JP2009178104A - Method for homogenizing specific region of diploid cell chromosome using lose of heterozygosity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an objective heterotype structural gene and/or regulatory gene existing in a specific position in a diploid cell using lose of heterozygosity. <P>SOLUTION: The method for producing a diploid cell having a homologous chromosome being a homo-type with respect to an objective hetero-type gene from a diploid cell having the objective heterotype gene in a specific region of a homologous chromosome comprises (1) a process for insertion and/or substitution of a first marker gene in any of hetero-type homologous chromosomes in a diploid cell, (2) a process for culturing the diploid cell obtained in the process (1) and selecting the diploid cell to which the first marker gene is transferred with expression of the first marker gene as an index and (3) a process for culturing the diploid cell obtained by the process (2) and obtaining a diploid cell having a homologous chromosome being a homo-type with respect to the objective structural gene and/or regulatory gene in the specific region with the drop of the first marker gene as an index. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for homogenizing heterozygosity existing at a specific position in a diploid cell utilizing loss of heterozygosity with a target genotype. More particularly, the present invention utilizes a loss of heterozygosity to destroy both alleles (or sequences) in diploid cells and target genes on both homologous chromosomes in diploid cells. It relates to a method of introducing (or sequence).

  Various gene recombination methods have been developed to date, and most of them insert a target gene into only one homologous chromosome when the cell into which the gene is introduced is a diploid cell. For example, sake yeast is a safe brewing microorganism, produces high concentrations of ethanol, has high ethanol tolerance, is resistant to various stresses, can be fermented at low temperatures, grows fast, and has stable characteristics. Have industrially useful characteristics. On the other hand, sake yeast hardly spores despite being diploid, making it difficult to obtain recessive mutants. This hinders its use as a host for genetic engineering.

  Mutation treatment is an example of a method for obtaining an inferior mutant strain. Although diploid yeast is not as good as haploid yeast, recessive mutant strains can be obtained by mutation treatment. In particular, there is a report that mutant strains could be obtained efficiently when mutation treatment was performed by UV irradiation. However, there is a risk that the mutation treatment is introduced into an undesired portion of the mutation treatment, and it is considered that it is not desirable to perform the mutation treatment more than once. There are various reports on gene disruption in yeast. For example, a gene disruption method by gene targeting using a PCR product (one-step gene disruption), a gene disruption method (two-step gene disruption) in which a selectable marker is recovered after introducing a PCR product or a plasmid, and the like. However, these are the results of studies with haploid yeast. When this method is applied to diploid yeast, hetero-disrupted strains are obtained, and recessive mutations do not appear as phenotypes. In order to prepare a homo-disrupted strain from this hetero-disrupted strain, it can be generally obtained by forming a spore in yeast and multiplying the haploid disrupted strains together. However, sake yeast hardly spores, so this homogenization method cannot be applied. Therefore, Kitamoto et al. Created a TRP1 heterologous strain of sake yeast, and after culturing it, after searching for strains that had comprehensively changed to Trp requirement from about 10,000 strains, two strains were acquired. There are also reports. In this way, if an auxotrophic strain is used, it is possible to obtain the desired gene-disrupted strain even if it takes time and effort. However, it takes a long time to confirm the phenotype, or the genes that cannot confirm the phenotype are covered. It is almost impossible to obtain the gene-disrupted strain by the genetic screening.

  On the other hand, in order to introduce a gene into sake yeast, a method of incorporating 1 to 2 copies into a specific site of a chromosome is adopted (because a selection pressure is not required and a 2 μm multicopy type plasmid cannot be maintained in the microbial cells). In addition, sake yeast is diploid (has two homologous chromosomes), so it should be able to incorporate genes into both of them, but in reality it is almost always incorporated into only one. Since heterogeneous regions are generally considered to be genetically unstable, it may be desirable to use homotypes for gene introduction in diploid yeast. It is suggested that it is effective to produce a homozygous integration strain in order to increase the copy number for further enhancing the effect of gene transfer and improve the stability of the trait.

  In diploid yeast, homointegration from heterointegrated strains can also be obtained by generally sporulating yeast and crossing haploid integrated strains. The low spore-forming ability of sake yeast has hindered this, and acquisition of homo-integrated strains has to rely on comprehensive screening.

Thus, by homogenizing the heterologous region, producing a homo-disrupted strain or a homo-gene-incorporated strain can increase the utility value of sake yeast and construct an industrially useful strain. Therefore, we selected efficiently strain heterogeneous region is homo of developed "H ighly E fficient L oss o f H eterozygosity method (HELOH method)", diploid sake yeast hardly sporulation Gene disruption and homozygous integration strains were prepared.
JP 2004-49171 A Hashimoto, et al., Appl. Environ. Microbiol., Vol. 71, p. 312-319, 2005 Kitamono et al., Agic. Biol. Chem. 54, 2979-2987 (1990)

  The present invention relates to a method of homogenizing a heterogeneous target structural gene and / or regulatory gene present at a specific position in a diploid cell using loss of heterozygosity, more specifically, heterozygous To provide a method for disrupting both alleles (or sequences) in diploid cells and a method for introducing target genes (or sequences) into both homologous chromosomes in diploid cells using loss Objective.

  In view of the current situation as described above, the present inventors have conducted extensive research, and as a result, it is possible to detect the introduction and removal of marker genes (particularly by complementing the sensitivity or requirement of diploid cells). A marker gene or a combination of two marker genes) and loss of heterozygosity (hereinafter referred to as LOH), so that at least about the genotype from the heterozygous diploid cell. One group found that diploid cells that are homozygous can be obtained very efficiently, leading to the completion of the present invention. That is, the present invention provides a method for producing a diploid cell in which a heterogeneous target structural gene and / or regulatory gene is homogenized in the following specific regions.

Item 1. A diploid cell having a hetero-type structural gene and / or regulatory gene of interest in a specific region of the homologous chromosome has a homologous chromosome homozygous for the structural gene and / or regulatory gene of interest in the specific region A method for producing a diploid cell comprising the following steps:
(1) inserting and / or replacing the first marker gene into any of heterozygous homologous chromosomes in diploid cells;
(2) a step of culturing the diploid cell obtained in step (1) and selecting a diploid cell into which the first marker gene has been introduced using the expression of the first marker gene as an index;
(3) The diploid cell obtained in step (2) is cultured, and the homologous homologous to the target structural gene and / or regulatory gene in the specific region, using the dropout of the first marker gene as an index Obtaining a diploid cell having a chromosome.
Item 2. Item 2. The method according to Item 1, wherein a diploid cell having a heterogeneous target structural gene and / or regulatory gene in a specific region of a homologous chromosome is obtained by a method comprising the following steps:
(1) inserting and / or replacing a second marker gene and a target structural gene and / or regulatory gene into a specific region of a homologous chromosome of a diploid cell;
(2) Culturing the diploid cell obtained in step (1), and using the expression of the second marker gene as an index, the target structural gene and / or regulatory gene of the heterotype is placed in a specific region of the homologous chromosome. Obtaining diploid cells.
Item 3. Item 3. The method according to Item 2, wherein the first marker gene and the second marker gene are different marker genes.
Item 4. Item 3. The method according to Item 1 or 2, wherein the marker gene is a marker gene that complements the sensitivity or requirement of diploid cells.
Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the marker gene is capable of detecting both complementarity of sensitivity and / or requirement and return to sensitivity and / or requirement.
Item 6. Item 4 or 5 wherein the marker gene is one or more marker genes selected from the group consisting of URA3, URA5, LYS2, LYS5, LEU2, HIS3, KanMX, AUR1-R, CYH2, CAN1, TRP1, GAP1, and GIN11M86 The method described in 1.
Item 7. Item 6. The method according to Item 4 or 5, wherein the sensitivity is a sensitivity selected from the group consisting of drug sensitivity, temperature sensitivity, UV sensitivity, and radiation sensitivity.
Item 8. Item 8. The method according to any one of Items 1 to 7, wherein the diploid cell is yeast.
Item 9. Yeast is Saccharomyces genus, Candida genus, Torulopsis genus, Zygosaccharomyces genus, Schizosaccharomyces genus, Pichia genus, Yarrowia genus, Hansenula genus, Kluyveromyces genus, Debaryomyces genus, Geotrichum genus, Wickerhamia sp The method described in 1.
Item 10. Item 10. The method according to Item 9, wherein the yeast of the genus Saccharomyces is Saccharomyces cerevisiae.
Item 11. Item 11. The method according to Items 1 to 10, wherein the target structural gene is one or more selected from the group consisting of LEU2, HIS3, PEP4, and SED1 genes.
Item 12. Item 12. The method according to any one of Items 1 to 11, wherein the target regulatory gene is a region containing a promoter and / or terminator.
Item 13. Item 13. The method according to Item 12, wherein the promoter is a SED1 promoter.
Item 14. Item 14. The method according to any one of Items 1 to 13, wherein the target structural gene is a structural gene connected in a form that can be expressed downstream of the SED1 promoter.
Item 15. Item 15. The method according to any one of Items 1 to 10 and Items 12 to 14, wherein the target structural gene is one or more selected from the group consisting of BGL1, BGL7, ATF1, and ATF2.
Item 16. Item 16. A gene disrupted yeast obtained by the method according to any one of Items 1 to 15.
Item 17. Item 16. A gene high expression yeast obtained by the method according to any one of Items 1 to 15.

  By using the method of the present invention, from a diploid cell having a heterogeneous gene, i.e. a structural gene of interest and / or a regulatory gene present in only one of the homologous chromosomes, a homotype (i.e. A diploid cell having the genotype in at least one set of homologous chromosomes can be efficiently produced. Therefore, by the method of the present invention, obtaining a diploid cell in which a specific genotype is completely destroyed (for at least one set of homologous chromosomes), or introducing a new gene into at least one set of homologous chromosomes. This can be done much more efficiently than before.

  For example, Kitamoto et al. Created a TRP1 heterologous strain of sake yeast, and after culturing it, after searching for a strain that had changed from about 10,000 strains to Trp requirement comprehensively, two strains were acquired. I am reporting. In this case, it was necessary to examine the Trp requirement for all about 10,000 shares, which was very laborious and time consuming. On the other hand, in the method of the present invention, by using LOH, it is possible to easily obtain a large number of homozygous mutants from one plate by positive selection. Also, converting heterozygosity of homologous chromosomes to homozygosity means that the genotype is more stably maintained in the cell.

In one embodiment, the present invention relates to a target structural gene and / or regulatory gene in a specific region from a diploid cell having a hetero-type target structural gene and / or regulatory gene in a specific region of a homologous chromosome. A method for producing a diploid cell having a homologous homologous chromosome, comprising the following steps.
(1) inserting and / or replacing the first marker gene into any of heterozygous homologous chromosomes in diploid cells;
(2) a step of culturing the diploid cell obtained in step (1) and selecting a diploid cell into which the first marker gene has been introduced using the expression of the first marker gene as an index;
(3) The diploid cell obtained in step (2) is cultured, and the homologous homologous to the target structural gene and / or regulatory gene in the specific region, using the dropout of the first marker gene as an index Obtaining a diploid cell having a chromosome.

  In the present invention, the specific region means not only a gene encoding a protein but also an arbitrary region on a chromosome having some function such as a promoter and an enhancer. The specific region also includes a region that has gained, lost, or changed function due to mutation. The specific region may be either a wild type or a mutant type, and the function of the gene may be not only a currently known function but also an unknown function. Specific examples of functions of proteins encoded in specific regions include amino acid synthesis systems, nucleic acid synthesis systems, proteases, cell wall proteins, and transcription factors.

  In the present invention, the structural gene is a base sequence encoding a protein, and the regulatory gene means a base sequence having some gene expression regulation function such as a promoter and an enhancer. The introduction may be either a structural gene or a regulatory gene, or a combination of a regulatory gene and a structural gene. Specific examples of functions of proteins encoded by structural genes include amino acid synthesis systems, nucleic acid synthesis systems, proteases, cell wall proteins, and transcription factors. Specific examples of regulatory genes include promoters, terminators, enhancers, transcription factor binding regions, and the like base sequences necessary for regulating gene expression levels.

  In the present invention, the heterotype means that structural genes and / or regulatory genes contained in a specific region on a pair of homologous chromosomes are different from each other at the base sequence level. Similarly, the homozygous means that structural genes and / or regulatory genes contained in a specific region on a pair of homologous chromosomes are identical to each other at the base sequence level.

  Any of the marker genes used in the art may be used for the first marker gene and the second marker gene used in the present invention. A preferred first marker gene is a bifunctional marker gene. Here, the bifunctional marker gene is a function that can detect that the marker gene has been introduced when the marker gene is introduced into a diploid cell (introduction detection function), and the once introduced marker gene is removed. It means a marker gene having a function (removal detection function) capable of directly detecting the host (cell) when it is performed. The bifunctional marker gene may be a combination of a marker gene having only an introduction detection function and a separate marker gene having only a removal detection function, but from the viewpoint of efficient handling, the introduction detection function and the removal detection function. A single bifunctional marker provided with one marker gene for both of these is preferred. Preferred examples of the single bifunctional marker gene include URA3, URA5, LYS2 and LYS5.

  As an introduction detection function marker that can be used when a marker having an introduction detection function and a marker having a removal detection function are combined to form a bifunctional marker, the introduction detection function marker usually used in the art has an introduction detection function Any marker can be used as long as it is a marker, and examples thereof include drug resistance genes and genes called reporter genes such as genes encoding fluorescent proteins and enzymes that catalyze specific reactions. Examples of drug resistance genes include known G418 resistance genes, aureobasidin resistance genes, kanamycin resistance genes, neomycin resistance genes, hygromycin resistance genes, phleomycin resistance genes, tetracycline resistance genes, zeocin resistance genes, ampicillin resistance genes, and the like. Drug resistance genes can be used, but are not limited to these. Examples of the fluorescent protein include a green fluorescent protein gene and a luciferase gene.

  The sensitivity in the present invention includes drug sensitivity, temperature sensitivity, ultraviolet sensitivity, and radiation sensitivity. Drug sensitivity means no drug resistance, and the drug resistance gene can be used as a complementary marker gene. By introducing a drug-sensitive complementary gene, the host cell can grow even in the presence of the drug.

  Temperature sensitivity means a trait that cannot grow above or below a specific temperature within a temperature range in which normal cells can grow, and low temperature sensitivity and high temperature sensitivity are known. A known temperature-sensitive complementary gene can be used as a gene that complements these sensitivities. By introducing a temperature-sensitive complementary gene, the host cell can grow at a temperature at which normal cells can grow.

  UV sensitivity and radiosensitivity mean a trait that is defective in damage repair function of chromosomal genes caused by irradiation with UV light and radiation, and a known gene such as RAD54 gene having gene damage repair function can be used as a complementary gene . By introducing UV-sensitive and radiation-sensitive complementary genes, host cells can grow after UV and radiation irradiation.

  The requirement in the present invention means an auxotrophy, that is, a trait that cannot grow in the absence of specific amino acids, vitamins, nucleic acids, or the like, or that grows very slowly. Such requirements include uracil requirement and lysine requirement. , Leucine requirement, histidine requirement, tryptophan requirement, methionine requirement, biotin requirement, adenine requirement and the like are known. As a marker gene that complements these requirements, URA3 gene, URA5 gene, LYS2 gene, LYS5 gene, LEU2 gene, HIS3 gene, TRP1 gene, MET1 gene, MET3 gene, BIO6 gene and other known genes can be used. . By introducing an auxotrophic complementary gene, host cells can grow in the absence of specific amino acids, vitamins, nucleic acids and the like.

  In the present invention, “reversion” means that the sensitivity / requirement of the host cell is complemented by introducing a gene that complements the host cell, and then the sensitivity / requirement before introduction is again shown by mutation treatment. Say the state.

  The marker gene of the present invention has a promoter sequence that functions in the diploid cell at the 5 'end side and a terminator sequence at the 3' end so that it can be expressed in the diploid cell. By introducing the marker gene downstream of the promoter inherent in the diploid cell, the marker gene can be expressed using the promoter inherent in the diploid cell. In this case, the promoter on the 5 'end side of the marker gene can be omitted.

  The marker gene of the present invention is introduced into the chromosome of a diploid cell by homologous recombination. Therefore, the marker gene (including promoter and terminator) is a vector having a pair of base sequences homologous to chromosomal DNA of diploid cells (hereinafter also referred to as homologous sequences) on its 5 ′ end side and 3 ′ end side. Introduced into diploid cells in the form.

  When this homologous sequence and chromosomal DNA in the diploid cell undergo homologous recombination, the bifunctional marker gene is introduced into the chromosome of the diploid cell by insertion or replacement. By designing a homologous sequence based on the chromosomal DNA of a diploid cell whose base sequence is clear, a marker gene can be introduced into a specific position (region) of the chromosome.

  At that time, a diploid cell in a form in which the 5′-side base sequence of a specific region on the chromosome is connected to the 5 ′ side of the marker gene and the 3′-side base sequence is connected to the 3 ′ side of the marker gene. When introduced into, substitutional homologous recombination occurs, and a specific region on the chromosome can be replaced with the target structural gene and / or regulatory gene.

  On the other hand, when a 3 ′ internal base sequence of a specific region on a chromosome is introduced into a diploid cell in a form in which the 5 ′ internal base sequence is connected to the 5 ′ side of the marker gene and the 3 ′ internal base sequence is connected to the 3 ′ side of the marker gene. Insertion-type homologous recombination occurs, and the target structural gene and / or regulatory gene can be inserted into a specific region on the chromosome.

  A vector for introducing a marker gene into a diploid cell may be circular like a plasmid, or may be a linear DNA fragment. Introduction of a vector into a diploid cell can be performed using a known gene transfer technique. Examples thereof include a particle gun method, an electroporation method, a polyethylene glycol (PEG) method, a calcium phosphate method, a calcium chloride method, a DEAE dextran method, a microinjection method, a lipofection method, and a lithium acetate method. These methods can be appropriately selected and used according to the type of diploid cell. When the diploid cell is diploid yeast, for example, the lithium acetate method is preferably used.

  A method for producing a diploid cell having the gene of interest in both homologous chromosomes from a diploid cell having the gene of interest present only in one of the homologous chromosomes of the present invention comprises loss of heterozygosity (LOH ). Here, LOH is a state in which specific sequences in a specific region of a pair of homologous chromosomes in a diploid cell differ (heterozygosity). It is a phenomenon in which a specific region in a pair of homologous chromosomes changes to the same state (homozygosity) at the base sequence level due to gene conversion, deletion, point mutation or the like. For example, in the case of Saccharomyces cerevisiae, it is known that LOH occurs at a frequency of 1 to 1 million cells and 1 to 10,000 cells.

  In the present invention, using a bifunctional marker gene, a diploid cell having a target gene (target gene) existing only in one of homologous chromosomes and having the target gene in both homologous chromosomes The principle of obtaining is as follows and is schematically shown in FIG. Bifunctional markers are introduced into homologous chromosomes that lack the target gene in diploid cells that have the target gene on only one of the homologous chromosomes. A diploid cell in which a bifunctional marker is introduced into the target homologous chromosome is detected using the introduction detection function of the marker gene as an index. For example, when uracil-requiring sake yeast is used as a diploid cell and the URA3 marker gene is used as a bifunctional marker gene, it grows on a selective medium without uracil after the marker gene introduction operation to form a colony Yeast is yeast that incorporates the URA3 marker gene into its chromosome. Therefore, by selecting a yeast that grows on a medium not added with uracil in this way, a yeast having a bifunctional marker on a homologous chromosome can be obtained. The obtained yeast has a pair of homologous chromosomes consisting of a chromosome having a target gene and a chromosome having a bifunctional marker gene, and when LOH is generated in this homologous chromosome, both chromosomes forming the homologous chromosome are Have the target gene. In this case, since the chromosome on the side where the marker gene exists is excluded, the bifunctional marker gene is removed. On the other hand, when LOH does not occur or when LOH is generated in such a manner that the chromosome on the side having the marker gene is doubled, the target gene exists only on one side of the homologous chromosome or does not exist on any chromosome Conversely, it has a bifunctional marker gene on either or both chromosomes. Undesired diploid cells having such homologous chromosomes can be excluded using the bifunctional marker gene removal detection function as an index. For example, when URA3 is used as a bifunctional marker gene as described above, since only yeast from which the marker gene has been removed by LOH can grow on a 5-FOA medium, a yeast that grows on a 5-FOA medium is selected. By doing so, a diploid yeast having a homologous homologous chromosome for the target gene of interest can be obtained.

  The presence of the target gene (target gene) in both homologous chromosomes can be confirmed by any method. For example, confirmation can be performed based on the size of the amplified fragment by performing PCR on a chromosome region sandwiching the target gene by confirming the homologous target gene by the phenotype. Moreover, you may detect using a probe specific to a target gene. For auxotrophic strains, the phenotype can also be confirmed by inoculating the medium.

  In the present invention, the marker gene can be introduced at any position as long as it is in the homologous chromosome on the side where the target gene does not exist. Preferably, it is introduced into a region corresponding to a specific region where the gene of interest exists in the allelic homologous chromosome, and most preferably, it is introduced into a location corresponding to the position where the gene of interest exists in the allelic homologous chromosome.

In the present invention, a diploid cell (heterotype) having a target gene only in one of homologous chromosomes to be inserted with a marker gene can be prepared by a known genetic recombination method, and the method is particularly limited. Is not to be done. For example, a diploid cell having a hetero-type structural gene of interest and / or a regulatory gene in a specific region of a homologous chromosome can be obtained by a method including the following steps.
(1) inserting and / or replacing a second marker gene and a target structural gene and / or regulatory gene into a specific region of a homologous chromosome of a diploid cell;
(2) Culturing the diploid cell obtained in step (1), and using the expression of the second marker gene as an index, the target structural gene and / or regulatory gene of the heterotype is placed in a specific region of the homologous chromosome. Obtaining diploid cells.

  Here, the second marker gene may be any of the marker genes shown above.

  In the present invention, a diploid cell means a cell having a chromosome number (nuclear phase) of 2n and having a pair of homologous chromosomes derived from parents. The diploid cell includes any organism that is a diploid, and examples thereof include yeast, mold, plant, and animal cells. Preferred are cells that can be cultured relatively easily, and most preferred is diploid yeast.

In the embodiment of the present invention, a diploid cell having a hetero target allele in which one gene on a homologous chromosome is a gene having a specific function and the other gene is a gene lacking the function Can be prepared from homozygous diploid cells for the target gene using known methods such as gene targeting, and includes a method comprising the following steps as one aspect:
(1) (a) loop-out forming sequence, (b) promoter functioning in diploid cells, (c) bifunctionality capable of detecting both introduction and removal of a marker gene under the control of said promoter sequence A vector comprising a marker and a pair of homologous recombination sequences arranged on the 5 ′ end side and 3 ′ end side with the above (a) to (c) in between is a homotype having a function with respect to the target allele Introducing into a polyploid cell and causing homologous recombination with one homologous chromosome;
(2) culturing the cells obtained in step (1) and selecting diploid cells having chromosomes homologously recombined using the expression of the bifunctional marker gene as an index;
(3) A step of culturing the cell obtained in step (2) to obtain a diploid cell from which the bifunctional marker gene has been removed.

  Here, the homologous recombination sequence arranged on the 5 ′ end side is a sequence homologous to the base sequence on the 5 ′ end side of the target gene, and the homologous recombination sequence arranged on the 3 ′ end side is the target gene. Is a sequence homologous to the base sequence on the 3 ′ end side, and when the loop-out forming sequence is on the 5 ′ end side with respect to the bifunctional marker gene sequence, the loop-out forming sequence is 3 of the target gene. When the sequence is more homologous to the base sequence located on the 3 ′ end side than the base sequence located on the “terminal side”, and the loop-out forming sequence is located on the 3 ′ end side with respect to the bifunctional marker gene sequence, The loop-out forming sequence is a base sequence that is further on the 5 ′ end side than the base sequence on the 5 ′ end side of the target gene. A schematic diagram of an embodiment using this method is shown in FIG.

  Here, loop-out refers to a phenomenon in which homologous sequences on the same chromosome undergo recombination and the sequence between them falls off.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these.
Bacterial strain and medium Escherichia coli DH5α [F ^- endA1 hsdR17 (r _K ⁻ / m _K ⁻ ) supE44 thi-1 λ ^- recA1 gyrA96 DlacU169 (φ80lacZΔM15)] was used as a host for recombinant DNA manipulation. E. coli was cultured at 37 ° C. in LB (+ Amp) medium (10 g / l polypeptone, 5 g / l yeast extract, and 10 g / l sodium chloride and 100 mg / l ampicillin). As a sake yeast used for host and template genomic DNA extraction, Kyokai No. 9 (Brewing Association), GRI-117-U (Japanese Patent Laid-Open No. 2007-189909), GRI-117-UK (Biochemical Engineering Journal Volume 33, Issue 3, March 2007) , Pages 232-237). Depending on the situation, yeast can be used in YPD medium (non-selective medium: 10 g / l yeast extract, 20 g / l polypeptone and 20 g / l glucose), SD medium (auxotrophic strain selective medium: 6.7 g / l yeast). nitrogen base without amino acids, 20 g / l glucose and appropriate supplements), 5-FOA medium (Ura ^- strains for selection: 6.7 g / l yeast nitrogen base without amino acids, 20 g / l glucose, 1 g / l 5- FOA and 20 g / l uracil and appropriate supplements), α-AA medium (Lys ^- strain selection: 1.6 g / l yeast nitrogen base without amino acids and ammonium sulfate, 20 g / l glucose, 2 g / l DL-α -AA and 50 mg / l lysine-HCl) at 30 ° C. In agar medium, add 2% agar and culture at 30 ° C.

Example 1
Preparation of LYS2-disrupted strain DNA fragments used for homologous recombination were synthesized by junction PCR. Using the primers shown in Table 1 (SEQ ID NOs: 26 to 33), using yeast genomic DNA as a template, LYS2 (−250 to 220) as a 5′-end homologous recombination sequence, and LYS2 (1881 to 2380) as a loop-out forming sequence ), URA3 marker as a marker gene, and each DNA fragment of LYS2 (1381 to 1880) as a homologous recombination sequence on the 3 ′ end side was amplified by PCR. After purifying these fragments, the respective fragments are mixed and subjected to a second PCR using primers of 1F (SEQ ID NO: 26) and 4R (SEQ ID NO: 33), whereby a 2,602 bp DNA fragment for gene substitution lys2- URA3 was made. Sake yeast GRI-117-U (requiring uracil) was transformed with this fragment to obtain a strain in which LYS2 of one chromosome was replaced with lys2-URA3. When this strain was cultured for 3 days in YPD and applied to a 5-FOA plate, colonies appeared at a frequency of 1.0 × 10 ⁻⁴ . The bacterial cells forming the colony are those obtained by removing the URA3 marker gene from the chromosome. PCR was performed directly from the colonies using the primers shown in Table 3 (SEQ ID NOs: 80 and 81), and two strains with amplified fragments of 2.6 kb and 0.9 kb were removed from the Lys2 gene and the marker gene URA3 by loop-out. Selected as a stock. The obtained strain was designated as LYS2 heterologous strain GKT1000. GKT1000 has the Lys2 gene on only one of the homologous chromosomes. When this GKT1000 was cultured in YPD for 3 days and applied to an α-AA plate, colonies appeared with a frequency of 1.1 × 10 ⁻⁴ . The colony that appeared was the selective marker URA3 disappeared by LOH (showing uracil requirement) and lysine requirement, and this was designated as LYS2 homo-disruption strain GKT1001.

Example 2
Preparation of LEU2-disrupted strain DNA fragments used for homologous recombination were synthesized by junction PCR. Using the primers shown in Table 1 (SEQ ID NOs: 34 to 41), using yeast genomic DNA as a template, LEU2 (−150 to −1) as a homologous recombination sequence on the 5 ′ end side, and LEU2 (643 to 643 as a loopout forming sequence) 1198), each DNA fragment of URA3 marker as a marker gene and LEU2 (493 to 642) as a homologous recombination sequence on the 3 ′ end side was amplified by PCR. After purifying these fragments, the respective fragments are mixed and subjected to a second PCR using primers of 1F (SEQ ID NO: 34) and 4R (SEQ ID NO: 41), whereby a 1,987 bp DNA fragment leu2- URA3 was made. Sake yeast GKT1001 was transformed with this fragment to obtain a strain in which LEU2 on one chromosome was replaced with leu2-URA3. When this strain was cultured in YPD for 3 days and applied to a 5-FOA plate, colonies appeared at a frequency of 1.6 × 10 ⁻⁵ . PCR was performed directly from the colonies using the primers shown in Table 3 (SEQ ID NOs: 34 and 82), and colonies with amplified fragments of 1.4 kb and 0.8 kb were selected as strains from which the selection marker URA3 had been removed by loop-out. The obtained strain was designated LEU2 heterologous strain GKT2000.

In order to homogenize the disrupted region of LEU2 of GKT2000, it was marked with plasmid pRSU-LEU2 containing the URA3 marker by homologous recombination. The construction of pRSU-LEU2 is as follows. First, using the primers shown in Table 2 (SEQ ID NOs: 66 to 69), PCR was performed using yeast genomic DNA as a template, followed by junction PCR. The obtained DNA fragment was purified, treated with restriction enzymes with EcoR I and Sal I, and inserted into pRS406 treated with EcoR I and Sal I. After pRSU-LEU2 was digested with Hpa I and linearized, it was introduced into GKT2000 by the lithium acetate method to obtain a strain GKT2000 / pRSU-LEU2 marked with a non-destructive LEU2 region. When GKT2000 / pRSU-LEU2 was cultured in YPD for 3 days and applied to a 5-FOA plate, colonies appeared with a frequency of 6.5 × 10 ⁻⁵ . When the method of the present invention is used, colonies can be detected with a single plate, but when using conventional exhaustive screening, about 100 plates are required. The appearing colonies were directly PCR from the colonies using the primers shown in Table 3 (SEQ ID NOs: 34 and 82), and colonies with an amplified fragment of only 0.8 kb were selected as strains in which the selection marker URA3 had disappeared due to LOH. About 40% of the colonies that appeared were strains in which LEU2 was homo-disrupted, and this was designated as GKT2001. It requires uracil, lysine, and leucine.

Example 3
Preparation of HIS3-disrupted strain DNA fragments used for gene replacement were synthesized by junction PCR. Using the primers shown in Table 1 (SEQ ID NOs: 42 to 49), using yeast genomic DNA as a template, HIS3 (-400 to -251) as a homologous recombination sequence on the 5 'end side, and HIS3 (251 to as a loopout forming sequence) 750), each DNA fragment of URA3 marker as a marker gene and HIS3 (101-250) as a homologous recombination sequence on the 3 ′ end side was amplified by PCR. After purifying these fragments, each fragment is mixed and subjected to a second PCR using primers of 1F (SEQ ID NO: 42) and 4R (SEQ ID NO: 49), whereby a 1,932 bp gene fragment DNA fragment his3- URA3 was made. Sake yeast GKT2001 was transformed with this fragment to obtain a strain in which HIS3 on one chromosome was replaced with his3-URA3. When this strain was cultured for 3 days in YPD and applied to a 5-FOA plate, colonies appeared with a frequency of 2.4 × 10 ⁻⁵ . PCR was carried out directly from the colonies using the primers shown in Table 3 (SEQ ID NOs: 42 and 83), and colonies with amplified fragments of 1.2 kb and 0.6 kb were selected as strains from which the selection marker URA3 had been removed by loop-out. The obtained strain was designated as HIS3 heterologous strain GKT3000.

In order to homogenize the HIS3 disruption region of GKT3000, it was marked with plasmid pRSU-HIS3 containing the URA3 marker by homologous recombination. The construction of pRSU-HIS3 is as follows. First, PCR was performed using yeast genomic DNA as a template, followed by junction PCR using the primers (SEQ ID NOs: 70 to 73) shown in Table 2. The obtained DNA fragment was purified, treated with restriction enzymes with EcoR I and Sal I, and inserted into pRS406 treated with EcoR I and Sal I. GKT3000 was treated with pRSU-HIS3 with restriction enzyme Hpa I, linearized, and then introduced into GKT3000 by the lithium acetate method to obtain a strain GKT3000 / pRSU-HIS3 in which the HIS3 nondestructive region was marked. When GKT3000 / pRSU-HIS3 was cultured in YPD for 3 days and applied to a 5-FOA plate, colonies appeared at a frequency of 3.2 × 10 ⁻³ . When the method of the present invention is used, colonies can be detected with a single plate, but when using conventional exhaustive screening, about 100 plates are required. The emerged colonies were directly PCR from the colonies using the primers shown in Table 3 (SEQ ID NOs: 42 and 83), and colonies having an amplified fragment of only 0.6 kb were selected as a strain in which the selection marker URA3 had disappeared due to LOH. Approximately 10% of the selected colonies were strains in which HIS3 was homo-disrupted, and this was designated as GKT3001. This strain is uracil, lysine, leucine auxotrophic.

Example 4
Preparation of PEP4-disrupted strain DNA fragments used for gene replacement were synthesized by junction PCR. Using the primers shown in Table 1 (SEQ ID NOs: 50 to 57), using yeast genomic DNA as a template, PEP4 (-160 to -1) as a homologous recombination sequence on the 5 'end side, and PEP4 (1219 to 1219 as a loop-out forming sequence) 1728), DNA fragments of URA3 marker as a marker gene and PEP4 (1069-1218) as a homologous recombination sequence at the 3 ′ end were amplified by PCR. After purifying these fragments, the respective fragments are mixed and subjected to a second PCR using primers of 1F (SEQ ID NO: 50) and 4R (SEQ ID NO: 57), whereby a 1,952 bp DNA fragment for gene replacement pep4- URA3 was made. Sake yeast GKT2001 was transformed with this fragment to obtain a strain in which PEP4 on one chromosome was replaced with pep4-URA3. When this strain was cultured in YPD for 3 days and applied to a 5-FOA plate, colonies appeared with a frequency of 5.3 × 10 ⁻⁵ . PCR was carried out directly from the colonies using the primers shown in Table 3 (SEQ ID NOs: 50 and 84), and colonies with amplified fragments of 1.9 kb and 0.7 kb were selected as strains from which the selection marker URA3 had been removed by loop-out. The obtained strain was designated as PEP4 hetero-destroying strain GKT2100.

In order to homogenize the disrupted region of PEP4 of GKT2100, it was marked with plasmid pRSU-PEP4 containing the URA3 marker by homologous recombination. The construction of pRSU-PEP4 is as follows. First, PCR was performed using yeast genomic DNA as a template using the primers (SEQ ID NOs: 74 and 75) shown in Table 2. The obtained DNA fragment was purified, treated with restriction enzymes with EcoR I and SalI, and inserted into pRS406 treated with restriction enzymes with EcoR I and Sal I. After pRSU-PEP4 was treated with restriction enzyme with Hpa I and linearized, it was introduced into GKT2100 by the lithium acetate method to obtain a strain GKT2100 / pRSU-PEP4 marked with a non-destructive region of PEP4. When GKT2100 / pRSU-PEP4 was cultured in YPD for 3 days and applied to a 5-FOA plate, colonies appeared with a frequency of 5.5 × 10 ⁻⁵ . When the method of the present invention is used, colonies can be detected with a single plate. However, when conventional exhaustive screening is performed, about 100 plates are required, and a target colony is selected from these plates. The selection of is almost impossible. The emerged colonies were directly PCR from the colonies using the primers shown in Table 3 (SEQ ID NOs: 50 and 84), and the colonies whose amplified fragments were only 0.7 kb were selected as strains in which the selection marker URA3 had disappeared due to LOH. Approximately 40% of the appearing colonies were strains in which PEP4 was homo-disrupted, and this was designated as GKT2101. This strain is uracil, lysine and leucine auxotrophic, and the PEP4 gene is disrupted in a homo form.

Example 5
Preparation of SED1-disrupted strain DNA fragments used for gene replacement were synthesized by junction PCR. Using the primers (SEQ ID NOs: 58 to 65) shown in Table 1, using yeast genomic DNA as a template, SED1 (-1334 to -1104) as a homologous recombination sequence on the 5 ′ end side, and SED1 (1260 as a loopout forming sequence) 1759), each DNA fragment of SED1 (1060 to 1259) as a URA3 marker as a marker gene and a homologous recombination sequence on the 3 ′ end side was amplified by PCR. After purifying these fragments, the respective fragments are mixed and subjected to a second PCR using primers of 1F (SEQ ID NO: 58) and 4R (SEQ ID NO: 65), whereby a 2,062 bp DNA fragment for gene replacement sed1- URA3 was made. Sake yeast GRI-117-UK was transformed with this fragment to obtain a strain in which SED1 of one chromosome was replaced with sed1-URA3. When this strain was cultured in YPD for 3 days and applied to a 5-FOA plate, colonies appeared at a frequency of 3.4 × 10 ⁻⁵ . PCR was performed directly from the colonies using the primers shown in Table 3 (SEQ ID NOs: 85 and 86), and colonies with amplified fragments of 3.2 kb and 0.9 kb were selected as strains from which the selection marker URA3 had been removed by loop-out. The obtained strain was designated as SED1 hetero-destroying strain GRI-117-UK (sed1 / SED1).

In order to homogenize the disrupted region of SED1 of this GRI-117-UK (sed1 / SED1), it was marked with plasmid pRSU-SED1 containing the URA3 marker by homologous recombination. The construction of pRSU-SED1 is as follows. First, using the primers (SEQ ID NOs: 76-79) shown in Table 2, PCR was performed using yeast genomic DNA as a template, followed by junction PCR. The obtained DNA fragment was purified, treated with restriction enzymes with EcoR I and Sal I, and inserted into pRS406 treated with EcoR I and Sal I. pRSU-SED1 is treated with restriction enzyme with Hpa I, linearized, and then introduced into GRI-117-UK (sed1 / SED1) by the lithium acetate method, so that the strain GRI-117- marked with the non-destructive region of SED1 UK (sed1 / SED1) / pRSU-SED1 was obtained. When GRI-117-UK (sed1 / SED1) / pRSU-SED1 was cultured in YPD for 3 days and applied to a 5-FOA plate, colonies appeared at a frequency of 3.8 × 10 ⁻⁵ . When the method of the present invention is used, colonies can be detected with a single plate. However, when conventional exhaustive screening is performed, about 100 plates are required, and a target colony is selected from these plates. The selection of is almost impossible. The appearing colonies were directly PCRed from the colonies using the primers shown in Table 3 (SEQ ID NOs: 85 and 86), and a strain in which the selection marker URA3 disappeared by LOH was selected (only amplified fragment 0.9 kb). Approximately 5% of the appearing colonies were strains in which SED1 was homo-disrupted, and this was designated as GRI-117-UK (sed1 / sed1). This strain is uracil and lysine auxotrophic, and the SED1 gene is disrupted in a homo form.

  The loopout and LOH frequencies in Examples 1-5 are shown in Tables 4 and 5 below.

  The genotypes of the mutant strains obtained in Examples 1 to 5 are shown in Table 6 below.

Example 6
Confirmation of the phenotype <Netrotrophic strain> When the obtained auxotrophic strains were inoculated on the SD, SD-Ura, SD-Lys, SD-Leu, and SD-His plates, the nutrients were as shown in FIG. The requirement was shown. Moreover, when the growth of each strain in YPD was measured with a biophoto recorder (manufactured by Advantech Toyo Co., Ltd.), the start of growth was slightly delayed with each addition of auxotrophy, but the growth was generally good ( FIG. 4).
<PEP4-disrupted strain> The PEP4-disrupted strain was evaluated according to Methods Enzymol., 194, 429-453 (1991). As a result, as shown in Table 7, about half of the parent strain was detected in the hetero-disrupted strain, and almost no activity was detected in the homo-disrupted strain.

<SED1-disrupted strain> Cells cultured in YPD for 3 days were suspended in 0.5 U / ml Zymolyase (pH 7.5) so that OD ₆₀₀ = 0.5, and reacted at 30 ° C. As a result, as shown in Shimoi et al. (J Bacteriol., 180, 3381-3387 (1998)), it was found that the sensitivity to Zymolyase was increased in the SED1 homozygous strain (FIG. 5).

Example 7
Confirmation of β-glucosidase activity Yeast was prepared by introducing one copy of an expression cassette (Japanese Patent Application No. 2007-228373) that displays bacilli b-glucosidase (BGL7) on the surface of the yeast cell. -As a result of measurement of glucosidase activity, the SED1 hetero-disrupted strain showed 1.11 times that of the parent strain, and the SED1 homo-disrupted strain showed 1.16 times that of the parent strain (Table 8).

Example 8
Production of β-glucosidase-secreting production strain In the present invention, detection of enzyme activity and measurement of enzyme activity were performed by the following methods.

<Quantification of β-glucosidase activity>
The β-glucosidase activity was determined by adding the culture supernatant of the test cells to 50 mM acetate buffer (pH 5.0) containing 1 mM 4-Nitrophenylβ-D-glucopyranoside and allowing to stand at 37 ° C. for 3 minutes. An equal amount of Na ₂ CO ₃ was added to stop the reaction, and the amount was determined from the amount of released 4-Nitrophenol. β-glucosidase activity was defined as 1 U = the amount of enzyme required to produce 1 μmol of 4-Nitrophenol per minute from 4-Nitrophenyl-β-D-glucopyranoside.

<Preparation of hetero-type expression strain>
pK112-BG1 (Japanese Patent Laid-Open No. 2007-28912) was used as a plasmid for expressing β-glucosidase. This plasmid has a promoter (P _SED1) and the yeast ADH1 gene terminator (T _ADH1) yeast SED1 gene, koji mold (Aspergillus oryzae) derived from β- glucosidase - is designed to overexpress synthase gene (BGL1) Yes. This plasmid was linearized by cutting one site with the restriction enzyme Sse8387I and introduced into the sake yeast Saccharomyces cerevisiae GKT1001 by the lithium acetate method obtained in Example 1. Culturing was carried out on an SD-U agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (MPBIO), and 2% glucose, and the grown yeast was selected. In this medium, plasmid pK112-BG1 is introduced into the chromosome, and only strains with complemented uracil requirement can grow.

  The pattern of integration of the plasmid pK112-BG1 into the chromosome was confirmed from the result of PCR using the primers shown below and the β-glucosidase activity.

Primer A 5'- ATGTGGCTGTGGTTTCAGGGTCCATAAAGC -3 '(SEQ ID NO: 1)
Primer B 5'- TCATTATAGAAATCATTACGACCGAGATTC -3 '(SEQ ID NO: 2)
Primer C 5'- GGTTACGCGCAGCGTGACCGCTACACTTGC -3 (SEQ ID NO: 3)
In the case of the combination of primers A and B, only a strain having a wild type (no pK112-BG1 incorporated) chromosome shows an amplified fragment of 1.1 kb. Only strains with a modified chromosome (incorporating pK112-BG1) are designed to show a 1.6 kb amplified fragment.

  Thus, the parent strain (WT / GKT1001 strain) amplified a 1.1 kb fragment by the combination of primers A and B, while the growing strain was 1.1 kb by the combination of primers A and B and the combination of primers A and C. And a 1.6 kb theoretical length fragment were amplified, but there were two types of β-glucosidase activity, high / low. This indicates that there are strains in which 1 copy of pK112-BG1 is incorporated in a hetero form and strains in which 2 copies are incorporated. A strain in which one copy of the plasmid was incorporated into the chromosome in the heterozygous form was designated as (× 1), and a strain in which the two copies of the plasmid was incorporated into the chromosome in the chromosome was designated as (× 2).

<Preparation of marking plasmid>
A LYS2 marker: LYS2 (dHpaI) in which the restriction enzyme HpaI recognition sequence present therein was destroyed was synthesized by junction PCR. 5'-gcg gacgtc CACTTGCAATTACATA-3 '(lower case is a preliminary sequence for improving the efficiency of subsequent AatII digestion, and lower case lowercase is AatII recognition sequence) and 5'-TATTTGAGTACGATCGTTCCTattaacAAC-3' (SEQ ID NO: 4) : Lower case part that disrupted HpaI recognition sequence), 5'-TTGAACGTTCAGCTACTAGTTgttaatAGG-3 '(Sequence number 5: Lower case part that disrupted HpaI recognition sequence) and 5'-CTGCACGTGATTTACAGTTCTTATTCAATA -3' (Sequence number 6: PCR was performed to obtain two DNA fragments, which were purified and mixed, and again 5′-gcg gacgtc CACTTGCAATTACATA-3 ′ (SEQ ID NO: 7) and 5′-CTGCACGTGATTTACAGTTCTTATTCAATA-3 ′ (SEQ ID NO: 8) LYS2 (dHpaI) was obtained by PCR using the primers of 1. A fragment obtained by treating LYS2 (dHpaI) with restriction enzymes Aat II was inserted into plasmid pRS406 treated with restriction enzymes Aat II and Nae I, and pAKK2 and did.

Next, the 5 ′ upstream region and 3 ′ downstream region of the URA3 gene on the yeast chromosome were amplified and joined by junction PCR. Specifically, 5'-ccc gtcgac GGGAATCTCGGTCGTAATGATTTCTA-3 '(SEQ ID NO: 9 is a preliminary sequence for improving the efficiency of later SalI digestion, underlined lowercase letters are SalI recognition sequences) and 5'-TCCATCTCATTAgttaacTATTGAATTTTGTTTGG-3' (SEQ ID NO: 10 : Lower case is HpaI recognition sequence), 5'-CAAAATTCAATAgttaacTAATGAGATGGAATCGG -3 '(SEQ ID NO: 11 lower case is HpaI recognition sequence) and 5'- ccc gaattc TATGGACCCTGAAACCACAGCCACAT -3' (Sequence number 12: Lower case makes EcoRI digestion more efficient PCR was performed using a preliminary sequence (underlined lowercase letters are EcoRI recognition sequences) to obtain two DNA fragments. Each fragment was purified and mixed, and PCR was performed again using primers of 5′-ccc gtcgac GGGAATCTCGGTCGTAATGATTTCTA-3 ′ (SEQ ID NO: 13 and 5′-ccc gaattc TATGGACCCTGAAACCACAGCCACAT-3 ′ (SEQ ID NO: 14). A fragment obtained by joining the 5 ′ upstream region and the 3 ′ downstream region of the obtained URA3 gene was designated as URA3-UTR.

  URA3-UTR was digested with restriction enzymes SalI and EcoRI and inserted into pAKK2 that was similarly digested with SalI and EcoRI to obtain a marking plasmid pURA3-UTR.

<Marking of hetero-type expression strain>
DNA linearized by cutting pURA3-UTR with the restriction enzyme HpaI was introduced into each of the x1 and x2 strains by the lithium acetate method. Culturing was carried out on an SD-UK agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.067% CSM-LYS-URA (MPBIO) and 2% glucose, and the grown yeast was selected. In this medium, only a strain (marking strain) in which plasmid pURA3-UTR is introduced into the chromosome and lysine requirement is complemented can grow.

  The β-glucosidase activity of the marking strain was measured, and it was confirmed that it had an enzyme activity comparable to that of the × 1, × 2 strain. The obtained marking strains were designated as × 1M and × 2M strains, respectively.

<Preparation of homo-type expression strain>
After × 1M and × 2M strains were cultured in YPD medium for 3 days, they were applied to α-AA plates. After culturing for 7 days, the growing yeast was selected to obtain a strain in which lysine requirement was restored. The obtained strain was subjected to PCR using the primers A and B, and confirmed to be the target homotype strain. The obtained homotype strains were designated as x1H and x2H strains, respectively.

  FIG. 6 shows a schematic diagram of the chromosomal plasmid integration site (near URA3) of the prepared strains of x1, x1H, x2, and x2H.

Example 9
Evaluation of β-glucosidase activity of homotype strains The × 1, × 1H, × 2, and × 2H strains prepared in Example 8 were cultured in YPD medium for 48 hours, and the β-glucosidase activity of the culture supernatant was measured. . At this time, the parent strain GKT1001 was used as a control (WT). As a result, the homozygous strain exhibited β-glucosidase activity approximately twice that of the heterozygous strain (× 1 → × 1H: 2.1 times, × 2 → × 2H: 1.8 times). This indicates that the enzyme productivity has been doubled, and together with the stability of the traits shown in Example 12, the usefulness of the present invention is shown (FIG. 7).

Example 10
Production of high production strain of isoamyl acetate < Production of hetero-type expression strain>
A DNA fragment used for gene replacement for overexpression of alcohol acetyltransferase was synthesized by junction PCR. First, using pK112 (Japanese Patent Laid-Open No. 2007-28912) as a template, 5'-TATTGCGGGATAATGAGTAAACGAATTCAAACGTCTTCAATTCAT-3 '(SEQ ID NO: 15) and 5'-CGTTAATTTTCTATATCCAAGGTACCAGGGTAATAACTGA-3' (SEQ ID NO: 16), PCR was performed using '(SEQ ID NO: 16) and 5'-ATGTGGTTCGTATCCTTCTATATCTTCCATCCTTAATAGAGCGAA-3' (SEQ ID NO: 17) to amplify the SED1 promoter.

After these fragments are purified, the respective fragments are mixed and subjected to the second PCR using 5′-CTACATTGAACTCTGTAGGCCACCGATAAATATTGCGGGATAATG-3 ′ (SEQ ID NO: 18) and 5′-ATGGCCACGGTCTATCAACTCTTGAGTGATATGTGGTTCGTATCC-3 ′ (SEQ ID NO: 19). As a result, a 2,030 bp gene replacement DNA fragment P _SED1 _ATF2 was prepared. This DNA fragment was introduced into the GKT1001 strain by the lithium acetate method, and yeast that had grown on the SD-U agar medium was selected. In this medium, P _{SED1_ATF2} can be replaced with the ATF2 promoter region on the chromosome, and only strains with complemented uracil requirement can grow.

PCR was performed using the following primers growing strain, P _SED1 _ATF2 was confirmed to be a strain replaced with the ATF2 promoter region on the chromosome in heterozygous.

Primer D 5'- CTACATTGAACTCTGTAGGCCACCGATAAA-3 '(SEQ ID NO: 20)
Primer E 5′-ATCCACTGACACCGTCGGAGCCGCAGTGGT-3 ′ (SEQ ID NO: 21)
The combination of primers D and E will amplify a 0.9 kbp fragment from the wild-type (P _SED1 _ATF2 not substituted) chromosome and a 2.5 kbp fragment from the recombinant (P _SED1 _ATF2 substituted) chromosome. Primers were designed (Figure 8). From the parent strain (WT / GKT1001 strain), a fragment of only 0.9 kbp was amplified, while in the growing strain, two fragments of 0.9 kbp and 2.5 kbp were amplified. From this result, it was found that this strain was a heterozygous strain in which P _{SED1_ATF2} was replaced with the ATF2 promoter region on the chromosome. This strain was defined as a hetero type strain.

<Preparation of marking plasmid>
Using yeast genomic DNA as a template, 5'-ggg aagctt ACGTCAGAAAAAGCAATATATAGTAA-3 '(SEQ ID NO: 22: lower case is a preliminary sequence for efficient HindIII digestion, underlined lower case is HindIII recognition sequence) and 5'-ggg tctaga AATTAACCTGGACAATTTTTATTGCT PCR was performed using -3 ′ (SEQ ID NO: 23: a lower case is a preliminary sequence for improving the efficiency of subsequent XbaI digestion, and an underlined lower case is an XbaI recognition sequence) to amplify the ATF2 promoter region. This DNA fragment was _designated as P _ATF2 (sequence 4). Next, P _ATF2 was digested with restriction enzymes HindIII and XbaI and inserted into pAKK2 that was similarly digested with SalI and EcoRI to obtain a marking plasmid pPATF2.

<Marking of hetero-type expression strain>
DNA linearized by cleaving pPATF2 with the restriction enzyme EcoRI was introduced into a hetero type strain by the lithium acetate method. Culturing was carried out on an SD-UK agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.067% CSM-LYS-URA (MPBIO) and 2% glucose, and the grown yeast was selected. In this medium, only a strain (marking strain) in which plasmid pPATF2 is introduced into the chromosome and lysine requirement is complemented can grow.

  The isoamyl acetate contained in the culture supernatant of the marking strain and heterozygous strain cultured in YPD medium for 24 hours was quantified by headspace solid-phase extraction gas chromatography (SPME-GC / VARIAN CP-3800). It was confirmed that isoamyl acetate was produced. The obtained strain was used as a marking strain.

<Preparation of homo-type expression strain>
The marking strain was cultured in YPD medium for 3 days and then applied to an α-AA plate. After culturing for 7 days, the growing yeast was selected to obtain a strain in which lysine requirement was restored. The obtained strain was subjected to PCR using the above primers D and E, and only the 2.5 kbp fragment was amplified, so that it was confirmed that the obtained strain was the intended homotype strain.

Example 11
<Evaluation of isoamyl acetate productivity of homozygous strain>
A small preparation test for sake was conducted with preparation (320 g pregelatinized rice, 86 g dried rice cake, 840 ml water, 600 OD 600 Unit yeast, 400 μl 90% lactic acid). At this time, a strain obtained by introducing the plasmid pRS406 (gene non-introduced plasmid) into the parent strain GKT1001 was used as a control strain. The charging bottle was left to stand at 15 ° C. for 20 days, and the sputum was sampled over time. The ethanol and aroma components of the sample were quantified by gas chromatography, and the fermentation performance and isoamyl acetate productivity of the strain were evaluated.

  FIG. 9 shows changes with time in the amounts of ethanol and isoamyl acetate. All the test strains fermented well and the final ethanol concentration reached about 19% (v / v). On the other hand, isoamyl acetate increased in the order of control <hetero type strain (control ratio 3 times) <homo type strain (6 times same). From this, it was clarified that the homotype strain in which the copy number was doubled by homogenization increased the expression level of ATF2, and the productivity of isoamyl acetate was improved compared to the heterotype strain. In a low-temperature and long-term culture environment that suppresses the initial bacterial body, such as sake brewing, it showed excellent substance productivity without applying selective pressure, demonstrating the industrial utility of homozygous strains. .

Example 12
<Evaluation of homozygous trait stability>
After single colonies of heterozygous and homozygous strains were inoculated three times with YPD medium, 10 ⁶ cells were plated on 5-FOA plates. As a result, a strain that grew on the 5-FOA plate was confirmed from the heterozygous strain (FIG. 10). This indicates that the URA3 marker that was supposed to have dropped out. As a result of examining the nucleotide sequences of the ATF2 upstream region of these 12 grown strains, it was confirmed that all had returned to the non-recombinant type. Since the heterozygous strain has a wild-type chromosome, it was considered that (normal) LOH was produced in the process of inoculation in YPD medium without selective pressure, and the strain did not have a recombinant chromosome. . From the number of colonies, the appearance frequency was expected to be about 10 ^-4 . It is thought that isoamyl acetate productivity of these strains has decreased to the same level as the parent strain. This indicates that heterozygous strains are unstable in substance productivity and that the acquired traits have not been lost in practice. It suggests that it needs to be managed. On the other hand, the homo-type strain had no growing strain, and it was confirmed that the incorporated trait was maintained extremely stably without applying selective pressure. This indicates that the homozygous mutant obtained by the present invention is very practical because it is resistant to planting and has high stability.

  The method of the present invention can be applied not only to a recessive trait that does not appear as a phenotype in the heterozygous form, but also to a DNA region in which it is difficult to confirm the phenotype as well as to obtain a homotypic strain exhibiting the phenotype. . Furthermore, even a useful hetero-recombinant strain can exhibit a more useful trait by making it a homo-type recombinant strain. For example, it is possible to confer auxotrophy to diploid cells with excellent practicality, making it easy to produce industrially useful recombinants. Protease-deficient strains are expected to improve useful protein production. In addition, by destroying cell wall proteins, the efficiency of surface layer presentation in surface layer display engineering, which has been researched in recent years, is improved. Furthermore, in this method, it is possible to further strengthen the useful traits present in the heterozygous form by homogenizing. For example, by homogenizing a genetic trait relating to the production of a useful substance introduced into a hetero form, the expression level can be increased and the productivity of the useful substance can be improved.

  Thus, by using the method of the present invention, the gene recombination operation of diploid cells having excellent practicality, which was difficult with the prior art, becomes extremely easy, and further the effect of gene recombination can be improved. Therefore, the present invention can greatly contribute to the creation of industrially useful genetic recombinants. Moreover, a useful substance can be produced in a larger amount than before by acquiring strains.

  Furthermore, unlike the hetero type strain, the homo type strain obtained by the method of the present invention does not lose the acquired character, has high stability, and has extremely high industrial practicality.

FIG. 1 schematically shows the outline of the present invention. FIG. 2 schematically illustrates one embodiment of the present invention. FIG. 3 shows the growth of each diploid sake yeast auxotrophic strain in each medium. FIG. 4 shows the growth of diploid sake yeast auxotrophic strains in YPD. FIG. 5 shows Zymolyase sensitivity of SED1-disrupted strains. FIG. 6 schematically shows the chromosomal plasmid integration position of the prepared strain. FIG. 7 shows the β-glucosidase activity of the prepared strain culture supernatant. FIG. 8 shows the principle of confirmation of the chromosomal gene replacement site (near the ATF2 promoter) of the prepared strain by PCR. FIG. 9 shows the result of the sake preparation test by the prepared strain. FIG. 10 shows the comparison results of the trait stability of the prepared strains.

Claims (17)

A diploid cell having a hetero-type structural gene and / or regulatory gene of interest in a specific region of the homologous chromosome has a homologous chromosome homozygous for the structural gene and / or regulatory gene of interest in the specific region A method for producing a diploid cell comprising the following steps:
(1) inserting and / or replacing the first marker gene into any of heterozygous homologous chromosomes in diploid cells;
(2) a step of culturing the diploid cell obtained in step (1) and selecting a diploid cell into which the first marker gene has been introduced using the expression of the first marker gene as an index;
(3) The diploid cell obtained in step (2) is cultured, and the homologous homologous to the target structural gene and / or regulatory gene in the specific region, using the dropout of the first marker gene as an index Obtaining a diploid cell having a chromosome. The method according to claim 1, wherein a diploid cell having a heterogeneous target structural gene and / or regulatory gene in a specific region of a homologous chromosome is obtained by a method comprising the following steps:
(1) inserting and / or replacing a second marker gene and a target structural gene and / or regulatory gene into a specific region of a homologous chromosome of a diploid cell;
(2) Culturing the diploid cell obtained in step (1), and using the expression of the second marker gene as an index, the target structural gene and / or regulatory gene of the heterotype is placed in a specific region of the homologous chromosome. Obtaining diploid cells. The method according to claim 2, wherein the first marker gene and the second marker gene are different marker genes. The method according to claim 1 or 2, wherein the marker gene is a marker gene that complements the sensitivity or requirement of diploid cells. The method according to any one of claims 1 to 4, wherein the marker gene is capable of detecting both sensitivity and / or requirement complementation and return to sensitivity and / or requirement. The marker gene is one or more marker genes selected from the group consisting of URA3, URA5, LYS2, LYS5, LEU2, HIS3, KanMX, AUR1-R, CYH2, CAN1, TRP1, GAP1, and GIN11M86. 5. The method according to 5. The method according to claim 4 or 5, wherein the sensitivity is a sensitivity selected from the group consisting of drug sensitivity, temperature sensitivity, UV sensitivity, and radiation sensitivity. The method according to any one of claims 1 to 7, wherein the diploid cell is yeast. Yeast is Saccharomyces, Candida, Torulopsis, Zygosaccharomyces, Schizosaccharomyces, Pichia, Yarrowia, Hansenula, Kluyveromyces, Debaryomyces, Geotrichum, Wickerhamia, Wellohames, Spo 9. The method according to 8. The method according to claim 9, wherein the yeast of the genus Saccharomyces is Saccharomyces cerevisiae. The method according to any one of claims 1 to 10, wherein the target structural gene is one or more selected from the group consisting of LEU2, HIS3, PEP4, and SED1 genes. The method according to any one of claims 1 to 11, wherein the target regulatory gene is a region containing a promoter and / or terminator. The method according to claim 12, wherein the promoter is a SED1 promoter. The method according to any one of claims 1 to 13, wherein the target structural gene is a structural gene connected in a form that can be expressed downstream of the SED1 promoter. The method according to any one of claims 1 to 10 and claims 12 to 14, wherein the target structural gene is one or more selected from the group consisting of BGL1, BGL7, ATF1, and ATF2. A gene disrupted yeast obtained by the method according to claim 1. A high gene expression yeast obtained by the method according to any one of claims 1 to 15.

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