JP4955358B2 - New Bacillus subtilis mutant - Google Patents

Description

  The present invention relates to a novel Bacillus subtilis mutant, and more particularly to a novel Bacillus subtilis mutant having improved secretion productivity of various proteins.

  Bacillus subtilis is not only widely used in molecular biology studies as a model of Gram-positive bacteria, but is also widely used in the fermentation industry, the pharmaceutical industry, and the like as a bacterium that produces various enzymes such as amylase and protease. Although the entire base sequence of the Bacillus subtilis genome has already been determined by the Japan-European Joint Genome Project, the functional identification of about 4100 genes existing in the Bacillus subtilis genome has not been completed.

To date, about 4100 types of disrupted strains of genes present in the Bacillus subtilis genome have been exhaustively studied, and it has been pointed out that 271 genes are essential for growth (Non-patent Document 1).
In addition, genes related to early sporulation such as Bacillus subtilis, protease genes, genes related to D-alanine addition to teichoic acid in the cell wall or cell membrane, and genes related to biosynthesis or secretion of surfactin alone are lacking. Lost or inactivated strains have been constructed (see Patent Literature 1, Patent Literature 2, Patent Literature 3, Patent Literature 4, Patent Literature 5, Patent Literature 6, Patent Literature 7, and Patent Literature 8). However, the improvement in protein productivity by these strains has not been sufficient, and to date, useful knowledge has been obtained regarding mutants derived from Bacillus subtilis having improved productivity of various proteins and comprehensive analysis of the mutants. It is not done.

K. Kobayashi et al., Proc. Natl. Acad. Sci. USA., 100, 4678-4683, 2003 JP-A-58-190390 JP 61-1381 WO89 / 04866 pamphlet Japanese National Patent Publication No. 11-509096 Patent No. 3210315 Special table 2001-527401 Special table 2002-520017 Special table 2001-503641

  Then, in view of the above-mentioned situation, the present invention aims to provide a novel Bacillus subtilis mutant strain excellent in productivity of various enzymes by comprehensively analyzing gene-disrupted strains derived from Bacillus subtilis.

  In order to solve the above problems, the present inventors comprehensively analyze mutants lacking a large region of the Bacillus subtilis genome, and obtain many Bacillus subtilis mutants excellent in productivity of various enzymes. It succeeded, and it came to complete invention.

  The Bacillus subtilis mutant strain according to the present invention has a genome structure in which the region described in the column of the deletion region shown in Table 1 below is deleted. In this Bacillus subtilis mutant, when the gene encoding the target protein is introduced so that it can be expressed, the secretory productivity of the target protein is significantly improved compared to the case where the gene is introduced into the wild type. ing. Further, the Bacillus subtilis mutant may be one into which a gene encoding the target protein has been introduced so as to be expressed. Furthermore, the gene encoding the target protein contains a base sequence encoding a region corresponding to the secretion signal, or is appropriately linked to DNA containing a base sequence encoding the region corresponding to the secretion signal upstream thereof. It may be. Here, the target protein may be at least one enzyme selected from the group consisting of cellulase, protease and amylase. Furthermore, the Bacillus subtilis mutant strain may be prepared using the Bacillus subtilis 168 strain as a wild strain. Furthermore, the genomic region described in the column of the defective region in Table 1 below may include a region sandwiched by the oligonucleotide sets in Table 2 below.

  According to the present invention, a novel Bacillus subtilis mutant strain excellent in productivity of various enzymes can be provided. By using the Bacillus subtilis mutant according to the present invention, it is possible not only to realize industrial production methods of various enzymes with excellent productivity, but also to provide biological materials useful for elucidating the production mechanisms of various enzymes. can do.

  Hereinafter, the present invention will be described in detail.

  The novel Bacillus subtilis mutant strain provided in the present invention can be obtained by deleting a large region from the Bacillus subtilis genome. These Bacillus subtilis mutants have improved secretion productivity of the target protein or polypeptide derived from the gene when the cloned gene is introduced. Here, the gene to be introduced is not limited as long as it encodes a protein, and may be exogenous or endogenous. The gene may include a base sequence encoding a region corresponding to a secretion signal, or appropriately linked to DNA including a base sequence encoding a region corresponding to a secretion signal upstream of the gene. It may be. Moreover, the said gene may be introduce | transduced into the genome of a Bacillus subtilis mutant, and may be introduce | transduced into a Bacillus subtilis mutant as an expression vector. Furthermore, the number of genes to be introduced may be one or plural. When a plurality of genes are introduced, the plurality of genes may be introduced in parallel on one DNA fragment, or the plurality of genes may be introduced as different DNA fragments. The method for introducing a gene is not particularly limited, and conventionally known transformation methods, transduction methods, and the like can be used.

  In addition, the gene to be introduced is not particularly limited, and examples thereof include secretory alkaline cellulase, secreted alkaline protease, and secreted alkaline amylase.

  Table 1 shows the names and deletion regions of the novel Bacillus subtilis mutants according to the present invention.

  In addition, the deletion region shown in Table 1 can be rephrased as a region sandwiched between a pair of oligonucleotide sets shown in Table 2.

  In addition, each Bacillus subtilis mutant strain according to the present invention has a genomic structure in which other regions are deleted from the genomic DNA of a standard wild strain such as Bacillus subtilis 168 in addition to the deletion region defined above. Also included. Examples of other regions include a gene region excluding a growth essential gene and a non-coding region, and a region that does not reduce the above-described secretory productivity even when deleted from the genome is preferable.

  The method for deleting the deletion region shown in Table 1 from the Bacillus subtilis genome is not particularly limited, but for example, the following method shown in FIG. 1 can be applied.

  That is, according to a method using a two-step single crossover method using a deletion plasmid inserted with a deletion DNA fragment prepared by the so-called SOE-PCR method (Gene, 77, 61 (1989)), Table 1 Is deleted from the Bacillus subtilis genome. The DNA fragment for deletion used in this method is a DNA fragment (downstream fragment) in which about 0.1 to 3 kb fragment adjacent to the upstream of the deletion target region (referred to as upstream fragment) and about 0.1 to 3 kb fragment adjacent to the downstream are bound. Are called DNA fragments. A DNA fragment in which a drug resistance marker gene fragment such as a chloramphenicol resistance gene is bound downstream or upstream of the DNA fragment can also be used.

  First, by the first PCR, an upstream fragment and a downstream fragment of a deletion target gene, and, if necessary, three fragments of a drug resistance marker gene fragment are prepared. At this time, a primer to which a sequence of 10 to 30 base pairs at the end of the DNA fragment to be bound is added is designed. For example, when the upstream fragment and the downstream fragment are joined in this order, a sequence corresponding to 10 to 30 bases upstream of the downstream fragment is added to the 5 ′ end of the primer located at the downstream end of the upstream fragment (annealing). In addition, a sequence corresponding to 10 to 30 bases downstream of the upstream fragment is added to the 5 ′ end of the primer located at the upstream end of the downstream fragment (annealing). When the upstream fragment and the downstream fragment are amplified using the primer set designed in this way, a region corresponding to the upstream side of the downstream fragment is added to the downstream side of the amplified upstream fragment. A region corresponding to the downstream side of the upstream fragment is added to the upstream side of the fragment.

  Next, the upstream fragment and the downstream fragment prepared in the first round are mixed to form a template, and a pair consisting of a primer located on the upstream side of the upstream fragment (annealed) and a primer located on the downstream side of the downstream fragment (annealed) Perform a second PCR using these primers. By this second PCR, a deletion DNA fragment in which the upstream fragment and the downstream fragment are combined in this order can be amplified.

  When the drug resistance marker gene fragment is linked to the deletion DNA fragment, the drug resistance marker gene fragment is amplified so that a region corresponding to the downstream side of the downstream fragment is added in the first PCR. Further, in the second PCR, a pair of primers consisting of a primer located upstream of the upstream fragment (annealing) and a primer located downstream of the drug resistance marker gene fragment (annealed) are used. As a result, the deletion DNA fragment in which the upstream fragment, the downstream fragment, and the drug resistance marker gene fragment are combined in this order can be amplified.

  Further, after amplifying the deletion DNA fragment obtained by joining the upstream fragment and the downstream fragment in this order by the second PCR, by inserting the deletion DNA fragment into the plasmid containing the drug resistance marker gene, the upstream fragment, A deletion DNA fragment having the downstream fragment and the drug resistance marker gene fragment in this order may 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, etc. when Bacillus subtilis is used as the host.

  Next, transformation of the host bacterium with the deletion plasmid is performed by the competent cell transformation method (J. Bacteriol. 93, 1925 (1967)), etc., and homology on the genome with the upstream or downstream fragment inserted into the plasmid. A transformant in which the deletion plasmid is fused into the host bacterial genomic DNA is obtained by homologous recombination between the regions. For selection of transformants, drug resistance by a marker gene such as a chloramphenicol resistance gene in a plasmid for deletion introduction may be used as an index.

  In the genome of the transformant thus obtained, there are duplicates of the upstream and downstream sequences of the drug resistance gene on the genome to be deleted that are derived from the host fungus genome and the deletion plasmid. ing. In this upstream region or downstream region, by causing homologous recombination in the genome in a region different from the region homologous recombination when obtaining the transformant, drug resistance on the genome together with the region derived from the deletion plasmid Deletion of the target gene to be deleted occurs, such as a gene. As a method for causing homologous recombination in the genome, for example, a method of inducing competence (J. Bacteriol. 93, 1925 (1967)) can be mentioned, but it can be induced spontaneously even during culturing in a normal medium. Homologous recombination occurs. Strains that have undergone homologous recombination within the genome as intended can be selected from strains that have become drug-sensitive because the drug resistance gene is lost at the same time and the drug resistance is lost. Genomic DNA may be extracted from these strains and the deletion of the target gene may be confirmed by PCR or the like.

When selecting a deletion strain of interest, 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 efficiently obtain the target deletion strain, it is desirable to devise measures such as increasing the ratio of drug-sensitive strains. 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.

  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. Furthermore, a Bacillus subtilis mutant strain having a genomic structure lacking a plurality of regions can be produced 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”. That is, in the LP transformation method, a protoplast having a cell wall lysed is provided as a donor DNA to a competent cell of a recipient strain. The added protoplast is considered to be destroyed by osmotic shock, and the donor DNA released into the culture medium is taken up into the competent cell of the recipient strain. In addition, according to the LP transformation method, DNA damage to be introduced is greatly reduced as compared with a general transformation method.

  By applying this LP transformation method, a Bacillus subtilis mutant strain having a genome structure in which a plurality of regions are deleted can be newly produced from a Bacillus subtilis mutant strain having a genome structure deleted alone. Specifically, first, a Bacillus subtilis mutant strain having a genomic structure lacking a specific region (referred to as a first deletion region) is protoplasted, and a different region (second deletion region) is deleted. And co-existing with competent cells of Bacillus subtilis mutants having the genomic structure. Thereby forming a pair of interchain exchange structures between genomic DNA having a first deletion region (donor DNA) and genomic DNA having a second deletion region (host DNA); Become. This set of interstrand exchange structures occurs at a position sandwiching the first deletion region in the donor DNA, whereby the first deletion region in the donor DNA is introduced into the host DNA. Thus, by applying the LP transformation method, a Bacillus subtilis mutant strain having a genomic structure in which the first deletion region and the second deletion region are deleted can be produced. By applying this method, a Bacillus subtilis mutant strain having a genomic structure in which a plurality of regions are deleted can be produced unless a gene essential for growth is deleted.

  The Bacillus subtilis mutant strain according to the present invention produced as described above is superior in the secretory production ability of the protein or polypeptide encoded by the introduced gene, compared to the wild standard strain such as Bacillus subtilis 168. It has the characteristics of being. The target protein or target polypeptide produced using the Bacillus subtilis mutant strain of the present invention is not particularly limited. For example, the industrial protein used in various industries such as detergents, foods, fibers, feeds, chemicals, medicines, and diagnostics. Examples include enzymes and physiologically active peptides, and industrial enzymes are particularly preferable. Industrial enzymes are classified according to their functions: oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthase (Ligase / Synthetase) and the like. Among these, the target protein produced using the Bacillus subtilis mutant strain of the present invention preferably includes hydrolases such as cellulase, α-amylase, and protease.

  For example, in a Bacillus subtilis mutant strain into which cellulase, protease, and amylase have been introduced as genes, the amount of enzyme produced secreted outside the cell body is significantly improved as compared to the case of introduction into a wild standard strain. The secretory productivity of these enzymes in the Bacillus subtilis mutant according to the present invention can be measured by applying conventionally known various techniques without limitation.

  Cellulase productivity can be measured, for example, as follows. First, a test Bacillus subtilis mutant is transformed using a vector having a cellulase gene. Next, after culturing the obtained transformant, a culture supernatant from which the cells are removed is obtained by centrifugation or the like. Then, for example, p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation) is added to the resulting supernatant as a substrate, reacted for a predetermined time, and the amount of p-nitrophenol released when the reaction is performed is 420 nm. Quantified by change in absorbance at OD420nm. Thereby, the productivity of the cellulase encoded by the cellulase gene introduced into the mutant strain of Bacillus subtilis can be measured. In addition, the cellulase productivity in a test Bacillus subtilis mutant can be evaluated as a relative value by measuring the cellulase productivity in a standard wild-type strain such as Bacillus subtilis 168.

  Cellulases include, for example, cellulases belonging to Family 5 in the polysaccharide hydrolase classification (Biochem. J., 280, 309, 1991), and among them, cellulases derived from microorganisms, particularly Bacillus bacteria are preferred. As a more specific example, an alkaline cellulase derived from the Bacillus genus bacterium KSM-S237 strain (FERM BP-7875) consisting of the amino acid sequence represented by SEQ ID NO: 116, or a Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 118 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, particularly preferably 98% or more. A cellulase consisting of an amino acid sequence having identity is exemplified. The alkaline cellulase having the amino acid sequence represented by SEQ ID NO: 116 and the alkaline cellulase having the amino acid sequence represented by SEQ ID NO: 118 showed about 92% identity in comparison between the amino acid sequences, both of which are used in the present invention. A specific example of cellulase is preferable, but alkaline cellulase having the amino acid sequence represented by SEQ ID NO: 116 is more preferable.

  In the production of such cellulase, among the Bacillus subtilis mutants of the present invention, the MGB653 strain, MGB683 strain, MGB781 strain, MGB723 strain, MGB773 strain, MGB822 strain, MGB834 strain, MGB846 strain, MGB872 strain in Table 1 MGB885, MGB913, MGB860, MGB874, MGB887, NED02021, NED0400, NED0600, NED0803, NED0804, NED1100, NED1200, NED1400, NED1500, NED1901, NED1901, NED2201 , NED2202, NED2402, NED2500, NED2602, NED2702, NED2802, NED3000, NED3200, NED3303, NED3701, NED3800, NED4000, NED4001, NED4002, and NED4100 More preferably, a mutant strain is used.

  As an example, the productivity of protease can be measured as follows. First, a test Bacillus subtilis mutant is transformed using a vector having a protease gene. Next, after culturing the obtained transformant, a culture supernatant from which the cells are removed is obtained by centrifugation or the like. Then, for example, Succinyl-L-Alanyl-L-Alanyl-L-Alanine p-Nitroanilide (STANA Peptide Laboratories) is added to the resulting supernatant as a substrate, reacted for a predetermined time, and released when the reaction is performed. The amount of p-nitroaniline to be measured is quantified by absorbance change at 420 nm (OD420 nm). Thereby, the productivity of the protease encoded by the protease gene introduced into the B. subtilis mutant can be measured. In addition, the protease productivity in a test Bacillus subtilis mutant can be evaluated as a relative value by measuring the protease productivity in a standard wild strain such as Bacillus subtilis 168.

  Specific examples of proteases include serine proteases, metal proteases, and the like derived from microorganisms, in particular, Bacillus bacteria. More specific examples include alkaline protease derived from Bacillus clausii KSM-K16 strain (FERM BP-3376) consisting of the amino acid sequence represented by SEQ ID NO: 119, 70%, preferably 80% of the amino acid sequence. More preferred is a protease comprising an amino acid sequence having an identity of 90% or more, more preferably 95% or more, particularly preferably 98% or more.

  In the production of such protease, among the Bacillus subtilis mutant strains of the present invention, MGB533 strain, MGB592 strain, MGB604 strain, MGB625 strain, MGB653 strain, MGB683 strain, MGB781 strain, MGB723 strain, MGB773 strain, MGB822 strain, MGB834 strain, B. subtilis mutation selected from MGB846, MGB872, MGB885, MGB913, MGB943, MGB860, MGB874, MGB887, NED0302, NED0400, NED0600, NED0803, NED1500, NED1902, and NED3200 More preferably, the strain is used.

  The productivity of alkaline amylase can be measured as follows as an example. First, a test Bacillus subtilis mutant is transformed using a vector having an alkaline amylase gene. Next, after culturing the obtained transformant, a culture supernatant from which the cells are removed is obtained by centrifugation or the like. Then, for example, the activity of alkaline amylase contained in the supernatant can be measured using Liquitech Amy EPS (Roche Diagnostics) which is an amylase activity measurement kit. Thereby, the productivity of the alkaline amylase encoded by the alkaline amylase gene introduced into the mutant strain of Bacillus subtilis can be measured. In addition, the alkaline amylase productivity in a test Bacillus subtilis mutant can be evaluated as a relative value by measuring the alkaline amylase productivity in a standard wild-type strain such as Bacillus subtilis 168.

  Specific examples of amylase include α-amylase derived from microorganisms, and liquefied amylase derived from Bacillus bacteria is particularly preferable. More specific examples include 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: 120, and 70%, preferably 80%, more preferably the amino acid sequence. Is an amylase consisting of an amino acid sequence having 90% or more, more preferably 95% or more, particularly preferably 98% or more.

  In the production of such α-amylase, among the Bacillus subtilis mutants of the present invention, MGB653 strain, NED0301, NED0302, NED0400, NED0600, NED0802, NED0804, NED0900, NED1002, NED1003, NED1100 More preferably, a Bacillus subtilis mutant selected from the following strains: NED1602, NED2602, NED2702, NED3402, NED3701 and NED3800.

The target protein or polypeptide gene to be introduced into the Bacillus subtilis mutant of the present invention has a control region involved in transcription, translation, and secretion of the gene upstream thereof, that is, a transcription initiation control region comprising a promoter and a transcription initiation site, It is desirable that at least one region selected from a translation initiation region containing a ribosome binding site and an initiation codon and a secretory signal peptide region be bound in an appropriate form. In particular, it is preferable that three regions consisting of a transcription initiation control region, a translation initiation control region, and a secretion signal region are combined, and the secretion signal peptide region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus , It is preferable that the start region and the translation start region are 0.6-1 kb region upstream of the cellulase gene and are linked to the target protein or polypeptide gene in an appropriate form. For example, Bacillus (Bacillus) bacteria as described in 2000-210081 and JP 4-190793 Patent Publication JP, i.e. KSM-S237 strain (FERM BP-7875), KSM -64 strain (FERM BP- It is desirable that the transcription initiation regulatory region, the translation initiation region, and the secretory signal peptide region of the cellulase gene derived from 2886) are appropriately combined with the structural gene of the target protein or polypeptide. More specifically, the base sequence of base numbers 1 to 659 of the base sequence represented by SEQ ID NO: 115, the base sequence of base numbers 1 to 696 of the cellulase gene consisting of the base sequence represented by SEQ ID NO: 117, and the base sequence Or a DNA fragment comprising a nucleotide sequence having an identity of 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, or any of the above bases It is desirable that a DNA fragment consisting of a base sequence from which a part of the sequence is deleted is appropriately bound to the structural gene of the target protein or polypeptide. Here, a DNA fragment consisting of a base sequence from which a part of the base sequence has been deleted is a part of the base sequence that has been deleted, but retains functions related to gene transcription, translation, and secretion. Means a DNA fragment.

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

  In this example, mutant strains were prepared in which various regions on the genome were deleted using Bacillus subtilis 168 as a wild strain. In addition, regarding the various primers used in this example, the correspondence between the primer name, the base sequence, and the sequence number is shown in Table 10 at the end.

[Example 1] Preparation of mutant strain lacking multiple regions <Preparation of strain lacking upp gene (including cat-upp cassette)>
As shown in FIG. 2, genomic DNA extracted from Bacillus subtilis 168 strain was used as a template, and upp genes on the genome (upp-AFW and upp-ARV, and upp-BFW and upp-BRV were used as primer sets ( A 1.0 kb fragment (a1) adjacent to the upstream of BG 13408; uracil phosphoribosyl-transferase) and a 1.0 kb fragment (b1) adjacent to the downstream were amplified by PCR. In addition, a 1.2 kb fragment (c1) containing an erythromycin resistance gene was prepared by PCR using plasmid pMutinT3 (Microbiology, 144, 3097, 1998) as a template and a set of Erm-FW and Erm-RV primers. In the PCR reaction, the reaction system was 20 μL, and LATaq polymerase (manufactured by Takara Bio Inc.) was used according to the attached protocol. For (a1) and (b1), 50 ng of the genome of Bacillus subtilis 168 strain, which is a template DNA, In the above, 1 ng of plasmid DNA, 200 nM each of the above primers, 200 μM each of dATP, dTTP, dCTP, and dGTP, LATaq 0.2 U, and the attached 10 × buffer solution were mixed so as to be 1 ×. For the amplification reaction, a PCR system (GeneAmp9700 manufactured by Applied Biosystems) was used. After heat denaturation at 95 ° C. for 5 minutes, constant temperature was maintained at 95 ° C. for 15 seconds, then 55 ° C. for 30 seconds, and then 72 ° C. for 60 seconds. This was repeated 25 times, and finally the extension was completed at 72 ° C. for 30 seconds. The three PCR amplification fragments (a1), (b1) and (c1) thus obtained were purified with Centricon (Millipore), mixed with 0.5 μL of each, and further added with primer upp- AFW and upp-BRV were added, and the constant temperature at 72 ° C. of the above PCR conditions was changed to 3 minutes, and SOE-PCR was performed. As a result of this PCR, a 3.2 kb DNA fragment (d1) was obtained in which the three fragments were combined in the order of (a1), (c1) and (b1). Using this DNA fragment, 168 strains of Bacillus subtilis were transformed by a competent method (J. Bacteeriol., 81, 741, 1960). That is, 168 strains of Bacillus subtilis were treated with SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.02% casamino acid ( Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan) at 37 ° C. until the degree 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% trisodium citrate dihydrate, 0.50% Inoculated with glucose, 0.01% casamino acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / ml tryptophan), and further cultured with shaking until the growth degree (OD600) is about 0.4. 168 strain competent cells were prepared. Next, 5 μL of the solution containing the DNA fragment (d1) (the reaction solution for the 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, The whole amount was smeared on LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing 0.5 g / mL erythromycin. After static culture at 37 ° C., the grown colonies were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR using this as a template that the upp gene was deleted from the genome and replaced with an erythromycin resistance gene. This strain is hereinafter referred to as 168Δupp. In addition, transformation by the competent method described later was performed in the same manner as described above except that the DNA to be used and the agar medium for selection of transformants were appropriately changed.

<Construction of cat-upp cassette DNA fragment>
As shown in FIG. 3, plasmid pSM5022 (Mol. Microbiol. 6, 309, 1992) whose transcription has been confirmed in Bacillus subtilis so that transcription of the upp gene and the chloramphenicol resistance gene (cat) is performed reliably. The upp gene was ligated downstream of the cat gene. That is, a 1.3-kb DNA fragment containing cat was amplified by PCR using pSM5022 as a template using a primer set of cat-Fw and cat-Rv. Further, PCR was performed using primers upp-Fw and upp-RV with the 168 strain genome as a template to obtain a 1.1-kb DNA fragment containing the upp gene. Next, these two fragments were purified and then combined by SOE-PCR to prepare a 2.4 kb ca-upp cassette DNA fragment (C) in which the upp gene was bound downstream of the cat gene. Further, this fragment was inserted into the ClaI cleavage site of plasmid pBR322 to obtain a recombinant plasmid pBRcatupp.

<Preparation of Pro1 region deletion strain (including cat-upp cassette fragment)>
As shown in FIG. 4, a 0.6 kb adjoining upstream of the Pro1 region by PCR using the 168 strain genome as a template using each of the Pro1-AFW and Pro1-ARV, Pro1-BFW and Pro1-BRV primer sets. Fragment (A) and a downstream adjacent 0.3 kb fragment (B) were prepared. In FIG. 4, the Pro1 region is described as “deletion target region”.

  Next, SOE-PCR using primers Pro1-AFW and Pro1-BRV was performed using the obtained PCR amplified fragments (A) and (B) and the above-described cat-upp cassette fragment (C) as a template to obtain 3 fragments. They were combined in the order of (A), (C) and (B). The obtained DNA fragment (D) was used to transform the 168Δupp strain shown in Example 2 by a competent method, and a transformant capable of growing on an LB agar medium containing 10 ppm of chloramphenicol was isolated. . It was confirmed by PCR that the Pro1 region was deleted from the genome and replaced with a cat-upp cassette DNA fragment by PCR. Furthermore, Cg + glucose agar medium (7% dipotassium hydrogen phosphate, 3% potassium dihydrogen phosphate, 0.5% sodium citrate) to which this transformant was added with various concentrations of 5FU (manufactured by Sigma-Aldrich) 1% ammonium sulfate, 0.1% magnesium sulfate, 0.05% glutamic acid, 0.5% glucose, 10 ng / mL L-tryptophan, 0.55 μg / mL calcium chloride, 0.17 μg / mL zinc chloride, 43 ng / mL Culture was performed on copper chloride dihydrate, 60 ng / mL cobalt chloride hexahydrate, 60 ng / mL sodium molybdate (IV) dihydrate, 1.5% agar), but 0.5 μg / mL No growth was observed in the medium supplemented with 5FU. On the other hand, growth of the 168Δupp strain, the parent strain of the transformant, was also observed in a medium supplemented with 5 μg / mL of 5FU under the same conditions. From the above results, it was estimated that the upp gene introduced into the transformant was expressed by transcription from the cat gene promoter and became sensitive to 5FU. The strain thus obtained was designated as ΔPro1 :: cat-upp strain. In FIG. 4, the ΔPro1 :: cat-upp strain is described as a Δ deletion target region :: cat-upp strain.

<Deletion of cat-upp cassette fragment (C) from Pro1 region deletion strain>
As shown in FIG. 5, using the ΔPro1 :: cat-upp strain genome as a template and upstream of the cat-upp cassette fragment (C) using the Pro1-AFW and Pro1-ERV and Pro1-FFW and Pro1-BRV primer sets. A 0.6 kb fragment adjacent to (E) and a 0.3 kb fragment adjacent to downstream (F) are amplified, and both DNA fragments obtained as templates are used as templates for SOE using Pro1-AFW and Pro1-BRV primer sets. -A 0.9 kb fragment (G) in which both fragments were bound was prepared by PCR. Using this fragment (G), the above-mentioned ΔPro1 :: cat-upp strain was transformed by a competent method to obtain a strain capable of growing on a Cg + glucose agar medium supplemented with 1 μg / mL / 5FU. In the obtained strain, chloramphenicol sensitivity was confirmed, and it was confirmed that the Pro1 region on the genome and the cat-upp cassette DNA fragment were deleted, and this was designated as ΔPro1 strain. In FIG. 5, ΔPro1 :: cat-upp strain and ΔPro1 strain are described as “Δ deletion target region :: cat-upp strain” and “Δ deletion target region strain”, respectively.

<Preparation of single region deletion strain 1>
According to the above-described production procedure of ΔPro1 strain, ΔPro2 :: cat-upp strain, ΔPro3 :: cat-upp strain, ΔPro4 :: cat-upp strain, ΔPro5 :: cat-upp strain are obtained by the method described in FIG. , ΔPro6 :: cat-upp strain, ΔPro7 :: cat-upp strain, ΔPBSX :: cat-upp strain, ΔSPβ :: cat-upp strain, ΔSKIN :: cat-upp strain, Δpks :: cat-upp strain and Δpps :: Cat-upp strain was prepared. Further, ΔPro2 strain, ΔPro3 strain, ΔPro4 strain, ΔPro5 strain, ΔPro6 strain, ΔPro7 strain, ΔPBSX strain, ΔSPβ strain, ΔSKIN strain, Δpks strain and Δpps strain were prepared by the method described in FIG. Table 3 below shows the primer sets used for amplifying fragment (A) to fragment (G) in the step of producing each of these strains.

<Construction of multiple deletion strains (MGB01 to MGB07)>
Next, a strain in which a plurality of regions were deleted (multiple deletion strain) was constructed using ΔPro7 strain (also referred to as MGB01 strain). First, a double deletion strain of Pro7 region and Pro6 region was constructed as follows. Specifically, ΔPro7 strain was transformed by competent method using the genomic DNA of ΔPro6 :: cat-upp strain in which Pro6 region was replaced with cat-upp cassette fragment, and the LB agar medium containing 10 ppm of chloramphenicol was used. Growing colonies were isolated as transformants. Next, the obtained chloramphenicol resistant transformant was transformed with ΔPro6 strain genomic DNA by the competent method and capable of growing on a Cg + glucose agar medium supplemented with 1 μg / mL of 5FU. Acquired shares. In the obtained strain, chloramphenicol sensitivity was confirmed, and both the Pro6 region and the Pro7 region were deleted, and a double deletion strain containing no cat-upp cassette fragment was isolated. This strain was designated as MGB02 strain.

  By repeating the same operation, an MGB03 strain in which the Pro7 region, the Pro6 region, and the Pro1 region were sequentially deleted was constructed. By repeating the same operation, the MGB04 strain in which the Pro7 region, Pro6 region, Pro1 region and Pro4 region were sequentially deleted was constructed. By repeating the same operation, the MGB05 strain in which the Pro7 region, the Pro6 region, the Pro1 region, the Pro4 region and the PBSX region were sequentially deleted was constructed. By repeating the same operation, the MGB06 strain in which the Pro7 region, the Pro6 region, the Pro1 region, the Pro4 region, the PBSX region, and the Pro5 region were sequentially deleted was constructed. By repeating the same operation, the MGB07 strain in which the Pro7 region, Pro6 region, Pro1 region, Pro4 region, PBSX region, Pro5 region and Pro3 region were sequentially deleted was constructed.

<Preparation 2 of each single region deletion strain>
In a method different from the <Preparation of single region deletion strain 1> described above, SPβ region, pks region, SKIN region, pps region, Pro2 region, Pro5 region, NED0302 region (ydcL-ydhU region), NED0803 region (yisB- yitD region), NED3200 region (yunA-yurT region), NED1902 region (cgeE-ypmQ region), NED0501 region (yeeK-yesX region), NED0400 region (ydiM-yebA region), NED1100 region (ykuS-ykqB region), NED4002 Region (pdp-rocR region), NED02021 region (ycxB-sipU region), SKIN-Pro7 region (spoIVCB-yraK region), NED3701 region (sbo-ywhH region), NED0600 region (cspB-yhcT region), NED4100 region (yybP) -yyaJ region), NED2702 region (ytxK-braB region) and NED1602 region (yncM-fosB region) were deleted alone.

  Here, a construction example of a strain in which the SPβ region is deleted alone will be described. As shown in FIG. 6, using a primer set of spB-AFW and spB-ARV2 and spB-BFW2 and spB-BRV, using a 168 strain genome as a template, a 0.6 kb adjoining upstream of the SPβ region by PCR. Fragment (H) and a downstream adjacent 0.3 kb fragment (I) were prepared. In FIG. 6, SPβ is described as “deletion target region”.

Further, a tetracycline resistance gene region fragment (J) was amplified using a primer set of tet-FW and tet-RV. Next, SOE-PCR using primers spB-AFW and spB-BRV was performed using the obtained PCR amplified fragments (H) (I) (J) as templates, and the three fragments were in the order of (H) (J) (I). It was made to combine. Using the obtained DNA fragment (K), the above-described 168Δupp strain was transformed by a competent method to isolate a transformant capable of growing on an LB agar medium containing 15 ppm tetracycline. The resulting transformant was confirmed by PCR to have the SPβ region deleted from the genome and replaced with a tetracycline resistance gene fragment, and this was designated as ΔSPβ :: tet strain. In FIG. 6, the ΔSPβ :: tet strain is described as “Δ deletion target region :: tet strain”.
Similarly, a strain in which each region described above was deleted alone was prepared. The prepared strain is referred to as “Δ deletion target region :: tet strain” in the same manner as the ΔSPβ :: tet strain.

<Deletion of SPβ region from MGB07 strain>
The ΔPro7 strain was transformed with the genomic DNA of the ΔSPβ :: tet strain prepared above to obtain a tetracycline resistant strain MGB07ΔSPβ :: tet strain. On the other hand, the tetracycline resistance gene fragment was removed from the genome as follows.

  As shown in FIG. 7, using the primer sets of spB-AFW and spB-ERV, and spB-FFW and spB-BRV, a 0.6 kb fragment adjacent to the upstream of the SPβ region by PCR using the 168 strain genome as a template (L) and a downstream adjacent 0.3 kb fragment (M) were prepared. In FIG. 7, SPβ is described as “deletion target region”.

Next, using the obtained PCR amplified fragment (L) (M) as a template, SOE-PCR was performed using primers spB-AFW and spB-BRV to bind the two fragments in the order of (L) (M). . The obtained DNA fragment (N) was inserted into the above-described sac I- Kpn I restriction enzyme site of pBRcatupp (blunted after cleavage) to construct a plasmid pBRcatuppΔSPβ for deletion of the tetracycline resistance gene fragment. In FIG. 7, pBRcatuppΔSPβ is described as “pBRcatuppΔ deletion target region”.

  By transforming the MGB07ΔSPβ :: tet strain with the constructed pBRcatuppΔSPβ, single crossover recombination occurred between the region upstream or downstream of SPβ on the plasmid and the region upstream or downstream of SPβ on the genome. A plasmid was introduced into the genome to obtain a strain MGB07ΔSPβ (pBR) strain exhibiting chloramphenicol resistance.

  The obtained transformant MGB07ΔSPβ (pBR) was inoculated into 50 mL of LB medium (500 mL Sakaguchi flask) containing 1.5 μg / mL of tetracycline so that OD600 = 0.3, and cultured at 37 ° C. with shaking for 1 hour. Ampicillin 15 mg (300 μg / mL) was added to the eye, and then the culture was continued while adding ampicillin 15 mg every 2 hours. After 8.5 hours of culture, the culture was washed with 2% sodium chloride aqueous solution, and the drug-free LB agar medium Smeared on. Among the grown colonies, those that became chloramphenicol sensitive as the plasmid region dropped out were selected.

  Using the genomic DNA of the selected strain as a template, PCR was performed to confirm that the SPβ region and the tetracycline resistance gene fragment were deleted, and the MGB08 strain was obtained.

<Deletion of pks region from MGB08 strain and restoration of Pro5 region>
Deletion of the pks region from the MGB08 strain prepared above was performed using the Δpks :: tet strain according to the method described above (FIG. 7). A strain in which the pks region was deleted from the MGB08 strain was named MGB09 strain. When the genomic DNA of the obtained MGB09 strain was confirmed by PCR, the pks region was deleted, but the presence of a sequence within the Pro5 region that was located close to the pks region on the genome was confirmed, and the Pro5 region was I found out that I have returned. When the MGB08 strain was transformed with the Δpks :: tet genomic DNA, the upstream region of the pks region on the Δpks :: tet strain genome was introduced simultaneously with the introduction of the tetracycline resistance gene accompanying the deletion of the pks region. And the region downstream of the Pro5 region were considered to be due to the occurrence of homologous recombination between the corresponding region on the genome of the MGB08 strain.

<Deletion of SKIN region from MGB09 strain and restoration of Pro7 region>
The deletion of the SKIN region from the MGB09 strain prepared above was performed using the ΔSKIN :: tet strain in accordance with the method described above (FIG. 7). A strain in which the SKIN region was deleted from MGB09 strain was designated as MGB10 strain. When the genomic DNA of the obtained MGB10 strain was confirmed by PCR, the SKIN region was deleted, but the presence of a sequence within the Pro7 region that was located close to the SKIN region and the genome was confirmed, and the Pro7 region was I found out that I have returned. When the MGB09 strain was transformed using the ΔSKIN :: tet genomic DNA, the upstream region of the SKIN region on the ΔSKIN :: tet strain genome was introduced simultaneously with the introduction of the tetracycline resistance gene accompanying the deletion of the SKIN region. And the region downstream of the Pro7 region were considered to be due to the occurrence of homologous recombination between the corresponding region on the genome of the MGB09 strain.

<Deletion of pps region from MGB10 strain>
Deletion of the pps region from the MGB10 strain prepared above was performed using the Δpps :: tet strain according to the method described above (FIG. 7). A strain in which the pps region was deleted from MGB10 strain was designated as MGB11 strain. When the obtained genomic DNA of the MGB11 strain was confirmed by PCR, the pps region was deleted without restoring other regions.

<Deletion of Pro2 region from MGB11 strain>
Deletion of the Pro2 region from the MGB11 strain prepared above was performed using the ΔPro2 :: tet strain in accordance with the method described above (FIG. 7). A strain in which the Pro2 region was deleted from the MGB11 strain was named MGB12 strain. When the obtained genomic DNA of the MGB12 strain was confirmed by PCR, the Pro2 region was deleted without restoring other regions.

<Deletion of Pro5 region from MGB12 strain>
In order to delete again the Pro5 region that was restored when the MGB09 strain was prepared, the Pro5 region was deleted from the MGB12 strain prepared above using the ΔPro5 :: tet strain according to the method described above (FIG. 7). I went. A strain in which the Pro5 region was deleted from MGB12 strain was designated as MGB11d strain. When the obtained genomic DNA of the MGB11d strain was confirmed by PCR, the Pro5 region was deleted without restoring other regions.

  MGB11d strain prepared as described above, in Bacillus subtilis 168 strain, Pro6 (yoaV-yobO) region, Pro1 (ybbU-ybdE) region, Pro4 (yjcM-yjdJ) region, PBSX (ykdA-xlyA) region, Pro5 (ynxB-dut) region, Pro3 (ydiM-ydjC) region, SPβ (yodU-ypqP) region, pks (pksA-ymaC) region, SKIN (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region and Pro2 (ydcL) -ydeJ) has a deleted genomic structure.

<Construction of Bacillus subtilis mutant strain according to the present invention>
The Bacillus subtilis mutant according to the present invention was prepared from the MGB11d strain prepared as described above (see FIG. 8). That is, according to the method described above (FIG. 7), deletion of the NED0302 region from the MGB11d strain was performed using the ΔNED0302 :: tet strain. A strain in which the NED0302 region was deleted from the MGB11d strain was named MGB533 strain. The acquired MGB533 strain has a genomic structure in which the NED0302 region is deleted in addition to the deletion region in the MGB11d strain.

  Thereafter, the NED0803 region, the NED3200 region, the NED1902 region, the NED0501 region, the NED0400 region, the NED1100 region, and the NED4002 region were deleted in this order to construct a mutant strain. The constructed mutant strains were named MGB559 strain, MGB592 strain, MGB604 strain, MGB625 strain, MGB653 strain, MGB683 strain and MGB781 strain, respectively.

  The NED3200 region includes the Pro2 region, and the actual region deleted from the MGB559 strain when constructing the MGB592 strain is the ydeK-ydhU region. The NED1902 region contains the SPβ region, and the actual regions deleted from the MGB592 strain when constructing the MGB604 strain are the cgeE-phy region and the yppQ-ypmQ region. Similarly, the NED0400 region includes the Pro3 region, and the actual region deleted from the MGB625 strain during the construction of the MGB653 strain is the gutR-yebA region.

  Next, deletion of the NED40002 region from the constructed MGB625 strain was performed using the ΔNED40002 :: tet strain in accordance with the method described above (FIG. 7). A strain in which the NED40002 region was deleted from MGB625 strain was designated as MGB723 strain.

  Thereafter, the NED02021 region, SKIN-Pro7 region, NED3701 region, NED0600 region, NED4100 region, NED2702 region, NED0400 region and NED1100 region were deleted in this order to construct a mutant strain. The constructed mutant strains were named MGB773 strain, MGB822 strain, MGB834 strain, MGB846 strain, MGB872 strain, MGB885 strain, MGB913 strain and MGB943 strain, respectively.

  The SKIN-Pro7 region contains the SKIN region, and the actual region deleted from the MGB773 strain when constructing the MGB822 strain is the yrkS-yraK region. Similarly, NED0400 contains the Pro3 region, and the actual region deleted from the MGB885 strain when constructing the MGB913 strain is the gutR-yebA region.

  Next, deletion of the NED4100 region from the constructed MGB834 strain was performed using the ΔNED4100 :: tet strain in accordance with the method described above (FIG. 7). A strain in which the NED4100 region was deleted from MGB834 strain was designated as MGB860 strain.

  Thereafter, the NED1602 region and the NED2702 region were deleted in order to construct a mutant strain. The constructed mutant strains were named MGB874 strain and MGB887 strain, respectively.

  Table 4 below shows the primer sets used when amplifying fragment (H) to fragment (N) in the step of preparing each strain.

[Example 2] Mutant strain lacking a single region In this example, a specific region in Bacillus subtilis 168 strain was replaced with a cat-upp cassette or replaced with a chloramphenicol resistance gene, so that the region was isolated. A mutant strain deleted in step 1 was prepared.

<Construction of a single region deletion strain by replacement with cat-upp cassette>
The regions to be replaced with the cat-upp cassette are summarized in Table 5 below.

  The NED0100 region includes a Pro1 region, the NED0302 region includes a Pro2 region, the NED1500 region includes a pks region, the NED1802 region includes a Pro6 region, and the NED2500 region includes a SKIN-Pro7 region.

  In this example, using the cat-upp cassette fragment prepared in Example 1, a mutant strain was constructed by substituting a specific region with the cat-upp cassette fragment according to the method shown in FIG. In this example, fragment (A), fragment (B), fragment (C) and fragment (D) in FIG. 4 are respectively represented as fragment (O), fragment (P), fragment (Q) and fragment (R). It is called. Table 6 below shows primer sets used for amplifying fragment (P) to fragment (R) in the step of preparing each strain.

<Construction of a single region deletion strain by substitution with a chloramphenicol resistance gene>
The region replacement with the chloramphenicol resistance gene was performed by first replacing the target region with the tetracycline resistance gene, and then replacing the central part of the tetracycline resistance gene with the chloramphenicol resistance gene. The regions to be replaced with the chloramphenicol resistance gene are summarized in Table 7 below.

  The NED0400 region includes a Pro3 region, the NED1002 region includes a Pro4 region, the NED1003 region includes a PBSX region, and the NED1902 region includes an SPβ region.

  Hereinafter, a method for deleting the NED0301 region will be described first. Using each primer set of NED0301-AFW and NED0301-ARV, NED0301-BFW and NED0301-BRV, using the 168 strain genome as a template, a 0.6 kb fragment adjacent to the upstream of the NED0301 region by PCR (S) and downstream An adjacent 0.3 kb fragment (T) was amplified. Further, a tetracycline resistance gene region fragment (U) was amplified using a primer set of tet-FW and tet-RV. . Next, SOE-PCR using primers NED0301-AFW and NED0301-BRV was performed using the obtained PCR amplified fragments (S), (T), and (U) as templates, and the three fragments were (S), (U), and (T) in that order. A fragment (V) bound in such a manner was obtained. Using the obtained fragment (V), the 168Δupp strain prepared in Example 1 was transformed by a competent method, and a transformant capable of growing on an LB agar medium containing 15 ppm tetracycline was isolated. It was confirmed by PCR that the NED0301 region was deleted from the genome and replaced with a tetracycline resistance gene fragment by PCR. Next, the upstream 0.5 kb fragment (W) and the downstream 0.5 kb fragment (X) of the tetracycline resistance gene were amplified using the tet-FW and tet-ARV and tet-BFW and tet-RV primer sets. did. Furthermore, using the plasmid pSM5022 used in Example 1 as a template, a 1.3 kb fragment (Y) containing a chloramphenicol resistance gene was amplified using cat-FW and cat-RV. Next, using the obtained PCR amplified fragments (W) (X) (Y) as a template, SOE-PCR using primers tet-FW and tet-RV was performed, and the three fragments were in the order of (W) (Y) (X). A fragment (Z) bound in such a manner was obtained. Using the resulting fragment (Z), the tetracycline resistant strain was transformed by a competent method to isolate a transformant capable of growing on an LB agar medium containing 10 ppm of chloramphenicol. It was confirmed by PCR that a part of the tetracycline resistance gene was deleted and replaced with a chloramphenicol resistance gene by PCR. The strain lacking the NED0301 region was named NED0301 strain.

  Similarly, a mutant strain in which each region shown in Table 7 above was deleted alone was prepared and named in the same manner as the NED0301 strain. Table 8 below shows the primer sets used when amplifying the fragment (S) to the fragment (V) in the step of producing each of these strains.

[Example 3] Evaluation of mutant strain In Example 3, the secretory productivity of the Bacillus subtilis mutant strain according to the present invention prepared in Examples 1 and 2 was evaluated. In this example, alkaline cellulase, alkaline protease and alkaline amylase were used as target proteins 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 (JP-A 2000-210081) 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 shake-cultured overnight at 37 ° C. in 10 mL of LB medium, and 0.05 mL of this culture 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 the measurement of cellulase activity, 50 μL of 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku) 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). The amount of p-nitrophenol liberated when mixed and reacted at 30 ° C. was quantified by the change in absorbance at 420 nm (OD420 nm). The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.

<Evaluation of alkaline protease secretion production>
Evaluation of secretion productivity of alkaline protease was carried out as follows. Specifically, using genomic DNA extracted from Bacillus clausii strain KSM-K16 (FERM BP-3376) as a template, PCR was performed using a primer set of S237pKAPpp-F and KAPter-R (BglII), and the amino acid sequence was determined. A 1.3 kb DNA fragment encoding an alkaline protease (Appl. Microbiol. Biotechnol., 43, 473, (1995)) was amplified. In addition, using genomic DNA extracted from Bacillus sp. KSM-S237 strain (FERM BP-7875) as a template, PCR was performed using the S237ppp-F2 (BamHI) and S237pKAPpp-R primer sets, and the alkaline cellulase gene A 0.6 kb DNA fragment containing the promoter region of JP-A-2000-210081 was amplified. Next, the two fragments obtained were mixed to serve as a template, and SOE-PCR using the S237ppp-F2 (BamHI) and KAPter-R (BglII) primer sets was performed, downstream of the promoter region of the alkaline cellulase gene. A 1.8 kb DNA fragment linked with an alkaline protease gene was obtained. The obtained 1.8 kb DNA fragment was inserted into the BamHI-BglII restriction enzyme cleavage point of shuttle vector pHY300PLK (Yakult) to construct a plasmid pHYKAP (S237p) for evaluating alkaline protease productivity.

The constructed plasmid pHYKAP (S237p) was introduced into each strain by protoplast transformation method. The recombinant strain thus obtained was subjected to shaking culture for 3 days under the same conditions as in the above <Evaluation of production of secreted alkaline cellulase>. After the culture, the alkaline protease activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of the alkaline protease secreted and produced outside the cells by the culture was determined. The activity of protease in the culture supernatant was measured as follows. That is, 75 mM boric acid-KCl buffer containing 7.5 mM Succinyl-L-Alanyl-L-Alanyl-L-Alanine p-Nitroanilide (STANA Peptide Institute) as a substrate in 50 μl of culture supernatant diluted appropriately with ₂ mM CaCl ₂ solution 100 μL of the solution (pH 10.5) was added and mixed, and the amount of p-nitroaniline released upon reaction at 30 ° C. was quantified by the change in absorbance at 420 nm (OD420 nm). The amount of enzyme that liberates 1 μmol of p-nitroaniline per minute was defined as 1 U.

<Evaluation of alkaline amylase secretion production>
The secretion productivity of alkaline amylase was evaluated as follows. Specifically, PCR was performed using a genomic DNA extracted from Bacillus sp. KSM-K38 strain (FERM BP-6946) as a template and a primer set of K38matu-F2 (ALAA) and SP64K38-R (XbaI). A 1.5 kb DNA fragment encoding alkaline amylase (Appl. Environ. Microbiol., 67, 1744, (2001)) was amplified. Also, using genomic DNA extracted from Bacillus sp. KSM-S237 strain (FERM BP-7875) as a template, PCR was performed using a primer set of S237ppp-F2 (BamHI) and S237ppp-R2 (ALAA), A 0.6 kb DNA fragment containing the promoter region of the alkaline cellulase gene (Japanese Patent Laid-Open No. 2000-210081) and the region encoding the secretory signal sequence was amplified. Next, the two fragments obtained were mixed and used as a template. By performing SOE-PCR using the S237ppp-F2 (BamHI) and SP64K38-R (XbaI) primer sets, the promoter region and secretion signal of the alkaline cellulase gene were used. A 2.1 kb DNA fragment was obtained in which an alkaline amylase gene was ligated downstream of the region encoding the sequence. The obtained 2.1 kb DNA fragment was inserted into the BamHI-XbaI restriction enzyme cleavage point of shuttle vector pHY300PLK (Yakult) to construct alkaline amylase productivity evaluation plasmid pHYK38 (S237ps).

  The constructed plasmid pHYK38 (S237ps) was introduced into each strain by protoplast transformation method. The recombinant strain thus obtained was subjected to shaking culture for 5 days under the same conditions as in the above <Evaluation of production of secreted alkaline cellulase>. After culturing, the alkaline amylase activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of amylase secreted and produced outside the cells by the culture was determined. Liquitech Amy EPS (Roche Diagnostics) was used to measure the amylase activity in the culture supernatant. In other words, 50 μL of a sample solution appropriately diluted with 1% NaCl-1 / 7.5 M phosphate buffer (pH 7.4 Wako Pure Chemical Industries) was added to 100 μL of R1 and R2 mixture (R1 (coupling enzyme): R2 (amylase substrate) ) = 5: 1 (Vol.)) Was added and mixed, and the amount of p-nitrophenol released when the reaction was performed at 30 ° C. was quantified by the change in absorbance (OD405 nm) at 405 nm. The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.

<Result>
Table 9 summarizes the secretory production capacities of alkaline cellulase, alkaline protease, and alkaline amylase. In Table 9, the secretory production ability of various enzymes is shown as a relative value when the amount of enzyme production in Bacillus subtilis 168 strain into which each gene is similarly introduced is defined as 100.

It is a schematic diagram for demonstrating an example of the method of deleting a predetermined area | region from the genome of Bacillus subtilis. It is a schematic diagram for demonstrating the procedure which produces 168 (DELTA) upp strain from Bacillus subtilis 168 strain. It is a schematic diagram for demonstrating the procedure which produces recombinant plasmid pBRcatupp formed by inserting a cat-upp cassette DNA fragment. FIG. 3 is a schematic diagram for explaining a procedure for preparing a Δ-deleted target region :: cat-upp strain. It is a schematic diagram for demonstrating the procedure which produces (DELTA) deletion target area | region strain | stump | stock. FIG. 3 is a schematic diagram for explaining a procedure for producing a Δ deletion target region :: tet strain. It is a schematic diagram for demonstrating the procedure of deleting the deletion target area | region in a predetermined mutant using a pBRcatupp (DELTA) deletion target area | region. It is a schematic diagram for demonstrating the preparation process of the Bacillus subtilis mutant which deleted the some deletion area | region which concerns on this invention.

Claims (7)

A Bacillus subtilis mutant strain having a genomic structure in which the region described in the defective region column shown in Table 1 is deleted.

  When a gene encoding a target protein is introduced so that it can be expressed, the secretory productivity of the target protein is significantly improved compared to the case where the gene is introduced into a wild type strain. The Bacillus subtilis mutant according to claim 1.   The Bacillus subtilis mutant according to claim 1 or 2, wherein a gene encoding the target protein is introduced so as to be expressed.   The gene encoding the target protein contains a base sequence encoding a region corresponding to a secretion signal, or is appropriately linked to DNA containing a base sequence encoding a region corresponding to a secretion signal upstream thereof. The Bacillus subtilis mutant according to claim 2 or 3.   The Bacillus subtilis mutant strain according to any one of claims 2 to 4, wherein the target protein is at least one enzyme selected from the group consisting of cellulase, protease and amylase.   6. The Bacillus subtilis mutant strain according to any one of claims 1 to 5, wherein 168 strains of Bacillus subtilis are used as wild strains. The Bacillus subtilis mutant strain according to any one of claims 1 to 6, wherein the genomic region described in the column of the defective region in Table 1 includes a region sandwiched between the oligonucleotide sets in Table 2.

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