────────────────── Cohesiveness (Note 1) − + FLO8 Relative transfer amount 1. 87 (Note 2) 1.00 FLO1 Relative transfer amount 0.006 1.00 ──────────────────────────────── ──── (Note 1): In the indication of cohesiveness, − represents non-cohesiveness and + represents cohesiveness. (Note 2): The FLO8 transcript of the YF191LEU strain lacks a function because it lacks the C-terminus.

Regarding the FLO8 gene, 10 μ of the YF191LEU strain was used.
Although the transcription amount of g per total RNA was 1.87 times that of the YF191 strain, the FLO8 gene of the YF191LEU strain had a carboxyl terminus.
Due to the 284 bp deletion, the FLO8 gene transcript of the YF191LEU strain had a slightly lower molecular weight than the FLO8 gene transcript of the YF191 strain. Regarding the FLO1 gene, 10 of the YF191LEU strain was used.
The transcription amount per μg total RNA was only 6% of that of the YF191 strain in which the FLO8 gene was not disrupted. From this, it was shown that when the carboxyl terminus of the FLO8 gene is deleted and its function is lost, the agglutinability is lost due to a large decrease in the transcription amount of the FLO1 gene.

Furthermore, between the promoter region of the 1.0 kb glyceraldehyde-3-phosphate dehydrogenase gene described by Yamano et al. And the terminator region of the 0.38 kb phosphoglycerate kinase gene (J. Biote
chnol., 32, 165-171 (1994)), a 1.1 kb Pst-XhoI fragment containing the G418 resistance gene of pUC4K (Pharmacia) was blunt-ended with Klenow DNA polymerase to prepare a ligated cassette. Yeast multicopy vector
YEp13K (Sone H. et al., Appl. Env. Microbiol., 54, 38-4
2, (1988)) at the BglII-Sal site. To the unique XhoI site of the thus obtained plasmid, the approximately 11.2 kb XhoI fragment in the pF415-1 insertion fragment containing the FLO8 gene was ligated to prepare pNEOF8. This plasmid
It was introduced into the non-aggregating yeast YPH500 strain (Stratagene) by the method of Chen et al. (Curr. Genet., 21, 83-84, (1992)). The transformant YNEOF8 thus obtained was converted into agglutinative.

Two strains of non-aggregating yeast YPH500 strain and aggregating yeast YNEOF8 strain obtained by introducing pNEOF8 into YPH500 strain were OD600
Was inoculated into SD medium (a leucine-free medium was used for culturing the transformant) so that the ratio was 0.15, and the mixture was cultured at 30 ° C. for 15 hours while shaking. After culturing, 100 × OD600 bacterial cells were collected, and the transcripts of the FLO8 gene and FLO1 gene were measured by the method described above. Table 3 shows the results.

[0066]

[Table 3] Table 3. Transcription levels of FLO8 and FLO1 in FLO8 gene-enhanced strains ─────────────────────────────────── YNEOF8 strain YPH500 strain ─────────────────────────────────── Aggregability (Note 1) ＋ − FLO8 Relative transfer amount 2. 25 1.00 FLO1 relative transfer amount 6.05 1.00 ──────────────────────────────────── (Note 1): In the display of cohesiveness, − represents non-cohesiveness and + represents cohesiveness.

Regarding the FLO8 gene, the transcription amount of the YNEOF8 strain was 2.25 times that of the YPH500 strain per 10 μg of total RNA.
Regarding the FLO1 gene, the transcription amount per 10 μg total RNA of the YNEOF8 strain was 6.05 times that of the YPH500 strain. From this, it was shown that when a large number of copies of the FLO8 gene were introduced into the non-aggregating YPH500 strain, the agglutination was imparted by increasing the transcription amount of the FLO1 gene.

From the two facts shown above, it was shown that the FLO8 gene imparts aggregating property to yeast by increasing the transcription amount of the FLO1 gene.

[Example 7] Acquisition of DNA chain that hybridizes with FLO8 gene from brewer's yeast According to the method described in JP-A-6-62849, a brewer's yeast chromosomal DNA library was prepared as follows.

Yeast was OD600 = 1 in 1 liter of YPD medium.
After culturing to 0, the cells were collected, washed once with sterile water, and 2 ml of solution A (1 M sorbitol, 0.125 M EDT) was added per 1 g of cells.
A, 0.1 M trisodium citrate (pH 7.0), 0.75% 2-mercaptoethanol, 0.01% Zymolyase 100T) was suspended and shaken gently at 37 ° C for 2 hours to protoplast the yeast. 3.5 ml of B solution (0.5 M Tris-HCl buffer (pH 9.0), 0.2 M EDTA, 3% SD per 1 g of cells)
S) was added, and after gently stirring, the mixture was placed at 65 ° C for 15 minutes to lyse the cells. After cooling this to room temperature, 23.5 ml of a 10% to 40% sucrose density gradient solution (0.8% NaCl, 0.02M Tris-HCl buffer (p
H8.0), 0.01M EDTA, 10% to 40% sucrose) were gently placed in 10 ml aliquots. Using this Hitachi Ultracentrifuge SCP85H, 4 ℃,
It was centrifuged at 26000 rpm for 3 hours. After centrifugation, 5 ml of each portion as close as possible to the bottom of the tube was collected using a Komagome pipette, and this was dialyzed overnight in 1 liter of TE buffer. The DNA solution thus obtained was subjected to the method of Frischauf et al. (Mehtods in Enzymology, 152, p.183, Academic pre
(ss1987), partially digest with the restriction enzyme Sau3AI, and
Place on a 0% to 40% sucrose density gradient solution, 20 ℃, 25000rp
Centrifugation was performed at m for 22 hours. After the ultracentrifugation is completed, make a hole in the bottom of the ultracentrifuge tube using an injection needle, collect about 0.5 ml of each falling sample, and electrophores a portion of each sample on an agarose gel for 15-20 kb. The fraction containing the DNA of 1. was collected by ethanol precipitation. 1μ from this fraction
Take a DNA solution corresponding to g and use λ-EMBL3 / BamHI vecto
r kit (Stratagene) λ-EMBL3 vector 1 μg
And ligated at 16 ° C overnight according to the protocol. This was packaged using GIGAPACK GOLD (Stratagene) according to its protocol. 50 μl of the obtained packaging solution was added to λ-EMBL3 / Bam
The host strain P2392 attached to the HIvector kit was infected. That is, 1 platinum loop of P2392 was added to 5 ml of TB medium (1% bactotryptone, 0.5% NaCl, 0.2% maltose, pH 7.4).
Incubate overnight in 50 ml of TB medium and incubate to 50 ml of TB medium until OD600 = 0.5. After cooling in an ice bath, collect the cells and collect 15 ml of ice-cold 10 mM sulfuric acid. It was suspended in magnesium liquid. To 1 ml of this bacterial solution, 0.95 ml of SM solution (0.1M
Sodium chloride, 10 mM magnesium sulfate, 50 mM Tris-hydrochloric acid buffer (pH 7.5), 0.01% gelatin) and 50 μl of the packaging solution were added, and the mixture was gently stirred and kept at 37 ° C. for 15 minutes. 200 μl of this solution was dispensed in 7 ml aliquots and BBL soft agar medium (1% Triptic
ase peptone (BBL), 0.5% NaCl, 0.5% agarose)
After gently stirring, the mixture was spread on a BBL plate having a diameter of 15 cm (the same as the BBL soft agar medium except that 1.5% agar was added instead of 0.5% agarose). this
After culturing at 37 ° C. for 8 hours, a phage library with a total size of about 30,000 was prepared.

Brewer's yeast chromosome prepared as described above
A DNA that hybridizes with the yeast ATCC60715 strain FLO8 gene obtained in Example 1 was cloned from the DNA library. Specifically, first, using the P2392 strain (Stratagene) as a host, a brewery yeast chromosomal DNA library was described in "Plant Biotechnology Experimental Manual, Cloning and Sequence" (Rural Culture Co., Ltd., (1989)). In a different way, NZ
The cells were cultured on a YM plate (1% NZ amine (Wako Pure Chemical Industries), 0.5% yeast extract, 0.5% NaCl, 10 mM MgSO4, lower layer was 1.5% agar, upper layer was 0.6% agarose). Total of about 2000
0 plaques were blotted onto nylon membrane Hybond N + (Amersham) according to the protocol. The 1574 bp HindIII fragment from pF415-1 (see Figure 1) was labeled by the method described in Example 1 and hybridized and washed according to the Hybond N + protocol. Autoradiography was performed on X-ray film by exposing the signal from the hybrid band to obtain a total of 10 positive plaques. From the plaques amplified in liquid culture from these plaques, DNA was prepared by the method described in "Plant Biotechnology Experimental Manual, Cloning and Sequencing" (Rural Culture Co., (1989)) and digested with the restriction enzyme HindIII. Electrophoresed on 1% agarose and observed, fragments with a common length were found, so it was judged that they all acquired the gene at the same locus. DNA of one plaque in it
Was digested with the restriction enzyme XhoI, and the 11.2 kb fragment was cloned into the XhoI site of pRS415 (Stratagene). The resulting plasmid pLF8 showed the same pattern of HindIII fragment length as pF415-1. pLF8 was added to the non-aggregating experimental yeast YPH500 strain (Stratagene) by the method of Ito et al. (J. Bacterio
l., 153, 163-168, (1983))
The transformant was transformed into agglutinative. From this, DN that hybridizes with the FLO8 gene obtained from brewer's yeast
A is judged to have the same aggregating ability as the FLO8 gene.

[0072]

[Sequence list]

SEQ ID NO: 1 Sequence length: 729 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Asn Cys Lys Asn Thr Leu Asn Glu Tyr Ile Phe Asp Phe Leu Thr 1 5 10 15 Lys Ser Ser Leu Lys Asn Thr Ala Ala Ala Phe Ala Gln Asp Ala His 20 25 30 Leu Asp Arg Asp Lys Gly Gln Asn Pro Ile Asp Gly Ser Lys Ser Lys 35 40 45 Glu Asn Asn Gly Asn Gln Asn Thr Phe Ser Lys Val Val Asp Thr Pro 50 55 60 Gln Gly Phe Leu Tyr Glu Trp Trp Gln Ile Phe Trp Asp Ile Phe Asn 65 70 75 80 Thr Ser Ser Ser Arg Gly Gly Ser Glu Phe Ala Gln Gln Tyr Tyr Gln 85 90 95 Leu Val Leu Gln Glu Gln Arg Gln Glu Gln Ile Tyr Arg Ser Leu Ala 100 105 110 Val His Ala Ala Arg Leu Gln His Asp Ala Glu Arg Arg Gly Glu Tyr 115 120 125 Ser Asn Glu Asp Ile Asp Pro Met His Leu Ala Ala Met Met Leu Gly 130 135 140 Asn Pro Met Ala Pro Ala Val Gln Met Arg Asn Val Asn Met Asn Pro 145 150 155 160 Ile Pro Ile Pro Met Val Gly Asn Pro Ile Val Asn Asn Phe Ser Ile 165 170 175 Pro Pro Tyr Asn Asn Ala Asn Pro Thr Thr Gly Ala Thr Ala Val Ala 180 185 190 Pro Thr Ala Pro Pro Ser Gly Asp Phe Thr Asn Val Gly Pro Thr Gln 195 200 205 Asn Arg Ser Gln Asn Val Thr Gly Trp Pro Val Tyr Asn Tyr Pro Met 210 215 220 Gln Pro Thr Thr Glu Asn Pro Val Gly Asn Pro Cys Asn Asn Asn Thr 225 230 235 240 Thr Asn Asn Thr Thr Asn Asn Lys Ser Pro Val Asn Gln Pro Lys Ser 245 250 255 Leu Lys Thr Met His Ser Thr Asp Lys Pro Asn Asn Val Pro Thr Ser 260 265 270 Lys Ser Thr Arg Ser Arg Ser Ala Thr Ser Lys Ala Lys Gly Lys Val 275 280 285 Lys Ala Gly Leu Val Ala Lys Arg Arg Arg Lys Asn Asn Thr Ala Thr 290 295 300 Val Ser Ala Gly Ser Thr Asn Ala Gly Ser Pro Asn Ile Thr Thr Pro 305 310 315 320 Gly Ser Thr Thr Ser Glu Pro Ala Met Val Gly Ser Arg Val Asn Lys 325 330 335 Thr Pro Arg Ser Asp Ile Ala Thr Asn Phe Arg Asn Gln Ala Ile Ile 340 345 350 Phe Gly Glu Glu Asp Ile Tyr Ser Asn Ser Lys Ser Ser Pro Ser Leu 355 360 365 Asp Gly Thr Ser Pro Ser Ala Leu Val Ser Lys Gln Pro Thr Lys Val 370 375 380 Arg Lys Asn Thr Lys LysAla Ser Thr Ser Ala Phe Pro Val Glu Ser 385 390 395 400 Thr Asn Lys Leu Gly Gly Asn Ser Val Val Thr Gly Lys Lys Arg Ser 405 410 415 Pro Pro Asn Thr Arg Val Ser Arg Arg Lys Ser Thr Pro Ser Val Ile 420 425 430 Leu Asn Ala Asp Ala Thr Lys Asp Glu Asn Asn Met Leu Arg Thr Phe 435 440 445 Ser Asn Thr Ile Ala Pro Asn Ile His Ser Ala Pro Pro Thr Lys Thr 450 455 460 Ala Asn Ser Leu Pro Phe Pro Gly IleAsn Leu Gly Ser Phe Asn Lys 465 470 475 480 Pro Ala Val Ser Ser Pro Leu Ser Ser Val Thr Glu Ser Cys Phe Asp 485 490 495 Pro Glu Ser Gly Lys Ile Ala Gly Lys Asn Gly Pro Lys Arg Ala Val 500 505 510 Asn Ser Lys Val Ser Ala Ser Ser Pro Leu Ser Ile Ala Thr Pro Pro 515 520 525 Ser Gly Asp Ala Gln Lys Gln Arg Ser Ser Lys Val Pro Gly Asn Val 530 535 540 Val Ile Lys Pro Pro His Gly Phe Ser Thr Thr Asn Leu Asn Ile Thr 545 550 555 560 Leu Lys Asn Ser Lys Ile Ile Thr Ser Gln Asn Asn Thr Val Ser Gln 565 570 575 Glu Leu Pro Asn Gly Gly Asn Ile Leu Glu Ala Gln Val Gly Asn Asp 580 585 590 Ser Arg Ser Ser Lys Gly Asn Arg Asn Thr Leu Ser Thr Pro Glu Glu 595 600 605 Lys Lys Pro Ser Ser Asn Asn Gln Gly Tyr Asp Phe Asp Ala Leu Lys 610 615 620 Asn Ser Ser Ser Leu Leu Phe Pro Asn Gln Ala Tyr Ala Ser Asn Asn 625 630 635 640 Arg Thr Pro Asn Glu Asn Ser Asn Val Ala Asp Glu Thr Ser Ala Ser 645 650 655 Thr Asn Ser Gly Asp Asn Asp Asn Thr Leu Ile Gln Pro Ser Ser Asn 660 665 670 Val Gly Thr Thr Leu Gly Pro Gln Gln Thr Ser Thr Asn Glu Asn Gln 675 680 685 Asn Val His Ser Gln Asn Leu Lys Phe Gly Asn Ile Gly Met Val Glu 690 695 700 Asp Gln Gly Pro Asp Tyr Asp Leu Asn Leu Leu Asp Thr Asn Glu Asn 705 710 715 720 Asp Phe Asn Phe Ile Asn Trp Glu Gly 725

SEQ ID NO: 2 Sequence length: 2846 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Saccharomyces cerevisiae
s cerevisiae) Strain name: ATCC60715 Sequence features Characteristic symbol: CDC Location: 267. ． 2456 method to determine the characteristics: E SEQ CACGAAGACG TTTATAGACA TAAATAAAGA GGAAACGCAT TCCGTGGTAG AATGAGTTAT 60 AAAGTGAATA GTTCGTATCC AGATTCAATT CCTCCCACGG AACAACCGTA CATGGCAAGC 120 CAGTATAAAC AAGATTTGCA GAGTAATATT GCAATGGCAA CGAATAGTGA ACAGCAGCGA 180 CAACAACAGC AGCAACAGCA GCAGCAGCAG CAGCAGCAGC AGTGGATAAA TCAACTACGG 240 CGGAAAATTC GGATTTGAAG GAAAAA ATG AAC TGC AAG AAT ACG CTC AAT GAG 293 Met Asn Cys Lys Asn Thr Leu Asn Glu 1 5 TAC ATA TTT GAC TTT CTT ACG AAG TCG TCT TTG AAA AAC ACT GCA GCA 341 Tyr Ile Phe Asp Phe Leu Thr Lys Ser Ser Leu Lys Asn Thr Ala Ala 10 15 20 25 GCC TTT GCT CAA GAT GCG CAC CTA GAT AGA GAC AAA GGC CAA AAC CCA 389 Ala Phe Ala Gln Asp Ala His Leu Asp Arg Asp Lys Gly Gln Asn Pro 30 35 40 ATC GAC GGA TCC AAA TCT AAA GAA AAC AAT GGT AAC CAG AAT ACG TTC 437 Ile Asp Gly Ser Lys Ser Lys Glu Asn Asn Gly Asn Gln Asn Thr Phe 45 50 55 TCG AAG GTA GTA GAT ACA CCT CAA GGC TTT TTG TAT GAA TGG TGG CAA 485 Ser Lys Val Val Asp Thr Pro Gln Gly Phe Leu Tyr Glu Trp Trp Gln 60 65 70 A TA TTC TGG GAC ATC TTT AAT ACC AGT TCT TCC AGA GGT GGC TCA GAG 535 Ile Phe Trp Asp Ile Phe Asn Thr Ser Ser Ser Arg Gly Gly Ser Glu 75 80 85 TTC GCT CAG CAA TAT TAT CAA CTA GTT CTT CAA GAA CAA AGG CAG GAA 581 Phe Ala Gln Gln Tyr Tyr Gln Leu Val Leu Gln Glu Gln Arg Gln Glu 90 95 100 105 CAA ATA TAT AGA AGC TTG GCT GTT CAT GCG GCA AGG CTA CAA CAC GAT 629 Gln Ile Tyr Arg Ser Leu Ala Val His Ala Ala Arg Leu Gln His Asp 105 110 115 GCA GAA CGA AGA GGG GAA TAT AGT AAC GAG GAC ATA GAC CCC ATG CAC 677 Ala Glu Arg Arg Gly Glu Tyr Ser Asn Glu Asp Ile Asp Pro Met His 125 130 135 TTG GCT GCT ATG ATG CTA GGA AAT CCT ATG GCA CCT GCG GTT CAA ATG 725 Leu Ala Ala Met Met Leu Gly Asn Pro Met Ala Pro Ala Val Gln Met 140 145 150 CGC AAT GTT AAT ATG AAC CCT ATA CCA ATT CCT ATG GTT GGT AAC CCT 773 Arg Asn Val Asn Met Asn Pro Ile Pro Ile Pro Met Val Gly Asn Pro 155 160 165 ATC GTT AAT AAT TTT TCC ATT CCA CCA TAC AAT AAT GCA AAC CCC ACG 821 Ile Val Asn Asn Phe Ser Ile Pro Pro Tyr Asn Asn Ala Asn Pro Thr 170 17 5 180 185 ACT GGT GCA ACT GCT GTT GCT CCC ACA GCG CCG CCT TCC GGC GAT TTT 869 Thr Gly Ala Thr Ala Val Ala Pro Thr Ala Pro Pro Ser Gly Asp Phe 190 195 200 ACA AAT GTA GGG CCA ACC CAG AAT CGG AGT CAA AAC GTT ACT GGC TGG 917 Thr Asn Val Gly Pro Thr Gln Asn Arg Ser Gln Asn Val Thr Gly Trp 205 210 215 CCA GTC TAT AAT TAT CCA ATG CAA CCC ACT ACG GAA AAT CCA GTG GGA 965 Pro Val Tyr Asn Tyr Pro Met Gln Pro Thr Thr Glu Asn Pro Val Gly 220 225 230 AAC CCG TGT AAC AAT AAT ACC ACA AAT AAT ACA ACT AAT AAC AAA TCT 1013 Asn Pro Cys Asn Asn Asn Thr Thr Asn Asn Thr Thr Asn Asn Lys Ser 235 240 245 CCA GTG AAC CAA CCT AAA AGT TTA AAA ACT ATG CAT TCA ACA GAT AAA 1061 Pro Val Asn Gln Pro Lys Ser Leu Lys Thr Met His Ser Thr Asp Lys 250 255 260 265 CCA AAT AAT GTC CCG ACG TCA AAA TCT ACA AGA AGT AGA TCT GCA ACC 1109 Pro Asn Asn Val Pro Thr Ser Lys Ser Thr Arg Ser Arg Ser Ala Thr 270 275 280 TCA AAA GCG AAG GGT AAA GTT AAA GCC GGT CTA GTG GCT AAG AGA CGA 1157 Ser Lys Ala Lys Gly Lys Val Lys Ala Gly Leu Val Al a Lys Arg Arg 285 290 295 AGA AAA AAT AAT ACC GCT ACA GTT TCC GCG GGA TCG ACG AAC GCT GGT 1205 Arg Lys Asn Asn Thr Ala Thr Val Ser Ala Gly Ser Thr Asn Ala Gly 300 305 310 TCG CCA AAT ATT ACC ACA CCA GGC TCA ACA ACA AGT GAA CCC GCT ATG 1253 Ser Pro Asn Ile Thr Thr Pro Gly Ser Thr Thr Ser Glu Pro Ala Met 315 320 325 GTA GGT TCA AGA GTA AAT AAG ACT CCA AGA TCA GAT ATT GCT ACT AAC 1301 Val Gly Ser Arg Val Asn Lys Thr Pro Arg Ser Asp Ile Ala Thr Asn 330 335 340 345 TTC CGC AAT CAA GCA ATA ATA TTT GGC GAG GAA GAT ATT TAT TCT AAT 1349 Phe Arg Asn Gln Ala Ile Ile Phe Gly Glu Glu Asp Ile Tyr Ser Asn 350 355 360 TCC AAA TCT AGC CCA TCG TTG GAT GGA ACA TCA CCT TCC GCT TTA GTT 1397 Ser Lys Ser Ser Pro Ser Leu Asp Gly Thr Ser Pro Ser Ala Leu Val 365 370 375 TCT AAA CAG CCC ACA AAG GTA AGG AAA AAT ACA AAA AAG GCA TCC ACC 1445 Ser Lys Gln Pro Thr Lys Val Arg Lys Asn Thr Lys Lys Ala Ser Thr 380 385 390 TCA GCT TTT CCA GTA GAG TCT ACG AAT AAA CTC GGT GGC AAC AGC GTG 1493 Ser Ala Phe Pro Val Glu Ser Thr Asn Lys Leu Gly Gly Asn Ser Val 395 400 405 GTG ACA GGT AAA AAG CGC AGT CCC CCT AAC ACT AGA GTG TCG AGG AGG 1541 Val Thr Gly Lys Lys Arg Ser Pro Pro Asn Thr Arg Val Ser Arg Arg 410 415 420 425 AAA TCC ACT CCT TCT GTT ATT CTG AAT GCT GAT GCC ACT AAG GAT GAG 1589 Lys Ser Thr Pro Ser Val Ile Leu Asn Ala Asp Ala Thr Lys Asp Glu 430 435 440 AAT AAT ATG TTA AGA ACA TTC TCG AAT ACT ATT GCT CCG AAT ATT CAT 1637 Asn Asn Met Leu Arg Thr Phe Ser Asn Thr Ile Ala Pro Asn Ile His 445 450 455 TCC GCT CCG CCC ACT AAA ACT GCG AAT TCT CTC CCT TTT CCA GGT ATA 1685 Ser Ala Pro Pro Thr Lys Thr Ala Asn Ser Leu Pro Phe Pro Gly Ile 460 465 470 AAT TTG GGA AGT TTC AAC AAG CCG GCT GTA TCC AGT CCA TTA TCT TCA 1737 Asn Leu Gly Ser Phe Asn Lys Pro Ala Val Ser Ser Pro Leu Ser Ser 475 480 485 GTG ACA GAG AGT TGC TTC GAT CCA GAA AGT GGC AAG ATT GCC GGA AAG 1781 Val Thr Glu Ser Cys Phe Asp Pro Glu Ser Gly Lys Ile Ala Gly Lys 490 495 500 505 AAT GGA CCC AAG CGA GCA GTA AAC TCA AAA GTT TCG GCA TCA TCC CCA 1829 Asn Gly Pro Lys Arg Ala Val Asn Ser Lys Val Ser Ala Ser Ser Pro 510 515 520 TTA AGC ATA GCA ACA CCT CCG TCT GGT GAC GCT CAG AAG CAA AGA AGT 1877 Leu Ser Ile Ala Thr Pro Pro Ser Gly Asp Ala Gln Lys Gln Arg Ser 525 530 535 TCT AAG GTA CCA GGA AAC GTG GTT ATA AAG CCG CCA CAT GGG TTT TCA 1925 Ser Lys Val Pro Gly Asn Val Val Ile Lys Pro Pro His Gly Phe Ser 540 545 550 ACC ACC AAT TTG AAT ATT ACT TTA AAG AAC TCT AAA ATA ATC ACT TCA 1973 Thr Thr Asn Leu Asn Ile Thr Leu Lys Asn Ser Lys Ile Ile Thr Ser 555 560 565 CAG AAT AAT ACA GTA TCC CAA GAA TTG CCG AAT GGG GGA AAC ATA CTG 2021 Gln Asn Asn Thr Val Ser Gln Glu Leu Pro Asn Gly Gly Asn Ile Leu 570 575 580 585 GAG GCG CAA GTA GGC AAT GAT TCA AGA AGT AGT AAA GGC AAT CGT AAC 2069 Glu Ala Gln Val Gly Asn Asp Ser Arg Ser Ser Lys Gly Asn Arg Asn 590 595 600 ACA TTA TCT ACT CCA GAG GAA AAA AAG CCG AGT AGT AAT AAT CAA GGA 2117 Thr Leu Ser Thr Pro Glu Glu Lys Lys Pro Ser Ser Asn Asn Gln Gly 605 610 615 TAT GAT TTT GAC GCC CTC AAA AAT TCA AGT TCT TTG TTG TTT CC T AAT 2165 Tyr Asp Phe Asp Ala Leu Lys Asn Ser Ser Ser Leu Leu Phe Pro Asn 620 625 630 CAA GCT TAT GCT TCT AAC AAT AGA ACA CCA AAC GAG AAT TCA AAT GTT 2213 Gln Ala Tyr Ala Ser Asn Asn Arg Thr Pro Asn Glu Asn Ser Asn Val 635 640 645 GCT GAT GAA ACC TCT GCA TCT ACA AAT AGT GGC GAT AAT GAT AAC ACA 2261 Ala Asp Glu Thr Ser Ala Ser Thr Asn Ser Gly Asp Asn Asp Asn Thr 650 655 660 665 TTA ATT CAG CCC TCA TCC AAT GTG GGT ACA ACT TTG GGT CCT CAG CAA 2309 Leu Ile Gln Pro Ser Ser Asn Val Gly Thr Thr Leu Gly Pro Gln Gln 670 675 680 ACC AGT ACT AAT GAA AAT CAG AAT GTA CAC TCT CAG AAC TTG AAG TTT 2357 Thr Ser Thr Asn Glu Asn Gln Asn Val His Ser Gln Asn Leu Lys Phe 685 690 695 GGG AAT ATT GGT ATG GTT GAA GAC CAA GGA CCG GAT TAC GAT CTC AAT 2405 Gly Asn Ile Gly Met Val Glu Asp Gln Gly Pro Asp Tyr Asp Leu Asn 700 705 710 GAA GGC TTA CTG GAT ACA AAT GAA AAT GAT TTC AAT TTT ATT AAT TGG 2453 Leu Leu Asp Thr Asn Glu Asn Asp Phe Asn Phe Ile Asn Trp Glu Gly 715 720 725 TGA AGTC ACGTGTTGAG TATTATAATA AAAAATAA AA ACTCTTAATG ACAGGAAACA 2510 TAATAAAAAG CTAGGAGAAT GGAGTTACGT ATCTGACATT TCGCTAAATT TGGGGATGG 2570 TTTAAGCCCT GTGAACTGAA CCATTTTCAC AGCAACGACT TTTTATAAAC GGTTTTTTA 2630 GATATTTGTT TTGTGATAGC TTTATTTTTC TTCCATGATA AGATAGAAGT GTTTAATTT 2690 TAGATAAGAT ATTGTACTTA TAAAAAGAAC ATTTGAACCA TTAAATACAG GCACTTTTC 2750 GGTTGATGTA CACTCATTGA AGTAGCTTGG CATAATGGTA AATTAACTTT TGACATTAC 2810 ATGATCATGA TTTACGATGA CCGTCTCGTC TCTTTT 2846

SEQ ID NO: 3 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GAGGGCTGAA TTAATGTGTT 10

SEQ ID NO: 4 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGAAAAATTA TTAACGATAG 10

SEQ ID NO: 5 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AAGTAGTAAA GGCAATCGTA 10

SEQ ID NO: 6 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TTTAATACCA GTTCTTCCAG 10

SEQ ID NO: 7 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTTGAGCTCA GCAGCAGCAG TGGATAAATC AACT 34

SEQ ID NO: 8 Sequence Length: 33 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GCTCTAGAAT TATGTTTCCT GTCATTAAGA GTT 33

SEQ ID NO: 9 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GATGAAACTG TCATTGTTGT CAAA 24

SEQ ID NO: 10 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCGTTTCAGC AGCTAAAGTA T 21

[Brief description of drawings]

FIG. 1 shows the insert fragment of plasmid pF415-1 (full length
A restriction map of 11.2kb) is shown.

FIG. 2 shows the procedure for integration mapping of plasmid p7H12.

FIG. 3 is a yeast YF191LEU in which the FLO8 gene has been disrupted.
The procedure for producing the strain is shown below.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 15/09 ZNA C12R 1: 865) (C12N 1/19 C12R 1: 865) C12R 1: 865 )

Claims (9)

[Claims] 1. A FLO8 protein having an amino acid sequence substantially as shown in SEQ ID NO: 1 in the sequence listing. 2. A FLO8 gene DNA containing a base sequence encoding a FLO8 protein having substantially the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing. 3. F having an activity of imparting aggregating property to yeast
A DNA containing a base sequence encoding LO8 protein and selected from the group consisting of (a) and (b) below. (a) Hybridizes with a DNA containing the sequence from the 267th base to the 2453th base of the base sequence shown in SEQ ID NO: 2 or its complementary strand, and (b) the DNA of (a) or its fragment. DNA to soybean 4. The plasmids pF415-1, p7H12, pNEOF8,
A DNA containing a nucleotide sequence encoding a FLO8 protein which is incorporated into pYESF8 or pLF8 and has an activity of imparting aggregating property to yeast. 5. The DNA according to claim 4, which is a DNA fragment of about 11.2 kb which is incorporated into the plasmid pF415-1 and has the following restriction map. Embedded image 6. D according to any one of claims 2 to 5.
A vector containing NA. 7. D according to any one of claims 2 to 5.
A method for producing a yeast having an imparted or enhanced cohesiveness, which comprises introducing NA. 8. D according to any one of claims 2 to 5.
A method for producing a yeast having a deletion or a decrease in agglutination property, which comprises introducing a DNA in which the ability to express FLO8 protein is deleted or decreased by destroying NA. 9. D according to any one of claims 2 to 5.
A method for deleting or reducing yeast aggregability by suppressing the expression of NA.