JP4721868B2 - Method for deleting a target region of host DNA deletion - Google Patents

Description

  The present invention relates to, for example, a method of using a Bacillus subtilis genome or the like as a host DNA to delete a predetermined region of the host DNA and at the same time deleting a selection marker gene used for selection.

  Conventionally, in the field of genetic engineering, selection marker genes such as drug resistance genes have been used as means for selecting transformants. These selectable marker genes are useful as a means for selecting transformants, but depending on the type of the selectable marker gene, there is a problem in that it causes adverse effects on the environment by remaining in the host. In addition, when a plurality of genetic modifications are performed on one host, the types of selectable marker genes that can be used are limited. Therefore, a method for removing the selection marker gene from the transformant is desired.

  On the other hand, several gene modification methods using a repressor and a promoter repressed by the repressor have been disclosed so far.

Non-Patent Document 1 uses the repressor gene blaI involved in the regulation of the expression of the Bacillus licheniformis β-lactamase gene blaP and the blaP promoter repressed by this repressor. A method for producing a Bacillus subtilis recombinant strain from which the selectable marker gene has been removed is disclosed. However, with this method, the efficiency of obtaining a Bacillus subtilis recombinant strain from which the selection marker gene has been removed was as low as 50%.

  Patent Document 1 discloses a gene modification method using a CI repressor and a promoter repressed by the repressor (see Examples in Patent Document 1). Specifically, λDNA into which the CI repressor gene has been inserted is introduced into the Bacillus subtilis genome. On the other hand, a promoter repressed by the CI repressor and a neomycin resistance gene are incorporated downstream thereof at other sites of the Bacillus subtilis genome. In this case, the promoter is repressed by the CI repressor, and neomycin resistance gene expression does not occur. When the CI repressor gene is replaced with other DNA and the CI repressor gene disappears, the promoter is driven and the downstream neomycin resistance gene is expressed. According to this principle, a transformant in which the CI repressor gene site is replaced with another DNA can be selected due to neomycin resistance.

  Until now, there has been no method for efficiently deleting a predetermined region of host DNA together with a selection marker gene used for selection of transformants.

JP 2000-316576 A A. Brans et al., "Applied And Environmental Microbiology", 2004, 70, 12, p.7241-7250

  In view of the above circumstances, an object of the present invention is to provide a method for deleting a deletion target region of a host DNA.

  As a result of intensive studies to solve the above-mentioned problems, it has a 5 ′ region outside the deletion target region of the host DNA, a 3 ′ region, and a first homologous recombination region of the host DNA. Homologous recombination (hereinafter referred to as “first homologous recombination”) between the 5 ′ region and the first homologous recombination region in a donor DNA having one selectable marker gene and a repressor gene and host DNA. In the host DNA after the first homologous recombination, the 3 ′ region outside the deletion target region derived from the host DNA and the deletion target region possessed by the host DNA derived from the donor DNA By performing homologous recombination (hereinafter sometimes referred to as “second homologous recombination”) with the 3 ′ region outside of the region, after the second homologous recombination, the region to be deleted from the host DNA And the above selection marker gene can be deleted simultaneously to complete the present invention. Was Tsu.

The present invention includes the following.
(1) A method for deleting a deletion target region of a host DNA including a step of performing homologous recombination between a donor DNA and the host DNA, wherein the deletion method includes a deletion target possessed by the host DNA. A donor DNA having a 5 'region outside the region, a 3' region and a first homologous recombination region of the host DNA, and further comprising a first selectable marker gene and a repressor gene; In the first step of performing homologous recombination (first homologous recombination) between the 5 ′ region and the first homologous recombination region, and the host DNA after the first homologous recombination, derived from the host DNA The second homologous recombination (second homologous recombination) is performed between the 3 ′ region outside the deletion target region and the 3 ′ region outside the deletion target region of the host DNA derived from the donor DNA. The first homologous recombination region includes (a) a region consisting of a part or all of the deletion target region, or (b) the 3 ′ side region. Of the host DNA, wherein the first selection marker gene, the repressor gene and the deletion target region are deleted from the host DNA after the second step. Deletion method of the target region.

  (2) In the host DNA, a second region downstream of a promoter containing an operator controlled by the repressor is located in a region other than the deletion target region, the 5 ′ side region and the 3 ′ side region outside the deletion target region. The method for deleting a deletion target region of host DNA according to (1), wherein a selection marker gene is present.

  (3) After the first step, using the first selection marker gene as an indicator, the 3 ′ region outside the deletion target region of the host DNA derived from the donor DNA, the first selection marker gene, and the repressor gene The method for deleting a deletion target region of host DNA according to (1), comprising the step of selecting a transformant having

  (4) After the first step, using the first selection marker gene and / or the second selection marker gene as an index, the 3 ′ region outside the deletion target region of the host DNA derived from the donor DNA and the first The method for deleting a deletion target region of host DNA according to (2), comprising a step of selecting a transformant having a selection marker gene and a repressor gene.

  (5) After the second step, the method includes a step of selecting a transformant lacking the first selection marker gene, the repressor gene, and the deletion target region using the first selection marker gene as an index. A method for deleting a deletion target region of a host DNA according to (1) or (3).

  (6) After the second step, using the first selection marker gene and / or the second selection marker gene as an index, a transformant lacking the first selection marker gene, repressor gene and deletion target region is selected. A method for deleting a deletion target region of a host DNA according to (2) or (4), which comprises a step.

  (7) The method for deleting a deletion target region of a host DNA according to any one of (1) to (6), wherein the selection marker gene is a drug resistance gene.

(8) The method for deleting a deletion target region of a host DNA according to any one of (1) to (7), wherein the repressor gene is an araR gene.

  (9) The host DNA deletion method according to any one of (1) to (8), wherein the host is Bacillus subtilis.

  (10) A transformant obtained by the method for deleting a deletion target region of a host DNA according to any one of (1) to (9).

  According to the present invention, there is provided a method for deleting a deletion target region of a host DNA capable of simultaneously deleting the deletion target region of the host DNA and the selection marker gene used for selection of the transformant.

Hereinafter, the present invention will be described in detail.
The deletion method of the deletion target region of the host DNA according to the present invention is a method of deleting the deletion target region in the host DNA and deleting the selection marker gene used for selection of the transformant. In this method, first, the host DNA has a 5′-side region, a 3′-side region outside the deletion target region, and a first homologous recombination region possessed by the host DNA. A donor DNA having a presser gene is prepared. The 5 'region outside the deletion target region of the host DNA in the donor DNA (hereinafter referred to as "5' outer region") is high enough to cause homologous recombination with the 5 'outer region of the host DNA. It is a region having the sameness. Similarly, the 3 ′ region outside the deletion target region of the host DNA in the donor DNA (hereinafter referred to as “3 ′ outer region”) can cause homologous recombination with the 3 ′ outer region in the host DNA. It is a region having a high degree of identity.

  The first homologous recombination region is a region where homologous recombination occurs in the first homologous recombination between the donor DNA and the host DNA together with the 5 ′ outer region described above. The first homologous recombination region possessed by the host DNA in the donor DNA is a region having high identity to the extent that homologous recombination can occur with the first homologous recombination region in the host DNA.

  The first homologous recombination region is (a) a region consisting of part or all of the deletion target region, or (b) a region including the 5 ′ end of the 3 ′ outer region. Examples of the region (b) include, for example, a partial region including the 5 ′ end of the 3 ′ outer region, the entire 3 ′ outer region, and a 3 ′ outer region and 3 ′ outer region in the host DNA. And a region including a side region. Furthermore, a part of the region including the 3 ′ end of the deletion target region or the entire deletion target region may be included.

  In the following description, the 5 ′ outer region, the 3 ′ outer region, and the first homologous recombination region of the host DNA in the donor DNA are represented in the same manner as the corresponding regions in the host DNA. 'The outer region and the first homologous recombination region.

  Here, the 5 ′ side and the 3 ′ side mean the direction following the 5 ′ end side and the direction continuing to the 3 ′ end side of the deletion target region, based on either strand in the DNA duplex. .

  The donor DNA is included in the order of 5 ′ outer region, 3 ′ outer region, first selection marker gene, repressor gene, and first homologous recombination region. Alternatively, the order of the first selection marker gene and the repressor gene may be reversed.

  Next, the donor DNA and the host DNA are allowed to coexist under conditions that cause homologous recombination. Under such conditions, a pair of interstrand exchange structures are easily formed between the 5 ′ outer region and the first homologous recombination region between the donor DNA and the host DNA. By forming this pair of interstrand exchange structures, double crossover homologous recombination (first homologous recombination) occurs between the donor DNA and the host DNA. The region, the first selection marker gene, and the repressor gene can be introduced into the host DNA. When there is a region sandwiched between the 5 ′ outer region and the first homologous recombination region in the host DNA, the region sandwiched with the host DNA (part of the deletion target region) is missing from the host DNA. To lose.

  Furthermore, two 3 ′ outer regions exist in the host DNA that has undergone homologous recombination with the donor DNA. One of these two 3 ′ outer regions is derived from host DNA and the other is derived from donor DNA. Therefore, the presence of host DNA under a predetermined condition forms one interstrand exchange structure between the 3 ′ outer regions in the host DNA, resulting in homologous recombination (second homologous recombination). Selectable marker gene, repressor gene, and deletion target region in host DNA (when there is a region sandwiched between the 5 ′ outer region and the first homologous recombination region, a part of the deletion target region is excluded. The remainder) will be deleted from the host DNA.

  For example, when the first homologous recombination region does not include the deletion target region in the region (b) above, the deletion target region is missing from the host DNA at the time of the first homologous recombination. Will be lost.

The deletion target region may be any region as long as it already exists in the host DNA before application of this method. For example, the deletion target region may be an originally existing region or artificially introduced before application of this method. It may be a region that has been made. Examples of the artificially introduced region include a region containing a selection marker gene and a reporter gene. In the host DNA deletion target region deletion method according to the present invention, the size of the deletion target region is not particularly limited, but is preferably 1 bp or more, more preferably 0.2 kb or more, and most preferably 0.5 kb or more. For example, the largest continuous region that does not contain a gene essential for growth (hereinafter referred to as “growth essential gene”) in the Bacillus subtilis genome is about 200 kb between the dltA gene and the yycH gene (Proc. Natl. Acad. Sci. USA, 100, 4679 (2003)). In consideration of not deleting the essential growth gene from this viewpoint, the size of the deletion target region is preferably 200 kb or less, more preferably 160 kb or less, further preferably 120 kb or less, particularly preferably 80 kb or less, and most preferably 40 kb or less. .

  Further, the distance between the 5 ′ outer region and the first homologous recombination region in the host DNA is not particularly limited, but is preferably 0 kb to 200 kb, particularly preferably 0 kb to 140 kb.

  In the present invention, the 5 ′ outer region, the 3 ′ outer region and the first homologous recombination region form an interstrand exchange structure between the donor DNA and the host DNA or in the process of homologous recombination in the host DNA. It is an area including a part. Accordingly, the donor DNA and the host DNA have a high degree of identity between the 5 ′ outer region, the 3 ′ outer region, and the first homologous recombination region so that an interstrand exchange structure can be formed. It is preferable to have.

  Specifically, the identity between each region is calculated by, for example, the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the unit size to compare (ktup) can be calculated as 2 based on the analysis. . According to the result of the calculation, it is sufficient that each region has 60% or more identity, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, 99% or more. Most preferably, they have the same identity.

  The sizes of the 5 ′ outer region, the 3 ′ outer region, and the first homologous recombination region are not particularly limited as long as they can form an interchain exchange structure, but are preferably 0.1 kb to 3 kb, for example. Furthermore, it is preferably 0.5 kb to 3 kb, particularly preferably 0.5 kb to 2 kb.

In the present invention, donor DNA means DNA containing a 5 ′ outer region, a 3 ′ outer region, a first homologous recombination region, a first selectable marker gene, and a repressor gene. The donor DNA can include a promoter that controls expression of the repressor gene. In the donor DNA, the position of the promoter with respect to the repressor gene is a position where the expression of the repressor gene can be controlled, and is introduced into the host DNA together with the repressor gene after the first homologous recombination. Between the region and the first homologous recombination region. For example, in the donor DNA, each region is arranged in the order of 5 ′ outer region, 3 ′ outer region, first selection marker gene, repressor gene, and first homologous recombination region. In the position between the first selection marker gene and the repressor gene, or in the donor DNA, each region is a 5 ′ outer region, a 3 ′ outer region, a repressor gene, a first selection marker gene, a first homologous recombination. When arranged in the order of the region, the position between the 3 ′ outer region and the repressor gene can be mentioned. Similarly, the donor DNA may contain a promoter that controls the expression of the first selectable marker gene. The location of the promoter relative to the first selectable marker gene in the donor DNA can be appropriately determined according to the determination of the location of the promoter relative to the repressor gene described above. Examples of donor DNA include DNA fragments such as PCR products, whole or part of genome, plasmid, shuttle vector, helper plasmid, phage DNA, viral vector, BAC (Proc. Natl. Acad. Sci. USA 87, 8242 (1990)) and YAC (Science 236, 806 (1987)), as well as plasmids, shuttle vectors, helper plasmids, phage DNA, viral vectors, BAC and YAC, etc. Etc. For example, the plasmid, Escherichia coli (Escherichia) derived plasmids (e.g., pET system such as pET30b, pBR system such as pBR322 and pBR325, pUC118, pUC119, pUC18 and pUC series such as pUC19, pBluescript, etc.), Bacillus subtilis (Bacillus subtilis) Examples include plasmids derived from (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp systems such as YEp13, YCp systems such as YCp50, etc.), and the like. Examples of the phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.) and PBS1 phage (J. Bacteriol. 90, 1575 (1965)). Furthermore, animal virus vectors such as retrovirus or vaccinia virus, plant virus vectors such as cauliflower mosaic virus, or insect virus vectors such as baculovirus can also be used. Alternatively, LP (lysis of protoplasts) transformation method (T. Akamatsu and J. Sekiguchi, Archives of Microbiology, 1987, 146, p.353-357; T. Akamatsu and H. Taguchi, Bioscience, Biotechnology, and Biochemistry, 2001 , 65, 4, p.823-829), for example, microorganisms such as Bacillus subtilis having donor DNA can be used.

  The selectable marker gene is not particularly limited. For example, a drug resistance gene (chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene and blast cycle gene can be used. Gin S resistant gene). Furthermore, it is possible to use a gene defect associated with auxotrophy as a selection marker gene. In the present invention, the first selection marker gene and the second selection marker gene are of different types.

In the present invention, a repressor means a protein that recognizes a specific operator and binds to it to suppress a group of genes linked to a promoter containing the operator, that is, a protein that negatively regulates the expression of an operon. To do. The operator means the binding site of the repressor. The operator exists in the middle of the promoter and the operon structural gene, or upstream of the operon so as to partially overlap the promoter. In other words, the operator may have an overlapping position with the promoter. As a repressor gene / promoter (including an operator) that can be used in the present invention, for example, an araR gene (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2) / araA gene promoter (SEQ ID NO: 2) derived from Bacillus subtilis. 3) or araE gene promoter (SEQ ID NO: 4), purR gene (base sequence: SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6) / purE gene promoter (SEQ ID NO: 7), ctsR gene (base sequence: SEQ ID NO: 8, amino acid) Sequence: SEQ ID NO: 9) / clpP gene promoter (SEQ ID NO: 10) or clpE gene promoter (SEQ ID NO: 11), lmrA gene (base sequence: SEQ ID NO: 12, amino acid sequence: SEQ ID NO: 13) / yxaG gene promoter (SEQ ID NO: 14) ), trer gene (nucleotide sequence: SEQ ID NO: 15, the amino acid sequence: SEQ ID NO: 16) / Trep gene promoter (SEQ ID NO: 17), exuR genetic (Nucleotide sequence: SEQ ID NO: 18, the amino acid sequence: SEQ ID NO: 19) / uxaC gene promoter (SEQ ID NO: 20), xylR gene (nucleotide sequence: SEQ ID NO: 21, amino acid sequence: SEQ ID NO: 22) / xylA gene promoter (SEQ ID NO: 23 ), DeoR gene (base sequence: SEQ ID NO: 24, amino acid sequence: SEQ ID NO: 25) / dra gene promoter (SEQ ID NO: 26), and iolR gene (base sequence: SEQ ID NO: 27, amino acid sequence: SEQ ID NO: 28) / iolA gene A promoter (SEQ ID NO: 29) can be mentioned. The base sequence of the above-described repressor gene and the corresponding amino acid sequence are not limited to the base sequence shown in the above SEQ ID No. and the corresponding amino acid sequence. Each repressor consists of an amino acid sequence in which one or several (for example, 1 to 10, 1 to 5) amino acids are substituted, deleted or added in the amino acid sequence shown in the above SEQ ID No., and the repressor Any function may be used. Similarly, the above-described promoter is not limited to the base sequence shown in the above SEQ ID NO. Each promoter consists of a base sequence in which one or several (for example, 1 to 10, 1 to 5) nucleotides are substituted, deleted or added in the base sequence shown in the above SEQ ID NO, and has a promoter function. It is sufficient if it has and is suppressed by the repressor (that is, has an operator function). In the following description, “promoter” means an operator.

  The host DNA means DNA having a deletion target region, a 5 ′ outer region and a 3 ′ outer region. Further, in the host DNA, a second selectable marker gene exists in a region other than the deletion target region, the 5 ′ outer region and the 3 ′ outer region, downstream of the promoter controlled by the above-described repressor. The location of the promoter and the second selection marker gene in the host DNA can be determined as appropriate according to the selection method.

Examples of host DNA include genomic DNA and mitochondrial DNA in a predetermined organism. As the host, bacteria belonging to the genus Bacillus such as Bacillus subtilis, Escherichia such as Escherichia coli and Pseudomonas ptida ( Pseudomonasputida ), Saccharomyces cerevisiae and Schizosaccharomyces pombe , Animal cells such as COS cells and CHO cells, insect cells such as Sf9, and the family Gramineae ( Oryza sativa , corn ( Zeamays )), Brassicaceae ( Arabidopsis thaliana ), Solanum ( Nicotianatabacum ) And legumes ( Glycine max ), etc. Bacillus subtilis is particularly preferred.

  In the host DNA deletion target region deletion method according to the present invention, first, a donor DNA comprising a 5 ′ outer region, a 3 ′ outer region, a first homologous recombination region, a first selectable marker gene, and a repressor gene. Prepare. The 5 ′ outer region, the 3 ′ outer region, the first homologous recombination region, the first selection marker gene and the repressor gene (hereinafter referred to as “region and gene”) are, for example, organisms having these region and gene, respectively. The DNA can be easily obtained as a DNA fragment by PCR using the genomic DNA or the like as a template and primers complementary to the nucleotide sequences at both ends of the region.

  Once the region and the base sequence of the gene, etc. are determined, the region is then synthesized by chemical synthesis, by PCR using the cloned probe as a template, or by hybridizing with a DNA fragment having the base sequence as a probe. In addition, genes and the like can be obtained as DNA fragments.

  Next, each DNA fragment containing these regions, genes and the like is ligated. As the ligation method, for example, a method of cleaving each purified DNA fragment with an appropriate restriction enzyme and ligating is adopted. Alternatively, DNA fragments containing these regions and genes, etc. each have a partially homologous region, and as a template, perform SOE (splicing by overlap extension) -PCR (Gene, 77, 61, (1989)) Each DNA fragment can be ligated. Alternatively, DNA fragments containing these regions and genes may be ligated by sequentially inserting them into an appropriate vector.

  On the other hand, in the host DNA, a promoter corresponding to the repressor to be used and a second selection marker gene are introduced downstream thereof. Preparation of the promoter and the second selectable marker gene can be performed by a method according to the preparation of the donor DNA described above. Next, a transformant is prepared by introducing a DNA fragment (such as a PCR product or a vector) containing the promoter and the second selection marker gene into the host.

  The method for introducing a DNA fragment into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method. When the host is Bacillus subtilis, competent cell transformation method (J. Bacterial. 93, 1925 (1967)) and LP transformation method (T. Akamatsu and J. Sekiguchi, Archives of Microbiology, 1987, 146, p.353-357; T. Akamatsu and H. Taguchi, Bioscience, Biotechnology, and Biochemistry, 2001, 65, 4, p.823-829).

  The method for introducing a DNA fragment into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method (electroporation method), a spheroplast method, and a lithium acetate method.

  When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, and the like are used. Examples of the method for introducing a DNA fragment into an animal cell include an electroporation method, a calcium phosphate method, and a lipofection method.

  When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing a DNA fragment into an insect cell include a calcium phosphate method, a lipofection method, and an electroporation method.

  When using a plant as a host, the whole plant body, plant organs (e.g. leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g. epidermis, phloem, soft tissue, xylem, vascular bundles, etc.) and plant culture Cells are used. Examples of the method for introducing a DNA fragment into a plant include an electroporation method, an Agrobacterium method, a particle gun method, and a PEG method.

  As described above, the DNA fragment containing the promoter and the second selection marker gene can be introduced into the host. In order to introduce the promoter and the second selection marker gene into a specific site of the host DNA, a homologous region to the host DNA is provided at both ends of the DNA fragment containing the promoter and the second selection marker gene. By introducing this DNA fragment into the host by the introduction method described above, homologous recombination occurs between the host DNA and the DNA fragment, and the promoter and the second selection marker gene can be incorporated at a predetermined site of the host DNA. it can.

  Whether or not the DNA fragment has been incorporated into the host can be confirmed using the second selection marker gene as an indicator. Alternatively, whether or not the DNA fragment has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. Thereafter, the amplified product is transformed by performing agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., staining with ethidium bromide, SYBR Green solution, etc., and detecting the amplified product as a band. Make sure. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, a method may be employed in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction.

  In the host DNA deletion target region deletion method according to the present invention, homologous recombination is performed between the donor DNA prepared as described above and the host DNA. First, under conditions where homologous recombination occurs, donor DNA and host DNA are brought into close proximity, and double crossover homology between the donor DNA and host DNA in the 5 ′ outer region and the first homologous recombination region. Cause recombination. As a result, the 3 ′ outer region of the donor DNA, the first selectable marker gene and the repressor gene are introduced between the 5 ′ outer region of the host DNA and the first homologous recombination region. Here, the conditions under which homologous recombination occurs means a state in which the function of an enzyme involved in the recombination reaction inherently possessed by the host is not lost. Examples of the recombination method between the donor DNA and the host DNA include, for example, a general competent cell transformation method (J. Bacterial. 93, 1925 (1967)) and a protoplast transformation method (Mol. Gen. Genet 168, 111 (1979)), electroporation (FEMS Microbiol. Lett. 55, 135 (1990)) or LP transformation (T. Akamatsu and J. Sekiguchi, Archives of Microbiology, 1987, 146, p. 353-357; T. Akamatsu and H. Taguchi, Bioscience, Biotechnology, and Biochemistry, 2001, 65, 4, p. 823-829). For example, when the host is Bacillus subtilis, transformation can be performed as follows according to a competent cell transformation method (J. Bacterial. 93, 1925 (1967)).

  The host strain was treated with SPI medium (0.20% (W / V) ammonium sulfate, 1.40% (W / V) dipotassium hydrogen phosphate, 0.60% (W / V) potassium dihydrogen phosphate, 0.10% (W / V) Trisodium acid dihydrate, 0.50% (W / V) glucose, 0.02% (W / V) casamino acid (Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan) at 37 ° C., for example Shake culture until the degree of growth (OD600) is about 1. After shaking culture, a portion of the culture broth was added 9 times the volume of SPII medium (0.20% ammonium sulfate, 1.40% (W / V) dipotassium hydrogen phosphate, 0.60% (W / V) potassium dihydrogen phosphate, 0.10% ( W / V) Trisodium citrate dihydrate, 0.50% (W / V) glucose, 0.01% (W / V) casamino acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / ml tryptophan) By inoculating and shaking culture until, for example, the degree of growth (OD600) is about 0.4, it can be induced from the host strain to competent cells.

The donor DNA is then added to the competent cell of the host strain. The amount of donor DNA to be added is not particularly limited, but 100 pg to 100 μg, preferably 1 ng to 10 μg is added. The competent cell is 10 ⁵ to 10 ⁹ CFU, preferably 10 ⁶ to 10 ⁸ CFU.

The homologous recombination between donor DNA and host DNA will be described more specifically with reference to FIG. In FIG. 1, the chloramphenicol resistance gene is the first selection marker gene, and the neomycin resistance gene is the second selection marker gene. The araR gene is a repressor gene, and the araA gene promoter is a promoter.

In FIG. 1, the donor DNA includes 5 ′ outer region, 3 ′ outer region, chloramphenicol resistance gene ( cat ), araR gene ( araR ), and first homologous recombination region in this order. In FIG. 1, the first homologous recombination region is a part of the deletion target region. On the other hand, in the host DNA, an araA gene promoter (P _ara ) and a neomycin resistance gene ( Nm ) are inserted in the region other than the deletion target region, the 5 ′ outer region and the 3 ′ outer region. A host (for example, Bacillus subtilis) having the host DNA is neomycin resistant (Nm ^R ). In FIG. 1, in the host DNA, the 5 ′ outer region and the first homologous recombination region are adjacent to each other.

First, by transforming a host having host DNA (for example, Bacillus subtilis) using the donor DNA, between the donor DNA and the host DNA, the 5 ′ outer region and the first homologous recombination region Double-crossover homologous recombination (first homologous recombination) occurs (in FIG. 1, the mode of homologous recombination is indicated by a broken line). As a result, the 3 ′ outer region of the donor DNA, the chloramphenicol resistance gene, and the araR gene can be introduced between the 5 ′ outer region of the host DNA and the first homologous recombination region. In the transformant obtained after the first homologous recombination, the chloramphenicol resistance gene is expressed. Therefore, the transformant becomes chloramphenicol resistant (Cm ^R ). The obtained transformant expresses the araR gene. The repressor protein encoded by the araR gene binds to the araA gene promoter and suppresses the expression of the neomycin resistance gene existing downstream of the promoter. Therefore, the obtained transformant becomes neomycin sensitive (Nm ^S ). The resulting transformant can be selected using chloramphenicol resistance (positive selection) and neomycin sensitivity (negative selection) as indicators. Alternatively, in the first homologous recombination in the transformant, PCR is performed using the host DNA of the transformant as a template and the 3 ′ outer region derived from the donor DNA, the chloramphenicol resistance gene and the araR gene. This can be confirmed.

Subsequently, the obtained transformant (for example, Bacillus subtilis) is cultivated for 0 to 48 hours, preferably 1 to 24 hours, particularly preferably 2 to 3 hours after single colonization. In the meantime, as shown in FIG. 1, homologous recombination occurs between two 3 ′ outer regions existing in the host DNA after the first homologous recombination (second homologous recombination). As a result, the chloramphenicol resistance gene and araR gene inserted into the host DNA and the deletion target region are deleted from the host DNA. The resulting transformant becomes chloramphenicol sensitive (Cm ^S ) due to the deletion of the chloramphenicol resistance gene. In addition, due to the deletion of the araR gene, the expression of the repressor protein disappears, and the neomycin resistance gene present downstream of the araA gene promoter is expressed. Therefore, the obtained transformant becomes neomycin resistant (Nm ^R ). The obtained transformant can be selected using chloramphenicol sensitivity (negative selection) and neomycin resistance (positive selection) as indicators. Alternatively, the second homologous recombination in the transformant can be confirmed by performing PCR on the 5 ′ outer region and the 3 ′ outer region using the host DNA of the transformant as a template.

On the other hand, in FIG. 1, when the 5 ′ outer region and the first homologous recombination region are separated from each other in the host DNA, first, in the first homologous recombination, the 3 ′ outer region of the donor DNA and chloramphenicol. A resistance gene and an araR gene are introduced into the host DNA. At the same time, a part of the deletion target region sandwiched between the 5 ′ outer region and the first homologous recombination region in the host DNA is deleted from the host DNA. Next, in the second homologous recombination, the remainder excluding the chloramphenicol resistance gene, the araR gene and a part of the deletion target region sandwiched between the 5 ′ outer region and the first homologous recombination region in the host DNA is the host. It will be deleted from the DNA.

Further, in FIG. 1, when the first homologous recombination region is, for example, one that does not include the deletion target region in the region (b), first, in the first homologous recombination, 5 ′ outside of the donor DNA The 3 ′ outer region adjacent to the region, the chloramphenicol resistance gene and the araR gene are introduced into the host DNA. At the same time, the deletion target region in the host DNA is deleted from the host DNA. Next, in the second homologous recombination, the chloramphenicol resistance gene and the araR gene are deleted from the host DNA.

  According to the host DNA deletion target region deletion method according to the present invention described above, the first selection marker gene used for selecting a transformant at the same time as deleting the DNA fragment of the host DNA. Can be reliably deleted. In the host DNA deletion target region deletion method according to the present invention, the ratio of transformants after the second homologous recombination is about 100% (eg, 98% or more) when the selection marker gene is used as an index. It is obtained by.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

  For the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, GeneAmp PCR System (Applied Biosystems) is used, and DNA amplification is performed using Pyrobest DNA Polymerase (Takara Bio) and attached reagents. went. The PCR reaction solution composition was 1 μL of appropriately diluted template DNA, 20 pmol of sense and antisense primers and 2.5 U of Pyrobest DNA Polymerase, respectively, and the total amount of the reaction solution was 50 μL. PCR reaction conditions were 98 ° C for 10 seconds, 55 ° C for 30 seconds and 72 ° C for 1 to 5 minutes (adjusted according to the target amplification product, 1 minute per 1 kb). After repeating, the reaction was carried out at 72 ° C. for 5 minutes. The primers used in PCR are those shown in Table 1 below.

  Further, in the following examples, upstream and downstream of a gene are not positions from the replication start point, but upstream is a region continuing on the 5 ′ side of the start codon of the gene that is regarded as a target in each operation / step. On the other hand, “downstream” indicates a region continuing 3 ′ of the stop codon of the gene taken as a target in each operation / step.

  Furthermore, the names of the genes and gene regions in the following examples are reported in Nature, 390, 249-256, (1997), and published on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http: //bacillus.genome.ad.jp/, updated March 10, 2004), based on genome data of Bacillus subtilis.

[Example 1] Preparation of a marker-free gene-modified strain
1. Production of cassette (PNm and CRC)
FIG 2, pnm used in this embodiment is shown a - - (araR cat) cassette (P araA neo) cassette and CRC. The PNm cassette is a cassette in which a neomycin resistance gene ( neo ) is linked downstream of the araA gene promoter ( ParaA ) (SEQ ID NO: 3) derived from Bacillus subtilis 168 strain. On the other hand, the CRC cassette is a cassette in which an araR gene (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2) is linked downstream of a chloramphenicol resistance gene ( cat ). An araR gene promoter (SEQ ID NO: 58) exists between the chloramphenicol resistance gene ( cat ) and the araR gene.

Below, the preparation methods of a PNm cassette and a CRC cassette are demonstrated.
(1) Preparation of PNm cassette Using the genomic DNA extracted from Bacillus subtilis 168 strain as a template, using the Paraf (SEQ ID NO: 30) and Para / Nmr (SEQ ID NO: 31) primer sets shown in Table 1, The promoter region 0.2 kb fragment (A) of the araA gene was amplified by PCR. In addition, a plasmid pUB110 (Plasmid 15, 93 (1986)) was used as a template, and a primer set of Nmf (SEQ ID NO: 32) and Nmr (SEQ ID NO: 33) shown in Table 1 was used to add a promoter-free 0.85 kb neomycin. Resistance gene (Nm) fragments were prepared by PCR.

Next, the obtained fragment (A) and Nm fragment were mixed and used as a template, and SOE (splicing by overlap extension) -PCR (Gene 77, using the above primers Paraf (SEQ ID NO: 30) and Nmr (SEQ ID NO: 33). 61 (1989)) to prepare a PNm cassette (1.1 kb) in which a neomycin resistance gene fragment (second selection marker gene) was linked downstream of the promoter region (operator) of the araA gene.

(2) Preparation of CRC cassette Using plasmid pC194 (J. Bacteriol. 150 (2) 815 (1982)) as a template, and using the catf (SEQ ID NO: 34) and catr (SEQ ID NO: 35) primer sets shown in Table 1 A chloramphenicol resistance gene (Cm) fragment (0.9 kb) was prepared by PCR. Further, using a genomic DNA extracted from Bacillus subtilis 168 strain as a template, using a primer set of araRf / Cm (SEQ ID NO: 36) and araRr (SEQ ID NO: 37) shown in Table 1, a promoter (SEQ ID NO: 58) in the genome is used. The araR gene region 1.3 kb fragment (B) containing) was amplified by PCR.

Next, the obtained Cm fragment and the fragment (B) were mixed and used as a template, and SOE-PCR using the above-described primers catf (SEQ ID NO: 34) and araRr (SEQ ID NO: 37) was carried out, whereby chloramphenicol resistance A CRC cassette (2.1 kb) in which the araR gene (repressor gene) was linked downstream of the gene (Cm) (first selection marker gene) was prepared.

2. Production of AR3 strain (a strain having host DNA into which an operator and a second selection marker gene have been introduced) As shown in FIG. 3, the AR3 strain, which is the base strain for constructing a marker-free deletion strain, is as follows: It was constructed. In FIG. 3, the symbols B, D, L, M, N, P, and Q are a predetermined L-arabinose utilization gene ( ara gene) group. _Para means the araA gene promoter.

Using the genomic DNA extracted from Bacillus subtilis 168 strain as a template, using the araRfw (SEQ ID NO: 38) and araR / PNmr (SEQ ID NO: 39) primer sets shown in Table 1, adjacent to the upstream of the araR gene in the genome A 1.0 kb fragment (C) was amplified by PCR. In addition, using the genomic DNA as a template, using the araR / PNmf (SEQ ID NO: 40) and araRrv (SEQ ID NO: 41) primer sets shown in Table 1, a 1.0 kb fragment adjacent to the downstream of the araR gene in the genome ( D) was amplified by PCR.

  Next, the obtained 1.0 kb fragment (C), 1.0 kb fragment (D) and 3 fragments of the PNm cassette obtained in 1 (preparation of cassette) above were mixed to provide a template as araRfw2 ( By performing SOE-PCR using the primer sets of SEQ ID NO: 42) and araRrv2 (SEQ ID NO: 43), 3 fragments are included in the order of 1.0 kb fragment (C), PNm cassette, 1.0 kb fragment (D). A kb PCR fragment was obtained.

  Furthermore, the Bacillus subtilis 168 strain was transformed using the obtained PCR fragment by a competent cell transformation method (J. Bacterial. 93, 1925 (1967)). After transformation, colonies that grew on LB agar medium containing neomycin (10 μg / mL) were isolated as transformants.

Genomic DNA was extracted transformants, Arar gene by PCR is deleted, Arar gene was confirmed that by substituting PNm cassette by homologous recombination. As described above, an AR3 strain was constructed in which a PNm cassette was inserted into the araR gene site of genomic DNA in Bacillus subtilis.

3. Production of AR31 strain and AR32 strain (transformants after the first homologous recombination)
(1) Production of AR31 strain Production of the AR31 strain will be described below with reference to FIG.
Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, 1.0 kb fragment containing iolR gene in the genome using iolfw (SEQ ID NO: 44) and iol / csbCUr (SEQ ID NO: 45) primer sets shown in Table 1 (E) (5 ′ outer region) was amplified by PCR. In addition, using the genomic DNA as a template, using a primer set of iol / CRf (SEQ ID NO: 46) and iolrv (SEQ ID NO: 47) shown in Table 1, a 1.0 kb fragment (F) containing the iolS gene in the genome (F) ( The first homologous recombination region) was amplified by PCR. Furthermore, using the genomic DNA as a template, the primer set of csbC-Df (SEQ ID NO: 48) and csbC / Cmr (SEQ ID NO: 49) shown in Table 1 was used, and 1.0 kb adjacent to the downstream of the csbC gene in the genome. Fragment (G) (3 ′ outer region) was amplified by PCR.

  Next, the obtained 1.0 kb fragment (G) and the two fragments of the CRC cassette obtained in 1 (preparation of the cassette) were mixed as a template, and csbC-Df (SEQ ID NO: 48) shown in Table 1 and By performing SOE-PCR using the primer set of araRr (SEQ ID NO: 37), a 3.1 kb PCR fragment (H) containing 2 kb fragments in the order of 1.0 kb fragment (G) and CRC cassette was obtained.

  Next, 3 fragments of the obtained 1.0 kb fragment (E), 3.1 kb fragment (H) and 1.0 kb fragment (F) were mixed and used as templates as iolfw2 (SEQ ID NO: 50) and iolrv2 ( By performing SOE-PCR using the primer set of SEQ ID NO: 51), a 5.1 kb PCR containing 3 fragments in the order of 1.0 kb fragment (E), 3.1 kb fragment (H), and 1.0 kb fragment (F) A fragment (donor DNA) was obtained.

  Furthermore, the AR3 strain was transformed using the 5.1 kb PCR fragment obtained by the competent cell transformation method (J. Bacterial. 93, 1925 (1967)). After the transformation, colonies that grew on the LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants (55 transformants / mL).

Genomic DNA was extracted transformants was confirmed by PCR that the csbC gene downstream 1.0kb fragment (G) and CRC cassette by homologous recombination between the iolR gene and iolS genes are inserted . The PCR results (electrophoresis photograph) are shown in FIG. In addition, in FIG. 5, each lane 1-4 shows the result of the PCR product corresponding to the area | region of 1-4 shown in FIG. For amplification of the regions 1 to 4, iolfw2 (SEQ ID NO: 50) and catr (SEQ ID NO: 35), csbC-Df (SEQ ID NO: 48), araRr (SEQ ID NO: 37), catf (SEQ ID NO: 34) and A primer set of iolrv2 (SEQ ID NO: 51), csbC-Df (SEQ ID NO: 48) and iolrv2 (SEQ ID NO: 51) was used. M lane indicates a molecular weight marker.

As shown in FIG. 5, the confirmed amplification of the expected PCR product, is csbC gene downstream 1.0kb fragment (G) and CRC cassette by homologous recombination between the iolR gene and iolS genes are inserted It was confirmed.
The AR31 strain was constructed as described above.

(2) Production of AR32 strain Production of the AR32 strain will be described below with reference to FIG.
Using the genomic DNA extracted from Bacillus subtilis 168 as a template and using the hutMfw (SEQ ID NO: 52) and hutM-Dr (SEQ ID NO: 53) primer sets shown in Table 1, adjacent to the upstream of the hutM gene in the genome A 1.0 kb fragment (I) (5 ′ outer region) was amplified by PCR. Further, using the genomic DNA as a template, the hutM / CRf (SEQ ID NO: 54) and hutMrv (SEQ ID NO: 55) primer sets shown in Table 1 were used, and a 1.0 kb fragment adjacent to the downstream of the hutM gene in the genome ( J) (first homologous recombination region) was amplified by PCR.

  Next, 3 kb fragments of the obtained 1.0 kb fragment (I), 3.1 kb fragment (H) obtained in the above 3 (1) (production of AR31 strain), and 1.0 kb fragment (J) were mixed to serve as a template. By performing SOE-PCR using the hutMfw2 (SEQ ID NO: 56) and hutMrv2 (SEQ ID NO: 57) primer sets shown in Table 1, three fragments were converted into 1.0 kb fragment (I) and 3.1 kb fragment (H). And a 5.1 kb PCR fragment (donor DNA) contained in the order of 1.0 kb fragment (J).

  Furthermore, the AR3 strain was transformed using the 5.1 kb PCR fragment obtained by the competent cell transformation method. After the transformation, colonies that grew on the LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants (20 transformants / mL).

Genomic DNA of the obtained transformant was extracted, and it was confirmed that the hutM gene was deleted by PCR and the hutM gene was replaced with a 3.1 kb PCR fragment (H) by homologous recombination. The PCR results (electrophoresis photograph) are shown in FIG. In addition, in FIG. 7, each lane 1-4 shows the result of the PCR product corresponding to the area | region of 1-4 shown in FIG. For amplification of the regions 1 to 4, hutMfw2 (SEQ ID NO: 56) and catr (SEQ ID NO: 35), csbC-Df (SEQ ID NO: 48), araRr (SEQ ID NO: 37), catf (SEQ ID NO: 34) and A primer set of hutMrv2 (SEQ ID NO: 57), csbC-Df (SEQ ID NO: 48) and hutMrv2 (SEQ ID NO: 57) was used. M lane indicates a molecular weight marker.

As shown in FIG. 7, it was confirmed that the expected PCR product was amplified, the hutM gene was deleted, and the hutM gene was replaced with a 3.1 kb PCR fragment (H) by homologous recombination.
The AR32 strain was constructed as described above.

4. Selection of marker-free deleted strains (AR31d and AR32d; transformants after the second homologous recombination)
(1) Selection of 3.8 kb deleted strain (AR31d strain) The selection of the AR31d strain will be described below with reference to FIG.

The AR31 strain obtained in 3 (1) above (preparation of AR31 strain) was inoculated into LB medium and cultured with shaking at 37 ° C. for 4 hours. The culture solution was appropriately diluted with LB medium, seeded on LB agar medium containing neomycin, and cultured at 37 ° C. overnight. After cultivation, the ratio of the grown colonies to the number of viable bacteria when they were plated on an agar medium was 1.76 × 10 ⁻⁴ . Next, 100 colonies were replicated on an LB agar medium containing chloramphenicol, all of which were sensitive to chloramphenicol. Furthermore, genomic DNA was extracted from 6 neomycin-resistant and chloramphenicol-sensitive colonies, and the csbC gene was converted from the iolS gene by PCR using the primer set of iolfw (SEQ ID NO: 44) and csbC / Cmr (SEQ ID NO: 49). It was confirmed that the previous 3.8 kb fragment (deletion target region) was deleted. The PCR results (electrophoresis photograph) are shown in FIG. In addition, in FIG. 8, each lane 1-6 shows the result of each PCR product of the six colonies mentioned above. M lane indicates a molecular weight marker.

As shown in FIG. 8, amplification of the expected 2 kb PCR product containing the iolR gene and 1.0 kb fragment (G) shown in FIG. 4 was confirmed, and the 3.8 kb fragment from the iolS gene to the csbC gene was deleted. I confirmed that. The strain thus obtained was named AR31d strain.

(2) Selection of 42 kb deletion strain (AR32d strain) Selection of the AR32d strain will be described below with reference to FIG.
The AR32 strain obtained in the above 3 (2) (production of AR32 strain) was inoculated into LB medium and cultured with shaking at 37 ° C. for 4 hours. The culture solution was appropriately diluted with LB medium, seeded on LB agar medium containing neomycin, and cultured at 37 ° C. overnight. After cultivation, the ratio of the grown colonies to the number of viable bacteria when they were plated on an agar medium was 1.18 × 10 ⁻⁴ . Next, 100 colonies were replicated on LB agar medium containing chloramphenicol, and 98 were chloramphenicol sensitive. Further, genomic DNA was extracted from 6 neomycin-resistant and chloramphenicol-sensitive colonies, and the csbC gene was obtained from the hutM gene by PCR using the primer set of hutMfw (SEQ ID NO: 52) and csbC / Cmr (SEQ ID NO: 49). It was confirmed that the 42 kb fragment (region to be deleted) was deleted. The PCR results (electrophoresis photograph) are shown in FIG. In addition, in FIG. 9, each lane 1-6 shows the result of each PCR product of the six colonies mentioned above. M lane indicates a molecular weight marker.

As shown in FIG. 9, the amplification of the expected 2 kb PCR product including the 1.0 kb fragment (I) and the 1.0 kb fragment (G) shown in FIG. 6 can be confirmed, and the 42 kb fragment from the hutM gene to the csbC gene. Was confirmed to be deleted. The strain thus obtained was named AR32d strain.

FIG. 1 shows a schematic diagram of homologous recombination between donor DNA and host DNA. 2, PNm (P araA - neo) cassette and CRC (cat - araR) showing a cassette. FIG. 3 shows the production of the AR3 strain (base strain). FIG. 4 shows the production of the AR31 strain and AR31d strain. FIG. 5 shows the PCR results (electrophoresis photograph) of the genomic DNA of the AR31 strain. FIG. 6 shows the production of the AR32 and AR32d strains. FIG. 7 shows the PCR results (electrophoresis photograph) of the genomic DNA of the AR32 strain. FIG. 8 shows the PCR results (electrophoresis photograph) of the genomic DNA of the AR31d strain. FIG. 9 shows the results (electrophoresis photograph) of PCR on genomic DNA of the AR32d strain.

  Sequence number 30-57 is a primer.

Claims (6)

A method for deleting a deletion target region of host DNA comprising a step of performing homologous recombination between donor DNA and host DNA,
The deletion method is
A donor having a 5 'region outside the region to be deleted of host DNA, a 3' region and a first homologous recombination region of host DNA, and further comprising a first selectable marker gene and a repressor gene A first step of performing homologous recombination between the 5 ′ region and the first homologous recombination region in DNA and host DNA;
In the host DNA after the homologous recombination, between the 3 ′ region outside the deletion target region derived from the host DNA and the 3 ′ region outside the deletion target region possessed by the host DNA derived from the donor DNA A second step of performing homologous recombination,
The donor DNA contains the region and the gene in the order of 5 ′ side region, 3 ′ side region, first selectable marker gene, repressor gene, and first homologous recombination region outside the region to be deleted, Or 5 ′ side region outside the region to be deleted, 3 ′ side region, repressor gene, first selection marker gene, first homologous recombination region,
The first homologous recombination region is
(a) a region consisting of part or all of the deletion target region, or
(b) a region containing the 5 ′ end of the 3 ′ region,
And
In the host DNA, a second selection marker gene downstream of a promoter containing an operator controlled by the repressor in a region other than the deletion target region, the 5 ′ side region and the 3 ′ side region outside the deletion target region Exists,
A method for deleting a deletion target region of a host DNA, wherein the first selection marker gene, the repressor gene and the deletion target region are deleted from the host DNA after the second step. After the first step, using the first selection marker gene and / or the second selection marker gene as an index, the 3 ′ region outside the deletion target region of the host DNA derived from the donor DNA and the first selection marker gene The method for deleting a deletion target region of a host DNA according to claim 1 , comprising the step of selecting a transformant having a repressor gene. After the second step, including a step of selecting a transformant lacking the first selection marker gene, the repressor gene, and the deletion target region using the first selection marker gene and / or the second selection marker gene as an index. The method for deleting a deletion target region of a host DNA according to claim 1 or 2, wherein The method for deleting a deletion target region of a host DNA according to any one of claims 1 to 3 , wherein the selection marker gene is a drug resistance gene. The method for deleting a deletion target region of a host DNA according to any one of claims 1 to 4 , wherein the repressor gene is an araR gene. The method for deleting a deletion target region of a host DNA according to any one of claims 1 to 5 , wherein the host is Bacillus subtilis.

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