JP2011160686A - New variant of bacillus subtilis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a variant of Bacillus subtilis from which a large region of genome is deleted based on a wild strain, and to provide a method for producing a gene product by using the variant. <P>SOLUTION: The variant of the Bacillus subtilis has a genome structure obtained by deleting any one of the following regions in the genome of Bacillus subtilis 168 strain from the genome region of MGB874 strain of the variant of the Bacillus subtilis: (a) ybbU-ybfI region; (b) ydjM-cotA region; (c) yefA-yesX region; (d) yfiB-yfiX region; (e) yhcE-yhcU region; (f) yhaU-yhaL region; (g) yjbX-yjlB region; (h) xkdA-ykcC region; (i) bpr-ylmA region; (j) flgB-cheD region; (k) ynfF-ppsA region; (l) yoxC-yobO region; (m) spoVAF-spoIIAA region; (n) spoIIIAH-yqhV region; (o) ytvB-ytqB region; (p) yteA-ytaB region; (q) yuaJ-yugO region; (r) yusJ-mrgA region; (s) gerAA-yvrI region; (t) yvaM-yvbK region; (u) araE-yveK region; (v) yvdE-yvcP region; (w) gerBA-ywsC region; (x) ywrK-ywqM region; (y) spoIIID-ywoB region; (z) slp-ylaF region; (aa) licH-sigY region; (ab) yqeF-yrhK region; (ac) yuzE-yukJ region; and (ad) yncM-yndN region. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel Bacillus subtilis mutant and a method for producing a target gene product using the Bacillus subtilis mutant.

  The industrial production of useful substances by microorganisms includes a wide variety of types including foods such as alcoholic beverages, miso and soy sauce, as well as amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, proteins, etc. In addition, its application has been extended to a wide range of fields from foods, pharmaceuticals, detergents, daily necessaries such as cosmetics to various chemical raw materials.

  In industrial production of useful substances by such microorganisms, improvement of productivity is one of the important issues, and breeding of produced bacteria by genetic techniques such as mutation has been performed as a technique. Particularly recently, with the development of microbial genetics and biotechnology, more efficient breeding of production bacteria using genetic recombination techniques has been carried out. Furthermore, in response to the rapid development of genome analysis technology in recent years, attempts have been made to decode genome information of target microorganisms and actively apply them to industry.

  In recent years, about Bacillus subtilis, about 4100 kinds of gene-disrupted strains present in the Bacillus subtilis genome have been comprehensively studied, and it has been pointed out that 271 genes are essential for growth (Non-patent Document 1). . In addition, the genes involved in the early stages of sporulation such as Bacillus subtilis, protease genes, genes involved in D-alanine addition to teichoic acid in the cell wall or cell membrane, and genes involved in biosynthesis or secretion of surfactin alone are missing. Lost or inactivated strains have been constructed (see Patent Documents 1 to 8).

  As for Bacillus subtilis, mutants having excellent productivity of various enzymes have been found by comprehensive analysis of mutants having large genomic regions or genes deleted. The MGB874 strain, one of the Bacillus subtilis mutant strains obtained in this study, is derived from Bacillus subtilis Marburg No.168 (Bacillus subtilis 168), and is a mutant strain in which a total of 874 kb of the genome has been deleted. However, the protein secretion productivity is significantly improved as compared to the wild-type 168 strain (Patent Document 9).

  On the other hand, microorganisms that maintain the same or higher gene product productivity compared to the wild type in spite of large genomic deletions are less robust. It is easy to do. For example, the MGB874 strain is a gene disruption strain as long as 874 kb, and is promising as a model for analyzing the gene product synthesis mechanism.

  However, there is a need for a Bacillus subtilis mutant strain with improved productivity in order to further improve productivity in industrial production of the target gene product. In addition, for more detailed analysis of the gene product synthesis mechanism of organisms, mutations that have the same gene product productivity as the wild type but have lost as much genome as possible compared to the wild type Stocks are sought.

JP-A-58-190390 JP 61-1381 WO89 / 04866 pamphlet Japanese National Patent Publication No. 11-509096 Japanese Patent No. 3210315 Special table 2001-527401 Special Table 2002-520017 Special table 2001-503641 Japanese Unexamined Patent Publication No. 2007-130013

K. Kobayashi et al., Proc. Natl. Acad. Sci. USA., 100, 4678-4683, 2003

  The present invention relates to providing a Bacillus subtilis mutant strain in which a large region of the genome is deleted from a wild strain, and a method for producing a target gene product using the Bacillus subtilis mutant strain.

  The present inventors have found that in a Bacillus subtilis mutant derived from Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168), a large region of the 168 genome has been deleted, further genomic regions are deleted. Tried. As a result, the inventors succeeded in obtaining a mutant strain that can maintain viability even when a large genomic region is further deleted from MGB874, which lacks a large genomic region compared to the wild strain. Furthermore, the present inventors have succeeded in obtaining a recombinant Bacillus subtilis mutant strain that is superior in productivity of the target product by incorporating a gene encoding the target product into the mutant strain.

That is, the present invention relates to a Bacillus subtilis mutant strain having a genome structure in which the genome region is further deleted from the genome region of the Bacillus subtilis mutant strain MGB874.
The present invention also relates to a recombinant Bacillus subtilis in which a gene encoding a target gene product has been introduced into the Bacillus subtilis mutant strain so that the gene can be expressed.
The present invention also relates to a method for producing a target gene product using the above recombinant Bacillus subtilis.

In one aspect, the present invention provides the following.
(1) Bacillus having a genomic structure in which any one of the following regions (a) to (ad) shown in the genome of Bacillus subtilis 168 is deleted from the genome region of Bacillus subtilis mutant MGB874 Fungal mutant:
(a) ybbU-ybfI region;
(b) ydjM-cotA region;
(c) yefA-yesX region;
(d) yfiB-yfiX region;
(e) the yhcE-yhcU region;
(f) yhaU-yhaL region;
(g) yjbX-yjlB region;
(h) the xkdA-ykcC region;
(i) bpr-ylmA region;
(j) flgB-cheD region;
(k) ynfF-ppsA region;
(l) yoxC-yobO region;
(m) spoVAF-spoIIAA region;
(n) spoIIIAH-yqhV region;
(o) ytvB-ytqB region;
(p) the yteA-ytaB region;
(q) yuaJ-yugO region;
(r) yusJ-mrgA region;
(s) gerAA-yvrI region;
(t) yvaM-yvbK region;
(u) araE-yveK region;
(v) yvdE-yvcP region;
(w) gerBA-ywsC region;
(x) ywrK-ywqM region;
(y) spoIIID-ywoB region;
(z) slp-ylaF region;
(aa) licH-sigY region;
(ab) yqeF-yrhK region;
(ac) yuzE-yukJ region; and
(ad) yncM-yndN region.
(2) The Bacillus subtilis mutant strain according to (1), wherein the region represented by (a) to (ad) is a region sandwiched between a pair of oligonucleotide sets represented by the following SEQ ID NO:
(a) SEQ ID NO: 51 and SEQ ID NO: 52;
(b) SEQ ID NO: 53 and SEQ ID NO: 54;
(c) SEQ ID NO: 55 and SEQ ID NO: 56;
(d) SEQ ID NO: 57 and SEQ ID NO: 58;
(e) SEQ ID NO: 59 and SEQ ID NO: 60;
(f) SEQ ID NO: 61 and SEQ ID NO: 62;
(g) SEQ ID NO: 63 and SEQ ID NO: 64;
(h) SEQ ID NO: 65 and SEQ ID NO: 66;
(i) SEQ ID NO: 67 and SEQ ID NO: 68;
(j) SEQ ID NO: 69 and SEQ ID NO: 70;
(k) SEQ ID NO: 71 and SEQ ID NO: 72;
(l) SEQ ID NO: 73 and SEQ ID NO: 74;
(m) SEQ ID NO: 75 and SEQ ID NO: 76;
(n) SEQ ID NO: 77 and SEQ ID NO: 78;
(o) SEQ ID NO: 79 and SEQ ID NO: 80;
(p) SEQ ID NO: 81 and SEQ ID NO: 82;
(q) SEQ ID NO: 83 and SEQ ID NO: 84;
(r) SEQ ID NO: 85 and SEQ ID NO: 86;
(s) SEQ ID NO: 87 and SEQ ID NO: 88;
(t) SEQ ID NO: 89 and SEQ ID NO: 90;
(u) SEQ ID NO: 91 and SEQ ID NO: 92;
(v) SEQ ID NO: 93 and SEQ ID NO: 94;
(w) SEQ ID NO: 95 and SEQ ID NO: 96;
(x) SEQ ID NO: 97 and SEQ ID NO: 98;
(y) SEQ ID NO: 99 and SEQ ID NO: 100;
(z) SEQ ID NO: 101 and SEQ ID NO: 102;
(aa) SEQ ID NO: 103 and SEQ ID NO: 104;
(ab) SEQ ID NO: 105 and SEQ ID NO: 106;
(ac) SEQ ID NO: 107 and SEQ ID NO: 108;
(ad) SEQ ID NO: 109 and SEQ ID NO: 110.
(3) The Bacillus subtilis mutation according to (2), which has a genomic structure in which at least three regions represented by (f), (j) and (m) are deleted from the genomic region of the Bacillus subtilis mutant MGB874 stock.
(4) A recombinant Bacillus subtilis obtained by introducing a gene encoding a target gene product into the Bacillus subtilis mutant strain according to any one of (1) to (3) so as to allow expression.
(5) The recombinant Bacillus subtilis according to (4), wherein at least one region selected from a transcription initiation control region, a translation initiation control region and a secretion signal region is operably linked upstream of a gene encoding a target gene product .
(6) The recombinant Bacillus subtilis according to (5), wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretory signal region are combined.
(7) A secretory signal region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus, and a transcription initiation control region and a translation initiation control region start from the initiation codon of the cellulase gene and have an upstream region of 0.6 to 1 kb in length The recombinant Bacillus subtilis according to (6), which is derived from.
(8) A DNA fragment consisting of the base sequence of base numbers 1 to 659 of the base sequence shown in SEQ ID NO: 1, wherein the three regions consisting of the transcription start control region, translation start control region and secretory signal region are shown in SEQ ID NO: 3 A base fragment of base numbers 1 to 696 of a cellulase gene comprising a base sequence, a DNA fragment comprising a base sequence having 70% or more identity with any of the base sequences, a DNA comprising any of the above base sequences and a stringent The recombinant Bacillus subtilis according to (6), which is a DNA fragment that hybridizes under the conditions described above or a DNA fragment comprising a base sequence from which any part of the base sequence has been deleted.
(9) The recombinant Bacillus subtilis according to any one of (4) to (8), wherein the target gene product is a heterologous protein or polypeptide.
(10) A method for producing a target gene product using the recombinant Bacillus subtilis according to any one of (4) to (9).

  According to the present invention, a Bacillus subtilis mutant strain excellent in productivity of various gene products is provided. By using the Bacillus subtilis mutant strain, industrial production of various enzymes and other useful substances having excellent productivity can be realized. Although the Bacillus subtilis mutant strain also has high gene product production ability, it is less robust in the gene product synthesis mechanism due to the small amount of genome and the intracellular mechanism being simplified. It can be a useful biological material for elucidating the mechanism.

The schematic diagram which shows an example of the method of deleting a predetermined area | region from the genome of Bacillus subtilis. The schematic diagram which shows the preparation procedure of a selection marker gene cassette DNA fragment. The schematic diagram which shows the procedure which removes a foreign drug resistance gene from a Bacillus subtilis genome.

  In the present invention, the identity of the base sequence and amino acid sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985). Specifically, it is calculated by performing an analysis with a unit size to compare (ktup) of 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

  The Bacillus subtilis mutant strain MGB874 is used as a parental microorganism for constructing the Bacillus subtilis mutant strain of the present invention. The MGB874 strain uses Bacillus subtilis Marburg No.168 (Bacillus subtilis 168 strain) as a wild strain, and its genome region is large, ie, prophage6 (yoaV-yobO) region, prophage1 (ybbU-ybdE) region, prophage4 (yjcM- yjdJ) region, PBSX (ykdA-xlyA) region, prophage5 (ynxB-dut) region, prophage3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) Region, pps (ppsE-ppsA) region, prophage2 (ydcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, The ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region are deleted (Patent Document 9). These deletion regions can also be represented as regions sandwiched by a pair of oligonucleotide sets shown in Table 1.

  The novel Bacillus subtilis mutant strain provided in the present invention has a genomic structure in which the genomic region is further deleted from the genomic region of the Bacillus subtilis mutant MGB874 strain. Specifically, in the Bacillus subtilis mutant strain of the present invention, at least one of the deletion regions shown in Table 2 on the genome of Bacillus subtilis 168 strain was deleted from the genome region of Bacillus subtilis mutant MGB874 strain. Has a genomic structure. Examples of the deletion region include gene regions shown in Table 2-1 to Table 2-2 and regions corresponding to the regions.

  The entire base sequence and gene of Bacillus subtilis 168 have been reported and published on the Internet (Nature, 390, 249-256, 1997 and BSORF Bacillus subtilis Genome Database [http://bacillus.genome.jp/ ], GenBank: AL009126.2 [http://www.ncbi.nlm.nih.gov/nuccore/38680335]). Those skilled in the art will be able to obtain MGB874 strain based on genomic information of Bacillus subtilis 168 strain obtained from these sources, for example, GenBank: AL009126.2 [http://www.ncbi.nlm.nih.gov/nuccore/38680335]. The gene regions shown in Table 2 above to be deleted from the genome can be found. Here, the gene region to be deleted is a natural or artificially induced deletion, substitution, insertion, or addition in one or more bases relative to the public base sequence of the Bacillus subtilis 168 gene. It may have a base sequence that may contain such mutations. The range of the number of bases that can be mutated is not particularly limited as long as the correspondence with the genome of Bacillus subtilis 168 can be recognized. For example, the identity of the base sequence of the genome region of Bacillus subtilis 168 is 50% or more. , Preferably more than 60% identity, more preferably more than 70% identity, more preferably more than 80% identity, even more preferably more than 90% identity, still more preferably more than 95% identity. The range which has is mentioned. The “addition” includes addition of a base to one end and both ends of a base sequence.

  Alternatively, examples of the deletion region shown in Table 2 include regions sandwiched between a pair of oligonucleotide sets shown in Table 2. That is, the Bacillus subtilis mutant strain of the present invention has a genomic structure in which at least one of the regions sandwiched between the pair of oligonucleotide sets shown in Table 2 is deleted from the genomic region of the Bacillus subtilis mutant strain MGB874. The region sandwiched between the pair of oligonucleotide sets shown in Table 2 above is, for example, that one end of the region is adjacent to the base sequence of the first oligonucleotide shown in Table 2-1 to Table 2-2 and its complementary sequence And the other end is a region adjacent to the base sequence of the second oligonucleotide shown in Table 2-1 to Table 2-2 and its complementary sequence.

  The Bacillus subtilis mutant strain of the present invention is also directly from the Bacillus subtilis 168 strain, the prophage6 (yoaV-yobO) region, prophage1 (ybbU-ybdE) region, prophage4 (yjcM-yjdJ) region, PBSX (ykdA-xlyA) region, prophage5 (ynxB-dut) region, prophage3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region, prophage2 (ydcL) -ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) It can be prepared by deleting at least one of the region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region, and the deletion region shown in Table 2-1 to Table 2-2. .

  Examples of Bacillus subtilis mutants of the present invention include Bacillus subtilis mutants shown in Tables 3-1 and 3-2 below.

  In addition to the genomic region of Bacillus subtilis strain 168 defined above, a Bacillus subtilis mutant strain having a genomic structure in which other regions are deleted is also included in the Bacillus subtilis mutant strain of the present invention. Other regions to be deleted include, for example, gene regions excluding essential growth genes and non-coding regions, etc., so that the viability of the bacteria can be maintained even when deleted from the genome, and the above-described target gene product is produced. A region that does not reduce the performance is preferred.

The method for deleting the region to be deleted from the Bacillus subtilis genome is not particularly limited. For example, the method shown in FIG. 1 can be applied.
That is, the region to be deleted is determined by the double crossover method using a deletion plasmid inserted with the deletion DNA fragment prepared by the so-called SOE-PCR method (Gene, 77, 61 (1989)). Delete from the genome. The deletion DNA fragment used in the present method includes an about 0.1 to 3 kb fragment adjacent to the upstream of the region to be deleted (referred to as upstream fragment) and an about 0.1 to 3 kb fragment adjacent to the downstream in the same manner. A DNA fragment ligated with a bound DNA fragment (referred to as a downstream fragment) can be used. In order to confirm the deletion of the target region, a chloramphenicol resistance gene is inserted between the upstream fragment and the downstream fragment. A DNA fragment combined with a drug resistance marker gene fragment such as is preferably used.

  First, an upstream fragment A and a downstream fragment B of the region to be deleted and three drug resistance marker gene fragment Cm as necessary are prepared by the first PCR (FIG. 1). At this time, in order to prepare the upstream and downstream fragments, a primer added with a sequence of 10 to 30 base pairs at the end of the DNA fragment to be bound is designed. For example, when the upstream fragment A, the drug resistance marker gene fragment Cm, and the downstream fragment B are combined in this order, the drug resistance marker gene fragment is located at the 5 ′ end of the primer located at the downstream end of the upstream fragment A (annealed). A sequence corresponding to 10 to 30 bases upstream of Cm is added (FIG. 1, primer DR1), and at the 5 ′ end of the primer located at the upstream end of the downstream fragment B (annealed), the drug resistance marker gene fragment Cm A sequence corresponding to 10 to 30 bases downstream is added (FIG. 1, primer DF2). When the upstream fragment A and the downstream fragment B are amplified by PCR using the primer set designed in this way, a region corresponding to the upstream side of the drug resistance marker gene fragment Cm is present on the downstream side of the amplified upstream fragment A ′. Thus, a region corresponding to the downstream side of the drug resistance marker gene fragment Cm is added to the upstream side of the amplified downstream fragment B ′.

  Next, the upstream fragment A ′ prepared by the first PCR, the drug resistance marker gene fragment Cm, and the downstream fragment B ′ are mixed to serve as a template, and a primer located at the upstream side of the upstream fragment (annealed) (FIG. 1, A second PCR is performed using a pair of primers consisting of primer DF1) and a primer (annealed) located downstream of the downstream fragment (FIG. 1, primer DR2). By this second round of PCR, a deletion DNA fragment D in which the upstream fragment A, the drug resistance marker gene fragment Cm, and the downstream fragment B are joined in this order can be amplified (FIG. 1).

  Alternatively, after amplifying a deletion DNA fragment in which the upstream fragment and the downstream fragment are combined, the deletion DNA fragment is inserted into a plasmid containing a drug resistance marker gene, so that the drug resistance is added to the upstream fragment and the downstream fragment. A deletion DNA fragment having a marker gene fragment can be prepared.

  Furthermore, the DNA fragment for deletion obtained by the above-mentioned method is inserted into plasmid DNA that cannot be easily amplified in a host bacterium using normal restriction enzymes and DNA ligase, or plasmid DNA that can be easily removed, such as a temperature-sensitive plasmid. Thus, a plasmid for introducing a deletion is constructed. Examples of plasmid DNA that is not amplified in the host bacterium include, but are not limited to, pUC18, pUC118, pBR322, and the like when Bacillus subtilis is used as the host.

  Next, transformation of the host bacterium with the deletion plasmid is performed by a conventional method such as the competent cell transformation method (J. Bacteriol. 93, 1925 (1967)), and the upstream and downstream fragments inserted into the plasmid. And the homologous recombination between the two homologous regions on the genome and the deletion plasmid is fused into the genomic DNA of the host fungus by the double crossover homologous recombination, so that the region to be deleted is replaced with a drug resistance marker gene. The obtained transformant is obtained (FIG. 1). The transformant may be selected using drug resistance by a marker gene such as a chloramphenicol resistance gene present in the DNA fragment for deletion as an index. For example, Bacillus subtilis can be added to SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50 Incubate with shaking at 37 ° C. in% glucose, 0.02% casamino acid (Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan until the value of growth (OD600) 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% tricitrate citrate). Sodium dihydrate, 0.50% glucose, 0.01% casamino acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / ml tryptophan), and the growth degree (OD600) is A competent cell of Bacillus subtilis is prepared by shaking culture to about 0.4. Next, 5 μL of the solution containing the above DNA fragment (the reaction solution of the above SOE-PCR) was added to 100 μL of the prepared competent cell suspension (culture medium in SPII medium), and after shaking culture at 37 ° C. for 1 hour, antibiotics ( Spread the entire amount on LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing chloramphenicol. After static culture at 37 ° C., the grown colonies are isolated as transformants. By extracting the genomic DNA of the transformant and using this as a template DNA, it can be confirmed that the target region has been deleted. Genomic DNA can be extracted by the method of Saito & Miura (Saito & Miura Biochem. Biophys. Acta. 72, 619-629 (1963)) or a commercially available genomic DNA extraction kit such as UltraClean Microbial DNA. Isolation Kit (MO BIO Laboratories, Inc.) can be used.

  From the transformant thus obtained, the foreign drug resistance marker gene inserted into the genomic DNA is further removed. The removal procedure is not particularly limited, but a two-step homologous recombination method using a DNA fragment prepared by the SOE-PCR method (Gene, 77, 61 (1989)) can be used (see FIG. 3). . The procedure will be described below.

  First, a DNA fragment (donor DNA) for the first homologous recombination is prepared. Although it does not specifically limit as a preparation method, The method etc. which bind | bond a desired fragment on the SOE-PCR method mentioned above and a plasmid, etc. are mentioned. Examples of the donor DNA include an about 0.1 to 3 kb fragment (upstream fragment) adjacent to the upstream of the foreign drug resistance marker gene region to be removed (ie, the deleted region) and about 0 adjacent to the downstream. A fragment in which a DNA fragment (downstream fragment) in which .1 to 3 kb fragments are sequentially bound and a DNA fragment in the downstream region of the foreign drug resistance marker gene is bound can be used. Preferably, a DNA fragment in which another marker gene or the like serving as an index of homologous recombination is inserted between the downstream fragment and the marker gene downstream region fragment is used (FIG. 3).

  Subsequently, the prepared donor DNA is introduced into the transformant by a conventional method such as competent cell transformation method, and the region corresponding to the upstream fragment of the transformant genomic DNA and the downstream region of the foreign drug resistance marker gene And homologous recombination between them (first homologous recombination). A transformant in which the desired homologous recombination has occurred can be selected using as an index the drug resistance due to another marker gene inserted into the donor DNA. In the genomic DNA of a transformant in which the first homologous recombination has occurred appropriately, an upstream fragment, a downstream fragment, and if necessary, another marker gene, a foreign drug resistance marker gene downstream region, and a downstream fragment are arranged in order. (See FIG. 3). In genomic DNA having such an arrangement, homologous recombination can occur spontaneously between the two downstream fragments (intragenomic homologous recombination). Due to this intra-genomic homologous recombination, the region located between the two downstream fragments is deleted, whereby the foreign drug resistance marker gene is removed from the transformant genome. Since a transformant that has undergone homologous recombination within the genome as intended has lost its resistance to drugs, the target transformant can be obtained by selecting a strain that has become drug-sensitive. Genomic DNA can be extracted from the obtained strain, and deletion of the target gene can be confirmed by PCR or the like.

When selecting a target deletion strain, it is difficult to directly select a strain that has changed from drug resistance to sensitivity, and homologous recombination in the genome is considered to occur at a low frequency of about 10 ⁻⁴ or less. Therefore, in order to obtain target deletion strains efficiently, devise methods such as increasing the abundance ratio of drug-sensitive strains and selecting target deletion strains using genes that are lethal under certain conditions. It is desirable.

  As a method for concentrating drug-sensitive strains, for example, a method of concentration utilizing methods that penicillin antibiotics such as ampicillin act on bactericidal cells while not acting on non-proliferating cells (Methods in Molecular Genetics). Cold Spring Harbor Labs, (1970)). In the case of concentration using ampicillin or the like, it is effective for deletion of a resistance gene for a drug that acts bacteriostatically on host cells such as tetracycline and chloramphenicol. In an appropriate medium containing an appropriate amount of such a bacteriostatic agent, a resistant strain carrying the drug resistance gene can grow, and a sensitive strain lacking the drug resistance gene does not grow or die. Under such conditions, when a suitable concentration of penicillin antibiotics such as ampicillin is added and cultured, resistant strains to be grown are killed, while sensitive strains are not affected by ampicillin or the like, resulting in The proportion of sensitive strains will increase. To efficiently select sensitive strains by smearing and culturing such a concentrated culture solution on an appropriate agar medium, and confirming the presence or absence of resistance to the marker drug of the colonies that appeared by replica method etc. Is possible.

  Alternatively, a lethal gene that works under certain conditions can be used as a method for selecting a target deletion strain. As the lethal gene, for example, genes encoding cell growth inhibitory proteins such as chpA gene (gene encoding ribonuclease) and ccdB gene (gene encoding an inhibitor of DNA gyrase) can be used. Two lethal genes such as chpA gene are linked downstream of an expression-inducing promoter such as spac promoter that induces expression in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG), and two downstream fragments in the donor DNA A lethal gene such as chpA gene can be expressed as necessary by inserting it between. Therefore, by culturing a transformant generated by the first homologous recombination in a medium containing IPTG, cells expressing a lethal gene such as the chpA gene are killed, and lethal such as the chpA gene is killed in the second homologous recombination step. Since a cell from which a gene has been dropped can proliferate and grow, a desired transformant can be selected using the presence or absence of colony formation as an index (see FIG. 3).

  As described above, a Bacillus subtilis mutant strain having a genome structure in which a predetermined region on the genome is deleted alone can be produced. A Bacillus subtilis mutant strain having a genomic structure in which a plurality of regions are deleted can be obtained by repeating the above steps, but can also be prepared by a so-called LP (lysis of protoplasts) transformation method. LP transformation methods are described in “T. Akamatsu and J. Sekiguchi,“ Archives of Microbiology ”, 1987, 146, p.353-357” and “T. Akamatsu and H. Taguchi,“ Bioscience, Biotechnology, and Biochemistry ”, 2001, Vol. 65, No. 4, p. 823-829”.

  The Bacillus subtilis mutant strain of the present invention thus obtained maintains its viability even though it lacks a large region of the genome compared to the parent strain Bacillus subtilis mutant MGB874. Yes. Moreover, the Bacillus subtilis mutant strain of the present invention has the same or higher gene product producing ability than the parent strain. Therefore, the Bacillus subtilis mutant strain of the present invention has the ability to produce gene products, but since the amount of genome is small due to deletion of the large genome region, the intracellular mechanism is further simplified, so the production mechanism of various gene products It can be a biological material useful for elucidation of Moreover, the recombinant Bacillus subtilis of the present invention in which the productivity of the target gene product is improved can be obtained by introducing the gene encoding the target gene product into the Bacillus subtilis mutant of the present invention in an expressible manner.

  In the present specification, “introducing a gene into a microorganism so that the gene can be expressed” refers to introducing the gene so that the gene can be expressed in the microorganism. For example, an expression vector in which a target gene is incorporated at an appropriate position, or a vector containing a target gene linked to a control region such as a transcription initiation control region or a translation start region can be transformed into a microorganism using a general transformation method. The target gene can be “expressably introduced” into the microorganism. Alternatively, a Bacillus subtilis mutant of the present invention can be obtained by directly incorporating a DNA fragment containing a target gene downstream of a control region on the microbial genome and a homologous region downstream of the control region. It can be introduced so that it can be expressed. Various techniques for introducing a gene into a cell as exemplified above so as to be expressed are well known to those skilled in the art.

  Therefore, in one embodiment, a gene encoding a target gene product to be introduced into the Bacillus subtilis mutant of the present invention has upstream a regulatory region involved in transcription, translation, and secretion of the gene, that is, a promoter and a transcription start point. It is preferably operably linked to at least one region selected from a transcription initiation control region containing, a ribosome binding site and a translation initiation region containing an initiation codon, and a secretory signal peptide region. More preferably, the three regions consisting of a transcription initiation control region, a translation initiation control region, and a secretion signal region, and more preferably, the secretion signal peptide region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus. The region and the translation initiation control region, which is an upstream region having a length of 0.6 to 1 kb starting from the initiation codon of the cellulase gene, are operably linked upstream of the gene encoding the target gene product.

  In the present specification, “operational linkage” between a transcription initiation control region, a translation initiation region, or a secretion signal and a gene means that the control region can induce transcription or translation of the gene, or is encoded in the gene. It is linked so that the produced protein or peptide is secreted by the action of a secretion signal. The procedure of “operable linkage” between the control region or secretion signal and a gene is well known to those skilled in the art.

  For example, the gene encoding the target gene product to be introduced into the Bacillus subtilis mutant strain of the present invention is a Bacillus (described in JP 2000-210081, JP 4-190793, etc.). Bacillus) bacteria, ie, KSM-S237 strain (FERM BP-7875) or KSM-64 strain (FERM BP-2886) cellulase gene transcription initiation regulatory region, translation initiation region and secretory signal peptide region are the target gene products It is desirable to be operatively connected to each other. More specifically, a DNA fragment comprising the base sequence of base numbers 1 to 659 of the base sequence represented by SEQ ID NO: 1, the base sequence of base numbers 1 to 696 of the cellulase gene comprising the base sequence of SEQ ID NO: 3, 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more of the nucleotide sequence. A DNA fragment having a function related to transcription, translation, or secretion, or a DNA that hybridizes with any of the above nucleotide sequences under stringent conditions and has a function related to transcription, translation, or secretion of a gene, or any of the above DNA fragment comprising a nucleotide sequence in which a part of the nucleotide sequence is deleted and having a function related to gene transcription, translation and secretion It is desirable that is operably linked to the structural gene of the target gene product. Here, a DNA fragment consisting of a base sequence from which a part of the base sequence has been deleted means a part of the base sequence, preferably several (for example, 1 to 50, preferably 1 to 30, More preferably, it means a DNA fragment having a function related to transcription, translation and secretion of a gene. The stringent conditions referred to herein include, for example, the conditions described in [Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989)]. For example, in a solution containing 6 × SSC (composition of 1 × SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA A condition for hybridization with a probe at 65 ° C. for 8 to 16 hours is mentioned.

  The gene encoding the target gene product to be introduced is not particularly limited, and may be an endogenous gene or a heterologous gene. For example, genes to be introduced include contractile proteins, transport proteins, signal proteins, biological defense proteins, receptor proteins, structural proteins, gene regulatory proteins, enzymes, genes encoding proteins such as storage proteins, functional RNA, etc. And a gene encoding the non-coding RNA.

  In one embodiment, the introduced gene is a gene encoding a heterologous protein or polypeptide. As a heterologous protein or polypeptide gene, any protein or polypeptide useful in various fields such as detergent, food, fiber, feed, chemicals, medicine, diagnosis, research, etc., for example, enzyme, bioactive peptide, marker, signal transduction Examples include genes encoding substances, structural proteins, and various types of regulation. In addition, the enzymes described above are classified according to their functions: oxidoreductase, transferase, hydrolase, lyase, isomerase, isomerase, and ligase. / Synthetase), etc., preferably hydrolyzing enzymes such as cellulase, α-amylase, protease and the like.

  Cellulases include, for example, cellulases belonging to Family 5 in the classification of polysaccharide hydrolases (Biochem. J., 280, 309, 1991). Among them, cellulases derived from microorganisms, preferably derived from bacteria belonging to the genus Bacillus are preferred. Can be mentioned. As a more specific example, an alkaline cellulase derived from the Bacillus bacterium strain KSM-S237 (FERM BP-7875) comprising the amino acid sequence represented by SEQ ID NO: 2 or a Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 4 Alkaline cellulase derived from KSM-64 strain (FERM BP-2886) or the amino acid sequence and 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more. A cellulase comprising an amino acid sequence having the same identity, or an alkaline cellulase comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 It is done.

  Specific examples of α-amylase include α-amylase derived from microorganisms, and preferably liquefied amylase derived from Bacillus bacteria. As a more specific example, alkaline amylase derived from the Bacillus genus bacterium KSM-K38 strain (FERM BP-6946) consisting of the amino acid sequence represented by SEQ ID NO: 5, or 70%, preferably 80%, more preferably the amino acid sequence. Is an amylase comprising an amino acid sequence having 90% or more, more preferably 95% or more, and even more preferably 98% or more identity, or one or several amino acids are substituted or missing in the amino acid sequence shown in SEQ ID NO: 5. Examples include alkaline amylase consisting of a deleted, inserted or added amino acid sequence.

  Specific examples of proteases include serine proteases, metal proteases and the like derived from microorganisms, preferably from Bacillus bacteria. As a more specific example, an alkaline protease derived from the Bacillus clausii strain KSM-KP43 (FERM BP-6532) consisting of the amino acid sequence shown in SEQ ID NO: 6, or 70%, preferably 80% with the amino acid sequence. More preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more, a protease comprising an amino acid sequence, or one or several amino acids in the amino acid sequence represented by SEQ ID NO: 6 Examples include alkaline protease consisting of an amino acid sequence substituted, deleted, inserted or added.

  In the present specification, the “amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added” has 1 to 50, preferably 1 to 30, more preferably 1 to 20, Preferably, an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, inserted or added is included, and the “addition” includes addition of amino acids to one end and both ends of the amino acid sequence.

  In another embodiment, the introduced gene is a gene encoding a non-coding RNA. Non-coding RNA is RNA that is transcribed from DNA and not translated into protein. Non-coding RNAs include functional RNAs such as untranslated regions including regulatory sequences, tRNAs, rRNAs, mRNA-type ncRNAs (mRNA-like non-coding RNAs), snRNAs (small nuclear RNAs), snRNAs (small nuclear RNAs). , MiRNA (microRNA), stRNA (Small Temporal RNA), siRNA (short-interfering RNA) and the like. These non-coding RNAs are responsible for a wide variety of mechanisms related to cell expression control, development, differentiation, and other life activities, including research, medicine, diagnosis, drug discovery, breeding and agricultural production such as agricultural production, It can be used in the livestock field and chemical production.

  Production of the target gene product using the recombinant Bacillus subtilis of the present invention is to inoculate the strain into a medium containing an assimilable carbon source, nitrogen source, and other essential components, and cultured by a normal microbial culture method. After completion of the culture, the target gene product may be collected and purified as necessary. And as shown in the below-mentioned Example, the improvement of the productivity of a target gene product is achieved compared with the case where the Bacillus subtilis mutant MGB874 strain which is a parent strain is used.

  Hereinafter, the present invention will be described in more detail with reference to examples.

  The primer used in the Example is described in Table 4, and the base sequence is described in Table 5-1 to Table 5-7.

[Example 1] Preparation of a single deletion strain of the genomic region In this example, mutant strains in which various regions on the genome of Bacillus subtilis MGB874 strain were deleted were produced. The flow is shown in FIG.

<Preparation of a single deletion strain of R25 region>
Using the R25 region shown in Table 2 on the genome of the Bacillus subtilis mutant MGB874 strain as a deletion target region, the region was deleted from the MGB874 strain genome.

  As shown in FIG. 1, genomic DNA extracted from the MGB874 strain was used as a template, 25-DF1 (SEQ ID NO: 155) and 25-DR1 (SEQ ID NO: 157), 25-DF2 (SEQ ID NO: 156) and 25-DR2 Using each primer set of (SEQ ID NO: 158), a 0.5 kb fragment (A) adjacent to the upstream of the R25 region on the genome and a 0.5 kb fragment (B) adjacent to the downstream were amplified by PCR. In addition, the plasmid pDLT3 (Morimoto et al., 2002) is used as a template to contain a chloramphenicol resistance gene using a primer set of rPCR-CmF (SEQ ID NO: 351) and rPCR-CmR (SEQ ID NO: 352). A 0 kb fragment (Cm) was prepared by PCR. The obtained three amplified DNA fragments were ligated by the SOE-PCR method (see, for example, Gene, 77, 61 (1989)) to prepare a DNA fragment for ligation deletion.

  The MGB874 strain was transformed with the deletion DNA fragment donor DNA obtained as described above. The conditions for transformation are in accordance with a competent cell transformation method (see, for example, J. Bacteriol. 93, 192 (1967)), and 1 μg or more of the PCR product (DNA fragment donor DNA for deletion) is added to 400 μl of competent cells. In addition, the cells were further cultured for 1.5 hours. This was applied to an LB agar medium containing 10 ppm of chloramphenicol, and the grown colonies were isolated as transformants by homologous recombination (see FIG. 1). The acquired strain was designated as MGB874-R25 :: cm strain.

<Preparation of other individual deletion strains>
In accordance with the procedure for producing a single deletion strain of R25 region described above, R02 region, R07 region, R08 region, R10N region, R12 region, R13 region, R15 region, R16 region, R20N region, R21 region, R22 region, R23 Region, R25 region, R26 region, R30 region, R31 region, R32 region, R33 region, R34 region, R35 region, R36 region, R37 region, R39 region, R40 region, R41 region, R45 region, R49A region, R50N region, Each single deletion strain of R53 region and Rd22 region (collectively referred to as R * region) was prepared. When constructing a single deletion strain of the above R * region, each region single deletion strain described in Table 4 and Tables 5-1 to 5-7 was prepared in the same manner as the preparation of the R25 region single deletion strain. A deletion DNA fragment was prepared using the primer set for the preparation, and a single deletion strain of the R * region was prepared.
A strain in which the R * region alone was deleted from the genome of the Bacillus subtilis mutant MGB874 strain was designated as an MGB874-R * :: cm strain.

[Example 2] Preparation of selectable marker gene cassette First, Bacillus subtilis 168 (aprE :: spec, in which a DNA fragment containing a spectinomycin resistance gene, lacI gene, and Pspac-chpA gene was inserted into the Bacillus subtilis 168 aprE gene region. (lacI, Pspac-chpA, cm) strain was prepared as follows.

As shown in FIG. 2, using the Bacillus subtilis expression vector pO2HC as a template, the region from lacI to the SD sequence downstream of the promoter containing the spac promoter is designated as pO2HC-lacF (SEQ ID NO: 355) and pO2HC-lacR (sequence). No. 356) was amplified by PCR using the primer set. In addition, the region from the start codon to the stop codon of the chpA gene in the genome using the primer set of chpA-F (SEQ ID NO: 358) and chpA-R (SEQ ID NO: 359) using the genome of Escherichia coli W3110 as a template. Amplified by PCR. The obtained two amplified DNA fragments were combined by the SOE-PCR method (see, for example, Gene, 77, 61 (1989)) to prepare a combined DNA fragment.
Next, the DNA fragment prepared above was digested with restriction enzymes ApaI and BamHI to obtain restriction enzyme-treated fragments. Similarly, the vector pAPNC213 (containing spectinomycin resistance gene) was digested with restriction enzymes ApaI and BamHI to obtain restriction enzyme-treated fragments. The restriction enzyme-treated fragments thus obtained were ligated using DNA ligase.
The resulting circular DNA was used to transform Bacillus subtilis 168 strain. The cells were cultured on an LB agar plate medium containing 100 μg / ml of spectinomycin, and the target transformant was selected to construct strain 168 (aprE :: spec, lacI, Pspac-chpA).
Next, PCR was performed using the obtained 168 (aprE :: spec, lacI, Pspac-chpA) strain chromosomal DNA as a template and using APNC-F primer (SEQ ID NO: 357) and chpA-R primer (SEQ ID NO: 359). The DNA fragment was amplified by (see FIG. 2). The amplified DNA fragment is referred to as a selectable marker gene cassette.

[Example 3] Insertion of DNA construct for removing foreign sequence into genome of single region deletion strain In this example, single region deletion strain MGB874-R25 :: cm strain constructed above and MGB874-R * A mutant was prepared by inserting a DNA construct for exogenous sequence removal into each genome of the :: cm strain. The flow of this example is shown in FIG.

<Insertion of DNA construct for removing foreign sequence into genome of R25 region-only deletion strain>
A mutant strain in which a DNA construct for exogenous sequence removal was inserted into the genome of the MGB874-R25 :: cm strain, which is a single R25 region-deleted strain, was produced as follows. The donor DNA shown in FIG. 3 was constructed as follows using the primers shown in Table 4 and Tables 5-1 to 5-7.

<Preparation of donor DNA>
Also, using the chromosomal DNA of Bacillus subtilis 168 strain as a template, two fragments of the 5 ′ outer region (fragment A) of the deletion target region and the 3 ′ outer region (fragment B) of the deletion target region were each 25-DF1 ( Amplified by PCR using primer sets of SEQ ID NO: 155) and 25-DR1.2 (SEQ ID NO: 316), and 25-DF2.2 (SEQ ID NO: 315) and 25-DR2 (SEQ ID NO: 158) (FIG. 3) . In addition, the plasmid pSM5022 (Mol. Microbiol. 6, 309, 1992) was used as a template to clone by PCR using a primer set of Cm-F (del) (SEQ ID NO: 353) and Cm-R (del) (SEQ ID NO: 354). The lamphenicol resistance gene (fragment Cm) was amplified (FIG. 3).

  Selectable marker gene cassettes obtained by these PCRs, 5 'outer region (fragment A), 3' outer region (fragment B) and 4 fragments of the internal sequence of the chloramphenicol resistance gene (fragment 'Cm'), and SOE-PCR (Gene, 77, 61 (1989)) was performed using a primer set of 25-DF1 (SEQ ID NO: 155) and Cm-R (del) (SEQ ID NO: 354). Thereby, as shown in FIG. 3, the 5 ′ outer region (fragment A), the 3 ′ outer region (fragment B), the selectable marker gene cassette and the chloramphenicol resistance gene upstream deletion sequence (fragment “Cm”) DNA fragments arranged in this order could be obtained. In this example, this DNA fragment was used as donor DNA.

<Transformation>
The donor DNA obtained as described above was used to transform an R25 region-only deletion strain (MGB874-R25 :: cm strain). According to the transformation method for competent cells (see, for example, J. Bacteriol. 93, 192 (1967)), 1 μg or more of PCR product (donor DNA) was added to 400 μl of competent cells, and 1 The first homologous recombination (FIG. 3) was performed after 5 hours of culture.

Transformants were selected using a recombinantly introduced spectinomycin resistance gene. Specifically, the cells after the above transformation treatment were cultured overnight at 37 ° C. on an LB agar plate medium containing 100 μg / ml spectinomycin to form colonies, and viable strains were obtained. By this culture, only Bacillus subtilis that has acquired the spectinomycin resistance by incorporating the donor DNA by homologous recombination grows and forms a colony.
The acquired strain was designated as MGB874 (R25 :: R25B, spec, lacI, Pspac-chpA, 'cm') strain.

<Insertion of DNA construct for removing foreign sequences into the genome of R * region-only deletion strain>
According to the procedure for inserting the DNA construct for removing foreign sequences into the genome of the R25 region-only deletion strain described above, the primers described in Table 4 and Tables 5-1 to 5-7 were used according to the method described in FIG. The DNA construct for removing foreign sequences was inserted into each genome of the MGB874-R * :: cm strain, which is a single deletion strain of the R * region. For the amplification of the 5 ′ outer region (fragment A) and 3 ′ outer region (fragment B) of the deletion target region for preparation of donor DNA, * -DF1 and * -DR1.2, and * -DF2.2 and * -DR2 primer sets (for example, 21-DF1 and 21-DR1.2 and 21-DF2.2 and 21-DR2 for the R21 region, respectively) The primer set was used.
The obtained strain was designated as MGB874 (R * :: R * B, spec, lacI, Pspac-chpA, 'cm') strain.

[Example 3] Construction of a multi-region deletion strain In this example, a multi-region deletion strain was constructed using the DNA construct insertion strain for exogenous sequence removal to each single deletion strain genome constructed above. .

<Homologous recombination in the genome of MGB874 (R25 :: R25B, spec, lacI, Pspac-chpA, 'cm') strain>
MGB874 (R25 :: R25B, spec, lacI, Pspac-chpA, 'cm') strain was cultured overnight in LB liquid medium. After dilution of the culture solution, it was applied to an LB agar plate supplemented with 1 mM IPTG, and colony formation was confirmed. Those transformed Bacillus subtilis that survived on the IPTG-containing LB agar plate and confirmed colony formation had a DNA construct for removing foreign sequences and a chloramphenicol resistance gene (fragment 'Cm') by genome homologous recombination. It has been deleted from DNA (see FIG. 3).
Furthermore, in this example, the deletion of the deletion target region (R25 region) is shown in Table 4 and Tables 5-1 to 5-7 for the single colony of transformed Bacillus subtilis obtained by the above experiment. This was confirmed using a -checkF primer (SEQ ID NO: 255) and a 25-checkR primer (SEQ ID NO: 256) (FIG. 3). The strain obtained in this experiment was named RGF880 strain.

<Deletion of R21 region from RGF880 strain genome>
An experiment was conducted to delete the R21 region shown in Table 2 from the genome of the RGF880 strain.
The genomic DNA of strain MGB874 (R21 :: R21B, spec, lacI, Pspac-chpA, 'cm') constructed by inserting a DNA construct for exogenous sequence insertion into the genome of MGB874-R21 :: cm strain was extracted, and RGF880 Transformation experiments were performed on the strains. According to the transformation method for competent cells (see, for example, J. Bacteriol. 93, 192 (1967)), 1 μg or more of PCR product (donor DNA) was added to 400 μl of competent cells, and 1 Incubated for 5 hours. This was applied to an LB agar medium containing 10 ppm of chloramphenicol, and the grown colonies were isolated as transformants by homologous recombination (see FIG. 3). The acquired strain was designated as RGF880 (R21 :: R21B, spec, lacI, Pspac-chpA, 'cm').

RGF880 (R21 :: R21B, spec, lacI, Pspac-chpA, 'cm') strain was cultured overnight in LB liquid medium. After dilution of the culture solution, it was applied to an LB agar plate supplemented with 1 mM IPTG, and colony formation was confirmed. Those transformed Bacillus subtilis that survived on the IPTG-containing LB agar plate and confirmed colony formation had a DNA construct for removing foreign sequences and a chloramphenicol resistance gene (fragment 'Cm') by genome homologous recombination. It has been deleted from DNA (see FIG. 3).
Further, in this example, deletion of the deletion target region (R21 region) is shown in Table 4 and Tables 5-1 to 5-7 for the single colony of transformed Bacillus subtilis obtained by the above experiment. This was confirmed using a -checkF primer (SEQ ID NO: 249) and a 21-checkR primer (SEQ ID NO: 250) (FIG. 3). The strain obtained in this experiment was named RGF905 strain.

<Deletion of R * region from each region deleted strain genome>
Multiple deletion strains were constructed from the RGF880 strain genome in the same manner as in the experiment for deleting the R21 region. An experiment for deleting the R * region from the genome of the RGFx strain was performed as follows, with the target region-deleted strain as the RGFx strain.

  The genomic DNA of strain MGB874 (R * :: R * B, spec, lacI, Pspac-chpA, 'cm') constructed by inserting a DNA construct for removing foreign sequences into the genome of MGB874-R * :: cm strain Extraction and transformation experiments were performed on the region deleted strain RGFx. According to the transformation method for competent cells (see, for example, J. Bacteriol. 93, 192 (1967)), 1 μg or more of PCR product (donor DNA) was added to 400 μl of competent cells, and 1 Incubated for 5 hours. This was applied to an LB agar medium containing 10 ppm of chloramphenicol, and the grown colonies were isolated as transformants by homologous recombination (see FIG. 3). The acquired strain was designated as RGFx (R * :: R * B, spec, lacI, Pspac-chpA, ‘cm’).

RGFx (R * :: R * B, spec, lacI, Pspac-chpA, 'cm') strains were cultured overnight in LB liquid medium. After dilution of the culture solution, it was applied to an LB agar plate supplemented with 1 mM IPTG, and colony formation was confirmed. Those transformed Bacillus subtilis that survived on the IPTG-containing LB agar plate and confirmed colony formation had a DNA construct for removing foreign sequences and a chloramphenicol resistance gene (fragment 'Cm') by genome homologous recombination. It has been deleted from DNA (see FIG. 3).
Furthermore, in the present Example, deletion of a deletion object area | region (R * area | region) is shown to Table 4 and Tables 5-1 to 5-7 about the single colony of the transformed Bacillus subtilis obtained by the above experiment. It confirmed using * -checkF primer and * -checkR primer (FIG. 3).
The above experiment was repeated to construct Bacillus subtilis mutants of the present invention shown in Table 2.

[Example 4] Production of recombinant Bacillus subtilis and evaluation of target gene product productivity The Bacillus subtilis mutants RGF916, RGF933, RGF1042, RGF1323, RGF1334, RGF1351 of the present invention prepared according to Examples 1 to 3 A recombinant Bacillus subtilis strain in which the gene encoding the target gene product was introduced into the GF1368 strain and RGF1370 was produced, and the ability to produce the target gene product was evaluated. In this example, alkaline cellulase was used as the target gene product to be introduced into the Bacillus subtilis mutant.

<Alkaline cellulase secretion production evaluation>
Evaluation of alkaline cellulase secretion productivity was performed as follows. That is, an alkaline cellulase gene fragment (3.1 kb) derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) was inserted into the BamHI restriction enzyme cleavage point of shuttle vector pHY300PLK. The resulting recombinant plasmid pHY-S237 was introduced into each strain by protoplast transformation. The recombinant strain thus obtained was subjected to shaking culture in 10 mL of LB medium overnight at 37 ° C., and 0.05 mL of this culture solution was further added to 50 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% % NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline), followed by shaking culture at 30 ° C. for 3 days. The alkaline cellulase activity of the culture supernatant from which the cells were removed by centrifugation was measured, and the amount of alkaline cellulase secreted and produced outside the cells by the culture was determined.

  For cellulase activity measurement, 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation) was added to 50 μL of a sample solution appropriately diluted with 1 / 7.5 M phosphate buffer (pH 7.4, Wako Pure Chemical Industries, Ltd.). 50 μL was added and mixed, and the amount of p-nitrophenol released upon reaction at 30 ° C. was quantified by the change in absorbance at 420 nm (OD 420 nm). The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.

<Result>
Table 6 summarizes the secretory productivity of alkaline cellulase. In Table 6, the secretory production ability of various enzymes is shown as a relative value when the amount of enzyme produced when each gene is similarly introduced into the parent strain MGB874 is 100.

  As shown in Table 6, the RGF916 strain, RGF933 strain, RGF1042, RGF1334 strain, RGF1351 strain, RGF1368 strain, and RGF1370 strain, which are the recombinant Bacillus subtilis of the present invention, have a secretion productivity of alkaline cellulase from the MGB874 strain. It became clear that it was improving.

Claims (10)

Bacillus subtilis mutant strain having a genomic structure in which any one of the following regions (a) to (ad) shown in the genome of Bacillus subtilis 168 strain is deleted from the genome region of Bacillus subtilis mutant strain MGB874 :
(a) ybbU-ybfI region;
(b) ydjM-cotA region;
(c) yefA-yesX region;
(d) yfiB-yfiX region;
(e) the yhcE-yhcU region;
(f) yhaU-yhaL region;
(g) yjbX-yjlB region;
(h) the xkdA-ykcC region;
(i) bpr-ylmA region;
(j) flgB-cheD region;
(k) ynfF-ppsA region;
(l) yoxC-yobO region;
(m) spoVAF-spoIIAA region;
(n) spoIIIAH-yqhV region;
(o) ytvB-ytqB region;
(p) the yteA-ytaB region;
(q) yuaJ-yugO region;
(r) yusJ-mrgA region;
(s) gerAA-yvrI region;
(t) yvaM-yvbK region;
(u) araE-yveK region;
(v) yvdE-yvcP region;
(w) gerBA-ywsC region;
(x) ywrK-ywqM region;
(y) spoIIID-ywoB region;
(z) slp-ylaF region;
(aa) licH-sigY region;
(ab) yqeF-yrhK region;
(ac) yuzE-yukJ region; and
(ad) yncM-yndN region. The Bacillus subtilis mutant strain according to claim 1, wherein the region represented by (a) to (ad) is a region sandwiched between a pair of oligonucleotide sets represented by the following SEQ ID NOs:
(a) SEQ ID NO: 51 and SEQ ID NO: 52;
(b) SEQ ID NO: 53 and SEQ ID NO: 54;
(c) SEQ ID NO: 55 and SEQ ID NO: 56;
(d) SEQ ID NO: 57 and SEQ ID NO: 58;
(e) SEQ ID NO: 59 and SEQ ID NO: 60;
(f) SEQ ID NO: 61 and SEQ ID NO: 62;
(g) SEQ ID NO: 63 and SEQ ID NO: 64;
(h) SEQ ID NO: 65 and SEQ ID NO: 66;
(i) SEQ ID NO: 67 and SEQ ID NO: 68;
(j) SEQ ID NO: 69 and SEQ ID NO: 70;
(k) SEQ ID NO: 71 and SEQ ID NO: 72;
(l) SEQ ID NO: 73 and SEQ ID NO: 74;
(m) SEQ ID NO: 75 and SEQ ID NO: 76;
(n) SEQ ID NO: 77 and SEQ ID NO: 78;
(o) SEQ ID NO: 79 and SEQ ID NO: 80;
(p) SEQ ID NO: 81 and SEQ ID NO: 82;
(q) SEQ ID NO: 83 and SEQ ID NO: 84;
(r) SEQ ID NO: 85 and SEQ ID NO: 86;
(s) SEQ ID NO: 87 and SEQ ID NO: 88;
(t) SEQ ID NO: 89 and SEQ ID NO: 90;
(u) SEQ ID NO: 91 and SEQ ID NO: 92;
(v) SEQ ID NO: 93 and SEQ ID NO: 94;
(w) SEQ ID NO: 95 and SEQ ID NO: 96;
(x) SEQ ID NO: 97 and SEQ ID NO: 98;
(y) SEQ ID NO: 99 and SEQ ID NO: 100;
(z) SEQ ID NO: 101 and SEQ ID NO: 102;
(aa) SEQ ID NO: 103 and SEQ ID NO: 104;
(ab) SEQ ID NO: 105 and SEQ ID NO: 106;
(ac) SEQ ID NO: 107 and SEQ ID NO: 108;
(ad) SEQ ID NO: 109 and SEQ ID NO: 110.   The Bacillus subtilis mutant according to claim 2, wherein the Bacillus subtilis mutant MGB874 has a genome structure in which at least three regions represented by (f), (j) and (m) are deleted from the genome region of the MGB874 strain.   A recombinant Bacillus subtilis obtained by introducing a gene encoding a target gene product into the Bacillus subtilis mutant strain according to any one of claims 1 to 3.   The recombinant Bacillus subtilis according to claim 4, wherein at least one region selected from a transcription initiation control region, a translation initiation control region and a secretion signal region is operably linked upstream of a gene encoding a target gene product.   The recombinant Bacillus subtilis according to claim 5, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretory signal region are combined.   The secretory signal region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus, and the transcription initiation control region and the translation initiation control region are derived from an upstream region of 0.6 to 1 kb in length starting from the start codon of the cellulase gene The recombinant Bacillus subtilis according to claim 6, which is   Three regions comprising a transcription initiation control region, a translation initiation control region and a secretion signal region are composed of a DNA fragment comprising the base sequence of base numbers 1 to 659 of the base sequence represented by SEQ ID NO: 1, and the base sequence represented by SEQ ID NO: 3 A DNA fragment comprising a base sequence of base numbers 1 to 696 of the cellulase gene, or a base sequence having 70% or more identity with any of the base sequences, under stringent conditions with DNA comprising any of the above base sequences The recombinant Bacillus subtilis according to claim 6, wherein the recombinant Bacillus subtilis is a DNA fragment comprising a hybridizing DNA fragment or a base sequence from which any part of the base sequence is deleted.   The recombinant Bacillus subtilis according to any one of claims 4 to 8, wherein the target gene product is a heterologous protein or polypeptide.   The manufacturing method of the target gene product using the recombinant Bacillus subtilis of any one of Claims 4-9.