JPH10234368A - Cohesive alcoholic fermentation yeast and breeding thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To breed a cohesive alcoholic fermentation yeast and rationalize the production of an alcohol without adversely affecting the alcohol productivity by transferring a specific cohesive gene expression cassette into an optional marker gene region on a cheomosome of an alcoholic fermentation yeast. SOLUTION: A cohesive gene expression cassette composed by arranging a cohesive gene containing a control region so as to segment a marker gene region or substitute the internal sequence thereof is transferred into an optional marker gene region on a chromosome of an alcoholic fermentation yeast to breed a cohesive alcoholic fermentation yeast such as an FSC27 strain (FERM P-16023). The marker gene is preferably a gene participating in the biosynthesis of uracil.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a cohesive alcohol-fermenting yeast, and more particularly to a cohesive alcohol-fermenting yeast in which a cohesive gene is introduced into an alcohol-fermenting yeast by genetic manipulation and a method of breeding the yeast.

[0002]

2. Description of the Related Art Alcohol production by fermentation is performed by using a fermentation yeast, Saccharomyces cerevisiae, which has a particularly high alcohol productivity (hereinafter referred to as alcohol fermentation yeast), based on a batch fermentation method. ing.

In a conventional batch fermentation method, first, a preculture solution of the alcohol-fermenting yeast is added to a fermenter together with a raw material such as molasses and cultured under a predetermined condition. Decomposed to produce alcohol. The alcohol produced here is distilled and collected by heating the culture solution containing the yeast. on the other hand,
The yeast in the culture solution after the alcohol has been collected will be killed by heating. Therefore, when alcohol is to be produced again, the process is started by preparing a preculture liquid to be added to the fermenter.

[0004] However, when the production scale is large, such as in industrial alcohol production, a considerable amount of this pre-culture solution is also required, so that its preparation requires considerable labor.
Therefore, various techniques have been developed to reduce this labor. One of them is a repetitive batch method using flocculent yeast.

[0005] The flocculating yeast has a property that individual cells non-sexually interact with each other to form clumps and sediment to the liquid bottom in a stationary medium (hereinafter, this property is referred to as flocculating property). Refers to yeast having. The repetitive batch method using the flocculating yeast utilizes the flocculating property of the flocculating yeast to separate the yeast and the culture supernatant before collecting the alcohol by distillation,
It is characterized in that the isolated yeast can be repeatedly used for the next alcohol production.

[0006] Specifically, as in the prior art, a raw material and yeast are added to a fermenter and cultured under predetermined conditions to produce alcohol. After completion of the culturing step, the yeast is aggregated and settled on the liquid bottom by allowing the culture solution to stand still. In the alcohol recovery step, only the supernatant of the culture solution is separated, and the supernatant is distilled to recover the alcohol. On the other hand, the yeast left in the fermenter is repeatedly used for the next alcohol production by adding a new raw material. As a result, it is possible to omit the preparation of the conventional pre-culture solution, so that it is possible to improve the working efficiency and production efficiency in alcohol production.

As described above, flocculent yeasts useful in alcohol production are mostly obtained from the natural world by screening using flocculence as an index. However, flocculent yeasts obtained from the natural world, although having flocculating properties, are often insufficient in terms of alcohol productivity, and lacked practicality as yeasts for alcohol production, especially industrial alcohol production. .

[0008] From such a problem, in recent years, attempts have been made to artificially produce yeast having both this cohesiveness and high alcohol productivity (hereinafter referred to as cohesive alcohol-fermented yeast). One example is disclosed in Japanese Patent Application Laid-Open No. 5-236.
No. 942 discloses a technique for breeding a cohesive alcohol-fermenting yeast using a cell fusion method. That is, a cohesive alcohol-fermenting yeast is produced by cell fusion of a conventionally used alcohol-fermenting yeast having a high alcohol-producing ability and a cohesive yeast having a cohesive property.

[0009]

However, the above-mentioned cell fusion method has a problem that generally, both cells to be fused are not always completely fused. for that reason,
For strains with high polyploidy, such as yeasts with high alcohol-producing ability used for alcohol production, etc., to confirm whether the genetic information bearing the high alcohol-producing ability of interest has been transmitted to the fused cells Therefore, a large number of fusion strains must be screened, which requires a great deal of labor.

[0010] Even if the genetic information of both yeasts is transmitted, unexpected properties other than the purpose provided for the flocculent yeast are expected to be transmitted to the fused yeast, and this property may affect alcohol production and the like. Is also conceivable.

On the other hand, with the recent development of molecular biology, gene level analysis of this cohesiveness has been promoted, and genes involved in this cohesiveness (hereinafter referred to as cohesive genes).
Have been isolated and identified. One of these genes is the FLO1 gene, the sequence of which is described in
No. 9372.

Accordingly, the present inventors have developed a practical cohesive alcohol-fermenting yeast in which only a cohesiveness is imparted to a yeast having a high alcohol-producing ability and a method of breeding the yeast.

[0013]

The method for breeding cohesive alcohol-fermenting yeast of the present invention is carried out by introducing a cohesive gene expression cassette, which is a foreign DNA, into an arbitrary marker gene region on the chromosome of alcohol-fermenting yeast. I do.

The agglutinating gene expression cassette used in this method is constituted by arranging aggregating genes including a control region so as to divide the predetermined gene region or replace the internal region thereof. In arranging the aggregating gene, the transcription direction of the aggregating gene and the marker gene may be either the same direction or the opposite direction, but when the expression efficiency of the aggregating gene differs depending on the direction, The direction in which the expression of the aggregating gene is efficiently performed is selected and used.

As described above, by providing the sequence of the gene region to be separated or replaced at both ends of the expression cassette, homologous recombination occurs in the yeast with the marker gene region on the chromosome, and the aggregating gene is converted into the target gene. It becomes possible to introduce efficiently into the area.

According to the above-mentioned method, since only the target genetic information, that is, only the aggregating gene, is reliably introduced into the alcohol-fermenting yeast, there is a possibility that the alcohol-producing effect may be affected as in the conventional cell fusion method. It is possible to prevent the introduction of certain other genetic information. According to this method, there is no problem even if the introduced yeast is polyploid,
Rather, since the number of target marker gene regions is increased, the efficiency of introducing an aggregating gene may be increased.

Furthermore, according to the present invention, unlike the case where the aggregating gene is present on the chromosome as in the conventional cell fusion method, the aggregating gene is present on a cassette comprising a relatively short DNA sequence. Therefore, the agglutinating gene on this cassette can be modified into a mutant form having an appropriate aggregating property by variously modifying the cassette before introduction.

As the aggregating gene on the above-mentioned expression cassette, various known aggregating genes can be used. However, for the reason described later, it is preferable to use an aggregating gene derived from a homologous yeast, ie, Saccharomyces cerevisiae. preferable. In the case of an aggregating gene derived from Saccharomyces cerevisiae, for example, the above-mentioned FLO1L gene (Japanese Patent Application Laid-Open No. 7-509372) can be suitably used.

In addition, various control regions that regulate the expression of the aggregating gene can be selected. The control region that can be used here can be arbitrarily selected from existing control regions, but it is preferable to use a control region derived from the homologous yeast Saccharomyces cerevisiae for the reasons described below. Examples of those already known as control regions derived from Saccharomyces cerevisiae include, for example, ADH1
(Alcohol dehydrogenase 1).

The method of breeding yeast for cohesive alcohol fermentation of the present invention is based on so-called genetic recombination. It needs to be implemented under equipment. In addition, when alcohol is produced using the cohesive alcohol-fermenting yeast produced here, it is necessary to implement the method according to the Ministry of International Trade and Industry's guidelines for industrializing recombinant DNA technology.

However, when the foreign DNA to be introduced, that is, the aggregating gene expression cassette is derived from an organism that belongs to the taxonomically the same species as the alcohol-fermenting yeast as the receptor, it is treated as a self-cloning system, This is outside the framework of genetic recombination described above. As a result, the implementation of the above-described breeding method and the implementation of the alcohol production method using the yeast bred by this method can be performed under general facilities, that is, under existing facilities. For this reason, in the present invention, it is preferable to prepare an aggregating gene expression cassette from DNA derived from Saccharomyces cerevisiae of the same species as alcohol fermentation yeast,
It is economical.

On the other hand, with respect to marker genes on the chromosome of alcohol-fermenting yeast, the expression cassette is introduced into the phenotype so that the transductant into which the aggregating gene expression cassette has been introduced can be easily screened. Is preferably a selectable marker gene that changes. That is, by introducing the expression cassette, the marker gene is destroyed, and as a result, the phenotype of the transformant changes, and the change can be easily selected using the change as an index. Changes in this phenotype include:
These include changes in auxotrophy, changes in sensitivity to particular drugs, and the like. More preferably, as the marker gene, a gene that can be positively selected to acquire resistance to a specific drug or the like when disrupted by introduction of an aggregating gene expression cassette or the like is used. That is,
Here, the positive selection refers to selecting a gene that has acquired resistance to a specific drug and has become viable by modification of a specific gene (here, a marker gene).

When a gene capable of positive selection is used, the gene sequence is disrupted by transforming and culturing on a medium containing a specific drug, for example, by introducing an aggregating gene expression cassette. Only the yeast strain becomes viable. As a result, the desired transformed cells can be easily obtained.

The genes which can be selected positively include, for example, URA3 gene, LYS2 gene,
Examples include the CAN1 gene and the CYH2 gene, and specific drugs for these genes correspond to 5-fluoroorotic acid (5-FOA), α-aminoadipic acid, canavanine, and cycloheximide, respectively.

The drugs listed here have the property of inducing mutations in the corresponding genes, and are therefore particularly ploidy yeasts (in many cases, such as alcohol-fermenting yeasts used for alcohol production). (Ploid yeast).

That is, in the present invention, when the alcohol-fermenting yeast to be transduced is, for example, a diploid, it is not necessary to introduce an aggregating gene expression cassette into each marker gene on a plurality of chromosomes, and at least one marker The introduction of the cassette into the gene is sufficient for imparting cohesiveness. However, since the other marker gene remains in an intact state, it is difficult to select such a yeast, but by effectively utilizing the above properties, the yeast can be easily selected. Becomes That is, the cassette is introduced into one marker gene, and a mutation is introduced into the other marker gene at a fixed rate based on the drug. As a result, a transformed yeast in which both marker genes are disrupted is formed to acquire resistance to the drug, and it is possible to select the yeast from other yeasts on a medium containing the drug.

At this time, yeast cells into which the cassette has not been introduced cannot grow on a medium containing the drug unless mutations occur in all marker genes on a plurality of chromosomes. Expected to be low. That is, it can be said that the strain that grows in the medium after the cassette introduction operation has a very high probability that the cassette has been introduced as intended into at least one position of the marker gene on the chromosome.

In the cohesive alcohol-fermenting yeast bred by the above method, the cohesive gene expression cassette is introduced into the marker gene of the alcohol-fermenting yeast, and only the cohesiveness is added while maintaining the original alcohol-producing ability. Will be. For example, in the case of a yeast having a high alcohol-producing ability, cohesiveness is further added while maintaining its properties, so that a yeast more useful for alcohol production and the like is formed.

[0029]

Embodiments of the present invention will be described below with reference to the drawings.

(1) Preparation of an aggregating gene expression cassette An aggregating expression cassette can be prepared by a conventional method (for example, Molecular Clon
ing, A LABORATORY MANUAL, Cold Spring Harbor Labor
According to atory Press (1989)), an aggregating gene and a control DNA for expressing the aggregating gene can be appropriately linked and constructed so as to cleave the marker gene or replace it with an internal sequence.

As the aggregating gene, several known aggregating genes or modified versions of the aggregating gene can be used, but the present invention is not limited thereto. From the viewpoint of self-cloning, the aggregating gene used here is preferably derived from Saccharomyces cerevisiae. Aggregating genes of Saccharomyces cerevisiae include the FLO1, FLO5, and FLO8 genes. These aggregating genes may be prepared by isolating them from the chromosome, or may conveniently be those already isolated on a plasmid such as the FLO1 gene. Further, if the cohesiveness is effectively maintained, the existing cohesive gene can be modified and used.

The control region for expressing the aggregating gene contains a promoter necessary for the expression of the aggregating gene, a terminator required for terminating the expression, and if necessary, an enhancer for increasing the expression from the promoter. Can also be included. As such a control region, one that functions in Saccharomyces cerevisiae can be appropriately selected from the control regions of the aggregating gene itself and a number of currently known control regions. Similarly to the above, from the viewpoint of self-cloning, the control region used here is preferably prepared from Saccharomyces cerevisiae. Control regions derived from Saccharomyces cerevisiae include, for example, ADH1, GAP
DH, PHO, GAL, PGK, ENO, TRP, HI
A regulatory region of a gene such as P can be used.

As the marker gene, a selectable gene whose phenotype, for example, auxotrophy or sensitivity to a drug is changed when its gene sequence is disrupted, is preferably used. More preferably, a gene that can be positively selected so as to obtain resistance to a specific drug by disrupting its sequence is used. Such positively selectable genes include, for example, U
RA3 gene, LYS2 gene, CAN1 gene, CY
H2 gene and the like. Specific drugs for these genes include 5-fluoroorotic acid (5-FOA), α-aminoadipate, canavanine,
Cycloheximide corresponds to each. The URA
The auxotrophy of the 3 gene and the LYS2 gene also changes due to disruption of their sequences.

When the above-mentioned aggregating gene and control DNA are linked to a marker gene to form an aggregating gene expression cassette, for example, it can be performed in the following order. First, the control DNA is connected to the aggregating gene to form a unit capable of expressing the aggregating gene. Next, the unit formed here is connected so as to divide the marker gene region or replace the internal sequence thereof. In other words, the units may be connected in any order as long as the units can be connected inside the marker gene sequence while leaving the marker gene sequences on both sides.

As described above, if a marker gene sequence is provided on both sides, the connection direction between the unit and the marker gene may be the same direction or the opposite direction, and the expression efficiency depends on the selected DNA material. It is preferable to select a good connection direction.

The length of the marker gene provided on both sides may be changed within the range as long as the marker gene has at least a length capable of homologous recombination with the marker gene on the chromosome in yeast. be able to. In Saccharomyces cerevisiae, it has been shown that the minimum length required for recombination is 38-50 bp (Gene, 158, 113-117 (1995)).

When the expression cassette is amplified for use in a transformation method described later, it is convenient to insert the expression cassette into an appropriate plasmid and amplify it as a plasmid. Then, a portion of the expression cassette is cleaved from the amplified plasmid with a restriction enzyme, and the cleaved fragment is purified according to a conventional method to prepare a large amount of the expression cassette.

When a plasmid containing at least one of an aggregating gene, a control region and a marker gene already exists, the above-mentioned expression cassette is constructed on this plasmid, and finally the expression cassette is obtained from this plasmid. Can be prepared by excision and purification.

(2) Preparation of alcohol-fermenting yeast As the alcohol-fermenting yeast, an existing alcohol-fermenting yeast or a strain of Saccharomyces cerevisiae having any alcohol-producing ability can be selected and used. For example, the 396-9-6V strain (micro-organization accession number FE)
RMP-12804) can be used preferably.

When the above-mentioned alcohol-fermenting yeast is used for transformation described below, the alcohol-fermenting yeast is cultured in an appropriate liquid medium until an appropriate growth stage, and suspended in an appropriate solution to form a suspension. .

(3) Transformation of Aggregative Gene Expression Cassette into Alcohol Fermentation Yeast Transformation can be carried out according to a conventional method using any method such as a protoplast method, a lithium acetate method, or an electroporation method. it can.

The screening of the transformants is preferably carried out using a change in the phenotype based on the marker gene as an indicator. Here, when the marker gene is, for example, a gene involved in auxotrophy, it can be carried out on the basis of a change in the auxotrophy, and in the case of a gene involved in sensitivity to a drug, The change can be performed using the change in sensitivity to the drug as an index.

In order to carry out the above screening efficiently, it is desirable to use a gene that can be positively selected, such as the above-mentioned URA3 gene. For example, U
When the RA3 gene was used, the transformed yeast group was cultured on a medium containing 5-FOA, whereby
Only transformants in which the RA3 gene has been disrupted can grow on the medium. As a result, according to this method, it is possible to easily select only the transformant.

(4) Evaluation of Alcohol-Producing Ability The evaluation of the alcohol-producing action of the obtained transformant was carried out using a predetermined amount of a fermentation test medium (for example, synthetic medium; 1).
% Yeast extract, 2% peptone, 24% glucose,
And molasses medium; total molasses concentration 24% Indonesia molasses)
After inoculating a yeast culture solution having a predetermined concentration into the medium and shaking the culture at 32 ° C. for 6 days, the concentration of ethanol in the culture supernatant can be measured by gas chromatography.

The fermentation rate can be measured based on the amount of CO ₂ gas generated by this culture.

(5) Evaluation of agglutinability The agglutinability of the obtained transformant was evaluated by Johnston
(Yeast Genetics: Fundamental and Applied
Aspects, 205-224 (1983)).

(6) Genetic confirmation of cohesive alcohol-fermenting yeast In a transformant that has been confirmed to have alcohol-producing ability and cohesiveness, it is possible to confirm the presence of the introduced cohesive gene at the gene level. desirable. In this case,
For example, Southern hybridization or PCR (Po
The lymerase chain reaction) method can be suitably used.

In order to confirm that the aggregating gene is transcribed in the introduced yeast, for example, Northern hybridization can be suitably used. The above methods are used for Molecular Cloning, A LABORATOR
Y MANUAL, Cold Spring Harbor Laboratory Press (198
It can be implemented appropriately according to 9).

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0050]

EXAMPLE In this example, Saccharomyces cerevisiae strain 396-9-6V, which is an industrially high-alcohol-producing yeast, was used as an alcohol-fermenting yeast. -The FLO1L gene of S. cerevisiae was used.

[Example 1] Construction of an aggregating gene expression cassette An aggregating gene expression cassette was prepared from the YIp-dU-FLO1L plasmid shown in FIG. This YIp-dU-FLO1L is a restriction enzyme NcoI-Stu in the URA3 gene of the YIp5 plasmid.
The I region was constructed by replacing the ADH1 promoter-FLO1L-ADH1 terminator unit.

Specifically, as shown in FIG. 1, the YIp5 plasmid was digested with NcoI / StuI restriction enzymes, blunt-ended, and BAP-treated. Thereafter, the resultant was separated and extracted by agarose electrophoresis to obtain a 5.3 kb fragment.
YRpGL10-ADH1-FLO1L (Watari et al., Agricultural Biologic
al Chemistry, vol. 55, 1547 (1991)), digested with BamHI, blunt-ended similarly, and separated and extracted by agarose electrophoresis to obtain a 6.6 kb fragment. And the above 5.3k
The b fragment and this 6.6 kb fragment were ligated with T4 DNA ligase to construct (FIG. 1). Although not shown in detail in FIG. 1, this plasmid has a URA3 transcription direction and F
They are connected in the direction opposite to the transfer direction of L01L. Next, this plasmid was transformed into E. coli SURE (Stratagene
And digested with restriction enzymes PstI and SmaI to collect a cohesive gene expression cassette which is a linear DNA fragment.

[Example 2] Introduction of an aggregating gene expression cassette into a URA3 site on a chromosome [0053] Fig. 2 schematically shows a predicted mechanism that occurs in yeast in this transformation.

When the above DNA fragment is introduced into the 396-9-6V strain (A), the fragment is integrated into the URA3 region on one chromosome, and a uracil non-auxotrophic transformant (B) is formed. You. When this strain is selected with 5-FOA (fluoroorotic acid), a mutation is introduced into URA3 on another chromosome, and a uracil-requiring transformant (C) is formed.

Using the DNA fragment prepared above,
Transformation of ploidy alcohol-fermenting yeast strain 396-9-6V was carried out by Ito et al. (Journal of Bacteriology, Vol. 153, 163 (19).
83)) was carried out by partially modifying the lithium acetate method.

That is, 396-9-6V strain was added to 10 ml of Y
PD medium (1% yeast extract, 2% polypeptone,
The cells were cultured with shaking in 2% glucose) and collected by centrifugation during the logarithmic growth phase. The cells were washed with 5 ml of TE buffer and suspended in 0.25 ml of the same buffer. After adding an equal volume of a 0.2 M lithium acetate solution to the suspension, the suspension was gently shaken at 30 ° C. for 1 hour. 0.1 ml of this suspension was collected and transferred to a 1.5 ml microtube, and a DNA solution containing the above DNA fragment (5 μg
/ Μl) 10 μl was added. After the addition, the mixture was allowed to stand at 30 ° C. for 30 minutes. Furthermore, 70% PEG 4000 is added to this mixture.
Was added and mixed well, and allowed to stand at 30 ° C. for 1 hour. Next, the microtube containing the mixed solution was placed in the 42
It moved to a constant temperature water bath of ° C, and was left still for 5 minutes. The microtube was placed at room temperature, and the cells were allowed to cool to room temperature. After cooling, the cells were washed with sterile water and finally resuspended in 2 ml of YPD medium.

Example 3 Selection of Transformants First, the resuspension was cultured with shaking at 30 ° C. overnight, and only those strains that sedimented early after stirring, that is, those strains suggesting cohesiveness, were placed in a new medium. Transformation was repeated 10 times to concentrate the transformants. This concentrate was then purified with SD containing 1 mg / ml 5-fluoroorotic acid and 50 μg / ml uracil.
Medium (FOA plate: 0.67% yeast nitrogen b
ase without amino acids, 2% glucose, 2% aga
In r), the cells were cultured at 30 ° C. for one week. By this culture, 31 uracil-requiring and FOA-resistant colonies were obtained. The frequency of transformation at this time was 0.62 colonies / μg DNA. From the 31 strains, 28 strains were selected as strains having stable uracil requirement and aggregability, and the following fermentation test was performed.

As described above, when an expression cassette in which the transcription directions of URA3 and FL01 were reversed was used, the result was that the transformation frequency was 30 times higher than that of the expression cassette in the same direction.

Example 4 Evaluation of Ability to Produce Alcohol In conducting a fermentation test, first, preculture was performed to prepare a fresh culture solution. Preculture was performed using YM medium (2 ml) in a test tube.
Then, one loopful of fresh yeast cells was inoculated and shake-cultured at 30 ° C. overnight.

The fermentation test is performed at the test tube level (mini scale).
I went in. 10 ml of fermentation test medium (synthetic medium: 1% yeast extract, 2% peptone, 2%) in an L-shaped test tube
4% glucose; molasses medium: Indonesian molasses with a total sugar concentration of 24%) was prepared, 100 μl of a preculture solution was inoculated therein, and shaking culture was carried out at 30 ° C. for 7 days. One strain having excellent fermentation performance was selected and named this strain FSC27. The strain was then examined for cohesiveness. This strain was subcultured for 2 weeks using a YPD liquid medium, and it was confirmed from the degree of sedimentation that the strain had stable cohesiveness.

Next, it was examined whether or not this aggregation was dependent on Ca ²⁺ which is a characteristic of aggregation by the FLO1 gene.

Example 5 Evaluation of Aggregative Ca ²⁺ Dependency 1 ml of 0.5 mM EDTA solution (p
H8.0), and the mixture was stirred well.
Aggregation buffer containing ²⁺ (0.51 g / ml CaS
O ₄ , 6.8 g / ml CH ₃ COONa, 4.0 g / m
1 CH ₃ COOH, pH 4.5), and the presence or absence of cohesiveness was confirmed from the degree of sedimentation at that time.

As a result, it was shown that the agglutinability of the transformant was Ca ²⁺ -dependent. Next, the gene analysis of this FSC27 strain was performed.

Example 6 Gene Analysis of FSC27 Strain The presence of an aggregating gene expression cassette on the URA3 gene of the chromosome of FSC27 strain was confirmed by Southern hybridization. In this Southern hybridization method, a URA3 gene fragment and a FLO1 gene fragment were used as probes.

First, total DNA was prepared from the FSC27 strain. This total DNA was prepared according to the method of Philippsen et al. (Methods in Enzymology, Vol. 194, 169 (1990)). In addition, the Southern hybridization method and the labeling of the probe were performed using Amersham's ECL direct nucleic acid.
Performed according to the acid and detection system. The result is shown in FIG.

FIG. 3A shows the results when the URA3 probe was used, and FIG. 3B shows the results when the FLO1 probe was used. FIG. 3C shows the results when pBR322 was used as a probe as a negative control.

The lane 1 contains the plasmid YIp-dU-FLO1 shown in FIG. 1, and each lane 2 contains the parent strain 396-9-FLO1.
The total DNA of the 6V strain was electrophoresed in each lane 3, and the total DNA of the FSC27 strain was electrophoresed. The DNA sample in each lane was electrophoresed after Hind III digestion.

In FIG. 3A, in the parent strain (lane 2), U
Using the RA3 probe, a signal of about 0.8 kb was detected. On the other hand, in the FSC27 strain (lane 3), a signal of about 2.0 kb longer than that of the parent strain was detected. This indicated that the FSC27 strain had an aggregating gene expression cassette inserted into the URA3 gene region.

In FIG. 3B, when the FLO1 probe was used, the FSC27 strain (lane 3) had an introduced FLO of about 3 kb which was not found in the parent strain (lane 2).
The signal considered to be derived from No. 1 was detected. Therefore, it was shown that the cohesiveness confirmed in the FSC27 strain was derived from the introduced FLO1 gene. The signal detected in the parent strain is considered to be derived from a sequence similar to FL01 and not involved in aggregation.

In FIG. 3C, the plasmid YIp-dU-FLO1
Using the pBR322 probe, which is the parent of
It was analyzed whether a DNA sequence other than the cohesive gene expression cassette was introduced into the seven strains. As a result, a signal was detected only in the plasmid YIp-dU-FLO1 (lane 1), but not in lanes 2 and 3. That is, it was shown that only the expression cassette was introduced into the FSC27 strain. This expression cassette contains 396-
Since the strain was composed of Saccharomyces cerevisiae of the same species as the 9-6V strain, it was confirmed that this FSC27 strain was a self-cloning strain.

Next, the flocculence of this FSC27 strain was determined by FL
To make sure that it is derived from the O1 gene,
The transcription of the LO1 gene was analyzed by Northern hybridization.

Example 7 FL in 27 strains of FSC
Transcriptional analysis of O1 gene Transcriptional analysis of the FLO1 gene was performed by Northern hybridization using FLO1 as a probe.
The total RNA was recovered from the FSC27 strain according to the method of Schmitt et al. (Nucleic Acids Research, Vol. 18,309 (1990)). Northern hybridization operation and probe labeling were performed using Boehringer Meiheim's DI
This was performed using a G system. As a positive control, plasmid YCpHF19L (FL01 under the control region of FL01 itself)
A strain in which the gene was expressed) was introduced into the YJW6 strain, the FLO1 gene was expressed on the plasmid, and the same operation as described above was performed.

FIG. 4 shows the result. Lane 1 shows the total RNA of the positive control strain, and lane 2 shows the total RNA of the parent strain.
Total RNA from 96-9-6V strain, FS in lane 3
Total RNA of the C27 strain was electrophoresed.

As shown in FIG. 4, a signal was detected at the same position as in lane 1 as a positive control in the FSC27 strain. From this, it was confirmed that the introduced FLO1 gene was expressed in the FSC27 strain.
Therefore, it was confirmed that the aggregation of the FSC27 strain was derived from the FLO1 gene.

Next, the fermentation performance (alcohol production capacity) of this FSC27 strain was analyzed.

Example 8 Fermentation Performance Test of FSC27 Strain The fermentation performance of the FSC27 strain was analyzed by comparing it with the fermentation performance of the parent strain.

The fermentation performance test was conducted on a flask level scale. In conducting a fermentation test, first, FSC2
Fresh precultures were prepared for each of the 7 strains and the parent strain 396-9-6V in the same manner as described above. Then
The above mentioned 24% in each Erlenmeyer flask with fermentation tube
A pre-culture solution (15 m
l) was inoculated and cultured at 32 ° C. for 6 days while stirring with a stirrer such as a magnetic stirrer. After completion of the culture, the culture was centrifuged at 12000 rpm.
, Centrifuged for 5 minutes, and the supernatant was collected. The ethanol concentration in the supernatant was determined by gas chromatography (Shimadzu GC).
-8A, column 2m × 3mmφ, gas chromatography pack 54
＠) was measured. Table 1 shows the results.

Since the FSC27 strain is auxotrophic for uracil, the uracil concentration is 0-100 μm in a 24% molasses medium.
g / ml, and the effect of uracil on ethanol production was also tested.

As shown in Table 1, in the medium to which uracil was not added, the ethanol concentration of the parent strain was 12.09%, whereas that of the FSC27 strain was 11.73%, which was higher than that of the parent strain. Performance was slightly inferior. But,
In the medium supplemented with uracil (50 μg / ml, 100 μg / ml), the FSC27 strain was about 0.5% less than the parent strain.
%. This showed a similar result in the fermentation rate.

The ratios shown in the table show the ethanol concentration in each uracil-containing medium of the parent strain and the ethanol concentration in each uracil-containing medium of the FSC27 strain, when the ethanol concentration in the uracil-free medium of the parent strain is 100%. Was. As shown from this ratio,
In the parent strain, no effect was observed on the fermentation performance due to the addition of uracil to the medium, whereas in the FSC27 strain, it was shown that the fermentation performance was increased by the addition of uracil to the medium.

[0081]

[Table 1] In addition, in this FSC27 strain, it was shown that the addition of uracil to the medium increased the fermentation rate. The fermentation rate was measured based on the amount of CO ₂ gas generated during the culture. The results are shown in FIG. In FIG. 5, ５:
396-9-6V strain (uracil 0 μg / ml), Δ: 396
-9-6V strain (uracil 50 μg / ml), □: 396-9
-6V strain (uracil 100 μg / ml), ●: FSC27 strain (uracil 0 μg / ml), ▲: FSC27 strain (uracil 5
0 μg / ml), black painted □: strain FSC27 (uracil 100
μg / ml).

As shown in FIG. 5, in the FSC27 strain, the fermentation rate was slower in the uracil-free medium than in the parent strain. On the other hand, in each uracil-containing medium, FS
Although the fermentation rate of the C27 strain was slightly delayed in the initial stage as compared to the parent strain, the fermentation rate of the FSC27 strain exceeded the parent strain rather than the middle stage (72 hours).

Example 9 Confirmation of Aggregation of FSC27 Strain At the same time as measuring the alcohol-producing ability, the aggregation was also measured.

The photograph of FIG. 6 shows the 396-9-6V strain and F
Each of the SC27 strains is cultured in a YPD medium at 30 ° C. overnight, and then shows agglutination when left for 1 minute. From these results, it was shown that the FSC27 strain acquired an agglutinability that was not present in the parent strain (396-9-6V strain) by introducing the aggregative gene expression cassette.

The evaluation of the cohesiveness was carried out according to the method of Johnston et al. (Yeast Genetics: Fundamental and Applied Aspe
cts, 205-224 (1983)). That is, the degree of cohesion (DF value) is evaluated on a scale of 0 to 5.
In addition, 0: non-aggregation, 1: very weak aggregation, 2: weak aggregation, 3: medium aggregation, 4: strong aggregation, 5: very strong aggregation.

As shown in Table 1, the parent strain 396-9-6
In the case of the V strain, the degree of agglutination was "0", ie, non-aggregating, whereas in the case of the FSC27 strain, the degree of aggregating was "5", ie, very strong aggregating.

In order to further clarify the above results, a standard strain (ABXL-1D,
Using ABXR-11B), the degree of agglutination of the FSC27 strain was measured in comparison with these standard strains. In addition, ABXL-
1D strain is ABX as a standard strain of the aggregating gene FLO1
The R-11B strain was used as a standard strain for the aggregating gene FLO5.

[0088]

[Table 2] Table 2 shows the results. In the ABXL-1D strain having the same aggregating gene as the FSC27 strain, the degree of agglutination was "3" (medium agglutinability), whereas the FS
The C27 strain was “5”. In addition, compared to the ABXR-11B strain having a different aggregating gene FLO5,
The agglutinability of the SC27 strain was high.

This FSC27 strain was shown to have a considerably higher flocculating property than the currently existing flocculating yeast.

Example 10 Comparison with Existing Cohesive Alcohol-Fermenting Yeast Further, in this example, the cohesive alcohol-fermenting yeast (F-5 strain) created by the cell fusion described above (Japanese Patent Laid-Open No.
236942) and the above-mentioned FSC27 strain. Table 3 shows the results.

The F-5 strain is derived from the same parent strain as the FSC27 strain, the 396-9-6V strain, and was created by fusing yeast having the FLO5 gene, which is an aggregating gene, to this parent strain. is there.

As shown in Table 3, in the medium containing no uracil, the FSC27 strain had a concentration of produced ethanol lower by about 0.3% as compared with the F-5 strain. However, in the uracil-containing medium (50 μg / ml, 100 μg / ml), the FSC27 strain was shown to be about 0.3% higher in ethanol concentration than the F-5 strain.

Further, regarding the fermentation rate, the CO
₂ Measurement was performed based on the amount of gas generated. The result is shown in FIG. In the figures, 図: F-5 strain (uracil 0 μg /
ml), Δ: F-5 strain (uracil 50 μg / ml), □: F-
5 strains (uracil 100 μg / ml), ●: FSC27 strain (uracil 0 μg / ml), ▲: FSC27 strain (uracil 50 μg / ml)
g / ml), black □: FSC27 strain (uracil 100 μg /
ml).

As shown in FIG. 7, in the FSC27 strain, the fermentation rate was slower in the uracil-free medium than in the F-5 strain.
The fermentation rate of the C27 strain was similar to that of the F-5 strain.

Regarding the agglutinability, as shown in Table 3, the degree of agglutination was "5" in the FSC27 strain, while "3" (medium) was lower in the F-5 strain than in the FSC27 strain. Cohesiveness). It is unclear whether this result is a difference derived from the difference in the method of creation or a difference in the DNA material used such as the aggregating gene and control region used.

The above FSC27 strain was obtained from the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-160.
No. 23 has been deposited.

[0097]

[Table 3]

[0098]

As described above, according to the present invention, since only an aggregating gene can be introduced into an alcohol-fermenting yeast, it can be aggregated into an alcohol-fermenting yeast without affecting the inherently high alcohol-producing ability. Property can be imparted.

Therefore, according to the present invention, it is possible to provide a more practical cohesive alcohol-fermenting yeast in alcohol production, and to use the cohesive alcohol-fermented yeast of the present invention in an alcohol production process. Thus, it is possible to increase production efficiency and rationalize the production process.

[Brief description of the drawings]

FIG. 1 is a diagram showing a plasmid carrying the cohesive gene expression cassette of Example 1.

FIG. 2 is a schematic diagram showing a predictive mechanism of transformation in Example 2.

FIG. 3 is a photograph showing the results of Southern hybridization analysis of a transformant (FSC27 strain).

FIG. 4 is a photograph showing the result of transcription analysis of a transformant (FSC27 strain).

FIG. 5. Transformant (FSC27 strain) and parent strain (396
9 is a graph comparing the fermentation rate with (−9-6V strain).

FIG. 6. Transformant (FSC27 strain) and parent strain (396
9 is a photograph comparing the aggregability with (-9-6V strain).

FIG. 7 is a graph comparing the fermentation rates of a transformant (FSC27 strain) and a known cohesive alcohol-fermenting yeast (F-5 strain).

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] May 14, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Figure 3

[Correction method] Change

[Correction contents]

FIG. 3 shows D recovered from a transformant (FSC27 strain).
After electrophoresis of NA, URA3 probe (A), FLO1
Probe (B), the electrophoresis path when performing the <br/> Southern hybridization using PBR322 probe (C)
It is a photograph showing a turn .

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Fig. 4

[Correction method] Change

[Correction contents]

FIG. 4 shows R recovered from a transformant (FSC27 strain).
After electrophoresis of NA, Northern blotting was performed using FLO1 as a probe.
It is a photograph which shows the electrophoresis pattern at the time of performing hybridization .

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] Fig. 6

[Correction method] Change

[Correction contents]

FIG. 6. Transformant (FSC27 strain) and parent strain (396
-9-6V strain) (organisms)
3 is a photograph showing the form of the present invention.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/19 C12R 1: 865) (72) Inventor Keiko Fujii 4-5-1, Inage-Higashi, Inage-ku, Chiba-shi, Chiba New Energy and Industry Research and Development Center, Alcohol Business Division, Technology Development Organization (72) Inventor Shingo Goto 4-5-1, Inage-Higashi, Inage-ku, Chiba-shi, Chiba Prefecture, Research and Development Center for Alcohol Business Division, New Energy and Industrial Technology Development Organization (72) Inventor Hiroki Sugiyama, Sapporo Lobby Brewery Research Institute, 10 Oka Tome, Yaizu City, Shizuoka Prefecture

Claims (7)

[Claims] 1. A method for breeding an aggregating alcohol-fermenting yeast by introducing an aggregating gene expression cassette into an arbitrary marker gene region on a chromosome of an alcohol-fermenting yeast, wherein the aggregating gene expression cassette comprises the marker A method for breeding a cohesive alcohol-fermenting yeast, wherein a cohesive gene containing a control region is arranged and configured so as to divide a gene region or replace an internal sequence thereof. 2. The method for breeding a cohesive alcohol-fermenting yeast according to claim 1, wherein the control region and the cohesive gene are derived from the same yeast as the alcohol-fermenting yeast. 3. The aggregation according to claim 1, wherein the marker gene is a gene that can be positively selected to acquire resistance to a predetermined drug when its sequence is disrupted. Breeding method for fermenting alcoholic yeast. 4. The method according to claim 1, wherein the marker gene is a gene involved in uracil biosynthesis.
The method for breeding cohesive alcohol-fermenting yeast according to 1. 5. An agglutinating alcohol-fermenting yeast, which is bred by transducing an aggregating gene expression cassette into an arbitrary marker gene region on a chromosome of the alcohol-fermenting yeast, wherein the aggregating gene expression cassette comprises A cohesive alcohol-fermenting yeast, wherein a cohesive gene containing a control region is arranged and configured so as to divide or replace the gene region of the present invention. 6. The cohesive alcohol-fermenting yeast according to claim 5, wherein the cohesive gene expression cassette is prepared from the same kind of yeast as the alcohol-fermenting yeast. 7. The method according to claim 7, wherein the cohesive alcohol-fermenting yeast is FSC.
The cohesive alcohol-fermenting yeast according to claim 6, which is 27 strains (FERMP-16023).