Method for producing insecticidal protein in bacillus subtilis

Abstract

Provided are: a host Bacillus subtilis strain that, with a gene that encodes crystal protein (Cry protein) introduced thereto, is capable of producing a recombinant Bacillus subtilis suitable for producing inactivated Bacillus with cytosolic crystals (IBaCC); a recombinant Bacillus subtilis obtained by introducing a gene encoding Cry protein into the host Bacillus subtilis strain such that the protein is expressed; and a method for producing Cry protein and IBaCC using the recombinant Bacillus subtilis. The host Bacillus subtilis strain, which is for producing Cry protein, has a genome in which: a specific region is deleted; all genes of epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE and aprX are deleted or inactivated; sigF gene is deleted or inactivated; lytC gene is deleted or inactivated; and at least one gene selected from the group consisting of sdpA gene, sdpB gene and sdpC gene is deleted or inactivated.

Description

Method for producing insecticidal proteins using Bacillus subtilis

[0001] The present invention relates to a method for producing insecticidal crystal proteins in Bacillus subtilis.

[0002] Bacillus thuringiensis is a microorganism that produces various insecticidal crystal proteins (hereinafter referred to as "Cry proteins") and is used as a biopesticide (Non-Patent Document 1). However, many commercially available B. thuringiensis strains contain the enterotoxin gene associated with B. cereus food poisoning. Because significant regulatory hurdles exist in using live B. thuringiensis, a related species of B. cereus, as a therapeutic agent for intentional consumption, a method using crystals contained within dead vegetative cells (inactivated Bacillus with cytosolic crystals; IBaCC) instead of using live B. thuringiensis was developed (Non-Patent Document 2). IBaCC is an excellent agent in terms of stability, cost, scale, and ease of production. In this report, the nematicidal Cry protein Cry5B was first expressed in asporogenous B. thuringiensis to form cytosolic crystals (BaCC), and then the BaCC was inactivated with food-grade essential oil to prepare inactivated BaCC (IBaCC). The efficacy of IBaCC was then confirmed in vitro and in vivo. In addition to using IBaCC itself as an active ingredient, purified cytosolic crystals (PCC) can also be prepared from IBaCC as a raw material by extracting Cry protein crystals from the cells (Non-Patent Document 3).

[0003] Generally, for industrial production of target substances such as proteins using microorganisms, mutant strains that have been modified in various ways to improve production efficiency are used. For example, when producing proteins as target substances, wild-type microorganisms have a wide variety of endogenous proteolytic enzymes (proteases, peptidases), which may degrade the target protein and hinder the production of foreign proteins. Therefore, attempts have been made to prevent the degradation of the target protein by using mutant microorganisms that have been modified, for example, by deleting these protease genes. For example, it has been reported that a B. subtilis mutant strain deleted in nine protease genes, i.e., epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX, is suitable as a host for producing substances such as useful proteins (Non-Patent Document 4). However, it has been reported that in cells with extremely low protease activity due to multiple protease gene deficiencies, cell wall-lytic enzymes are not degraded by proteases and accumulate on the cell surface, resulting in bacteriolysis during the stationary phase (Non-Patent Document 5).

[0004] Examples of lytic enzymes include lysozyme, glucosaminidase, amidase, L,D-endopeptidase, and D,L-endopeptidase. It has been reported that inactivating the cwlB (lytC) gene, which encodes the major cell wall lytic enzyme CwlB (LytC) (N-acetylmuramoyl-alanine amidase) of Bacillus subtilis, suppresses bacteriolysis (Non-Patent Document 6). However, cell wall lytic enzymes are said to play an important role in cell growth, such as cell division and motility, and their deletion or inactivation may significantly alter cell growth and affect protein or polypeptide production. For example, although this is preliminary data, there is a report that inactivating the cwlB (lytC) gene or cwlG (lytD) gene, which encodes a cell wall lytic enzyme, suppresses protein secretion (Non-Patent Document 7).

[0005] Furthermore, cannibalism, a strategy used by B. subtilis to induce lysis, is known to delay energy-requiring sporulation (Non-Patent Document 8). Cannibalism in B. subtilis generally involves the production and secretion of two known cannibalistic toxins: sporulation delaying protein (SDP) and sporulation killing factor (SKF). These cannibalistic toxin proteins are thought to prevent sporulation by lysing susceptible coexisting sibling cells to obtain nutrients. For example, the skf operon contains eight genes (i.e., skfABCDEFGH), where the skfA gene encodes the toxin SKF, and the sdp operon contains three genes (i.e., sdpABC), where the sdpC gene encodes the toxin SPD.

[0006] Many genes are involved in microbial sporulation. These genes include genes encoding sporulation-phase-specific σ factors, genes involved in the expression of the σ factor genes and the activation of σ factors, genes that promote spore formation, and genes that are transcribed by the σ factors and are involved in promoting sporulation. For example, in Bacillus subtilis, these include the sigF gene and sigG gene that encode fospore-specific σ factors. It is known that protein or polypeptide productivity in microorganisms can be improved by deleting or inactivating these genes (Patent Document 1).

[0007] (Patent Document 1) Patent No. 4336082 (Non-Patent Document 1) Agaisse H. and Lereclus D, J. of Bacteriology 177(21), 6027-6032 (1995) (Non-Patent Document 2) Li H, Abraham A, Gazzola D, Hu Y, Beamer G, Flanagan K, Soto E, Rus F, Mirza Z, Draper A, Vakalapudi S, Stockman C, Bain P, Urban JF Jr, Ostroff GR, Aroian RV. Antimicrob Agents Chemother. 2021 Mar; 65(3): e01469-20 (Non-Patent Document 3) Chicca J, Cazeault NR, Rus F, Abraham A, Garceau C, Li H, et al., Microbiol Spectr. 10(4): e02356-22 (2022) (Non-licensed Reference 4) Kodama T, Endo K, Sawada K, Ara K, Ozaki K, Kakeshita H et al., J. Biosci. Bioeng. 104(2), 135-143 (2007) (Non-licensed Reference 5) Kodama T, Endo K, Ara K, Ozaki K, Kakeshita H, Yamane K et al. J. Biosci. Bioeng. 103(1), 13-21 (2007) (Non-licensed Reference 6) Kuroda A and Sekiguchi J, J. Bacteriol. 173, 7304-7312 (1991) (Non-licensed Reference 7) Smith TJ, Blackman SA and Foster SJ, Microbiology 146, 249-262 (2000) (Non-licensed reference 8) Gonzalez-pastor JE, Hobbs EC and Losick R, Science 301(5632), 510-513 (2003)

[0008] The present invention relates to the following 1) to 7): 1) A host Bacillus subtilis strain for producing a Cry protein, comprising a prophage6 region, a prophage1 region, a prophage4 region, a PBSX region, a prophage5 region, a prophage3 region, a spb region, a pks region, a skin region, a pps region, a prophage2 region, a ydcL-ydeK-ydhU region, a yisB-yitD region, a yunA-yurT region, a cgeE-ypmQ region, a yeeK-yesX region, a pdp-rocR region, a ycxB-sipU region, a SKIN-Pro7 region, a sbo-ywhH region, a yyb 1) A host Bacillus subtilis strain having a genome in which at least one region selected from the group consisting of the P-yyaJ region and the yncM-fosB region is deleted, and in which all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes are deleted or inactivated, the sigF gene is deleted or inactivated, the lytC gene is deleted or inactivated, and at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene is deleted or inactivated. 2) A recombinant Bacillus subtilis for producing a Cry protein, wherein a gene encoding a Cry protein is introduced into the host Bacillus subtilis strain according to 1) in an expressible manner. 3) Inactivated Bacillus with Cytosolic Crystals (IBaCC) containing Cry protein, which is the inactivated bacterial cell of the recombinant Bacillus subtilis described in 2).4) A method for producing a host Bacillus subtilis strain for producing Cry protein, comprising: prophage6 region, prophage1 region, prophage4 region, PBSX region, prophage5 region, prophage3 region, spb region, pks region, skin region, pps region, prophage2 region, ydcL-ydeK-y dhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and ync A method for detecting a Bacillus subtilis mutant having a genome in which at least one region selected from the group consisting of the M-fosB region is deleted, the method comprising deleting or inactivating all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, deleting or inactivating the sigF gene, deleting or inactivating the lytC gene, and deleting or inactivating at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene.5) A method for producing a recombinant Bacillus subtilis for producing a Cry protein, comprising: a prophage6 region, a prophage1 region, a prophage4 region, a PBSX region, a prophage5 region, a prophage3 region, a spb region, a pks region, a skin region, a pps region, a prophage2 region, a ydcL-ydeK-ydhU region, a yisB-yitD region, a yunA-yurT region, a cgeE-ypmQ region, a yeeK-yesX region, a pdp-rocR region, a ycxB-sipU region, a SKIN-Pro7 region, a sbo-ywhH region, a yybP-yyaJ region, and a yncM-fosB region. a method for producing a Cry protein-containing IBaCC in a Bacillus subtilis mutant strain having a genome in which at least one selected region is deleted, the method comprising deleting or inactivating all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, deleting or inactivating the sigF gene, deleting or inactivating the lytC gene, and deleting or inactivating at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene, and introducing a gene encoding a Cry protein in an expressible manner. 6) A method for producing a Cry protein in bacterial cells or a culture containing the same, the method comprising culturing the recombinant Bacillus subtilis according to 2). 7) A method for producing an IBaCC containing a Cry protein, the method comprising culturing the recombinant Bacillus subtilis according to 2), and treating the bacterial cells after culture with a fungicide.

[0009] 1 is a schematic diagram showing the preparation of a DNA fragment for gene deletion by SOE-PCR, and a method for deleting a target gene (region) using the DNA fragment (replacing it with a drug resistance gene). 2 is a schematic diagram showing a method for deleting a target gene (replacing it with a drug resistance gene) using a gene deletion plasmid. 3 is a schematic diagram showing a method for constructing a Cry protein (Cry5B) expression plasmid. 4 is a schematic diagram showing the procedure for the marker-free deletion method using a mazF cassette. 5 is a schematic diagram showing a method for markerless introduction of a Cry gene (cry5B gene) into the genome of Bacillus subtilis. Detailed Description of the Invention

[0010] The names of the genes and genome regions of Bacillus subtilis described herein are based on the Bacillus subtilis genome data published on the Internet at GenBank: AL009126.2 ([www.ncbi.nlm.nih.gov / nuccore / 38680335]).

[0011] Herein, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing an analysis using the Search homology program in the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0012] Unless otherwise defined herein, "one or several" when used in reference to deletion, substitution, addition, or insertion of amino acids or nucleotides in an amino acid sequence or nucleotide sequence means, for example, 1 to 60, preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3, for amino acid sequences, and 1 to 180, preferably 1 to 90, more preferably 1 to 30, even more preferably 1 to 15, and even more preferably 1 to 9, for nucleotide sequences. Furthermore, as used herein, "addition" of an amino acid or nucleotide includes addition of one or several amino acids or nucleotides to one or both ends of a sequence.

[0013] As used herein, "stringent conditions" for hybridization include those described in Molecular Cloning—A Laboratory Manual Third Edition (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001), and may be, for example, conditions in which a solution containing 6×SSC (1×SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhardt's, and 100 mg / mL herring sperm DNA is incubated together with a probe at 65° C. for 8 to 16 hours to allow hybridization.

[0014] As used herein, the terms "upstream" and "downstream" of a gene or region refer to the regions following the 5' and 3' sides of the gene or region under consideration, respectively. Unless otherwise defined, the terms "upstream" and "downstream" of a gene are not limited to the upstream region from the translation initiation point or transcription initiation point of the gene and the downstream region from the stop codon.

[0015] As used herein, the term "regulatory region of a gene" refers to a region that functions to regulate the expression of a downstream gene in a cell, preferably a region that functions to constitutively or highly express the downstream gene. Specifically, it can be defined as a region that is located upstream of the coding region of the gene and that functions to interact with RNA polymerase to regulate the transcription of the gene. Preferably, the term "regulatory region of a gene" refers to a region approximately 200 to 600 nucleotides upstream of the coding region of the gene. The regulatory region includes the transcription initiation control region and / or translation initiation control region of the gene, or the region extending from the transcription initiation control region to the translation initiation control region. The transcription initiation control region is a region that includes a promoter and a transcription start point, and the translation initiation control region is a region corresponding to the Shine-Dalgarno (SD) sequence that forms a ribosome binding site together with the initiation codon (Shine, J., Dalgarno, L., Proc. Natl. Acad. Sci. USA., 1974, 71:1342-1346).

[0016] As used herein, "operably linking" a gene encoding a Cry protein (a gene of interest) to a regulatory region means arranging the regulatory region and the gene of interest on DNA so that the function of the regulatory region to control gene expression acts on the gene of interest. Methods for operably linking a gene of interest to a regulatory region include linking the gene of interest downstream of the regulatory region.

[0017] In the present invention, Cry protein refers to a crystalline insecticidal protein produced by Bacillus thuringiensis. An insecticidal protein refers to a protein that causes death or prevents normal growth of invertebrates inoculated with the protein. In the present invention, IBaCC (Inactivated Bacillus with Cytosolic Crystals) refers to inactivated Bacillus subtilis cells containing crystalline proteins, particularly Cry proteins, in the cytoplasm.

[0018] The application of IBaCC is expected to become more feasible by using B. subtilis, which does not have an enterotoxin gene, instead of B. thuringiensis as a host for producing Cry proteins. However, nothing is known about what modifications of B. subtilis will affect IBaCC production. Therefore, the present inventors conducted an investigation using various modified Bacillus subtilis mutant strains. As a result, they used a Bacillus subtilis mutant host lacking nine protease genes, i.e., epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX, the sporulation-related gene sigF gene, and the lysis-related gene lytC gene, to produce recombinant Bacillus subtilis by introducing a gene encoding a Cry protein. They then cultured the recombinant Bacillus subtilis to produce the Cry protein within the bacterial cells, and inactivated the recombinant Bacillus subtilis cells after culture to attempt to produce IBaCC. They found that while Cry protein productivity was good, there was a problem in that the efficiency of IBaCC production was low. Therefore, the present invention relates to providing a host Bacillus subtilis strain capable of producing recombinant Bacillus subtilis suitable for producing IBaCC by introducing a gene encoding a Cry protein, a recombinant Bacillus subtilis in which a gene encoding a Cry protein has been introduced into the host Bacillus subtilis strain in an expressible manner, and a method for producing a Cry protein and IBaCC using the recombinant Bacillus subtilis.

[0019] The present inventors have discovered that a Bacillus subtilis mutant strain in which a large region of the genome has been deleted, in addition to the deletion of the nine protease genes, the sigF gene, and the lytC gene, and in which the cannibalism-related gene sdpABC gene has been deleted, is a host Bacillus subtilis strain that can be used to produce recombinant Bacillus subtilis suitable for IBaCC production by introducing a gene encoding a Cry protein into the host Bacillus subtilis strain in an expressible manner, and that by using a recombinant Bacillus subtilis in which a gene encoding a Cry protein has been introduced into the host Bacillus subtilis strain in an expressible manner, the production efficiency of IBaCC can be significantly improved without compromising the productivity of the Cry protein.

[0020] According to the present invention, there are provided a host Bacillus subtilis strain for producing a recombinant Bacillus subtilis suitable for producing IBaCC containing a Cry protein, and a recombinant Bacillus subtilis obtained by introducing a gene encoding a Cry protein into the host Bacillus subtilis strain in an expressible manner, and by using the recombinant Bacillus subtilis, a Cry protein or an IBaCC containing the Cry protein can be efficiently produced.

[0021] [1. Host Bacillus subtilis Strain for Producing Cry Protein] As shown in the Reference Examples and Examples below, a Bacillus subtilis mutant host lacking nine protease genes, i.e., epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX, the sporulation-associated gene sigF gene, and the lysis-associated gene lytC gene, was used to produce recombinant Bacillus subtilis by introducing a gene encoding a Cry protein. The recombinant Bacillus subtilis was cultured to produce the Cry protein within the bacterial cells, and the recombinant Bacillus subtilis cells after culture were inactivated to attempt to produce IBaCC. As a result, although the productivity of the Cry protein was good, the production efficiency of IBaCC was low. Next, a similar study was performed using a Bacillus subtilis mutant host strain that, in addition to the deletion of the nine protease genes, the sigF gene, and the lytC gene, also lacked a large region of the genome unnecessary for survival, growth, and protein production. The Cry protein productivity was good and the IBaCC production efficiency improved, but considering actual production, further improvement in IBaCC production efficiency was required. Therefore, a similar study was performed using a Bacillus subtilis mutant host strain that had a large region of the genome deleted and, in addition to the deletion of the nine protease genes, the sigF gene, and the lytC gene, also lacked the sdpABC gene, a cannibalism-related gene. The Cry protein productivity was good and the IBaCC production efficiency was significantly improved. Therefore, such a Bacillus subtilis mutant strain is useful as a host Bacillus subtilis strain for producing Cry proteins.

[0022] The host Bacillus subtilis strain of the present invention is a host Bacillus subtilis strain for producing a Cry protein, and has a genome in which a large region of the genome of a wild-type Bacillus subtilis strain has been deleted, and in which all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes have been deleted or inactivated, the sigF gene has been deleted or inactivated, the lytC gene has been deleted or inactivated, and at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene has been deleted or inactivated. Here, the term "host Bacillus subtilis strain for producing a Cry protein" refers to a Bacillus subtilis mutant strain that can produce a Cry protein by introducing a gene encoding a Cry protein into it so that it can be expressed. The host Bacillus subtilis strain of the present invention can be prepared, for example, by adding to a Bacillus subtilis mutant strain in which a large region of the genome has been deleted modifications to delete or inactivate all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, modifications to delete or inactivate the sigF gene, modifications to delete or inactivate the lytC gene, and modifications to delete or inactivate at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene.

[0023] [1-1. Bacillus subtilis Mutant Strain Having a Deletion of a Genomic Region] A Bacillus subtilis mutant strain having a deletion of a large region of its genome, which can serve as a parent strain for the host Bacillus subtilis strain of the present invention, has a genome in which a large region of the genome has been deleted compared to the genome of a wild-type Bacillus subtilis strain (e.g., Bacillus subtilis Marburg No. 168; hereinafter referred to as Bacillus subtilis strain 168 or simply strain 168, or wild-type strain), and examples thereof include the mutant strain described in Japanese Patent No. 4,955,358.For example, the prophage6 (yoaV-yobO) region, prophage1 (ybbU-ybdE) region, prophage4 (yjcM-yjdJ) region, PBSX (ykdA-xlyA) region, pr ophage5 (ynxB-dut) region, prophage3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (pp At least one region selected from the group consisting of a prophage2 (ydcL-ydeJ) region, a prophage3 (ydcL-ydeK-ydhU) region, a prophage4 (ydcL-ydeK) region, a prophage5 (ydcL-ydeK) region, a prophage6 (ydcL-ydeK) region, a prophage7 (ydcL-ydeK) region, a prophage8 (ydcL-ydeK) region, a prophage9 (ydcL-ydeK) region, a prophage10 (ydcL-ydeK) region, a prophage11 (ydcL-ydeK) region, a prophage12 (ydcL-ydeK) region, a prophage13 (ydcL-ydeK) region, a prophage14 (ydcL-ydeK) region, a prophage15 (ydcL-ydeK) region, a prophage16 (ydcL-ydeK) region, a prophage17 (ydcL-ydeK) region, a prophage18 (ydcL-ydeK) region, a prophage19 ...9 (ydcL-ydeK) region, Examples of such mutants include Bacillus subtilis mutants lacking the following regions: the prophage6 (yoaV-yobO) region, the prophage1 (ybbU-ybdE) region, the prophage4 (yjcM-yjdJ) region, the PBSX (ykdA-xlyA) region, the prophage5 (ynxB-dut) region, the prophage3 (ydiM-ydjC) region, the spb (yodU-ypqP) region, the pks (pksA-ymaC) region, and the skin (spoIVCB-spoIIIC) region. An example is the Bacillus subtilis MGB874 strain, which lacks all of the following regions: 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, prophage7 (yrkS-yraK) region, sbo-ywhH region, yybP-yyaJ region, and yncM-fosB region. The MGB874 strain is available for purchase from the National BioResource Project (NBRP) of the National Institute of Genetics (http: / / www.shigen.nig.ac.jp / bsub / kaoListAction.do).The term "region deleted" used herein means that the regions flanked by the above-described genes on both ends are deleted, including the genes on both ends.

[0024] A B. subtilis mutant strain lacking a large region of the genome can be prepared by deleting the aforementioned genomic region from any B. subtilis strain, such as strain 168 (NBRC 111470). The target genomic region to be deleted can be determined, for example, by comparing the genomic sequence of the B. subtilis strain with the published genomic sequence of B. subtilis strain 168. The complete nucleotide sequence and genome information of B. subtilis strain 168 are available from GenBank: AL009126.2 ([www.ncbi.nlm.nih.gov / nuccore / 38680335]). Those skilled in the art can determine the target genomic region to be deleted based on the genome information of B. subtilis strain 168 obtained from these sources. Here, the genomic region to be deleted may be a genomic region having a nucleotide sequence containing a mutation such as a naturally or artificially induced deletion, substitution, insertion, or addition of one or several (for example, 1 to 100, preferably 1 to 50, more preferably 1 to 30, even more preferably 1 to 10, and even more preferably 1 to 5) nucleotides relative to the nucleotide sequence of the above-mentioned genomic region of the published Bacillus subtilis strain 168. Alternatively, the genomic region to be deleted may be a genomic region that is preferably 80% or more identical, more preferably 90% or more identical, and even more preferably 95% or more identical in nucleotide sequence to the above-mentioned genomic region of the published Bacillus subtilis strain 168.

[0025] Alternatively, the genomic region to be deleted can be expressed as a region flanked by a pair of oligonucleotide sets shown in Table 1. Methods for deleting the regions listed in Table 1 from the Bacillus subtilis genome are not particularly limited, and examples include a double-crossover method using a deletion DNA fragment prepared by SOE-PCR (splicing by overlap extension PCR: Gene, 1989, 77:61-68). The procedure for producing a mutant strain in which a predetermined genomic region has been deleted from a Bacillus subtilis wild-type strain using this method is described in detail in JP 2007-130013 A, but will be outlined below.

[0026]

[0027] An overview of the procedures for preparing deletion DNA (SOE-PCR) fragments by SOE-PCR and deleting a target region by double-crossover using the deletion DNA fragments is shown in Figure 1. First, a DNA fragment is prepared by SOE-PCR, linking a fragment corresponding to an approximately 0.1-3 kb region adjacent to the upstream of the target region to be deleted (referred to as the "upstream fragment") with a fragment corresponding to an approximately 0.1-3 kb region adjacent to the downstream of the same region (referred to as the "downstream fragment"). Preferably, a DNA fragment is prepared by further linking a marker gene fragment such as a drug resistance gene between the upstream and downstream fragments to confirm the deletion of the target region.

[0028] First, three fragments are prepared by a first PCR: an upstream fragment and a downstream fragment of the region to be deleted, and, if necessary, a marker gene fragment (the drug resistance gene fragment in Figure 1). When PCR amplifying the upstream and downstream fragments, primers are used that contain the sequence of 10 to 30 nucleotides at the end of the fragment to be ligated later. For example, when the upstream fragment, drug resistance gene fragment, and downstream fragment are ligated in this order, a sequence corresponding to 10 to 30 nucleotides upstream of the drug resistance gene fragment is added to the 5' end of the primer that binds to the downstream end of the upstream fragment, and a sequence corresponding to 10 to 30 nucleotides downstream of the drug resistance gene fragment is added to the 5' end of the primer that binds to the upstream end of the downstream fragment. When the upstream and downstream fragments are amplified by PCR using a primer set designed in this way, a region corresponding to the upstream side of the drug resistance gene fragment will be added downstream of the amplified upstream fragment, and a region corresponding to the downstream side of the drug resistance gene fragment will be added upstream of the amplified downstream fragment.

[0029] Next, the upstream fragment, drug resistance gene fragment, and downstream fragment prepared in the first PCR are mixed together and used as a template to perform a second PCR using a pair of primers consisting of a primer that binds upstream of the upstream fragment and a primer that binds downstream of the downstream fragment. This second PCR can amplify a deletion DNA fragment in which the upstream fragment, drug resistance gene fragment, and downstream fragment are linked in this order.

[0030] The DNA fragment for deletion obtained by the above-mentioned method or the like is inserted into a plasmid using a conventional restriction enzyme and DNA ligase to construct a plasmid for introducing a deletion. Alternatively, a DNA fragment for deletion can be prepared by directly linking the upstream fragment and the downstream fragment, and then the DNA fragment for deletion can be inserted into a plasmid containing a drug resistance gene to construct a plasmid for introducing a deletion containing the drug resistance gene fragment in addition to the upstream fragment and the downstream fragment.

[0031] The deletion-introducing plasmid constructed according to the above procedure is introduced into the Bacillus subtilis strain in which the genomic region is to be deleted using a conventional method, such as competent cell transformation. Introduction of the plasmid results in double-crossover homologous recombination between the upstream and downstream fragments on the plasmid and their homologous regions in the Bacillus subtilis genome, resulting in a transformant in which the region to be deleted has been replaced with a drug resistance gene (Figure 1). Transformants can be selected using the expression of a marker gene, such as a drug resistance gene, present in the deletion DNA fragment as an indicator. For example, bacteria transformed with a chloramphenicol resistance gene fragment can be cultured in a medium containing an antibiotic (such as chloramphenicol) and grown colonies recovered to obtain a transformant in which the region of interest has been deleted and replaced with a chloramphenicol resistance gene. Furthermore, deletion of the region of interest can be confirmed by extracting genomic DNA from the transformant and performing PCR using this as a template.

[0032] Next, the marker gene inserted into the genomic DNA is removed from the resulting transformant. The removal procedure is not particularly limited, but a two-step homologous recombination method can be used (Japanese Patent Laid-Open Publication No. 2009-254350). In this method, a DNA fragment (donor DNA) for the first homologous recombination is first prepared. The preparation method is not particularly limited, but examples include the SOE-PCR method described above. As the donor DNA, for example, a DNA fragment in which a fragment containing an approximately 0.1-3 kb fragment (upstream fragment) corresponding to the region adjacent to the upstream side of the marker gene region to be removed (i.e., the deleted region) and an approximately 0.1-3 kb fragment (downstream fragment) corresponding to the region adjacent to the downstream side of the marker gene region to be removed is ligated with a fragment in the downstream region of the marker gene to be removed can be used. Preferably, a DNA fragment in which a second marker gene or the like, which serves as an indicator of homologous recombination, is inserted between the downstream fragment and the fragment in the downstream region of the first marker gene to be removed, is used.

[0033] The prepared donor DNA is then introduced into the transformant by a conventional method such as competent cell transformation, causing homologous recombination between the upstream fragment and a region in the transformant genome corresponding to the downstream region of the first marker gene (first homologous recombination). Transformants in which the desired homologous recombination has occurred can be selected using the expression of the second marker gene inserted into the donor DNA as an indicator. In the genomic DNA of a transformant in which the first homologous recombination has properly occurred, the upstream fragment, the downstream fragment, and optionally the second marker gene, the downstream region of the first marker gene, and the downstream fragment are arranged in this order. In genomic DNA with this arrangement, spontaneously induced homologous recombination can occur between the two downstream fragments (intragenomic homologous recombination). This intragenomic homologous recombination results in the deletion of the region located between the two downstream fragments, thereby removing the first marker gene from the transformant genome.

[0034] A method for selecting transformants that have undergone intragenomic homologous recombination includes selecting bacteria that do not have drug resistance when the first marker gene is a drug resistance gene. Penicillin antibiotics have a bactericidal effect on growing cells but not on non-growing cells. Therefore, by culturing bacteria in the presence of a drug and a penicillin antibiotic, it is possible to selectively enrich for drug-resistant bacteria that do not grow in the presence of the drug (Methods in Molecular Genetics, Cold Spring Harbor Labs, 1970). Another method includes introducing a lethal gene. For example, if a lethal gene such as the chpA (mazF) gene is introduced into bacteria as the second marker, bacteria that have not undergone intragenomic homologous recombination will die due to the action of the lethal gene, allowing the selection of transformants that have undergone intragenomic homologous recombination. Genomic DNA can be extracted from the selected strain and used as a template for PCR to confirm that the target region has been deleted (see JP 2009-254350 A).

[0035] In this manner, a Bacillus subtilis mutant strain lacking a specific region on the genome can be prepared. Furthermore, by repeating this procedure, a Bacillus subtilis mutant strain lacking part or all of the aforementioned genomic region can be prepared.

[0036] [1-2. Deletion or Inactivation of Various Genes] In the host Bacillus subtilis strain of the present invention, in addition to the deletion of large regions of the genome described above, all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes are deleted or inactivated, the sigF gene is deleted or inactivated, the lytC gene is deleted or inactivated, and at least one gene selected from the group consisting of the sdpA, sdpB, and sdpC genes is deleted or inactivated. Here, the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes are genes encoding extracellular proteases of Bacillus subtilis. The sigF gene is a gene encoding the fospore-specific σF factor of Bacillus subtilis. The lytC (cwlB) gene encodes the major cell wall lytic enzyme (N-acetylmuramoyl-L-alanine amidase) of Bacillus subtilis. The sdpA, sdpB, and sdpC genes are a group of genes involved in the production and secretion of sporulation delaying protein (SDP), a type of cannibalistic toxin in Bacillus subtilis, and form the sdpABC operon. In the host Bacillus subtilis strain of the present invention, it is preferred that all of the sdpA, sdpB, and sdpC genes are deleted or inactivated.

[0037] Furthermore, in the host Bacillus subtilis strain of the present invention, in addition to the deletion or inactivation of the above genes, it is preferable that the spoIIE gene is also deleted or inactivated in order to improve productivity of the Cry protein. Here, the spoIIE gene is a gene that activates the foaspore-specific σF factor in Bacillus subtilis.

[0038] The gene names, gene numbers, and functional descriptions of the encoded proteins of the above genes are shown in Table 2. Those skilled in the art can identify these genes based on the SubtiList Web Server (pasteur.fr) http: / / genolist.pasteur.fr / SubtiList / or KEGG: Kyoto Encyclopedia of Genes and Genomes https: / / www.kegg.jp / kegg / .

[0039]

[0040] The genes listed in Table 2 may be, for example, genes having the same function as the genes, but consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, or added in the nucleotide sequence of the genes. Furthermore, genes having the same function as the genes listed in Table 2 and / or having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more identity in nucleotide sequence to each gene in Table 2 are also considered to be genes corresponding to the genes listed in Table 2, and are included in the genes that can be deleted or inactivated in the present invention.

[0041] Means for deleting or inactivating the gene include mutating one or more nucleotides in the nucleotide sequence of the gene, substituting or inserting another nucleotide sequence into the nucleotide sequence, or deleting part or all of the gene sequence. Means for introducing a mutation that inhibits transcription of the gene include mutating the promoter region of the gene, or inactivating the promoter by substituting or inserting another nucleotide sequence. Specific techniques for mutagenesis or substituting or inserting a nucleotide sequence include ultraviolet irradiation, site-specific mutagenesis, and the SOE-PCR and homologous recombination methods described above in [1-1. Bacillus subtilis mutant strains with deleted genome regions]. The location and nucleotide sequence of the gene to be deleted or inactivated on the Bacillus subtilis genome can be confirmed at the above-mentioned SubtiList Web Server (pasteur.fr) http: / / genolist.pasteur.fr / SubtiList / or KEGG: Kyoto Encyclopedia of Genes and Genomes https: / / www.kegg.jp / kegg / .

[0042] The host Bacillus subtilis strain of the present invention can be prepared by the above-mentioned procedures, and the host Bacillus subtilis strain is not particularly limited as long as the microorganism finally obtained has the above-mentioned deletion of a large region of the genome, deletion or inactivation of all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, deletion or inactivation of the sigF gene, deletion or inactivation of the lytC gene, and deletion or inactivation of at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene.

[0043] [2. Recombinant Bacillus for Producing Cry Protein] The recombinant Bacillus for producing the Cry protein of the present invention is a recombinant Bacillus in which a gene encoding a Cry protein of interest (hereinafter referred to as "target gene") has been further introduced into the host Bacillus strain described above so that it can be expressed. Note that the recombinant Bacillus for producing a Cry protein in which the target gene of the present invention has been introduced so that it can be expressed includes Bacillus subtilis in which the target gene is originally expressed and which has been subjected to the genetic modification related to the present invention. The recombinant Bacillus subtilis may be a microorganism finally obtained in which the above-mentioned deletion of a large region of the genome, deletion or inactivation of all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, deletion or inactivation of the sigF gene, deletion or inactivation of the lytC gene, and deletion or inactivation of at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene, and introduction of a gene of interest capable of expression have been achieved, and the order of each gene modification and introduction step is not particularly limited. Although not limited thereto, a genome deletion strain in which a large region of the genome has been deleted can be preferably produced by using a host Bacillus subtilis strain as a parent strain in which all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes have been deleted or inactivated, the sigF gene has been deleted or inactivated, the lytC gene has been deleted or inactivated, and at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene has been deleted or inactivated, and introducing a gene of interest into the parent strain so that it can be expressed.

[0044] [2-1. Cry Proteins] Cry proteins are classified into classes Cry1 to Cry75 based on the primary structure of the protein (Microbiology and Molecular Biology Reviews (1998) 62, 807-813. Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins, http: / / www.lifesci.sussex.ac.uk / Home / Neil_Crickmore / Bt / . (December 7, 2017)), and each class is further classified into subclasses based on the degree of sequence similarity. For example, there are more than 100 species that belong to the Cry1 class. The major Cry proteins are shown in Tables 3-1 to 3-3 below. The accession numbers shown in the tables are GenBank Accession Nos.

[0045]

[0046]

[0047]

[0048] As the Cry proteins of the present invention, Cry1A protein, Cry1Ca protein, Cry1F protein, Cry2A protein, Cry34A protein, Cry35A protein, Cry3A protein, Cry3B protein, Cry21 protein, Cry14A protein, Cry6A protein, Cry13 protein, Cry5B protein, Cry4Aa protein, Cry4Ba protein, Cry11Aa protein, Cry14Ab protein, and Cry21Aa protein are preferred, Cry1A protein, Cry1Ca protein, Cry3A protein, Cry11Aa protein, Cry21Aa protein, Cry5B protein, Cry4Aa protein, Cry4Ba protein, and Cry11Aa protein are more preferred, and Cry5B protein is even more preferred.

[0049] The Cry5B protein is a nematicidal protein known to be effective against soil-transmitted helminthiasis (Cappello M et al. Proc. Natl. Acad. Sci. USA 103:15154-15159, Hu Y et al. PLoS Negl. Trop. Dis. 4:e614). An example of the amino acid sequence of Cry5B is the amino acid sequence shown in SEQ ID NO:2, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO:1.

[0050] Cry1A protein and Cry1C protein are known as insecticidal toxins effective against Lepidoptera and Diptera (Palma L. et al. Toxins 6, 3296-3325 (2014)). An example of the amino acid sequence of Cry1A is the amino acid sequence shown in SEQ ID NO: 231, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 230. An example of the amino acid sequence of Cry1Ca is the amino acid sequence shown in SEQ ID NO: 235, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 234.

[0051] Cry3A is known as an insecticidal toxin effective against Coleoptera (Palma L. et al. Toxins 6, 3296-3325 (2014)). An example of the amino acid sequence of Cry3A is the amino acid sequence shown in SEQ ID NO: 204, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 203.

[0052] Cry4Aa protein, Cry4Ba protein, and Cry11Aa protein are known as insecticidal toxins effective against Diptera (Palma L. et al. Toxins 6, 3296-3325 (2014)). An example of the amino acid sequence of Cry4Aa is the amino acid sequence shown in SEQ ID NO: 206, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 205. An example of the amino acid sequence of Cry4Ba is the amino acid sequence shown in SEQ ID NO: 208, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 207. An example of the amino acid sequence of Cry11Aa is the amino acid sequence shown in SEQ ID NO: 210, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 209.

[0053] Cry14A and Cry21A are known as insecticidal toxins effective against nematodes (Palma L. et al. Toxins 6, 3296-3325 (2014)). An example of the amino acid sequence of Cry14Ab is the amino acid sequence shown in SEQ ID NO: 212, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 211. An example of the amino acid sequence of Cry21Aa is the amino acid sequence shown in SEQ ID NO: 215, and an example of the nucleotide sequence of the gene encoding the protein is the nucleotide sequence shown in SEQ ID NO: 214.

[0054] It is well known that among such natural proteins, there exist mutant proteins with one to several amino acid mutations due to genetic mutations caused by differences in ecotype, etc., or the existence of similar isozymes, etc. Therefore, in addition to the Cry proteins shown in Table 3, the Cry proteins of the present invention also include mutants of the Cry proteins in which one to several amino acid residues are added or substituted, or one to several amino acid residues are deleted, in the amino acid sequence constituting the protein, and which have similar insecticidal activity.

[0055] Furthermore, it is known that most Cry proteins are composed of three domains: domain I, which contributes to membrane perforation; domain II, which is involved in toxin-receptor interaction; and domain III, which is involved in receptor binding and pore formation. Furthermore, some Cry proteins form a protoxin structure consisting of domains IV, V, VI, and VII (Palma L. et al. Toxins 6, 3296-3325 (2014)). In addition to commonly known mutations, domain swapping methods that focus on the similarity in domain structure between Cry proteins are known to alter the properties of Cry proteins. Domain swapping methods are well known to those skilled in the art and can be performed by using DNA homology between Cry proteins to induce homologous recombination, linking Cry proteins digested with restriction enzymes, or exchanging corresponding domains or loops connecting the domains of two or more Cry proteins. Examples have been reported in which domain swapping between Cry1Aa and Cry1Ac resulted in an activity improvement of up to 37-fold, domain swapping between Cry1Ca and Cry1Ac resulted in an activity improvement of up to 172-fold, and domain swapping between Cry11A and Cry11B resulted in an activity improvement of up to 6-fold (Vilchez, S. Toxins 12, 600 (2020)). Therefore, the Cry proteins of the present invention include domain-swapped proteins of the Cry proteins shown in Table 3 or mutants of the Cry proteins, i.e., domain-swapped proteins in which at least one domain in the Cry proteins or mutants of the Cry proteins shown in Table 3 is swapped with a domain of another species of Cry protein or mutant of the Cry protein shown in Table 3, and which have insecticidal activity. Proteins having improved insecticidal activity compared to the parent protein are preferred as domain-swapped proteins of such Cry proteins.

[0056] For example, in the case of a Cry5B protein, a Cry1A protein, a Cry1Ca protein, a Cry3A protein, a Cry4Aa protein, a Cry4Ba protein, a Cry11Aa protein, a Cry14Ab protein, or a Cry21Aa protein, the following (A) to (D) are encompassed. (A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235. (B) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235, and having insecticidal activity. (C) A protein consisting of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235, and having insecticidal activity. (D) A domain-swapped protein in which at least one domain in any one of the proteins (A) to (C) is swapped with a domain from any one of the proteins (A) to (C) of another species, and which has insecticidal activity.

[0057] The gene encoding the Cry protein of the present invention (also referred to as the cry gene) is not particularly limited in type, and may be any of naturally occurring DNA, recombinant DNA, or chemically synthesized DNA, and may be any of a genomic DNA clone and a cDNA clone.

[0058] The cry genes of the present invention typically refer to the cry genes shown in Table 3 above, but it is well known to those skilled in the art that natural genes contain a small number of mutations due to differences in ecotype, etc., or due to the presence of similar isozymes. Also known are so-called domain-swapped proteins in which the domains of Cry proteins are swapped. Therefore, the cry genes of the present invention are not limited to only the genes shown in Table 3, but include all genes encoding the above-mentioned Cry proteins.

[0059] For example, in the case of the cry5B gene, cry1A gene, cry1Ca gene, cry3A gene, cry4Aa gene, cry4Ba gene, cry11Aa gene, cry14Ab gene, and cry21Aa gene, the following (a) to (h) are encompassed. (a) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, 203, 205, 207, 209, 211, 214, 230 or 234; (b) a polynucleotide consisting of a nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more identity to the nucleotide sequence shown in SEQ ID NO: 1, 203, 205, 207, 209, 211, 214, 230 or 234, and encoding a protein having insecticidal activity; (c) a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, 203, 205, 207, 209, 211, 214, 230 or 234, and encoding a protein having insecticidal activity. (d) A polynucleotide consisting of a nucleotide sequence in which the nucleotide sequence encoding at least one domain in any one of the polynucleotides (a) to (c) is swapped with a nucleotide sequence encoding a domain in any one of the polynucleotides (a) to (c) of another species, and encoding a protein having insecticidal activity. (e) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235. (f) A polynucleotide encoding a protein having insecticidal activity, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235.(g) A polynucleotide encoding a protein having an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235, and having insecticidal activity. (h) A polynucleotide encoding a protein having an amino acid sequence of a domain-swapped protein in which at least one domain in any of the proteins described in (e) to (g) is swapped with a domain of any of the proteins described in (e) to (g) of another species, and having insecticidal activity.

[0060] In the above, the "insecticidal activity" of a protein can be evaluated using the mortality rate when the protein is contacted with an appropriate insect as an indicator. For example, a 24-well flat-bottom plate is used, and 10 5-day-old mosquito larvae of Aedes albopictus, 50 μg / mL of Cry protein expressed in Bacillus subtilis, and water are added to each well to adjust the total volume to 1 mL. After leaving the plate at 25 ° C. for 24 hours, the mortality rate is calculated based on the number of dead mosquito larvae, and the insecticidal activity of the Cry protein against Diptera can be evaluated using the mortality rate as an indicator (Leetachewa et al., BMB reports, 47 (2014), 546-551). Alternatively, for example, a 48-well flat-bottom plate can be used, and 20 μL of a 1 g / 35 mL E. coli solution, 10 L1 larvae of the nematode (Caenorhabditis elegans), and an appropriate amount of Cry protein expressed in Bacillus subtilis (e.g., 5 μg / mL) can be added to each well. After culturing at 20°C for 3 days, the insecticidal activity of the Cry protein against nematodes can be evaluated using the growth inhibition status of the nematodes in each well as an indicator (Patent No. 7218090).

[0061] [2-2. Expressible Introduction of a Gene Encoding a Cry Protein] Expressible introduction of a gene encoding a Cry protein (a gene of interest) means that the gene of interest is placed in at least an expressible state in the host Bacillus subtilis strain of the present invention, regardless of whether the gene is expressed in the host Bacillus subtilis strain of the present invention. Expressible introduction of a gene of interest can be achieved, for example, by introducing a gene of interest operably linked to a regulatory region, preferably a strong regulatory region, into the genome or plasmid of the host Bacillus subtilis strain. Alternatively, it can also be achieved by modifying the host Bacillus subtilis strain so that multiple genes of interest are present in an expressible manner. An example of a procedure for expressibly introducing a gene of interest is described below.

[0062] (1) A DNA fragment is constructed in which a gene of interest is operably linked to a regulatory region and further ligated to a fragment (referred to as the "upstream fragment") corresponding to a region adjacent to the upstream side of the site of introduction of the gene of interest in the genome of a host Bacillus subtilis strain, and a fragment (referred to as the "downstream fragment") corresponding to a region adjacent to the downstream side of the site of introduction of the gene of interest. For example, a fragment is prepared in which, from upstream, a regulatory region (e.g., the regulatory region of the cellulase gene in KSM-S237) and a gene of interest (e.g., the cry5B gene) are arranged in this order, and the upstream fragment is ligated upstream of the fragment and the downstream fragment is ligated downstream of the fragment to prepare a DNA fragment. The DNA fragment may optionally contain a marker gene such as a drug resistance gene. Next, by introducing this DNA fragment into a host Bacillus subtilis strain, the DNA fragment is inserted into the genome of the host Bacillus subtilis strain, thereby transforming the host Bacillus subtilis strain. The gene of interest is expressed in the resulting transformant.

[0063] In step (1), a marker gene such as a drug resistance gene is simultaneously introduced into the genome of the host Bacillus subtilis strain, allowing for easy selection of the desired transformant based on the expression of the marker gene. The marker gene is preferably removed from the resulting transformant to prevent its propagation. The procedure for removing the marker gene is not particularly limited, and examples include the two-step homologous recombination method and the method of introducing a lethal gene described above in [1-1. Bacillus subtilis mutant strain lacking a genomic region] (see, for example, Figure 5). The final transformant does not contain the marker gene, and the gene of interest is expressed.

[0064] (2) A vector is constructed containing a gene of interest operably linked to a regulatory region. For example, a vector is prepared containing DNA in which, from upstream, a regulatory region (e.g., the regulatory region of the cellulase gene of KSM-S237) and a gene of interest (e.g., the cry5 gene) are arranged. This vector may optionally contain a marker gene such as a drug resistance gene. This vector can then be introduced into a host Bacillus subtilis strain to transform the host Bacillus subtilis strain. The gene of interest is expressed in the resulting transformant.

[0065] The regulatory region that can be used for expression of a gene of interest is preferably a regulatory region that has the function of enhancing expression of a downstream gene of interest in a host, more preferably a regulatory region that has the function of constitutively expressing or highly expressing the downstream gene of interest, and is also preferably a regulatory region that can enhance expression of the gene of interest (strong regulatory region) compared to the wild-type regulatory region of the gene of interest.

[0066] Examples of regulatory regions that can be used for the expression of a gene encoding a Cry protein include the regulatory region of an α-amylase gene derived from a bacterium of the genus Bacillus, the regulatory region of a protease gene, the rrnO operon regulatory region, the tufA gene regulatory region, the aprE gene regulatory region, the spoVG gene regulatory region, the regulatory region of a cellulase gene of Bacillus sp. strain KSM-S237, the regulatory region of a kanamycin resistance gene derived from Staphylococcus aureus, and the regulatory region of a chloramphenicol resistance gene (see JP 2009-089708 A for all of these).

[0067] Preferred examples include a 0.4 to 1.0 kb region (SEQ ID NO: 202) upstream of the translation initiation point of the cellulase gene, which is the regulatory region of the cellulase gene of Bacillus sp. KSM-S237 strain, and a base sequence having 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more identity in nucleotide sequence to said region and having a gene expression regulatory function equivalent to that of said region.

[0068] Introduction of a target gene or control region into a host Bacillus subtilis strain can be achieved by newly introducing the gene or control region into the intracellular genome or plasmid. The vector used may be any vector commonly used for transformation, such as a plasmid. The type of vector can be selected as appropriate, but a vector capable of self-replicating within the host Bacillus subtilis strain (e.g., a plasmid) is preferred, and a multi-copy vector is more preferred. Furthermore, the copy number of the plasmid relative to the genome (chromosome) of the host Bacillus subtilis strain is 2 to 100 copies, preferably 2 to 50 copies, and more preferably 2 to 30 copies. Examples of preferred plasmids include pT181, pC194, pUB110, pE194, pSN2, and pHY300PLK.

[0069] Insertion of a target gene or a control region into a vector can be performed according to conventional methods in the field. For example, fragments of the target gene and the control region can be amplified by PCR or the like, and these fragments can be inserted and ligated into a vector such as a plasmid using a restriction enzyme method or the like. Alternatively, a fragment in which the target gene and the control region fragment are ligated in advance can be prepared and inserted into a vector such as a plasmid. In this case, the control region fragment and the target gene fragment are ligated in this order from upstream on the vector.

[0070] Introduction of a DNA fragment or vector into a host Bacillus subtilis strain can be carried out according to conventional techniques, such as the protoplast method (Mol. Gen. Genet., 1979, 168:111-115) or the competent cell method (J. Bacteriol., 1963, 86:392-400; J. Bacteriol., 1960, 81:741-746).

[0071] 3. Production of Cry Protein or Culture Containing the Same The recombinant Bacillus subtilis of the present invention thus obtained has good Cry protein-producing ability. Therefore, by culturing the recombinant Bacillus subtilis of the present invention in a nutrient medium, Cry protein can be expressed (produced) intracellularly. The nutrient medium preferably contains a carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of Bacillus subtilis (transformant). Examples of carbon sources include glucose, dextran, soluble starch, sucrose, and methanol. Examples of inorganic nitrogen sources or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. If desired, the nutrient medium may also contain other nutrients (e.g., inorganic salts (e.g., sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (e.g., tetracycline, neomycin, kanamycin, spectinomycin, erythromycin, etc.)). The culture is carried out by a method known in the art. Culture conditions, such as temperature, aeration and agitation conditions, pH of the medium, and culture time, are appropriately selected so as to produce a large amount of the Cry protein of the present invention.

[0072] The culture obtained by the above-described culturing, which contains the Cry protein of the present invention, can be obtained by collecting the cells by procedures such as centrifugation or filtration, and suspending them in an appropriate buffer solution (for example, a buffer solution such as Tris buffer, phosphate buffer, HEPES buffer, or MES buffer having a concentration of about 10 M to 100 mM (preferably a pH range of 5.0 to 9.0)) or water. Furthermore, the cells can be disrupted by an appropriate combination of known cell disruption methods, such as lysozyme, freeze-thawing, ultrasonic treatment, French press, bead crushing, etc., and the Cry protein can be recovered by centrifugation.

[0073] The recovered Cry protein can be appropriately purified using sucrose density gradient methods, recrystallization, ion exchange chromatography, gel filtration, hydrophobic chromatography, isoelectric focusing chromatography, affinity columns using polyclonal antibodies against Cry protein as ligands, etc.

[0074] [4. Production of IBaCC] In a culture of the recombinant Bacillus subtilis of the present invention, the recombinant Bacillus subtilis of the present invention encapsulates the produced Cry protein in the cytoplasm in the form of crystals (Bacillus with Cytosolic Crystals; BaCC). Therefore, by treating the cultured recombinant Bacillus subtilis cells of the present invention with a fungicide, inactivated Bacillus cells encapsulating the Cry protein (inactivated Bacillus with cytosolic crystals; IBaCC) can be obtained. The IBaCC of the present invention is an inactivated recombinant Bacillus subtilis cell having a genome in which a large region of the genome of a wild-type strain of Bacillus subtilis has been deleted, in which the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes have all been deleted or inactivated, the sigF gene has been deleted or inactivated, the lytC gene has been deleted or inactivated, and at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene has been deleted or inactivated, and in which a gene encoding a Cry protein has been introduced in an expressible manner, and which contains the Cry protein.

[0075] The disinfectant is not particularly limited, but is preferably an agent that can be used in food, for example, terpenes. Terpenes can be used alone or as a plant-derived essential oil containing terpenes as the main component. Examples of terpenes include carvacrol, thymol, eugenol, geraniol, citral, etc., and carvacrol is particularly preferred. Terpenes can be used alone or in combination of two or more.

[0076] The amount of the disinfectant used can be appropriately determined depending on the type of disinfectant and the amount of bacterial cells. For example, in the case of carvacrol, the amount is preferably 0.125 to 50 mg / mL, and more preferably 0.25 to 2.0 mg / mL, relative to bacterial cells suspended at OD600<250.

[0077] Treatment of the cultured recombinant Bacillus subtilis cells of the present invention with a disinfectant is carried out by contacting the cells with the disinfectant at a predetermined temperature for a predetermined period of time. The contact temperature is preferably 1 to 40°C, more preferably 4 to 30°C. The contact time is preferably 10 to 300 minutes, more preferably 15 to 60 minutes. The contact can be carried out, for example, by suspending the cultured recombinant Bacillus subtilis cells of the present invention in an appropriate buffer solution and then adding the disinfectant to the suspension.

[0078] It is desirable to wash away the bactericide after treatment. For example, it is preferable to collect the treated recombinant Bacillus subtilis cells of the present invention by centrifugation, filtration, or the like, and then wash and suspend them in an appropriate buffer solution (for example, a buffer solution having a concentration of about 10 M to 100 mM, such as Tris buffer, phosphate buffer, HEPES buffer, or MES buffer (preferably in the pH range of 5.0 to 9.0)) or water.

[0079] The production efficiency of IBaCC using the recombinant Bacillus subtilis of the present invention can be evaluated by observing the resulting processed product under a microscope. Specifically, the IBaCC rate (number of IBaCC / (number of IBaCC+number of free crystals)×100) (%), which indicates the ratio of the number of IBaCC to the number of IBaCC and free crystals of Cry protein, can be calculated and used as an indicator for evaluation. The IBaCC rate obtained by the recombinant Bacillus subtilis of the present invention is higher than the IBaCC rate obtained by, for example, a recombinant Bacillus subtilis in which nine protease genes, the sigF gene, and the lytC gene have been deleted, in other words, a recombinant Bacillus subtilis that is the same as the recombinant Bacillus subtilis of the present invention except that the prophage6 (yoaV-yobO) region to the yncM-fosB region of the genome have not been deleted and the sdpA gene, the sdpB gene, and the sdpC gene have not been deleted or inactivated; or it is higher than the IBaCC rate obtained by a recombinant Bacillus subtilis in which the prophage6 (yoaV-yobO) region to the yncM-fosB region of the genome, the nine protease genes, and the sigF gene have been deleted, in other words, a recombinant Bacillus subtilis that is the same as the recombinant Bacillus subtilis of the present invention except that the lytC gene, the sdpA gene, the sdpB gene, and the sdpC gene have not been deleted or inactivated. The IBaCC rate of the recombinant Bacillus subtilis of the present invention is preferably 125% or more, more preferably 150% or more, and even more preferably 175% or more, when the IBaCC rate of a recombinant Bacillus subtilis that is the same as the recombinant Bacillus subtilis of the present invention except that the lytC gene, sdpA gene, sdpB gene, and sdpC gene are not deleted or inactivated is taken as the reference IBaCC rate (100%).

[0080] Since the bacterial cells in IBaCC are killed, IBaCC is highly stable in production, storage, and use, and has the advantage of passing through the stomach stably, making it useful as a pharmaceutical ingredient.

[0081] The Cry protein produced by the recombinant Bacillus subtilis of the present invention, a culture containing the Cry protein, or an IBaCC containing the Cry protein (hereinafter referred to as the IBaCC of the present invention, etc.) can be an anthelmintic agent having insecticidal activity according to the type of Cry protein, and can also be used to manufacture an anthelmintic agent. The IBaCC of the present invention can also be used for anthelmintics. Here, the use can be administration to humans or non-human animals, or use in specimens derived therefrom, and can be therapeutic or non-therapeutic. The term "non-therapeutic" does not include medical procedures, i.e., methods of surgery, treatment, or diagnosis of humans; more specifically, it does not include methods of surgery, treatment, or diagnosis performed on humans by a physician or a person under the direction of a physician.

[0082] The IBaCC etc. of the present invention can be used as an antiparasitic drug or quasi-drug by itself, or can be used as a material or preparation to be mixed with such a drug or quasi-drug.

[0083] When the anthelmintic agent of the present invention is used as a pharmaceutical (including quasi-drugs), the pharmaceutical can be administered in any dosage form. Examples of dosage forms include oral administration using tablets, capsules, granules, powders, syrups, etc., or parenteral administration using injections, suppositories, inhalants, transdermal absorbents, topical preparations, etc., with oral administration being preferred. These various dosage forms of pharmaceutical preparations can be prepared by appropriately combining the IBaCC of the present invention with other pharmaceutically acceptable excipients, binders, fillers, disintegrants, diluents, thickeners, emulsifiers, lubricants, dispersants, coating agents, surfactants, coating agents, osmotic pressure adjusters, buffers, pH adjusters, preservatives, stabilizers, antioxidants, colorants, flavorings, odorants, fragrances, etc.

[0084] The content of the IBaCC of the present invention in the above-mentioned pharmaceuticals (including quasi-drugs) varies depending on the target of anthelmintic treatment, the target of administration, and the route of administration, and is therefore not particularly limited and can be selected appropriately from a wide range.

[0085] The dosage and administration schedule of the anthelmintic agent of the present invention may be appropriately determined by those skilled in the art depending on the species, weight, sex, age, condition, and other factors of the subject.

[0086] The anthelmintic agent of the present invention can be administered to both humans and non-human animals. Non-human animals include non-human mammals, such as apes, other primates, mice, rats, horses, cows, pigs, sheep, dogs, cats, hamsters, and companion animals. Preferably, the anthelmintic agent of the present invention is administered to humans in need thereof.

[0087] The following are further disclosed as exemplary embodiments of the present invention, although the present invention is not limited to these embodiments. <1> A host Bacillus subtilis strain for producing a Cry protein, comprising: a prophage6 region, a prophage1 region, a prophage4 region, a PBSX region, a prophage5 region, a prophage3 region, a spb region, a pks region, a skin region, a pps region, a prophage2 region, a ydcL-ydeK-ydhU region, a yisB-yitD region, a yunA-yurT region, a cgeE-ypmQ region, a yeeK-yesX region, a pdp-rocR region, a ycxB-sipU region, a SKIN-Pro7 region, a sbo-ywhH region, a yyb <2> A host Bacillus subtilis strain having a genome in which at least one region selected from the group consisting of the P-yyaJ region and the yncM-fosB region is deleted, in which all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes are deleted or inactivated, the sigF gene is deleted or inactivated, the lytC gene is deleted or inactivated, and at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene is deleted or inactivated. <3> prophage6 area, prophage1 area, prophage4 area, PBSX area, prophage5 area, prophage3 area area, spb area, pks area, skin area, pps area, prophage2 area, ydcL-ydeK-ydhU area, yisB-yitD area, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 The host Bacillus subtilis strain according to <1> or <2>, which has a genome in which the sbo-ywhH region, yybP-yyaJ region, and yncM-fosB region are deleted. <4> The host Bacillus subtilis strain according to any one of <1> to <3>, in which the sdpA gene, the sdpB gene, and the sdpC gene are deleted or inactivated.

[0088] <5> A recombinant Bacillus subtilis for producing a Cry protein, wherein a gene encoding a Cry protein is introduced into the host Bacillus subtilis strain according to any one of <1> to <4> in an expressible manner. <6> The recombinant Bacillus subtilis according to <5>, wherein the Cry protein is any one selected from Cry5B, Cry1A, Cry1Ca, Cry3A, Cry4Aa, Cry4Ba, Cry11Aa, Cry14Ab, and Cry21Aa. <7> The recombinant Bacillus subtilis according to <5> or <6>, wherein the Cry protein is Cry5B. <8> The recombinant Bacillus subtilis according to <5> or <6>, wherein the Cry protein is Cry1A or Cry1Ca. <9> The recombinant Bacillus subtilis according to <5> or <6>, wherein the Cry protein is Cry3A. <10> The recombinant Bacillus subtilis according to <5> or <6>, wherein the Cry protein is Cry4Aa, Cry4Ba, or Cry11Aa. <11> The Cry protein is any of the following (A) to (D): (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235; (B) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235, and having insecticidal activity; (C) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235, and having insecticidal activity; (D) a domain-swapped protein in which at least one domain in any of the proteins (A) to (C) is swapped with a domain of any of the proteins (A) to (C) of another species, and having insecticidal activity, The recombinant Bacillus subtilis according to <5> or <6>, which is selected from the group consisting of:

[0089] <12> Inactivated Bacillus with Cytosolic Crystals (IBaCC) containing a Cry protein, which is an inactivated recombinant Bacillus subtilis cell according to any one of <5> to <11>. <13> An anthelmintic agent containing the IBaCC according to <12> as an active ingredient. <14> Use of the IBaCC according to <12> for anthelmintic purposes. <15> Use of the IBaCC according to <12> for the manufacture of an anthelmintic agent. <16> An anthelmintic method comprising administering the IBaCC according to <12> to a subject in need thereof.

[0090] <17> A method for producing a host Bacillus subtilis strain for producing Cry protein, comprising: prophage6 region, prophage1 region, prophage4 region, PBSX region, prophage5 region, prophage3 region, spb region, pks region, skin region, pps region, prophage2 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 region, sbo-ywhH region, yybP-yyaJ region, and yncM-fosB regions, in a Bacillus subtilis mutant strain having a genome in which at least one region selected from the group consisting of: deleting or inactivating all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes; deleting or inactivating the sigF gene; deleting or inactivating the lytC gene; and deleting or inactivating at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene.<18> A method for producing a recombinant Bacillus subtilis for producing a Cry protein, comprising: a recombinant Bacillus subtilis comprising a Cry protein selected from the group consisting of a prophage6 region, a prophage1 region, a prophage4 region, a PBSX region, a prophage5 region, a prophage3 region, a spb region, a pks region, a skin region, a pps region, a prophage2 region, a ydcL-ydeK-ydhU region, a yisB-yitD region, a yunA-yurT region, a cgeE-ypmQ region, a yeeK-yesX region, a pdp-rocR region, a ycxB-sipU region, a SKIN-Pro7 region, a sbo-ywhH region, a yybP-yyaJ region, and a yncM-fosB region. <19> A method according to <17> or <18>, further comprising deleting or inactivating the spoIIE gene, the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, the sigF gene, the lytC gene, and at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene, and introducing a gene encoding a Cry protein in an expressible manner in a Bacillus subtilis mutant strain having a genome in which at least one selected region has been deleted. <20> The method according to any one of <17> to <19>, wherein the Bacillus subtilis mutant has a genome in which the prophage6 region, the prophage1 region, the prophage4 region, the PBSX region, the prophage5 region, the prophage3 region, the spb region, the pks region, the skin region, the pps region, the prophage2 region, the ydcL-ydeK-ydhU region, the yisB-yitD region, the yunA-yurT region, the cgeE-ypmQ region, the yeeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yyaJ region, and the yncM-fosB region are deleted. <21> The method according to any one of <17> to <20>, comprising deleting or inactivating the sdpA gene, the sdpB gene, and the sdpC gene.<22> The method according to any one of <17> to <21>, wherein the Cry protein is any one selected from Cry5B, Cry1A, Cry1Ca, Cry3A, Cry4Aa, Cry4Ba, Cry11Aa, Cry14Ab and Cry21Aa. <23> The method according to any one of <17> to <22>, wherein the Cry protein is Cry5B. <24> The method according to any one of <17> to <22>, wherein the Cry protein is Cry1A or Cry1Ca. <25> The method according to any one of <17> to <22>, wherein the Cry protein is Cry3A. <26> The method according to any one of <17> to <22>, wherein the Cry protein is Cry4Aa, Cry4Ba or Cry11Aa. <27> The Cry protein is any of the following (A) to (D): (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231, or 235; (B) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231, or 235, and having insecticidal activity; (C) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231, or 235, and having insecticidal activity; (D) a domain-swapped protein in which at least one domain in any of the proteins (A) to (C) is swapped with a domain of any of the proteins (A) to (C) of another species, and having insecticidal activity, <22> The method according to any one of <17> to <22>, wherein the method is selected from the following:

[0091] <28> A method for producing a Cry protein in bacterial cells or a culture containing the same, comprising culturing the recombinant Bacillus subtilis according to any one of <5> to <11>. <29> A method for producing an IBaCC containing a Cry protein, comprising culturing the recombinant Bacillus subtilis according to any one of <5> to <11> and treating the cultured bacterial cells with a fungicide. <30> The method according to <29>, wherein the fungicide is a terpene, preferably carvacrol. <31> An IBaCC containing a Cry protein obtained by the method according to <29> or <30>.

[0092] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0093] In the following examples, polymerase chain reaction (PCR) for amplifying DNA fragments was performed by DNA amplification using PrimeSTAR Max DNA polymerase (Takara Bio Inc.) and the accompanying reagents with a GeneAmp PCR System (Applied Biosystems). The PCR reaction solution was prepared by adding 1 μL of appropriately diluted template DNA, 20 pmol each of sense and antisense primers, and 2.5 U of PrimeSTAR Max DNA polymerase, and adjusting the total reaction volume to 50 μL. The PCR reaction conditions were 30 cycles of three temperature changes: 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 5 to 50 seconds (adjusted depending on the target amplification product, approximately 5 seconds per 1 kb), followed by a 5-minute reaction at 72°C.

[0094] Furthermore, in the following Reference Examples and Examples, upstream and downstream of a gene do not refer to positions from the origin of replication, but rather upstream refers to the region following the 5' side of the start codon of the gene that is the target of each operation / step, while downstream refers to the region following the 3' side of the stop codon of the gene that is the target of each operation / step.

[0095] Transformation of Bacillus subtilis was carried out by the competent cell method (J. Bacteriol. 93, 1925 (1967)). Specifically, Bacillus subtilis strains were cultured with shaking in SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.02% casamino acids (Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / mL tryptophan; % is (w / v)%) at 37°C until the growth index (OD600) reached approximately 1. After the culture with shaking, a portion of the culture medium was The culture was inoculated into a 9-fold volume of SPII medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.01% casamino acids (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / mL tryptophan), and further cultured with shaking until the growth rate (OD600) reached approximately 0.4, thereby preparing competent cells of a Bacillus subtilis strain.

[0096] Next, 5 μL of a solution containing various DNA fragments (such as a SOE-PCR reaction solution) was added to 100 μL of the prepared competent cell suspension (culture solution in SPII medium), and after shaking culture at 37°C for 1 hour, the entire amount was spread on LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing an appropriate agent. After static culture at 37°C, grown colonies were isolated as transformants. The genome of the obtained transformant was extracted, and PCR was performed using this as a template to confirm that the desired genome structure had been modified.

[0097] The gene encoding the desired protein or polypeptide was introduced into the host microorganism by any of the following methods: competent cell transformation (J. Bacteriol. 93, 1925 (1967)), electroporation (FEMS Microbiol. Lett. 55, 135 (1990)), or protoplast transformation (Mol. Gen. Genet. 168, 111 (1979)).

[0098] For culturing protein production by recombinant microorganisms, LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), 2xYT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl), 2xL-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate), or CSL fermentation medium (2% yeast extract, 0.5% corn steep liquor (CSL), 0.05% magnesium chloride heptahydrate, 0.6% urea, 0.2% L-tryptophan, 10% glucose, 0.15% sodium dihydrogen phosphate, 0.35% disodium hydrogen phosphate, pH 7.2) was used.

[0099] Reference Example 1: Construction of a plasmid for epr gene deletion. Using genomic DNA extracted from Bacillus subtilis strain 168 as a template, a 0.6 kb fragment (A) adjacent to the upstream side of the epr gene on the genome and a 0.5 kb fragment (B) adjacent to the downstream side were prepared using the primer sets eprfw1 and eprUPr, and eprDNf and eprrv-rep shown in Table 4-1. Separately, a 1.2 kb fragment (C) was prepared by ligating the promoter region (Nucleic Acids Res. 17, 4410 (1989)) of the repU gene from plasmid pUB110 (Plasmid 15, 93 (1986)) upstream of the chloramphenicol resistance gene from plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)). Next, the resulting three fragments (A), (B), and (C) were mixed and used as a template in SOE-PCR using primers eprfw2 and Cmrv2 in Table 4-1 to ligate the three fragments in the order (A), (B), and (C), yielding a 2.2-kb DNA fragment (Figure 2). The ends of this DNA fragment were blunted and 5'-phosphorylated, and the fragment was inserted into the SmaI restriction enzyme site of the plasmid pUC118 (Methods Enzymol. 153, 3 (1987)) to construct the epr gene deletion plasmid pUC118-CmrΔepr. The 1.2 kb fragment (C) was prepared by mixing a 0.4 kb fragment (D) containing the repU gene promoter region, which was prepared using the repUfw and repUr-Cm primer set (Table 4-1) and the plasmid pUB110 as a template, with a 0.8 kb fragment (E) containing the chloramphenicol resistance gene, which was prepared using the CmUf-rep and Cmrv1 primer set (Table 4-1) and the plasmid pC194 as a template, and performing SOE-PCR using the primers repUfw and Cmrv1 shown in Table 4-1.

[0100]

[0101]

[0102] Reference Example 2: Construction of epr gene-deleted strain using deletion plasmid. The epr gene deletion plasmid pUC118-CmrΔepr constructed in Reference Example 1 was introduced into Bacillus subtilis strain 168 by competent cell transformation (J. Bacteriol. 93, 1925 (1967)). Transformants in which the epr gene was fused with genomic DNA by single-crossover homologous recombination between the upstream or downstream regions of the epr gene were obtained using chloramphenicol resistance as an indicator. The resulting transformants were inoculated into LB medium and cultured at 37°C for 2 hours. Competence induction was then repeated to induce intragenomic homologous recombination between the duplicated upstream or downstream regions of the epr gene on the genome. As shown in Figure 2, if homologous recombination occurs in a region different from that at the time of plasmid introduction, the epr gene is deleted with the loss of the chloramphenicol resistance gene and pUC118 vector region derived from the plasmid. Next, to increase the proportion of strains that became chloramphenicol-sensitive, ampicillin concentration was performed as follows. The culture solution after competent cell induction was inoculated into 1 mL of LB medium containing a final concentration of 5 ppm chloramphenicol and a final concentration of 100 ppm ampicillin sodium so that the turbidity at 600 nm (OD600) was 0.003. After 5 hours of cultivation at 37°C, 10 μL of a 10,000 ppm ampicillin sodium aqueous solution was added, and the cultivation continued for an additional 3 hours. After cultivation, the cells were centrifuged and washed with a 2% sodium chloride aqueous solution, then suspended in 1 mL of a 2% sodium chloride aqueous solution, and 100 μL of the suspension was spread on LB agar medium. The culture was incubated at 37°C for approximately 15 hours, and strains that became chloramphenicol-sensitive due to the loss of the plasmid region were selected from the grown strains. Using the genomic DNA of the selected strain as a template, PCR was carried out using the primers eprfw2 and eprrv-rep shown in Table 4-1 to confirm deletion of the epr gene, and an epr gene-deleted strain was obtained.

[0103] Reference Example 3: Construction of a protease gene octuple deletion strain and a protease 9-fold deletion strain The next deletion, the wprA gene, was carried out in the epr gene deletion strain in the same manner as in Reference Example 1. That is, a wprA gene deletion plasmid, pUC118-CmrΔwprA, was constructed in the same manner as in Reference Example 1, and a double deletion strain of the epr and wprA genes was obtained by introducing the constructed plasmid into genomic DNA and subsequently deleting the wprA gene by intragenomic homologous recombination. By repeating the same procedures thereafter, the mpr, nprB, bpr, nprE, vpr, and aprE genes were sequentially deleted, constructing a protease 8-fold deletion strain in which eight protease genes were deleted, and this strain was designated Δ8prt. The same procedure was repeated to construct a protease 9-fold deletion strain in which the aprX gene was deleted, and this strain was named Δ9prt. The sequences of the primers used for each deletion are shown in Tables 4-1 and 4-2, and the correspondence between each primer and the primer used for the epr gene deletion shown in Reference Example 1 is shown in Table 5.

[0104]

[0105] Reference Example 4: Deletion of the lytC (cwlB) gene, PBSX gene cluster, and lytE (cwlF) gene from a protease 9-fold deletion strain In the same manner as in Reference Example 3, the cwlB gene was deleted from the Δ9prt strain using the primer set for cwlB gene deletion shown in Tables 4-2 and 5, to construct a Δ9B strain in which nine protease genes and the cwlB gene were deleted. Next, the PBSX gene cluster was deleted. In the present invention, the PBSX gene cluster refers to the 38 genes from the xlyB gene to the spoIISA gene that are present consecutively on the Bacillus subtilis genome (Table 6). The PBSX gene cluster was deleted from the Δ9B strain using the primer set for PBSX gene cluster deletion shown in Tables 4-2 and 5, to construct the Δ9BP strain. The Δ9BF strain was constructed by deleting the cwlF gene from the Δ9B strain using the primer set for cwlF gene deletion shown in Tables 4-2 and 5. The Δ9BPF strain was constructed by deleting the cwlF gene from the Δ9BP strain using the primer set for cwlF gene deletion shown in Tables 4-2 and 5.

[0106]

[0107] Reference Example 5: Deletion of spoIIIC gene from Δ9BPF strain A method for deleting the spoIIIC gene in the genome using a drug resistance gene will be described with reference to Figure 1. The spoIIIC gene encodes the sigma factor SigK, which functions specifically during the sporulation stage of Bacillus subtilis together with the spoIVCB gene.

[0108] Using genomic DNA extracted from Bacillus subtilis strain 168 as a template, a 1.0 kb fragment (A) adjacent to the upstream of the spoIIIC gene in the genome was amplified by PCR with the primer set of spoIIIC-FW and spoIIIC / Em-R shown in Table 4-2. Also, using the above genomic DNA as a template, a 1.0 kb fragment (B) adjacent to the downstream of the spoIIIC gene in the genome was amplified by PCR with the primer set of spoIIIC / Em-F and spoIIIC-RV.

[0109] Furthermore, a 1.3 kb erythromycin (Em) resistance gene region (C) was prepared by PCR using the plasmid pMUTIN4 (Microbiology. 144, 3097 (1998)) as a template and the primer set of emf2 and emr2 shown in Table 4-2.

[0110] Next, as shown in FIG. 1 , the resulting three fragments, i.e., the 1.0 kb fragment (A), the 1.0 kb fragment (B), and the Cm resistance gene region (C), were mixed and used as a template for SOE-PCR using the primer set of spoIIIC-FW2 and spoIIIC-RV2 shown in Table 4-2 to obtain a 3.3 kb DNA fragment (D) containing the three fragments, in the order of the 1.0 kb fragment (A), the Em resistance gene region (C), and the 1.0 kb fragment (B).

[0111] The Δ9BPF strain was then transformed using the resulting DNA fragment (D) by competent cell transformation. After transformation, colonies grown on LB agar medium containing erythromycin (1 μg / mL) and lincomycin (25 μg / mL) were isolated as transformants.

[0112] Genomic DNA of the resulting transformant was extracted, and PCR was used to confirm that the spoIIIC gene had been deleted and replaced with the Em resistance gene. In this way, a Δ9BPFC strain was constructed.

[0113] Reference Example 6: Deletion of the sigD gene from the Δ9BPFC strain A method for deleting the sigD gene in the genome using a drug resistance gene will be described with reference to Figure 1. The sigD gene encodes the sigma factor SigD, which controls the expression of genes involved in cell wall lysis, flagellum formation, chemotaxis, etc. of Bacillus subtilis.

[0114] Using genomic DNA extracted from Bacillus subtilis strain 168 as a template, a 1.0 kb fragment (A) adjacent to the upstream of the sigD gene in the genome was amplified by PCR with the primer set of sigD-FW and sigD / Cm-R shown in Table 7. Also, using the above genomic DNA as a template, a 1.0 kb fragment (B) adjacent to the downstream of the sigD gene in the genome was amplified by PCR with the primer set of sigD / Cm-F and sigD-RV.

[0115] Furthermore, a 0.85 kb chloramphenicol (Cm) resistance gene region (C) was prepared by PCR using the catf and catr primer set shown in Table 7 and the plasmid DNA pC194 as a template.

[0116] Next, as shown in Figure 1, the resulting three fragments, i.e., the 1.0 kb fragment (A), the 1.0 kb fragment (B), and the Cm resistance gene region (C), were mixed and used as a template, and a 2.8 kb DNA fragment (D) containing the three fragments, in the order 1.0 kb fragment (A), the Cm resistance gene region (C), and the 1.0 kb fragment (B), was obtained by SOE-PCR using the primer set of sigD-FW2 and sigD-RV2 shown in Table 7.

[0117] Furthermore, the Δ9BPFC strain was transformed using the resulting DNA fragment (D) by competent cell transformation. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.

[0118] Genomic DNA of the resulting transformant was extracted, and PCR was used to confirm that the sigD gene had been deleted and replaced with a Cm resistance gene. In this way, a Δ9BPFCD strain (Δ9lyt strain) was constructed.

[0119] Reference Example 7: Deletion of sigF gene A method for deleting the sigF gene in the genome using a drug resistance gene will be described with reference to Figure 1. The sigF gene encodes the sigma factor SigF, which controls the expression of genes involved in sporulation of Bacillus subtilis.

[0120] Using genomic DNA extracted from Bacillus subtilis strain 168 as a template, a 1-kb fragment (A) adjacent to the upstream of the sigF gene in the genome was amplified by PCR with the primer set of sigFfw and sigF-cat-r shown in Table 7. Furthermore, using the above genomic DNA as a template, a 1-kb fragment (B) adjacent to the downstream of the sigF gene in the genome was amplified by PCR with the primer set of sigF-cat-f and sigFrv.

[0121] Furthermore, a 1 kb Cm resistance gene region (C) was prepared by PCR using the catf and catr primer set shown in Table 7 and the plasmid DNA pC194 as a template.

[0122] Next, the obtained three fragments, fragment (A), fragment (B), and Cm resistance gene region (C), were mixed and used as a template for SOE-PCR using the primer set of sigFfw2 and sigFrv2 shown in Table 7 to obtain DNA fragment (D) containing the three fragments in the order (A), (C), and (B) (Figure 1).

[0123] Furthermore, the obtained DNA fragment (D) was used by competent cell transformation to transform the Bacillus subtilis mutant Δ9prt strain constructed in Reference Example 3. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.

[0124] Genomic DNA of the resulting transformants was extracted, and PCR confirmed that the sigF gene had been deleted and replaced with a Cm resistance gene. In this manner, the Δ9F strain was constructed. The same procedures were then repeated to transform the Bacillus subtilis mutant strains Δ9B, Δ9BP, and Δ9BPF constructed in Reference Example 4, the Bacillus subtilis mutant strain Δ9BPFC constructed in Reference Example 5, and the Bacillus subtilis mutant strain Δ9BPFCD constructed in Reference Example 6, to construct the sigF-deleted Δ9BF, Δ9BPF, Δ9BPFF, Δ9BPFCF, and Δ9BPFCDF strains, respectively.

[0125]

[0126] Reference Example 8: Synthesis of artificial gene The insecticidal protein gene cry5B (GenBank: CP005935.1, SEQ ID NO: 1) derived from Bacillus thuringiensis YBT-1518 was artificially synthesized by GenScript, Inc. (USA). The 3738 bp of the synthesized gene was cloned into the KpnI and HindIII sites of pUC57 to obtain pUC57-cry5B.

[0127] Reference Example 9: Construction of Expression Plasmid In constructing the Cry5B expression plasmid, pHY300PLK was used as the vector, and sequences derived from the S237 cellulase gene (Hakamada et al., Biosci. Biotechnol. Biochem., 64 (2000), 2281-2289) were used as the promoter and terminator to prepare the cry5B gene expression plasmid (FIG. 3). The construction method followed the protocol for the In-Fusion (registered trademark) HD ​​EcoDry (trademark) Cloning Kit (Clontech).

[0128] Using the primer set vect+pF and vect+tR shown in Table 8, a vector containing the S237 promoter and terminator regions was amplified using pHYS237 DNA as a template. Next, the cry5B gene insert was amplified by PCR using the primer set cry5BpF and cry5BtR with pUC57-cry5B DNA as a template. Subsequently, the vector and insert were ligated using the In-Fusion HD EcoDry Cloning Kit (Clontech) and then transformed into E. coli HB101 competent cells (Takara Bio Inc.). Transformants selected for tetracycline resistance were confirmed by colony PCR and named pPscry5B (Figure 3). The extracted plasmid was further confirmed by PCR, and the digestion pattern of the plasmid was confirmed using the restriction enzymes EcoRI, SpeI, and XbaI.

[0129]

[0130] The constructed plasmids were sequenced. A sequence template was prepared by PCR, and 5' and 3' fragments were prepared using the frag1-F and frag1-R or frag2-F and frag2-R primers shown in Table 8. These PCR products were subjected to sequence analysis using the 10 primers SEQ-P1 to SEQ-P10 shown in Table 8. The analysis showed that no mutations were found in any of the plasmids, confirming that the plasmids were constructed as designed.

[0131] Reference Example 10: Plasmid introduction into Bacillus subtilis host and cultivation of the resulting transformants Plasmid pPscry5B constructed in Reference Example 9 was introduced into the Δ9F, Δ9BF, Δ9BPF, Δ9BPFF, Δ9BPFCF, and Δ9BPFCDF (Δ9lytF) strains constructed in Reference Example 7 by protoplast transformation. The resulting transformants were cultured in 2xL / mal medium for 4 days at 30°C with shaking at 250 rpm. 0.2 mL of the culture was centrifuged at 15,000 rpm at 4°C to separate the culture supernatant and cells. The cell pellet was washed with 1x PBS, then suspended in 1 mL of 1x PBS, and 2 mg / mL lysozyme was added and incubated at 37°C for 1 hour. Subsequently, the cells were disrupted by sonication using a Biorupter for 30 seconds x 20 times, followed by centrifugation at 15,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the precipitate was suspended in 1 mL of 1x PBS to obtain a cell disruption solution.

[0132] Reference Example 11: Quantification of Cry5B Protein Protein quantification was performed by SDS-polyacrylamide electrophoresis (SDS-PAGE). 10 μL of the prepared cell lysate was mixed with an equal volume of Laemmli Sample buffer (BIO-RAD) containing 100 mM DTT, heated at 100°C for 10 minutes, and cooled on ice for 3 minutes. An appropriate amount was then applied to a Mini-Protean TGX Gel Stain-Free 7.5% (BIO-RAD) and electrophoresed at 200 V for 35 minutes. After completion, the gel was washed with ion-exchanged water for 5 minutes, and the protein bands were analyzed using a ChemiDoc™ MP Imaging System (BIO-RAD). A calibration curve was created using BSA as a standard protein, and the amount of Cry5B protein was calculated by multiplying by a coefficient. Because this method results in fluorescence intensity that varies depending on the Trp content of the protein, a coefficient was calculated by comparing it with the Coomassie staining method. Table 9 shows the Cry5B productivity of each strain. The Δ9BF strain had equivalent Cry5B productivity to the Δ9F strain, but it was confirmed that the productivity of strains from Δ9BPF onward, in which PBSX was deleted, was reduced (Table 9).

[0133]

[0134] Reference Example 12: IBaCC Rate Confirmation IBaCC (Inactivated Bacillus with Cytosolic Crystals) refers to inactivated cells containing intracellular crystal proteins. IBaCC was prepared with reference to International Publication No. WO 2017 / 123946. 20 mL of culture medium was centrifuged (8000 rpm, 15 minutes) to collect the cell pellet and then suspended in 1x PBS buffer to an OD600 < 250. Carvacrol (> 98%, Sigma) was added at 1 mg / g (w / w) and the mixture was shaken at room temperature for 15 minutes (so as to wet the entire inner wall of the container). IBaCC were then recovered by centrifugation (8,000 rpm, 15 minutes). The pellet was washed three times with 20 mL of 1x PBS buffer or ultrapure water, and then resuspended in 20 mL of 1x PBS buffer for use in SDS-PAGE analysis, microscopic observation, etc. If not used immediately, the sample was stored frozen at -80°C. The IBaCC rate (%) was determined by microscopic examination, counting the number of IBaCC and free crystals within the field of view (100 or more), and calculating the formula: IBaCC number / (IBaCC number + free crystal number) x 100.

[0135] The Δ9BF strain showed productivity equivalent to that of the Δ9F strain, but the IBaCC rate was low at 50%. Taking into account that Cry5B productivity significantly decreases in subsequent strains, we decided to use the MGB874 strain to investigate the improvement of the IBaCC rate.

[0136] Example 1: Construction of strain 874dpr7 (874P) A strain deficient in extracellular proteases was constructed using a Bacillus subtilis mutant strain (MGB874 strain; Japanese Patent No. 4955358) in which a large region of the Bacillus subtilis wild-type genome had been deleted. Except for wprA and aprX, which had already been deleted during the large region deletion, the seven protease genes epr, mpr, nprB, bpr, nprE, vpr, and aprE were deleted in the same manner as in Reference Examples 2 and 3 to construct strain 874dpr7 (874P).

[0137] Example 2: Construction of 874PΔsigF(874PF) strain The sigF gene involved in sporulation was deleted from the 874P strain constructed in Example 1 in the same manner as in Reference Example 7 to construct the 874PΔsigF(874PF) strain.

[0138] Example 3: Construction of 874PΔsigFΔlytC (874PF1) strain The lysis-related gene lytC was deleted from the 874PF strain constructed in Example 2. This mutant strain was constructed by a marker-free deletion method using a cassette fusing an IPTG regulatory region with the mazF gene, a free mRNA-cleaving ribonuclease, as shown in Figure 4 (Genes Genet Syst. 2009, 84(4):315-8). First, using genomic DNA extracted from the 874 strain as a template, fragment (A), which is a region adjacent to the upstream start codon of the lytC gene, was amplified by PCR using primers lytC-DF1 and lytC-DR1 listed in Table 7. Using the same genomic DNA as a template, fragment (B), which is a region downstream of the stop codon of the lytC gene, was amplified by PCR using primers lytC-DF2 and lytC-DR2 listed in Table 7. Furthermore, using the same genomic DNA as a template, fragment (C), which is a region homologous to the lytC gene ORF, was amplified by PCR using primers lytC-DF and lytC-DR listed in Table 7. Furthermore, using the Bacillus subtilis mutant TOM31 (Genes Genet Syst. 2009, 84(4):315-8) as a template, fragment (D), which is a mazF cassette (mazF-lacI-spc) containing the spectinomycin resistance gene (spc), was amplified by PCR using primers casf and casr listed in Table 7.

[0139] Next, the resulting fragments (A), (B), (D), and (C) were ligated in this order by SOE-PCR using primers lytC-DF1 and lytC-DR shown in Table 7 to obtain a final DNA fragment. The resulting DNA fragment was used to transform Bacillus subtilis strain 874PF by the competent method, and colonies grown on LB agar medium containing spectinomycin but not IPTG were isolated as transformants (lytC::mazF cassette).

[0140] Finally, to remove the mazF cassette, the strain was cultured on LB agar medium containing IPTG, and grown colonies were selected. The resulting mutant strain was mutant 874PF1, in which the mazF cassette had been removed by intracellular homologous recombination in fragment (B). This mutant strain lacked the region from the start codon to the stop codon of the lytC gene on the genome, and the drug selection marker had also been removed. PCR and sequencing analysis using the genome of the resulting mutant strain 874PF1 confirmed the introduction of the cassette on the genome and the deletion of the lytC gene.

[0141] Example 4: 874PΔsigFΔlytCΔsdp (874PFL) strain and 874PΔsigFΔlytCΔsdpΔspoIIE (874PFLE) strain construction In the same manner as in the construction of the 874PF1 strain in Example 3, the sdpABC gene cluster was deleted, and the 874PF2 strain (874PFΔlytCΔsdpABC) was constructed. Furthermore, using the same method, the Cm marker introduced when deleting the sigF gene was deleted from the 874PF2 strain, and a markerless 874PFL strain was constructed. Next, for the 874PFL strain, the spoIIE gene was markerlessly deleted in the same manner as in Example 3, and the 874PFLE strain was constructed. The correspondence between the primers used in the construction of the 874PF2, 874PFL, and 874PFLE strains and the primers used in the construction of the 874PF1 strain is shown in Table 10.

[0142]

[0143] Example 5: Plasmid introduction into MGB874 mutant strain and cultivation of the resulting transformant Plasmid pPscry5B constructed in Reference Example 9 was introduced into strains 874PF, 874PF1, 874PFL, and 874PFLE constructed in Examples 2 to 4 by protoplast transformation. The resulting transformants were cultured in 2xL / ml medium for 4 days at 30°C with shaking at 250 rpm. 0.2 mL of the culture medium was centrifuged at 15,000 rpm at 4°C to separate the culture supernatant and cells. The cell pellet was washed with 1x PBS and then suspended in 1 mL of 1x PBS. 2 mg / mL lysozyme was added and the mixture was incubated at 37°C for 1 hour. Subsequently, the cells were disrupted by sonication using a Biorupter for 30 seconds x 20 times, followed by centrifugation at 15,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the precipitate was suspended in 1 mL of 1x PBS to obtain a cell disruption solution.

[0144] Example 6: Quantification of Cry5B Protein Protein quantification was performed by SDS-polyacrylamide electrophoresis (SDS-PAGE). 10 μL of the prepared cell lysate was mixed with an equal volume of Laemmli Sample buffer (BIO-RAD) containing 100 mM DTT, heated at 100°C for 10 minutes, and cooled on ice for 3 minutes. An appropriate amount was then applied to a Mini-Protean TGX Gel Stain-Free 7.5% (BIO-RAD) and electrophoresed at 200 V for 35 minutes. After completion, the gel was washed with ion-exchanged water for 5 minutes, and the protein bands were analyzed using a ChemiDoc MP Imaging System (BIO-RAD). A calibration curve was created using BSA as a standard protein, and the amount of Cry5B protein was calculated by multiplying by a coefficient. Cry5B protein productivity is shown in Table 11.

[0145]

[0146] Example 7: Confirmation of IBaCC rate The IBaCC rates of the 874PF strain, 874PF1 strain, and 874PFL strain were confirmed in the same manner as in Reference Example 12, and the results are shown in Table 11. It was confirmed that the 874PFL strain, in which lysis-related genes (lytC, sdpABC) were deleted, maintained high Cry5B productivity and also showed a significant improvement in the IBaCC rate.

[0147] Example 8: Synthesis of artificial gene A sequence (SEQ ID NO: 134) in which a chloramphenicol resistance gene (cat) and a promoter capable of expression in Bacillus subtilis were linked was artificially synthesized by Eurofins Inc. 1930 bp of the synthesized gene was inserted into a pUC-based plasmid to obtain pUC-CatP2.

[0148] Example 9: Introduction of cry5B into the genome using a Cm marker The first copy of cry5B was inserted between the yliA and hutP genes. A method for introducing cry5B simultaneously with the drug resistance gene cat will be described with reference to Figure 5. Using genomic DNA extracted from the Bacillus subtilis MGB874 strain as a template, a 1.0 kb fragment (upstream) adjacent to the upstream of the insertion site in the genome was amplified by PCR using the primer set yxiAfw2 and In3-cry5B-R shown in Table 12. Furthermore, using the genomic DNA as a template, a 1.0 kb fragment (downstream) adjacent to the downstream of the insertion site in the genome was amplified by PCR using the primer set In3-cry5B-F and hutPrv2.

[0149] Furthermore, using the plasmid DNA pUC-CatP2 as a template and the primer set Ter-recA-F and pro-cry5B-rv shown in Table 12, a 1.9 kb chloramphenicol resistance gene region (cat) and promoter region (cat-promoter) were prepared by PCR.

[0150] Furthermore, using the plasmid DNA pUC57-cry5B as a template and the primer set of cry5BaF and Ter-TS43-R shown in Table 12, a 3.7 kb cry5B gene region (cry5B) was prepared by PCR.

[0151] Next, as shown in FIG. 5, the four fragments obtained, 1.0 kb fragment (upstream), 1.0 kb fragment (downstream), 1.9 kb fragment (cat-promoter) and cry5B gene region (cry5B), were mixed and used as a template. Using the primer set of yxiAfw1 and hutPrv1 shown in Table 12, a 7.6 kb PCR fragment for introducing cry5B was obtained, containing the four fragments in the order of 1.0 kb fragment (upstream), 1.9 kb fragment (cat-promoter), 3.7 kb cry5B gene region (cry5B) and 1.0 kb fragment (B).

[0152] Furthermore, the obtained PCR fragment for introducing cry5B was used to transform the 874PFL strain obtained in Example 4 by the competent cell transformation method. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.

[0153] Genomic DNA of the resulting transformant was extracted, and PCR was used to confirm that cat-promoter-cry5B had been inserted between the yliA and hutP genes. In this way, the 874PFL-cry5B1c strain was constructed. Similarly, the 874PFLE-cry5B1c strain was constructed from the 874PFLE strain.

[0154]

[0155] Example 10: Deletion of Cm marker from 874PFL-cry5B1c strain The chloramphenicol resistance gene (cat) was deleted from the 874PFL-cry5B1c strain constructed in Example 9 based on Figure 5. Using genomic DNA extracted from the Bacillus subtilis MGB874 strain as a template and the primer set yxiAfw2 and In3-Psp64-R shown in Table 12, a 1.0 kb fragment (upstream) adjacent to the upstream of the cry5B insertion site in the genome was amplified by PCR.

[0156] Next, a 2.8-kb mazF cassette (mazF-lacI-spc) fragment containing a spectinomycin resistance gene (spc) was amplified by PCR using the Bacillus subtilis mutant strain TOM310 (Genes Genet Syst. 2009, 84(4):315-8) as a template and primers casf and casr shown in Table 12.

[0157] Furthermore, a 1.1-kb promoter fragment was amplified by PCR using the plasmid DNA pUC-CatP2 as a template and the primer set of Psp64-fw and sp64rv shown in Table 12. Furthermore, a 1-kb chloramphenicol resistance gene region (cat) was prepared by PCR using the primer set of cas-catr and catf shown in Table 12.

[0158] Next, as shown in FIG. 5 , the four fragments obtained, i.e., the 1.0 kb fragment (upstream), the 1.1 kb promoter fragment, the 2.8 kb mazF cassette fragment, and the 1 kb chloramphenicol resistance gene region (cat), were mixed and used as a template in SOE-PCR using the primer set of yxiAfw1 and catf shown in Table 12 to obtain a 5.9 kb PCR fragment for cat deletion containing the four fragments in the following order: the 1.0 kb fragment (upstream), the 1.1 kb fragment (promoter), the 2.8 kb mazF cassette fragment, and the 1.0 kb chloramphenicol resistance gene region (cat).

[0159] Furthermore, the 874PFL-cry5B1c strain obtained in Example 9 was transformed using the resulting cat deletion PCR fragment by competent cell transformation. After transformation, colonies grown on LB agar medium containing spectinomycin (100 μg / mL) were isolated as transformants. PCR was used to confirm that the mazF cassette had been inserted into the desired position in this Spc-resistant transformant. Next, this transformant was cultured overnight in LB liquid medium, and then colonies grown on LB agar medium containing IPTG (0.1 mM) were isolated as marker-free transformants.

[0160] Genomic DNA of the resulting marker-free transformant was extracted, and deletion of the cat gene region was confirmed by PCR. In this manner, the 874PFL-cry5B1 ​​strain was constructed. Similarly, the cat gene was deleted from the 874PFLE-cry5B1c strain to construct the 874PFLE-cry5B1 ​​strain.

[0161] Example 11: Genome introduction of the second copy of cry5B using a Cm marker Using the 874PFL-cry5B1 ​​strain and 874PFLE-cry5B1 ​​strain constructed in Example 10 as hosts, the 874PFL-cry5B2 strain and 874PFLE-cry5B2 strain were constructed by inserting the second copy of cry5B between the yweA gene and the spsL gene in the same manner as in Examples 9 and 10. The correspondence between the primers used in constructing 874PFL-cry5B2 (874PFLE-cry5B2) and 874PFL-cry5B1 ​​(874PFLE-cry5B1) is shown in Table 13.

[0162]

[0163] Example 12: Cultivation of cry5B genome-introduced strains The 874PFL-cry5B1, 874PFLE-cry5B1, 874PFL-cry5B2, and 874PFLE-cry5B2 strains constructed in Examples 10 and 11 were cultured in 2xL / mal medium for 4 days at 30°C with shaking at 250 rpm. 0.2 mL of the culture was centrifuged at 15,000 rpm at 4°C, and the culture supernatant and cells were separated. The cell pellet was washed with 1x PBS, then suspended in 1 mL of 1x PBS, and 2 mg / mL lysozyme was added and incubated at 37°C for 1 hour. Subsequently, the cells were disrupted by sonication using a Biorupter for 30 seconds x 20 times, followed by centrifugation at 15,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the precipitate was suspended in 1 mL of 1x PBS to obtain a cell disruption solution.

[0164] Example 13: Quantification of Cry5B Protein Protein quantification was performed by SDS-polyacrylamide electrophoresis (SDS-PAGE). 10 μL of the prepared cell lysate was mixed with an equal volume of Laemmli Sample buffer (BIO-RAD) containing 100 mM DTT, heated at 100°C for 10 minutes, and cooled on ice for 3 minutes. An appropriate amount was then applied to a Mini-Protean TGX Gel Stain-Free 7.5% (BIO-RAD) and electrophoresed at 200 V for 35 minutes. After completion, the gel was washed with ion-exchanged water for 5 minutes, and the protein bands were analyzed using a ChemiDoc MP Imaging System (BIO-RAD). A calibration curve was created using BSA as a standard protein, and the amount of Cry5B protein was calculated by multiplying by a coefficient. The Cry5B protein productivity in the plasmid expression system is shown in Table 14, and the Cry5B protein productivity in the genome expression system is shown in Table 15. The results confirmed that spoIIE deletion significantly improved productivity in both the plasmid expression system and the genome expression system.

[0165]

[0166]

[0167] Example 14: Confirmation of IBaCC rate The IBaCC rates of the 874PFLE-cry5B1 ​​strain and the 874PFLE-cry5B2 strain were confirmed in the same manner as in Reference Example 12, and the results are shown in Table 16. It was confirmed that in strains in which the lysis-related genes (lytC, sdpABC) and the spoIIE gene were deleted, Cry5B productivity was improved and the IBaCC rate was maintained at a high level.

[0168]

[0169] Example 15: Obtaining Cry genes Insecticidal protein genes cry3A (SEQ ID NO: 202, CDS: SEQ ID NO: 203, amino acid sequence: SEQ ID NO: 204, GenBank: WP_052574943.1), cry4Aa (SEQ ID NO: 205, amino acid sequence: SEQ ID NO: 206, GenBank: WP_0122114.1), cry4Ba (SEQ ID NO: 207, amino acid sequence: SEQ ID NO: 208, GenBank: WP_012211099.1), Cry11Aa (SEQ ID NO: 209, amino acid sequence: SEQ ID NO: 210, GenBank: WP_000390241.1), cry14Ab (SEQ ID NO: 211, amino acid sequence: SEQ ID NO: 212, GenBank: WP_103655240.1), cry21Aa (SEQ ID NO: 213, CDS: SEQ ID NO: 214, amino acid sequence: SEQ ID NO: 215, GenBank: WP_197201698.1) were artificially synthesized by GenScript. A PCR reaction was performed using an artificially synthesized gene fragment as a template, and a cry3A fragment was obtained using a combination of primers having SEQ ID NOs: 216 and 217, a cry4Aa fragment was obtained using a combination of primers having SEQ ID NOs: 218 and 219, a cry4Ba fragment was obtained using a combination of primers having SEQ ID NOs: 220 and 221, a cry11Aa fragment was obtained using a combination of primers having SEQ ID NOs: 222 and 223, a cry14Ab fragment was obtained using a combination of primers having SEQ ID NOs: 224 and 225, and a cry21Aa fragment was obtained using a combination of primers having SEQ ID NOs: 226 and 227. In addition, a PCR reaction was performed using Bacilex wettable powder (SDS Biotech Co., Ltd.) as a template, and a cry1A fragment (sequence number 230, amino acid sequence: sequence number 231, 99% identical to GenBank: WP_259384207.1) was obtained using a combination of primers of sequence numbers 228 and 229, and a cry1Ca fragment (sequence number 234, amino acid sequence: sequence number 235, 99% identical to GenBank: QEU48942.1) was obtained using a combination of primers of sequence numbers 232 and 233.

[0170] Example 16: Construction of expression plasmid A PCR reaction was performed using the plasmid pPscry5B as a template, and a vector fragment was amplified using a combination of primers of SEQ ID NO: 236 and SEQ ID NO: 237. The cry1A fragment, cry1Ca fragment, cry3A fragment, cry4Aa fragment, cry4Ba fragment, cry11Aa fragment, cry14Ab fragment, and cry21Aa fragment were each ligated to the resulting vector using an In-Fusion HD Cloning Kit (Clontech), and expression plasmids for each cry gene were obtained via cloning in E. coli.

[0171] Example 17: Introduction of plasmids into Bacillus subtilis host and cultivation of the obtained transformants In the same manner as in Example 5, the expression plasmids of each of the constructed cry genes were introduced into the 874PΔsigF (874PF) strain, the 874PΔsigFΔlytCΔsdp (874PFL) strain, and the 874PΔsigFΔlytCΔsdpΔspoIIE (874PFLE) strain, and the obtained transformants were cultured to obtain a cell lysate.

[0172] Example 18: Quantification of Cry proteins Each Cry protein was quantified in the same manner as in the quantification of Cry5B protein in Reference Example 11 and Example 6. The Cry protein productivity of each strain is shown in Table 17. As a result, it was confirmed that the spoIIE deletion improved Cry protein productivity in all strains expressing the Cry gene (Table 17).

[0173]

[0174] Example 19: Confirmation of IBaCC rate IBaCC was prepared for each strain and the IBaCC rate was calculated in the same manner as in Reference Example 12. As a result, it was confirmed that the 874PFL strain, in which lysis-related genes (lytC, sdpABC) were deleted from the 874PF strain, and the 874PFLE strain, in which the spoIIE gene was further deleted, maintained a higher IBaCC rate than the 874PF strain (Table 18).

[0175]

Claims

1. A host Bacillus subtilis strain for producing Cry protein, comprising: prophage6 region, prophage1 region, prophage4 region, PBSX region, prophage5 region, prophage3 region, spb region, pks region, skin region, pps region, prophage2 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 region, sbo-ywhH region, yyb A host Bacillus subtilis strain having a genome in which at least one region selected from the group consisting of the P-yyaJ region and the yncM-fosB region is deleted, in which the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE and aprX genes are all deleted or inactivated, the sigF gene is deleted or inactivated, the lytC gene is deleted or inactivated, and at least one gene selected from the group consisting of the sdpA gene, the sdpB gene and the sdpC gene is deleted or inactivated.

2. The host Bacillus subtilis strain of claim 1, further comprising a deleted or inactivated spoIIE gene.

3. A recombinant Bacillus subtilis for producing a Cry protein, which is obtained by introducing a gene encoding a Cry protein into the host Bacillus subtilis strain described in claim 1 or 2 in an expressible manner.

4. The recombinant Bacillus subtilis according to claim 3, wherein the Cry protein is any one selected from Cry5B, Cry1A, Cry1Ca, Cry3A, Cry4Aa, Cry4Ba, Cry11Aa, Cry14Ab and Cry21Aa.

5. The Cry protein is one of the following (A) to (D): (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235; (B) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235, and which has insecticidal activity; (C) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2, 204, 206, 208, 210, 212, 215, 231 or 235, and which has insecticidal activity; (D) a domain-swapped protein in which at least one domain in any of the proteins (A) to (C) is swapped with a domain from any of the proteins (A) to (C) of another species, and which has insecticidal activity, The recombinant Bacillus subtilis according to claim 3, which is selected from the group consisting of:

6. Inactivated Bacillus with Cytosolic Crystals (IBaCC) containing Cry protein, which is the inactivated recombinant Bacillus subtilis cell according to claim 3.

7. A method for producing a host Bacillus subtilis strain for producing Cry protein, comprising: prophage6 region, prophage1 region, prophage4 region, PBSX region, prophage5 region, prophage3 region, spb region, pks region, skin region, pps region, prophage2 region, ydcL-ydeK-y dhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and ync A method for detecting a Bacillus subtilis mutant having a genome in which at least one region selected from the group consisting of the M-fosB region is deleted, the method comprising deleting or inactivating all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, deleting or inactivating the sigF gene, deleting or inactivating the lytC gene, and deleting or inactivating at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene.

8. A method for producing a recombinant Bacillus subtilis for producing a Cry protein, comprising: a recombinant Bacillus subtilis comprising a gene selected from the group consisting of a prophage6 region, a prophage1 region, a prophage4 region, a PBSX region, a prophage5 region, a prophage3 region, a spb region, a pks region, a skin region, a pps region, a prophage2 region, a ydcL-ydeK-ydhU region, a yisB-yitD region, a yunA-yurT region, a cgeE-ypmQ region, a yeeK-yesX region, a pdp-rocR region, a ycxB-sipU region, a SKIN-Pro7 region, a sbo-ywhH region, a yybP-yyaJ region, and a yncM-fosB region. A method for expressing a Bacillus subtilis mutant strain having a genome in which at least one selected region is deleted, the method comprising deleting or inactivating all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, deleting or inactivating the sigF gene, deleting or inactivating the lytC gene, and deleting or inactivating at least one gene selected from the group consisting of the sdpA gene, the sdpB gene, and the sdpC gene, and introducing a gene encoding a Cry protein in an expressible manner.

9. A method for producing a Cry protein in bacterial cells or a culture containing the same, which comprises culturing the recombinant Bacillus subtilis according to claim 3.

10. A method for producing IBaCC containing a Cry protein, comprising culturing the recombinant Bacillus subtilis according to claim 3 and treating the cultured cells with a bactericide.

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