Method for producing transformant

[0001] The present application claims priority from Japanese patent application JP 2020-200752 filed on Dec. 3, 2020, the content of which is hereby incorporated by reference into this application.

[0002] The present disclosure relates to a method for producing a transformant comprising deleting a particular region from a host genome with the use of a site-specific recombinase and a recognition sequence thereof.

[0003] A site-specific recombinase is an enzyme that has activity of recognizing a particular, short, homologous pair of nucleotide sequences and causing homologous recombination between the pair of nucleotide sequences. When homologous recombination takes place between a pair of homologous nucleotide sequences aligned in the same direction, a region flanked by such pair of nucleotide sequences would be cleaved. When homologous recombination takes place between a pair of homologous nucleotide sequences aligned in the opposite direction, in contrast, a region flanked by such pair of nucleotide sequences would be inverted.

[0004] A site-specific recombinase and a recognition sequence thereof may be used to delete (knock out) a particular region from the host genome, or a selection marker gene may be located between a pair of nucleotide sequences to remove the selection marker gene. According to a technique involving the use of a site-specific recombinase and a recognition sequence thereof, a transformant or a gene recombinant that has traits different from the original traits can be produced. By efficiently producing a transformant or a gene recombinant with the utilization of such technique, for example, synthetic biology-based microbial metabolic engineering can be advanced and efficiency thereof can be promoted. Synthetic biology is a technique that is achieved by rapidly advancing a cycle of design, construction, evaluation, and learning of a production host. In synthetic biology involving the use of a yeast host, in particular, efficient host construction; i.e., efficient preparation of a recombinant yeast, is critical.

[0005] Transformation techniques involving the use of the yeast hosts can be roughly classified into a method involving the use of a cyclic plasmid comprising a target gene integrated therein and a method involving the use of a linear vector comprising a target gene. A target gene can be easily introduced into a yeast host using a cyclic plasmid, and a transgenic yeast can be prepared with high efficiency of approximately 10⁻² (Gietz, R. D., et al., “High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method,” Nature Protocols, 2, 2007: 31-34). When a target gene is introduced into a yeast host using a linear vector, in contrast, it is necessary that the target gene be integrated into the genome via homologous recombination. Thus, efficiency for preparing a transgenic yeast would be approximately 10⁻⁶ at most (Storici, F, et al., “Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast,” Proc. Natl. Acad. Sci., U.S.A., 100, 2003: 14994-14999).

[0006] When a target gene is to be integrated into a particular site of genome DNA, the target gene is integrated into a site between a pair of homologous recombination sequences that enable homologous recombination with an upstream region and a downstream region of the site. As shown in FIG. 15, a nucleic acid fragment 100 containing an upstream region of the site, a nucleic acid fragment 101 containing a target gene, and a nucleic acid fragment 102 containing a downstream region of the site are introduced simultaneously, so that the target gene can be integrated into the particular site. In this case, homologous recombination regions are provided at the end of the nucleic acid fragment 100 containing the upstream region and at the end of the nucleic acid fragment 101 containing the target gene, so that the nucleic acid fragment 100 can be linked to the nucleic acid fragment 101 via homologous recombination. Also, homologous recombination regions are provided at the end of the nucleic acid fragment 101 and at the end of the nucleic acid fragment 102.

[0007] Because of the construction as shown in FIG. 15, the nucleic acid fragment 101 containing a target gene can be used in common, regardless of the site of the genome DNA into which such fragment is to be integrated. That is, the nucleic acid fragment 100 and the nucleic acid fragment 102 may be designed in accordance with the sites of the genome DNA into which the fragments are to be integrated. Thus, the nucleic acid fragment 101 containing a target gene can be used in common.

[0008] With the use of the site-specific recombinase and a recognition sequence thereof, a target gene included in the nucleic acid fragment 101 can be cleaved from genome DNA (not shown). When a selection marker gene is designated as a target gene, for example, a nucleic acid fragment may be first introduced, and a selection marker gene may then be removed (i.e., a marker recycle method).

[0009] When a plurality of nucleic acid fragments including a nucleic acid fragment having a target gene are introduced into a host, however, a complicated step, such as a nucleic acid amplification reaction, was necessary to determine whether or not the plurality of nucleic acid fragments had been integrated into genome DNA as designed or the target gene had been accurately cleaved. Under the above circumstances, the present disclosure provides a method for producing a transformant that enables evaluation as to accurate integration of a plurality of nucleic acid fragments including a nucleic acid fragment having a target gene into the host genome DNA or accurate cleaving of a target gene in a simple manner, so that a transformant can be efficiently produced.

[0010] The present disclosure encompasses the following.

• • [0011] (1) A method for producing a transformant comprising steps of:
    • [0012] introducing a group of nucleic acid fragments comprising a nucleic acid fragment having a target gene, a nucleic acid fragment having either one of the pair of homologous recombination sequences corresponding to a particular region of genome DNA, and a nucleic acid fragment having the other of the pair of homologous recombination sequences into a host cell; and
    • [0013] selecting a host cell in which the target gene flanked by the pair of recognition sequences recognized by a site-specific recombinase had been cleaved from genome DNA,
    • [0014] wherein nucleic acid fragments constituting the group of nucleic acid fragments can be linked to each other via a homologous recombination sequence provided at the end of each nucleic acid fragment, and
    • [0015] wherein at least one of the pair of recognition sequences is provided in a nucleic acid fragment other than the nucleic acid fragment having a target gene among the group of nucleic acid fragments.
  • [0016] (2) The method for producing a transformant according to (1), wherein the transformant lacks the particular region of genome DNA upon integration of nucleic acid fragments constituting the group of nucleic acid fragments therein.
  • [0017] (3) The method for producing a transformant according to (1), wherein the target gene is a selection marker gene.
  • [0018] (4) The method for producing a transformant according to (1) further comprising steps of:
    • [0019] introducing a group of nucleic acid fragments comprising a nucleic acid fragment having a target gene, a nucleic acid fragment having either one of the pair of homologous recombination sequences corresponding to the other region of genome DNA, and a nucleic acid fragment having the other of the pair of homologous recombination sequences into the selected host cell; and
    • [0020] selecting a host cell in which the target gene flanked by the pair of recognition sequences recognized by a site-specific recombinase had been cleaved from genome DNA,
    • [0021] wherein the nucleic acid fragment having the target gene introduced in the above step has the same homologous recombination sequence as the homologous recombination sequence in the nucleic acid fragment having a target gene introduced into the host cell in the previous step.

[0022] In the method for producing a transformant according to the present disclosure, at least one recognition sequence of a pair of recognition sequences recognized by a site-specific recombinase is provided in a nucleic acid fragment other than a nucleic acid fragment having a target gene. In the case that a group of nucleic acid fragments are not accurately integrated into genome DNA, accordingly, it is not possible to cleave the target gene from genome DNA by the method of the present disclosure. According to the method for producing a transformant according to the present disclosure, whether or not a group of nucleic acid fragments were accurately integrated into genome DNA can be determined based on the target gene expression. As descried above, whether or not a group of nucleic acid fragments were accurately integrated into genome DNA can be determined in a very simple manner according to the method for producing a transformant of the present disclosure. Thus, a transformant can be produced in a very efficient manner.

[0038] Hereafter, the present disclosure is described in greater detail with reference to the drawings and the examples.

[0039] In the method for producing a transformant according to the present disclosure (hereafter, referred to as “the method of the present disclosure”), a group of nucleic acid fragments comprising a target genes (i.e. a group of nucleic acid fragments comprising a nucleic acid fragment comprising the target gene and other nucleic acid fragments) are introduced into a particular position of the host genome DNA, so that a particular region is deleted from the genome DNA or a target gene is integrated into a particular position of the genome DNA, and genetic properties of the host are altered as a consequence. According to the method of the present disclosure, specifically, expression of the integrated target gene and deletion of a particular region from the genome DNA or expression of the integrated target gene would alter the genetic properties of the host.

[0040] A group of nucleic acid fragments is composed of a plurality of nucleic acid fragments. At least one nucleic acid fragment of a group of nucleic acid fragments comprises a target gene. A plurality of nucleic acid fragments constituting a group of nucleic acid fragments each have homologous sequences at both ends, so that the fragments can be integrated into the genome DNA or can be linked to each other via homologous recombination. Nucleic acid fragments located at the both ends when such plurality of nucleic acid fragments are linked to each other via homologous recombination have homologous sequences at the ends so as to integrate a group of nucleic acid fragments into a particular region of genome DNA. That is, a group of nucleic acid fragments comprises a nucleic acid fragment having either one of the pair of homologous recombination sequences corresponding to a particular region of genome DNA and a nucleic acid fragment having the other of the pair of homologous recombination sequences.

[0041] In the method of the present disclosure, in particular, a site-specific recombinase and a pair of recognition sequences recognized by such enzyme are used to cleave a target gene from the genome DNA. Thus, whether or not a group of nucleic acid fragments having target genes was accurately introduced into a particular region of the genome DNA can be determined. In the method of the present disclosure, specifically, a host cell in which the target gene flanked by the pair of recognition sequences recognized by the site-specific recombinase is cleaved from the genome DNA is selected from among the host cells into which the group of nucleic acid fragments had been introduced. According to the method of the present disclosure, at least one recognition sequence of a pair of recognition sequences is provided in a nucleic acid fragment other than the nucleic acid fragment comprising a target gene among the group of nucleic acid fragments. When the group of nucleic acid genes are inaccurately integrated into the genome DNA, accordingly, a target gene would not be flanked by a pair of recognition sequences, and a target gene would remain in the genome DNA. Therefore, whether or not the target gene has been cleaved from the genome DNA and whether or not a particular region has been accurately deleted from the genome DNA can be determined on the basis of the target gene expression.

[0042] In the method of the present disclosure, a site-specific recombinase is an enzyme that has activity of targeting a recognition sequence comprising a particular nucleotide sequence and causing recombination between a pair of recognition sequences. An example of a representative site-specific recombinase is a Cre recombinase, which is Type I topoisomerase derived from bacteriophage P1 (it may be simply referred to as “Cre”). Cre catalyzes site-specific recombination of DNA between a pair of loxP sites comprising particular sequences (i.e., Cre recognition sequences). A loxP recognition sequence is a 34-bp sequence comprising a 8-bp spacer sequence that determines the orientation and two 13-bp inverted repeat sequences that flank the spacer sequence. Cre is capable of recognizing the lox511 sequence comprising a nucleotide sequence different from the loxP recognition sequence and various variant lox sequences, such as lox2272 and loxFAS (Siegel, R. W. et al., 2001, FEBS letters, 499 (1-2), 147-53) and catalyzing site-specific recombination between such variant lox sequences.

[0043] In addition to Cre recombinase, examples of system of site-specific recombinases and recognition sequences include Flp recombinase and FRT sequence derived from the yeast 2μ plasmid (Broach, J. R. et al., 1982, Cell, 29 (1), 227-34), Dre recombinase and rox sequence derived from the Enterobacteria phage D6 (U.S. Pat. No. 7,422,889), R recombinase and RS sequence derived from a soy sauce yeast (Zygosaccharomyces rouxii) (Araki, H. et al., 1985, Journal of molecular biology, 182 (2), 191-203), and Gin recombinase and gix sequence derived from bacteriophage Mu (Maeser, S., et al., Molecular & general genetics: MGG, 230 (1-2), 170-6).

[0044] When two recognition sequences of a site-specific recombinase are present toward the same direction, a region between the recognition sequences would be circularly cleaved. When two enzyme recognition sequences are present toward opposite directions, the site-specific recombinase would invert a region between the recognition sequences.

[0045] A target gene refers to a nucleic acid to be introduced into the host genome. Accordingly, a target gene is not limited to a nucleotide sequence encoding a particular protein, and it encompasses nucleic acids comprising various nucleotide sequences, such as a nucleotide sequence encoding siRNA, a nucleotide sequence of a transcription regulatory region, such as a promoter or enhancer, that regulates the timing of transcription and the amount of production of transcription products, and a nucleotide sequence encoding transfer RNA (tRNA) or ribosome RNA (rRNA).

[0046] An example of a target gene is a so-called selection marker gene. Examples of selection marker genes include a drug-resistant gene that provides a host having susceptibility to a particular drug with resistance against such drug, a gene that encodes a fluorescent protein, a gene that encodes an enzyme catalyzing a coloring reaction, and an auxotrophic marker gene. Examples of drug-resistant genes include, but are not particularly limited to, antibiotic-resistant genes, such as a G418-resistant gene, a nourseothricin-resistant gene, a kanamycin-resistant gene, a hygromycin-resistant gene, a neomycin-resistant gene, an ampicillin-resistant gene, and an Aureobasidin A-resistant gene.

[0047] A target gene is not limited to the marker genes mentioned above, and it may be a site-specific recombinase gene. Specific examples include genes encoding the Cre recombinase, the Dre recombinase, and the Flp or Gin recombinase mentioned above. A site-specific recombinase gene is adequately selected in accordance with the recognition sequence used in the method of the present disclosure.

[0048] A target gene is integrated into genome DNA in an expressible state. A site-specific recombinase gene may be integrated into genome DNA together with a target gene in an expressible state, or a site-specific recombinase gene may be integrated into an expression vector and then introduced into the host in an expressible state. In an expressible state, a target gene or a site-specific recombinase gene is linked to a promoter, so that the gene is expressed under the control of a particular inducible promoter or a constitutive expression promoter in a host organism. In addition, cis elements, such as a promoter, a terminator, and, according to need, an enhancer, a splicing signal, a poly A addition signal, or a ribosome-binding (SD) sequence, can be ligated to the target gene.

[0049] A promoter for a target gene or a site-specific recombinase gene may be an inducible promoter or a constitutive expression promoter. An inducible promoter has functions of inducing expression under specific conditions. Examples of inducible promoters include, but are not particularly limited to, a promoter that induces expression in the presence of a particular substance, a promoter that induces expression under particular temperature conditions, and a promoter that induces expression in response to various types of stress. A promoter can be adequately selected in accordance with a host to be transformed.

[0050] Examples of inducible promoters include galactose-inducible GAL1 and GAL10 promoters, Tet-on/Tet-off system promoters that induce expression with the addition or removal of tetracycline or its derivative, and promoters of genes encoding heat shock proteins (HSP) such as HSP10, HSP60, and HSP90. As an inducible promoter, a CUP1 promoter that is activated with the addition of copper ions can be used. When a host is a prokaryotic cell such as E. coli, in addition, examples of inducible promoters include an IPTG-inducible lac promoter, a cold-shock-inducible cspA promoter, and an arabinose-inducible araBAD promoter.

[0051] A method for regulating target gene or site-specific recombinase gene expression is not limited to a method involving the use of a promoter, such as an inducible promoter or a constitutive expression promoter. For example, a method involving the use of a DNA recombinase may be employed. An example of a method that turns gene expression on or off with the use of a DNA recombinase is the FLEx switch method (A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range Circuit Mapping, Atasoy et al., The Journal of Neuroscience, 28, 7025-7030, 2008). According to the FLEx switch method, a DNA recombinase is used to cause recombination that alters the promoter sequence orientation and turns the gene expression on or off.

[0052] FIG. 1 schematically shows an embodiment of the method of the present disclosure. In the method shown in FIG. 1, a first nucleic acid fragment 1 containing, as a target gene, a selection marker gene S, a second nucleic acid fragment 2 containing one recognition sequence M1 of a pair of recognition sequences recognized by a site-specific recombinase, and a third nucleic acid fragment 3 containing the other recognition sequence M2 are introduced into the host. As long as the nucleotide sequences of the first nucleic acid fragment 1 to the third nucleic acid fragment 3 are determined, each of such fragments can be prepared as a double-stranded DNA via chemical synthesis in accordance with a conventional technique. In the method of the present disclosure, a site-specific recombinase gene is introduced into the host in an expressible manner, although it is not shown in FIG. 1. A site-specific recombinase gene may be contained in the first nucleic acid fragment 1, or it may be contained in an expression vector separately prepared.

[0053] The second nucleic acid fragment 2 comprises a first homologous recombination sequence 4 that can undergo homologous recombination with the host genome DNA at one end and a second homologous recombination sequence 5 that can undergo homologous recombination with the first nucleic acid fragment 1 at the other end. The first nucleic acid fragment 1 comprises a third homologous recombination sequence 6 that can undergo homologous recombination with the second nucleic acid fragment 2 at one end and a fourth homologous recombination sequence 7 that can undergo homologous recombination with the third nucleic acid fragment 3 at the other end. The third nucleic acid fragment 3 comprises a fifth homologous recombination sequence 8 that can undergo homologous recombination with the first nucleic acid fragment 1 at one end and a sixth homologous recombination sequence 9 that can undergo homologous recombination with the host genome DNA at the other end.

[0054] The first homologous recombination sequence 4 of the second nucleic acid fragment 2 and the sixth homologous recombination sequence 9 of the third nucleic acid fragment 3 can be designed based on a region to be deleted from the genome DNA. When a region R is to be deleted from the genome DNA in FIG. 1, for example, the first homologous recombination sequence 4 is designed based on the sequence of a recombinant region 10 upstream of the region R, and the sixth homologous recombination sequence 9 is designed based on the sequence of a recombinant region 11 downstream of the region R.

[0055] The first homologous recombination sequence 4 of the second nucleic acid fragment 2 and the sixth homologous recombination sequence 9 of the third nucleic acid fragment 3 can be designed as an upstream region in a particular region of genome DNA and a downstream region continuous to the upstream region. Specifically, the recombinant region 10 and the recombinant region 11 can be designed as continuous regions (i.e., the region R is not present), and the first homologous recombination sequence 4 and the sixth homologous recombination sequence 9 can be designed to correspond to such regions. In such a case, the original genome DNA would not be deleted of any region, and a group of nucleic acid fragments comprising target genes would be inserted into the genome DNA.

[0056] The second homologous recombination sequence 5 of the second nucleic acid fragment 2 and the fifth homologous recombination sequence 8 of the third nucleic acid fragment 3 can be designed based on the nucleotide sequences at the both ends of the first nucleic acid fragment 1. The second homologous recombination sequence 5 of the second nucleic acid fragment 2 and the third homologous recombination sequence 6 of the first nucleic acid fragment 1 can be designed as any nucleotide sequences as long as such sequences can undergo homologous recombination. Also, the fifth homologous recombination sequence 8 of the third nucleic acid fragment 3 and the fourth homologous recombination sequence 7 of the first nucleic acid fragment 1 can be designed as any nucleotide sequences as long as such sequences can undergo homologous recombination. Specifically, the first nucleic acid fragment 1 can be designed to comprise sequences at the both ends other than the target gene (i.e., the third homologous recombination sequence 6 and the fourth homologous recombination sequence 7) as any common nucleotide sequences, regardless of the region R to be deleted and the selection marker gene S.

[0057] A region between the first homologous recombination sequence 4 of the second nucleic acid fragment 2 and the recombinant region 10 and a region between the sixth homologous recombination sequence 9 of the third nucleic acid fragment 3 and the recombinant region 11 have sequence identity that is high enough to undergo homologous recombination (crossing). Nucleotide sequence identity in each of the regions can be calculated using a conventional sequence comparison software, such as blastn. Nucleotide sequence identity in each of the regions may be 60% or higher. In some embodiments, sequence identity may be 80% or higher, 90% or higher, 95% or higher, and 99% or higher.

[0058] The length of the first homologous recombination sequence 4 of the second nucleic acid fragment 2 may be the same with or different from that of the sixth homologous recombination sequence 9 of the third nucleic acid fragment 3. The length of the first homologous recombination sequence 4 and that of the sixth homologous recombination sequence 9 need to be long enough to undergo homologous recombination (crossing) with the genome DNA. For example, such length may be 0.1 kb to 3 kb, 0.5 kb to 3 kb, and 0.5 kb to 2 kb.

[0059] The lengths of the second homologous recombination sequence 5 to the fifth homologous recombination sequence 8 of the first nucleic acid fragment 1 to the third nucleic acid fragment 3 may be the same with or different from one another, provided that these sequences can undergo homologous recombination. The lengths of the second homologous recombination sequence 5 to the fifth homologous recombination sequence 8 may be, for example, 30 b to 300 b. In some embodiments, the length may be 40 b to 200 b, or 50 b to 100 b.

[0060] Sequence identity between the third homologous recombination sequence 6 of the first nucleic acid fragment 1 and the second homologous recombination sequence 5 of the second nucleic acid fragment 2 and sequence identity between the fourth homologous recombination sequence 7 of the first nucleic acid fragment 1 and the fifth homologous recombination sequence 8 of the third nucleic acid fragment 3 need to be high enough to undergo homologous recombination (crossing). Nucleotide sequence identity in each of the regions can be calculated using a conventional sequence comparison software, such as blastn. Nucleotide sequence identity in each of the regions may be 60% or higher. In some embodiments, sequence identity may be 80% or higher, 90% or higher, 95% or higher, and 99% or higher.

[0061] The first nucleic acid fragment 1, the second nucleic acid fragment 2, and the third nucleic acid fragment 3 configured as described above are introduced into the host. As a result, homologous recombination takes place between the first nucleic acid fragment 1 and the genome DNA, between the first nucleic acid fragment 1 and the second nucleic acid fragment 2, between the first nucleic acid fragment 1 and the third nucleic acid fragment 3, and between the third nucleic acid fragment 3 and the genome DNA, as schematically shown in FIG. 1. Thus, the first nucleic acid fragment 1, the second nucleic acid fragment 2, and the third nucleic acid fragment 3 would be integrated into the genome DNA. This would result in deletion of the region R from the genome DNA.

[0062] Upon expression of the site-specific recombinase gene contained in the first nucleic acid fragment 1 or an expression vector, a region between the pair of recognition sequences M1 and M2 would be deleted. More specifically, the third homologous recombination sequence 6, the selection marker gene S, and the fourth homologous recombination sequence 7 between the pair of recognition sequences M1 and M2 would be deleted from the genome DNA, as shown in FIG. 2.

[0063] In the method of the present disclosure, the recognition sequence M1 is located on the second nucleic acid fragment 2, and the recognition sequence M2 is located on the third nucleic acid fragment 3. When the first nucleic acid fragment 1 to the third nucleic acid fragment 3 are not accurately integrated into the genome DNA, accordingly, the selection marker gene S would not be flanked by the pair of recognition sequences M1 and M2. Even if the site-specific recombinase gene is expressed, the selection marker gene S would remain in the genome DNA.

[0064] When the second nucleic acid fragment 2 is not integrated but the first nucleic acid fragment 1 and the third nucleic acid fragment 3 are integrated into a region other than the region R in the genome DNA as shown in FIG. 3, for example, the selection marker gene S would not be deleted and it would remain in the genome DNA even if the site-specific recombinase gene is expressed. With the use of a phenotype resulting from the selection marker gene S, such as drug resistance or fluorescence, as the indicator, whether or not the first nucleic acid fragment 1 to the third nucleic acid fragment 3 had been accurately integrated into the genome DNA, in other words, whether or not the region R had been accurately deleted, can be evaluated.

[0065] When the pair of recognition sequences M1 and M2 are present in the same nucleic acid fragment (the first nucleic acid fragment 1 in FIG. 1) to flank the selection marker gene as shown in FIG. 4, in contrast, the selection marker gene S flanked by the pair of recognition sequences M1 and M2 would be deleted from the genome DNA upon expression of the site-specific recombinase gene, regardless of whether a plurality of nucleic acid fragments had been integrated into the genome DNA accurately or inaccurately. In such a case, it is not possible to evaluate as to whether or not a plurality of nucleic acid fragments had been accurately integrated into the genome DNA using a phenotype resulting from the selection marker gene S, such as drug resistance or fluorescence, as the indicator. When the pair of recognition sequences M1 and M2 are present in the same nucleic acid fragment to flank the selection marker gene S as shown in FIG. 4, accordingly, it is necessary to subject the plurality of nucleic acid fragments to a nucleic acid amplification reaction, such as PCR, to verify that the target region had been deleted from the genome DNA.

[0066] As described above, the method of using the site-specific recombinase and its recognition sequence to remove the selection marker gene S from the genome DNA can be applied to the so-called marker recycle method. According to the marker recycle method, a plurality of genes are successively introduced or deleted, a selection marker gene used for a single instance of gene introduction or deletion is removed, and the same selection marker gene is used for the subsequent gene introduction or deletion.

[0067] The method of the present disclosure shown in FIGS. 1 to 3 can be applied to the marker recycle method. As shown in FIGS. 1 and 2, specifically, a transformant from which the particular region R had been removed is selected using the phenotype resulting from the expression of the selection marker gene S as an indicator, and the selection marker gene S is then removed using the site-specific recombinase and its recognition sequence. As described above, a transformant lacking a particular region R does not have the selection marker gene S. Thus, the first nucleic acid fragment 1 having the selection marker gene S may be used again to delete a region other than the region R. According to the marker recycle method, as shown in FIG. 3, it is possible to verify that the first nucleic acid fragment 1 to the third nucleic acid fragment 3 were not accurately integrated based on the expression of the selection marker gene S.

[0068] In the marker recycle method involving the use of the method of the present disclosure, in particular, the first nucleic acid fragment 1 having the selection marker gene S can be repeatedly used. According to the method of the present disclosure, as described above, the selection marker gene S is removed with the use of the site-specific recombinase and its recognition sequence, and the third homologous recombination sequence 6 and the fourth homologous recombination sequence 7 are removed at the same time. When the pair of recognition sequences M1 and M2 are present in the same nucleic acid fragment to flank the selection marker gene S as shown in FIG. 4, in contrast, the selection marker gene S may be deleted from a region between the pair of recognition sequences M1 and M2 with the aid of a site-specific recombinase. In such a case, the homologous recombination sequences 20 and 21 at the both ends of the nucleic acid fragment would remain in the genome DNA. When another region is to be deleted with the use of the nucleic acid fragment comprising the pair of recognition sequences M1 and M2 and the selection marker gene S again, as shown in FIG. 5, a newly introduced nucleic acid fragment may undergo homologous recombination with the homologous recombination sequences 20 and 21 remaining in the genome DNA.

[0069] According to the method of the present disclosure, in contrast, the third homologous recombination sequence 6 and the fourth homologous recombination sequence 7 in the first nucleic acid fragment 1 comprising the selection marker gene S would not remain in the genome DNA at the same time. Even if a region other than the region R is deleted with the use of the first nucleic acid fragment 1 comprising the selection marker gene S again, accordingly, it is possible to prevent a problem such that the first nucleic acid fragment 1 would be integrated into a site where the region R had existed. In the marker recycle method, accordingly, at least one recognition sequence of the pair of recognition sequences M1 and M2 may be provided in a nucleic acid fragment other than the first nucleic acid fragment 1 having the selection marker gene S. Thus, the first nucleic acid fragment 1 having the selection marker gene S can be repeatedly used.

[0070] The first nucleic acid fragment 1 can be repeatedly used. In other words, it is not necessary to prepare the first nucleic acid fragment 1 every time when the selection marker gene S is used a plurality of times in the marker recycle method. When the pair of recognition sequences M1 and M2 is present in the same nucleic acid fragment to flank the selection marker gene S as shown in FIG. 4, it is necessary to prepare nucleic acid fragments designed to constitute the homologous recombination sequences 20 and 21 comprising different nucleotide sequences in order to prevent the problem as shown in FIG. 5. Since the first nucleic acid fragment 1 can be repeatedly used in the method of the present disclosure, a procedure can be simplified when such method is applied to the marker recycle method.

[0071] According to the method shown in FIGS. 1 and 2, the second nucleic acid fragment 2 and the third nucleic acid fragment 3 were designed to comprise the recognition sequences M1 and M2, respectively. That is, both of the pair of recognition sequences M1 and M2 were designed to be located in nucleic acid fragments other than the first nucleic acid fragment 1 comprising the selection marker gene S. In the method of the present disclosure, in contrast, it is sufficient if the pair of recognition sequences M1 and M2 flanks the selection marker gene S, at least one of the pair of recognition sequences M1 and M2 is located on a nucleic acid fragment other than the first nucleic acid fragment 1 comprising the selection marker gene S, and the other thereof is located on the first nucleic acid fragment 1. For example, the recognition sequence M1 of the pair of recognition sequences M1 and M2 may be located on the first nucleic acid fragment 1 comprising the selection marker gene S, and the other recognition sequence M2 may be located on the third nucleic acid fragment 3, as shown in FIG. 6. In such a case, as shown in FIG. 7, the selection marker gene S and the fourth homologous recombination sequence 7 are deleted from a region between the pair of recognition sequences M1 and M2 by the action of the site-specific recombinase, but the third homologous recombination sequence 6 of the first nucleic acid fragment 1 would remain in the genome DNA.

[0072] Even if the third homologous recombination sequence 6 of the first nucleic acid fragment 1 remains in the genome DNA as shown in FIG. 7, as with the case shown in FIGS. 1 to 3, a phenotype resulting from the selection marker gene S, such as drug resistance or fluorescence, may be used as the indicator to evaluate as to whether or not the first nucleic acid fragment 1 to the third nucleic acid fragment 3 had been accurately integrated into the genome DNA, in other words, whether or not the region R had been accurately deleted. Even if the third homologous recombination sequence 6 of the first nucleic acid fragment 1 remains in the genome DNA as shown in FIG. 7, the first nucleic acid fragment 1 having the selection marker gene S would not be erroneously integrated into the same position. As described above, accordingly, it is possible to prevent erroneous integration of the first nucleic acid fragment 1 having the selection marker gene S in the marker recycle method.

[0073] In the method of the present disclosure, a plurality of nucleic acid fragments are not limited to the first nucleic acid fragment 1 to the third nucleic acid fragment 3, and 4 or more nucleic acid fragments may be used to introduce a target gene into a particular region of genome DNA. When the pair of recognition sequences M1 and M2 flanks the selection marker gene S and at least one of the pair of recognition sequences M1 and M2 is located on a nucleic acid fragment other than the first nucleic acid fragment 1 comprising the selection marker gene S, a phenotype resulting from the selection marker gene S, such as drug resistance or fluorescence, may be used as the indicator to evaluate as to whether or not a plurality of nucleic acid fragments had been accurately integrated into the genome DNA, in other words, whether or not the region R had been accurately deleted. As described above, accordingly, it is possible to prevent erroneous integration of the first nucleic acid fragment 1 having the selection marker gene S in the marker recycle method.

[0074] The method of the present disclosure can be applied to any host cell without particular limitation. Examples of host cells include fungi such as filamentous fungi and yeast, bacteria such as E. coli and Bacillus subtilis, plant cells, and animal cells including mammalian and insect cells. In particular, yeast host cells may be used. Examples of yeast include, but are not particularly limited to, yeast of Saccharomyces, yeast of Kluyveromyces, yeast of Candida, yeast of Pichia, yeast of Schizosaccharomyces, and yeast of Hansenula. More specifically, the method of the present disclosure can be applied to yeast species of Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces boulardii.

[0075] Expression vectors comprising site-specific recombinase genes are not particularly limited. Examples of expression vectors that can be used include: YCp-type E. coli-yeast shuttle vectors such as pRS413, pRS414, pRS415, pRS416, YCp50, pAUR112, and pAUR123; Yep-type E. coli-yeast shuttle vectors such as YES2 and YEp13; YIp-type E. coli-yeast shuttle vectors such as pRS403, pRS404, pRS405, pRS406, pAUR101, and pAUR135; E. coli-derived plasmids, such as ColE-type plasmids such as pBR322, pBR325, pUC18, pUC19, pUC118, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396, and pTrc99A, p15A-type plasmids such as pACYC177 and pACYC184, and pSC101-type plasmids such as pMW118, pMW119, pMW218, and pMW219; Agrobacterium-derived plasmids such as pBI101; and Bacillus subtilis-derived plasmids such as pUB110 and pTP5.

[0076] In the method of the present disclosure, the first nucleic acid fragment 1 to the third nucleic acid fragment 3 or an expression vector can be introduced into a host cell by any method without particular limitation. An adequate method may be selected from among conventional techniques, such as the calcium chloride method, the competent cell method, the protoplast or spheroplast method, and the electropulse method.

[0077] In order to express a site-specific recombinase gene under the control of an inducible promoter, adequate conditions are determined in accordance with the inducible promoter. In the case that a galactose-inducible promoter, such as GAL1- or GAL10-inducible promoter, is used, for example, galactose is added to a medium used for culturing a host cell comprising the first nucleic acid fragment 1 to the third nucleic acid fragment 3 or the expression vector introduced therein, or the host cell is transferred to and cultured in a galactose-containing medium. Thus, a site-specific recombinase gene can be induced to express. In the case that a promoter of a gene encoding a heat shock protein (HSP) is used as an inducible promoter, heat shock is applied at a desired timing when culturing a host cell comprising the first nucleic acid fragment 1 to the third nucleic acid fragment 3 or the expression vector introduced therein. Thus, a site-specific recombinase gene can be induced to express at a desired timing.

[0078] Under the conditions in which an inducible promoter can induce gene expression, the first nucleic acid fragment 1 to the third nucleic acid fragment 3 or the expression vector may be introduced into a host cell, and a site-specific recombinase gene may be expressed under the control of the inducible promoter. In such a case, it is not necessary to transfer the host cell to an expression-inducible condition. Thus, a transformant can be obtained more easily.

[0079] In the method of the present disclosure, the first homologous recombination sequence 4 of the second nucleic acid fragment 2 and the sixth homologous recombination sequence 9 of the third nucleic acid fragment 3 are designed to comprise nucleotide sequences that are highly homologous to an upstream region and a downstream region of a particular gene. In such a case, a nucleic acid fragment comprising a target gene would be integrated into the genome via homologous recombination and the particular gene would be deleted from the genome. By observing a phenotype resulting from deletion of the particular gene, accordingly, whether or not a nucleic acid fragment comprising a target gene had been integrated into the genome can be determined. When the ADE1 gene is used as a particular gene, for example, the ADE1 gene would be deleted from the genome upon integration of a group of nucleic acid fragments comprising target genes into the genome. As a result, 5-aminoimidazole riboside is accumulated in the host, and a transformant is colored red by the polymerized polyribosyl aminoimidazole. By detecting a red color, accordingly, whether or not a group of nucleic acid fragments having target genes had been integrated into the host genome can be determined.

[0080] Hereafter, the present disclosure is described in greater detail with reference to the examples, although the technical scope of the present disclosure is not limited to the following examples.

[0081] In this example, the haploid experimental yeast strain, S. cerevisiae BY4742, was used as a host.

Preparation of a GRE3-Destructive Linear Vector Set in which loxP and a Marker are Contained in the Same DNA Fragment

[0082] In this example, as shown in FIG. 8, 3 types of plasmids each comprising a DNA fragment containing the 5′-homologous recombination region of the GRE3 gene (homologous sequence 1) and a DNA sequence to perform homologous recombination (homologous sequence 2); a DNA fragment containing a gene sequence having a G418-resistant gene (G418 marker) and a DNA recombinase Cre gene (not shown in FIG. 8) to perform loxP-sequence-site-specific recombination flanked by loxP sequences and, at both ends, DNA sequences to perform homologous recombination (homologous sequences 2 and 3); and a DNA fragment containing the DNA sequence to perform homologous recombination (homologous sequence 3) and a 3′-homologous recombination region of the GRE3 gene (homologous sequence 4) inserted therein were prepared. The homologous sequence 2 and the homologous sequence 3 were both of approximately 60 bp and designed on a primer sequence. Upon expression of the Cre gene, the G418 marker and the Cre gene flanked by two loxP sequences can be removed. DNA sequences can be amplified via PCR. In order to bind DNA fragments to each other, primers were artificially synthesized with the addition of a DNA sequence to overlap with an adjacent DNA sequence by approximately 15 bp (Table 1).

[0083] With the use of the primers described above and the genome or synthetic DNA of the S. cerevisiae BY4742 strain as a template, the target DNA fragments were amplified, the DNA fragments were successively bound to each other using the In-Fusion HD Cloning Kit or the like, and the resultant was cloned into the pUC19 vector to prepare the 3 types of target plasmids. The resulting plasmids were designated as pUC-5U_GRE3-homologous sequence 2, pUC-homologous sequence 2-loxP-P_ERG1-G418-T_URA3-T_CYC1-Cre-P_GAL1-loxP-homologous sequence 3, and pUC-homologous sequence 3-3U_GRE3. A set of the 3 types of plasmids was designated as the pUC-gre3::loxPin-G418-Cre vector set.

[0084]

TABLE 1
Amplified DNA fragment	Primer sequence (5′-3′)	SEQ ID NO:
pUC-5U_GRE3-homologous sequence 2
Fragment containing GRE3 5′-	GTGGCCCGGGAGAGACAGTTTAGTAGTGACTCGCGGCCAGTTGGGG	1
homologous recombination	CTAAACGAGATTTGGAATATTG
sequence and homologous	CAATCACAGGGCGGGAAATAAGCTACAATTAACGCCAAAAAATCTT	2
sequence 2	TAAAAAATTTCCAATTTTCCTTTACGATTTATATTACAG
Fragment containing pUC19	CCCGCCCTGTGATTGAGGCGGGATGGTGTCCCCACAGTCACGACGT	3
and homologous sequence 2	TGTAAAACG
	TCTCTCCCGGGCCACGACGCTAGGCCAGTACCTCCACTCTCAGTAC	4
	AATCTGCTCTGATG
pUC-homologous sequence 2-loxP-P_REG1-G418-T_URA3-T_CY1-Cre-P_GAL1-loxP-homologous
sequence 3
Fragment containing	GGGATGGTGTCCCCACAGTTACCGTTCGTATAGCATACATTATACG	5
homologous sequence 2, loxP	AAGTTATGTCTCTTGCAGAACACAATAAGTG
sequence, and ERG1 promoter	GTTGAATATGGCTCATCCTTGTATTACTCGTTTGTTCTGTTTCTAT	6
Fragemnt containing G418	ATGAGCCATATTCAACGGGAAAC	7
	TTTAGTAGACATGCATTACAACCAATTAACCAATTCTG	8
Fragment containing URA3	TGCATGTCTACTAAACTCACAAATTAGAGCTTCAATT	9
terminator	GGGTAATAACTGATATAATTAAATTGAAGCTC	10
Fragment containing CYC1	TATCAGTTATTACCCAGCTTGCAAATTAAAGCCTTCG	11
terminator	TTAGTTATGTCACGCTTACATTCACG	12
Fragment containing Cre gene	GCGTGACATAACTAATCAATCACCATCTTCCAACAATC	13
(posterior region) and COX5B	CCATTTACTAACATTCGAGGTGTACAAGCACAAGTTTTGCAGGCTT	14
intron	TCGAAAGAACTGATTTCGATC
Fragment containing COX5B	GAATGTTAGTAAATGGATATCTTTTAAAACTCAAAACAAAAATCAG	15
intron and Cre gene	TGTTTGTTATACATGCCAAAGCCTGCTTAGCTCTTTCAC
(anterior region)	CAAGGAGAAAAAACCATGTCTAACTTGTTGACTGTTC	16
Fragment containing GAL1	TGGTTTTTTCTCCTTGACGTTAAAGTATAG	17
promoter, loxP sequence, and	CAGTCGTAGATGCGTAAAATACCGTTCGTATAATGTATGCTATACG	18
homologous sequence 3	AAGTTATACGGATTAGAAGCCGCCG
Fragment containing pUC19	ACGCATCTACGACTGTGGGTCCCGTGGAGAAATGTATGAAACCCTG	19
and homologous sequences	TATGGAGAGTGATTCAGTCACGACGTTGTAAAACGAC
2 and 3	TGGGGACACCATCCCGCCTCAATCACAGGGCGGGAAATAAGCTACA	20
	ATTAACGCCAAACTCTCAGTACAATCTGCTCTG
pUC-homologous sequence 3-3U_GRE3
Fragment containing GRE3 3′-	CGTGGAGAAATGTATGAAACCCTGTATGGAGAGTGATTCAGTTCCA	21
homologous recombination	GCCAGTAAAATCCATACTC
sequence and homologous	AGTGCGTCGGCAGTACCGGATCCTAAAGCCGATTCAAGAAAAGTCT	22
sequence 3	TTTTGCCAGCCAGTCC
Fragment containing pUC19	TACTGCCGACGCACTTTAGAACGGCCACCGTCCTCAGTCACGACGT	23
and homologous sequenc 3	TGTAAAACG
	ATACATTTCTCCACGGGACCCACAGTCGTAGATGCGTACTCTCAGT	24
	ACAATCTGCTCTG

Preparation of a GRE3-Destructive Linear Vector Set (3 Fragments) in which loxP and a Marker are Contained in Different DNA Fragments

[0085] In this example, as shown in FIG. 9, 3 types of plasmids each comprising a DNA fragment containing the 5′-homologous recombination region of the GRE3 gene (homologous sequence 1), one loxP sequence and a DNA sequence to perform homologous recombination (homologous sequence 5); a DNA fragment containing a gene sequence having a G418-resistant gene (G418 marker), the DNA recombinase Cre gene, and, at both ends, DNA sequences to perform homologous recombination (homologous sequences 5 and 6); and a DNA fragment containing the DNA sequence to perform homologous recombination (homologous sequence 6), the other loxP sequence, and a 3′-homologous recombination region of the GRE3 gene (homologous sequence 4) inserted therein were prepared. The homologous sequence 2 and the homologous sequence 3 were both of approximately 60 bp and designed on a primer sequence. The G418 marker and the Cre gene are flanked by two loxP sequences located on adjacent fragments. Upon expression of the Cre gene, the G418 marker, the Cre gene, and the homologous sequences 2 and 3 can be removed. DNA sequences can be amplified via PCR. In order to bind DNA fragments to each other, primers were synthesized with the addition of a DNA sequence so as to overlap with the adjacent DNA sequence by approximately 15 bp (Table 2).

[0086] With the use of the primers described above and the genome or synthetic DNA of the S. cerevisiae BY4742 strain as a template, the target DNA fragments were amplified, the DNA fragments were successively bound to each other using the In-Fusion HD Cloning Kit or the like, and the resultant was cloned into the pUC19 vector to prepare the 3 types of target plasmids. The resulting plasmids were designated as pUC-5U_GRE3-loxP-homologous sequence 5, pUC-homologous sequence 5-P_REG1-G418-T_URA3-T_CYC1-Cre-P_GAL1-homologous sequence 6, and pUC-homologous sequence 6-loxP-3U_GRE3. A set of the 3 types of plasmids was designated as the pUC-gre3::loxPout-G418-Cre vector set.

[0087]

TABLE 2
Amplified DNA fragment	Primer sequence (5′-3′)	SEQ ID NO:
pUC-5U_GRE3-loxP-homologous sequence 5
Fragment containing GRE3 5′-	GTGGCCCGGGAGAGACAGTTTAGTAGTGACTCGCGGCCAGTTGGGGC	25
homologous recombination	TAAACGAGATTTGGAATATTGGGGCTAAACGAGATTTGGAATATTG
sequence, loxP, and	GCCTTCACATATAGTATAACTTCGTATAATGTATGCTATACGAACGG	26
homologous sequence 5	TAAAAATCTTTAAAAAATTTCCAATTTTCCTTTACGATTTATATTAC
	AGTATTTTCC
Fragment containing pUC19	ACTATATGTGAAGGCATGGCTATGGCACGGCAGACATTCCGCCAGAT	27
and homologous sequence 5	CATCAATAGGCACCAGTCACGACGTTGTAAAACG
	TCTCTCCCGGGCCACGACGCTAGGCCAGTACCTCCACTCTCAGTACA	28
	ATCTGCTCTGATG
pUC-homologous sequence 5-P_REG1-G418-T_URA3-T_CYC1-Cre-P_GAL1-homologous
sequence 6
Fragment containing	TGGCACGGCAGACATTCCGCCAGATCATCAATAGGCACCAGTGTCTC	29
homologous sequence 5 and	TTGCAGAACACAATAAG
ERG1 promoter	GTTGAATATGGCTCATCCTTGTATTACTCGTTTGTTCTGTTTCTATT	30
	C
Fragment containing G418	ATGAGCCATATTCAACGGGAAAC	31
	TTTAGTAGACATGCATTACAACCAATTAACCAATTCTG	32
Fragment containing URA3	TGCATGTCTACTAAACTCACAAATTAGAGCTTCAATT	33
terminator	GGGTAATAACTGATATAATTAAATTGAAGCTC	34
Fragment containing CYC1	TATCAGTTATTACCCAGCTTGCAAATTAAAGCCTTCG	35
terminator	TTAGTTATGTCACGCTTACATTCACG	36
Fragment containing Cre gene	GCGTGACATAACTAATCAATCACCATCTTCCAACAATC	37
(posterior region) and	CCATTTACTAACATTCGAGGTGTACAAGCACAAGTTTTGCAGGCTTT	38
COX5B intron	CGAAAGAACTGATTTCGATC
Fragment containing COX5B	GAATGTTAGTAAATGGATATCTTTTAAAACTCAAAACAAAAATCAGT	39
intron and Cre gene	GTTTGTTATACATGCCAAAGCCTGCTTAGCTCTTTCAC
(anterior region)	CAAGGAGAAAAAACCATGTCTAACTTGTTGACTGTTC	40
Fragment containing GAL1	TGGTTTTTTCTCCTTGACGTTAAAGTATAG	41
promoter and homologous	GAATCATTTAGACACGGGCATCGTCCTCTCGAAAGGTGAAAAACGGA	42
sequence 6	TTAGAAGCCGC
Fragment containing pUC19	GTGTCTAAATGATTCGACCAGCCTAAGAATGTTCAACCAGTCACGAC	43
and homologous sequences	GTTGTAAAACG
5 and 6	ATGTCTGCCGTGCCATAGCCATGCCTTCACATATAGTACTCTCAGTA	44
	CAATCTGCTCTG
pUC-homologous sequence 6-loxP-3U_GRE3
Fragment containing GRE3 3′-	CTAAGAATGTTCAACATAACTTCGTATACGCATACATTATACGAACG	45
homologous recombination	GTACAGTTCCAGCCAGTAAAATCC
sequence, loxP, and	AGTGCGTCGGCAGTACCGGATCCTAAAGCCGATTCAAGAAAAGTCTT	46
homologous sequence 6	TTTGCCAGCCAGTCC
Fragment containing pUC19	TACTGCCGACGCACTTTAGAACGGCCACCGTCCTCAGTCACGACGTT	47
and homologous sequence 3	GTAAAACG
	GTTGAACATTCTTAGGCTGGTCGAATCATTTAGACACGGGCATCGTC	48
	CTCTCGAAAGGTGACTCTCAGTACAATCTGCTC

Preparation of a GRE3-Destructive Linear Vector Set (5 Fragments) in which loxP and a Marker are Contained in Different DNA Fragments

[0088] In this example, as shown in FIG. 10, a GRE3-destructive linear vector set comprising 5 fragments was prepared. In this example, 2 types of plasmids each comprising a DNA fragment containing the loxP sequence and, at both ends, the homologous sequence 2 and the homologous sequence 5 and a DNA fragment containing the loxP sequence and, at both ends, the homologous sequence 6 and the homologous sequence 3 inserted therein were prepared. A 3′-downstream region of the ALD4 gene is inserted in a region between the homologous sequence 2 and the loxP sequence, and a 3′-downstream region of the ALD6 gene is inserted in a region between the loxP sequence and the homologous sequence 3 as dummy sequences so as to extend the length of the DNA fragment. DNA sequences can be amplified via PCR. In order to bind DNA fragments to each other, primers were synthesized with the addition of a DNA sequence so as to overlap with the adjacent DNA sequence by approximately 15 bp (Table 3).

[0089] With the use of the primers described above and the genome or synthetic DNA of the S. cerevisiae BY4742 strain as a template, the target DNA fragments were amplified, the DNA fragments were successively bound to each other using the In-Fusion HD Cloning Kit or the like, and the resultant was cloned into the pUC19 vector to prepare the 2 types of target plasmids. The resulting plasmids were designated as pUC-homologous sequence 2-loxP-dummy 1-homologous sequence 5 and pUC-homologous sequence 6-loxP-dummy 2-homologous sequence 3. A set of the 5 plasmids comprising pUC-homologous sequence 2-dummy 1-loxP-homologous sequence 5, pUC-homologous sequence 6-loxP-dummy 2-homologous sequence 3, pUC-5U_GRE3-homologous sequence 2 designed in advance (FIG. 8), pUC-homologous sequence 5-P_REG1-G418-T_URA3-T_CYC1-Cre-P_GAL1-homologous sequence 6 (FIG. 9), and pUC-homologous sequence 3-3U_GRE3 (FIG. 8) was designated as the pUC-gre3::loxPout-G418-Cre (5 fragments) vector set.

[0090]

TABLE 3
Amplified DNA fragment	Primer sequence (5′-3′)	SEQ ID NO:
pUC-homologous sequence 2-dummy 1-loxP-homologous sequence 5
Fragment containing	ATTTCCCGCCCTGTGATTGAGGCGGGATGGTGTCCCCACAGTGG	49
homologous sequence 2, loxP,	TCATCAATAAGCCTGG
3′-downstream region of ALD4	GCCTTCACATATAGTATAACTTCGTATAATGTATGCTATACGAA	50
gene, and homologous	CGGTAAAAAGATATTTCCAGTGCTAAGGTCAAC
sequence 5
Fragment containing pUC19,	ACTATATGTGAAGGCATGGCTATGGCACGGCAGACATTCCGCCA	51
homologous sequence 2, and	GATCATCAATAGGCACCAGTCACGACGTTGTAAAAC
homologous sequence 5	CACAGGGCGGGAAATAAGCTACAATTAACGCCAAACTCTCAGTA	52
	CAATCTGCTC
pUC-homologous sequence 6-loxP-dummy 2-homologous sequenc 3
Fragment containing	CTAAGAATGTTCAACATAACTTCGTATAGCATACATTATACGAA	53
homologous sequence 6, loxP,	CGGTACAGTTGTACCAACCTGCATTTC
3′-downstream region of ALD6	ATACATTTCTCCACGGGACCCACAGTCGTAGATGCGTAAAAGCT	54
gene, and homologous	CACTTGTTTCTTGATTTTTTAG
sequence 3
Fragment containing pUC19,	CGTGGAGAAATGTATGAAACCCTGTATGGAGAGTGATTCAGTCA	55
homologous sequence 6, and	CGACGTTGTAAAACG
homologous sequence 3	GTTGAACATTCTTAGGCTGGTCGAATCATTTAGACACGGGCATC	56
	GTCCTCTCGAAAGGTGACTCTCAGTACAATCTGCTCTG

Preparation of an ADE1-Destructive Linear Vector Set in which loxP and a Marker are Contained in the Same DNA Fragment

[0091] In this example, as shown in FIG. 11, 3 types of plasmids each comprising a DNA fragment containing a 5′-homologous recombination region of the ADE1 gene (homologous sequence 7) and a DNA sequence to perform homologous recombination (homologous sequence 2); a DNA fragment containing the nourseothricin-resistant gene (nat1: nat marker) and the DNA recombinase Cre gene (not shown) flanked by the loxP sequences and, at both ends, DNA sequences to perform homologous recombination (homologous sequences 2 and 3); and a DNA fragment containing a 3′-homologous recombination region of the ADE1 gene (homologous sequence 8) and a DNA sequence to perform homologous recombination (homologous sequence 3) inserted therein were prepared. Upon expression of the Cre gene, the nat marker and the Cre gene flanked by a pair of loxP sequences can be removed. DNA sequences can be amplified via PCR. In order to bind DNA fragments to each other, primers were synthesized with the addition of a DNA sequence so as to overlap with the adjacent DNA sequence by approximately 15 bp (Table 4).

[0092] With the use of the primers described above and the genome or synthetic DNA of the S. cerevisiae BY4742 strain as a template, the target DNA fragments were amplified, the DNA fragments were successively bound to each other using the In-Fusion HD Cloning Kit or the like, and the resultant was cloned into the pUC19 vector to prepare the 3 types of target plasmids. The resulting plasmids were designated as pUC-5U_ADE1-homologous sequence 2, pUC-homologous sequence 2-loxP-P_PMA1-nat1-T_LEU2-P_GAL1-Cre-T_CYC1-loxP-homologous sequence 3, and pUC-homologous sequence 3-3U_ADE1. A set of the 3 types of plasmids was designated as the pUC-ade1::loxPin-nat1-Cre vector set.

[0093]

TABLE 4
Amplified DNA fragment	Primer sequence (5′-3′)	SEQ ID NO:
pUC-5U_ADE1-homologous sequence 2
Fragment containing ADE1 5′-	CGTGGCCCGGGAGAGACAGTTTAGTAGTGACTCGCGGCCAGTA	57
homologous recombination	TGAGTCGGGCAATTCCG
sequence and homologous	CAATCACAGGGCGGGAAATAAGCTACAATTAACGCCAAAAAAT	58
sequence 2	ATCGTTAATATTTCGTATGTGTATTCTTTGTAAG
Fragment containing pUC19	CCCGCCCTGTGATTGAGGCGGGATGGTGTCCCCACAGTCACGA	59
and homologous sequence 2	CGTTGTAAAACG
	CTCTCCCGGGCCACGACGCTAGGCCAGTACACTCTCAGTACAA	60
	TCTGCTC
Fragment containing pUC19 and homologous sequence 2
Fragment containing	GGGATGGTGTCCCCACAGTTACCGTTCGTATAGCATACATTAT	61
homologous sequence 2, loxP	ACGAAGTTATGTTACTCTCACACTCTTTAGTTC
sequence, and PMA1 promoter	TCACCGAAATCTTCATATTGATATTGTTCGATAATTAAATCTT	62
	TCTTATC
Fragment containing nat1	ATGAAGATTTCGGTGATCCCTG	63
	TTAGGCGTCATCCTGTGCTC	64
Fragment containing LEU2	CAGGATGACGCCTAAAAAGATTCTCTTTTTTTATGATATTTGT	65
terminator	AC
	AGGAATCATAGTTTCATGATTTTCTGTTAC	66
Fragment containing GAL1	GAAACTATGATTCCTACGGATTAGAAGCCGCCG	67
promoter	TGGTTTTTTCTCCTTGACGTTAAAGTATAG	68
Fragment containing Cre gene	CAAGGAGAAAAAACCATGTCTAACTTGTTGACTGTTC	69
(anterior region) and COX5B	CCATTTACTAACATTCGAGGTGTACAAGCACAAGTTTTGCAGG	70
intron	CTTTCGAAAGAACTGATTTCGATC
Fragment containing Cre gene	GTTTGTTATACATGCCAAAGCCTGCTTAGCTCTTTC	71
(posterior region)	CAAGGAGAAAAAACCATGTCTAACTTGTTGACTGTTC	72
Fragment containing CYC1	TGGTTTTTTCTCCTTGACGTTAAAGTATAG	73
terminator, loxP sequence,	CAGTCGTAGATGCGTAAAATACCGTTCGTATAATGTATGCTAT	74
and homologous sequence 3	AC
Fragment containing pUC19	ACGCATCTACGACTGTGGGTCCCGTGGAGAAATGTATGAAACC	75
and homologous sequences	CTGTATGGAGAGTGATTCAGTCACGACGTTGTAAAACGAC
2 and 3	TGGGGACACCATCCCGCCTCAATCACAGGGCGGGAAATAAGCT	76
	ACAATTAACGCCAAACTCTCAGTACAATCTGCTCTG
pUC-homologous sequence 3-3U_ADE1
Fragment containing ADE1 3′-	CGTGGAGAAATGTATGAAACCCTGTATGGAGAGTGATTCAGTA	77
homologous recombination	TTCATACATACCCGTATG
sequence and homologous	AGTGCGTCGGCAGTACCGGATCCTAAAGCCGATTCAAGAAAAT	78
sequence 3	GACCGGATGAAACCACCG
Fragment containing pUC19	TACTGCCGACGCACTTTAGAACGGCCACCGTCCTCAGTCACGA	79
and homologous sequence 3	CGTTGTAAAACG
	ATACATTTCTCCACGGGACCCACAGTCGTAGATGCGTACTCTC	80
	AGTACAATCTGCTCTG

Preparation of an ADE1-Destructive Linear Vector Set in which loxP and a Marker are Contained in Different DNA Fragments

[0094] In this example, as shown in FIG. 12, 3 types of plasmids each comprising a DNA fragment containing a 5′-homologous recombination region of the ADE1 gene (homologous sequence 7), the loxP sequence, and a homologous sequence to perform homologous recombination (homologous sequence 2); a DNA fragment containing a gene sequence having the nourseothricin-resistant gene (nat1) (nat marker), the DNA recombinase Cre gene, and, at both ends, DNA sequences to perform homologous recombination (homologous sequences 2 and 3); and a DNA fragment containing a 3′-homologous recombination region of the ADE1 gene (homologous sequence 8), the loxP sequence, and a homologous sequence to perform homologous recombination (homologous sequence 3) inserted therein were prepared. The nat marker and the Cre gene are flanked by the loxP sequences contained in the adjacent fragments. Upon expression of the Cre gene, the nat marker and the Cre gene can be removed. DNA sequences can be amplified via PCR. In order to bind DNA fragments to each other, primers were synthesized with the addition of a DNA sequence so as to overlap with the adjacent DNA sequence by approximately 15 bp (Table 5).

[0095] With the use of the primers described above and the genome or synthetic DNA of the S. cerevisiae BY4742 strain as a template, the target DNA fragments were amplified, the DNA fragments were successively bound to each other using the In-Fusion HD Cloning Kit or the like, and the resultant was cloned into the pUC19 vector to prepare the 3 types of target plasmids. The resulting plasmids were designated as pUC-5U_ADE1-loxP-homologous sequence 2, pUC-homologous sequence 2-P_PMA1-nat1-T_LEU2-P_GAL1-Cre-T_CYC1-homologous sequence 3, and pUC-homologous sequence 3-loxP-3U_ADE1. A set of the 3 types of plasmids was designated as the pUC-ade1::loxPout-nat1-Cre vector set.

[0096]

TABLE 5
Amplified DNA fragment	Primer sequence (5′-3′)	SEQ ID NO:
pUC-5U_ADE1-loxP-homologous sequence 5
Fragment containing ADE1 5′-	CGTGGCCCGGGAGAGACAGTTTAGTAGTGACTCGCGGCCAG	81
homologous recombination	TATGAGTCGGGCAATTCCG
sequence, loxP, and	GCCTTCACATATAGTATAACTTCGTATAATGTATGCTATAC	82
homologous sequence 5	GAACGGTAAAATATCGTTAATATTTCGTATGTGTATTCTTT
	GTAAGCAATG
Fragment containing pUC19	ACTATATGTGAAGGCATGGCTATGGCACGGCAGACATTCCG	83
and homologous sequence 5	CCAGATCATCAATAGGCACCAGTCACGACGTTGTAAAAC
	TCTCTCCCGGGCCACGACGCTAGGCCAGTACCTCCACTCTC	84
	AGTACAATCTGCTCTGATG
pUC-homologous sequence 5-P_PMA1-nat1-T_LEU2-P_GAL1-Cre-T_CYC1-homologus
sequence 6
Fragment containing	TGGCACGGCAGACATTCCGCCAGATCATCAATAGG	85
homologous sequence 5 and	TCACCGAAATCTTCATATTGATATTGTTCGATAATTAAATC	86
PMA1 promoter	TTTCTTATC
Fragment containing nat1	ATGAAGATTTCGGTGATCCCTG	87
	TTAGGCGTCATCCTGTGCTC	88
Fragment containing LEU2-	CAGGATGACGCCTAAAAAGATTCTCTTTTTTTATGATATTT	89
terminator	GTAC
	AGGAATCATAGTTTCATGATTTTCTGTTAC	90
Fragment containing GAL1	GAAACTATGATTCCTACGGATTAGAAGCCGCCG	91
promoter	TGGTTTTTTCTCCTTGACGTTAAAGTATAG	92
Fragment containing Cre gene	CAAGGAGAAAAAACCATGTCTAACTTGTTGACTGTTC	93
(anterior region) and	GAATGTTAGTAAATGGATATCTTTTAAAACTCAAAACAAAA	94
COX5B intron	ATCAGTGTTTGTTATACATGCCAAAGCCTGCTTAGCTCTTT
	CAC
Fragment containing COX5B	CCATTTACTAACATTCGAGGTGTACAAGCACAAGTTTTGCA	95
intron and Cre gene	GGCTTTCGAAAGAACTGATTTCGATC
(posterior region)	GCGTGACATAACTAATCAATCACCATCTTCCAACAATC	96
Fragment containing CYC1	TTAGTTATGTCACGCTTACATTCACG	97
terminator and homologous	GAATCATTTAGACACGGGCATCGTCCTCTCGAAAGGTGAAA	98
sequence 6	AAGCTTGCAAATTAAAGCCTTCG
Fragment containing pUC19	GTGTCTAAATGATTCGACCAGCCTAAGAATGTTCAACCAGT	99
and homologous sequences	CACGACGTTGTAAAACG
5 and 6	ATGTCTGCCGTGCCATAGCCATGCCTTCACATATAGTACTC	100
	TCAGTACAATCTGCTCTG
pUC-homologous sequence 6-loxP-3U_ADE1
Fragment containing ADE1 3′-	CTAAGAATGTTCAACATAACTTCGTATAGCATACATTATAC	101
homologous recombinant	G
sequence, loxP, and	AGTGCGTCGGCAGTACCGGATCCTAAAGCCGATTCAAGAAA	102
homologous sequence 6	ATGACCGGATGAAACCACCG
Fragment containing pUC19	TACTGCCGACGCACTTTAGAACGGCCACCGTCCTCAGTCAC	103
and homologous sequence 6	GACGTTGTAAAACG
	GTTGAACATTCTTAGGCTGGTCGAATCATTTAGACACGGGC	104
	ATCGTCCTCTCGAAAGGTGACTCTCAGTACAATCTGCTC

Preparation of an ADE1-Destructive Linear DNA Fragment Set (5 Fragments) in which loxP and a Marker are Contained in Different DNA Fragments

[0097] In this example, as shown in FIG. 13, 2 types of plasmids each comprising a DNA fragment containing the loxP sequence and, at both ends, the homologous sequence 2 and the homologous sequence 5; and a DNA fragment containing the loxP sequence and, at both ends, the homologous sequence 6 and the homologous sequence 3 inserted therein were prepared. In these DNA fragments, a 5′-upstream region of the PDC6 gene is inserted in a region between the homologous sequence 2 and the loxP sequence, and a 5′-upstream region of the SED1 gene is inserted in a region between the loxP sequence and the homologous sequence 3 as dummy sequences so as to merely extend the length. DNA sequences can be amplified via PCR. In order to bind DNA fragments to each other, primers were synthesized with the addition of a DNA sequence so as to overlap with the adjacent DNA sequence by approximately 15 bp (Table 6).

[0098] With the use of the primers described above and the genome or synthetic DNA of the S. cerevisiae BY4742 strain as a template, the target DNA fragments were amplified, the DNA fragments were successively bound to each other using the In-Fusion HD Cloning Kit or the like, and the resultant was cloned into the pUC19 vector to prepare the 2 types of target plasmids. The resulting plasmids were designated as pUC-homologous sequence 2-dummy 3-loxP-homologous sequence 5 and pUC-homologous sequence 6-loxP-dummy 4-homologous sequence 3. A set of the 5 types of plasmids comprising pUC-homologous sequence 2-dummy 3-loxP-homologous sequence 5, pUC-homologous sequence 6-loxP-dummy 4-homologous sequence 3, pUC-5U_ADE1-homologous sequence 2 designed in advance (Table 4, FIG. 11), pUC-homologous sequence 2-P_PMA1-nat1-T_LEU2-P_GAL1-Cre-T_CYC1-homologous sequence 3 (Table 5, FIG. 12), and pUC-homologous sequence 3-3U_ADE1 (Table 4, FIG. 11) was designated as the pUC-ade1::loxPout-nat1-Cre (5 fragments) vector set.

[0099]

TABLE 6
Amplified DNA fragment	Primer sequence (5′-3′)	SEQ ID NO:
pUC-homologous sequence 2-dummy 3-loxP-homologous sequence 5
Fragment containing	GGGATGGTGTCCCCACAGTTGTCCAGACTACGTCGAATCCTT	105
homologous sequence 2, loxP,	AC
5′-upstream region of PDC6	GCCTTCACATATAGTATAACTTCGTATAATGTATGCTATACG	106
gene, and homologous	AACGGTAAAAATTTTGGCCAAATGCCACAGC
sequence 5
Fragment containing pUC19,	ACTATATGTGAAGGCATGGCTATGGCACGGCAGACATTCCGC	107
homologous sequence 2, and	CAGATCATCAATAGGCACCAGTCACGACGTTGTAAAACG
homologous sequence 5	TGGGGACACCATCCCGCCTCAATCACAGGGCGGGAAATAAGC	108
	TACAATTAACGCCAAACTCTCAGTACAATCTGCTCTG
pUC-homologous sequence 6-loxP-dummy 4-homologous sequence 3
Fragment containing	CTAAGAATGTTCAACATAACTTCGTATAGCATACATTATACG	109
homologous sequence 6, loxP,	AACGGTACAGTAGTGAAGGCGTCATCCTC
3′-downstream region of SED1	CAGTCGTAGATGCGTAAAATCGAAAGAAGGCCGCATGAC	110
gene, and homologous
sequence 3
Fragment containing pUC19,	ACGCATCTACGACTGTGGGTCCCGTGGAGAAATGTATGAAAC	111
homologous sequence 6, and	CCTGTATGGAGAGTGATTCAGTCACGACGTTGTAAAACGAC
homologous sequence e	GTTGAACATTCTTAGGCTGGTCGAATCATTTAGACACGGGCA	112
	TCGTCCTCTCGAAAGGTGACTCTCAGTACAATCTGCTCTG

Transformation

[0100] With the use of the plasmids prepared in the manner described above; i.e., the pUC-gre3::loxPin-G418-Cre vector set; the pUC-gre3::loxPout-G418-Cre vector set; and the pUC-gre3::loxPout-G418-Cre (5 fragments) vector set, as templates, DNA fragments contained in the plasmids were amplified via PCR. The primer sets used in PCR are shown in Table 7. With the use of the amplified DNA fragments, the BY4742 strains were transformed, the transformants were applied to a G418-containing YPD agar medium, and the grown colonies were then naturalized. Strains in which deletion of the GRE3 gene from the chromosome was observed were selected via PCR. Further, the stains were cultured in a YPGa medium (10 g/l yeast extract, 20 g/l peptone, and 20 g/l galactose), Cre gene expression was induced, and the G418 marker and the Cre gene were removed via Cre/loxP site-specific recombination. The resulting strains were designated as Uz3444, Uz3445, and Uz3760 strains. Transformation was performed in accordance with the method of Akada et al. (Akada, R. et al., “Elevated temperature greatly improves transformation of fresh and frozen competent cells in yeast,” BioTechniques 28, 2000: 854-856).

[0101] With the use of the plasmids; i.e., the pUC-ade1::loxPin-nat1-Cre vector set or the pUC-ade1::loxPout-nat1-Cre vector set, as templates, DNA fragments contained in the plasmids were amplified via PCR. The primer sets used in PCR are shown in Table 7. With the use of the amplified DNA fragments, the Uz3444 or Uz3445 strains were transformed, the transformants were applied to a nourseothricin-containing YPD medium, and colonies were allowed to grow.

[0102] With the use of the plasmids; i.e., the pUC-ade1::loxPout-nat1-Cre (5 fragments) vector set, as templates, DNA fragments contained in the plasmids were amplified via PCR. The primer sets used in PCR are shown in Table 7. With the use of the amplified DNA fragments, the BY4742 or Uz3760 strains were transformed, the transformants were applied to a nourseothricin-containing YPD medium, and colonies were allowed to grow. The ADE1 gene is a gene of the adenine biosynthetic pathway. In ADE1-deficient strains, adenine intermediary metabolites; i.e., 5-aminoimidazole riboside, are accumulated, and polymerized polyribosyl aminoimidazole is colored red. Thus, ADE1-deficient strains can be easily identified. The red grown colonies and the white grown colonies were separately counted to inspect the efficiency of the strain comprising the vector set genome introduced thereinto for destructing the ADE1 gene.

[0103]

TABLE 7
Amplified DNA fragment	Primer sequence (5′-3′)	SEQ ID NO:
pUC-5U_GRE3-homologous	TGGGGCTAAACGAGATTTGG	113
sequence 2	TGGGGACACCATCCCGC	114
pUC-homologous sequence 2-loxP-	AATCACTCTCCATACAGGGTTTCATACATTTCTCC	116
P_REG1-G418-T_URA3-T_CYC1-Cre-
P_GAL1-loxP-homologous sequenc 3
pUC-homologous sequence	ACGCATCTACGACTGTGGGTCC	117
3-3U_GRE3	GTCTTTTTGCCAGCCAGTCC	118
pUC-5U_GRE3-loxP-homologous	TGGGGCTAAACGAGATTTGG	119
sequence 5	GTGCCTATTGATGATCTGGCGGAATGTCTG	120
pUC-homologous sequence 5-P_REG1-	ACTATATGTGAAGGCATGGCTATGGCACG	121
G418-T_URA3-T_CYC1-Cre-P_GAL1-	GTTGAACATTCTTAGGCTGGTCGAATCATTTAGAC	122
homologous sequence 6
pUC-homologous sequence 6-loxP-	CACCTTTCGAGAGGACGATGCCCG	123
3U_GRE3	GTCTTTTTGCCAGCCAGTCC	124
pUC-homologous sequence 2-dummy	TTGGCGTTAATTGTAGCTTATTTCCC	125
1-loxP-homologous sequence 5	GTGCCTATTGATGATCTGGCGGAATGTCTG	126
pUC-homologous sequence 6-loxP-	CACCTTTCGAGAGGACGATGCCCG	127
dummy 2-homologous sequence 3	AATCACTCTCCATACAGGGTTTCATACATTTCTCC	128
pUC-5U_ADE1-homologous sequence 2	ATGAGTCGGGCAATTCCG	129
	TGGGGACACCATCCCGC	130
pUC-homologous sequence 2-loxP-	TTGGCGTTAATTGTAGCTTATTTCCC	131
P_PMA1-nat1-T_LEU2-P_GAL1-Cre-	AATCACTCTCCATACAGGGTTTCATACATTTCTCC	132
T_CYC1-loxP-homologous sequence 3
pUC-homologous sequence	ACGCATCTACGACTGTGGGTCC	133
3-3U_ADE1	TGACCGGATGAAACCACC	134
pUC-5U_ADE1-loxP-homologous	ATGAGTCGGGCAATTCCG	135
sequence 5	GTGCCTATTGATGATCTGGCGGAATGTCTG	136
pUC-homologous sequence 5-P_PMA1-	ACTATATGTGAAGGCATGGCTATGGCACG	137
nat1-T_LEU2-P_GAL1-Cre-T_CYC1-	GTTGAACATTCTTAGGCTGGTCGAATCATTTAGAC	138
homologous sequence 6
pUC-homologous sequence 6-loxP-	CACCTTTCGAGAGGACGATGCCCG	139
3U_ADE1	TGACCCGGATGAAACCACC	140
pUC-homologous sequence 2-dummy	TTGGCGTTAATTGTAGCTTATTTCCC	141
3-loxP-homologous sequence 5	GTGCCTATTGATGATCTGGCGGAATGTCTG	142
pUC-homologous sequence 6-loxP-	CACCTTTCGAGAGGACGATGCCCG	143
dummy 4-homologous sequence 3	AATCACTCTCCATACAGGGTTTCATACATTTCTCC	144

Results and Discussion

[0104] According to the method described in the example, it is necessary that DNA fragments containing loxP accurately undergo homologous recombination to flank the marker gene. Otherwise, it is impossible to remove the marker gene through site-specific recombination with the aid of Cre recombinase (see FIG. 3). According to the method described in the example, whether or not the marker gene had been removed was determined by simply observing the phenotype resulting from marker gene expression without a confirmation process such as PCR.

[0105] Meanwhile, according to the method described in the example, the effects achieved by a so-called marker recycle method were verified in the manner described below. Specifically, loxP is provided in each of the DNA fragments adjacent to the DNA fragment that would undergo homologous recombination via the homologous sequence, not in a DNA fragment containing a marker gene. As a result, the marker gene and the homologous sequences flanking the marker gene would be removed from the genome DNA through site-specific recombination by the Cre recombinase. In the case that loxP sequences are provided to flank a marker gene in a DNA fragment comprising the marker gene, in contrast, the marker gene would be removed through site-specific recombination by Cre recombinase, but the homologous sequences flanking the marker gene would not be removed from the genome DNA. When a DNA fragment having the homologous sequences and the marker gene is repeatedly used, accordingly, in the second homologous recombination and thereafter, a homologous sequence remaining after the previous homologous recombination may undergo homologous recombination with the DNA fragment to be repeatedly used. Such homologous recombination may be problematic in practical applications (see FIG. 5).

[0106] In order to examine the above problem, at the outset, the pUC-gre3::loxPin-G418-Cre vector set containing a DNA fragment comprising a marker gene flanked by a pair of loxP sequences, and the pUC-gre3::loxPout-G418-Cre vector set containing a DNA fragment comprising a marker gene, other DNA fragments comprising loxP positioned adjacent to the both sides of the aforementioned DNA fragment were introduced into the GRE3 gene locus, respectively, and the marker gene was removed via Cre/loxP site-specific recombination to prepare strains (Uz3444, Uz3445). Subsequently, the second homologous recombination was performed. That is, the pUC-ade1::loxPin-nat1-Cre vector set containing a DNA fragment in which a pair of loxP sequences flanks the marker gene, or the pUC-ade1::loxPout-nat1-Cre vector set containing a DNA fragment having a marker gene and DNA fragments positioned adjacent to the both sides of the aforementioned DNA fragment and comprising loxP was introduced into the ADE1 gene locus, and efficiency for homologous recombination was examined.

[0107] When a DNA fragment comprising a pair of loxP sequences flanking a marker gene was used, as a result, it was found that repetition of homologous recombination increases the frequency for inaccurate homologous recombination to the extent that practical problems would be caused (Table 8). When a DNA fragment comprising a marker gene and DNA fragments comprising loxP are used, in contrast, it was found that repetition of homologous recombination does not substantially lead to inaccurate homologous recombination. In Table 8, the efficiency for homologous recombination was determined in terms of the red colony count/total colony count.

[0108]

TABLE 8
	Marker gene and	Marker gene and
	loxP provided	loxP provided
	in the same	in different
	DNA fragment	DNA fragments
ADE1-deficient strain	50(0.532)	411(0.930)
Red colony count (homologous
recombination efficiency*)
Nourseothricin resistant,	44	31
non-ADE1-deficient strain
White colony

[0109] Meanwhile, in the case that the loxP sequences are provided in both DNA fragments adjacent to the DNA fragment comprising a marker gene, in contrast, even if the marker gene flanked by the pair of loxP sequences and homologous sequences are removed, the homologous sequences provided outside the pair of loxP sequences would remain in the genome DNA when the number of DNA fragments is 5 or more (see FIG. 10, the homologous sequences 2 and 3 in FIG. 10). When the marker gene is to be introduced into the ADE1 gene locus in the second recombination (see FIG. 13), homologous recombination may occur between the homologous sequences 2 and 3 remaining in the first GRE3-gene locus and the 3 DNA fragments, as shown in FIG. 14.

[0110] In order to examine the possibility described above, at the outset, the pUC-gre3::loxPout-G418-Cre (5 fragments) vector set was introduced into the GRE3 gene locus and the marker gene was removed through Cre/loxP site-specific recombination to prepare a strain of interest (Uz3760). Subsequently, the second homologous recombination was performed. That is, the pUC-ade1::loxPout-nat1-Cre (5 fragments) vector set was introduced into the ADE1 gene locus to prepare a strain of interest. For comparison, a strain selectively comprising the pUC-ade1::loxPout-nat1-Cre (5 fragments) introduced therein was prepared, and the efficiency for homologous recombination was compared.

[0111] As a result, no significant differences were observed in the efficiency for homologous recombination as shown in FIG. 9. This indicates that an extent of lowering in the efficiency for homologous recombination caused by repeated use of DNA fragments would be insignificant, provided that the marker gene flanked by a pair of loxP sequences and the homologous sequences are removed. Therefore, the method described in the example can be adopted even if the number of DNA fragments is 5 or more and the applicability of the method is considered extensive.

[0112]

TABLE 9
	An introduction	Introduction
	into one locus	into two loci
ADE1-deficient strain	92(0.876)	84(0.913)
Red colony count (homologous
recombination efficiency*)
Nourseothricin resistant,	13	8
non-ADE1-deficient strain
White colony