JP4479199B2 - Method for producing plasmid containing inserted DNA unit - Google Patents

Description

  The present invention provides a DNA having a plurality of DNA fragments linked and accumulated in a certain order and orientation by using the DNA uptake ability and homologous recombination ability of microorganisms such as Bacillus genus bacteria, and which can be amplified in the microorganism. The present invention relates to a method for obtaining easily and a method for obtaining a microorganism containing the DNA in genomic DNA.

  A number of genes are involved in a series of processes (bioprocesses) such as production of certain substances performed by living organisms, and these genes may exist relatively in the genome. Are often distributed. The entire genome of living organisms is being clarified as a result of recent genome projects, and efficient biotechnology can now be achieved by selecting gene groups necessary and sufficient for a certain bioprocess using these genomic gene information. It is necessary to reconstruct the process or to construct a new bioprocess with a combination of various genes. The average length of one gene is about 1 kb in bacteria and 10 to 100 kb in animals, and the combined length of these genes inevitably becomes a huge DNA of 10 kb or more. . In such a large DNA manipulation, a method has been adopted in which the DNA is divided into strands that can be recombined and then incorporated into a plasmid one by one. In the method, restrictions on the restriction enzyme site to be used for each integration become severe, and there are limitations.

  In order to solve these problems, a long-chain DNA continuous ligation method using a solid-layer method has been devised by Toyota Central Research Laboratory, Inc. (see Patent Document 1 and Non-Patent Document 1). After fixing one end of the E. coli vector on the bead and ligating the DNA fragment in the specified direction using the non-pasted sequence sticky end towards the other end, removing unreacted material and further removing the next fragment After ligating a plurality of DNA fragments in a desired order and orientation by repeating the operation of ligation, this is separated from the beads, the resulting linear DNA is ligated in a circular shape, and then transformed into E. coli. A large number of DNA fragments are accumulated on a plasmid in a desired direction and direction. However, this method requires first preparation for ligation of the E. coli vector to the beads, removal of unreacted substances at each step of the ligation operation, and finally separation of the DNA from the beads. Many operations are complicated and time-consuming in that they are connected in a ring shape. In particular, in the case of ligation, ie, intramolecular ligation, it is necessary to reduce the DNA concentration. However, the longer the length of the DNA, the lower the generation efficiency of the circular DNA theoretically (see Non-Patent Document 2). Therefore, the longer the total length of the DNA fragments to be ligated, the lower the efficiency of obtaining the DNA to which they are ligated.

On the other hand, a method for obtaining plasmid DNA using Bacillus subtilis is known. Bacillus subtilis appears in certain physiological states as cells (competent cells) having the ability to actively take up DNA. In this incorporation process, first, double-stranded DNA serving as a substrate is cleaved on competent cells. From this break point, it is known that the DNA strand on one side of the double strand is decomposed, and the other DNA strand is taken into Bacillus subtilis as a single-stranded DNA. The incorporated single-stranded DNA is repaired into a circular double-stranded DNA by associating with the complementary single-stranded DNA incorporated completely separately (see Non-Patent Document 3). Unlike the double-stranded circular DNA required in the E. coli plasmid transformation system in order for the plasmid DNA to be cleaved and incorporated in this manner, the substrate plasmid in plasmid transformation with Bacillus subtilis competent cells is different. The shape does not need to be circular DNA, but rather must be a structure (concatemer structure) in which the same DNA is arranged in multiple layers in the same direction so as to favor plasmid repair after incorporation. I knew it was. However, a plasmid containing an inserted DNA composed of a plurality of DNA fragments has not been obtained using such a method.
JP 2002-262870 A Koda et al., 2001, Japanese Society for Biotechnology, Abstracts of Lectures, p. 223 Dugaiczyk, A., et al. J. Mol. Biol. 96, 171-184. (1975) Canosi, U., et al. Mol. Gen. Genet., 181, 434-440. (1981)

  The present invention relates to a plasmid in which a plurality of DNA fragments are linked and accumulated in a certain order and orientation by using the DNA uptake ability and homologous recombination ability of microorganisms such as Bacillus bacteria, and can be amplified in the microorganism. The present invention relates to a method for easily obtaining DNA and a method for obtaining a microorganism containing a DNA sequence in which a plurality of DNA fragments are linked and accumulated in a certain order and orientation in genomic DNA.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have made use of the fact that the protruding end of 3 bases generated by digestion with the restriction enzyme SfiI of DNA can be designated as an arbitrary sequence, and the constituent elements to be accumulated So that the ends of the linear plasmid vector fragments having a replication mechanism effective in Bacillus subtilis cells can be ligated in order in a single DNA accumulation unit. The SfiI cleavage site was designed and prepared, and these SfiI fragments were mixed at the same concentration so as to be equimolar, and then ligated in the presence of polyethylene glycol and a salt to multiplex this DNA linking unit. It is desirable in Bacillus subtilis plasmids to generate linear macromolecular DNA having a repeating structure and transformed into Bacillus subtilis competent cells. And it succeeded in linking the DNA to turn and direction. Also, co-culturing Bacillus subtilis competent cells in which a common sequence with the sequence in the plasmid is inserted into genomic DNA and linear polymer DNA having a structure in which the DNA ligation unit obtained above is repeated repeatedly. As a result, the present invention was completed by successfully ligating DNA into Bacillus subtilis genomic DNA in the desired order and direction.

That is, according to the present invention,
(1) It consists of a DNA containing a replication origin effective in Bacillus subtilis and two or more genes, and
The inserted DNA unit containing the inserted DNA having a length of 10 kb or more is divided into unit DNAs having a structure that can be joined in order while utilizing the complementarity of the base sequences at the ends of each other, and the unit DNA is subjected to a ligation reaction. subjected, the cells by contacting the competent cells of the ligation reaction solution and Bacillus subtilis transformed, from the transformants obtained, and obtains the plasmid DN a comprising inserting DNA unit Method for preparing plasmid DNA for Bacillus subtilis cell transformation,
(2) The plasmid DNA containing the inserted DNA unit has two common sequences at both ends of the inserted DNA which are common to the base sequence on the Bacillus subtilis genome used when the inserted DNA is further inserted into the Bacillus subtilis genome by homologous recombination. A method for producing a plasmid DNA for Bacillus subtilis cell transformation according to (1) above, which is characterized in that
(3) Bacillus subtilis cell transformation according to (1) or (2) above, wherein the unit DNA is ligated by mixing equimolar amounts of each unit DNA in the presence of a salt and polyethylene glycol. For preparing plasmid DNA for
(4) The method for producing a plasmid DNA for Bacillus subtilis cell transformation according to any one of (1) to ( 3) above, wherein the inserted DNA contains a series of genes necessary for substance production,
(5) above (1) to (4) a Bacillus subtilis cell transformation plasmid DNA prepared by the method described in B. subtilis competent cells and Kyo培Nutrient insert DNA unit characterized in that A method for producing Bacillus subtilis comprising genomic DNA,
Is provided.

According to the present invention, a plurality of DNA fragments can be ligated by a single ligation operation using a competent cell transformation method such as Bacillus subtilis which is a kind of soil fine bacteria. In other words, it was shown that ligation and integration of a huge DNA into an amplifiable DNA fragment, which was difficult with the conventional method, can be performed very simply.
The DNA fragments to be accumulated are limited in the base sequence of the protruding part at the end, but do not require any special base sequence that induces or controls recombination in the base sequence of the other part. No homologous sequence with the host genome is required. That is, the present invention is expected to be widely applicable to general DNA accumulation regardless of prokaryote or eukaryote.

Furthermore, when producing a DNA fragment for microbial transformation, it is the first time according to the present invention that a plasmid finally obtained can be efficiently obtained by setting the molar ratio of each unit DNA to be accumulated to 1. It was revealed.
In addition, as shown in Example 2 below, the method of the present invention can be integrated without any problems even if the length of the unit DNA is large (minimum 0.3 kb, maximum 38 kb), and the integration unit is 50 kb. The above is possible. That is, it can be arbitrarily linked without considering the size of the unit DNA. In addition, many eukaryotic genes including mammals (including mice and humans) and plants have a length exceeding 10 kb, but it has been shown that these genes can also be handled as unit DNA.

  In addition, the pps encoding the prepastatin synthesis gene used in Example 2 below is a multigene integrated plasmid prepared by the method of the present invention, despite a sequence having 10 repeats every about 3 kb. It was also revealed that it was stably maintained and expressed in Bacillus subtilis. Repeated sequences are widely observed in eukaryotes including mammals (including mice and humans) and plants, but the method of the present invention can also be applied to DNA accumulation having repeated sequences.

Hereinafter, the present invention will be described in more detail.
(1) Preparation of DNA fragment for microbial transformation One of the present invention is a method for preparing DNA obtained by linking a plurality of DNA fragments in an arbitrary order and direction, and specifically, (1 A DNA comprising a replication origin effective in the host microorganism and an inserted DNA unit containing the inserted DNA are prepared as a plurality of unit DNAs having a structure capable of being repeatedly linked to each other while maintaining the order; (2) the unit After ligating DNA to prepare a DNA fragment having at least one inserted DNA unit and one or more unit DNAs overlapping, (3) co-culturing with the DNA fragment and competent cells of the host microorganism Then, by collecting plasmid DNA from the microorganism, a method is prepared in which a plurality of DNA fragments are linked in an arbitrary order and direction to accumulate DNA.

  The inserted DNA referred to in the present invention is DNA to be cloned, and the kind and size thereof are not particularly limited, but 10 kb or more is preferable. The type of DNA may be any of eukaryotic, including prokaryotic organisms, mammals or plants, genomic DNA fragments, already cloned DNA, and cDNA fragments, and is not particularly limited. In the method of the present invention, since independent DNA fragments can be ligated on a plasmid, it is particularly effective to use an inserted DNA containing two or more genes that exist independently on the genome. . Such a plurality of types of genes preferably include a series of genes necessary for production of a certain substance, and specific examples thereof include degQ, sfp, and pps genes constituting prepastatin. Furthermore, when the inserted DNA contains a series of genes necessary for such substance production, and when the suitable sequence for substance production is known with respect to the sequence of the genes, the distance between the genes, the base sequence, etc., It is preferred to prepare according to them.

  In addition, the DNA containing an effective replication origin in the host microorganism may be any DNA as long as DNA replication is performed in the microorganism transformed with the produced DNA fragment. As described later, Bacillus genus bacteria and the like are used as the host microorganism of the present invention. As a specific microorganism and a DNA containing a replication origin effective in the microorganism, for example, B. In the case of subtilis, Bacillus subtilis has a θ-type replication mechanism (Janniere, L., et al. Gene 8753-61. (1990)), specifically pTB19 (Imanaka, T., et al. J. Gen. Microbiol. 130, 1399-1408. (1984)), pLS32 (Tanaka, T. and Ogura, M. FEBS Lett. 422, 243-246. (1998)), pAMβ1 (Swinfield, TJ, et al. Gene 87, 79-90. (1990)) and other sequences such as replication origins included in plasmids. Moreover, the thing which can adjust the copy number of the produced plasmid freely is preferable. In the case of B. subtilis (Bacillus subtilis), such a DNA having a θ-type replication mechanism is a linearized pGETS109 vector (Japanese Agricultural Chemical Society Vol. 75, Special Issue p. 112). Used.

  In addition to this, the inserted DNA unit containing the inserted DNA and the replication origin may contain an appropriate base sequence as necessary. For example, when a plasmid for expressing a gene contained in an inserted DNA is prepared by the method of the present invention, a nucleotide sequence that controls transcription and translation such as a promoter, an operator, an activator, and a terminator may be included. As a promoter when Bacillus subtilis is used as a host, specifically, Pspac (Yansura, D. and Henner, DJ Proc. Natl. Acad. Sci. USA 81, whose expression can be controlled by IPTG (isopropyl sD-thiogalactopyranoside), 439-443. (1984)), or Pr promoter (Itaya, M. Biosci. Biotechnol. Biochem. 63, 602-604. (1999)).

  The inserted DNA unit is prepared as a plurality of unit DNAs. The unit DNA has a structure that can be repeatedly connected to each other while maintaining the order, and a DNA fragment that is connected one by one means a DNA fragment that becomes one inserted DNA unit. Ligating while maintaining the order of each other means that unit DNAs having sequences adjacent to each other on the inserted DNA unit are linked while maintaining their order and orientation, and repeatedly ligating means 5 ′ of the inserted DNA unit. It means that the 5 ′ end of a unit DNA having an end base sequence is linked to the 3 ′ end of a unit DNA having a 3 ′ end base sequence of an inserted DNA. Specific examples of such unit DNA include, for example, those having ends that can be repeatedly linked to each other while maintaining their order using the complementarity of the base sequences of the protruding ends of the fragments. The structure of this protruding end is not particularly limited. However, if a restriction enzyme recognition sequence or the like may be inserted into the ligation site of the unit DNA, the restriction enzyme recognition sequence can be confirmed by confirming the structure of the DNA fragment for microbial transformation by digestion with a restriction enzyme, This is preferable because a protruding end can be prepared by digestion with a restriction enzyme during the preparation of DNA. As a restriction enzyme recognition sequence, when using an enzyme that recognizes multiple types of sequences, such as SfiI and BstXI, the protruding ends of the unit DNA fragments can be different at each ligation site. This is convenient because it can be digested with one type of restriction enzyme when confirming the structure of the DNA for microbial transformation produced with a restriction enzyme or when producing a unit DNA.

  Examples of the unit DNA include a series of DNA fragments described as the unit DNA in FIG. BsR1st has the base sequence of the blasticidin S gene in the direction of the arrow, CmR2nd has the base sequence of the chloramphenicol gene in the direction of the arrow, and EmR3rd has the base sequence of the erythromycin gene in the direction of the arrow , NmR4th has the base sequence of the tetracycline gene in the direction of the arrow, and SpR5th has the base sequence of the spectinomycin gene in the direction of the arrow. PGETS109SfiI has the base sequence of the repA gene including the replication origin.

  Each unit DNA is prepared so as to be repeatedly ligated while maintaining the order shown in FIG. 1 and the base sequence direction. Specifically, the 3 ′ end of BsR1st is a protruding end that specifically links to the 5 ′ end of CmR2nd, and the 5 ′ end is a protruding end that specifically binds to the 5 ′ end of pGETS109Sfil. It has become. Similarly, the 3 ′ end of CmR2nd and the 5 ′ end of EmR3rd, the 3 ′ end of EmR3rd and the 5 ′ end of NmR4th, the 3 ′ end of NmR4th and the 5 ′ end of SpR5th, and the 5 ′ end of SpR5th and 5 ′ of pGETS109SfiI The end is a protruding end that specifically binds. In addition, each protruding end is designed to be a recognition sequence for SfiI when linked.

The DNA chain length of the unit DNA is not particularly limited, but preferably 0.1 to 100 kb. Further, it may be composed of a sequence that divides the inserted DNA unit. However, when a restriction enzyme recognition sequence is arranged at the end, it is preferable that the base sequence of the gene contained in the inserted DNA unit is not disrupted.
The method for producing the unit DNA may be any method that can produce the unit DNA of the present invention. Preferably, a restriction enzyme recognition sequence that constitutes each protruding end in the base sequence on the template DNA. There is a method in which a DNA fragment is amplified by polymerase chain reaction (PCR) using a primer to which is added, and the obtained DNA fragment is digested with the restriction enzyme. The unit DNA having a protruding end obtained by digestion with a restriction enzyme is preferably purified using an appropriate method. Here, when SfiI is used as a restriction enzyme, the restriction enzyme recognition sequence 5'-ggccnnnnngggcc-3 '(where n is any one of a, c, g, and t) PCR is performed with primers to which different sequences such as 5′-ggccatgtgggg-3 ′, 5′-ggccagagtgggg-3 ′, 5′-ggccagagtggggcc-3 ′, and the like are added.

  The unit DNA thus obtained can be ligated with a DNA ligase or the like to produce a DNA fragment for microbial transformation. The ligation method is not particularly limited, but when the unit DNA has a long chain length, it is preferable to carry out the reaction by mixing equimolar amounts of each unit DNA in the presence of polyethylene glycol and a salt. More preferable examples of the salt include monovalent alkali metal salts. Further, it is more preferable to carry out with a ligation reaction solution containing 10% polyethylene glycol 6000 and 150 mM sodium chloride. The concentration of each unit DNA in the reaction solution is not particularly limited, but is preferably a concentration of 0.1 fmol / μl or more and equimolar. The reaction temperature and time for ligation are not particularly limited. Preferably, the temperature is from room temperature to 37 ° C. and from 5 minutes to 30 minutes.

  The DNA fragment for microbial transformation thus obtained has at least one inserted DNA unit and one or more unit DNAs. Specific examples of the shortest chain length include DNA fragments I to II described in the DNA fragment for microbial recombination in FIG. Moreover, what consists of a several DNA fragment which has unit DNA which mutually overlaps can also be used as such a DNA fragment. Specifically, as shown in FIG. 2, a fragment linked to pGETS109SfiI-BsR1st-CmR2nd-EmR3rd, a fragment linked to CmR2nd-EmR3rd-NmR4th-SpR5th, and an NmR4th-SpR5th-pGETS109S linked The thing which consists of the fragment which has been mentioned. Each of these fragments has at least one inserted DNA unit and does not overlap with one or more unit DNAs. However, each unit DNA of each of these fragments has at least one inserted DNA unit as a whole. And one or more unit DNAs overlapping.

  It is not particularly necessary to select and purify them from the DNA fragments obtained by ligating the unit DNAs. This is because a DNA fragment that does not have the above structure due to transformation of a microorganism with the DNA fragment described later cannot become a plasmid in the microorganism.

(2) Method for producing plasmid DNA containing an insertion unit using a DNA fragment for microbial transformation The DNA fragment and a host cell's competent cell (hereinafter, sometimes simply referred to as “competent cell”) By culturing and recovering plasmid DNA from the microorganism, it is possible to obtain DNA containing the inserted DNA and the replication origin, that is, DNA that can replicate in the microorganism.

  The host microorganism is not particularly limited as long as it has a natural transformation ability. Examples of such microorganisms include those having a natural transformation ability to be processed and incorporated into single-stranded DNA when DNA is incorporated. Specific examples include Bacillus genus bacteria, Streptococcus genus bacteria, Haemophilus genus bacteria, Neisseria genus bacteria, and the like. Examples of Bacillus bacteria include B. subtilis, Bacillus subtilis. megaterium (Bacteria), B. stearothermophilus (medium thermophile) and the like. Among these, the most preferred microorganism is Bacillus subtilis excellent in its natural transformation ability and recombination ability.

However, if there is a base sequence homologous to the inserted DNA in the microbial genome, it is highly possible that the sequence will be homologous recombined with the sequence. When integrating into the genome, there is no particular problem even if a sequence having homology with the sequence at the end of the inserted DNA unit is present.
As a method for making microbial cells competent, a known method suitable for each microorganism can be selected. Specifically, for example, in the case of Bacillus subtilis, the method described in Anagnostopoulos, C. and Spizizen, JJ Bacteriol. 81, 741-746. (1961) is preferably used. As the transformation method, a known method suitable for each microorganism can be used. There is no particular limitation on the amount of ligation product to be given to competent cells. Preferably, the amount is from 1/20 to the equivalent amount, more preferably half the amount of the competent cell culture solution. As a method for purifying the plasmid from the transformant, a known method can be used.

  It can be confirmed by the Southern blotting method, PCR method and the like that are known per se that the plasmid thus obtained has the inserted DNA. In addition, if the inserted DNA is prepared so as to contain a marker gene, such as a drug resistance gene, it can also be confirmed by screening with the drug.

(3) Method for Producing Microorganism Having Insertion DNA Unit in Genomic DNA The second aspect of the present invention has an insertion DNA unit in the genomic DNA by inserting the DNA prepared above into the genomic DNA of the host microorganism. A method for producing a microorganism and the microorganism.

  As a specific method, the insertion DNA unit according to (I) (1), wherein the DNA containing the replication origin effective in the host microorganism is further divided into two common sequences for recombination. A plasmid DNA containing the inserted DNA unit according to (2), which has at both ends thereof (hereinafter sometimes referred to as “DNA fragment for homologous recombination”), DNA having a replication origin effective in the host microorganism further having two different common sequences for recombination at both ends thereof (hereinafter, this may be referred to as “plasmid for homologous recombination”) Co-culture with competent cells of microorganisms whose consensus sequences for recombination exist adjacent to each other in genomic DNA to induce homologous recombination and arbitrarily select multiple DNA fragments A method for producing a microorganism having genomic DNA linked and accumulated in the order and direction (hereinafter sometimes referred to as “indirect method”), or (II) two different commons for recombination An insertion DNA unit comprising a DNA having a sequence at both ends thereof and an insertion DNA unit containing the insertion DNA is prepared as a plurality of unit DNAs having a structure that can be repeatedly connected to each other while maintaining the order, and the unit DNAs are ligated to insert DNA A DNA fragment for microbial cell transformation characterized by producing a DNA fragment having at least one unit and at least one unit DNA overlapping (hereinafter referred to as “DNA fragment for homologous recombination that does not self-replicate”) And a consensus sequence for recombination exists adjacent to the genomic DNA in the same manner as described in (2). A method of producing a microorganism having genomic DNA containing DNA in which homologous recombination is induced by co-culturing with microbial competent cells and a plurality of DNA fragments are linked in an arbitrary order and direction (hereinafter referred to as this) May be referred to as “direct method”).

  The inserted DNA can be the same as in (1) above. In the case of the indirect method, the inserted DNA unit includes an inserted DNA, a replication origin effective in the host microorganism, and two different common sequences for homologous recombination with the genomic DNA of the host microorganism (hereinafter referred to as “ What contains DNA which may be called a "common sequence" at the both ends is used. As the common sequence, sequences at both ends of the inserted DNA can be used. Furthermore, when this is co-cultured with competent cells of the host microorganism, the plasmid DNA containing the inserted DNA unit produced has a structure such that the copy number is low, or a structure capable of controlling the copy number Those having the following are preferably used. Specifically, such a structure is a plasmid whose copy number can be adjusted according to the expression level of the protein required for replication by controlling the promoter with substances such as lactose and its derivative IPTG, xylose, and arabinose. And a temperature-sensitive replication origin and a plasmid whose copy number can be adjusted by temperature using a replication protein. In the direct method, a DNA containing inserted DNA and DNA having a common sequence is used.

  This common sequence is indispensable for the insertion of the inserted DNA into the genomic DNA of the host microorganism and should serve as a scaffold for inserting the inserted DNA into the genomic DNA of the host microorganism. The above common sequence can be incorporated in advance so as to be adjacent to the genomic DNA of the host microorganism such as Bacillus subtilis, or a sequence present in the genomic DNA of the host microorganism can also be used as the common sequence. Between the common sequences adjacent to the genomic DNA of this microbial cell is the insertion site of the inserted DNA unit.

  In the method of the present invention, homologous recombination is induced by coculturing a competent recombination cell of a host microorganism in which a consensus sequence for recombination exists adjacent to the genomic DNA and a plasmid or DNA fragment for homologous recombination. The inserted DNA unit is then introduced into the genomic DNA of the host. Homologous recombination refers to recombination that occurs in a portion having a homologous base sequence of a pair of double-stranded DNAs. As used herein, “a common sequence for recombination” means that the common sequence in the inserted DNA unit and the common sequence in the genomic DNA of the host microorganism are completely identical as long as homologous recombination can occur. There is no need to provide a substantially homologous sequence (for example, a sequence having 60% or more, preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more, most preferably 95% or more). The length of such a common sequence is not particularly limited, but is generally preferably 1 kb or more, for example, preferably about 5 kb to several tens of kb, more preferably about 10 kb to 25 kb. From about 5 to 15% of the length excluding one set of common sequences is optimal.

  As the host microorganism, those described in (2) above can be used. For details of homologous recombination by co-culture of a plasmid for homologous recombination with a host microorganism or DNA fragment for homologous recombination, Mitsuo Ogura, Bioscience and Industry, Vol. 57, No. 8, 532-535 (1999), Mitsutoshi Itaya, It is described in Japanese Journal of Agricultural Chemistry, Vol.67, No.4, 703-706 (1993) and Mitsutoshi Itaya, Journal of Japanese Agricultural Chemistry Society, Vol.68, No.11, 1545-1550 (1994) The method can be used. The recombinant microorganism thus produced is also included in the scope of the present invention.

  In the method of the present invention, the inserted DNA may be a DNA containing a series of genes necessary for substance production. In this case, a genomic DNA containing an intron is used as the inserted DNA. May be used, the gene contained in the genomic DNA containing introns cannot be expressed in Bacillus subtilis, that is, the target substance may not be produced. In such a case, a gene contained in the inserted DNA can be expressed by introducing a protein that induces splicing into the host microbial cell.

(4) DNA fragment suitable for target substance production in microbial cells, and method for selecting and obtaining microorganisms containing the same in genomic DNA The third of the methods of the present invention is suitable for target substance production in microbial cells. A method for selecting and obtaining a DNA fragment and a microorganism containing it in genomic DNA, comprising: (a) a replication origin effective in a host microorganism and / or a common sequence for two different recombination at both ends A plurality of inserted DNA units, each of which has a structure capable of repeatedly linking with each other while maintaining the order of the inserted DNA units including the inserted DNA having the DNA and the series of genes necessary for substance production and linking the genes in different orders (B) linking the unit DNAs to have at least one inserted DNA unit, and overlapping one or more unit DNAs. A step of producing a group of DNA fragments, (c) a step of producing a group of microorganisms containing each insertion unit in the genomic DNA by the method according to claim 12 or 13, using the group of DNA fragments, (d) This is a method comprising the steps of producing a target substance in a microbial cell and analyzing the amount of the target substance.

About the process of said (a)-(c), the method demonstrated in detail to said (1)-(3) can be used.
As a method for producing the target substance in the produced microbial cells, a known conventional method can be used. For example, when the host microorganism is Bacillus subtilis, a plasmid having a gene involved in production is used. Examples include a method of producing in a state where the copy number is increased, or a method of producing a substance in accordance with growth so that a promoter of a gene involved in production can be induced by an inducing substance or environmental factors such as temperature. . Moreover, the analysis of the amount of the target substance produced in the microbial cell can be appropriately selected according to the type of the target substance and the type of the host microorganism. Specifically, for example, when the target substance is an antibacterial substance and it is produced by Bacillus subtilis, the secreted product is obtained by using a method such as high performance liquid chromatography, mass spectrometry, or biological assay. In the case of a bacterial exocrine enzyme such as protease, the amount of production was the highest by measuring the amount of the substrate of the enzyme, such as by changing the absorbance of the substrate substance, etc. By selecting a microbial cell, it is possible to select and obtain a DNA fragment suitable for producing a target substance in the microbial cell, and a microorganism containing it in the genomic DNA.

The present invention will be described more specifically with reference to the following examples. However, the following examples are merely examples of the present invention, and the scope of the present invention is not limited by the following examples.
(material)
As Bacillus subtilis 168 strain and RM125 strain (Uozumi, T., et al. Mol. Gen. Genet., 152, 65-69 (1977)), those inherited in our laboratory were used. Plasmid pTB522, which is derived from moderately thermophilic bacteria and capable of replication in Bacillus subtilis, is described in Imanaka, T .; et al. , J .; Gen. Microbiol. , 131, 1753-1863 (1985). Plasmid pDH88 capable of regulating the expression of the cloned gene by the addition of IPTG is described in Henner, D. et al. H. Methods Enzymol. , 185, 223-228 (1990) were used. E. coli plasmid pBR322 purchased from Takara Shuzo was used. Plasmid pBEST305 carrying the tetracycline resistance gene cassette is described in Itaya, M .; Biosci. Biotech. Biochem. 56, 685-686 (1992). Plasmid pBEST4D carrying a cassette of chloramphenicol resistance gene is described in Itaya, M .; , Et al. J. et al. Biochem. 107, 799-801 (1990). Plasmid pBEST501 carrying the neomycin resistance gene is described in Itaya, M .; et al. Nucleic Acid Res. 17, 4410 (1989). Plasmid pBEST515C carrying the neomycin resistance gene (Pr-neo) under the control of the Pr promoter is disclosed in Itaya, M, Biosci. Biotech. Biochem. 56, 685-686 (1992). Plasmids pCISP310B and pCISP311B are described in Itaya, M .; et al. J. et al. Biochem. , 128, 869-875 (2000).

  Ampicillin, chloramphenicol, erythromycin, tetracycline, spectinomycin, and neomycin antibiotics were purchased from Sigma. The restriction enzyme SfiI was purchased from NEB. The restriction enzyme NotI was purchased from Takara Shuzo. Other restriction enzymes, Alkaline Phosphatase (E. coli), T4 Polynucleotide Kinase, and T4 DNA ligase were purchased from Toyobo. Takara Shuzo's DNA Blunting Kit and DNA Ligation Kit were used for the smoothing and ligation of the DNA fragments used for plasmid preparation using E. coli. For gene amplification by PCR, TaKaRa Ex Taq HS manufactured by Takara Shuzo Co., Ltd. was used according to the attached instructions. PCR reaction conditions were 98 ° C., 5 minutes, and 30 cycles of the following temperature cycle were performed. 98 ° C., 20 seconds; 60 ° C., 30 seconds; 72 ° C., 1 minute per kb amplification length. For cloning of PCR products, TOPO XL PCR Cloning Kit from Invtrogen was used. A lysozyme manufactured by Seikagaku Corporation was used. As the medium component and agar of LB medium, those manufactured by Becton Dickington were used. IPTG (isopropyl s-D-thiogalactopyranoside) was from Sigma. All other biochemical reagents were from Sigma and Nacalai Tesque.

  For construction of plasmids other than those specifically mentioned, Escherichia coli JA221 strain (CGSC #: 7091) was used. Southern hybridization used Roche's DIG DNA Labeling and Detection Kit. Standard protocols (Sambrook J, et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)) are used for procedures such as extraction, assay, and mass preparation of the constructed plasmid. Transformation of competent E. coli JA221 and competent Bacillus subtilis was performed as previously reported (Itaya, M. Mol. Gen. Genet., 241, 287-297 (1993)).

Example 1 Accumulation of Five Drug Resistance Gene Fragments in Plasmid (1) Preparation of Unit DNA A unit DNA containing a drug resistance gene and having a SfiI recognition sequence at the end was prepared as follows. Using the plasmid pCISP310B (Itaya, M. et al. J. Biochem., 128, 869-875 (2000)) having the gene for resistance to blasticidin S, chloramphenicol, and spectinomycin as a template and primers BsR-Sfi1F The blasticidin S resistance gene was amplified by PCR using BsR-Sfi1R (SEQ ID NOs: 8 and 9) and cloned into the pCR-XL-TOPO (Invitrogen) vector to construct plasmid pCRBsR1st. In addition, the chloramphenicol gene was amplified by PCR using cat-SfiI2F and cat-SfiI2R (SEQ ID NOs: 10 and 11) using the plasmid as a template, and a plasmid pCRCmR2nd was constructed in which the chloramphenicol resistance gene was cloned. . Further, a spectinomycin resistance gene was amplified by PCR using spc-SfiI5F and spc-SfiI5R (SEQ ID NOs: 16 and 17), and a plasmid pCRSpR5th was constructed in which the spectinomycin resistance gene was cloned. In addition, erm-SfiI3F and erm-SfiI3R (SEQ ID NOs: 12 and 13) are used using pCISP311B (Itaya, M. et al. J. Biochem., 128, 869-875 (2000)) having an erythromycin resistance gene as a template. The plasmid pCREmR3rd was constructed by amplifying the erythromycin resistance gene from the PCR and cloning the erythromycin resistance gene. Further, pBEST501 (Itaya, M. et al., Nucleic. Acid. Res., 17, 4410 (1989)) plasmid having a neomycin resistance gene is used as a template for neo-SfiI4F and neo-SfiI4R (SEQ ID NOs: 14 and 15). The neomycin resistance gene was amplified by the PCR used, and the plasmid pCRNmR4th obtained by cloning the neomycin resistance gene was obtained.

  In addition, pGETS109SfiI, which is an E. coli-Bacillus subtilis shuttle plasmid having an effective replication origin in Bacillus subtilis and capable of controlling the copy number, and having an SfiI recognition sequence, was constructed as follows. After digesting plasmid pTB522 (Imanaka, T., et al., J. Gen. Microbiol., 131, 1753-1763 (1985)) with restriction enzymes EcoRI and MluI, DNA Blunting Kit (manufactured by TAKARA) was used. This site was smoothed. In addition, plasmid pDH88 (Henner, DH, Methods Enzymol., 185, 223-228 (1990)) was cleaved with restriction enzymes HindIII and BglII and dephosphorylated, and then this site was used using DNA Blunting Kit (manufactured by TAKARA). Was smoothed. Both fragments were ligated to construct pDHrepAF. To clone the terminator, PCR was performed with primers rrnBterm2F and rrnBtermR (SEQ ID NOs: 1 and 2) using the genomic DNA of E. coli JA221 (CGSC #: 7091) as a template, and the amplified fragment was plasmid pCR XL TOPO (manufactured by Invitrogen) Cloning into plasmid pCRrrnBTerm2 was constructed. A fragment obtained by digesting pCRrrnBTerm2 with EcoRI and MunI was ligated to pDHrepAF digested with EcoRI and dephosphorylated to construct plasmid pDHrepAT1. pBR322 (manufactured by TAKARA) was cleaved with the restriction enzyme PvuII and dephosphorylated, and then a fragment obtained by cleaving pDHrepAT1 with EcoRI and BamHI and blunting the ends was ligated to construct a plasmid pBRDHrepAT1F. tetracycline resistance obtained by digesting pBRDHrepAT1F with SmaI and dephosphorylating, then digesting pBEST305 (Itaya, M. Biosci. Biotech. Biochem., 56,685-686. (1992)) with EcoRI and PstI Plasmid pGETS109 was constructed by ligating gene cassettes. A linker prepared by annealing VSfiKpn-1F and VSfiKpn-1R (SEQ ID NOs: 3 and 4) was inserted into the HindIII site of plasmid pGETS109 to prepare plasmid pGETS109SfiI.

  Each plasmid is digested with SfiI, electrophoresed in low-melting point agarose, and the band is cut out to purify unit DNAs having drug resistance genes, BsR1st, CmR2nd, EmR3rd, NmR4th, SpR5th, and pGETS109SfiI. The concentration was examined by electrophoresis of the part. The sequences at the ends of these SfiI fragments are as shown in the unit DNA of FIG.

(2) Ligation of unit DNA
Each unit DNA of BsR1st, CmR2nd, EmR3rd, NmR4th, SpR5th and pGETS109SfiI prepared in (1) above has a final concentration of 0.25 fmol / μl in a volume of 22 μl at the time of ligation, that is, a molar ratio of 1. After adding TE buffer so that the total amount becomes 10 μl, 2 × Linear ligation buffer (132 mM Tris-HCl (pH 7.6), 13.2 mM MgCl2, 20 mM Dithiothreithol, 0.2 mM ATP, 300 Mix 11 μl of mM NaCl, 20% (w / v) polyethylen glycol), add 1 μl of T4 DNA Ligase (4 weiss unit / μl: NEB), and incubate at 37 ° C for 30 minutes. Ligated with. At this time, as shown in FIG. 1, the ligation product is a high-molecular linear DNA in which unit DNA is multiply repeated. Let this sample be (i).

  In addition, in order to investigate the effect on transformation efficiency when the molar ratio of each fragment deviates from 1, (ii) to (ii) among the unit DNA, CmR2nd was not added at all, (Iii) CmR2nd added 8 times compared to other unit DNA (final concentration 2 fmol / μl), (iv) 8 times the concentration of unit DNApGETS109SfiI compared to other unit DNA (final concentration 2 fmol / μl) ) The added ones were also adjusted and ligated in the same manner as in (i) above.

(3) Transformation of Bacillus subtilis 7 μl of the ligation product obtained in (2) above was transferred to Bacillus subtilis RM125 strain (Uozumi, T., et al., Mol. Gen. Genet., 152, 65-69 (1977). )) In a competent cell culture medium (Itaya, M., et al., Mol. Gen. Genet., 241, 287-297 (1993)), respectively, and incubated at 37 ° C. for 30 minutes. Then, in order to express the drug resistance gene contained in each unit DNA, 200 μl of LB medium was added, then cultured at 37 ° C. for 1 hour, spread on LB agar medium containing 10 μg / ml tetracycline (manufactured by Sigma). And cultured at 37 ° C. overnight. The obtained tetracycline resistant colonies were 246 from the ligation product of (i), 12 from (ii), 129 from (iii), and 59 from (iv). For most of these strains, each drug contains the presence or absence of resistance to 5 drugs (blastosidin S, chloramphenicol, erythromycin, neomycin, spectinomycin) whose resistance genes contained in the unit DNA are resistant When examined by replicating with a toothpick on the plate, the proportion of colonies showing resistance to all drugs was (i) 74%, (ii) 0%, (iii) 77%, (iv) 59% It was.

  Among the transformants obtained from the ligation product (i) above, arbitrarily selected 12 colonies were transformed into 10 μg / ml tetracycline (manufactured by SIGMA) and 1 mM to increase the plasmid copy number. Of IPTG (isopropyl sD-thiogalactopyranoside: manufactured by SIGMA) was inoculated into 2 ml of LB medium, cultured at 37 ° C. overnight, and then purified on a small scale. The obtained plasmid was digested with SfiI, subjected to agarose gel electrophoresis, and stained with ethidium bromide. The results are shown in FIG. As a result, BsR1st, CmR2nd, EmR3rd, NmR4th, SpR5th, which are unit DNAs prepared in (1) above, and 8 strains having a plasmid in which the presence of pGETS109SfiI was confirmed. The results were completely consistent with the resistance results with each drug plate described above. From these facts, it was found that ligation efficiency was high when equimolar amounts of each unit DNA were mixed.

  In order to investigate the structure of this plasmid in more detail, one of the plasmids confirmed to contain each unit DNA as described above was transformed into Escherichia coli JA221 (CGSC #: 7091) and As a result of preparing plasmids and examining the cleavage patterns of various restriction enzymes in detail, it was confirmed that fragments of BsR1st, CmR2nd, EmR3rd, NmR4th, and SpR5th were accumulated in this order in the plasmid pGETS109SfiI.

Example 2 Accumulation of three genes degQ, sfp, and pps involved in production of antifungal substance prepastatin in Bacillus subtilis plasmid (1) Gene for prepastatin production Lipopeptide antibacterial substance prepa produced by Bacillus subtilis To date, it has been shown that three genes, degQ (0.3 kb), sfp (1 kb), and pps (38 kb) are required for the production of statins (Tsuge, K., et al. Antimicrob Agents Chemother. 43, 2183-2192. (1999)). Bacillus subtilis 168 strain (Uozumi, T., et al., Mol. Gen. Genet., 152, 65-69 (1977)) used as a host in this example is a mutant degQ gene (degQ0 gene) Displacement-type sfp gene (sfp0 gene) does not produce prepastatin because both degQ gene and sfp gene are dysfunctional due to a single base mutation compared to wild-type degQ gene and sfp gene (Tsuge, K., et. al. Antimicrob. Agents Chemother. 43, 2183-2192. (1999)). Therefore, in this example, an attempt was made to introduce an active form of the same gene into the host as a plasmid and to produce prepastatin in the host.

(2) Removal of mutant degQ sfp and pps genes from Bacillus subtilis 168 strain genome Bacillus subtilis that becomes the host for accumulation of three DNA fragments containing active degQ, sfp and pps genes in the Bacillus subtilis plasmid If these genes remain, the instability of the genome and the plasmid may be caused by homologous recombination between the genome and the plasmid. Therefore, prior to performing the above gene accumulation, first, Bacillus subtilis 168 Mutant degQ, sfp, and pps gene DNA present in the strain genome was completely removed.

To remove mutant degQ gene DNA from the Bacillus subtilis 168 genome, plasmid pCRdegQ (prepared in (3) (ii) described later) was templated with primers degQ2F-Bgl and degQ2R-Bgl (SEQ ID NOs: 18, 19). As described above, pCRΔdegQ was constructed by performing PCR in the opposite direction from both ends of the degQ gene DNA, digesting the obtained fragment with BglII and self-closing. This plasmid has a 0.3 kb degQ gene DNA removed compared to the original plasmid pCRdegQ, and a BglII site is present instead. The region at both ends of the degQ gene cloned in pCRΔdegQ was excised with EcoRI, and cloned into the EcoRI site of pBR322 (TAKARA) to prepare pBRΔdegQF. The plasmid pBEST515C (Itaya, M. Biosci. Biotech. Biochem., 56,685-) was obtained by cleaving the BglII site in the region at both ends of degQ of this plasmid, followed by dephosphorylation and blunt end treatment. The plasmid pBRΔdegQF :: Pr-neo30F was constructed by inserting the blunt-ended Pr-neo fragment obtained by EcoRI digestion of 686. (1992)). This plasmid was digested with PstI to linearize it, and then Bacillus subtilis 168 strain (Uozumi, T., et al. Mol. Gen. Genet., 152, 65-69
(1977)) was transformed into the method described in Itaya, M. Mol. Gen. Genet., 241, 287-297 (1993) and selected for neomycin resistance to completely lack degQ gene DNA. B. subtilis BEST8635 strain having Pr-neo, a neomycin resistance gene, was constructed in that region.

  Next, in order to delete the sfp gene DNA, the 5 'upstream DNA of the sfp gene is used as primers sfp2F-Bam and sfp2R-Bam (SEQ ID NOs: 20, 21), and the 3' downstream DNA is used as the primer sfp3F-Bam. And sfp3R-Bam (SEQ ID NOs: 22 and 23) and amplified by PCR and cloned into pCR-XL-TOPO vector (manufactured by Invirogen). The plasmid containing the 5 'upstream DNA fragment of the sfp gene was designated pCRupsfp, and the plasmid obtained by cloning the 3' downstream DNA was designated pCRdownsfp. pCRdownsfp was cleaved with BamHI, and the resulting DNA fragment containing the 3 ′ downstream region of the sfp gene was inserted into the BamHI site of pCISP310B (Itaya, M. et al. J. Biochem., 128, 869-875 (2000)). The constructed plasmid pC [downsfp /] was constructed. Next, a fragment containing the 5 'upstream region of the sfp gene obtained by cutting the plasmid pCRupsfp with EcpRI was inserted into the EcoRI site of this plasmid to construct a plasmid pC [downsfp / upsfp]. This plasmid was transformed into the BEST8635 strain in the same manner as described above to obtain the BEST8646 strain.

  In the genomic DNA of this strain, there is no sfp gene, but instead a cI repressor gene and a spectinomycin resistance gene cassette are inserted, and the introduced cI repressor gene suppresses the expression of the Pr promoter. Although this strain is a neomycin sensitive strain, when the cI repressor gene disappears, it changes to neomycin resistance (Japanese Patent Laid-Open No. 2001-321170).

  Next, in order to delete the cI repressor gene and spectinomycin resistance gene cassette from the genomic DNA of the BEST8646 strain, a DNA fragment was constructed by linking the 5 'upstream and 3' downstream region DNAs of the sfp gene. First, pCRupsfp was cleaved with PstI, and then an about 1 kb sfp gene 5 'upstream fragment of PstI-EcoT22I was obtained by partial digestion with EcoT22I, which was introduced into the PstI site of pCRdownsfp to obtain pCRdownsfp / upsfp. This plasmid was transformed into Bacillus subtilis BEST8646 strain to obtain BEST8647 strain in which the cassette of cI repressor gene and spectinomycin resistance gene was deleted by recombination by double crossing of sfp gene 5 'upstream and downstream regions.

  Furthermore, in order to erase the pps gene DNA, the 5 ′ upstream region of the pps gene is primer ppsA2F-Bam and ppsA2R-Bam (SEQ ID NOS: 24, 25), and the 3 ′ downstream region is primer ppsE2F-Bam and ppsE2R. -Plasmids pCRupppsA and pCRdownppsE obtained by PCR using the genomic DNA of Bacillus subtilis 168 strain as a template with -Bam (SEQ ID NO: 26, 27) and cloned into the pCR-XL-TOPO vector (Invitrogen), respectively. did. An adapter prepared by annealing the oligo DNAs RVICeuPstF and RVICeuPstR (SEQ ID NO: 28, 29) was inserted between NotI-PstI of pCRdownppsE to prepare pCRdownppsE-ICeuI. This plasmid was obtained by digesting MluI and PstI. The DNA fragment in the 3 ′ downstream region of the pps gene was partially digested with EcoT22I and then ligated to the fragment obtained by digestion with MluI to construct plasmid pCRupppsA / downppsE-ICeuI.

  This plasmid was further partially digested with EcoT22I, and then the fragment obtained with PstI was self-cycled to construct plasmid pCRupppsA / downppsE-ICeuI-last. This plasmid was inserted into the EcoRV site and linearized at the PvuII site of pCISP303 # 6 to construct pCRupppsA / downppsE-ICeuI-last / CISPR3. This plasmid was introduced into the above Bacillus subtilis BEST8647 strain in the same manner as described above to obtain a spectinomycin resistant strain, whereby the pps gene was completely deleted. Instead, the plasmid pCISP303 # 6 (Itaya, M., et al., J. Biochem., 128, 869-875 (2000)) constructed Bacillus subtilis BEST8652 strain inserted in this part. This strain was transformed with pCRupppsA / downppsE-ICeuI-last in the same manner as described above to obtain a neomycin resistant strain, thereby preparing a Bacillus subtilis BEST8666 strain in which the pps gene was completely deleted. This strain is a strain that is completely deficient in the degQ, sfp, and pps genes on the genomic DNA as compared with the original 168 strain, that is, can be expressed as follows. trpC2 ΔdegQ :: Pr-neo Δsfp Δpps.

(3) Acquisition of wild-type degQ, sfp, and pps genes and addition of overhanging ends (i) Acquisition of wild-type degQ and sfp genes First, 1.6 kb containing mutant degQ0 gene for the purpose of obtaining wild-type degQ gene DNA The DNA fragment was amplified by PCR using primers degQF and degQR (SEQ ID NOs: 30 and 31) using the Bacillus subtilis 168 strain genomic DNA as a template and cloned into the pCR-XL-TOPO vector (Invitrogen) to construct plasmid pCRdegQ0. .

  Using this plasmid as a template, PCR was performed using point mutation-introducing primers degQFmut and degQRmut (SEQ ID NOs: 32 and 33) phosphorylated with T4 Polynucleotide kinase in advance. This primer has a base sequence that converts a point mutation of the mutant degQR gene of Bacillus subtilis 168 strain into a wild type. A plasmid pCRdegQ having a wild type degQ gene DNA was constructed by self-closing the PCR product. Furthermore, a DNA fragment containing a 0.3 kb degQ gene was amplified by PCR using primers degQF-BamHI and degQR-BamHI (SEQ ID NOs: 34 and 35) using this plasmid as a template, and this was amplified into a pCR-TL-TOPO vector (Invitrogen Plasmid pCRdegQBamHI11 was constructed.

  Next, functional sfp gene DNA was obtained by a procedure substantially similar to the method for obtaining a DNA fragment containing the degQ gene described above. First, a 1.6 kb region containing a mutant sfp gene was amplified by PCR using primers sfpF and sfpR (SEQ ID NOs: 36 and 37) and Bacillus subtilis 168 genomic DNA as a template, and pCR-XL-TOPO vector (Invitrogen) Plasmid pCRsfp0 cloned in was constructed. Amplify the full length of the PCR plasmid using pCRsfp0 as a template using point mutation-introducing primers sfpF3 and sfpR1 (SEQ ID NOs: 38 and 39) having a sequence for converting a mutant sfpR gene phosphorylated by T4 Polynucleotide kinase into a wild type. Then, by self-closing the PCR product, a plasmid pCRsfp having a wild type sfp gene DNA was constructed. Using this plasmid as a template, a DNA fragment containing a 1 kb sfp gene was amplified by PCR using primers sfpF-BamHI and sfpR-BamHI (SEQ ID NOs: 40 and 41) and cloned into the pCR-TL-TOPO vector. Plasmid pCRsfpBamHI33 was constructed.

(Ii) Preparation of unit DNA by adding SfiI site to degQ and sfp gene The linker HindIII-NotI (SEQ ID NO: 5) was introduced into the HindIII site of plasmid pBR322 to construct plasmid pBR322NotI. The oligonucleotides 1st-F and 1st-R having the nucleotide sequences shown in SEQ ID NOs: 42 and 43 and the oligonucleotides 2nd-F and 2nd-R having the nucleotide sequences shown in SEQ ID NOs: 44 and 45 were annealed, respectively. Adapters 1st and 2nd having two sites were prepared, and plasmids pBR-1st and pBR-2nd were constructed by incorporating them into the above-described pBR322NotI, respectively. These plasmids have a BglII site between two SfiI sites (6 bp). A fragment containing degQ obtained from BamHI digestion of plasmid pCRdegQBamHI11 prepared in (3) (i) above was introduced into the BglII site, and pBR-degQ1stF and pBR-degQ1stR in the opposite direction were introduced from pBR-1st. Moreover, pBR-degQ2ndF and the reverse pBR-degQ2ndR were constructed from pBR-2nd, respectively. Similarly, a fragment containing sfp obtained by BamHI digestion of the plasmid pCRsfpBamHI33 prepared in (3) (i) above was introduced into the BglII site of pBR-1st or pBR-2nd, and pBR- sfp1stF and its reverse pBR-sfp1stR were constructed, and pBR-sfp2ndF and its reverse pBR-sfp2ndR were constructed from pBR-2nd, respectively.

(Iii) Acquisition of pps gene and production of unit DNA with SfiI site added
The pps gene DNA fragment was obtained by the method of recombination transfer (JP 2002-017350, Tsuge, K. and Itaya, MJ Bacteriol. 183,5453-5458. (2001)) as follows. In this case, the whole was prepared so as to be obtained as a fragment of SfiI. First, using the primers ppsAF-SfiI and ppsAR-NotI (SEQ ID NO: 46, 47) and primers ppsEF-NotI and ppsER-SfiI (SEQ ID NO: 48, 49) The plasmids pCRppsA and pCRppsE were obtained by PCR using Bacillus subtilis 168 genomic DNA as a template and cloned into pCR-XL-TOPO (Invitrogen).

  The plasmid pGETS109NotI in which the HindIII site of pGETS109 prepared in Example 1 (1) was cleaved and a HindIII-NotI linker (SEQ ID NO: 5) was inserted was prepared. A linker prepared by annealing V-3rd-F and V-3rd-R (SEQ ID NOs: 6 and 7) was inserted into the NotI site of this plasmid to prepare plasmid pGETS109V-3rdF. Furthermore, pBRDHrepAT1F prepared in Example 1 (1) was cleaved with SmaI and dephosphorylated, and then pBEST4D (Itaya, M., et al. J. Biochem., 107,799-801. (1990)) was digested with SalI. The plasmid pGETS113 was constructed by ligating the chloramphenicol resistance gene cassette obtained by blunting the ends.

  pCRppsE was digested with HindIII and cloned into the HindIII site of the plasmid pGETS113 to prepare pGETS113ppsE. The fragment obtained by digesting pCRppsA with NotI was cloned into the NotI site of this plasmid, and pGETS113ppsAE was cloned. It was constructed. The boundary between the two ends incorporated into the plasmid pGETS113ppsAE is a NotI site, which is cut with NotI and transformed into Bacillus subtilis 168, and 38 kb in the genome of Bacillus subtilis 168 by recombination transfer. The pps region of was transferred into a plasmid as follows.

  1 μg of pGETS113 ppsAE prepared in large quantities by the cesium chloride density gradient method was reacted with 10 U of restriction enzyme NotI at 37 ° C. for 1 hour to form a linear plasmid containing 100 μl of competent cells of Bacillus subtilis 168 strain The medium was mixed well in a 1.5 ml tube and incubated at 37 ° C. for 30 minutes. 300 μl of LB medium was added to this and mixed well, followed by further incubation at 37 ° C. for 1 hour. Select a chloramphenicol resistant transformant by applying the entire volume of this culture to a 1.5% agar LB plate containing chloramphenicol (final concentration 5 μg / ml) and culturing overnight at 37 ° C. did. This colony was inoculated into 100 ml of LB liquid medium containing chloramphenicol (final concentration 5 μg / ml) and cultured overnight at 37 ° C. (17 hours). IPTG (isopropyl s-D-thiogalactopyranoside: manufactured by SIGMA) was added to this culture solution to a final concentration of 1 mM, and further cultured at 37 ° C. for 6 hours. After completion of the culture, the cells were collected, lysed with lysozyme, solubilized with sarkosyl, and then fractions containing the plasmid were collected by ethidium bromide-cesium chloride density gradient ultracentrifugation. This was purified again by ethidium bromide-cesium chloride density gradient ultracentrifugation to obtain plasmid pGETS113ppsABCDE. In this plasmid, 38 kb ppsABCDE (hereinafter abbreviated as pps) can be excised as an SfiI fragment.

(4) Preparation of unit DNA containing wild-type degQ, sfp, and pps genes All DNA fragments having 3 ′ terminal overhangs of specific 3 bases, which are unit DNAs prepared in (3) above, are obtained by digestion with SfiI. Designed to be obtained. The plasmid pGETS113ppsABCDE was digested with SfiI, followed by electrophoresis in a low-melting point agarose gel. The desired fragment was excised from the gel and purified to obtain a 38 kb pps fragment. The vector pGETS109V-3rdF was also digested with SfiI and the vector portion was purified. PBR-degQ1stF, pBR-degQ2ndF, pBR-sfp1stF, pBR-sfp1stR, pBR-sfp2ndF, and pBR-sfp2ndR were similarly digested with SfiI from the plasmids containing each unit DNA prepared in (3) and (iii) above. Thereafter, it was separated from the vector by agarose gel electrophoresis to obtain fragments degQ1stF, degQ2ndF, sfp1stF, sfp1stR, sfp2ndF, and sfp2ndR. The ends of these fragments are as shown in FIG. The concentration of these fragments was determined by electrophoresis after partial purification, and then adjusted with TE buffer to 2.5 fmol / μl.

(5) Construction of unit DNA containing degQ, sfp, and pps genes In order to analyze the influence on the production of prepastatin in transformed Bacillus subtilis by the difference in the order and direction of ligation in the unit DNA , Four plasmids (a) pGETS-pps-degQ1stF-sfp2ndF, (b) pGETS-pps-degQ1stF-sfp2ndR, (c) pGETS-pps-sfp1stF-degQ2ndF, (d) pGETS-pps-sfp1stR-degQ2ndF (Figure 4) ) Was prepared by the following procedure. (A) 0.5 g of each degQ1stF, sfp2ndF, pps, and pGETS109V-3rdF SfiI fragments obtained in (4) above at 2.5 fmol / μl and 2 × Linear ligation buffer (132 mM Tris-HCl (pH 7.6 ), 13.2 mM MgCl2, 20 mM Dithiothreithol, 0.2 mM ATP, 300 mM NaCl, 20% (w / v) polyethylen glycol (2.5 μl) are mixed, and T4 DNA Ligase (4 weiss unit / μl: manufactured by NEB) is mixed there. Was added, and ligated by incubating at 37 ° C. for 30 minutes. For the rest of the plasmid production, the SfiI fragment of degQ1stF and sfp2ndF added to (a) was changed to degQ1stF and sfp2ndR in (b), sfp1stF and degQ2ndF in (c), and sfp1stR and degQ2ndF in (d), respectively. And ligated as in (a). These ligation products were added to 50 μl of competent cell culture solution of Bacillus subtilis BEST8666 strain, respectively, and incubated at 37 ° C. for 30 minutes. Thereafter, in order to express the drug resistance gene, 200 μl of LB medium was added, cultured at 37 ° C. for 1 hour, spread on LB agar medium containing 10 μg / ml tetracycline, and cultured at 37 ° C. overnight.

(6) Structure confirmation of constructed unit DNA The number of tetracycline resistant strains obtained from each ligation product was 15 to 47. Select 6 strains from each and add 10 μg / ml tetracycline (SIGMA) and 1 mM IPTG (isopropyl sD-thiogalactopyranoside: SIGMA) to increase plasmid copy number 2 ml The LB medium was inoculated and cultured at 37 ° C. overnight, and the plasmid was purified on a small scale. FIG. 5 shows the result of digesting the obtained plasmid with SfiI, performing pulse field gel electrophoresis, and staining with ethidium bromide. In addition, degQ and sfp fragments, which are difficult to confirm by ethidium bromide staining, were confirmed by Southern hybridization using degQ or sfp probes (FIG. 5: PFGE sfp probe, PFGE degQ probe). . As a result, 3 or more of 6 samples each had pps, degQ, sfp and pGETS109V-3rd of the correct size (a clone with a circle on the lane in FIG. 5).

(7) Confirmation of gene expression of library plasmid In order to confirm whether the genes in the prepared plasmids (a) to (d) function, a strain having the plasmids (a) to (d) Each strain was selected and No. 3 medium supplemented with 10 μg / ml tetracycline (manufactured by SIGMA) and 1 mM IPTG (manufactured by SIGMA) (10 ml of Bacto Tryptone (manufactured by Becton Dickinson) 10 g, glucose 10 g, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.5 g dissolved, adjusted to pH 6.8 with sodium hydroxide) and shaken at 30 ° C for 5 days After culturing, the methanol extract of the acidic precipitate in the culture was analyzed by high performance liquid chromatography. As shown in FIG. 6, the Bacillus subtilis BEST8666 strain does not produce prepastatin because it does not have pps, degQ, or sfp, but among the strains in which the above plasmid was constructed, It was confirmed that pripastatin production was confirmed and gene accumulation was performed correctly.

Example 3 Insertion of three genes degQ, sfp, pps involved in production of antifungal substance prepastatin into Bacillus subtilis genomic DNA via gene accumulation by plasmid (1) Preparation of unit DNA of degQ, sfp, pps genes The unit DNA sfp1stF, sfp1stR, sfp2ndF, sfp2ndF, pps and vector pGETS109V-3rd used in this Example are the same as those prepared in Example 2 (4). pGETS109V-3rd is a plasmid derived from pGETS109. The average copy number in Bacillus subtilis cells is about 1 copy, but the expression level of the gene repA encoding the plasmid replication protein is expressed by Isopropyl 1-thio-β-galactoside (IPTG). When IPTG is added to the medium, the copy number of the plasmid increases according to the expression level of RepA protein, and is amplified to about 10 copies when 1 mM IPTG is added. When confirming the structure of plasmid DNA, add IPTG and culture, but when removing the plasmid from the plasmid-bearing strain, culture for about 10 generations without IPTG and without applying drug selection pressure. Can easily obtain a plasmid-depleted strain. The unit DNA of the degQ gene was newly prepared as follows, unlike that of Example 2.

  Using a primer degQ2BamHF and degQR-BamHI (SEQ ID NO: 50, 35), a DNA fragment (hereinafter referred to as degQ ') containing a 0.6 kb degQ gene was amplified by PCR, and this was pCR-XL-TOPO vector (Invitrogen) To obtain plasmid pCRdegQ'BamHI. Thereafter, the fragment containing degQ 'obtained from the BamHI digest of pCRdegQ'BamHI was cloned into the BglII sites of pBR-1st and pBR-2nd in the same manner as in Example 2 (3) ii. PBR-degQ'1stF was constructed from pCR-1st and pBR-degQ'2ndF was constructed from pCR-2nd. After preparing each plasmid by the method of Example 2 (3) iii, after digesting the above plasmid with SfiI by the same method as in Example 2 (4), the plasmid was separated from the vector by agarose gel electrophoresis and fragment degQ '1stF and degQ'2ndF were obtained. These fragments were prepared with TE buffer so that the concentration was 2.5 fmol / ml after measuring the concentration by electrophoresis of a part after purification.

(2) Preparation of Host Microbial Strain that Accepts Inserted DNA Unit BEST8666 strain prepared by Example 2 (3) is DdegQ :: Pr-neo, Dsfp, Dpps. In order to introduce a common sequence (pBR322) for introducing an accumulation unit into the bacterium by recombination between homologous sequences and a marker gene unit for detecting recombination, pCRupppsA prepared in Example 2 (2) An I-PpoI adapter prepared by annealing SEQ ID NOS: 51 and 52 was inserted into the EcoRV site of / downppsE-ICeuI-last, and pCRupppsA / downppsE-ICeuI-last-IPpoIF and pCRupppsA / downppsE-ICeuI depending on the insertion direction. Two plasmids -last-IPpoIR were constructed. Similarly, pCISP303 # 6-IPpoIR was constructed by inserting an I-PpoI adapter prepared by annealing SEQ ID NOs: 51 and 52 into the PvuII site of pCISP303 # 6 used in Example 2 (2). pCISP303 # 6-IPpoIR linearized at the I-PpoI site at the I-PpoI site of pCRupppsA / downppsE-ICeuI-last-IPpoIR and pCR [upppsA / downppsE-ICeuI-last-IPpoI] B- 3 was constructed, and this was inserted using the homology of the upstream and downstream regions of Dpp of Bacillus subtilis BEST8666 strain to construct BEST8824 strain that became Dpps :: pBR322: :( cI spc). Similarly, pCISP303 # 6-IPpoIR linearized at the I-PpoI site was introduced into the I-PpoI site of pCRupppsA / downppsE-ICeuI-last-IPpoIF, and pCR [upppsA / downppsE-ICeuI-last-IPpoI ] A28-3 was constructed and inserted into Dpps of Bacillus subtilis BEST8666 strain to construct BEST8827 strain that became Dpps :: pBR322: :( cI spc). That is, pBR322: :( cI spc) of BEST8824 strain and BEST8827 strain is cassetteized by the I-PpoI site, and the direction of the cassette is different between both strains. The structures of these plasmids are shown in FIG. In the figure, squares with vertical stripes indicate common sequences for recombination.

(3) Preparation of inserted DNA unit BEST8824 and BEST8827 strains were analyzed in order to analyze the effect on the production of prepastatin in transformed Bacillus subtilis due to differences in ligation in the unit DNA and in the genome. As a library construction reflecting the difference in the order and direction of gene fragments in the multigene accumulation by the method of Example 2 (5), using the two strains of (A) pGETS-pps-degQ'1stF-sfp2ndF, (b) pGETS-pps-degQ'1stF-sfp2ndR, (c) pGETS-pps-sfp1stF-degQ'2ndF, (d) pGETS-pps-sfp1stR- degQ′2ndF (FIG. 7) was prepared by the following procedure. (A) is 2.5 fmol / ml degQ'1stF obtained in (1) and sfp2ndF, pps, pGETS109V-3rdF obtained in Example 2 (4), 0.5 ml each, 2 × Linear. Mix 2.5 ml of ligation buffer (132 mM Tris-HCl (pH 7.6), 13.2 mM MgCl ₂ , 20 mM Dithiothreithol, 0.2 mM ATP, 300 mM NaCl, 20% (w / v) polyethylen glycol), and add T4 DNA. Ligase (4 weiss unit / ml) 0.5 ml was added, and ligation was performed by incubating at 37 ° C. for 30 minutes. In the rest of the plasmid preparation, the SfiI fragment of degQ'1stF and sfp2ndF added to (a), (b) to degQ'1stF and sfp2ndR, (c) to sfp1stF and degQ'2ndF, (d) to sfp1stR And ligQ as in (a) with degQ'2ndF. These ligation products were added to 50 ml of competent cell cultures of Bacillus subtilis BEST8824 and BEST8827, respectively, and incubated at 37 ° C. for 30 minutes. Thereafter, in order to express the drug resistance gene, 200 ml of LB medium was added, cultured at 37 ° C. for 1 hour, spread on LB agar medium containing 10 mg / ml tetracycline, and cultured at 37 ° C. overnight.

(4) Insertion of constructed insertion DNA unit into host genomic DNA The number of tetracycline resistant strains obtained from each ligation product was 13 to 519 colonies. Select 6 strains from each, inoculate 2 ml of LB medium containing 10 mg / ml tetracycline, and add 1 mM IPTG to increase the copy number of the plasmid. After overnight culture at 37 ° C., the plasmid was purified on a small scale from the culture broth containing IPTG. By electrophoresing the purified plasmid as it is without restriction digestion, select each clone that has correctly accumulated unit DNA in the plasmid using the size as an index, and add IPTG in the culture solution of this strain. The culture without culturing was diluted 100-fold or 10000-fold with LB medium, 100 ml was smeared on a plate containing 3 mg / ml neomycin, and cultured at 30 ° C. overnight. CI sandwiched between two consensus sequences by recombination between the recombination consensus sequence (pBR322 sequence) inserted in the Dpps region and the pBR322 sequence present in the vector part of the integrated plasmid. A strain that became resistant to neomycin was obtained by replacing the repressor gene with an accumulation unit. This structure is shown in FIG. In the figure, Nm represents neomycin, Sp represents spectinomycin, Tc represents tetracycline, R represents resistance, and S represents sensitivity.

The frequency of appearance of the neomachine resistant strain was one in 10 ⁴ to 10 ⁶ plasmid-bearing strains. 25 of each were replicated on a plate containing 10 mg / ml tetracycline, a plate containing 3 mg / ml neomycin, and a plate containing 50 mg / ml spectinomycin, and the accumulation unit in the plasmid was When introduced into the genome using the homologous region of the common sequence, the cI repressor gene and the spectinomycin resistance gene disappear, and the phenotype shown when no plasmid remains is neomycin resistance, tetracycline sensitivity 1 to 25 neomycin sensitive strains were obtained. After culturing these strains, genomic DNA was prepared in an agarose gel block, digested with restriction enzyme I-PpoI, and analyzed by pulse field gel electrophoresis. As a result, an accumulation unit was inserted into the original host. A band of about 44 kb was detected (FIG. 9). Furthermore, we confirmed that degQ, sfp, and pps of the three genes were linked in the order specified by PCR and Southern hybridization using genomic DNA as a template. Four strains having the integration unit were named BUSY8120, 8121, 8345, 8124 strains, and four strains having the integration units (a) to (d) in the BEST8827 strain genome were named BUSY8127, 8128, 8352, 8243 strains.

(5) Comparison of gene expression levels in the library 4 strains having the integrated units (a) to (d) in the BEST8824 strain genome prepared in Example 3 (4) and (a) to ( d) A total of 8 libraries including 4 strains with accumulation units were pre-cultured overnight at 37 ° C. in LB medium, and then ACS medium (100 g sucrose, 11.7 g citric acid, sodium sulfate per liter of medium) 4 g, yeast extract 5 g, diammonium hydrogen phosphate 4.2 g, potassium chloride 0.76 g, magnesium chloride hexahydrate 0.42 g, zinc chloride 0.0104 g, ferric chloride hexahydrate 0.0245 g, manganese chloride・ Dissolve 0.0181 g of tetrahydrate, adjust to pH 6.9 with ammonia water, and inoculate in autoclave), shake culture at 30 ° C for 5 days, and then rapidly extract the methanol extract of the acidic precipitate in the culture. Production of prepastatin in each strain by liquid chromatography Measured, it was confirmed all of the production of plipastatin than stock. Each prepastatin production amount is BUSY8243 (56 mg / ml), BUSY8127 (55 mg / ml), BUSY8128 (54 mg / ml), BUSY8352 (53 mg / ml), BUSY8345 (45 mg / ml) ), BUSY8124 strain (43 mg / ml), BUSY8120 strain (41 mg / ml), BUSY8121 strain (36 mg / ml) in this order. Productivity depends on the order of the 3 genes in the integrated unit and the orientation in the genome. Was different.

Example 4 Direct insertion of three genes degQ, sfp, and pps, which are involved in the production of the antifungal substance prepastatin, into the Bacillus subtilis genome. In the present example, the insertion unit after ligation was directly transformed into the host fungus, and then the DNA unit directly inserted into the genome was selected.
(1) Preparation of unit DNA The same one as in Example 3 was used.
(2) Host strains that accept the inserted DNA unit BEST8824 strain and BEST8827 strain prepared in Example 3 were used.
(3) Preparation of Inserted DNA Unit and Integration into Host Genome An integrated unit pps-degQ′1stF-sfp2ndF (corresponding to (a) of Example 3) was prepared by the following procedure. 2.5 ml each of SfiI fragments of degQ'1stF obtained in (1) and sfp2ndF, pps, and pGETS109V-3rdF obtained in Example 2 (4), 2 × Linear ligation buffer (132 mM) Mix 2.5 ml of Tris-HCl (pH 7.6), 13.2 mM MgCl ₂ , 20 mM Dithiothreithol, 0.2 mM ATP, 300 mM NaCl, 20% (w / v) polyethylen glycol, and add T4 DNA Ligase (4 weiss unit) Ligation was performed by adding 0.5 ml / ml) and incubating at 37 ° C. for 30 minutes. The ligation product was added to 50 ml of competent cell cultures of Bacillus subtilis BEST8824 and BEST8827 strains, respectively, and incubated at 37 ° C. for 30 minutes. Thereafter, in order to express the drug resistance gene, 200 ml of LB medium was added, and cultured at 37 ° C. for 1 hour, then spread on LB agar medium containing 3 mg / ml neomycin, and cultured at 37 ° C. overnight. 95 strains were obtained from BEST8824 strain, and 181 neomycin resistant strains were obtained from BEST8827 strain. Replicate all strains to a plate containing 10 mg / ml tetracycline, a plate containing 3 mg / ml neomycin, and a plate containing 50 mg / ml spectinomycin. By introducing the sequence homology region into the genome, the cI repressor gene and the spectinomycin resistance gene disappear, and the phenotype shown when the vector pGETS109V-3rdF does not remain, neomycin resistance, 24 and 58 tetracycline sensitive and neomycin sensitive strains were obtained, respectively (FIG. 10). In general, these candidate strains include mutants that naturally lack the cI repressor gene and the spectinomycin resistance gene, so the resulting neomycin resistance, tetracycline susceptibility and neomycin sensitivity strains have the pps gene. Is screened by colony PCR using primers ppsE-F and ppsE-R (SEQ ID NOs: 53 and 54). One strain from the BEST8824 strain and eight strains from the BEST8827 transformant A positive strain was obtained. From each of these strains, one strain (BEST8830 strain and BEST8831 strain, respectively) was arbitrarily selected, and genomic DNA was prepared in gel, then cleaved with restriction enzyme I-PpoI, and analyzed by pulse field gel electrophoresis. An accumulation unit of about 44 kb was confirmed from both strains (FIG. 11).

(4) Confirmation of gene expression of insertion unit In order to confirm whether the genes accumulated in the genome function, BEST8830 strain and BEST8831 strain were treated at 37 ° C. in LB medium by the same method as in Example 3 (5). After overnight pre-culture, inoculated into ACS medium, and after 5 days of shaking culture at 30 ° C, the acidic precipitate methanol extract of the culture was analyzed by high performance liquid chromatography. Production was confirmed and each unit DNA was confirmed to function.

According to the present invention, using a competent cell transformation method such as Bacillus subtilis which is a kind of fine soil bacteria, it is possible to obtain a plasmid in which a plurality of DNA fragments are linked by a single ligation operation. In other words, it was shown that ligation and integration of a huge DNA into an amplifiable DNA fragment, which was difficult with the conventional method, can be performed very simply.
In addition, as shown in Example 2, the method of the present invention can be integrated without any problem even if the length of the unit DNA is large (minimum 0.3 kb, maximum 38 kb), and the integration unit can be 50 kb or more. Therefore, it can be arbitrarily linked without considering the size of the unit DNA at all. Many eukaryotic genes including mammals (including mice and humans) and plants have a length exceeding 10 kb, but these genes can also be handled as unit DNA.

  In addition, the pps encoding the prepastatin synthesis gene used in Example 2 is a multigene integrated plasmid prepared by the method of the present invention, despite a sequence having 10 repeats every about 3 kb. It was also revealed that it was stably maintained and expressed in Bacillus subtilis. Such a repetitive sequence is widely found in eukaryotes including mammals (including mice and humans), plants, etc., but the method of the present invention can also be applied to DNA accumulation having repetitive sequences.

  That is, the method of the present invention or the microbial cell in which the inserted DNA is inserted into the genomic DNA, and the useful material etc. that could not be incorporated into the host cell due to the large size of the gene, are artificially and efficiently produced. It becomes possible.

It is a figure which shows the structure of the plasmid DNA acquired from the microorganisms transformed by five types of unit DNA, the DNA fragment for microorganisms transformation which consists of these, and this DNA fragment. It is a figure which shows the structure of the DNA fragment for microorganisms transformation which consists of several DNA fragments which have unit DNA which mutually overlaps, and the structure of the plasmid DNA acquired from the microorganisms transformed by this DNA fragment. It is the photograph of the agarose gel electrophoresis which shows the analysis result of the plasmid which a transformant has. It is a figure which shows the base-base sequence of the terminal of each fragment of degQ, sfp, and pps of three genes which are concerned with the production of pripastatin, and the structure of the library constructed | assembled by these. It is the photograph which shows the result of having performed the agarose gel electrophoresis after cut | disconnecting the plasmid obtained from six transformants each with restriction enzyme SfiI, and the photograph which shows the result of the Southern hybridization which used degQ and sfp as a probe. It is a graph which shows the result of having analyzed the productivity of the prepastatin of Bacillus subtilis which has each plasmid of a library by the high performance liquid chromatography. It is a figure which shows the structure of four types of insertion DNA, the genome structure of the host microorganism which integrated it, and the figure which shows the result of the productivity of the prepastatin of the obtained library. It is a figure which shows the procedure which integrates an insertion unit in the genome of the microorganism strain which has the plasmid of the insertion DNA which consists of three types of unit DNA. It is a figure which shows that the insertion DNA unit was integrated in the genomic DNA of the host microorganism. It is a figure which shows the procedure for integrating the polymer linear DNA which the insertion unit repeated repeatedly in the microorganism genome without going through a plasmid structure. If the double crossover recombination site is between distant insertion units, a second recombination occurs between redundant regions. It is a figure which shows that the insertion DNA unit was integrated in the genomic DNA of the host microorganism.

Claims (5)

It consists of DNA containing a replication origin effective in the host Bacillus subtilis , two or more genes, and 1
An inserted DNA unit containing an inserted DNA having a length of 0 kb or more is divided into unit DNAs having a structure that can be joined in order while utilizing the complementarity of the base sequences of each other, and the unit DNA is subjected to a ligation reaction. subjected, the cells by contacting the competent cells of the ligation reaction solution and Bacillus subtilis transformed, from the transformants obtained, and obtains the plasmid DN a comprising inserting DNA unit the method for manufacturing a B. subtilis cell transformation plasmid DN a. The plasmid DNA containing the inserted DNA unit has two common sequences at both ends of the inserted DNA which are common to the base sequence on the Bacillus subtilis genome used when the inserted DNA is further inserted into the Bacillus subtilis genome by homologous recombination. The method for producing plasmid DNA for Bacillus subtilis cell transformation according to claim 1. The method for producing plasmid DNA for Bacillus subtilis cell transformation according to claim 1 or 2 , wherein the unit DNA is ligated by mixing equimolar amounts of each unit DNA in the presence of a salt and polyethylene glycol. . The method for producing plasmid DNA for Bacillus subtilis cell transformation according to any one of claims 1 to 3 , wherein the inserted DNA contains a series of genes necessary for substance production. Including B. subtilis cells transformed plasmid DNA prepared by the method of claim 1 to 4, the insert DNA unit characterized in that competent cells and Kyo培nourishing the B. subtilis genome DNA A method for producing Bacillus subtilis .

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