Plasmid for transformation assistance, method for producing transformant using the same, and transformation method

Abstract

To produce simply and efficiently a stable transformant in which a gene of interest is incorporated into the genome.SOLUTION: A method for producing a transformant, comprises a step of introducing, into a host, linear genome-introduced nucleic acid fragment(s) comprising a gene of interest and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragment(s), and then selecting a transformant, in which the gene of interest is incorporated into a predetermined position in the host genome and the gene of interest is expressed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a transformation-assisting plasmid used when introducing a target gene into a host, a method for producing a transformant using the transformation-assisting plasmid, and a transformation method using the transformation-assisting plasmid. [Background technology]

[0002] In general, the technology of introducing a target gene into a host cell from the outside is called transformation or genetic recombination, and the cell into which the target gene has been introduced is called a transformant or recombinant. By efficiently producing transformants using transformation technology, for example, it is possible to accelerate and streamline microbial metabolic engineering using synthetic biology techniques. Here, synthetic biology techniques are technologies that are established by quickly rotating the cycle of designing, constructing, evaluating, and learning a production host. In particular, in synthetic biology using yeast as a host, one of the important challenges is efficient host construction, that is, efficient production of recombinant yeast.

[0003] Transformation of yeast as a host can be broadly divided into two methods: one using a circular plasmid incorporating a target gene, and the other using a linear vector containing a target gene. It is easy to introduce a target gene into yeast using a circular plasmid, and it has been around for about 10 -2 On the other hand, when a target gene is introduced into yeast using a linear vector, the target gene needs to be integrated into the genome by homologous recombination, which requires a high degree of efficiency of about 10 -6 However, it is only possible to produce transformed yeast with an efficiency of about 100% (Non-Patent Document 2).

[0004] The method of introducing a target gene into yeast using a circular plasmid is efficient as described above, but the circular plasmid may be lost, making it impossible to produce a stable recombinant yeast. On the other hand, the method of introducing a target gene into yeast using a linear vector is stable because the target gene is integrated into the genome, but as described above, it cannot be said to be an efficient method.

[0005] To improve the introduction efficiency of a target gene into a genome, a technique is known in which a target sequence of a target-specific endonuclease such as a homing endonuclease is previously introduced into a site to be introduced in the genome to cleave the double strand at that site (Non-Patent Document 2). Further, instead of the target-specific endonuclease, a technique is known in which a technique capable of cleaving an arbitrary base sequence such as CRISPR-Cas9 or TALEN is used to similarly cleave the double strand at the site to be introduced in the genome (Non-Patent Document 3). Thus, by cleaving the double strand at the site where the target gene is to be introduced, the homologous recombination efficiency can be improved to about 10 -2 ~10 -1 times.

[0006] However, in these methods for improving the introduction efficiency of target genes, it is necessary to previously introduce the target sequence of the endonuclease into the site to be introduced in the genome, or it is necessary to prepare a guide RNA or the like for the target site. Thus, these methods for improving the introduction efficiency of target genes are complex methods that require various steps in addition to preparing a DNA fragment for homologous recombination containing the target gene and transforming using this.

[0007] Further, Patent Document 1 discloses a plasmid having a selection marker having an intron configured to sandwich a homing endonuclease target sequence with a telomere seed sequence. The plasmid disclosed in Patent Document 1 is converted from a circular plasmid into a linear molecule by the expression of the homing endonuclease and can stably exist due to the terminal telomere seed sequence.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009] [Non-Patent Document 1] Gietz, R.D., et al. “High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method.” Nature Protocols. 2 (2007): 31-34. [Non-Patent Document 2] Storici, F, et al. “Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast.” Proc. Natl. Acad. Sci. USA. 100 (2003): 14994-14999. [Non-Patent Document 3] DiCarlo, J.E., et al. “Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.” Nucleic Acids Res. 41 (2013): 4336-4343. [Summary of the Invention] [Problems to be Solved by the Invention]

[0010] However, any of the above-described methods has a problem that a stable transformant in which a target gene is integrated into the genome cannot be easily and efficiently produced. Therefore, in view of the above-described circumstances, the present invention aims to provide a method for producing a transformant and a transformation method capable of easily and efficiently producing a stable transformant in which a target gene is integrated into the genome, and a plasmid for assisting transformation that can be used in these methods. [Means for Solving the Problems]

[0011] The present invention that has achieved the above-described object includes the following. (1) A step of introducing into a host one or more linear genomic introduction nucleic acid fragments comprising a target gene for introduction at a predetermined position on the genome and a transformation assisting plasmid having a pair of homologous recombination sequences for incorporating the linear genomic introduction nucleic acid fragment, wherein the linear genomic introduction nucleic acid fragment is incorporated into the transformation assisting plasmid, and a pair of homologous recombination sequences for homologous recombination with the outside of the target gene and a predetermined position on the genome are arranged, and a pair of endonuclease target sequences are arranged outside the pair of homologous recombination sequences. A step of selecting a transformant in which the target gene is incorporated at the predetermined position in the genome of the host and the target gene is expressed. A method for producing a transformant. (2) The method for producing a transformant according to (1), wherein the transformation assisting plasmid comprises a pair of homologous recombination sequences that homologous recombine with the outside of the target gene in the linear genomic introduction nucleic acid fragment, and a pair of endonuclease target sequences arranged on the opposite side of the position where the linear genomic introduction nucleic acid fragment is incorporated via the homologous recombination sequences. (3) The method for producing a transformant according to (1), wherein the linear genomic introduction nucleic acid fragment comprises a pair of homologous recombination sequences for incorporation at the predetermined position on the genome that sandwich the target gene, a pair of endonuclease target sequences outside the pair of homologous recombination sequences, and a pair of homologous recombination sequences for homologous recombination with the transformation assisting plasmid outside the pair of endonuclease target sequences. (4) The method for producing a transformant according to (1), wherein the transformation assisting plasmid is capable of expressing a target-specific endonuclease gene that specifically cleaves the double strand of the endonuclease target sequence. (5) The method for producing a transformant according to (4), wherein the target-specific endonuclease gene is a homing endonuclease gene. (6) The method for producing a transformant according to (5), wherein the endonuclease target sequence is a sequence specifically recognized by a homing endonuclease. (7) The method for producing a transformant according to (4), wherein the plasmid for transformation assistance has an inducible promoter that controls the expression of the target-specific endonuclease gene. (8) The method for producing a transformant according to (1), wherein the plurality of linear genomic introduction nucleic acid fragments consist of a first linear genomic introduction nucleic acid fragment to an n-th linear genomic introduction nucleic acid fragment (n is an integer of 2 or more), and the 3'-terminal side of the m-th linear genomic introduction nucleic acid fragment (m is an integer satisfying 1 ≦ m ≦ n - 1) has a sequence that undergoes homologous recombination with the 5'-terminal side of the (m + 1)-th linear genomic introduction nucleic acid fragment.

[0012] (9) A step of introducing into a host a transformant assistance plasmid having one or more linear genomic introduction nucleic acid fragments comprising a target gene for introduction at a predetermined position on the genome and a pair of homologous recombination sequences for incorporating the linear genomic introduction nucleic acid fragment, wherein the linear genomic introduction nucleic acid fragment is in a state incorporated into the transformant assistance plasmid and has a pair of homologous recombination sequences for homologous recombination with the outside of the target gene and a predetermined position on the genome, and a pair of endonuclease target sequences are arranged outside the pair of homologous recombination sequences, and the target gene is expressed, which is a transformation method. (10) The transformation method according to (9), wherein the transformant assistance plasmid comprises a pair of homologous recombination sequences that undergo homologous recombination with the outside of the target gene in the linear genomic introduction nucleic acid fragment, and a pair of endonuclease target sequences arranged on the opposite side of the position where the linear genomic introduction nucleic acid fragment is incorporated via the homologous recombination sequences. (11) The transformation method according to (9), wherein the linear genomic introduction nucleic acid fragment comprises a pair of homologous recombination sequences for incorporation at a predetermined position on the genome that sandwich the target gene, a pair of endonuclease target sequences outside the pair of homologous recombination sequences, and a pair of homologous recombination sequences for homologous recombination with the transformant assistance plasmid outside the pair of endonuclease target sequences. (12) The plasmid for assisting transformation is characterized by being capable of expressing a target-specific endonuclease gene that specifically cleaves the double strand of the endonuclease target sequence, according to the transformation method described in (9). (13) The transformation method according to (12), wherein the target-specific endonuclease gene is a homing endonuclease gene. (14) The transformation method according to (13), wherein the endonuclease target sequence is a sequence specifically recognized by a homing endonuclease. (15) The transformation method according to (12), wherein the plasmid for assisting transformation has an inducible promoter that controls the expression of the target-specific endonuclease gene. (16) The plurality of linear genomic introduction nucleic acid fragments consist of the first linear genomic introduction nucleic acid fragment to the nth linear genomic introduction nucleic acid fragment (n is an integer of 2 or more), and the 3'-terminal side of the mth linear genomic introduction nucleic acid fragment (m is an integer satisfying 1 ≦ m ≦ n - 1) has a sequence that undergoes homologous recombination with the 5'-terminal side of the (m + 1)th linear genomic introduction nucleic acid fragment, according to the transformation method described in (9).

[0013] (17) A plasmid for assisting transformation, which can incorporate a linear genomic introduction nucleic acid fragment comprising a target gene to be introduced at a predetermined position on the genome by homologous recombination, and has a pair of homologous recombination sequences that undergo homologous recombination with the outside of the target gene in the linear genomic introduction nucleic acid fragment, and a pair of endonuclease target sequences arranged on the opposite side of the position where the linear genomic introduction nucleic acid fragment is incorporated via the homologous recombination sequence. (18) The plasmid for assisting transformation according to (17), which is capable of expressing a target-specific endonuclease gene that specifically cleaves the double strand of the endonuclease target sequence. (19) The plasmid for assisting transformation according to (18), wherein the target-specific endonuclease gene is a homing endonuclease gene. (20) The endonuclease target sequence of the above is a sequence specifically recognized by a homing endonuclease, and is characterized by the transformation assisting plasmid according to (19). (21) The transformation assisting plasmid according to (18), characterized by having an inducible promoter that controls the expression of the above target-specific endonuclease gene.

Effects of the Invention

[0014] In addition, in the method for producing a transformant according to the present invention, since the linear genome-introducing nucleic acid fragment containing the target gene is incorporated into the transformation assisting plasmid and the target gene is sandwiched between a pair of endonuclease target sequences, a transformant obtained by incorporating the target gene into the host genome can be efficiently produced.

[0015] Furthermore, in the transformation method according to the present invention, since the linear genome-introducing nucleic acid fragment containing the target gene is incorporated into the transformation assisting plasmid and the target gene is sandwiched between a pair of endonuclease target sequences, excellent transformation efficiency for producing a transformant obtained by incorporating the target gene into the host genome can be achieved.

[0016] By using the transformation assisting plasmid according to the present invention, a transformant obtained by incorporating the target gene into the host genome can be efficiently produced.

Brief Description of the Drawings

[0017]

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Embodiments for Carrying Out the Invention

[0018] Hereinafter, the present invention will be described in more detail with reference to the drawings and examples. In the method for producing a transformant and the transformation method (hereinafter collectively referred to as this method) according to the present invention, a linear genome-introducing nucleic acid fragment having a target gene to be integrated into the genome of a host and a plasmid for transformation assistance that integrates the linear genome-introducing nucleic acid fragment by homologous recombination are introduced into the host. In this method, the linear genome-introducing nucleic acid fragment is integrated into the plasmid for transformation assistance by homologous recombination. Then, in the host, the target gene sandwiched between a pair of homologous recombination sequences is excised by a predetermined endonuclease, and the target gene can be integrated into the genome by homologous recombination with the host genome.

[0019] At this time, by arranging the target gene sandwiched between a pair of homologous recombination sequences so as to be sandwiched between a pair of endonuclease target sequences, the target gene sandwiched between the pair of homologous recombination sequences can be excised by an endonuclease that specifically recognizes the endonuclease target sequences. That is, as schematically shown in FIG. 1, the fragment excised from the plasmid by the endonuclease has a pair of homologous recombination sequences at both ends, and the target gene is sandwiched by the pair of homologous recombination sequences. Then, as a result of homologous recombination occurring between the pair of homologous recombination sequences and the host genome, the target gene can be incorporated into the host genome.

[0020] Here, the pair of endonuclease target sequences may be arranged in advance in the linear genomic introduction nucleic acid fragment to be introduced into the host, or may be arranged in advance in the plasmid for transformation assistance. Alternatively, one of the pair of endonuclease target sequences may be arranged in advance in the linear genomic introduction nucleic acid fragment, and the other may be arranged in advance in the plasmid for transformation assistance.

[0021] [First Embodiment] Hereinafter, a form in which a pair of endonuclease target sequences are arranged in a plasmid for transformation assistance will be described. As shown in FIG. 2, the plasmid for transformation assistance according to the present invention includes a pair of homologous recombination sequences for incorporating a linear genomic introduction nucleic acid fragment, and a pair of endonuclease target sequences arranged on the opposite side of the position where the linear genomic introduction nucleic acid fragment is incorporated via the homologous recombination sequences. In other words, when the plasmid for transformation assistance is cut at the position where the linear genomic introduction nucleic acid fragment is incorporated to make it linear, it has a pair of homologous recombination sequences at both ends, and each homologous recombination sequence is followed by an endonuclease target sequence.

[0022] As shown in Fig. 3, the plasmid for transformation assistance can incorporate a linear genomic introduction nucleic acid fragment containing a target gene by homologous recombination via the pair of homologous recombination sequences. Here, the linear genomic introduction nucleic acid fragment has a target gene and a pair of homologous recombination sequences sandwiching the target gene. That is, by homologous recombination occurring between the homologous recombination sequence in the linear genomic introduction nucleic acid fragment and the homologous recombination sequence in the plasmid for transformation assistance, the linear genomic introduction nucleic acid fragment can be incorporated into the plasmid for transformation assistance. Also, by homologous recombination occurring between the homologous recombination sequence in the linear genomic introduction nucleic acid fragment and a predetermined position in the genome, the target gene can be incorporated into the genome (see Fig. 1).

[0023] The target gene means a nucleic acid to be introduced into the host genome. Therefore, the target gene is not limited to a base sequence encoding a specific protein, and includes nucleic acids consisting of any base sequences such as a base sequence encoding siRNA or the like, a base sequence of a transcriptional regulatory region such as a promoter or enhancer that controls the transcription timing and production amount of a transcript, and a base sequence encoding transfer RNA (tRNA), ribosomal RNA (rRNA), or the like.

[0024] Also, the target gene is preferably incorporated into the above site in an expressible state. The expressible state means that the target gene and the promoter are linked so as to be expressed under the control of a predetermined promoter in the host organism.

[0025] Furthermore, a promoter, a terminator, cis - elements such as an enhancer if desired, a splicing signal, a poly - A addition signal, a selection marker, a ribosome - binding sequence (SD sequence), etc. can be linked to the target gene. Examples of the selection marker include antibiotic resistance genes such as an ampicillin resistance gene, a kanamycin resistance gene, and a hygromycin resistance gene.

[0026] A pair of homologous recombination sequences means a pair of nucleic acid regions having homology to a predetermined region in the host genome. When a pair of homologous recombination sequences in a linear genome-introduced nucleic acid fragment cross with the homologous host genome respectively, a target gene sandwiched between the pair of homologous recombination sequences can be incorporated into the host genome. Therefore, as a pair of homologous recombination sequences, although there is no limitation to a specific base sequence, for example, it can be a base sequence having high homology with the upstream region and the downstream region of a predetermined gene existing in the host genome. In this case, when homologous recombination occurs between the linear genome-introduced nucleic acid fragment and the host genome, since the gene is deleted from the host genome, the success or failure of homologous recombination can be determined by observing the phenotype due to the deletion of the gene.

[0027] For example, as a pair of homologous recombination sequences, it can be a region upstream of the coding region of the ADE1 gene involved in the adenine biosynthesis pathway and a region downstream of the coding region of the ADE1 gene. In this case, when homologous recombination occurs between a pair of homologous recombination sequences in the linear genome-introduced nucleic acid fragment and the host genome, 5-aminoimidazole riboside, an intermediate metabolite of adenine, accumulates, and the transformant is colored red due to the polymerized polyribosylaminoimidazole. Therefore, by detecting this red coloring, it can be determined that homologous recombination has occurred between a pair of homologous recombination sequences in the linear genome-introduced nucleic acid fragment and the host genome.

[0028] Here, between a pair of homologous recombination sequences in the linear genome-introduced nucleic acid fragment and the recombination region of the host genome, there is a high sequence identity to such an extent that homologous recombination (crossing) can occur. The identity of the base sequences between each region can be calculated using conventionally known sequence comparison software such as blastn. The base sequences between each region only need to have an identity of 60% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably have an identity of 99% or more.

[0029] In addition, the pair of homologous recombination sequences in the linear genome-introducing nucleic acid fragment may have the same length or different lengths. The pair of homologous recombination sequences in these linear genome-introducing nucleic acid fragments only need to be of a length that can undergo homologous recombination (crossing) with the genome. For example, each is preferably 0.1 kb to 3 kb, more preferably 0.5 kb to 3 kb, and particularly preferably 0.5 kb to 2 kb.

[0030] Incidentally, the plasmid for assisting transformation according to the present invention has a pair of homologous recombination sequences for incorporating the above-described linear genome-introducing nucleic acid fragment. The homologous recombination sequences in the plasmid for assisting transformation only need to undergo homologous recombination with the homologous recombination sequences in the linear genome-introducing nucleic acid fragment, and may have the same length or different lengths as the homologous recombination sequences in the linear genome-introducing nucleic acid fragment. The homologous recombination sequences in the plasmid for assisting transformation are nucleotide sequences having homology with the homologous recombination sequences in the linear genome-introducing nucleic acid fragment, and can be, for example, 30 b to 300 b, preferably 40 b to 200 b, and more preferably 50 b to 100 b.

[0031] In addition, in the plasmid for assisting transformation according to the present invention, the pair of homologous recombination sequences for incorporating the linear genome-introducing nucleic acid fragment means both those that can directly undergo homologous recombination with the pair of homologous recombination sequences in the linear genome-introducing nucleic acid fragment and those that can indirectly undergo homologous recombination with the pair of homologous recombination sequences in the linear genome-introducing nucleic acid fragment via one or more linear nucleic acid fragments. Here, the one or more linear nucleic acid fragments are fragments in which one nucleic acid fragment or a plurality of nucleic acid fragments are linked by homologous recombination, and one end has a sequence capable of undergoing homologous recombination with the homologous recombination sequence in the plasmid for assisting transformation, and the other end has a sequence capable of undergoing homologous recombination with the homologous recombination sequence in the linear genome-introducing nucleic acid fragment.

[0032] In addition, the plasmid for assisting transformation according to the present invention has an endonuclease target sequence following the pair of homologous recombination sequences described above. The endonuclease target sequence means a base sequence recognized by an endonuclease.

[0033] The endonuclease is not particularly limited and broadly means an enzyme having an activity of recognizing a predetermined base sequence and cleaving double-stranded DNA. Examples of the endonuclease include restriction enzymes, homing endonucleases, Cas9 nuclease, meganucleases (MN), zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and the like. In addition, the homing endonuclease includes both an endonuclease encoded by an intron (prefixed with I-) and an endonuclease contained in an intein (prefixed with PI-). More specific examples of the homing endonuclease include I-Ceu I, I-Sce I, I-Onu I, PI-Psp I, and PI-Sce I. Note that the target sequences specifically recognized by these specific endonucleases, that is, the endonuclease target sequences, are known and can be appropriately obtained by those skilled in the art.

[0034] In addition, as shown in FIG. 4, the plasmid for assisting transformation according to the present invention may contain an inducible promoter and an endonuclease gene. Note that the expression of the endonuclease gene is not limited to an inducible promoter, and a constitutive expression promoter may be used.

[0035] This endonuclease gene encodes an enzyme having an activity of specifically recognizing the pair of endonuclease target sequences described above and cleaving the double strand. That is, examples of the endonuclease gene include restriction enzyme genes, homing endonuclease genes, Cas9 nuclease genes, meganuclease genes, zinc finger nuclease genes, transcription activator-like effector nuclease genes, and the like.

[0036] An inducible promoter means a promoter having a function of inducing expression under specific conditions. The inducible promoter is not particularly limited, and examples thereof include a promoter that induces expression in the presence of a specific substance, a promoter that induces expression under specific temperature conditions, a promoter that induces expression in response to various stresses, etc. The promoter to be used can be appropriately selected according to the host to which the trait is added.

[0037] For example, as the inducible promoter, there can be mentioned galactose-inducible promoters such as GAL1 and GAL10, the Tet-on system / Tet-off system promoter induced by the addition or removal of tetracycline or its derivatives, the promoter of a gene encoding a heat shock protein (HSP) such as HSP10, HSP60, and HSP90. In addition, as the inducible promoter, the CUP1 promoter activated by the addition of copper ions can also be used. Furthermore, as the inducible promoter, when the host is a prokaryotic cell such as Escherichia coli, there can be mentioned the lac promoter induced by IPTG, the cspA promoter induced by cold shock, the araBAD promoter induced by arabinose, etc.

[0038] In addition, the control of endonuclease gene expression is not limited to methods using promoters such as inducible promoters or constitutive expression promoters. For example, a method using a DNA recombinase may also be applied. As a method of turning gene expression on and off using a DNA recombinase, for example, 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.) can be mentioned. In the FLEx switch method, by causing recombination that changes the orientation of the promoter sequence with a DNA recombinase, the on and off of gene expression can be achieved.

[0039] On the other hand, the plasmid for transformation assistance according to the present invention can be prepared based on conventionally known and available plasmids. Examples of such plasmids include YCp-type Escherichia coli-yeast shuttle vectors such as pRS413, pRS414, pRS415, pRS416, YCp50, pAUR112 or pAUR123; YEp-type Escherichia coli-yeast shuttle vectors such as pYES2 or YEp13; YIp-type Escherichia coli-yeast shuttle vectors such as pRS403, pRS404, pRS405, pRS406, pAUR101 or pAUR135; plasmids derived from Escherichia coli (ColE-type plasmids such as pBR322, pBR325, pUC18, pUC19, pUC118, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396 or pTrc99A; p15A-type plasmids such as pACYC177 or pACYC184; pSC101-type plasmids such as pMW118, pMW119, pMW218 or pMW219, etc.), plasmids derived from Agrobacterium (for example, pBI101, etc.), plasmids derived from Bacillus subtilis (for example, pUB110, pTP5, etc.).

[0040] The plasmid for transformation assistance according to the present invention can further contain an origin of replication, an autonomous replication sequence (ARS), and a centromere sequence (CEN). By including these, it can replicate stably after being introduced into a host cell. Also, the plasmid for transformation assistance according to the present invention can contain a selection marker. The selection marker is not particularly limited, and examples thereof include a drug resistance marker gene and an auxotrophic marker gene. By including these selection markers, host cells into which the plasmid for transformation assistance has been introduced can be efficiently selected.

[0041] By using the plasmid for transformation assistance configured as described above, a stable transformant in which the target gene is integrated into the genome can be produced simply and efficiently. To produce a transformant, first, a linear genomic introduction nucleic acid fragment having the target gene and the plasmid for transformation assistance are introduced into a host cell according to a conventional method. At this time, the linear genomic introduction nucleic acid fragment is integrated into the plasmid for transformation assistance by homologous recombination and becomes a circular plasmid (see Figure 3). Then, as schematically shown in Figure 1, the double strand is cleaved at a pair of endonuclease target sequences by an endonuclease, and a linear genomic introduction nucleic acid fragment containing the target gene sandwiched between a pair of homologous recombination sequences is excised. The excised linear genomic introduction nucleic acid fragment crosses between a pair of homologous recombination sequences in the linear genomic introduction nucleic acid fragment and homologous recombination sequences in the host genome and is integrated into the genome. Thereby, a stable transformant in which the target gene is integrated into the genome can be produced.

[0042] At this time, the method for introducing the linear genomic introduction nucleic acid fragment having the target gene and the plasmid for transformation assistance into the host cell is not particularly limited, and conventionally known methods such as the calcium chloride method, the competent cell method, the protoplast or spheroplast method, and the electropulse method can be appropriately used. Then, when the plasmid for transformation assistance has a selection marker, host cells into which the plasmid for transformation assistance has been introduced can be selected using the selection marker.

[0043] In addition, to express an endonuclease under the control of an inducible promoter, appropriate conditions are set according to the inducible promoter. For example, when using a galactose-inducible promoter such as GAL1 and GAL10 as the inducible promoter, galactose is added to the medium for culturing the host cell into which the transformation plasmid has been introduced, or the host cell is transferred to a galactose-containing medium and cultured, thereby inducing the expression of the endonuclease. When using the promoter of a gene encoding a heat shock protein (HSP) as the inducible promoter, a heat shock is applied at a desired timing when culturing the host cell into which the transformation plasmid has been introduced, thereby inducing the expression of the endonuclease at that timing.

[0044] However, under the conditions in which the inducible promoter induces expression, a treatment of introducing the linear genomic introduction nucleic acid fragment and the transformation assisting plasmid into the host cell may be performed to express the endonuclease under the control of the inducible promoter. In this case, the treatment of shifting to the expression induction conditions is unnecessary, and a transformant can be obtained more simply.

[0045] In addition, in the above-described plasmid for transformation assistance, when a pair of homologous recombination sequences are nucleotide sequences highly homologous to the upstream region and the downstream region of a predetermined gene, a linear genomic introduction nucleic acid fragment containing the target gene is integrated into the genome by homologous recombination, and the predetermined gene is deleted from the genome. Therefore, by observing the phenotype resulting from the deletion of the predetermined gene, it is possible to determine whether or not a linear genomic introduction nucleic acid fragment containing the target gene has been integrated into the genome. For example, when the ADE1 gene is used as the predetermined gene, if a linear genomic introduction nucleic acid fragment containing the target gene is integrated into the genome, the ADE1 gene will be deleted from the genome. As a result, 5-aminoimidazole riboside accumulates in the host, and the transformant is colored red due to the polymerized polyribosylaminoimidazole. Therefore, by detecting this red coloring, it is possible to determine that a linear genomic introduction nucleic acid fragment containing the target gene has been integrated into the host genome.

[0046] In the above-described example, the plasmid for transformation assistance has a configuration having an inducible promoter and an endonuclease gene. However, the plasmid for transformation assistance according to the present invention may have a configuration that does not have an inducible promoter and an endonuclease gene. In this case, an expression vector having an inducible promoter and an endonuclease gene may be separately prepared and introduced into a host cell together with a linear genomic introduction nucleic acid fragment having a target gene and the plasmid for transformation assistance according to the present invention. Even in this case, in a host cell into which an expression vector having an inducible promoter and an endonuclease gene, a linear genomic introduction nucleic acid fragment, and a plasmid for transformation have been introduced, the endonuclease gene is expressed under the control of the inducible promoter, so that, as shown in FIG. 1, a linear genomic introduction nucleic acid fragment containing the target gene sandwiched between a pair of homologous recombination sequences is excised, and a transformant in which the target gene is integrated into the genome can be produced.

[0047] On the one hand, by using the plasmid for assisting transformation according to the present invention, a plurality of linear genome-introducing nucleic acid fragments can be arranged in series and integrated into the host genome. If a plurality of types of linear genome-introducing nucleic acid fragments are designated as the first linear genome-introducing nucleic acid fragment to the nth linear genome-introducing nucleic acid fragment (n is an integer of 2 or more), then by setting the 3'-terminal side of the mth linear genome-introducing nucleic acid fragment (m is an integer satisfying 1 ≦ m ≦ n - 1) and the 5'-terminal side of the (m + 1)th linear genome-introducing nucleic acid fragment as homologous recombination sequences, the first to nth linear genome-introducing nucleic acid fragments can be ligated in this order by homologous recombination. As an example, as shown in FIG. 5, when integrating the first to third linear genome-introducing nucleic acid fragments into the host genome, the 3'-terminal side of the first linear genome-introducing nucleic acid fragment and the 5'-terminal side of the second linear genome-introducing nucleic acid fragment are set as homologous recombination sequence 2, and the 3'-terminal side of the second linear genome-introducing nucleic acid fragment and the 5'-terminal side of the third linear genome-introducing nucleic acid fragment are set as homologous recombination sequence 3, so that the first to third linear genome-introducing nucleic acid fragments can be ligated in this order by homologous recombination. The fragment obtained by ligating the first to third linear genome-introducing nucleic acid fragments is integrated into the plasmid for assisting transformation and the host genome by homologous recombination via homologous recombination sequences 1 and 4 between the plasmid for assisting transformation and the host genome.

[0048] By the way, in order to arrange a plurality of linear genome-introducing nucleic acid fragments in series by homologous recombination, homologous recombination sequences are provided between adjacent linear genome-introducing nucleic acid fragments. These homologous recombination sequences only need homologous recombination to occur between them and the homologous recombination sequences in adjacent linear genome-introducing nucleic acid fragments, and they may have the same length or different lengths as the homologous recombination sequences in adjacent linear genome-introducing nucleic acid fragments. This homologous recombination sequence is a base sequence having homology with the homologous recombination sequences in adjacent linear genome-introducing nucleic acid fragments, and can be, for example, 30b to 300b, preferably 40b to 200b, and more preferably 50b to 100b.

[0049] As described above, by using the plasmid for transformation assistance according to the present invention, a plurality of linear genomic-introducing nucleic acid fragments can be arranged in series and integrated into the host genome. Here, each of the plurality of linear genomic-introducing nucleic acid fragments may have a target gene, or only some of the linear genomic-introducing nucleic acid fragments may have a target gene.

[0050] In addition, the transformation method and the method for producing a transformant using the plasmid for transformation assistance according to the present invention are not particularly limited and can be applied to any host cell. Examples of host cells include fungi such as filamentous fungi and yeasts, bacteria such as Escherichia coli and Bacillus subtilis, plant cells, and animal cells including mammals and insects. Among these, it is preferable to use yeast as the host cell. The yeast is not particularly limited, and examples thereof include yeasts belonging to the genus Saccharomyces, yeasts belonging to the genus Kluyveromyces, yeasts belonging to the genus Candida, yeasts belonging to the genus Pichia, yeasts belonging to the genus Schizosaccharomyces, and yeasts belonging to the genus Hansenula. More specifically, it can be applied to yeasts belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces boulardii.

[0051] [Second Embodiment] Hereinafter, a form in which a pair of endonuclease target sequences are arranged in a linear genomic-introducing nucleic acid fragment having a target gene will be described. In the following description, the same terms as those used in the description of the first embodiment are used, and detailed descriptions of the configuration and the like are omitted.

[0052] In the second embodiment, as shown in FIG. 6, the linear genome-introducing nucleic acid fragment has, at both ends, a pair of first homologous recombination sequences capable of undergoing homologous recombination with the transformation assisting plasmid, an endonuclease target sequence inside the first homologous recombination sequence, and a pair of second homologous recombination sequences capable of undergoing homologous recombination with the host genome inside the endonuclease target sequence, and a target gene inside the pair of second homologous recombination sequences. In this embodiment, the transformation assisting plasmid includes a pair of third homologous recombination sequences for integrating the linear genome-introducing nucleic acid fragment. Further, although not shown, the transformation assisting plasmid may include an inducible promoter and an endonuclease gene downstream of the inducible promoter as shown in FIG. 4 in the first embodiment.

[0053] Note that also in the second embodiment, the pair of third homologous recombination sequences in the transformation assisting plasmid and the pair of first homologous recombination sequences in the linear genome-introducing nucleic acid fragment may directly undergo homologous recombination or may indirectly undergo homologous recombination via one or more linear nucleic acid fragments. Here, the one or more linear nucleic acid fragments are nucleic acid fragments in which one nucleic acid fragment or a plurality of nucleic acid fragments are linked by homologous recombination, and one end has a sequence capable of undergoing homologous recombination with the third homologous recombination sequence in the transformation assisting plasmid, and the other end has a sequence capable of undergoing homologous recombination with the first homologous recombination sequence in the linear genome-introducing nucleic acid fragment.

[0054] By using the linear genome-introducing nucleic acid fragment and the plasmid for transformation assistance configured as described above, a stable transformant with the target gene integrated into the genome can be easily and efficiently produced. To produce a transformant, first, the linear genome-introducing nucleic acid fragment having the target gene described above and the plasmid for transformation assistance are introduced into a host cell according to a conventional method. At this time, homologous recombination occurs between the first homologous recombination sequence in the linear genome-introducing nucleic acid fragment and the third homologous recombination sequence in the plasmid for transformation assistance, and the linear genome-introducing nucleic acid fragment is incorporated into the plasmid for transformation assistance to form a circular plasmid (see Fig. 7). Then, as schematically shown in Fig. 7, the double strand is cleaved at a pair of endonuclease target sequences by an endonuclease, and a fragment containing the target gene sandwiched between a pair of second homologous recombination sequences is excised. The excised fragment crosses between the pair of second homologous recombination sequences in the fragment and the fourth homologous recombination sequence in the host genome and is incorporated into the genome. Thereby, a stable transformant with the target gene integrated into the genome can be produced.

[0055] Also, in this embodiment, a plurality of linear genome-introducing nucleic acid fragments can be arranged in series and incorporated into the host genome. As an example, as shown in Fig. 8, when the first to third linear genome-introducing nucleic acid fragments are incorporated into the host genome, the 3'-terminal side of the first linear genome-introducing nucleic acid fragment and the 5'-terminal side of the second linear genome-introducing nucleic acid fragment are used as homologous recombination sequence 2, and the 3'-terminal side of the second linear genome-introducing nucleic acid fragment and the 5'-terminal side of the third linear genome-introducing nucleic acid fragment are used as homologous recombination sequence 3, so that the first to third linear genome-introducing nucleic acid fragments can be ligated in this order by homologous recombination. The ligated fragment is incorporated into the plasmid for transformation assistance by homologous recombination between its first homologous recombination sequence and the third homologous recombination sequence in the plasmid for transformation assistance. Also, the fragment excised by the endonuclease at the endonuclease target sequence is incorporated into the host genome by homologous recombination between its second homologous recombination sequence and the fourth homologous recombination sequence in the genome.

Example

[0056] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to the following examples.

[0057] [Example 1] In this example, the haploid experimental yeast S. cerevisiae BY4742 strain was used as the test strain. <Preparation of vectors> The three types of vectors prepared were the YEp-type yeast shuttle vector pRS436(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce (see Fig. 9), which is composed of a DNA fragment containing a pair of homologous recombination sequences for genome introduction inserted between the I-SceI (SCEI gene, NCBI accession No 854590), a homing endonuclease derived from S. cerevisiae induced by galactose, and a pair of I-SceI target sequences (endonuclease target sequences), and the YCp-type yeast shuttle vector pRS436cen(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce, and the YEp-type yeast shuttle vector pRS436(SAT)-P_GAL1-OnuIi-T_CYC1-Onu-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Onu, which is composed of a DNA fragment containing a pair of homologous recombination sequences for genome introduction inserted between the I-OnuI gene (NCBI accession No AY275136.2), a homing endonuclease derived from Ophiostoma novo-ulmi subsp. americana induced by galactose, and a pair of I-OnuI target sequences (endonuclease target sequences).

[0058] In pRS436(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce, the SCEI gene (the sequence in which the intron of the COX5B gene was inserted and the full length was codon-converted according to the codon usage frequency of the yeast nuclear genome, SEQ ID NOs: 1 and 2) with the GAL1 promoter and the CYC1 terminator added, a gene sequence containing the nourseothricin resistance gene (nat marker), as a homologous recombination sequence for genome introduction, the gene sequence of the region approximately 1000 bp upstream from the 5'-terminal of the ADE1 gene (5U_ADE1) and the DNA sequence of the region approximately 950 bp downstream from the 3'-terminal of the ADE1 gene (3U_ADE1), and as a marker gene for homologous recombination, a gene sequence containing the G418 resistance gene with the TEF1 promoter and the TEF1 terminator from Ashbya gossypii added (G418 marker) were inserted into the vector obtained by removing the URA3 gene, the TDH3 promoter, and the CYC1 terminator from pRS436GAP (NCBI accession No. AB304862), a YEp-type yeast shuttle vector. Note that the 5U_ADE1, 3U_ADE1, and G418 marker sequences are inserted between two homing endonuclease I-SceI target sequences and can be excised by the SCEI gene added to the GAL1 promoter induced in a medium with galactose as the carbon source.

[0059] Each DNA sequence can be amplified by PCR. To ligate each DNA fragment, primers were synthesized with DNA sequences added so as to overlap approximately 15 bp with the adjacent DNA sequences (Table 1). Using these primers, the target DNA fragments were amplified with the S. cerevisiae OC-2 strain genome or synthetic DNA as a template, the DNA fragments were sequentially ligated using an In-Fusion HD Cloning Kit or the like, and cloned into the pRS436GAP vector to prepare the final target plasmid.

[0060] pRS436cen(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce is a vector whose copy number in cells is maintained at one copy. It is obtained by deleting the replication origin derived from the 2μm plasmid from pRS436(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce and inserting an autonomously replicating sequence (ARS) and a centromere sequence (CEN) instead. This vector can be prepared by amplifying the target DNA fragment using pRS436(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce or the S. cerevisiae OC-2 strain genome as a template (the primers used are shown in Table 1) and ligating the DNA fragments using an In-Fusion HD Cloning Kit or the like.

[0061] pRS436(SAT)-P_GAL1-OnuIi-T_CYC1-Onu-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Onu is obtained by replacing the SCEI gene with the I-OnuI gene and further replacing the I-SceI target sequence with the I-OnuI target sequence in pRS436(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce. This vector can be prepared by amplifying the target DNA fragment using pRS436(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce or the synthetic gene of the I-OnuI gene as a template (the primers used are shown in Table 1) and ligating the DNA fragments using an In-Fusion HD Cloning Kit or the like.

[0062]

Table 1

[0063] <Preparation of Linear Genome-introducing Nucleic Acid Fragment for ADE1 Destruction> In this example, among the vectors prepared above, using the YEp-type yeast shuttle vector pRS436(SAT)-P_MET25-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce as a template and the primers shown in Table 2, a fragment containing the 5' homologous recombination sequence of the ADE1 gene, a fragment containing the 3' homologous recombination sequence of the ADE1 gene, and a fragment containing the G418 marker were amplified by PCR. Specifically, as schematically shown in FIG. 9, a fragment containing the 5' homologous recombination sequence of the ADE1 gene was amplified using primers P1 and P2, a fragment containing the G418 marker was amplified using primers P3 and P4, and a fragment A containing the 3' homologous recombination sequence of the ADE1 gene was amplified using primers P5 and P6. The primers were designed so that each fragment overlapped by about 60 bp. The nucleotide sequences of each primer are shown in Table 2.

[0064]

Table 2

[0065] <Preparation of Plasmid for Transformation Assistance> In this example, using the YEp-type yeast shuttle vector pRS436(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce, the YCp-type yeast shuttle vector pRS436cen(SAT)-P_GAL1-SCEI-T_CYC1-Sce-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Sce, or the YEp-type yeast shuttle vector pRS436(SAT)-P_GAL1-OnuIi-T_CYC1-Onu-5U_ADE1-P_AgTEF1-G418-T_AgTEF1-3U_ADE1-Onu, which are the vectors prepared above, as templates, and using the primers shown in Table 3 that overlap by approximately 60 bp with the linear genomic introduction nucleic acid fragment containing the 5’ or 3’ homologous recombination sequence of ADE1, the plasmid for transformation assistance was amplified. Specifically, as schematically shown in Fig. 10, the plasmid for transformation assistance was amplified by PCR using primers P7 and P8. The nucleotide sequences of each primer are shown in Table 3.

[0066]

Table 3

[0067] <Transformation Using Linear Genomic Introduction Nucleic Acid Fragment and Plasmid for Transformation Assistance> Using the three types of linear genomic introduction nucleic acid fragments prepared as described above and the plasmid for transformation assistance at a concentration of 2 fmol / μl each, transformation of the S. cerevisiae BY4742 strain was performed (10 6 cells / μl). After culturing in YPGa (carbon source: 2% galactose) liquid medium for 7 hours, the cell concentration was measured with a spectrophotometer, and YPGa agar medium containing G418 (10 6 and 10 7It was applied to (cells / plates), and the grown colonies were counted. Transformation was carried out according to the method of Akada [Akada, R. et al. “Elevated temperature greatly improves transformation of fresh and frozen competent cells in yeast“ BioTechniques 28 (2000): 854-856].

[0068] In the medium containing galactose, the homing endonuclease I-SceI is induced, three kinds of linear genomic introduced nucleic acid fragments are excised in a ligated state, homologous recombination occurs at the ADE1 locus, and it is considered that the ADE1 gene is disrupted. The ADE1 gene is a gene in the adenine biosynthesis pathway, and in its disrupted strain, 5-aminoimidazole riboside, an intermediate metabolite of adenine, accumulates, and the polymerized polyribosylaminoimidazole is colored red, so the ADE1 gene disrupted strain can be easily discriminated. The homologous recombination efficiency at the ADE1 locus was calculated by the following formula. ADE1 gene disruption efficiency (%) = number of red colonies grown on agar medium / number of cells spread on agar medium For comparison, transformation was carried out using only the three kinds of linear genomic introduced nucleic acid fragments prepared as described above without using the plasmid for transformation assistance, and the ADE1 gene disruption efficiency was similarly calculated.

[0069] <Results and Discussion> Table 4 shows the results of calculating the ADE1 gene disruption efficiency when using three kinds of linear genomic introduced nucleic acid fragments and three kinds of plasmids for transformation assistance and the ADE1 gene disruption efficiency when using only three kinds of linear genomic introduced nucleic acid fragments.

[0070]

Table 4

[0071] As can be seen from Table 4, when using the plasmid for transformation assistance, the ADE1 disruption strain was obtained with an efficiency approximately 50 to 240 times higher than when using only the linear genomic introduction nucleic acid fragment. From this, it became clear that by using a plasmid for transformation assistance comprising a pair of homologous recombination sequences for introducing a target gene into the host genome and a pair of endonuclease target sequences sandwiching the pair of homologous recombination sequences, the introduction efficiency of the linear genomic introduction nucleic acid fragment into the genome can be improved.

Claims

The method for producing a transformant according to (1) or (2), comprising: (1) A step of introducing into a host yeast a transformant-assisting plasmid having one or more linear genomic-introducing nucleic acid fragments comprising a target gene to be introduced at a predetermined position on the genome, and a pair of homologous recombination sequences A for incorporating the linear genomic-introducing nucleic acid fragments, without performing selection using a selection marker, on a circular plasmid formed by incorporating the linear genomic-introducing nucleic acid fragments into the transformant-assisting plasmid in the host yeast, a step of arranging the pair of homologous recombination sequences A and a pair of endonuclease target sequences outside the pair of homologous recombination sequences A; The pair of endonuclease target sequences are base sequences specifically recognized by a target-specific endonuclease. In the host yeast, the circular plasmid is cleaved at the positions of the pair of endonuclease target sequences by the target-specific endonuclease, a fragment having the pair of homologous recombination sequences A at its ends and containing the target gene is excised in the host yeast, and the fragment containing the target gene is incorporated into the predetermined position on the genome of the host yeast via the pair of homologous recombination sequences A, and a step of selecting a transformant in which the target gene is expressed, or (2) A step of introducing into a host yeast a transformant-assisting plasmid having one or more linear genomic-introducing nucleic acid fragments comprising a target gene to be introduced at a predetermined position on the genome, and a pair of homologous recombination sequences A for incorporating the linear genomic-introducing nucleic acid fragments, without performing selection using a selection marker, on a circular plasmid formed by incorporating the linear genomic-introducing nucleic acid fragments into the transformant-assisting plasmid in the host yeast, a step of arranging a pair of homologous recombination sequences B for homologous recombination at the outside of the target gene and at a predetermined position on the genome, and a pair of endonuclease target sequences outside the pair of homologous recombination sequences B; The pair of endonuclease target sequences are base sequences specifically recognized by a target-specific endonuclease. In the host yeast, the circular plasmid is cleaved by the target-specific endonuclease at the positions of the pair of endonuclease target sequences, a fragment having the pair of homologous recombination sequences B at its ends and containing the target gene is excised in the host yeast, and the fragment containing the target gene is integrated into the predetermined position in the genome of the host yeast via the pair of homologous recombination sequences B, and a step of selecting a transformant in which the target gene is expressed. The method for producing a transformant according to the above (1) or (2). **Claim 2** The method for producing a transformant according to claim 1, wherein the plasmid for transformation assistance comprises a pair of homologous recombination sequences A that recombine homologously with the outside of the target gene in the linear genome-introducing nucleic acid fragment, and a pair of endonuclease target sequences arranged on the opposite side of the position where the linear genome-introducing nucleic acid fragment is integrated via the pair of homologous recombination sequences A. **Claim 3** The method for producing a transformant according to claim 1, wherein the linear genome-introducing nucleic acid fragment comprises a pair of homologous recombination sequences B for integrating into a predetermined position of the genome at a position sandwiching the target gene, a pair of endonuclease target sequences outside the pair of homologous recombination sequences B, and a pair of homologous recombination sequences A for recombining homologously with the plasmid for transformation assistance outside the pair of endonuclease target sequences. **Claim 4** The method for producing a transformant according to claim 1, wherein the plasmid for transformation assistance can express a gene encoding the target-specific endonuclease that specifically cleaves the double strand of the endonuclease target sequence. **Claim 5** The method for producing a transformant according to claim 4, wherein the gene encoding the target-specific endonuclease is a homing endonuclease gene. **Claim 6** The method for producing a transformant according to claim 5, wherein the endonuclease target sequence is a sequence specifically recognized by a homing endonuclease. **Claim 7** The method for producing a transformant according to claim 4, wherein the plasmid for transformation assistance has an inducible promoter that controls the expression of the gene encoding the target-specific endonuclease.

8. The method for producing a transformant according to claim 1, wherein the plurality of linear genomic introduced nucleic acid fragments consist of a first linear genomic introduced nucleic acid fragment to an n-th linear genomic introduced nucleic acid fragment (n is an integer of 2 or more), and the 3'-terminal side of the m-th linear genomic introduced nucleic acid fragment (m is an integer satisfying 1 ≦ m ≦ n - 1) has a sequence that undergoes homologous recombination with the 5'-terminal side of the (m + 1)-th linear genomic introduced nucleic acid fragment.

9. A transformation method according to (1) or (2), (1) A step of introducing into a host yeast a plasmid for transformation assistance having one or more linear genomic introduced nucleic acid fragments comprising a target gene to be introduced at a predetermined position on the genome and a pair of homologous recombination sequences A for incorporating the linear genomic introduced nucleic acid fragment, without performing selection using a selection marker, and arranging the pair of homologous recombination sequences A and a pair of endonuclease target sequences outside the pair of homologous recombination sequences A on a circular plasmid formed by incorporating the linear genomic introduced nucleic acid fragment into the plasmid for transformation assistance in the host yeast, The pair of endonuclease target sequences are base sequences specifically recognized by a target-specific endonuclease, the circular plasmid in the host yeast is cleaved at the positions of the pair of endonuclease target sequences by the target-specific endonuclease, a fragment having the pair of homologous recombination sequences A at its ends and containing the target gene is excised in the host yeast, and the fragment containing the target gene is incorporated into the genome of the host yeast via the pair of homologous recombination sequences A, characterized in that the target gene is expressed, or (2) A step of introducing into a host yeast one or more linear genomic introduction nucleic acid fragments comprising a target gene to be introduced at a predetermined position on the genome and a transformation assisting plasmid having a pair of homologous recombination sequences A for incorporating the linear genomic introduction nucleic acid fragment, wherein selection using a selection marker is not performed, and on a circular plasmid formed by incorporating the linear genomic introduction nucleic acid fragment into the transformation assisting plasmid in the host yeast, a pair of homologous recombination sequences B for homologous recombination at a position outside the target gene and a predetermined position on the genome, and a pair of endonuclease target sequences are arranged outside the pair of homologous recombination sequences B, The pair of endonuclease target sequences are base sequences specifically recognized by a target-specific endonuclease. In the host yeast, the circular plasmid is cleaved at the positions of the pair of endonuclease target sequences by the target-specific endonuclease, and a fragment having the pair of homologous recombination sequences B at its ends and containing the target gene is excised in the host yeast, and the fragment containing the target gene is incorporated into the genome of the host yeast via the pair of homologous recombination sequences B, characterized in that the target gene is expressed, The transformation method according to (1) or (2) above.

10. The transformation assisting plasmid according to claim 9, characterized in that it comprises a pair of homologous recombination sequences A that homologous recombine with the outside of the target gene in the linear genomic introduction nucleic acid fragment and a pair of endonuclease target sequences arranged on the opposite side of the position where the linear genomic introduction nucleic acid fragment is incorporated via the pair of homologous recombination sequences A.

11. The linear genomic introduction nucleic acid fragment according to claim 9, characterized in that it comprises a pair of homologous recombination sequences B for incorporating into a predetermined position on the genome at a position sandwiching the target gene, a pair of endonuclease target sequences outside the pair of homologous recombination sequences B, and a pair of homologous recombination sequences A for homologous recombination with the transformation assisting plasmid outside the pair of endonuclease target sequences.

12. The transformation method according to claim 9, wherein the plasmid for assisting transformation can express a gene encoding the target-specific endonuclease that specifically cleaves the double strand of the endonuclease target sequence.

13. The transformation method according to claim 12, wherein the gene encoding the target-specific endonuclease is a homing endonuclease gene.

14. The transformation method according to claim 13, wherein the endonuclease target sequence is a sequence specifically recognized by a homing endonuclease.

15. The transformation method according to claim 12, wherein the plasmid for assisting transformation has an inducible promoter that controls the expression of the gene encoding the target-specific endonuclease.

16. The transformation method according to claim 9, wherein the plurality of linear genome-introducing nucleic acid fragments consist of a first linear genome-introducing nucleic acid fragment to an n-th linear genome-introducing nucleic acid fragment (n is an integer of 2 or more), and the 3'-terminal side of the m-th linear genome-introducing nucleic acid fragment (m is an integer satisfying 1 ≦ m ≦ n - 1) has a sequence that undergoes homologous recombination with the 5'-terminal side of the m + 1-th linear genome-introducing nucleic acid fragment.

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