US-12618084-B2 - Method for producing transformant

US12618084B2US 12618084 B2US12618084 B2US 12618084B2US-12618084-B2

Abstract

The present disclosure concerns evaluation as to whether or not a nucleic acid fragment having a target gene had been accurately integrated into the host genome. A group of nucleic acid fragments comprising a nucleic acid fragment having a target gene is introduced into host cells, and host cells in which the target gene had been cleaved from the genome DNA by the action of a site-specific recombinase are selected.

Inventors

Toru Onishi

Assignees

TOYOTA JIDOSHA KABUSHIKI KAISHA

Dates

Publication Date: 20260505
Application Date: 20211201
Priority Date: 20201203

Claims (6)

1 . A method for producing a transformant comprising steps of: i) introducing into at least one host cell a first group of three or more nucleic acid fragments having homologous recombination sequences each located at their 5′ and 3′ ends, wherein the first group of nucleic acid fragments comprises: a first nucleic acid fragment comprising a target gene, wherein the target gene is a selection marker gene; a second nucleic acid fragment in which the homologous recombination sequence at 5′ end is either one of a pair of homologous recombination sequences corresponding to a particular region of genome DNA; and a third nucleic acid fragment in which the homologous recombination sequence at 3′ end is the other of the pair of homologous recombination sequences; wherein the nucleic acid fragments constituting the first group of nucleic acid fragments can be linked to each other via the homologous recombination sequence at the end of each nucleic acid fragment, and wherein each of a pair of recognition sequences recognized by a site-specific recombinase is comprised in nucleic acid fragments among the first group of nucleic acid fragments, but none of the pair of recognition sequences is comprised in the first nucleic acid fragment comprising the target gene; ii) selecting a host cell in which the target gene flanked by the pair of recognition sequences recognized by the site-specific recombinase had been cleaved from genome DNA.
2 . The method for producing a transformant according to claim 1 , wherein the transformant lacks the particular region of genome DNA upon integration of nucleic acid fragments constituting the first group of nucleic acid fragments therein.
3 . The method for producing a transformant according to claim 1 , wherein one of the pair of recognition sequences recognized by the site-specific recombinase is comprised in the second nucleic acid fragment, and/or the other of the pair is comprised in the third nucleic acid fragment.
4 . The method for producing a transformant according to claim 1 , wherein the first nucleic acid fragment comprises the target gene between first and second homologous recombination sequences, wherein the second nucleic acid fragment comprises the one of the pair of recognition sequences recognized by the site-specific recombinase between third and fourth homologous recombination sequences, wherein the third homologous recombination sequence is at 5′ end of the second nucleic acid fragment and the fourth homologous recombination sequence will cause homologous recombination with the first homologous recombination sequence, wherein the third nucleic acid fragment comprises the other of the pair of recognition sequences recognized by the site-specific recombinase between fifth and sixth homologous recombination sequences, wherein the sixth homologous recombination sequence is at 3′ end of the third nucleic acid fragment and the fifth homologous recombination sequence will cause homologous recombination with the second homologous recombination sequence.
5 . The method for producing a transformant according to claim 1 , wherein the first group of nucleic acid fragments further comprises fourth and fifth nucleic acid fragments having homologous recombination sequences each located at their 5′ and 3′ ends, wherein the first nucleic acid fragment comprises the target gene between first and second homologous recombination sequences, wherein the second nucleic acid fragment comprises a third and fourth homologous recombination sequences at 5′ and 3′ end of the second nucleic acid fragment, wherein the third nucleic acid fragment comprises a fifth and sixth homologous recombination sequences at 5′ and 3′ end of the third nucleic acid fragment, wherein the fourth nucleic acid fragment comprises the one of the pair of recognition sequences recognized by the site-specific recombinase between seventh and eighth homologous recombination sequences, wherein the eighth homologous recombination sequence will cause homologous recombination with the first homologous recombination sequence, and the seventh homologous recombination sequence will cause homologous recombination with the fourth homologous recombination sequence, wherein the fifth nucleic acid fragment comprises the other of the pair of recognition sequences recognized by the site-specific recombinase between ninth and tenth homologous recombination sequences, wherein the ninth homologous recombination sequence will cause homologous recombination with the second homologous recombination sequence, and the tenth homologous recombination sequence will cause homologous recombination with the fifth homologous recombination sequence.
6 . The method for producing a transformant according to claim 1 further comprising: i) introducing into the selected host cell a second group of three or more nucleic acid fragments having homologous recombination sequences each located at their 5′ and 3′ ends, wherein the second group of nucleic acid fragments comprises: a fourth nucleic acid fragment comprising a target gene, wherein the homologous recombination sequences in the fourth nucleic acid fragment are the same as the homologous recombination sequence in the first nucleic acid fragment; a fifth nucleic acid fragment in which the homologous recombination sequence at 5′ end is either one of a pair of homologous recombination sequences corresponding to the other region of genome DNA, and a sixth nucleic acid fragment in which the homologous recombination sequence at 3′ end is the other of the pair of homologous recombination sequences; wherein the nucleic acid fragments constituting the second group of nucleic acid fragments can be linked to each other via the homologous recombination sequence at the end of each nucleic acid fragment, and wherein each of the pair of recognition sequences recognized by the site-specific recombinase is comprised in nucleic acid fragments among the second group of nucleic acid fragments, but none of the pair of recognition sequences is comprised in the fourth nucleic acid fragment comprising the target gene; ii) selecting a host cell in which the target gene flanked by the pair of recognition sequences recognized by the site-specific recombinase had been cleaved from genome DNA.

Description

CROSS REFERENCE TO RELATED APPLICATIONS 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. BACKGROUND Technical Field 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. Background Art 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. 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. 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). 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. 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. 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