JP5495468B2 - Method for batch replacement of host DNA using inversion mutation - Google Patents

Description

  The present invention relates to a method for batch replacement of host DNA using, for example, inversion mutation.

  Conventional transformation methods include, for example, competent cell transformation methods (J. Bacterial. 93, 1925 (1967)) in which recombinant plasmids or DNA fragments are introduced into a host such as Escherichia coli. Depending on the host, recombinant plasmids and DNA fragments can be transformed into the host by, for example, electroporation (electroporation), Agrobacterium method, particle gun method, calcium phosphate method and lipofection method. .

  However, in the conventional transformation method as described above, it has been difficult to introduce a DNA fragment having a large size of, for example, 100 kb or more into host DNA.

Therefore, a transformation method called LP (lysis of protoplasts) transformation method has been developed in order to introduce a large-sized DNA fragment into host DNA (Non-patent Documents 1 and 2). In the LP transformation method, in place of the extracted DNA fragment or recombinant plasmid, for example, a protoplast in which the cell wall of a Bacillus subtilis strain is dissolved is used as a donor DNA, which is provided to a competent cell of a recipient strain. . The added protoplast is considered to be destroyed by osmotic shock, and the donor DNA released into the culture medium is taken up into the competent cell of the recipient strain. In addition, according to the LP transformation method, DNA damage to be introduced is greatly reduced as compared with a general transformation method. When the LP transformation method is used, it is relatively easy to introduce a DNA fragment of 100 kb or more into host DNA.

  However, in the LP transformation method, as the size of the target DNA fragment increases, homologous recombination may occur at multiple sites in the homologous region between the protoplast donor DNA and the recipient strain host DNA. . That is, with the LP transformation method, it has been difficult to accurately introduce a large-sized target DNA fragment into host DNA.

  Therefore, a method for accurately introducing a large-sized DNA fragment into host DNA is desired as a transformation method.

On the other hand, for example, a method of mutating an arbitrary Bacillus subtilis genome region in the reverse direction (hereinafter referred to as “inversion mutation method”) has been developed (Non-patent Document 3). This inversion mutation method is used to analyze the influence of the relative positional relationship between the replication start site ( oriC ) and the replication termination site ( terC ) on the genomic DNA. This technology was developed for the purpose of constructing mutant strains. Alternatively, the inversion mutation method is a technology that has been developed for the purpose of modification within the same genome as a technology for adjoining cooperating genes that are dispersed and present on the genome. It is. According to the inversion mutation method, the target DNA fragment in the genomic DNA of Bacillus subtilis can be inverted.

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, 823-829 T. Toda et al., "Bioscience, Biotechnology, and Biochemistry", 1996, Vol. 60, No. 5, p.773-778

  An object of the present invention is to provide a method for accurately introducing a large-sized DNA fragment into a host DNA in view of the above-described circumstances.

  As a result of diligent research to solve the above-mentioned problems, after reversing the substitution target region of one of donor DNA and host DNA, recombination between donor DNA and host DNA is performed. The replacement target region of the host DNA is accurately replaced by the replacement target region of the donor DNA, and one of the replacement target regions of the donor DNA and the host DNA is inverted, and the donor DNA and the host DNA After subjecting the other replacement target region and the region outside the replacement target region to inversion mutation, recombination between the donor DNA and the host DNA makes the host DNA replacement target region the donor DNA. As a result, the present invention has been completed.

  The present invention includes the following.

  (1) including a first step of inversion mutation of one of the replacement target regions of the donor DNA and the host DNA, and a second step of performing recombination between the donor DNA and the host DNA, A method for replacing a host DNA, wherein the replacement target region of the host DNA is replaced with a replacement target region of the donor DNA.

  (2) After the second step, the method includes a step of selecting a transformant having a region to be replaced with donor DNA using a selection marker gene present in donor DNA and / or host DNA as an index. (1) The method for replacing a host DNA according to (1).

  (3) The method for replacing a host DNA according to (1) or (2), wherein the donor DNA is a protoplast and the host is a competent cell.

  (4) The host DNA replacement method according to any one of (1) to (3), wherein the host is Bacillus subtilis.

  (5) The method for replacing a host DNA according to any one of (1) to (4), wherein the replacement target region of the donor DNA has a DNA size of 20 kb or more.

  (6) Inversion mutation of one of the replacement target regions of the donor DNA and the host DNA, and reverse mutation of the other replacement target region of the donor DNA and the host DNA and a region outside the replacement target region And a second step of recombination between the donor DNA and the host DNA. In the second step, the replacement target region of the host DNA is replaced with the replacement target region of the donor DNA. A method for replacing a host DNA.

  (7) After the second step, the method includes a step of selecting a transformant having a replacement target region of the donor DNA using a selection marker gene present in the donor DNA and / or host DNA as an index. (6) The method for replacing a host DNA according to (6).

  (8) The method for replacing a host DNA according to (6) or (7), wherein the donor DNA is a protoplast and the host is a competent cell.

  (9) The method for replacing a host DNA according to any one of (6) to (8), wherein the host is Bacillus subtilis.

  (10) The host DNA replacement method according to any one of (6) to (9), wherein the donor DNA replacement target region has a DNA size of 20 kb or more.

  (11) A transformant in which the region to be replaced has been replaced by the host DNA replacement method according to any one of (1) to (10) above.

  According to the present invention, there is provided a method for replacing a host DNA capable of accurately introducing a large-sized DNA fragment into the host DNA. In the host DNA replacement method according to the present invention, the replacement target region of the donor DNA can be introduced into the host DNA without causing homologous recombination between the replacement target regions.

  Hereinafter, the present invention will be described in detail.

  In the host DNA replacement method according to the present invention, one of the replacement target regions of the donor DNA and the host DNA is inverted, and then the two regions in the region outside the replacement target region between the donor DNA and the host DNA. This is a method of performing homologous recombination at multiple crossings (hereinafter referred to as “first substitution method”). In addition, the host DNA replacement method according to the present invention includes reverse substitution mutation of one of the replacement target regions of the donor DNA and the host DNA, and the other replacement target region of the donor DNA and the host DNA and the replacement target region. And then double-crossing homologous recombination in the region outside the region to be replaced between the donor DNA and the host DNA (hereinafter referred to as “second substitution method”). "). The homologous recombination in the first substitution method and the second substitution method comprises a pair of interstrand exchange structures between a region outside the replacement target region of the donor DNA and a region outside the replacement target region of the host DNA. It arises by forming. According to the first substitution method and the second substitution method, the replacement target region of the host DNA is replaced with the replacement target region of the donor DNA by double crossover homologous recombination between the host DNA and the donor DNA. Can do. Hereinafter, the first replacement method and the second replacement method according to the present invention are collectively referred to as “replacement method according to the present invention”.

  Here, the replacement target region means a region to be replaced by homologous recombination. Therefore, the region to be replaced means a region present in each of the host DNA and the donor DNA and sandwiched between regions that form a pair of interstrand exchange structures in the process of homologous recombination between the host DNA and the donor DNA. To do. The host DNA replacement target region and the donor DNA replacement target region may have the same base sequence, or may have different base sequences. For example, the replacement target region of the donor DNA may consist of a base sequence in which one to several mutations are introduced into the base sequence of the host DNA replacement target region, or the host DNA replacement target region. It may be one into which a predetermined DNA fragment is introduced. In addition, the mutation here is, for example, one in which one to several bases are replaced with another base, one to several bases deleted, or one to several bases added Or one in which a continuous base sequence such as one to several genes is deleted.

  Further, the region outside the region to be replaced means the region continuing to the 3 ′ end side and the region continuing to the 5 ′ end side in the region to be replaced with one strand as a reference, and host DNA and donor DNA Means a region containing a site that forms a pair of interchain exchange structures in the process of homologous recombination.

Donor DNA means DNA having a target replacement target region to be introduced into host DNA and a region outside the replacement target region. Donor DNA includes DNA fragments, 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)) and the like, and further, plasmids, shuttle vectors, helper plasmids, phage DNAs, viral vectors, DNA fragments cloned into BAC and YAC, genomic DNA parts, and the like can be mentioned. For example, plasmids derived from Escherichia coli (for example, pET systems such as pET30b, pBR systems such as pBR322 and pBR325, pUC systems such as pUC118, pUC119, pUC18 and pUC19, pBluescript, etc.), Bacillus ( Bacillus ) subtilis ) -derived plasmids (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. Furthermore, in the replacement method according to the present invention, when the LP transformation method described below is used, for example, microorganisms such as Bacillus subtilis can be used as donor DNA.

The host DNA means DNA having a region (substitution target region) into which a donor DNA replacement target region is introduced and a region outside the replacement target region. Examples of host DNA include genomic DNA and mitochondrial DNA in a predetermined organism. Examples of the host include bacteria belonging to the genus Bacillus such as Bacillus subtilis, Escherichia such as Escherichia coli and Pseudomonas putida, Pseudomonas putida, Saccharomyces cerevisiae and Schizosaccharomyces pombe Yeast, COS cells and CHO cells, insect cells such as Sf9, and Gramineae ( Oryza sativa , corn ( Zea mays )), Brassicaceae ( Arabidopsis thaliana )), Solanum (Tobacco) ( Nicotiana tabacum ) and legumes (soybean ( Glycine max )), etc. Bacillus subtilis is particularly preferable.

  On the other hand, in the replacement method according to the present invention, the size of the replacement target region of the host DNA and donor DNA is not particularly limited, but the size of the replacement target region of the donor DNA is 20 kb or more, preferably 20 kb to 1000 kb. More preferably, the DNA size can be 20 kb to 600 kb, more preferably 100 kb to 600 kb, particularly preferably 250 kb to 600 kb. On the other hand, there is no problem if the size of the host DNA replacement target region is the same as that of the donor DNA replacement target region or is reduced due to deletion of the gene region, etc. There is no problem even when the region is larger than the region to be replaced in the donor DNA. For example, the size difference between the substitution target regions between the donor DNA and the host DNA can be 600 kb or less, preferably 250 kb or less, more preferably 100 kb or less, and particularly preferably 20 kb or less.

  In the first substitution method according to the present invention, first, one substitution target region of donor DNA and host DNA is inverted. In other words, in the first replacement method, first, donor DNA and host DNA in which one of the replacement target regions of the donor DNA and the replacement target region of the host DNA are inverted are prepared.

  Next, in the first substitution method according to the present invention, the donor DNA and the host DNA are brought into close proximity under conditions that cause homologous recombination. As a result, a pair of interchain exchange structures is formed between the region outside the replacement target region in the donor DNA and the region outside the replacement target region in the host DNA. Then, double crossover homologous recombination occurs between the donor DNA and the host DNA, and the replacement target region of the donor DNA is replaced with the replacement target region of the host DNA.

  In the first replacement method, when the region outside the replacement target region is in a homologous positional relationship, the replacement target region of the host DNA and the replacement target region of the donor DNA are non-homologous to each other. Even if the donor DNA and the host DNA come close to each other in a state where the regions outside the region to be replaced are in a homologous positional relationship under the resulting conditions, an interchain exchange structure may be formed inside the region to be replaced. Absent. Therefore, according to the first replacement method, the replacement target region of the donor DNA is selected by selecting a region in which homologous recombination has occurred in the region outside the replacement target region using a selection marker gene such as a drug resistance gene. Can be surely replaced with the replacement target region of the host DNA. In addition, according to the first replacement method, even when the replacement target region has a length of 20 kbp or more, particularly 600 kbp or more, homologous recombination does not occur inside the replacement target region, and the replacement target region can be reliably and collectively Substitutions can be made between donor DNA and host DNA.

  Furthermore, in the first replacement method according to the present invention, a transformant having a replacement target region of donor DNA can be selected using a selection marker gene present in donor DNA and / or host DNA as an index. By using the selectable marker gene, a transformant generated by homologous recombination at an undesired location such as inside the region to be replaced or between homologous sites other than the region outside the region to be replaced is excluded, and the donor A transformant having a DNA replacement target region can be reliably selected. Here, the selection marker gene is not particularly limited, and examples thereof include drug resistance genes (chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene and blast Saidin S resistance gene etc.). Furthermore, it is possible to use a gene defect associated with auxotrophy as a selection marker gene. The location of the selection marker gene in the donor DNA or host DNA can be appropriately determined according to the selection method.

  For example, when a drug resistance gene is present in the replacement target region of donor DNA or at the end of the replacement target region, a transformant having the donor replacement target region can be selected using the drug resistance as an index. On the other hand, when a drug resistance gene is present in the replacement target region of the host DNA or at the end of the replacement target region, a transformant having the donor replacement target region can be selected using the drug sensitivity as an index.

  Alternatively, when the LP transformation method described below is used, cells that are not completely protoplasted may be contained in the protoplast solution that is the donor DNA. Therefore, when the selection of the transformant is attempted using only the selection marker gene contained in the donor DNA as an index, the donor strain itself may be selected. Therefore, by combining selection with a selectable marker gene that is present at a position other than the region to be replaced in the host DNA and the region outside the region to be replaced, the region to be replaced in the host DNA is replaced with the region to be replaced in the donor DNA. The transformant can be selected with certainty. In addition, the presence of a selection marker gene such as a drug resistance gene at a position other than the replacement target region and the region outside the replacement target region of the host DNA causes homologous recombination at a location other than the region outside the replacement target region. No transformants can be selected. In particular, it is preferable that the selection marker gene is present at a position other than the replacement target region of the host DNA and the region outside the replacement target region and the selection marker gene is present at an appropriate position of the donor DNA. The position where the selection marker gene of the donor DNA is present is preferably within or at the end of the replacement target region, and particularly preferably at the end.

  Inversion mutation methods that can be used in the first substitution method according to the present invention include, for example, the NEO inversion mutation system (Non-patent Document 3), and applications thereof such as tetracycline resistance gene, erythromycin resistance gene, chloramphenic acid. A method of using a call resistance gene, a spectinomycin resistance gene or a blasticidin S resistance gene in place of the neomycin resistance gene in the NEO inversion mutation system, and an auxotrophy such as trpC2 (Microbiology 145, 3319 (1999)) Mutant strains include a method using a gene (such as trpC) that complements auxotrophy.

  The NEO inversion mutation system targeting Bacillus subtilis genomic DNA is described below with reference to FIG.

  As shown in FIG. 1, for example, an NE cassette composed of a front region (N; 470 bp) and a middle region (E; 521 bp) of a neomycin resistance gene, and a middle region (E) and a rear region (O; 323 bp). Using an EO cassette, a specific region of Bacillus subtilis genomic DNA can be inverted. As shown in FIG. 1, drug resistance genes other than the neomycin resistance gene (for example, chloramphenicol resistance gene (Cm) and erythromycin resistance gene (Em)) can be incorporated into the NE cassette and EO cassette. . By incorporating a drug resistance gene, a transformant introduced with the NE cassette or EO cassette can be selected using drug resistance as an index.

In the NEO inversion mutation system, in the Bacillus subtilis genomic DNA, the replication start point ( ori ) of the region to be inverted (hereinafter referred to as “inversion mutation target region”) (corresponding to the replacement target region in the present invention) ) Insert an EO cassette at the end far from the center so that the middle region (E) is directly linked to the inversion mutation target region. On the other hand, in the Bacillus subtilis genomic DNA, the NE cassette is inserted so that the front region (N) is directly linked to the end near the ori of the inversion mutation target region. In addition, as a method of inserting the EO cassette or NE cassette into Bacillus subtilis genomic DNA, a plasmid having an EO cassette or NE cassette or a DNA fragment containing an EO cassette or NE cassette prepared by PCR, for example, a common Competent cell transformation method (J. Bacterial. 93, 1925 (1967)), protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)) or electroporation method (FEMS Microbiol. Lett. 55, 135 (1990)). Further, as described above, by incorporating a drug resistance gene into the NE cassette or EO cassette, a transformant introduced with the NE cassette or EO cassette can be selected using drug resistance as an index. Bacillus subtilis itself constructed as described above is sensitive to neomycin because the neomycin resistance gene exists in an incomplete state. Subsequently, homologous recombination occurs between the front region (E) of the NE cassette and the front region (E) of the EO cassette by inducing intragenomic homologous recombination in Bacillus subtilis genomic DNA. By the homologous recombination in the genome, the inversion mutation target region is completely reversed from the original sequence, and at the same time, a complete neomycin resistance gene NEO is formed. The obtained inverted mutant can be selected using neomycin resistance as an index.

  By the method as described above, one of the replacement target regions of the donor DNA and the host DNA can be inverted.

  Next, in the first replacement method according to the present invention, the donor DNA and the host DNA are brought close to each other under conditions under which homologous recombination occurs, and the region outside the replacement target region in the donor DNA and the replacement target region in the host DNA A double crossover homologous recombination with the outer region of the DNA, and the replacement target region of the donor DNA is replaced with the replacement target region of the host DNA. 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 method (FEMS Microbiol. Lett. 55, 135 (1990)) or LP transformation method (Non-patent Documents 1 and 2). Among these, the LP transformation method in which competent cell transformation is performed using protoplastized cells as donor DNA is particularly preferred. According to the LP transformation method, damage to the donor DNA can be greatly reduced as compared with a general transformation method.

  As an example, LP transformation between donor DNA and host DNA constructed by the NEO inversion mutation system between Bacillus subtilis will be described with reference to FIG.

  As shown in FIG. 2, the donor DNA constructed by the above-described NEO inversion mutation system has the inversion-mutated replacement target region and the complete neomycin resistance gene NEO. Furthermore, for example, when the chloramphenicol resistance gene (Cm) is incorporated into the NE cassette and the erythromycin resistance gene (Em) is incorporated into the EO cassette, the donor DNA contains chloramphenicol resistance. There is a gene (Cm) and an erythromycin resistance gene (Em).

  On the other hand, as shown in FIG. 2, a drug resistance gene (for example, a spectinomycin resistance gene (Sp)) different from the drug resistance gene present in the donor DNA can be present in the host DNA.

  In LP transformation of a recipient strain having a host DNA using a donor strain having a donor DNA, protoplasts are first prepared from the donor strain. Protoplasts can be prepared from donor strains according to the method of Chang et al. (Mol. Gen. Genet. 168, 111 (1979)) as follows.

The donor strain is cultured with shaking in LB medium at 37 ° C. until, for example, the degree of growth (OD ₆₀₀ ) is about 1. After shaking culture, the cells collected by centrifugation from the culture were used as SMMP (3.50% Antibiotic Medium 3 (Difco) containing 4 mg / mL lysozyme (Sigma), 17.15% sucrose, 0.16% disodium maleate, 0.20% magnesium chloride. Suspend in hexahydrate, 0.10% bovine serum albumin (adjusted to pH 6.5)) buffer and incubate at 37 ° C. for 1-2 hours. After incubation, the protoplasts are collected by centrifugation and resuspended in SMMP buffer. Furthermore, the suspension can be appropriately concentrated or diluted in consideration of the transformation efficiency and the risk of homologous recombination occurring at locations other than the region outside the region to be replaced. For example, the above suspension after resuspension is concentrated 1 to 100 times (for example, 10 times) or diluted 1 to 1,000,000 times (for example, 10 times) with SMMP buffer, and used as a protoplast suspension. Can do. In general, a suspension obtained by diluting the obtained suspension after resuspension with an SMMP buffer 1 to 100 times can be used as a protoplast suspension.

  On the other hand, a recipient strain can be performed, for example, according to the method of Anagnostopoulos et al. (J. Bacteriol. 81, 741 (1961)) as follows.

Recipient strains in SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.02% casamino acid (Difco) , 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan) at 37 ° C. with shaking until, for example, the degree of growth (OD ₆₀₀ ) is about 1. After shaking culture, a portion of the culture broth was added to 9 times the amount of SPII medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% By inoculating glucose, 0.01% casamino acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / ml tryptophan), and further, for example, by shaking culture until the value of growth (OD ₆₀₀ ) is about 0.4, It can be derived from a recipient strain into a competent cell.

  The protoplast suspension is then added to the competent cell of the recipient strain. The amount of the protoplast suspension to be added is not particularly limited, but may be 1/10000 to 10 times, preferably 1/10 to 1 times the competent cell of the recipient strain. The added protoplast is considered to be destroyed by osmotic shock, and the donor DNA released into the culture medium is taken up into the competent cell of the recipient strain.

  As described above, when LP transformation is performed between a donor strain and a recipient strain, donor DNA and host DNA are brought into close proximity. In addition, due to the action of the enzyme involved in the homologous recombination inherent in the recipient strain, a pair of interchain exchange structures is formed between the regions outside the replacement target region present in the donor DNA and the host DNA. Formed, resulting in double crossover homologous recombination (in FIG. 2, the mode of homologous recombination is indicated by a dotted line). As a result, the region to be replaced in the host DNA is replaced with the region to be replaced that has been inverted in the donor DNA. The transformant is a drug resistance gene (in FIG. 2, chloramphenicol resistance gene (Cm), erythromycin resistance gene (Em)) or NEO inversion inserted at the end of the replacement target region in which donor DNA is inverted. Drug resistance based on the neomycin resistance gene inserted by the mutation system can be selected as an index. Further, as shown in FIG. 2, drug resistance based on other drug resistance genes (eg, spectinomycin resistance gene) present in the host DNA eliminates the donor strain itself, and transformant DNA (ie, In the host DNA), it can be confirmed that homologous recombination has not occurred in a region other than the region outside the region to be replaced. Alternatively, homologous recombination in the transformant can be confirmed by performing PCR on the region to be replaced with the inverted mutation.

  On the other hand, in the host DNA or donor DNA, genes outside the region to be replaced are deleted in advance before LP transformation. Next, after LP transformation, by confirming the presence or absence of the deleted gene in the DNA of the transformant by PCR, it is confirmed that no homologous recombination has occurred in any place other than the region outside the replacement target region. Can be confirmed.

  In addition, the transformation efficiency of the recipient strain with donor DNA is the frequency of appearance of the transformant relative to the number of recipient strains used in the transformation ((number of transformants) ÷ (number of recipient strains). ).

  In addition, when the host DNA replacement target region is inverted, the host DNA inversion target replacement region is replaced with the donor DNA replacement target region by LP transformation of the donor and recipient strains. The In this case, in the obtained transformant DNA, the replacement target region derived from the donor DNA is replaced with the original orientation.

  In the second substitution method according to the present invention, first, one of the replacement target regions of the donor DNA and the host DNA is inverted and the other replacement target region and the region outside the replacement target region are inverted. . In other words, in the second substitution method, first, a donor DNA in which the replacement target region is inverted and a host DNA in which the replacement target region and the region outside the replacement target region are inverted are prepared. Alternatively, in the second substitution method, first, a host DNA in which the replacement target region is inverted and a donor DNA in which the replacement target region and a region outside the replacement target region are inverted are prepared.

  In the second substitution method according to the present invention, next, as in the first substitution method, the donor DNA and the host DNA are brought into close proximity under conditions under which homologous recombination occurs. As a result, a pair of interchain exchange structures is formed between the region outside the replacement target region in the donor DNA and the region outside the replacement target region in the host DNA. Then, double crossover homologous recombination occurs between the donor DNA and the host DNA, and the replacement target region of the donor DNA is replaced with the replacement target region of the host DNA.

  In the second replacement method, when the region outside the replacement target region is in a homologous positional relationship, the replacement target region of the host DNA and the replacement target region of the donor DNA are non-homologous, and the replacement target region in the host DNA Since the region outside the region outside of the region and the region outside the region to be replaced in the donor DNA are non-homologous, the region outside the region to be replaced under the conditions where homologous recombination occurs. Even if the donor DNA and the host DNA are close to each other when the regions are in a homologous positional relationship, an interchain exchange structure is formed in the region to be replaced and in the region further outside the region to be replaced. There is no. In other words, in the second replacement method, only the region outside the replacement target region in the donor DNA is homologous to the region outside the replacement target region in the host DNA, and the region outside the limited replacement target region An interchain exchange structure is formed in the region.

  Furthermore, in the second replacement method according to the present invention, as in the first replacement method, transformation having a replacement target region of the donor DNA using the selection marker gene present in the donor DNA and / or host DNA as an index. The body can be selected. The location of the selection marker gene in the donor DNA or host DNA can be appropriately determined according to the selection method, as in the first substitution method. In particular, it is preferable that the selection marker gene is present at a position other than the replacement target region of the host DNA and the region outside the replacement target region and the selection marker gene is present at an appropriate position of the donor DNA. The position where the selection marker gene of the donor DNA is present is preferably within or at the end of the region to be replaced, and particularly preferably at the end.

  Therefore, according to the second replacement method, by using a selectable marker gene such as a drug resistance gene, one that has undergone homologous recombination in a region outside the limited replacement target region is selected. The replacement target region can be surely replaced with the replacement target region of the host DNA. Further, according to the second substitution method, even when the replacement target region has a length of 20 kbp or more, particularly 600 kbp or more, the replacement target region is located between the donor DNA and the host DNA, as in the first replacement method. Can be surely replaced at once. The inversion mutation method that can be used in the second substitution method may be the same as that that can be used in the first substitution method according to the present invention described above. In the second substitution method according to the present invention, the DNA size of the region outside the substitution target region to be subjected to the inversion mutation is 0.15 kb to 500 kb at each end, more preferably 0.5 kb to 500 kb, and even more preferably 0.5. kb to 20 kb, particularly preferably 0.5 kb to 10 kb. Since the region outside the region to be replaced that is the target of the inversion mutation has a DNA size in the above range, the region to be replaced can be surely replaced collectively between the donor DNA and the host DNA.

  The recombination method between the donor DNA and the host DNA in the second replacement method according to the present invention may be the same as the recombination method used in the first replacement method according to the present invention described above. The LP transformation method is particularly preferable.

  As an example, FIG. 3 schematically shows LP transformation of donor DNA and host DNA constructed by the NEO inversion mutation system between Bacillus subtilis in the second substitution method according to the present invention. In FIG. 3, the regions outside the replacement target region are denoted as “adjacent region (L)” and “adjacent region (R)”.

  In FIG. 3, in the donor DNA, the replacement target region, the adjacent region (L), and the adjacent region (R) are inverted by the NEO inversion mutation system. On the other hand, in the host DNA, the region to be substituted is inverted by the NEO inversion mutation system. Furthermore, the complete neomycin resistance gene NEO exists in donor DNA and host DNA constructed by the NEO inversion mutation system. For example, when the chloramphenicol resistance gene (Cm) is incorporated into the NE cassette and the erythromycin resistance gene (Em) is incorporated into the EO cassette, There is a ramphenicol resistance gene (Cm) and an erythromycin resistance gene (Em). Furthermore, in the donor DNA, a drug resistance gene different from these drug resistance genes (e.g., tetracycline resistance gene (Tc), spectinomycin resistance gene (Sp), etc.), for example, in the replacement target region or the replacement target region It can introduce | transduce between the area | regions outside the said substitution object area | region.

  When performing LP transformation between a donor strain having the donor DNA as described above and a recipient strain having the host DNA, in the donor DNA, in the replacement target region or outside the replacement target region A transformant is selected using drug resistance based on a drug resistance gene (for example, a tetracycline resistance gene, a spectinomycin resistance gene, etc.) existing between these regions as an index. After selection, from the obtained transformant, for example, when the NEO inversion mutation system is used, the resistance to chloramphenicol used as an index for selection of the transformant introduced with the NE cassette or EO cassette Selection is performed using as an index the drug sensitivity associated with the deletion of the gene (Cm) and the erythromycin resistance gene and / or the neomycin resistance gene introduced by the NEO inversion mutation system. By selection using drug resistance or drug sensitivity as an index as described above, an interstrand exchange structure is formed in adjacent regions (L) and (R) present in the donor DNA and the host DNA, as shown in FIG. A transformant in which double crossover homologous recombination has occurred can be selected (in FIG. 3, the mode of homologous recombination is indicated by a dotted line). As a result, the region to be replaced with the inverted mutation in the host DNA is replaced with the region to be replaced with the inverted mutation in the donor DNA. In the obtained transformant DNA (that is, host DNA), the replacement target region derived from the donor DNA is replaced in the original orientation. As described above, in the second replacement method according to the present invention, in the donor DNA, in addition to the region to be replaced, the region outside the region to be replaced is also inverted so that the position where homologous recombination occurs is replaced. It can be limited to the area outside the target area. Further, according to the second substitution method, the substitution target region derived from the donor DNA can be introduced in the original orientation.

  In addition, the occurrence of the desired homologous recombination in the transformant can be confirmed by performing PCR on the replacement target region derived from the donor DNA.

  Furthermore, in the host DNA or donor DNA, a gene existing in a region further outside the region outside the replacement target region is previously deleted. Next, after LP transformation, by confirming the presence or absence of the deleted gene in the DNA of the transformant by PCR, it is confirmed that no homologous recombination has occurred in any place other than the region outside the replacement target region. Can be confirmed.

  On the other hand, in the second substitution method according to the present invention, similarly, the replacement target region of the host DNA and the region outside the replacement target region are inverted and the replacement target region of the donor DNA is inverted. Thus, by combining selection using a selection marker gene such as a drug resistance gene as an index, the position where homologous recombination occurs can be limited to a region outside the region to be replaced.

  According to the substitution method according to the present invention described above, a large-sized DNA fragment can be reliably and collectively introduced into host DNA. Further, in the replacement method according to the present invention, the replacement target region of the donor DNA can be introduced into the host DNA without causing homologous recombination within the replacement target region.

  In particular, in the second replacement method according to the present invention, the position where homologous recombination occurs can be limited to a region outside the replacement target region.

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

The polymerase chain reaction (PCR) for amplification of DNA fragments in the following examples uses GeneAmp PCR System (Applied Biosystems) and DNA amplification using Pyrobest DNA Polymerase (Takara Bio) and the 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.

  Furthermore, in the following examples, upstream / downstream of a gene is not a position from the replication start point, but upstream refers to 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.

In addition, the names of each gene and gene region 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] First substitution method according to the present invention-When a region to be replaced on the genome of a donor strain (donor DNA) is inverted.
(1) Construction of donor strains (E600NR, E250NR, E100NR and E50NR)
1-1. Construction of Inverted Mutation Cassette Plasmids pBNEC and pBEOC As shown in FIG. 4, plasmids pBNEC and pBEOC incorporating the inverted mutation cassette were constructed as follows.

Using the plasmid pUB110 (Plasmid 15, 93 (1986)) as a template and the primer set of rneof-SE (SEQ ID NO: 1) and repUr-Nm (SEQ ID NO: 2) shown in Table 1, the repU gene promoter (Nucleic A region (rep fragment) (0.4 kb) containing Acids Res. 17, 4410 (1989)) was prepared by PCR. In addition, using the plasmid pUB110 as a template and using the primer set of NmUf-rep (SEQ ID NO: 3) and Ner-XH (SEQ ID NO: 4) shown in Table 1, the front middle region (nE fragment) of the neomycin resistance gene (0.6 kb) was prepared by PCR. Next, the obtained rep fragment and nE fragment were mixed to form a template, and SOE (splicing by overlap extension) -PCR (PCR) using the above-described primers rneof-SE (SEQ ID NO: 1) and Ner-XH (SEQ ID NO: 4). Gene 77, 61 (1989)) was used to prepare an NE fragment (1.0 kb) in which an nE fragment was ligated downstream of the promoter region of the repU gene.

  Furthermore, using plasmid pC194 (J. Bacteriol. 150 (2) 815 (1982)) as a template and using the primer set of catf-XH (SEQ ID NO: 5) and catr-SL (SEQ ID NO: 6) shown in Table 1. A chloramphenicol resistance gene Cm fragment (0.9 kb) was prepared by PCR.

Next, the NE fragment was treated with Spe I and Xho I and the Cm fragment was treated with Xho I and Sal I, respectively, and then inserted into the Spe I- Sal I restriction enzyme site of pBluescript II SK (+) (Stratagene). The NE cassette plasmid pBNEC in which the NE fragment and the Cm fragment are adjacent on the plasmid was constructed.

  On the other hand, using plasmid pMutinT3 (Microbiology 144, 3079 (1998)) as a template and the primer set of emf2-SE (SEQ ID NO: 7) and emr2-XH (SEQ ID NO: 8) shown in Table 1, the erythromycin resistance gene Em A fragment (1.3 kb) was prepared by PCR. In addition, using the plasmid pUB110 as a template and using the primer set of EOF-XH (SEQ ID NO: 9) and neor-SL (SEQ ID NO: 10) shown in Table 1, the middle rear region EO fragment (0.8 kb) of the neomycin resistance gene Was prepared by PCR.

Next, the Em fragment was treated with Spe I and Xho I and the EO fragment was treated with Xho I and Sal I, respectively, and then inserted into the Spe I- Sal I restriction enzyme site of pBluescript II SK (+) (Stratagene). The EO cassette plasmid pBEOC in which the Em fragment and the EO fragment are adjacent on the plasmid was constructed.

1-2. Construction of a 168EOC reference strain for donor strain construction As shown in FIG. 5, a reference strain for constructing a donor strain was constructed as follows.
Using the genomic DNA extracted from Bacillus subtilis 168 strain as a template, using the primer set of yxnB FW (SEQ ID NO: 11) and yxnB / Em R (SEQ ID NO: 12) shown in Table 1, upstream of the yxnB gene in the genome The adjacent 1.0 kb fragment (A) was amplified by PCR. In addition, using the genomic DNA as a template, a primer set of yxnB / Ne F (SEQ ID NO: 13) and yxnB RV (SEQ ID NO: 14) shown in Table 1 was used to adjoin 1.0 kb downstream of the yxnB gene in the genome. Fragment (B) was amplified by PCR.

  On the other hand, a 2.1 kb EO cassette (C) was prepared by PCR using the EO cassette plasmid pBEOC as a template and the primer set of emf2 (SEQ ID NO: 15) and neor (SEQ ID NO: 16) shown in Table 1.

  Next, the obtained 1.0 kb fragment (A), 1.0 kb fragment (B), and EO cassette (C) 3 fragments were mixed and used as templates to prepare yxnB FW2 (SEQ ID NO: 17) and yxnB RV2 shown in Table 1. By performing SOE-PCR using the primer set of (SEQ ID NO: 18), a 4.1 kb PCR containing 3 fragments in the order of 1.0 kb fragment (A), EO cassette (C), and 1.0 kb fragment (B) A 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 the LB agar medium containing erythromycin (2 μg / mL) were isolated as transformants.

Genomic DNA was extracted transformants, YxnB gene by PCR is deleted, YxnB gene was confirmed that by replacing the EO cassette (C) by homologous recombination. As described above, the 168EOC strain in which the EO cassette (C) was inserted into the yxnB gene site of the genomic DNA in Bacillus subtilis was constructed.

1-3. Construction of Donor Strain E600NR As shown in FIG. 6, using genomic DNA extracted from Bacillus subtilis 168 as a template, yvfU FW (SEQ ID NO: 19) and yvfU / Cm3 R (SEQ ID NO: 20) shown in Table 1 were used. ), A 1.0 kb fragment (D) adjacent to the downstream of the yvfU gene in the genome was amplified by PCR. Further, using the genomic DNA as a template, a 1.0 kb fragment (E) adjacent to the upstream of the yvfU gene in the genome using a primer set of yvfU / Ne5 F (SEQ ID NO: 21) and yvfU RV (SEQ ID NO: 22). Amplified by PCR.

  On the other hand, a 1.9 kb NE cassette (F) was prepared by PCR using the NE cassette plasmid pBNEC as a template and the primer set of rneof (SEQ ID NO: 23) and catr (SEQ ID NO: 24) shown in Table 1.

  Next, 3 fragments of the obtained 1.0 kb fragment (D), 1.0 kb fragment (E) and NE cassette (F) were mixed and used as a template to prepare yvfU FW2 (SEQ ID NO: 25) and yvfU RV2 shown in Table 1. By performing SOE-PCR using the primer set of (SEQ ID NO: 26), a 3.9 kb PCR fragment containing 3 fragments in the order of 1.0 kb fragment (D), NE cassette (F) and 1.0 kb fragment (E) Got.

  Furthermore, the Bacillus subtilis 168EOC strain obtained in 1-2 above was transformed by the competent cell transformation method using the obtained PCR fragment. After transformation, colonies that grew on the LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.

Genomic DNA of the obtained chloramphenicol-resistant strain was extracted, deleted YvfU gene 168EOC strain by PCR, YvfU gene was confirmed to be substituted with NE cassette (F) by homologous recombination. This strain was named EOC600R.

  Next, the EOC600R strain was cultured with shaking in 1 mL of LB medium at 37 ° C. for 4 hours. After shaking culture, 100 μL of the obtained culture solution was smeared on an LB agar medium containing neomycin (20 μg / mL), and the grown colonies were separated.

  The genomic DNA of the resulting neomycin resistant strain was extracted, and the primer set of yvfU FW2 (SEQ ID NO: 25) and yxnB FW2 (SEQ ID NO: 17) shown in Table 1, yvfU RV2 (SEQ ID NO: 26) and yxnB RV2 (sequence) No. 18), a primer set of yvfU FW2 (SEQ ID NO: 25) and emf2 (SEQ ID NO: 15), and a primer set of rneof (SEQ ID NO: 23) and yxnB RV2 (SEQ ID NO: 18), respectively. As a result of PCR, DNA fragments of 4.6 kb, 3.3 kb, 3.6 kb and 2.3 kb could be amplified, respectively. These DNA fragments could not be amplified in the EOC600R strain before the inversion mutation.

From the above results, it was confirmed that the 600 kb genomic region (replacement target region) between the yvfU gene and the yxnB gene was inverted. The obtained inverted mutant was named E600NR and used as a donor strain in the genome large region bulk replacement experiment described later. The efficiency of the inversion mutation was evaluated by the ratio of the number of colonies appearing on the LB agar medium containing neomycin to the number of colonies appearing on the LB agar medium containing no drug. As a result, the inversion mutation efficiency in the construction of E600NR was 8.33 × 10 ⁻⁷ .

1-4. Construction of donor strains E250NR, E100NR and E50NR
In the same way as E600NR, construct inversion mutants E250NR (250kb genomic region inversion mutation), E100NR (100kb genomic region inversion mutation) and E50NR (50kb genomic region inversion mutation). Used as donor strain in large area bulk replacement experiments. In E250NR, the 250 kb genomic region between the speE gene and the yxnB gene is inverted. In E100NR, the 100 kb genomic region between the yxjH gene and the yxnB gene is inverted. In E50NR, the 50 kb genomic region between the hutM gene and the yxnB gene is inverted.

  The reverse mutants E250NR, E100NR and E50NR were constructed in the same manner as the construction of E600NR described in 1-3 above. In Table 2, the primers used for constructing each reverse mutant were shown corresponding to the primers used for constructing E600NR.

Further, the efficiency of the inversion mutation was evaluated by the ratio of the number of colonies appearing on the LB agar medium containing neomycin to the number of colonies appearing on the LB agar medium containing no drug. As a result, the inversion mutation efficiencies during the construction of E250NR, E100NR and E50NR were 1.60 × 10 ⁻⁶ , 1.75 × 10 ⁻⁶ and 1.99 × 10 ⁻⁶ , respectively.

(2) Construction of recipient strain (host DNA) The parent strain for recipient strain construction includes eight genes encoding extracellular proteases of Bacillus subtilis ( aprE , nprB , nprE , bpr , vpr , mpr , epr , wprA ) Was deleted from the protease gene 8-fold deletion. In the following, each protease gene was deleted in sequence, the protease gene 8-deficient strain (ΔP8) was constructed, and the amyE gene on the constructed ΔP8 genome was replaced with a spectinomycin resistance gene, and the recipient strain was The constructed process will be described.

2-1. Construction of an 8-fold protease deletion strain
(a) Construction of plasmid for deletion of epr gene As shown in FIG. 7, genomic DNA extracted from Bacillus subtilis 168 strain was used as a template, and eprfw1 (SEQ ID NO: 45) and eprUpr (SEQ ID NO: 46) shown in Table 1 A 0.6 kb fragment (G) adjacent to the upstream of the epr gene on the genome was prepared by PCR using the primer set. In addition, using the genomic DNA as a template and using the eprDNf (SEQ ID NO: 47) and eprrv-rep (SEQ ID NO: 48) primer sets shown in Table 1, a 0.5 kb fragment adjacent to the downstream of the epr gene on the genome ( H) was prepared by PCR.

On the other hand, the promoter region (Nucleic of the repU gene derived from plasmid pUB110 (Plasmid 15, 93 (1986)) upstream of the chloramphenicol resistance gene derived from plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) Acids Res. 17, 4410 (1989)) and a 1.2 kb fragment Cm cassette (I) was prepared.

Next, 3 fragments of the obtained 0.6 kb fragment (G), 0.5 kb fragment (H) and Cm cassette (I) were mixed to form a template, and primers eprfw2 (SEQ ID NO: 49) and Cmrv2 (SEQ ID NO: 49) in Table 1 were used. 50) was used to obtain a 2.2 kb DNA fragment in which three fragments were bound in the order of a 0.6 kb fragment (G), a 0.5 kb fragment (H), and a Cm cassette (I). The ends of this DNA fragment were blunted and 5′-phosphorylated, inserted into the Sma I restriction enzyme site of plasmid pUC118 (Methods Enzymol. 153, 3 (1987)), and the plasmid pUC118-CmrΔepr for epr gene deletion was It was constructed.

The 1.2 kb fragment Cm cassette (I) is a repU gene promoter region prepared by PCR using the repUfw (SEQ ID NO: 51) and repUr-Cm (SEQ ID NO: 52) primer sets shown in Table 1 and the plasmid pUB110 as a template. And a chloramphenicol resistance gene prepared by PCR using the primer set of CmUf-rep (SEQ ID NO: 53) and Cmrv1 (SEQ ID NO: 54) in Table 1 and the plasmid pC194 as a template. A 0.8 kb fragment (K) containing was used as a template and prepared by performing SOE-PCR using the primers repUfw (SEQ ID NO: 51) and Cmrv1 (SEQ ID NO: 54) shown in Table 1.

(b) Construction of an epr gene deletion strain using a deletion plasmid As shown in FIG. 7, it was constructed in the above (a) by a competent cell transformation method (J. Bacteriol. 93, 1925 (1967)). The plasmid pUC118-CmrΔepr for deletion of the epr gene was introduced into Bacillus subtilis 168 strain, and the transformed strain fused with the genomic DNA by homologous recombination between the corresponding upstream or downstream regions of the epr gene was cloned. Lambphenicol resistance was obtained as an index.

After inoculating the obtained transformant into LB medium and culturing at 37 ° C. for 2 hours, the competent induction operation is performed again, so that the region between the upstream region and the downstream region of the epr gene overlapping in the genome is obtained. Intragenomic homologous recombination was induced.

As shown in FIG. 7, when homologous recombination occurs in a region different from that at the time of plasmid introduction, the epr gene is deleted along with the loss of the plasmid-derived chloramphenicol resistance gene and pUC118 vector region. Become.

  Next, in order to increase the abundance ratio of strains that became chloramphenicol-sensitive, ampicillin concentration was performed as follows.

  The culture solution after induction of competent cells was inoculated into 1 mL of LB medium containing chloramphenicol having a final concentration of 5 ppm and sodium ampicillin having a final concentration of 100 ppm so that the turbidity (OD600) at 600 nm was 0.003. After culturing at 37 ° C. for 5 hours, 10 μL of 10,000 ppm ampicillin sodium aqueous solution was added to the mixed solution containing the culture solution, and further cultured for 3 hours. After completion of the culture, the cells were centrifuged and washed with a 2% aqueous sodium chloride solution, suspended in 1 mL of a 2% aqueous sodium chloride solution, and 100 μL of the suspension was smeared on an LB agar medium. The medium smeared with the suspension was incubated at 37 ° C. for about 15 hours. After incubation, strains that became chloramphenicol sensitive as the plasmid region dropped out were selected from the grown strains.

Genomic DNA of Screened strain as a template, by PCR performing using primers eprfw2 shown in Table 1 (SEQ ID NO: 49) and eprrv-rep (SEQ ID NO: 48), confirms the epr gene deletion, epr gene A deletion strain was obtained.

(c) Deletion of other protease genes For the epr gene deletion strain constructed in (b) above, the deletion of the wprA gene was performed in the same manner as the deletion of the epr gene as the next deletion. That is, in the same manner as in the above (a), to construct plasmid pUC118-CmrΔwprA for wprA gene deletion, the introduction into the genomic DNA of plasmids constructed by deletion of the wprA gene by genomic internal phase homologous recombination followed by epr A double deletion strain of the gene and the wprA gene was obtained. By repeating the same procedure, the mpr , nprB , bpr , nprE , vpr , and aprE genes were deleted in sequence, and a protease 8-deficient strain ΔP8 was finally constructed in which 8 types of protease genes were deleted. did.

Table 1 shows the primer sequences used for each gene deletion. In Table 3, the primers used for deleting each gene are shown in correspondence with the primers used for deleting the epr gene in (a) and (b) above.

2-2. Replacement of amyE gene with spectinomycin resistance gene Using a genomic DNA extracted from Bacillus subtilis 168 as a template, amyE FW (SEQ ID NO: 90) and amyE / Sp R (SEQ ID NO: 91) shown in Table 1 A 1.0 kb fragment (L) adjacent to the upstream of the amyE gene in the genome was amplified by PCR using the primer set. In addition, using the genomic DNA as a template, a primer set of amyE / Sp F (SEQ ID NO: 92) and amyE RV (SEQ ID NO: 93) shown in Table 1 was used, and the adjacent 1.0 kb downstream of the amyE gene in the genome. Fragment (M) was amplified by PCR.

  On the other hand, a plasmid pDG1727 (Gene, 167, 335, (1995)) DNA was used as a template and a 1.2 kb spectino was prepared using the spf (SEQ ID NO: 94) and spr (SEQ ID NO: 95) primer sets shown in Table 1. The mycin resistance gene region (N) was prepared by PCR.

  Next, 3 fragments of the obtained 1.0 kb fragment (L), 1.0 kb fragment (M) and spectinomycin resistance gene region (N) were mixed and used as a template to prepare amyE FW2 (SEQ ID NO: 96) shown in Table 1. ) And amyE RV2 (SEQ ID NO: 97) by performing SOE-PCR using a primer set consisting of a 1.0 kb fragment (L), a spectinomycin resistance gene region (N) and a 1.0 kb fragment (M). A 3.2 kb PCR fragment included in sequence was obtained.

  Furthermore, colonies grown on LB agar medium containing spectinomycin (100 μg / mL) after transformation of the protease 8-fold deletion strain ΔP8 using the obtained PCR fragment by the competent cell transformation method Was isolated as a transformant.

Genomic DNA was extracted transformants, amyE gene deletion by PCR, amyE gene by homologous recombination was confirmed that replaced the spectinomycin resistance gene. The recipient strain ΔP8SP was constructed as described above.

(3) Explanation of homologous region in genomic DNA between donor strain and recipient strain Deleted in the inversion region of each donor strain E600NR, E250NR, E100NR and E50NR and recipient strain ΔP8SP in genomic DNA A schematic diagram of the protease gene is shown in FIG. In FIG. 8, eight genes ( aprE , nprB , nprE , bpr , vpr , mpr , epr , wprA ) encoding the extracellular protease of Bacillus subtilis surrounded by a square are deleted in the recipient strain ΔP8SP. Yes.

As shown in FIG. 8, between the donor strain E600NR, E250NR, E100NR or E50NR constructed in (1) above and the recipient strain ΔP8SP constructed in (2) above, in the genome of the donor strain, YvfU gene - 600 kb between yxnB gene (E600NR), speE gene - 250 kb between yxnB gene (E250NR), yxjH gene - among yxnB gene 100kb (E100NR) or hutM gene - among yxnB gene The 50 kb (E50NR) region is reversed, while the outside of each region (the region outside the replacement target region) is a completely homologous region. That is, between the donor strain and the recipient strain, homologous recombination is caused in a region outside each region, so that regions opposite to each other can be surely replaced together. At this time, by performing selection using a selection marker gene inserted at the end of the region on the donor strain side, a strain in which the target large genomic region is efficiently replaced at once can be obtained.

  In the following sections (4) to (7), explanation will be made based on the donor strain E600NR.

(4) Preparation of Protoplast from Donor Strain Protoplasts from a donor strain were prepared as follows according to the method of Chang et al. (Mol. Gen. Genet. 168, 111 (1979)).

The donor strain E600NR was cultured with shaking at 37 ° C. in a LB medium until the degree of growth (OD ₆₀₀ ) reached about 1. After shaking culture, the cells collected from 1 mL of the culture solution by centrifugation were suspended in 0.5 mL of SMMP buffer containing 20 mg / mL lysozyme (Sigma) and incubated at 37 ° C. for 2 hours. After incubation, the protoplasts were collected by centrifugation and resuspended in 0.5 mL SMMP buffer. Further, the suspension was diluted 10-fold with SMMP buffer and used for transformation.

(5) LP transformation
LP transformation was performed according to the method of Akamatsu et al. This method is characterized by drastically reducing DNA damage by providing donor cell protoplasts as donor DNA to the competent cells of the recipient strain instead of the extracted DNA. is there. 0.1 mL of the protoplast solution of the donor strain E600NR prepared in (4) above was added to 1 mL of the culture medium of the recipient strain ΔP8SP in which competence was induced. Thereafter, normal transformation operations were performed. It is considered that the added protoplast is destroyed by osmotic shock, and the genomic DNA released into the culture solution is taken into the competent cell of the recipient strain ΔP8SP.

(6) Selection of transformants using drug resistance as an index Transformants were selected using drug resistance as an index. FIG. 9 schematically shows inversion mutation regions (substitution target regions) in each donor DNA and homologous recombination in transformants of each donor strain and recipient strain.

  First, transformants were selected using spectinomycin resistance as a drug marker of recipient strain ΔP8SP and neomycin resistance as a drug marker present at the end of the inverted mutation region of donor strain E600NR as indices. Next, chloramphenicol (Cm) resistance and erythromycin (Em) resistance at the other end of the inverted mutation region of the donor strain E600NR were confirmed using a replica method. The selected transformants (50) were all resistant to spectinomycin, neomycin, chloramphenicol and erythromycin. Thus, in the transformant of the recipient strain ΔP8SP when using the donor strain E600NR, substitution by homologous recombination was performed with 100% efficiency.

In addition, the transformation efficiency was evaluated by the appearance frequency of the transformant relative to the number of recipient strains used in the transformation ((number of transformants) ÷ (number of recipient strains)). As a result, the transformation efficiency of the recipient strain ΔP8SP when using the donor strain E600NR was 0.47 × 10 ⁻⁵ .

(7) PCR confirmation of homologous recombination in transformant Genomic DNA was extracted from the transformant confirmed to have drug resistance in (6) above, and PCR confirmation was performed. Primer set of yvfU FW2 (SEQ ID NO: 25) and yxnB FW2 (SEQ ID NO: 17) shown in Table 1, primer set of yvfU RV2 (SEQ ID NO: 26) and yxnB RV2 (SEQ ID NO: 18), yvfU FW2 (SEQ ID NO: 18) 25) and emf2 (SEQ ID NO: 15) and rneof (SEQ ID NO: 23) and yxnB RV2 (SEQ ID NO: 18) primer sets, respectively. As a result of PCR, 4.6 kb and 3.3 kb were obtained, respectively. Thus, DNA fragments of 3.6 kb and 2.3 kb could be amplified. These fragments could not be amplified in the recipient strain ΔP8SP.

Further, as shown in FIG. 8, YvfU gene - For epr gene and vpr genes located between yxnB gene, primer set of the Eprfw2 (SEQ ID NO: 49) and eprrv-rep (SEQ ID NO: 48) shown in Table 1, And vprfw2 (SEQ ID NO: 84) and vprrv-rep (SEQ ID NO: 83) were confirmed by PCR. As a result, it was confirmed that the epr gene and vpr gene were present in the genomic DNA of the transformant. It was. From the above, it was confirmed that the 600 kb genomic region between the yvfU gene and the yxnB gene was derived from the E600NR strain which is a donor strain.

Furthermore, regarding the deletion of the aprE gene, mpr gene, wprA gene, nprE gene, nprB gene and bpr gene which are the traits of the ΔP8SP strain which is the recipient strain, aprEfw2 (SEQ ID NO: 89) and aprErv-rep ( Primer set with SEQ ID NO: 88), primer set with mprfw2 (SEQ ID NO: 64) and mprrv-rep (SEQ ID NO: 63), primer set with wprAfw2 (SEQ ID NO: 59) and wprArv-rep (SEQ ID NO: 58), nprEfw2 (SEQ ID NO: 79) and nprErv-rep (SEQ ID NO: 78) primer set, nprBfw2 (SEQ ID NO: 69) and nprBrv-rep (SEQ ID NO: 68) primer set, and bprfw2 (SEQ ID NO: 74) and bprrv-rep ( Each of the genomic regions other than between the yvfU gene and the yxnB gene was confirmed to be the same as the ΔP8SP strain that is the recipient strain (see FIG. 8).

(8) Replacing the speE gene- yxnB gene (250 kb), yxjH gene- yxnB gene (100 kb) and hutM gene- yxnB gene (50 kb) regions In the same manner as in (4) to (7) above, the donor strain LP transformation of recipient strain ΔP8SP was performed using E250NR, E100NR or E50NR, and selection of transformants using drug resistance as an index and PCR confirmation of homologous recombination in the transformants were performed.

  Transformants selected for LP transformation of recipient strain ΔP8SP using donor strain E250NR, E100NR or E50NR (50 per transformant) were all spectinomycin, neomycin, chloramphenicol and erythromycin. It was resistant to it. Thus, in the transformant of the recipient strain ΔP8SP obtained using each donor strain E250NR, E100NR or E50NR, substitution by homologous recombination was performed with 100% efficiency.

In addition, the transformation efficiency was evaluated by the appearance frequency of the transformant relative to the number of recipient strains used in the transformation ((number of transformants) ÷ (number of recipient strains)). The transformation efficiency of the recipient strain ΔP8SP when using the donor strain E250NR was 0.84 × 10 ⁻⁵ . The transformation efficiency of the recipient strain ΔP8SP when using the donor strain E100NR was 1.85 × 10 ⁻⁵ . Moreover, the transformation efficiency of the recipient strain ΔP8SP when using the donor strain E50NR was 2.68 × 10 ⁻⁵ .

Furthermore, was confirmed by PCR for homologous recombination in the transformants, speE gene in the case of using E250NR strain as a donor strain - 250 kb between YxnB gene, YxjH gene in the case of using the E100NR strain - YxnB intergenic When the E50NR strain was used, 50 kb between the hutM gene and the yxnB gene was confirmed to be replaced in a state opposite to the corresponding region of each of the ΔP8SP strains as the recipient strain. In addition, the same primers as those used in (1) for confirming the inversion mutation were used for PCR for confirming the end of the region to be replaced in the genomic DNA of the transformant (see Table 2 for the correspondence of each primer). Table 4 below shows the primer sets and PCR product sizes used in PCR for confirmation of the replacement target region ends in transformants of the recipient strain ΔP8SP using each donor strain E250NR, E100NR or E50NR.

In addition, when E250NR strain was used, the presence of the epr gene and vpr gene in the transformant genomic DNA and the deletion of the aprE gene, mpr gene, wprA gene, nprE gene, nprB gene and bpr gene were confirmed. did. Furthermore, when E100NR strain or E50NR strain was used, it was confirmed that all eight types of protease genes were deleted from the genomic DNA of the transformant. In the transformants prepared using the respective donor strains, it was confirmed that the genomic region other than the replacement target region was the same as the ΔP8SP strain that is the recipient strain (see FIG. 8).

[Example 2] Second replacement method according to the present invention--reverse mutation of a region to be replaced on the genome of a donor strain (donor DNA) and a region outside the region to be replaced, and a recipient strain (host DNA) ) When the region to be replaced on the genome is inverted

(1) Construction of donor strain
1-1. Construction of ΔP81 strain and ΔP82 strain Using the genomic DNA extracted from Bacillus subtilis 168 strain as a template, primer sets of yxnB FW (SEQ ID NO: 11) and yxnB / Sp R (SEQ ID NO: 98) shown in Table 1 were used. Using, a 1.0 kb fragment (O) adjacent to the upstream of the yxnB gene in the genome was amplified by PCR. In addition, using the genomic DNA as a template, using the primer set of yxnB / Sp F (SEQ ID NO: 99) and yxnB RV (SEQ ID NO: 14), a 1.0 kb fragment (P) adjacent to the downstream of the yxnB gene in the genome Amplified by PCR.

  On the other hand, using the plasmid pDG1727 DNA as a template, using the spf (SEQ ID NO: 94) and spr (SEQ ID NO: 95) primer sets shown in Table 1, a 1.2 kb spectinomycin (Sp) resistance gene region (N) was obtained. Prepared by PCR.

  Next, 3 fragments of the obtained 1.0 kb fragment (O), 1.0 kb fragment (P) and spectinomycin (Sp) resistance gene region (N) were mixed and used as a template to produce yxnB FW2 ( SEQ ID NO: 17) and yxnB RV2 (SEQ ID NO: 18) were subjected to SOE-PCR using the primer set, and the three fragments were 1.0 kb fragment (O), spectinomycin (Sp) resistance gene region (N) and 1.0 kb fragment ( A 3.2 kb PCR fragment contained in the order of P) was obtained.

  Furthermore, the PCR fragment obtained by the competent cell transformation method was used to transform the protease 8-deficient strain ΔP8 constructed in Example 1 (2) above, and spectinomycin (100 μg / mL) was transformed. The colonies that grew on the LB agar medium containing) were isolated as transformants.

Genomic DNA was extracted transformants, YxnB gene deletion by PCR, yxnB gene by homologous recombination was confirmed that replaced the spectinomycin resistance gene. As described above, was constructed ΔP81 strain inserting the spectinomycin resistance gene yxnB gene site.

Further, a ΔP82 strain was constructed by inserting a tetracycline resistance gene into the yxeA gene site in the same manner as the insertion of the spectinomycin resistance gene into the ΔP81 strain. In addition, pHY300PLK (Yakult Co., Ltd.) was used as a template for the preparation of the tetracycline resistance gene PCR fragment. Table 5 shows the primers used when the tetracycline resistance gene was inserted in correspondence with the primers used when the spectinomycin resistance gene was inserted. Furthermore, Table 5 shows the size of the PCR product obtained by PCR performed when the tetracycline resistance gene was inserted.

1-2. Construction of ΔP83 strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, using the primer set of yxnA FW (SEQ ID NO: 108) and yxnA / Em R (SEQ ID NO: 109) shown in Table 1, A 1.0 kb fragment (Q) adjacent to the upstream of the yxnA gene in the genome was amplified by PCR. Further, using the genomic DNA as a template, a primer set of yxnA / Ne F (SEQ ID NO: 110) and yxnA RV (SEQ ID NO: 111) shown in Table 1 was used, and 1.0 kb adjacent to the downstream of the yxnA gene in the genome. Fragment (R) was amplified by PCR.

  On the other hand, using the EO cassette plasmid pBEOC prepared in Example 1 (1) as a template, and using the primer set of emf2 (SEQ ID NO: 15) and neor (SEQ ID NO: 16) shown in Table 1, a 2.1 kb EO cassette (C) was prepared by PCR.

  Next, the obtained 1.0 kb fragment (Q), 1.0 kb fragment (R), and EO cassette (C) 3 fragments were mixed and used as templates to prepare yxnA FW2 (SEQ ID NO: 112) and yxnA RV2 shown in Table 1. By SOE-PCR using the primer set of (SEQ ID NO: 113), a 4.1 kb PCR fragment containing 3 fragments in the order of 1.0 kb fragment (Q), EO cassette (C) and 1.0 kb fragment (R) was obtained. .

  Furthermore, the ΔP82 strain was transformed by the competent cell transformation method using the obtained PCR fragment, and colonies that grew on the LB agar medium containing erythromycin (2 μg / mL) were isolated as transformants. .

Genomic DNA was extracted transformants, YxnA gene deletion by PCR, yxnA gene was confirmed that by replacing the EO cassette by homologous recombination. As described above, a ΔP83 strain in which an EO cassette was inserted into the yxnA gene site of Bacillus subtilis was constructed.

1-3. Construction of donor strains RSD1, RSD2, and RSD3 Primers of aldY FW (SEQ ID NO: 114) and aldY / Cm3 R (SEQ ID NO: 115) shown in Table 1 using genomic DNA extracted from Bacillus subtilis 168 as a template Using the set, a 1.0 kb fragment (R) adjacent to the upstream of the aldY gene in the genome was amplified by PCR. In addition, using the genomic DNA as a template, a primer set of aldY / Ne5 F (SEQ ID NO: 116) and aldY RV (SEQ ID NO: 117) shown in Table 1 was used, and the adjacent 1.0 kb downstream of the aldY gene in the genome. Fragment (S) was amplified by PCR.

  On the other hand, using the NE cassette plasmid pBEOC prepared in Example 1 (1) as a template, using the primer set of rneof (SEQ ID NO: 23) and catr (SEQ ID NO: 24) shown in Table 1, a 1.9 kb NE cassette (F) was prepared by PCR.

  Next, the obtained 1.0 kb fragment (R), 1.0 kb fragment (S), and NE cassette (F) 3 fragments were mixed and used as templates to obtain aldY FW2 (SEQ ID NO: 118) and aldY RV2 shown in Table 1. By SOE-PCR using the primer set of (SEQ ID NO: 119), a 3.9 kb PCR fragment containing 3 fragments in the order of 1.0 kb fragment (R), NE cassette (F) and 1.0 kb fragment (S) was obtained. .

  Further, by using the obtained PCR fragment by the competent cell transformation method, the Bacillus subtilis ΔP83 strain constructed in 1-2 above was transformed, and an LB agar medium containing chloramphenicol (10 μg / mL) The colonies grown above were isolated as transformants.

Genomic DNA was extracted transformants, Aldy gene deletion by PCR, Aldy gene was confirmed that by replacing the NE cassette by homologous recombination. This strain was designated as ΔP84 strain.

  Next, ΔP84 strain was cultured with shaking in 1 mL of LB medium at 37 ° C. for 4 hours. After shaking culture, 100 μL of the obtained culture solution was inoculated on an LB agar medium containing neomycin (20 μg / mL), and the grown colonies were isolated as inverted mutants.

  Genomic DNA of the obtained inverted mutant was extracted, and a primer set of aldY FW2 (SEQ ID NO: 118) and yxnA FW2 (SEQ ID NO: 112) shown in Table 1, aldY RV2 (SEQ ID NO: 119) and yxnA RV2 ( A primer set with SEQ ID NO: 113), a primer set with aldY FW2 (SEQ ID NO: 118) and emf2 (SEQ ID NO: 15), and a primer set with rneof (SEQ ID NO: 23) and yxnA RV2 (SEQ ID NO: 113), respectively. PCR was performed. As a result, amplification was confirmed for DNA fragments of 4.6 kb, 3.3 kb, 3.6 kb and 2.3 kb, respectively. These fragments were not amplified in the ΔP84 strain before the inversion mutation occurred.

From the above results, it was confirmed that the 120 kb genomic region between the aldY gene and the yxnA gene (the region to be replaced and the region outside the region to be replaced) was inverted. The obtained inverted mutant was named RSD1 strain and used as a donor strain in the following experiments. The efficiency of the inversion mutation was evaluated by the ratio of the number of colonies appearing on the LB agar medium containing neomycin to the number of colonies appearing on the LB agar medium containing no drug. As a result, the inversion mutation efficiency in the construction of the RSD1 strain was 6.77 × 10 ⁻⁷ .

Furthermore, as with the construction of the RSD1 strains, albC gene - YxnA donor strain 270kb region is inverted between genes RSD2, and yvbT gene - region of 620kb between YxnA gene was constructed inverted donor strain RSD3. Table 6 shows the primers used when the donor strains RSD2 and RSD3 were constructed in correspondence with the primers used when the RSD1 strain was constructed.

The efficiency of the inversion mutation was evaluated by the ratio of the number of colonies appearing on the LB agar medium containing neomycin to the number of colonies appearing on the LB agar medium containing no drug. As a result, the inversion mutation efficiencies during the construction of donor strains RSD2 and RSD3 were 4.7 × 10 ⁻⁷ and 2.36 × 10 ⁻⁷ , respectively.

  In the following sections (2) to (7), explanation will be made based on the donor strain RSD3 and the recipient strain E600NR.

(2) Construction of recipient strain In this example, experiments were performed using the donor strain E600NR constructed in Example 1 (1) as the recipient strain.

(3) Preparation of protoplast from donor strain Protoplast was prepared using donor strain RSD3 in the same manner as in Example 1 (4).

(4) Explanation of homologous region in genomic DNA of donor strain and recipient strain FIG. 10 shows an inversion mutation region in each donor DNA, an inversion mutation region in each host DNA, and each donor strain and recipient strain. The homologous recombination in a transformant is shown schematically. In FIG. 10, panel (a) shows the inverted mutation regions in the genomic DNA of donor strains RSD1, RSD2 and RSD3. Panel (b) shows homologous recombination in transformants of donor and recipient strains. Panel (c) shows the inverted mutation regions in the genomic DNA of recipient strains E100NR, E250NR and E600NR. Furthermore, the numerical position shown in panel (a) indicates the gene site at the numerical position in the donor DNA shown in panel (b). The numerical position shown in panel (c) indicates the gene site at the numerical position in the host DNA shown in panel (b).

  Furthermore, in FIG. 11, the genomic structure near the region to be replaced specifically with donor strain RSD3 and recipient strain E600NR is schematically represented by a straight line.

As shown in FIGS. 10 and 11, in the recipient strain E600NR, the region between the yvfU gene and the yxnB gene is inverted. On the other hand, the donor strain RSD3, yvfU gene - YvbT gene outside each 10kb away between yxnB gene - region between yxnA genes are inversions mutation. Between RSD3 strain and E600NR strain, YvfU gene - if YxnB intergenic region (replacement area) are in a positional relationship opposite to each other, YxnB gene - 10 kb region and yvbT gene between yxnA gene - YvfU intergenic The 10 kb region (region outside the region to be replaced) is a region homologous to each other, and all other regions are reversed. That is, the yvfU gene by double crossing (formation of a pair of interchain exchange structures and double crossover homologous recombination) limited between the two 10 kb regions by combining selection with drug resistance described below as an index -The yxnB intergenic region can be replaced.

(5) LP transformation In the same manner as in Example 1 (5), LP transformation was performed using the RSD3 strain as the donor strain and the E600NR strain as the recipient strain.

(6) Selection of transformants using drug resistance as an index Transformants were selected using drug resistance as an index. First, a spectinomycin resistant strain (192 strains) was selected using a spectinomycin resistant gene inserted into the yxnB gene site of RSD3 strain which is a donor strain. Next, using the replica method, all marker genes (chloramphenicol, erythromycin and neomycin resistance genes) on both sides of the inversion target region of both the RSD3 strain and the recipient strain E600NR are deleted. Then, 59 strains that became drug-sensitive were selected.

  As a result of examining tetracycline resistance for the selected 59 transformants, all 59 were resistant to tetracycline. Thus, after selecting a spectinomycin resistant strain, in a strain selected using the sensitivity of chloramphenicol, erythromycin or neomycin as an index, between the limited regions of the donor strain RSD3 genome and the recipient strain E600NR genome. The region to be replaced was 100% completely replaced by homologous recombination.

Moreover, the transformation efficiency was evaluated using the number of spectinomycin-resistant and erythromycin-sensitive transformants with respect to the viable count of the recipient strain used for transformation as an index. The transformation efficiency when using donor strain RSD3 and recipient strain E600NR was 1.22 × 10 ⁻⁵ .

(7) PCR confirmation for homologous recombination in transformant Genomic DNA is extracted from the transformant selected in (6) above, and both ends and replacement of two 10 kb homologous recombination regions (regions outside the region to be replaced) In order to confirm the genomic structure at both ends of the tetracycline resistance gene in the target region, the primer set of yvfU FW2 (SEQ ID NO: 25) and yvfU RV2 (SEQ ID NO: 26) shown in Table 1, yxeA FW2 (SEQ ID NO: 106) and tetr Primer set with (SEQ ID NO: 105), primer set with tetf (SEQ ID NO: 104) and yxeA RV2 (SEQ ID NO: 107), primer set with yxnB FW2 (SEQ ID NO: 17) and spr (SEQ ID NO: 95), spf ( SEQ ID NO: 94) and yxnB RV2 (SEQ ID NO: 18) primer set, yvbT FW2 (SEQ ID NO: 130) and yvbT RV2 (SEQ ID NO: 131) primer set, and yxnA FW2 (SEQ ID NO: 112) and yxnA RV2 (sequence) Primer with number 113) PCR was performed using each of the set. As a result of PCR, amplification of 2.6 kb, 2.6 kb, 2.6 kb, 2.2 kb, 2.2 kb, 3.0 kb and 3.0 kb DNA fragments was confirmed as expected by using each of the above primer sets.

FIG. 12 shows the genomic structure of donor strain RSD3 and the inverted mutation region of recipient strain E600NR. In FIG. 12, the region between the yvbT gene and the yxnA gene shown at the top shows the genomic structure of donor strain RSD3. The lower part shows the inversion mutation region of the recipient strain E600NR.

Regarding the epr gene and vpr gene located between the yvfU gene- yxnB gene shown in FIG. 12 in the genomic DNA of the transformant, the eprfw2 (SEQ ID NO: 49) and eprrv-rep (SEQ ID NO: 48) shown in Table 1 As a result of PCR using the primer set and the primer set of vprfw2 (SEQ ID NO: 84) and vprrv-rep (SEQ ID NO: 83), it was confirmed that both the epr gene and the vpr gene were deleted. It was done. Therefore, it was confirmed that the 600 kb genomic region between the yvfU gene and the yxnB gene was derived from the RSD3 strain which is a donor strain.

Furthermore, as shown in FIG. 12, the presence of the aprE gene, mpr gene, wprA gene, nprE gene, nprB gene, and bpr gene as traits of the recipient strain E600NR strain in the genomic DNA of the transformant, Primer set of aprEfw2 (SEQ ID NO: 89) and aprErv-rep (SEQ ID NO: 88), primer set of mprfw2 (SEQ ID NO: 64) and mprrv-rep (SEQ ID NO: 63) shown in Table 1, wprAfw2 (SEQ ID NO: 59) ) And wprArv-rep (SEQ ID NO: 58), nprEfw2 (SEQ ID NO: 79) and nprErv-rep (SEQ ID NO: 78) primer set, nprBfw2 (SEQ ID NO: 69) and nprBrv-rep (SEQ ID NO: 68) And a primer set of bprfw2 (SEQ ID NO: 74) and bprrv-rep (SEQ ID NO: 73), respectively. As a result, in the genomic DNA of the transformant, it was confirmed that the genomic region other than the region between the yvfU gene and the yxnB gene was the same as that of the recipient strain E600NR.

(8) Replacing the speE gene- yxnB gene (250 kb) and yxjH gene- yxnB gene (100 kb) regions LP of the donor strain RSD2 and the recipient strain E250NR in the same manner as in (2) to (7) above For the transformation and LP transformation of the donor strain RSD1 and the recipient strain E100NR, selection of transformants using drug resistance as an index and PCR confirmation of homologous recombination in the transformants were performed.

For transformation using donor strain RSD2 and recipient strain E250NR, select a spectinomycin resistant strain (192 strains) using the spectinomycin resistant gene inserted in the yxnB gene site of donor strain RSD2 did. Next, using the replica method, all of the marker genes (chloramphenicol, erythromycin and neomycin resistance genes) on both sides of the inversion target region of both the RSD2 strain and the recipient strain E250NR are deleted. Then, 110 transformants that became drug-sensitive were selected. As a result of examining tetracycline resistance for the selected 110 transformants, all 110 strains were tetracycline resistant. Thus, after selecting a spectinomycin resistant strain, in a strain selected using chloramphenicol, erythromycin or neomycin sensitivity as an index, between the limited regions of the donor strain RSD2 genome and the recipient strain E250NR genome. The region to be replaced was 100% completely replaced by homologous recombination.

In addition, the transformation efficiency when using donor strain RSD2 and recipient strain E250NR was evaluated using as an index the number of transformants resistant to spectinomycin and erythromycin sensitive to the number of recipient strains used for transformation. . The transformation efficiency when using donor strain RSD2 and recipient strain E250NR was 2.97 × 10 ⁻⁵ .

On the other hand, in the transformation using donor strain RSD1 and recipient strain E100NR, a spectinomycin resistant strain (178 strains) using the spectinomycin resistant gene inserted in the yxnB gene site of donor strain RSD1 strain Selected. Next, using the replica method, all the marker genes (chloramphenicol, erythromycin and neomycin resistance genes) on both sides of the inverted mutation regions of both the RSD1 strain and the recipient strain E100NR are deleted. Then, the transformant 106 strain that became drug-sensitive was selected. As a result of examining tetracycline resistance for the selected 106 transformants, all 106 were resistant to tetracycline. Thus, after selecting a spectinomycin-resistant strain, in a strain selected using the sensitivity of chloramphenicol, erythromycin or neomycin as an index, between the limited regions of the donor strain RSD1 genome and the recipient strain E100NR genome. The region to be replaced was 100% completely replaced by homologous recombination.

In addition, the transformation efficiency when using donor strain RSD1 and recipient strain E100NR was evaluated using the number of spectinomycin-resistant and erythromycin-sensitive transformants relative to the number of recipient strains used for transformation as an index. . The transformation efficiency when using donor strain RSD1 and recipient strain E100NR was 2.36 × 10 ⁻⁵ .

As shown in FIG. 10, when the RSD2 strain is used as the donor strain and the E250NR strain is used as the recipient strain in the same manner as (2) to (7) above, the 10 kb region between the yxnB gene and the yxnA gene and albC gene - speE intergenic homologous recombination results in a limited area in the 10kb region of, speE gene - was confirmed that replacement of 250kb region between yxnB gene occurs. Furthermore, regarding the protease gene in the genomic DNA of the obtained transformant, the epr gene and vpr gene are deleted, and the aprE gene, mpr gene, wprA gene, nprE gene, nprB gene and bpr gene are present. Confirmed to do.

Furthermore, as shown in FIG. 10, RSD 1 strain as a donor strain, in the case of using the E100NR strain as a recipient strain, YxnB gene - is limited to 10kb region between yxjH gene - 10kb region and aldY gene between yxnA gene result of homologous recombination regions, YxjH gene - was confirmed that replacement of 100kb region between yxnB gene occurs. Further, it was confirmed that all eight types of protease genes in the genomic DNA of the obtained transformant were present (see FIG. 12).

In addition, in the genomic DNA of the transformant (transformant of donor strain RSD2 and recipient strain E250NR, or transformant of donor strain RSD1 and recipient strain E100NR), both ends of two 10 kb homologous recombination regions And the primers used in the PCR for confirming the genomic structure at both ends of the tetracycline resistance gene in the replacement target region, the transformant (transformant of donor strain RSD3 and recipient strain E600NR) in (7) above. The genomic DNA is shown in Table 7 below in correspondence with the primers used for confirmation of the region replacement between the yvfU gene and the yxnB gene. In addition, the same primers as used in (7) above were used for PCR performed to confirm the presence or absence of the protease gene.

FIG. 1 shows the NEO inversion mutation system. FIG. 2 schematically shows LP transformation of a donor strain and a recipient strain constructed by the NEO inversion mutation system in the first replacement method according to the present invention. FIG. 3 schematically shows LP transformation between a donor strain and a recipient strain constructed by the NEO inversion mutation system in the second replacement method according to the present invention. FIG. 4 shows the construction of plasmids pBNEC and pBEOC incorporating the cassette for inversion mutation. FIG. 5 shows the construction of the reference strain 168EOC for donor strain construction in Example 1. FIG. 6 shows the construction of the EOC600R strain. FIG. 7 shows the construction of an epr gene deletion strain. FIG. 8 shows the inversion mutation regions of the donor strains E600NR, E250NR, E100NR and E50NR in Example 1 and the protease gene deleted in the recipient strain ΔP8SP. FIG. 9 shows the inversion mutation region in each donor DNA in Example 1, and homologous recombination in transformants of each donor strain and recipient strain. FIG. 10 shows the inversion mutation region in each donor DNA in Example 2, the inversion mutation region in each host DNA, and homologous recombination in transformants of each donor strain and recipient strain. FIG. 11 shows the genomic structure near the region to be replaced in donor strain RSD3 and recipient strain E600NR in Example 2. FIG. 12 shows the genomic structure of donor strain RSD3 in Example 2 and the inverted mutation region of recipient strain E600NR.

  SEQ ID NOs: 1-131 are primers.

Claims (10)

In the replacement of the replacement target region of the donor DNA comprising the replacement target region consisting of a base sequence introduced with one to several mutations into the base sequence of the host DNA replacement target region, and the host DNA replacement target region,
A first step of inversion mutation of one of the replacement target regions of donor DNA and host DNA;
A second step of recombination between the donor DNA and the host DNA,
In the second step, the host DNA replacement method is characterized in that a host DNA replacement target region is replaced with a donor DNA replacement target region.   The method further comprises a step of selecting, after the second step, a transformant having a region to be replaced with the donor DNA using the selection marker gene present in the donor DNA and / or the host DNA as an index. The method for replacing a host DNA according to 1. A DNA of the donor DNA is protoplasts, and characterized in that the host is competent cells, replacement method of host DNA according to claim 1 or 2, wherein.   The method for replacing a host DNA according to any one of claims 1 to 3, wherein the host is Bacillus subtilis.   The method for replacing a host DNA according to any one of claims 1 to 4, wherein the region to be replaced of the donor DNA has a DNA size of 20 kb or more. In the replacement of the replacement target region of the donor DNA comprising the replacement target region consisting of a base sequence introduced with one to several mutations into the base sequence of the host DNA replacement target region, and the host DNA replacement target region,
A first step in which one of the replacement target regions of the donor DNA and the host DNA is inverted, and the other replacement target region of the donor DNA and the host DNA and a region outside the replacement target region are inverted. ,
A second step of recombination between the donor DNA and the host DNA,
In the second step, the host DNA replacement method is characterized in that a host DNA replacement target region is replaced with a donor DNA replacement target region.   The method further comprises a step of selecting, after the second step, a transformant having a region to be replaced with the donor DNA using the selection marker gene present in the donor DNA and / or the host DNA as an index. 7. The method for replacing a host DNA according to 6. The donor DNA is DNA in protoplasts, and characterized in that the host is competent cells, replacement method of host DNA according to claim 6 or 7, wherein.   The method for replacing a host DNA according to any one of claims 6 to 8, wherein the host is Bacillus subtilis.   The method for replacing a host DNA according to any one of claims 6 to 9, wherein the replacement target region of the donor DNA has a DNA size of 20 kb or more.

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