JP4820921B2 - Amino acid sequence, DNA and method for growing yeast - Google Patents

Description

  The present invention relates to a method for growing yeast by genetic modification by amino acid sequence specific to yeast, its DNA, transformation and the like.

  Aggregation between cells is known to result from sexual aggregation between a-type cells and α-type cells, unseparation of budding daughter cells from mother cells, non-sexual aggregation, and the like. Of these, the purpose is to control non-sexual aggregation. As a model for explaining the mechanism of nonsexual aggregation, a lectin hypothesis that a lectin-like protein on the cell surface of an aggregating yeast and an adjacent yeast are bound by a sugar chain (see, for example, Non-Patent Document 1). ) Is powerful, but no lectin-like protein has been identified. This is also a factor that makes it difficult to control yeast aggregation.

Known genes involved in yeast aggregability include a family of genes encoding specific lectin-like proteins of FLO1, FLO5, FLO9, and FLO10, which are present in the chromosome end region (region around telomere). On the other hand, it has been reported that a gene encoding the protein FLO11 / MUC1 associated with agglutinability, membrane formation, invasive growth, and substrate adhesion exists in the non-chromosomal terminal region.
J. et al. Bacteriol. , 150, 878 (1982)

  Known FLO proteins (FLO1, 5, 9) have any of the 9 amino acid sequences shown in SEQ ID NOs: 1 to 5, or a combined sequence 1 to 3 times.

  The repetitive sequence constituting this sequence site is SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 5 three times each in FLO1, SEQ ID NO: 2, SEQ ID NO: 3 twice in FLO9, In YAL065C, there are two repetitions of SEQ ID NO: 2 and SEQ ID NO: 3 each time, and in FLO5, only SEQ ID NO: 2 is configured with no repetition once.

  Further, FLO8, FLO10, MUC1 (FLO11), YAR061W, YAR062W, and YHR213W did not have the above arrangement.

  The above-mentioned conventional fermentation production method of alcohol using yeast has the disadvantage that it is inefficient and alcohol productivity is insufficient because it is difficult to separate the yeast and the product. The cause of this is the limit of the cohesiveness of yeast. As a result of elucidating this problem, we have found that the inclusion of a sequence that repeats any of the amino acid sequences four or more times contributes to the improvement of the cohesiveness, leading to the present invention. It was.

Therefore, the problems of the present invention are as follows.
(1) To provide an amino acid sequence having an activity of imparting aggregability to yeast;
(2) to provide a genetic DNA encoding an amino acid sequence having an activity of imparting aggregability to yeast;
(3) Providing a method for breeding yeast with enhanced or enhanced aggregation by using the above gene DNA;

  The other subject of this invention becomes clear by the following description.

  The above problems are solved by the following inventions.

Invention of Claim 1 consists of an amino acid sequence which has the repetition of the arrangement | sequence shown by either of sequence number 1-3 4 times or more, and is a polypeptide which has the activity which provides aggregation property to yeast .

  Invention of Claim 2 is DNA which codes the amino acid sequence of Claim 1.

  ADVANTAGE OF THE INVENTION According to this invention, the amino acid sequence which has the activity which provides the aggregability to yeast can be provided, The gene DNA which codes the amino acid sequence which has the activity which provides the aggregability to yeast can be provided, The above-mentioned By using the gene DNA, it is possible to provide a method of breeding yeast with imparted or enhanced aggregation.

The figure which showed the measurement result of the expression intensity | strength of FLO9 gene in yeast AM12 strain and non-aggregation yeast S288C strain The figure which shows the preparation procedure and primer position of plasmid pAUR123-FLO1 / FLO9 chimera by In-Fusion method The photograph which shows the sedimentation property after leaving still for 1 minute about three types of yeast strain | stump | stock which each introduce | transduced the plasmid of pAUR123-FLO1, pAUR123-FLO1 / FLO9 chimera, and pAUR123 which does not have FLO1 gene.

  First, the repeating sequence of the amino acid sequence in the present invention will be described.

  The aggregation of Saccharomyces cerevisiae has been reported to involve a special cell surface lectin-like protein (or FLOCculin protein) that can directly bind the mannose residue of the mannan molecule on the cell surface between adjacent cells. This cell-to-cell interaction causes many cells to assemble and ultimately yield yeast sedimentation. In addition, two phenotypes with different susceptibility to sugar are known for the aggregation of Saccharomyces cerevisiae, FLO1 shows mannose sensitivity, and NewFLO (FLONS) shows sensitivity to mannose and glucose.

  As a gene involved in yeast aggregation, a family of genes encoding specific lectin-like proteins of FLO1, FLO5, FLO9, and FLO10 has been proposed and is present in the chromosome end region (telomere peripheral region). On the other hand, it has been reported that a gene encoding the protein FLO11 / MUC1 associated with agglutinability, membrane formation, invasive growth, and substrate adhesion exists in the non-chromosomal terminal region.

  All these FLO proteins are modified by a type of glycolipid called glycosylphosphatidylinositol (GPI) and are tethered to the cell membrane by GPI. Such a protein is called a GPI-anchored protein because GPI acts like a cocoon.

  The FLO protein is composed of three common domain regions, and includes an N-terminal lectin domain, a central domain containing a repetitive sequence containing many serine and threonine residues, and an anchor sequence with a C-terminal glycosylphosphatidylinositol. Is a domain.

  Some repeat sequences have been reported to be involved in the difference in sensitivity to sugar between mannose-sensitive FLO1 and mannose and glucose-sensitive NewFLO (FLONS). Little is known about the function.

  As molecular level studies of genes involved in the aggregability of these yeasts, the FLO1 gene has been isolated and analyzed (YEAST, 9, 423 (1993) and YEAST, 10, 211 (1994)). The FLO1 protein is a protein localized in the cell surface and is a factor related to the aggregation of yeast cells (Bony et al., J. Bacteriol., 179: 4929-4936 (1997)). From its N-terminal side, it has a secretory signal region, a region related to the aggregation of yeast cells, and a GPI anchor-binding region in the C-terminal region. This amino acid sequence has 13 sugar chain binding sites.

  The FLO protein (FLO1, 5, 9) existing in nature has any one of the 9 amino acid sequences of SEQ ID NOs: 1 to 3 or a combined sequence 1 to 3 times.

  The present inventors have found that there is a site having 4 repetitions of 9 amino acid sequences in the FLO protein (AM12 FLO9) derived from yeast AM12 having high aggregation properties.

  FLO having 4 repeats of 9 amino acids has not been reported so far and was first isolated in AM12.

  In the C-terminal region of the peptide chain of the AM12 FLO protein, there is a sequence site where SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 3 are repeated four times.

  As described above, the repetitive sequences constituting this sequence site are as follows: SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 5 for each 3 times in FLO1, and SEQ ID NO: 2 and SEQ ID NO: 3 for each time in FLO9. In YAL065C, there are two repetitions of SEQ ID NO: 2 and SEQ ID NO: 3 each time, and in FLO5, only SEQ ID NO: 2 is configured once and no repetition.

  Further, FLO8, FLO10, MUC1 (FLO11), YAR061W, YAR062W, and YHR213W did not have the above arrangement.

  As a result of analyzing these sequences, it was found that the aggregation of yeast can be improved by arbitrarily selecting SEQ ID NOs: 1 to 3 and incorporating a sequence repeated four or more times in the vicinity of the C-terminus of the FLO protein. .

  When the example of the arrangement | sequence repeated 4 times or more is given, sequence number 11 can be illustrated.

  By introducing the repetitive gene DNA of the present invention, it is possible to obtain a yeast imparted with or enhanced aggregability. As a method for repeatedly introducing a genetic DNA, a method commonly used in the field of genetic engineering may be used, and it may be carried out in accordance with conventional standards (ANALYTICAL BIOCHEMISTRY 163.391 (1987), etc.). Specifically, a method of incorporating a desired DNA into a vector and introducing it into yeast, a method of introducing it directly into yeast without incorporating into a vector, and the like can be mentioned. For industrial use, a chromosomally introduced strain is preferable to a vector-carrying strain.

  In the method of incorporating the above DNA into a vector and introducing it into yeast, usable vectors include, for example, the YRp system (multicopy vector for yeast using the ARS sequence of yeast chromosome as the origin of replication), YEp system (yeast Multicopy vector for yeast having 2 μm DNA replication origin), YCp system (single copy vector for yeast having yeast chromosome ARS sequence as replication origin and centromere DNA sequence of yeast chromosome), YIp system (yeast All known ones can be used, such as a yeast chromosome integration vector that does not have a replication origin. These vectors are described in literature (published by Medical Publishing Center, “New Biotechnology of Yeast”, p. 284), and can be easily prepared.

  A typical method for introducing DNA directly into yeast without incorporating it into a vector is a co-transformation method in which yeast is transformed simultaneously with a plasmid having a marker gene such as a drug resistance gene and the DNA sequence to be introduced. (Japanese Patent Publication No. 5-60918). In the method as described above, in order to express the introduced gene DNA in yeast or to increase the expression, a promoter which is a unit controlling transcription and translation is added to the 5′-upstream region of the DNA strand of the present invention. In addition, a terminator may be incorporated in the 3′-downstream region. As the promoter and terminator, in addition to those derived from the repetitive gene itself, an alcohol dehydrogenase gene (J. Biol. Chem., 257, 3018 (1982)), a phosphoglycerate kinase gene (Nucleic Acids Res., 10, 7791). (1982)), a gene derived from a known gene such as the glycerol aldehyde-3-phosphate dehydrogenase gene (J. Biol. Chem., 254, 9839 (1979)), or an artificially modified version thereof. Is possible. More specifically, promoters and terminators such as ADH (aka ADC), GAPDH (aka GPD), PHO, GAL, PGK, ENO, TRP, and HIP can be used.

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

  The present invention also includes a method for further improving the aggregability of yeast having originally aggregability by replacing the gene DNA repeated 5 times or more with the 4 times gene DNA of AM12 bacteria.

  This reverse conversion is also possible with the repetitive gene DNA provided by the present invention. That is, by disrupting the repetitive gene DNA of the present invention and introducing a DNA having the ability to express a repetitive gene protein deleted or reduced, a yeast with reduced or reduced aggregation can be obtained. The disruption of repetitive gene DNA can be achieved by adding or deleting single or multiple bases in the region involved in repetitive gene protein expression of the repetitive gene, for example, the promoter region or the coding region, or deleting these entire regions. Can be performed. By repeatedly disrupting the gene in this manner, DNA that has been deleted or reduced in ability to express the repetitive gene protein can be introduced into yeast by the same method as the DNA introduction method described above. By the introduction, homologous recombination occurs between the repetitive gene in the chromosomal DNA of the host yeast and the introduced DNA, and the ability of the recombination gene of the host yeast to be disrupted to express the repetitive gene protein is lost or reduced, As a result, it is considered that the aggregation property of the host yeast is lost or reduced.

  The present invention also includes a method for deleting or reducing the cohesiveness of yeast by suppressing the expression of the above-mentioned repetitive gene DNA. Examples of such methods include a method of introducing DNA in which the ability to express a repetitive gene protein is deleted or reduced by disrupting repetitive gene DNA, an antisense RNA method, and the like.

  In the present invention, the yeast to be transformed, that is, the host yeast, may be any taxonomically capable of entering the category of yeast. Yeast imparted with high cohesiveness by the present invention is useful in the process of bioethanol production. The brewed product comprising culturing the yeast of the present invention includes alcoholic beverages including beer, sake, shochu, wine, whiskey and brandy, seasonings such as soy sauce, miso and mirin, and alcohol for fuel. Include. The method for producing a brewed product in the present invention includes a brewing process relating to the brewed product.

  The yeast aggregation effect can also be obtained by simply mixing the protein of the present invention with a solution containing non-aggregable yeast as a cell aggregating agent. Further, the present invention can be applied not only to cell-cell aggregation but also to a case where cells are fixed on a substrate (such as a biosensor).

  Examples of the present invention will be described below, but the present invention is not limited to such examples.

Example 1 (Isolation of FLO gene fragment from RNA derived from yeast AM12 strain)
(1-1) Extraction of Total RNA Saccharomyces cerevisiae AM12 strain was cultured in YPD medium (1% Yeast extract, 2% Polypeptone, 2% D-glucose) at 30 ° C. for 8 hours, and the cells were collected. Thereafter, total RNA was extracted using a High Pure RNA Isolation kit manufactured by Roche Diagnostics.

(1-2) Acquisition of PCR product by 3′-RACE method Using the extracted total RNA as a template, 3′-Full RACE Core Set manufactured by Takara Bio Inc. was used for reverse transcription reaction, followed by PCR reaction. . Total RNA was heated at 65 ° C. for 10 minutes and then rapidly cooled on ice, and 1 μg was used for the reverse transcription reaction (reaction volume 20 μl). The reverse transcription reaction solution composition was performed according to the protocol of the kit. The reaction conditions of the PCR thermal cycler were 30 ° C. for 10 minutes, 42 ° C. for 60 minutes, 95 ° C. for 5 minutes, and 5 ° C. for 5 minutes.

  Next, 1 μl of the obtained reverse transcription reaction solution was used to carry out PCR reaction under the following reaction solution composition and reaction conditions. As an enzyme for PCR, Toyobo's highly accurate polymerase KOD-Plus-Ver. 2 was used. As a result, a PCR fragment of about 0.6 kbp was obtained. PCR reaction conditions were 94 ° C. for 2 minutes, followed by 30 cycles of 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 5 minutes, and 4 ° C. after completion of the reaction.

As a Forward primer, c00258-F1 was used (SEQ ID NO: 6).
5′-CTGCTCGAGCTCGGCCTACTGTGAATGATGTGTG-3 ′

  As the reverse primer, 3 sites Adapter Primer of 3'-Full RACE Core Set manufactured by Takara Bio Inc. was used.

(1-3) Recovery of PCR product As a result of electrophoresis of the PCR product on a 1% TAE agarose gel, a band was observed at a position of about 0.6 kbp. This band was cut out from the gel and extracted and purified using a Qiagen quick Gel extraction kit manufactured by Qiagen.

(1-4) TA Cloning Using TARGET Clone ^™ -Plus-kit manufactured by Toyobo Co., Ltd., the PCR product was incorporated into a pTA2 vector (having an ampicillin resistance gene as a marker) and transformed into E. coli DH5α. Next, plasmid extraction was performed from the obtained Escherichia coli showing ampicillin resistance using MagExtractor-Plasmid- manufactured by Toyobo. The extracted plasmid was treated with the restriction enzyme EcoRI and subjected to 1% TAE agarose electrophoresis. As a result, it was confirmed that the PCR product was incorporated into the pTA2 vector.

(1-5) Base sequence determination A Dye Terminator cycle sequence reaction was performed using a pTA2 vector in which a PCR product was incorporated as a template. Next, the nucleotide sequence was determined using CEQ8000 DNA Analysis System manufactured by BECKMAN COULTER. Both sides of the PCR product were decoded using universal primers M13 Primer M3 and M13 Primer RV. Next, the sequence inside the PCR product was determined using the following primers designed based on the obtained base sequence. Finally, for the pTA2 vector into which the PCR product obtained from two independent experiments was incorporated, the full-length sequence of the gene fragment was decoded and it was confirmed that the two were in agreement.

<Primers used for sequencing>
M13 Primer M3 (SEQ ID NO: 7)
5'-GTAAAACGACGGCCAGGT-3 '
M13 Primer RV (SEQ ID NO: 8)
5′-CAGGAAACAGCTATGAC-3 ′
c00258-F3 (SEQ ID NO: 9)
5′-CTGCTCGAGCTCATGAACAGTGCTACCAGTGAG-3 ′
c00258-F4 (SEQ ID NO: 10)
5′-ACAGTAGTCACCCTTCTGCT-3 ′

(1-6) Conversion from Base Sequence to Amino Acid and Comparative Analysis The conversion from the obtained base sequence to amino acid was carried out by Gene Information Co., Ltd., GENETYX (registered trademark) Ver. 9 Performed using a Windows® version.

  The sequence of the FLO gene fragment from the obtained RNA derived from the yeast AM12 strain is shown in SEQ ID NO: 12.

Example 2
<Comparison with known sequence>
Compared with known sequences, the BLAST search (BLASTN) of the base sequence to base sequence database, the BLAST search (BLASTP) of the protein sequence to protein sequence database, and the attached homology analysis function and multiple alignment function via the Internet It was performed using.

  As a result of comparison with known genes, the homology was highest with FLO1, and other homology was also observed with FLO9, YAL065C, and FLO5.

  The gene fragment is located at 1376 to 1554 on the C-terminal side of FLO1 (1537 amino acids). FLO9 and 5 were also located on the C-terminal side, and YAL065C was found to correspond to the full length.

  By further comparison, it was found that the isolated amino acid sequence has a sequence repeated four times of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 3.

  At corresponding sites in other genes, similar amino acid sequences were found 3 times in FLO1, twice in FLO9 and YAL065C, and once in FLO5.

Example 3 (Isolation of FLO9 gene from genomic DNA derived from yeast AM12 and determination of partial sequence)
(3-1) Preparation of fosmid genomic library After culturing AM12 strain in YPD medium at 30 ° C. for 8 hours and collecting the cells, genomic DNA Buffer Set & Genomic-tip (manufactured by Qiagen) was used to obtain genomic DNA. Extracted. Genomic DNA is randomly fragmented with a DNA fragmentation device, the ends of the DNA fragments are smoothed using Mighty Cloning Blut End (manufactured by Takara Bio Inc.), and a DNA having a size of 33 to 48 kb is gelled by pulse field electrophoresis. It fractionated from. DNA was purified from the excised gel fragment and used as insert DNA. The prepared insert DNA fragment and vector pCC1FOS (manufactured by EPICENTER) were subjected to ligation reaction overnight at 4 ° C. using T4 DNA ligase (manufactured by Takara Bio). After vector ligation, in vitro packaging was performed using MaxPlax Lambda packaging Extract (manufactured by EPICENTER). A portion of the library was used to transform into host E. coli EPI300 and titer was measured. Further, Fosmid DNA was extracted from 16 randomly selected white colonies, treated with restriction enzyme with Not I, and confirmed to have an insert size of 30 Kb or more by pulse field electrophoresis.

(3-2) Preparation of Clone Pool Glycerol Stock A library solution of about 10,000 clones was introduced into host E. coli EPI300 to prepare a transformant. In the method, a single colony of host E. coli EPI300 was inoculated into an LB medium supplemented with 10 mM MgSO ₄ and 0.2% (w / v) maltose, and cultured with shaking at 37 ° C. for 3 to 5 hours. The cells were collected by centrifugation, the supernatant was discarded, and the cells were suspended in 10 mM MgSO ₄ in the same amount as the supernatant.

SM buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgSO ₄ , 0.01% gelatin) was added and diluted to add 100 μl of the library solution to 100 μl of the cell suspension and mixed. The culture was allowed to stand at 1 ° C. for 1 hour. The LB agar medium (100 μmol / ml IPTG, 40 μg / ml X-Gal) supplemented with chloramphenicol was inoculated to a final concentration of 12.5 μg / ml and cultured at 37 ° C. overnight. A transformant was added to one well of a 96-well plate containing a chloramphenicol-resistant agar medium so that 100 clones were formed, followed by stationary culture at 37 ° C. overnight. The number of clones actually contained was measured by plating the same amount as that added to one well at the same time on a petri dish agar medium.

  Chloramphenicol LB liquid medium containing 20% glycerol was added to each well of the 96-well plate after culture. After suspension, the colonies were transferred to a new 96-well plate to obtain a clone pool glycerol stock plate. The titer of the produced glycerol stock solution was confirmed.

(3-3) Clone pool DNA preparation A part of the prepared clone pool glycerol stock plate was inoculated into LB medium, and after cultivation, fosmid DNA was prepared using the alkali-SDS method, and the sterilized distilled water was added to 20 μL / well. Dissolved.

(3-4) Primary screening Using the prepared clone pool DNA of each well as a template, LA Taq polymerase (manufactured by Takara Bio Inc.), FLO9-3F primer (SEQ ID NO: 14), and FLO9-2R primer (SEQ ID NO: 15) Primary screening PCR (annealing temperature 55 ° C., 30 cycles) was performed. The PCR product was electrophoresed and the well in which a band was observed was used as a primary positive clone.

FLO9-3F (SEQ ID NO: 14)
5′-TGGTCAAGCAGTTATAGTGTA-3 ′
FLO9-2R (SEQ ID NO: 15)
5'-AGTTATCAAAGCATTCGCCAA-3 '

(3-5) Sequence Determination A sequence was directly performed from the obtained PCR product of the primary positive clone by the primer walking method, and a part of the base sequence of the obtained clone was determined.

  A part of the FLO9 gene sequence derived from the yeast AM12 and the predicted translation sequence are shown in SEQ ID NO: 16. The putative ORF was determined from the homology with the reference sequence (Accession No. U12980 Saccharomyces cerevisiae chromosome I left arm sequence). Further, the FLO gene was the FLO9 gene because of the homology of the 3 'untranslated region. Furthermore, it was found that the sequence on the 3 ′ side of the ORF was identical to the sequence obtained by reverse transcription from the yeast extracted RNA in Example 1.

Example 4 (Expression analysis of FLO9 gene derived from yeast AM12 strain)
In order to examine whether the FLO9 gene is expressed in the yeast AM12 strain and the expression intensity thereof, DNA microarray analysis was performed. The AM12 strain and the S288C strain as a non-aggregating yeast were cultured in a YPD medium at 30 ° C. for 3 hours, and the cells were collected. Then, in the same manner as in Example 1, using a High Pure RNA Isolation kit (manufactured by Roche Diagnostics) RNA extraction was performed. DNA microarray experiments were performed using the extracted RNA. For the DNA chip, Yeast Oligo Microarray (V2) (Agilent Order Number 251507210525) (manufactured by Agilent) was used, and a single color labeling method was used. This DNA chip contains oligo DNA corresponding to all 6000 genes of the standard strain S288C, and the expression intensity of each gene can be measured by hybridization with RNA extracted from yeast. For FLO9, the probe A_06_P1087 was used.

  The analysis results are shown in FIG. In AM12 strain, the FLO9 gene was strongly expressed. On the other hand, in the S288C strain, which is a strain having no aggregability, the expression of the FLO9 gene remained at a low value.

Example 5 (flocculation test of FLO gene-introduced yeast)
(5-1) Preparation of FLO1 gene yeast expression vector (pAUR123-FLO1) BG1805-amp vector inserted with ORF region (including stop codon from start codon) of YAR050W (FLO1) derived from Saccharomyces cerevisiae Y258 strain E. coli strain DH5α was obtained from the Yeast ORF collection of Open Biosystems (catalog No. YSC3867-9520537). The sequence of the FLO1 gene derived from the Y258 strain and the predicted translation sequence are shown in SEQ ID NO: 18. A plasmid was extracted from Escherichia coli according to a conventional method, and PCR was performed using the c02553-F2 primer (SEQ ID NO: 20) and c00258-R3 primer (SEQ ID NO: 21) as a template to amplify the FLO1 ORF region. The obtained PCR product was treated with the restriction enzyme Xho I, and after electrophoresis, a band of the desired size was excised from the gel and extracted using the MagExtractor-PCR & Gel Clean up-kit (manufactured by Toyobo) to obtain a FLO1 fragment. On the other hand, the pAUR123 vector (manufactured by Takara Bio Inc.) was treated with the restriction enzyme XhoI, CIAP-treated, and after electrophoresis, the target band was excised from the gel, extracted using the MagExtractor-PCR & Gel Clean up-kit (manufactured by Toyobo), and pAUR123 -Xho I fragment. The pAUR123-Xho I fragment and the FLO1 fragment were ligated using T4 ligase, and the ligated product was transformed into E. coli HST08 Premium (manufactured by Takara Bio Inc.) to obtain a colony showing ampicillin resistance. After culturing the colony, the plasmid was extracted and purified according to a conventional method to obtain plasmid pAUR123-FLO1. Sequence analysis confirmed that the insertion direction of the FLO1 fragment was correct.

c02553-F2 primer (SEQ ID NO: 20)
5′-CTGCTCGAGCTCATGACAATGCCTCATCGCTA-3 ′
c00258-R3 primer (SEQ ID NO: 21)
5′-CGACTCGAGTTTAAATAATTGCCCAGCAATAAGGAGCGC-3 ′

(5-2) Yeast expression vector of FLO1 / FLO9 chimeric gene (production of pAUR123-FLO1 / FLO9) A fragment of FLO9C terminal derived from the yeast AM12 strain obtained in Example 1 (171 amino acids) was FLO1 derived from Y258 strain (907 amino acids, This corresponds to the position from the 746th amino acid to the stop codon on SEQ ID NO: 18) Therefore, the region from the 746th to the stop codon of FLO1 derived from the Y258 strain was replaced with the fragment (171 amino acids) of the FLO9C terminal derived from the AM12 strain. A plasmid capable of expressing the FLO1 / FLO9 chimeric protein was prepared.

  Y258 strain FLO1 gene translation product (SEQ ID NO: 18) In the newly produced FLO1 / FLO9 chimeric gene translation product (SEQ ID NO: 22), the first to 745th amino acids are exactly the same. From the 746th amino acid onward, the 778th and subsequent amino acids have a sequence that repeats three times of SEQ ID NO: 4, SEQ ID NO: 1, and SEQ ID NO: 5, and the latter includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, There is a 4 repeat sequence of number 3.

  Although there are 3 amino acid differences at other sites, the translation product of the newly prepared FLO1 / FLO9 chimeric gene (SEQ ID NO: 22) is substantially 778 of the translation product of the Y258 strain FLO1 gene (SEQ ID NO: 18). The third and subsequent repeat sequences are replaced with a four repeat sequence (SEQ ID NO: 11) consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 3.

  A specific manufacturing method is as follows. The pTA vector having the C-terminal gene fragment of FLO9 (FLO9C) derived from the AM12 strain prepared in Example 1 was treated with the restriction enzyme XhoI, and the FLO9C fragment part and the vector part were separated by electrophoresis. A band was cut out and extracted using MagExtractor-PCR & Gel Clean up-kit (manufactured by Toyobo Co., Ltd.) to obtain a FLO9C fragment. Using the pAUR123-XhoI fragment prepared above, the fragment was ligated using the FLO9C fragment and T4 ligase, and the ligation product was transformed into E. coli HST08 Premium (manufactured by Takara Bio Inc.) to obtain a colony showing ampicillin resistance. After culturing the colony, a plasmid was prepared according to a conventional method, and designated as plasmid pAUR123-FLO9C. Sequence analysis confirmed that the insertion direction of the FLO9C fragment was correct.

  Next, plasmid pAUR123-FLO1 / FLO9 chimera was prepared as shown in FIG. PCR was performed using the plasmid pAUR123-FLO9C as a template and the F9C-F1 primer (SEQ ID NO: 24) and p123-R4 primer (SEQ ID NO: 25) to amplify the full-length plasmid region including the vector portion. After electrophoresis of the obtained PCR product, a band of the desired size was excised from the gel, and extracted using MagExtractor-PCR & Gel Clean up-kit (manufactured by Toyobo) to obtain a pAUR123-FLO9C fragment. On the other hand, the F1N-F primer (SEQ ID NO: 26) and F9C-R1 primer (SEQ ID NO: 27) were prepared using the plasmid extract of the FLO1 ORF region (catalog No. YSC3867-9520537) of the above-mentioned Open Biosystems Yeast ORF collection as a template. PCR was performed to amplify the region from the 1st to the 745th amino acid of the Y258FLO1 ORF. After electrophoresis of the obtained PCR product, a band of the desired size was cut out from the gel, and extracted using MagExtractor-PCR & Gel Clean up-kit (manufactured by Toyobo) to obtain a FLO1-1-745 fragment. The pAUR123-FLO9C fragment and the FLO1-1-745 fragment are ligated using an In-Fusion kit (Takara Bio), and the ligation product is transformed into Escherichia coli HST08 Premium (Takara Bio) to show ampicillin resistance. Won. After culturing the colony, a plasmid was prepared according to a conventional method to obtain a plasmid pAUR123-FLO1 / FLO9 chimera. By sequence analysis, it was confirmed that the sequence of the FLO1 / FLO9 chimeric fragment and the direction of insertion were correct, and used for the next experiment.

F9C-F1 primer (SEQ ID NO: 24)
5′-CTGTGAATGATGTTGTTACG-3 ′
p123-R4 primer (SEQ ID NO: 25)
5'-CAGTTGATTGTTATGCTTGGT-3 '
F1N-F primer (SEQ ID NO: 26)
5'-GCATACAATCAACTGATGGACAATGCCTCATCGCTA-3 '
F9C-R1 primer (SEQ ID NO: 27)
5'-CAACATCATTCAGAGTAGCC-3 '

(5-3) Introduction of each expression vector into non-aggregating yeast Non-aggregating Saccharomyces cerevisiae BY1994 strain (DKD-5D, SH1994) provided by the Ministry of Education, Culture, Sports, Science and Technology NBRP "yeast" was used as a host. In addition, the above three plasmids, pAUR123-FLO1, pAUR123-FLO1 / FLO9 chimera, and pAUR123 were used for introduction into yeast.

The specific experiment is as follows. The BY1994 strain was cultured overnight in a YPD liquid medium at 30 ° C. as a preculture solution. The preculture was inoculated into 40 ml of YPD liquid medium and cultured at 30 ° C. until the cell turbidity reached about 1 at OD ₆₀₀ . The cultured cells are collected by centrifugation, suspended in 20 ml of a 0.1 M lithium acetate solution (0.1 M lithium acetate, 0.1 M DTT, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) at room temperature. And left for 1 hour. Centrifugation was performed again, and the cells were collected, washed twice with sterilized water, and further washed once with 1M sorbitol. Finally, 50 μl of 1M sorbitol was added to suspend the bacteria. 100 μl of yeast suspension and 0.1 μg to 1 μg of plasmid DNA were mixed, dispensed into a 0.2 cm gap cuvette, and allowed to stand in ice for 10 minutes. The cuvette was set in a gene pulser system (manufactured by Bio-Rad), and electroporation was performed under the conditions of 1.5 kV, 25 μF, 200Ω, and one pulse. The bacterial solution after electroporation was diluted with 1M sorbitol and smeared on a YPD agar plate medium supplemented with 1 μg / ml of aureobasidin. After culturing at 30 ° C. for 3 to 4 days, colonies showing aureobasidin resistance were obtained. After culturing colonies in YPD liquid medium added at a final concentration of 1 μg / ml of aureobasidin, genomic DNA was prepared according to a conventional method, and pAUR123F primer (Takara Bio) and pAUR123R primer (Takara Bio) were used as templates. PCR product (annealing temperature 60 ° C., 30 cycles) was used, and a plasmid-carrying strain was confirmed that amplification was confirmed.

(5-4) Comparison of Aggregation Precipitation of Each Vector-Introduced Yeast Three types of yeast strains into which the above-mentioned pAUR123-FLO1, pAUR123-FLO1 / FLO9 chimera, and pAUR123 plasmid without FLO1 gene (manufactured by Takara Bio Inc.) were introduced. Was inoculated into 5 ml of YPD liquid medium added so that the final concentration of aureobasidin was 1 μg / ml, followed by shaking culture at 30 ° C. for 2 days. After incubation, the test tube was allowed to stand, and the aggregation and sedimentation properties of each strain were compared visually.

  FIG. 3 is a photograph of the test tube after standing for 1 minute. In FIG. 3, the left is the yeast introduced with the pAUR123 vector, the middle is the yeast introduced with pAUR123-FLO1, and the right is the yeast introduced with the pAUR123-FLO1 / FLO9 chimera. In the yeast into which the pAUR123 vector was introduced, no bacterial cell sedimentation was observed even after standing for 10 minutes or longer. Also about the yeast which introduce | transduced pAUR123-FLO1, the sedimentation property which can be discriminate | determined slightly visually is only a grade. On the other hand, in the yeast introduced with the pAUR123-FLO1 / FLO9 chimera, sedimentation to the bottom of the test tube was observed after standing for 1 minute. This indicates that the gene aggregation modification of the gene so that any of the repetitive sequences represented by SEQ ID NOs: 1 to 3 is repeated four times enhances the aggregation and sedimentation of yeast produced by the function of the FLO gene. It was done.

Claims (2)

A polypeptide comprising an amino acid sequence having four or more repeats of the sequence represented by any of SEQ ID NOs: 1 to 3, and having an activity of imparting aggregability to yeast .   DNA encoding the amino acid sequence according to claim 1.

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