JP5495469B2 - Method for batch replacement of host DNA in which the region to be replaced has been deleted in advance - Google Patents

Description

  The present invention relates to a host DNA replacement method in which, for example, a Bacillus subtilis genome or the like is used as a host DNA, and a predetermined region of the host DNA is replaced with a corresponding region of a donor DNA.

  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, protoplast transformation, electroporation (electroporation), Agrobacterium, particle gun, calcium phosphate, and lipofection. Can be converted.

  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.

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

  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 problems, the donor DNA is recombined between the donor DNA and the host DNA after part or all of the replacement target region of the host DNA is deleted. It was found that the replacement target region was accurately introduced into the host DNA, and the present invention was completed.

The present invention includes the following.
(1) It includes a first step of deleting part or all of the replacement target region of the host DNA, and a second step of recombination between the donor DNA and the host DNA. In the second step, the donor A method for replacing a host DNA, wherein a region to be replaced is introduced into the host 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 host DNA replacement method according to any one of (1) to (4), wherein the donor DNA replacement target region has a DNA size of 20 kb or more.

(6) The host DNA replacement target region contains a gene essential for growth, and the first step is performed by the following method 1) or 2): (1) to (5) The method for replacing a host DNA according to any one of (1).
1) A method of substituting a replacement target region of host DNA with a DNA fragment containing at least the gene essential for growth.
2) A method of deleting a replacement target region other than the gene essential for the growth.

  (7) A transformant in which the region to be replaced is replaced by the host DNA replacement method according to any one of (1) to (6) 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, that is, collectively. 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.
The host DNA replacement method according to the present invention is a method of replacing a replacement target region in host DNA with a replacement target region in donor DNA. In this method, first, a part or all of the replacement target region of the host DNA is deleted. Next, the host DNA from which part or all of the replacement target region is deleted and the donor DNA having the replacement target region are allowed to coexist under conditions that allow homologous recombination to occur. At this time, the host DNA has substantially or no region corresponding to the replacement region of the donor DNA, but there is a region corresponding to the region outside the replacement region of the donor DNA. . For this reason, the host DNA and the donor DNA form a pair of interchain exchange structures in the region outside the replacement target region. By forming this pair of interstrand exchange structures, double crossover homologous recombination between the host DNA and the donor DNA occurs, and the replacement target region of the donor DNA can be introduced into the host DNA. it can.

  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. The mutation here is, for example, one in which one to several bases are replaced with another base, one in which one to several bases are deleted, one in which one to several bases are added, Or the thing from which the continuous base sequences, such as 1 to several genes, were deleted is shown.

Further, in the host DNA replacement method according to the present invention, when part of the host DNA replacement target region is deleted, part of the host DNA replacement target region is the growth of the host in the host DNA replacement target region. It means a region excluding genes essential for ligation (hereinafter referred to as “growth essential genes”) and genes desired to remain in the host DNA. The growth essential gene is not particularly limited as long as it is a gene essential for the growth of the host. For example, in Bacillus subtilis, the growth shown in Proc. Natl. Acad. Sci. USA, 100, 4679 (2003) is used. 271 essential genes, more specifically genes involved in DNA replication such as dnaA, genes involved in transcription such as rpoB and sigA, genes involved in translation such as fus and tufA , and proteins such as prsA Examples include genes involved in secretion. Moreover, the gene which wants to remain | survive in host DNA is not limited at all, and means that a gene which is not essential for growth is included.

  Further, the region outside the replacement target region means a region continuing to the 3 ′ end side and a region continuing to the 5 ′ end side in the replacement target region with respect to one strand, and includes 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. Therefore, it is preferable that the region outside the region to be replaced in the host DNA and the region outside the region to be replaced in the donor DNA have high homology to such an extent that an interchain exchange structure can be formed.

  Specifically, the homology between the region outside the region to be replaced in the host DNA and the region outside the region to be replaced in the donor DNA is determined by, for example, the Lipman-Pearson method (Science, 227, 1435, (1985)). Calculated. Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the unit size to compare (ktup) can be calculated as 2 based on the analysis. . According to the result of the calculation, the region outside the replacement target region in the host DNA and the region outside the replacement target region in the donor DNA may have 60% or more homology, and 80% or more. 90% or more is more preferable, 95% or more is particularly preferable, and 99% or more homology is most preferable.

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 Examples include YAC (Science 236, 806 (1987)), plasmids, shuttle vectors, helper plasmids, phage DNAs, viral vectors, DNA fragments cloned into BACs and YACs, parts of genomic DNA, and the like. 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 host DNA 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, with the replacement method according to the present invention, it is possible to accurately replace even a large size DNA, which has conventionally been difficult to accurately replace. 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. According to the conventional LP transformation method, as shown in the following comparative example, when the size of the replacement target region increases, the regions at both ends of the replacement target region of the donor strain are sandwiched between the recipient strains. The ratio (simultaneous conversion rate) at which the cultivated region is simultaneously retained in the recipient strain decreases. On the other hand, in the replacement method according to the present invention, since the simultaneous conversion rate is 100% regardless of the size of the replacement target region, the improvement effect increases as the size of the replacement target region increases. Therefore, in the present invention, the size of the replacement target region of the host DNA and donor DNA is preferably 20 kb or more, more preferably 30 kb or more, particularly preferably 60 kb or more, and most preferably 100 kb or more. For example, the largest continuous region that does not contain a growth essential gene in the Bacillus subtilis genome is about 200 kb between the dltA gene and the yycH gene (Proc. Natl. Acad. Sci. USA, 100, 4679 ( 2003)). In consideration of not deleting a growth essential gene from this viewpoint, the size of the replacement target region of the host DNA and donor DNA is preferably 200 kb or less, more preferably 180 kb or less, particularly preferably 160 kb or less, and most preferably 140 kb or less. . Furthermore, 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 200 kb or less, preferably 140 kb or less, more preferably 100 kb or less, and particularly preferably 20 kb or less.

  In the method for replacing a host DNA according to the present invention, since the host DNA does not have a region to be replaced under conditions in which homologous recombination occurs, even if the donor DNA and the host DNA are close to each other, No interchain exchange structure is formed inside. Therefore, according to the host DNA replacement method of the present invention, homologous recombination occurs only in the region outside the replacement target region, and the replacement target region of the donor DNA can be reliably introduced into the host DNA. In addition, according to the host DNA replacement method of the present invention, even when the replacement target region has a length of 20 kbp or more, particularly 140 kbp or more, homologous recombination does not occur in the replacement target region, and it is ensured collectively. In addition, the replacement target region of the donor DNA can be introduced into the host DNA.

  Furthermore, in the host DNA replacement method according to the present invention, a transformant having a donor DNA replacement target region can be selected using the donor DNA and / or the selection marker gene present in the host DNA as an index. By using the selectable marker gene, the transformant generated by homologous recombination at an undesired site such as between homologous sites other than the region outside the region to be replaced is excluded, and the region to be replaced in the donor DNA is excluded. The transformant which has can be selected reliably. 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 or at the end of the donor DNA replacement target region, the host DNA (transformant) into which the donor DNA replacement target region has been introduced is selected using the drug resistance as an index. be able to. On the other hand, in the host DNA, the drug resistance gene is introduced into the site of the replacement target region simultaneously with the deletion of the replacement target region or after the replacement target region is deleted. Next, according to the host DNA replacement method of the present invention, homologous recombination occurs only in the region outside the replacement target region, and the drug resistance gene present in the host DNA is replaced with the replacement target region of the donor DNA. It will be. Therefore, host DNA into which a donor DNA replacement target region has been introduced 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 donor DNA is located at the site of the region to be replaced in the host DNA. The introduced transformant can be reliably selected. 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.

  As an example, FIG. 1 shows a method for introducing a drug resistance gene into Bacillus subtilis donor DNA or host DNA. In FIG. 1, it is assumed that gene A, gene B, and gene C exist in succession on the donor DNA or host DNA of Bacillus subtilis. On the other hand, a drug resistance gene is present in the plasmid. In the donor DNA or host DNA, the direction from the 5 'side to the 3' side is the direction from the gene A to the gene C with reference to one strand.

  First, using the donor DNA or the host DNA as a template, the primer 1 designed based on the 5 ′ end portion of gene A, and the 3 ′ end portion of gene A and the 5 ′ end portion of the drug resistance gene were designed. A PCR product A having the 5 ′ end portion of gene A and drug resistance gene is prepared by PCR using a primer set with primer 2. Similarly, using the donor DNA or host DNA as a template, primer 5 designed based on the 5 ′ end portion of gene C and the 3 ′ end portion of the drug resistance gene, and design based on the 3 ′ end portion of gene C The PCR product C having the 3 ′ end portion of the gene C and drug resistance gene is prepared by PCR using the primer set with the primer 6. On the other hand, a PCR product B of the drug resistance gene is prepared by PCR using a primer set (primers 3 and 4) designed based on the drug resistance gene using a plasmid containing the drug resistance gene as a template. Next, the obtained PCR products A, B and C were mixed and used as a template, and SOE (splicing by overlap extension) -PCR (Gene, 77, 61, (1989)) using a primer set (primers 1 and 6) I do. As a result of SOE-PCR, PCR product D in which the above three PCR products are included in the order of PCR products A, B and C is obtained.

  Furthermore, by performing transformation of Bacillus subtilis using the obtained PCR product D (for example, competent cell transformation method (J. Bacterial. 93, 1925 (1967))), PCR product D and donor DNA are obtained. Alternatively, double crossover homologous recombination occurs in the gene A region and the gene C region with the host DNA (in FIG. 1, the mode of homologous recombination is indicated by a solid line). As a result, the gene B present on the donor DNA or host DNA is deleted, and the drug resistance gene present in the PCR product D can be introduced into the gene B region.

  On the other hand, examples of a method for deleting all of the host DNA replacement target regions that can be used in the host DNA replacement method according to the present invention include, for example, the aforementioned Bacillus subtilis donor DNA or host DNA resistance gene. The method similar to the introduction method of, etc. are mentioned.

  In the following, as an example of a method for deleting the entire region to be replaced in the host DNA, a method similar to the method for introducing the drug resistance gene into the donor DNA or host DNA of Bacillus subtilis described above will be described with reference to FIG. . In FIG. 2, areas outside the replacement target area are denoted as “adjacent area (L)” and “adjacent area (R)”. On the other hand, a drug resistance gene is present in the plasmid. In the host DNA, the direction from the 5 'side to the 3' side is the direction from the adjacent region (L) to the adjacent region (R) with respect to one strand.

  Using the host DNA as a template, designed based on the primer 1 designed based on the 5 ′ end portion of the adjacent region (L), the 3 ′ end portion of the adjacent region (L), and the 5 ′ end portion of the drug resistance gene A PCR product having a flanking region (L) and a 5 ′ end portion of a drug resistance gene is prepared by PCR using a primer set with primer 2. Similarly, using host DNA as a template, primer 5 designed based on the 3 ′ end part of the drug resistance gene and the 5 ′ end part of the adjacent region (R), and the 3 ′ end part of the adjacent region (R) A PCR product having the flanking region (R) and the 3 ′ end portion of the drug resistance gene is prepared by PCR using the designed primer set with primer 6. On the other hand, a PCR product of a drug resistance gene is prepared by PCR using a primer set (primers 3 and 4) designed based on the drug resistance gene using a plasmid containing the drug resistance gene as a template. Next, the three PCR products obtained above were mixed and used as a template, and by SOE-PCR using a primer set (primers 1 and 6), the three PCR products were converted into adjacent regions (L), drug resistance genes A PCR product ligated in the order of the adjacent region (R) is obtained.

  Furthermore, by transforming a host having host DNA (for example, Bacillus subtilis) using the obtained PCR product, an adjacent region (L) and an adjacent region (R) are obtained between the PCR product and the host DNA. Double crossover homologous recombination occurs in FIG. 2 (FIG. 2 shows the mode of homologous recombination as a solid line). As a result, it is possible to delete all of the replacement target region present on the host DNA and introduce the drug resistance gene into the site of the replacement target region. Alternatively, an appropriate restriction enzyme cleavage sequence is provided in each primer sequence used for the preparation of the above PCR product. Next, each of the obtained PCR products is cleaved with a restriction enzyme corresponding to the restriction enzyme cleavage sequence in the primer sequence, and sequentially inserted into an appropriate plasmid vector. In this way, it is possible to construct a plasmid incorporating a DNA fragment prepared using a PCR product in which the above-mentioned flanking region (L), drug resistance gene and flanking region (R) are linked in this order. Next, host DNA (host) can be transformed using the constructed plasmid, the host DNA replacement target region can be completely deleted, and a drug resistance gene can be introduced into the replacement target region.

By the method as described above, the entire region to be replaced in the host DNA can be deleted.
Alternatively, in the host DNA replacement method according to the present invention, part of the host DNA replacement target region is deleted. For example, host transformation is performed according to the method shown in FIG. 2 using a plasmid containing a growth essential gene in the replacement target region. According to the transformation, in the host DNA, a growth essential gene existing in the replacement target region remains, and the other replacement target regions are deleted.

  More specifically, FIGS. 3 and 4 show an example of a method for partially deleting the replacement target region of the host DNA. 3 and 4, the areas outside the replacement target area are denoted as “adjacent area (L)” and “adjacent area (R)”. On the other hand, a drug resistance gene is present in the plasmid. In the host DNA, the direction from the 5 'side to the 3' side is the direction from the adjacent region (L) to the adjacent region (R) with respect to one strand. Furthermore, the mode of homologous recombination is shown by a solid line.

  In the method shown in FIG. 3, the essential growth genes in the replacement target region of the host DNA are left in an orientation opposite to the original orientation, and other replacement target regions other than the essential growth gene are deleted. First, using the host DNA as a template, primer 1 designed based on the 5 ′ end portion of the adjacent region (L), and the 3 ′ end portion of the adjacent region (L) and the 3 ′ end portion of the essential growth gene A PCR product having a flanking region (L) and the 3 ′ end portion of a growth essential gene is prepared by PCR using the designed primer set with primer 2. Similarly, using host DNA as a template, primer 7 designed based on the 3 ′ end portion of the drug resistance gene and the 5 ′ end portion of the adjacent region (R), and the 3 ′ end portion of the adjacent region (R) A PCR product having a flanking region (R) and the 3 ′ end portion of the drug resistance gene is prepared by PCR using the designed primer set with primer 8. Furthermore, using host DNA as a template, primer 3 designed based on the 5 ′ end portion of the essential growth gene and the 5 ′ end portion of the drug resistance gene and primer 4 designed based on the 3 ′ end portion of the growth essential gene A PCR product having a 5 'terminal portion of a growth essential gene and a drug resistance gene is prepared by PCR using a primer set. On the other hand, a PCR product of a drug resistance gene is prepared by PCR using a primer set (primers 5 and 6) designed based on the drug resistance gene using a plasmid containing the drug resistance gene as a template. Next, the four PCR products obtained above were mixed and used as a template, and by SOE-PCR using the primer set (primers 1 and 8), the four PCR products were converted into adjacent regions (L), essential genes for growth. A PCR product in which the drug resistance gene and the adjacent region (R) are linked in this order is obtained. In the PCR product, the essential growth gene exists in the opposite orientation to the original orientation with respect to the adjacent regions (L) and (R). Next, a host having host DNA (for example, Bacillus subtilis) is transformed using the obtained PCR product. In the transformation, double crossover homologous recombination occurs in the flanking region (L) and flanking region (R). As a result, in the host DNA, the essential growth genes existing in the replacement target region remain in the opposite orientation to the other regions in the host DNA, and the other replacement target regions are deleted. The Rukoto.

  On the other hand, in the method shown in FIG. 4, the upstream and downstream deletions of the essential growth gene in the replacement target region are divided into two stages in the host DNA, and other replacement target regions other than the essential growth gene are missing. To lose. First, in the first stage, using the host DNA as a template, primer 1 designed based on the 5 ′ end portion of the adjacent region (L), 5 ′ of the 3 ′ end portion of the adjacent region (L) and the drug resistance gene A 5 A PCR product having a flanking region (L) and the 5 ′ end portion of drug resistance gene A is prepared by PCR using a primer set with primer 2 designed based on the “end portion”. Similarly, primer 5 designed based on the 3 'end part of drug resistance gene A and the 5' end part of the essential growth gene, and the primer designed based on the 3 'end part of the essential growth gene using host DNA as a template A PCR product having the 3 ′ end portion of the growth essential gene and drug resistance gene A is prepared by PCR using the primer set of No. 6. On the other hand, a PCR product of the drug resistance gene A is prepared by PCR using a primer set (primers 3 and 4) designed based on the drug resistance gene A using a plasmid containing the drug resistance gene A as a template. Next, the three PCR products obtained above were mixed and used as a template, and by SOE-PCR using a primer set (primers 1 and 6), the three PCR products were converted into adjacent regions (L), drug resistance genes A PCR product in which A and a growth essential gene are ligated in this order is obtained. Further, a host having host DNA (for example, Bacillus subtilis) is transformed using the obtained PCR product. In the transformation, double crossover homologous recombination occurs in the adjacent region (L) and the essential growth gene. As a result, in the host DNA, the region between the adjacent region (L) and the essential growth gene (that is, the replacement target region existing upstream of the essential growth gene) is deleted.

  In the second stage, using the host DNA obtained in the first stage as a template, primer 7 designed based on the 5 ′ end portion of the essential growth gene, 3 ′ end portion of the essential growth gene and 5 of the drug resistance gene B A PCR product having the 5 'end portion of the growth essential gene and drug resistance gene B is prepared by PCR using a primer set with primer 8 designed based on the' end portion. Similarly, using the host DNA as a template, primer 11 designed based on the 5 ′ end portion of the adjacent region (R) and the 3 ′ end portion of the drug resistance gene B and the 3 ′ end portion of the adjacent region (R) A PCR product having a flanking region (R) and the 3 ′ end portion of drug resistance gene B is prepared by PCR using a primer set with primer 12 designed based on the above. On the other hand, a PCR product of drug resistance gene B is prepared by PCR using a primer set (primers 9 and 10) designed based on drug resistance gene B using a plasmid containing drug resistance gene B as a template. Next, the three PCR products obtained were mixed and used as a template, and by SOE-PCR using a primer set (primers 7 and 12), the three PCR products were converted into a growth essential gene, a drug resistance gene B and A PCR product ligated in the order of the adjacent regions (R) is obtained. Further, a host (for example, Bacillus subtilis) having the host DNA obtained in the first step is transformed using the obtained PCR product. In the transformation, double crossover homologous recombination occurs in the essential growth gene and the adjacent region (R). As a result, a region between the growth essential gene and the adjacent region (R) in the host DNA (that is, a replacement target region existing downstream of the growth essential gene) is deleted. By performing upstream and downstream deletion of essential growth genes in the replacement target region in the two-steps described above, the essential growth genes existing in the replacement target region remain in the host DNA, and other substitutions are made. The region of interest will be deleted. In addition, when a growth essential gene is present at the end of the replacement target region in the replacement target region of the host DNA, other substitutions other than the growth essential gene can be performed by deleting only upstream or downstream of the growth essential gene. The region of interest can be deleted.

  Next, in the host DNA replacement method according to the present invention, the donor DNA and the host DNA are brought into close proximity under conditions under which homologous recombination occurs, and the region outside the replacement target region in the donor DNA and the deletion in the host DNA A double crossover homologous recombination is generated with a region outside the replacement target region, and the replacement target region of the donor DNA is introduced into the site of the replacement target region in 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.

  LP transformation of recipient strains with host DNA using donor strains with donor DNA is described based on FIG. In FIG. 5, regions outside the replacement target region are denoted as “adjacent region (L)” and “adjacent region (R)”.

  As shown in FIG. 5, for example, when the neomycin resistance gene (Ne) and the tetracycline resistance gene (Tc) are incorporated in the donor DNA, the donor DNA contains the neomycin resistance gene (Ne) and the tetracycline. There is a resistance gene (Tc).

  On the other hand, as shown in FIG. 5, the host DNA lacking the region to be replaced contains a drug resistance gene (eg, chloramphenicol resistance gene (Cm)) different from the drug resistance gene present in the donor DNA. It can exist at the site of the replacement target region that has been deleted.

  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 possibility 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 competent cell of a recipient strain can be prepared, 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, Competent cells of the recipient strain can be prepared.

  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 (FIG. 5 shows the mode of homologous recombination in solid lines). As a result, the replacement target region in the donor DNA is introduced into the deleted replacement target region in the host DNA. The transformant can be selected using as an index the drug resistance based on the drug resistance gene (in FIG. 5, neomycin resistance gene (Ne), tetracycline resistance gene (Tc)) inserted in the donor DNA replacement target region. In addition, as shown in FIG. 5, in the host DNA, the transformant using the drug sensitivity based on another drug resistance gene (eg, chloramphenicol resistance gene) present at the site of the replacement target region deleted as an index. Can be selected. Alternatively, homologous recombination in the transformant can be confirmed by performing PCR on the replacement target region derived from the donor DNA.

  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 the obtained transformant DNA, the replacement target region derived from the donor DNA is replaced in the original orientation.

  In addition, when the essential growth gene is left in the replacement target region of the host DNA using the method described based on FIGS. 3 and 4, the essential growth gene is compared with the region outside the replacement target region. Because it is far shorter, even if it remains in the same orientation as the original, in the homologous recombination between the host DNA and the donor DNA, the replacement target of the donor essential DNA and the donor DNA that remains in the replacement target region of the host DNA It is extremely unlikely that homologous recombination will occur because an interchain exchange structure is formed with a growth essential gene present in the region. In addition, when the essential growth gene is left in the reverse orientation of the original orientation in the host DNA replacement target region by the method shown in FIG. 3, in the homologous recombination between the host DNA and the donor DNA, When the region outside the replacement target region is homologous to each other, the growth essential gene remaining in the replacement target region of the host DNA and the growth essential gene present in the replacement target region of the donor DNA are not homologous. Thus, homologous recombination does not occur between the essential growth genes.

  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. In addition, in the replacement method according to the present invention, the replacement target region of the host DNA is deleted in advance, so that the replacement target region of the donor DNA is introduced into the host DNA without causing homologous recombination within the replacement target region. Can be introduced.

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

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

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

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

[Comparative Example 1] Conventional LP transformation
(1) Preparation of donor strains (D4-1 strain, D4-3 strain and D4-4 strain) Based on FIG. 6, the donor strains (D4-1 strain, D4-3 strain and D4-4 strain) were The production will be described. Each drug resistance gene is derived from the plasmid shown below. Chloramphenicol (Cm) resistance gene: pC194, erythromycin (Em) resistance gene: pMutinT3, neomycin (Ne) resistance gene: pUB110, and tetracycline (Tc) resistance gene: pHY300PLK.

1-1. Preparation of strain D4-1 Using genomic DNA extracted from Bacillus subtilis 168 as a template and using the primer set of Dlytf (SEQ ID NO: 1) and Dlytcmr (SEQ ID NO: 2) shown in Table 1, the lytT gene in the genome A downstream 1.0 kb fragment (A) was amplified by PCR. In addition, a 1.0 kb fragment (B) adjacent to the upstream of the lytS gene in the genome was amplified by PCR using the above genomic DNA as a template and the primer set of Dlytcmf (SEQ ID NO: 3) and Dlytr (SEQ ID NO: 4). . In the genomic DNA derived from Bacillus subtilis 168 strain, the lytT gene and the lytS gene are present continuously (indicated as lytTS in FIG. 6).

  Furthermore, using plasmid pC194 (J. Bacteriol. 150 (2) 815 (1982)) DNA as a template, and using the catf (SEQ ID NO: 5) and catr (SEQ ID NO: 6) primer sets shown in Table 1, 0.9 kb The chloramphenicol (Cm) resistance gene region (C) was prepared by PCR.

  Next, 3 fragments of the obtained 1.0 kb fragment (A), 1.0 kb fragment (B) and Cm resistance gene region (C) were mixed and used as templates to prepare Dlytf (SEQ ID NO: 1) and Dlytr shown in Table 1. By performing SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, (1989)) using the primer set of (SEQ ID NO: 4), 3 fragments became 1.0 kb fragment (A), Cm resistance gene A 2.9 kb DNA fragment contained in the order of region (C) and 1.0 kb fragment (B) was obtained.

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

The genomic DNA of the obtained transformant was extracted, and the primer sets of Dlytf (SEQ ID NO: 1) and catr (SEQ ID NO: 6) and catf (SEQ ID NO: 5) and Dlytr (SEQ ID NO: 4) shown in Table 1 were used. by PCR using, LytT and lytS genes are deleted, LytT and lytS gene was confirmed that replaced the Cm resistance gene (Cm marker) by homologous recombination. The strain thus obtained was named 168CM strain.

Next, as shown in FIG. 6, with respect to 168CM strain, displacement by erythromycin resistance gene yshB and yshA genes apart 30kb downstream of LytT gene (Em marker), LytT gene and lytS gene by the Cm marker This was carried out in the same manner as in the replacement. In the genomic DNA of the 168CM strain, the yshB gene and the yshA gene are present continuously (indicated as yshBA in FIG. 6). Similarly, as shown in FIG. 6, four genes of leuD gene, leuC gene, leuB gene and leuA gene (indicated as leuDCBA in FIG. 6) are replaced with a tetracycline resistance gene (Tc marker), while argH (in Figure 6, denoted as ArgHG) gene and argG gene was replaced with the neomycin resistance gene (Ne marker) and finally to prepare a D4-1 strain.

The Em marker region was prepared using pMutinT3 (Microbiology 144, 3079 (1998)) as a template. The Tc marker region was prepared using pHY300PLK (Yakult) as a template. The Ne marker was prepared using repU prepared using the primer set of rneof (SEQ ID NO: 23) and repUr-Nm (SEQ ID NO: 24) shown in Table 1 and plasmid pUB110 (Plasmid 15, 93 (1986)) as a template. Structural gene of neomycin resistance gene prepared using 0.4 kb fragment including gene promoter region, primer set of NmUf-rep (SEQ ID NO: 25) and neor (SEQ ID NO: 26) shown in Table 1 and plasmid pUB110 as template A 0.8 kb fragment containing the region was mixed and used as a template, and prepared by performing SOE-PCR using the primers rneof (SEQ ID NO: 23) and neor (SEQ ID NO: 26) shown in Table 1.

Table 1 shows the base sequences of the primers used for inserting the drug resistance genes (drug markers) with deletion of 2 or 4 genes, respectively, onto the genome. Also, in Table 2, the primers used for the deletion of 2 or 4 genes and the insertion of drug markers are the primers used for the deletion of the lytT gene and the lytS gene and the accompanying insertion of the Cm marker. It was shown corresponding to.

1-2. Preparation of D4-3 strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, and using the ysfCUf (SEQ ID NO: 27) and ysfCTcr (SEQ ID NO: 28) primer sets shown in Table 1, the ysfC gene in the genome A 2.0 kb fragment (A) containing an upstream adjacent region and a 5 kb 1.0 kb region of the ysfC gene was amplified by PCR. Further, using the genomic DNA as a template and using a primer set of ysfCTcf (SEQ ID NO: 29) and ysfCDr (SEQ ID NO: 30), a region adjacent to the downstream of the ysfC gene in the genome and a 0.1 kb region on the 3 ′ side of the ysfC gene A 2.0 kb fragment (B) containing and was amplified by PCR.

  Furthermore, using the plasmid pHY300PLK DNA as a template and the primer set of tetf (SEQ ID NO: 17) and tetr (SEQ ID NO: 18) shown in Table 1, a 1.6 kb tetracycline (Tc) resistance gene region (C) was obtained by PCR. Prepared.

  Next, the obtained 2.0 kb fragment (A), 2.0 kb fragment (B), and 3 fragments of the Tc resistance gene region (C) were mixed and used as templates to prepare ysfCUf (SEQ ID NO: 27) and ysfCDr shown in Table 1. A 5.6 kb DNA fragment containing 3 fragments in the order of 2.0 kb fragment (A), Tc resistance gene region (C), and 2.0 kb fragment (B) by the SOE-PCR method using the primer set of (SEQ ID NO: 30) Got.

  Further, the 168CM strain was transformed with the obtained DNA fragment by a competent cell transformation method. After transformation, colonies that grew on LB agar medium containing tetracycline (15 μg / mL) were isolated as transformants.

The genomic DNA of the obtained transformant was extracted, and each primer set of ysfCUf (SEQ ID NO: 27), tetr (SEQ ID NO: 18), tetf (SEQ ID NO: 17) and ysfCDr (SEQ ID NO: 30) shown in Table 1 was obtained. The PCR used confirmed that a Tc resistance gene (Tc marker) had been inserted into the ysfC gene site. The strain thus obtained was named 168CT strain.

Next, as shown in FIG. 6, the insertion of the neomycin resistance gene (Ne marker) into the araD gene region was performed on the 168CT strain in the same manner as the insertion of the Tc marker into the ysfC gene region. Built. Furthermore, an erythromycin resistance gene (Em marker) was inserted into the ysdC gene region for the 168CTN strain, and the D4-3 strain was finally produced.

Table 1 shows the base sequences of the primers used for inserting the drug marker into each gene region described above. Further, in Table 3, each primer used for inserting the drug marker into each gene region is shown in correspondence with the primer used when the Tc marker was inserted into the ysfC gene region.

1-3. Production of D4-4 strain As shown in FIG. 6, the erythromycin resistance gene (Em) of yshB gene and yshA gene (shown as yshBA in FIG. 6) was compared with the above-mentioned 168CTN strain in the same manner as the construction of D4-1 strain. D4-4 strain was prepared by replacement with a marker.

(2) Production of Recipient Strain Production of the recipient strain 168SP is described based on FIG.

Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, using the primer set of eprUf (SEQ ID NO: 39) and eprSpr (SEQ ID NO: 40) shown in Table 1, 1.0 kb adjacent to the upstream of the epr gene in the genome Fragment (A) was amplified by PCR. Furthermore, using the genomic DNA as a template, a 1.0 kb fragment (B) adjacent to the downstream of the epr gene in the genome was amplified by PCR using the eprSpf (SEQ ID NO: 41) and eprDr (SEQ ID NO: 42) primer sets. .

  Furthermore, using the plasmid pDG1727 DNA as a template and using the spf (SEQ ID NO: 43) and spr (SEQ ID NO: 44) primer sets shown in Table 1, a 1.2 kb spectinomycin (Sp) resistance gene region (C) was obtained. Prepared by PCR.

  Next, as shown in FIG. 7, three fragments of the obtained 1.0 kb fragment (A), 1.0 kb fragment (B) and Sp resistant gene region (C) were mixed and used as a template to prepare the eprUf shown in Table 1. By the SOE-PCR method using the primer set of (SEQ ID NO: 39) and eprDr (SEQ ID NO: 42), the 3 fragments were 1.0 kb fragment (A), Sp resistant gene region (C), and 1.0 kb fragment (B) in this order. The included 3.2 kb DNA fragment was obtained.

  Furthermore, 168 strains were transformed using the obtained DNA fragment by a competent cell transformation method. After the transformation, colonies that grew on the LB agar medium containing spectinomycin (100 μg / mL) were isolated as transformants.

The genomic DNA of the obtained transformant was extracted, and it was confirmed by PCR that the epr gene was replaced with the Sp resistance gene (Sp marker). The recipient strain 168SP was prepared as described above. In the genomic DNA of Bacillus subtilis 168 strain, the epr gene is present in the genome at a position 925 kb upstream from the argHG gene shown in FIG.

(3) 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)).

Donor strains (D4-1 strain, D4-3 strain, D4-4 strain) were cultured by shaking in LB medium at 37 ° C. until the degree of growth (OD ₆₀₀ ) was 1. After shaking culture, the cells were collected from 1 mL of the culture solution by centrifugation. 0.5 mL SMMP (3.50% Antibiotic Medium 3 (Difco), 17.15% sucrose, 0.16% disodium maleate, 0.20% magnesium chloride hexahydrate and 0.10% bovine serum albumin (pH 6. Adjusted to 5)) suspended in buffer and incubated at 37 ° C. for 2 hours. After incubation, the protoplasts were collected by centrifugation and resuspended in 0.5 mL SMMP buffer. The suspension was further diluted 10-fold with SMMP and used as donor DNA in LP transformation described below.

(4) Explanation of the homologous region in the genomic DNA of the donor strain and the recipient strain In the genomic DNA of the donor strain and the recipient strain, all except the introduced drug resistance gene and the gene deleted simultaneously with the introduction of the drug resistance gene Are the same area.

(5) LP transformation
LP transformation was performed according to the method of Akamatsu et al. This method is characterized in that, instead of the extracted DNA, a protoplast prepared from a donor strain as described in (3) above is provided as a donor DNA to a competent cell of a recipient strain.

  Add 0.1 mL of D4-1, D4-3, and D4-4 protoplasts to 1 mL of the recipient cell 168SP competent cell culture prepared by the competent cell transformation method. Conversion processing was performed. It is considered that the protoplast of the donor strain is destroyed in the competent cell culture solution by osmotic shock, and the genomic DNA released into the culture solution is taken into the competent cell of the recipient strain 168SP.

(6) Selection of transformants using drug resistance as an index and measurement of co-conversion rate After performing LP transformation as described in (5) above, transformants were selected using drug resistance as an index. Usually, when selecting a transformant based on drug resistance, the drug resistance gene of the recipient strain is used simultaneously with the drug resistance gene in the donor DNA. As a result, colony formation derived from a donor strain (non-transformant) remaining without protoplasting that appears when only the drug resistance gene in the donor DNA is used as an index can be suppressed. In addition, in the simultaneous conversion rate measurement described below, it is more convenient if an agar medium simultaneously containing drugs corresponding to a plurality of drug resistance genes in the donor DNA can be used, but on a medium containing three or more drugs. The colony formation rate is significantly reduced. Therefore, when obtaining the transformant, an agar medium containing a drug corresponding to each one drug resistance gene of the recipient strain and the donor strain was used.

  Here, it shows about the measurement of the simultaneous conversion rate by LP transformation based on LP transformation at the time of using 168SP strain as a recipient strain and D4-1 strain as a donor strain.

  First, 500 transformants were selected using the Sp marker introduced into the 168SP strain and the Tc marker introduced into the donor strain D4-1. Next, when the obtained transformants were examined for the presence or absence of a Cm marker using chloramphenicol (Cm) resistance as an index, 98 were Cm resistant. Furthermore, when transformants having Tc and Cm resistance were examined for the presence or absence of the Em marker using erythromycin (Em) resistance as an index, 18 were Em resistant. Here, the ratio of the number of transformants having Tc, Cm and Em resistance to the number of transformants having Tc and Cm resistance ((Tc, Cm and Em resistance) ÷ (Tc and Cm resistance) × 100%) was 18%. As shown in FIG. 6, the fact that the value is not 100% means that homologous recombination has occurred even in the region (between 60 kb) between the Tc marker and the Cm marker. As described above, when the regions at both ends of the replacement target region of the donor strain were introduced into the recipient strain, the ratio (simultaneous conversion rate) that the region sandwiched between them was simultaneously retained in the recipient strain was determined. . That is, for the number of transformants selected using two drug markers on the donor DNA as an index, the ratio of the number of transformants simultaneously holding the third drug resistance marker sandwiched between these two markers Simultaneous conversion rate. From the above results, the simultaneous conversion rate between 60 kb was calculated to be 18%.

  As shown in FIG. 6, similarly, using the D4-1 strain, the simultaneous conversion rate was determined for the region between 120 kb from the Tc marker to the Ne marker. Furthermore, as in the case of using the D4-1 strain, when using the D4-3 strain as a donor strain, it is between 10 kb (from Ne marker to Cm marker) and between 20 kb (from Tc marker to Cm marker). Regarding the region, when the D4-4 strain was further used as the donor strain, the simultaneous conversion rate was determined for the region between 20 kb (from Tc marker to Cm marker) and 30 kb (from Em marker to Cm marker).

(7) Evaluation and results of simultaneous conversion rate Table 4 shows the results of the simultaneous conversion rate of each fragment from 10 kb to 120 kb measured in (6) above.

  As shown in Table 4, a simultaneous conversion rate of 90% or more was shown up to 20 kb, but a significant decrease in the simultaneous conversion rate was confirmed at a distance of more than 20 kb. That is, it was shown that there is room for improvement in the conventional LP transformation method for simultaneous simultaneous replacement of 20 kb or more.

[Example 1] Host DNA replacement method according to the present invention-introduction of replacement target regions (PBSX region, Pro7 region and SPβ region) having different lengths
(1) Preparation of donor strain (XNT strain, 7NE strain and SPT strain)
1-1. Preparation of XNT strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, and using primer set of xNeUf (SEQ ID NO: 45) and xNeUr (SEQ ID NO: 46) shown in Table 1, upstream of xlyB gene in the genome A 1.5 kb fragment (A) containing the adjacent region and the 5 ′ region 0.08 kb of the xlyB gene was amplified by PCR. In addition, using the genomic DNA as a template and a primer set of xNeDf (SEQ ID NO: 47) and xNeDr (SEQ ID NO: 48), the region adjacent to the downstream of the xlyB gene in the genome and the 3 ′ region 0.8 kb of the xlyB gene A 1.5 kb fragment (B) containing was amplified by PCR.

  Further, as in (1) 1-1 of Comparative Example 1, a 1.3 kb neomycin (Ne) resistance gene was used using the primer set of rneof (SEQ ID NO: 23) and neor (SEQ ID NO: 26) shown in Table 1. Region (C) was prepared by PCR.

  Next, the obtained 1.5 kb fragment (A), 1.5 kb fragment (B) and neomycin (Ne) resistance gene region (C) 3 fragments were mixed and used as a template to prepare xNeUf (SEQ ID NO: 45) shown in Table 1. ) And xNeDr (SEQ ID NO: 48) primer set, SOE-PCR method included 3 fragments in the order of 1.5 kb fragment (A), neomycin (Ne) resistance gene region (C), 1.5 kb fragment (B) A 4.3 kb DNA fragment was obtained.

  Furthermore, Bacillus subtilis 168 strain was transformed with the obtained DNA fragment by a competent cell transformation method. After transformation, colonies grown on LB agar medium containing neomycin (20 μg / mL) were isolated as transformants.

The genomic DNA of the obtained transformant was extracted, and each primer set of xNeUf (SEQ ID NO: 45), neor (SEQ ID NO: 26), rneof (SEQ ID NO: 23) and xNeDr (SEQ ID NO: 48) shown in Table 1 was used. The used PCR was performed to confirm that the neomycin resistance gene was inserted into the xlyB gene region.

Next, a tetracycline (Tc) marker was inserted into the xkdK gene region of the strain in which the Ne marker was inserted in the same manner as in the insertion of the Ne marker to construct an XNT strain. Table 1 shows the base sequences of the primers used when inserting the Tc marker. In Table 5, the primers used for inserting the Tc marker are shown in correspondence with the primers used for inserting the Ne marker.

1-2. Preparation of 7NE strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, using the 7NeUf (SEQ ID NO: 53) and 7NeUr (SEQ ID NO: 54) primer sets shown in Table 1, the prophage in the genome A 1.5 kb fragment (A) containing a region adjacent to the upstream of the yrkM gene present in the region 7 and the 5 ′ side 0.04 kb of the yrkM gene was amplified by PCR. Further, using the genomic DNA as a template, and using a primer set of 7NeDf (SEQ ID NO: 55) and 7NeDr (SEQ ID NO: 56), a region adjacent to the downstream of the yrkM gene in the region of property 7 in the genome and yrkM A 1.5 kb fragment (B) containing 0.02 kb on the 3 ′ side of the gene was amplified by PCR.

  Further, in the same manner as (1) 1-1 of Comparative Example 1 described above, a 1.3 kb neomycin (Ne) was prepared using the primer set of rneof (SEQ ID NO: 23) and neor (SEQ ID NO: 26) shown in Table 1. The resistance gene region (C) was prepared by PCR.

  Next, the obtained 3 kb fragment (A), 1.5 kb fragment (B) and neomycin (Ne) resistance gene region (C) were mixed and used as a template to prepare 7NeUf (SEQ ID NO: 53) shown in Table 1. ) And 7NeDr (SEQ ID NO: 56) using the SOE-PCR method, 3 fragments are included in the order of 1.5 kb fragment (A), neomycin (Ne) resistance gene region (C), 1.5 kb fragment (B). A 4.3 kb DNA fragment was obtained.

  Furthermore, Bacillus subtilis 168 strain was transformed with the obtained DNA fragment by a competent cell transformation method. After transformation, colonies grown on LB agar medium containing neomycin (20 μg / mL) were isolated as transformants.

The genomic DNA of the obtained transformant was extracted, and each primer set of 7NeUf (SEQ ID NO: 53), neor (SEQ ID NO: 26), rneof (SEQ ID NO: 23) and 7NeDr (SEQ ID NO: 56) shown in Table 1 was used. It was confirmed by the PCR used that a neomycin resistance gene (Ne marker) was inserted into the yrkM gene region.

Next, the erythromycin resistance gene (Em marker) was inserted into the yraK gene region of the strain into which the Ne marker had been inserted in the same manner as the Ne marker, thereby constructing the 7NE strain. Table 1 shows the base sequences of the primers used for inserting the Em marker. In Table 6, the primers used for inserting the Em marker are shown in correspondence with the primers used for inserting the Ne marker.

1-3. Preparation of SPT strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template and using the primer set of yokBUf (SEQ ID NO: 61) and yokBUr (SEQ ID NO: 62) shown in Table 1, within the region of SPβ in the genome A 2 kb fragment (A) adjacent to the downstream of the existing yokB gene was amplified by PCR. In addition, using the above-described genomic DNA as a template and using the yokBDf (SEQ ID NO: 63) and yokBDr (SEQ ID NO: 64) primer sets shown in Table 1, adjacent to the upstream of the yokB gene present in the SPβ region of the genome A 2 kb fragment (B) containing the region to be synthesized and the 5 ′ 0.1 kb of the yokB gene was amplified by PCR.

  Furthermore, using the plasmid pHY300PLK DNA as a template and the primer set of tetf (SEQ ID NO: 17) and tetr (SEQ ID NO: 18) shown in Table 1, a 1.6 kb tetracycline (Tc) resistance gene region (C) was obtained by PCR. Prepared.

  Next, the obtained 2 kb fragment (A), 2 kb fragment (B) and tetracycline (Tc) resistance gene region (C) 3 fragments were mixed and used as a template to prepare yokBUf (SEQ ID NO: 61) shown in Table 1 and According to the SOE-PCR method using a primer set of yokBDr (SEQ ID NO: 64), 3 fragments are contained in the order of 2 kb fragment (A), tetracycline (Tc) resistance gene region (C), and 2 kb fragment (B). A DNA fragment was obtained.

  Furthermore, Bacillus subtilis 168 strain was transformed with the obtained DNA fragment by a competent cell transformation method. After transformation, colonies that grew on LB agar medium containing tetracycline (15 μg / mL) were isolated as transformants.

The genomic DNA of the obtained transformant was extracted, and each primer set of yokBUf (SEQ ID NO: 61), tetr (SEQ ID NO: 18), tetf (SEQ ID NO: 17) and yokBDr (SEQ ID NO: 64) shown in Table 1 was extracted. It was confirmed by PCR that the tetracycline resistance gene was inserted into the yokB gene region, and an SPT strain was prepared.

(2) Preparation of recipient strains by deleting each replacement target region on genomic DNA (ΔPBSX strain, ΔPro7 strain and ΔSPβ strain)
2-1. Preparation of ΔPBSX strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, and using pbsxFW (SEQ ID NO: 65) and pbsx / cmr (SEQ ID NO: 66) primer sets shown in Table 1, the xlyB gene in the genome An upstream adjacent 3.1 kb fragment (A) was amplified by PCR. In addition, using the genomic DNA as a template and a primer set of pbsx / cmf (SEQ ID NO: 67) and pbsxRV (SEQ ID NO: 68) shown in Table 1, a 3.3 kb fragment adjacent to the downstream of the xlyA gene in the genome ( B) was amplified by PCR. In the genomic DNA derived from Bacillus subtilis 168, the xlyB gene and the xlyA gene are genes located at the respective ends of the prophage PBSX region.

  Furthermore, using the plasmid pC194 DNA as a template and the primer set of catf (SEQ ID NO: 5) and catr (SEQ ID NO: 6) shown in Table 1, a 0.9 kb chloramphenicol (Cm) resistance gene region (C) Was prepared by PCR.

  Next, the obtained 3.1 kb fragment (A), 3.3 kb fragment (B) and chloramphenicol (Cm) resistance gene region (C) 3 fragments were mixed and used as a template to obtain pbsxFW ( 3 fragments were 3.1 kb fragment (A), chloramphenicol (Cm) resistance gene region (C), 3.3 kb fragment by SOE-PCR using the primer set of SEQ ID NO: 65) and pbsxRV (SEQ ID NO: 68) A 7.3 kb DNA fragment contained in the order of (B) was obtained.

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

  The genomic DNA of the obtained transformant was extracted, and each primer set of pbsxFW (SEQ ID NO: 65) and catr (SEQ ID NO: 6) and catf (SEQ ID NO: 5) and pbsxRV (SEQ ID NO: 68) shown in Table 1 was used. It was confirmed by the PCR used that the PBSX region was deleted and the PBSX region was replaced with the chloramphenicol resistance gene by homologous recombination. The strain thus obtained was named ΔPBSX strain.

2-2. Preparation of ΔPro7 strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template and using the primer set of pro7U2500F (SEQ ID NO: 69) and pro7CMUCR (SEQ ID NO: 70) shown in Table 1, upstream of yrkM gene in the genome An adjacent 2.5 kb fragment (A) was amplified by PCR. In addition, using the genomic DNA as a template and a primer set of pro7CMCDF (SEQ ID NO: 71) and pro7D2500R (SEQ ID NO: 72) shown in Table 1, a 2.5 kb fragment (B) adjacent to the downstream of the yraK gene in the genome Was amplified by PCR. In the genomic DNA derived from Bacillus subtilis 168, the yrkM gene and yraK gene are genes located at the respective ends of the prophage7 (Pro7) region.

  Furthermore, using the plasmid pC194 DNA as a template and the primer set of catf (SEQ ID NO: 5) and catr (SEQ ID NO: 6) shown in Table 1, a 0.9 kb chloramphenicol (Cm) resistance gene region (C) Was prepared by PCR.

  Next, 3 fragments of the obtained 2.5 kb fragment (A), 2.5 kb fragment (B) and chloramphenicol (Cm) resistance gene region (C) were mixed and used as a template to produce pro7U2500F ( 3 fragments were 2.5 kb fragment (A), chloramphenicol (Cm) resistance gene region (C), 2.5 kb fragment by SOE-PCR method using primer set of SEQ ID NO: 69) and pro7D2500R (SEQ ID NO: 72) A 5.9 kb DNA fragment contained in the order of (B) was obtained.

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

  The genomic DNA of the obtained transformant was extracted, and each primer set of pro7U2500F (SEQ ID NO: 69), pro7CMCR (SEQ ID NO: 73), pro7CMCF (SEQ ID NO: 74) and pro7D2500R (SEQ ID NO: 72) shown in Table 1 was obtained. PCR was performed to confirm the deletion of the Pro7 region and the insertion of the chloramphenicol resistance gene, and a ΔPro7 strain was prepared.

2-3. Production of ΔSPβ strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, and using the spbfw (SEQ ID NO: 75) and spb / cmr (SEQ ID NO: 76) primer sets shown in Table 1, the yotN gene in the genome A downstream 1.0 kb fragment (A) was amplified by PCR. In addition, using the genomic DNA as a template and a primer set of spb / cmf (SEQ ID NO: 77) and spbrv (SEQ ID NO: 78) shown in Table 1, a 1.0 kb fragment adjacent to the downstream of the yokA gene in the genome ( B) was amplified by PCR. In the genomic DNA derived from Bacillus subtilis 168 strain, the yotN gene and yokA gene are genes located at the respective ends of the prophage SPβ (SPβ) region. Since both the yotN gene and the 3 ′ side of the yokA gene are located outside the SPβ region, the outside of the SPβ region is a region adjacent to each downstream of the gene.

  Furthermore, using the plasmid pC194 DNA as a template and the primer set of catf (SEQ ID NO: 5) and catr (SEQ ID NO: 6) shown in Table 1, a 0.9 kb chloramphenicol (Cm) resistance gene region (C) Was prepared by PCR.

  Next, 3 fragments of the obtained 1.0 kb fragment (A), 1.0 kb fragment (B) and chloramphenicol (Cm) resistance gene region (C) were mixed and used as a template as a spbfw ( 3 fragments were 1.0 kb fragment (A), chloramphenicol (Cm) resistance gene region (C), 1.0 kb fragment by SOE-PCR using the primer set of SEQ ID NO: 75) and spbrv (SEQ ID NO: 78) A 2.9 kb DNA fragment contained in the order of (B) was obtained.

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

  The genomic DNA of the obtained transformant was extracted, and the primer sets of spbfw (SEQ ID NO: 75) and catr (SEQ ID NO: 6) and catf (SEQ ID NO: 5) and spbrv (SEQ ID NO: 78) shown in Table 1 were used. By the PCR used, deletion of the SPβ region and insertion of the chloramphenicol resistance gene were confirmed, and a ΔSPβ strain was prepared.

(3) Production of Protoplast from Donor Strain Protoplasts were produced in the same manner as (3) of Comparative Example 1 using donor strains XNT strain, 7NE strain and SPT strain. However, the protoplast solution recovered after lysozyme treatment and resuspended in the SMMP buffer was further diluted 100-fold with SMMP buffer for the XNT and 7NE strains, and diluted 5-fold for the SPT strain. It used for LP transformation mentioned later.

(4) Explanation of homologous region in genomic DNA of donor strain and recipient strain When XNT strain was used as the donor strain, ΔPBSX strain was used as the recipient strain. FIG. 8 shows the structure around the PBSX region in the genomic DNA of donor strain XNT and recipient strain ΔPBSX. As shown in FIG. 8, the PBSX region of about 30 kb is deleted in the genomic DNA of the ΔPBSX strain. Therefore, there is no homologous region in the PBSX region between the genomic DNAs of the XNT strain and the ΔPBSX strain, but all regions other than the region are completely the same. The PBSX region means a prophage PBSX region comprising 36 genes between the xlyB gene and the xlyA gene on the Bacillus subtilis genome.

In addition, when the 7NE strain was used as the donor strain, the ΔPro7 strain was used as the recipient strain. FIG. 9 shows the structure around the Pro7 region in the genomic DNA of the donor strain 7NE and the recipient strain ΔPro7. As shown in FIG. 9, the Pro7 region of about 42 kb is deleted in the genomic DNA of the ΔPro7 strain. Therefore, there is no homologous region in the Pro7 region between the genomic DNAs of the 7NE strain and the ΔPro7 strain, but all regions other than the region are completely identical. The Pro7 region means a prophage 7 region comprising 46 genes between the yrkM gene and the yraK gene on the Bacillus subtilis genome.

Further, when the SPT strain was used as the donor strain, the ΔSPβ strain was used as the recipient strain. FIG. 10 shows the structure around the SPβ region in the genomic DNA of the donor strain SPT and the recipient strain ΔSPβ. As shown in FIG. 10, the SPβ region of about 134 kb is deleted in the genomic DNA of the ΔSPβ strain. Accordingly, there is no homologous region in the SPβ region between the genomic DNAs of the SPT strain and ΔSPβ strain, but all regions other than the region are completely identical. The SPβ region means a prophage SPβ region comprising 185 genes between the yotN gene and the yraK gene on the Bacillus subtilis genome.

  In each of the above cases, homologous recombination is possible in the region outside the deletion region (substitution target region), but homologous recombination cannot occur in the deletion region.

(5) LP transformation
LP transformation was carried out in the same manner as (5) of Comparative Example 1 described above. Transformation was performed using the recipient strain ΔPBSX for the donor strain XNT, the recipient strain ΔPro7 for the donor strain 7NE, and the recipient strain ΔSPβ for the donor strain SPT.

(6) Selection of transformants using drug resistance as an index
6-1. Transformant of ΔPBSX strain (recipient strain) when using XNT strain (donor strain) As shown in FIG. 8, when transforming ΔPBSX strain (recipient strain) using XNT strain (donor strain) the were selected for Ne marker (XlyB inserted gene region) and ΔPBSX strain (inserted epr gene region) Sp markers with the transformants indicators with the XNT strain. That is, colonies that grew on an LB agar medium containing spectinomycin (100 μg / ml) and neomycin (20 μg / ml) were isolated as transformants. Next, using the Tc marker (inserted into the xkdK gene region), which is another drug resistance gene possessed by the XNT strain, as an index, all the transformants were obtained using the replica method for the 50 transformants obtained above. It was confirmed to be tetracycline resistant. Furthermore, it was confirmed that all 50 transformants were sensitive to chloramphenicol. That is, it was confirmed that the Cm marker inserted into the PBSX region of the genomic DNA of the ΔPBSX strain was deleted.

  Further, genomic DNA of the obtained transformant was extracted and confirmed by PCR as described in (7) below.

6-2. Transformant of ΔPro7 strain (recipient strain) when using 7NE strain (donor strain) As shown in FIG. 9, ΔPro7 strain (recipient strain) is transformed using 7NE strain (donor strain). At the time of transformation, transformants were selected using the Ne marker of 7NE strain (inserted into the yrkM gene region) and the Sp marker of ΔPro7 strain (inserted into the epr gene region) as indices. That is, colonies that grew on an LB agar medium containing spectinomycin (100 μg / ml) and neomycin (20 μg / ml) were isolated as transformants. Next, using the Em marker (inserted into the yraK gene region), which is another drug resistance gene possessed by the 7NE strain, as an index, all the transformants were obtained using the replica method for the 50 transformants obtained above. Confirmed resistance to erythromycin. Furthermore, it was confirmed that all 50 transformants were sensitive to chloramphenicol. That is, it was confirmed that the Cm marker inserted into the Pro7 region of the genomic DNA of the ΔPro7 strain was deleted.

  Further, genomic DNA of the obtained transformant was extracted and confirmed by PCR as described in (7) below.

6-3. Transformant of ΔSPβ strain (recipient strain) when using SPT strain (donor strain) As shown in FIG. 10, when transforming ΔSPβ strain (recipient strain) using SPT strain (donor strain) For selection, transformants were selected using the Tc marker possessed by the SPT strain (inserted into the ykoB gene region) and the Sp marker possessed by the ΔSPβ strain (inserted into the epr gene region) as indices. That is, colonies that grew on an LB agar medium containing spectinomycin (100 μg / ml) and neomycin (20 μg / ml) were isolated as transformants. Next, 50 transformants obtained above were confirmed to be sensitive to chloramphenicol using the replica method. That is, it was confirmed that the Cm marker inserted into the SPβ region of the genomic DNA of the ΔSPβ strain was deleted.

  Further, genomic DNA of the obtained transformant was extracted and confirmed by PCR as described in (7) below.

(7) Confirmation of PCR for homologous recombination in transformant Confirmation by PCR was performed on the genomic DNA of the transformant obtained in (6) above.
7-1. Transformant of ΔPBSX strain (recipient strain) when using XNT strain (donor strain)
Regarding the genomic DNA of the transformant obtained by transforming ΔPBSX strain (recipient strain) with XNT strain (donor strain), rneof (SEQ ID NO: 23), xNeDr (SEQ ID NO: 48), tetf (sequence) PCR was performed using each primer set of No. 17) and xTcDr (SEQ ID NO: 52) and xEmUf (SEQ ID NO: 79) and xEmDr (SEQ ID NO: 80). The PCR products amplified by using each primer set are shown in FIG. 8 as F1 (primer set of rneof (SEQ ID NO: 23) and xNeDr (SEQ ID NO: 48)), F2 (tetf (SEQ ID NO: 17). ) And xTcDr (SEQ ID NO: 52)) and F3 (xEmUf (SEQ ID NO: 79) and xEmDr (SEQ ID NO: 80) primer set).

  The PCR results (electrophoresis photograph) are shown in FIG. As shown in FIG. 11, DNA fragments of F1 (2.8 kb), F2 (3.1 kb) and F3 (3.0 kb), which are expected to be obtained when the PBSX region deleted in the ΔPBSX strain was restored, respectively. Amplification was confirmed. Transformants I and II are different transformants obtained. From the PCR results and the drug resistance confirmation results, it was confirmed that the PBSX region of the donor strain XNT was completely introduced at the position where the PBSX region of the recipient strain ΔPBSX was present. In addition, when both ends of the PBSX region of the donor strain XNT are introduced into the recipient strain ΔPBSX, the simultaneous conversion rate indicating the ratio that the region sandwiched between them is simultaneously retained in the recipient strain ΔPBSX is 100%. there were.

7-2. Transformant of ΔPro7 strain (recipient strain) when 7NE strain (donor strain) is used
Regarding the genomic DNA of the transformant obtained by transforming ΔPro7 strain (recipient strain) with 7NE strain (donor strain), rneof (SEQ ID NO: 23), 7NeDr (SEQ ID NO: 56), 7TcUf (sequence) No. 81) and 7TcDr (SEQ ID NO: 82), and 7EmUf (SEQ ID NO: 57) and emr2 (SEQ ID NO: 12) were used for PCR. The PCR products amplified by using each primer set are shown in FIG. 9 as F1 (primer set of rneof (SEQ ID NO: 23) and 7NeDr (SEQ ID NO: 56)), F2 (7TcUf (SEQ ID NO: 81)). ) And 7TcDr (SEQ ID NO: 82)) and F3 (7EmUf (SEQ ID NO: 57) and emr2 (SEQ ID NO: 12) primer set).

  The PCR results (electrophoresis photograph) are shown in FIG. As shown in FIG. 12, F1 (2.8 kb), F2 (3.0 kb) and F3 (2.8 kb) DNA fragments, which are expected to be obtained when the Pro7 region deleted in the ΔPro7 strain is restored, are obtained. Amplification was confirmed. Transformants I and II are different transformants obtained. From the PCR results and the drug resistance confirmation results, it was confirmed that the Pro7 region of the donor strain 7NE was completely introduced at the position where the Pro7 region of the recipient strain ΔPro7 was present. In addition, when the regions at both ends of the Pro7 region of donor strain 7NE were introduced into recipient strain ΔPro7, the simultaneous conversion rate indicating the proportion of the region sandwiched between them simultaneously retained in recipient strain ΔPro7 was 100%. there were.

7-3. Transformant of ΔSPβ strain (recipient strain) when using SPT strain (donor strain)
About transformant genomic DNA obtained when transforming ΔSPβ strain (recipient strain) using SPT strain (donor strain), tetf (SEQ ID NO: 17), tetr (SEQ ID NO: 18), yotMUf (SEQ ID NO: 83) and yotMUr (SEQ ID NO: 84), yokBUf (SEQ ID NO: 61) and yokBUr (SEQ ID NO: 62), 35kf (SEQ ID NO: 85) and 35kr (SEQ ID NO: 86), 70kf (SEQ ID NO: 87) and 70kr (SEQ ID NO: 88) ), And 105 kf (SEQ ID NO: 89) and 105kr (SEQ ID NO: 90) primer sets were used for PCR. The PCR products amplified by using each primer set are F1 (primer set of tetf (SEQ ID NO: 17) and tetr (SEQ ID NO: 18)) and F2 (yotMUf (SEQ ID NO: 83) shown in FIG. ) And yotMUr (SEQ ID NO: 84)), F3 (primer set of yokBUf (SEQ ID NO: 61) and yokBUr (SEQ ID NO: 62)), F4 (35kf (SEQ ID NO: 85) and 35kr (SEQ ID NO: 86)) Position of F5 (primer set of 70 kf (SEQ ID NO: 87) and 70kr (SEQ ID NO: 88)) and F6 (primer set of 105 kf (SEQ ID NO: 89) and 105kr (SEQ ID NO: 90)) It corresponds to.

  The PCR results (electrophoresis photograph) are shown in FIG. As shown in FIG. 13, F1 (1.6 kb), F2 (2.0 kb), F3 (1.8 kb), F4 (2.0 kb) expected to be obtained when the SPβ region deleted in the ΔSPβ strain was restored. Amplification of DNA fragments of kb), F5 (2.0 kb) and F6 (2.0 kb) was confirmed. Transformants I and II are different transformants obtained. From the PCR results and the drug resistance confirmation results, it was confirmed that the SPβ region of the donor strain SPT was completely introduced at the position where the SPβ region of the recipient strain ΔSPβ was present. In addition, when the regions at both ends of the SPβ region of the donor strain SPT are introduced into the recipient strain ΔSPβ, the simultaneous conversion rate indicating the ratio that the region sandwiched between them is simultaneously retained in the recipient strain ΔSPβ is 100%. there were.

FIG. 1 shows a method for introducing a drug resistance gene into donor DNA or host DNA of Bacillus subtilis. FIG. 2 shows a method for deleting a region to be replaced in host DNA. FIG. 3 shows a method in which a growth essential gene in the replacement target region of the host DNA is left in an orientation opposite to the original orientation and other replacement target regions other than the growth essential gene are deleted. FIG. 4 shows a method of performing deletion in upstream and downstream of essential growth genes in a replacement target region in two stages in the host DNA and deleting other replacement target regions other than the essential growth gene. . FIG. 5 shows LP transformation of recipient strains with host DNA using donor strains with donor DNA. FIG. 6 shows the production of donor strains (D4-1 strain, D4-3 strain, and D4-4 strain) in Comparative Example 1. FIG. 7 shows the production of the recipient strain 168SP in Comparative Example 1. FIG. 8 shows the structure around the PBSX region in the genomic DNA of the donor strain XNT and the recipient strain ΔPBSX in Example 1. FIG. 9 shows the structure around the Pro7 region in the genomic DNA of donor strain 7NE and recipient strain ΔPro7 in Example 1. FIG. 10 shows the structure around the SPβ region in the genomic DNA of the donor strain SPT strain and the recipient strain ΔSPβ strain in Example 1. FIG. 11 shows the PCR results for the genomic DNA of the transformant obtained when transforming the ΔPBSX strain (recipient strain) using the XNT strain (donor strain). FIG. 12 shows the results of PCR on the genomic DNA of the transformant obtained when the ΔPro7 strain (recipient strain) was transformed using the 7NE strain (donor strain). FIG. 13 shows the PCR results for the genomic DNA of the transformant obtained when transforming the ΔSPβ strain (recipient strain) using the SPT strain (donor strain).

  Sequence number 1-90 is a primer.

Claims (6)

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 deleting part or all of the region to be replaced in the host DNA;
A second step of recombination between the donor DNA and the host DNA,
In the second step, a host DNA replacement method, wherein a replacement target region of donor DNA is introduced into host DNA.   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. The replacement target region of the host DNA contains a gene essential for growth, and the first step is performed by the following method 1) or 2): A method for replacing the host DNA according to Item.
1) A method of substituting a replacement target region of host DNA with a DNA fragment containing at least the gene essential for growth.
2) A method of deleting a replacement target region other than the gene essential for the growth.

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