Method for producing insecticidal proteins from Bacillus subtilis

Abstract

The present invention provides a host Bacillus subtilis strain capable of producing recombinant Bacillus subtilis suitable for IBaCC production by introducing a gene encoding the Cry protein, recombinant Bacillus subtilis obtained by introducing a gene encoding the Cry protein into the host Bacillus subtilis strain so as to be expressible, and a method for producing Cry protein and IBaCC using the recombinant Bacillus subtilis. [Solution] A host Bacillus subtilis strain for producing Cry protein, having a genome in which a specific region is deleted, wherein 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, sdpB gene and sdpC gene is deleted or inactivated.

Description

Technical Field

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

Background Art

[0002] Bacillus thuringiensis produces various insecticidal crystal proteins (crystal protein, hereinafter referred to as "Cry protein") and is a microorganism used as a biological pesticide (Non-Patent Document 1). On the other hand, many commercially available B. thuringiensis contain enterotoxin genes related to B. cereus food poisoning. Since there are significant regulatory hurdles to using B. thuringiensis related to B. cereus as a live bacterium as a therapeutically administered drug that is intentionally ingested, a method has been developed to use crystals contained in inactivated vegetative cells (Inactivated Bacillus with Cytosolic Crystals; IBaCC) instead of using B. thuringiensis as a live bacterium (Non-Patent Document 2). IBaCC is an excellent agent in terms of stability, cost, scale, and ease of production. In this report, first, Cry5B of the Cry protein having nematocidal activity was expressed in sporeless B. thuringiensis to form cytoplasmic crystals (BaCC), and then inactivated BaCC (IBaCC) was prepared by inactivating BaCC with a food-grade essential oil. It has been reported that the effectiveness of this IBaCC was confirmed in vitro and in vivo. In addition to using IBaCC itself as an active ingredient, it is also possible to use IBaCC as a raw material to extract Cry protein crystals from cells and prepare them as purified cytoplasmic crystals (Purified Cytosolic Crystals; PCC) (Non-Patent Document 3).

[0003] Generally, in the industrial production of target substances such as proteins using microorganisms, mutant strains that have undergone various improvements to enhance production efficiency are used. For example, when producing proteins as the target substance, wild-type microorganisms possess a wide variety of endogenous proteases (proteases, peptidases), which can degrade the target protein and hinder the production of foreign proteins. Therefore, attempts are being made to prevent the degradation of the target protein produced by using mutant microorganisms that have been modified, such as by deleting these protease genes. For example, in Bacillus subtilis, mutant strains deficient in nine protease genes—epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX—have been reported to be suitable as hosts for the production of useful proteins and other substances (Non-Patent Literature 4). However, it has been reported that in cells with extremely low protease activity due to such multiple protease gene deletions, cell wall lytic enzymes are not degraded by proteases and accumulate on the cell surface, leading to cell lysis during the stationary phase (Non-Patent Literature 5).

[0004] Examples of lytic enzymes include lysozyme, glucosaminidase, amidase, L,D-endopeptidase, and D,L-endopeptidase. Suppression of lysis has been reported by inactivating the cwlB(lytC) gene, which encodes the major cell wall lytic enzyme CwlB(LytC) (N-acetylmuramoyl-alanine amidase) in Bacillus subtilis (Non-Patent Literature 6). However, cell wall lytic enzymes are said to play an important role in cell proliferation, such as cell division and motility, and there are concerns that their deletion or inactivation may cause significant changes in cell growth and affect protein or polypeptide production. For example, although it is preliminary data, there are reports that protein secretion was suppressed by inactivating the cwlB(lytC) gene or cwlG(lytD) gene, which encodes a cell wall lytic enzyme (Non-Patent Literature 7).

[0005] Furthermore, cannibalism is known as a strategy by Bacillus subtilis to induce lysis, in which case the energy-consuming process of spore formation is delayed (Non-Patent Literature 8). Cannibalism in Bacillus 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 spore formation by lysing susceptible 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 spore formation. These genes include a group of genes encoding spore-forming-specific σ factors, a group of genes involved in the expression and activation of these σ factor genes, a group of genes that promote spore formation, and a group of genes that are transcribed by these σ factors and are involved in promoting spore formation. For example, in Bacillus subtilis, examples include the sigF gene and sigG gene, which encode spore-specific σ factors. It is known that deleting or inactivating these genes improves the productivity of proteins or polypeptides in microorganisms (Patent Document 1). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 4336082 [Non-patent literature]

[0008] [Non-Patent Document 1] Agaisse H. and Lereclus D, J. of Bacteriology 177(21), 6027-6032 (1995)

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

[0009] It is thought that the application of IBaCC becomes more realistic by using Bacillus subtilis lacking the enterotoxin gene as a host for producing Cry protein, instead of B. thuringiensis. However, it is completely unknown what modifications of Bacillus subtilis affect IBaCC production. Therefore, the inventors investigated using Bacillus subtilis mutant strains with various modifications and created recombinant Bacillus subtilis by introducing the gene encoding Cry protein into a Bacillus subtilis mutant strain that lacked nine protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX), the spore formation-related gene (sigF gene), and the lysis-related gene (lytC gene) as a host. They then cultured the recombinant Bacillus subtilis to produce Cry protein within the cell and attempted to produce IBaCC by inactivating the cells of the recombinant Bacillus subtilis after culture. They found that while the productivity of Cry protein was good, the efficiency of IBaCC production was low. Therefore, the present invention relates to a host Bacillus subtilis strain capable of producing recombinant Bacillus subtilis suitable for IBaCC production by introducing a gene encoding the Cry protein, recombinant Bacillus subtilis obtained by introducing a gene encoding the Cry protein into the host Bacillus subtilis strain so as to be expressible, and a method for producing Cry protein and IBaCC using the recombinant Bacillus subtilis. [Means for solving the problem]

[0010] The inventors have found that a Bacillus subtilis mutant strain, in which a large area of ​​the genome is deleted in addition to the deletion of the nine protease genes, the sigF gene, and the lytC gene mentioned above, and the cannibalism-related gene sdpABC gene is deleted, is a host Bacillus subtilis strain capable of producing recombinant Bacillus subtilis suitable for IBaCC production by introducing a gene encoding the Cry protein into which it can be expressed. Furthermore, they have found that by using recombinant Bacillus subtilis in which a gene encoding the Cry protein is introduced into which it can be expressed, the production efficiency of IBaCC can be significantly improved without impairing the productivity of the Cry protein.

[0011] In other words, the present invention relates to the following 1) to 7). 1) A host Bacillus subtilis strain for producing Cry protein, 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 The genome has a deletion in at least one region selected from the group consisting of the P-yyaJ region and the yncM-fosB region, 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, sdpB gene and sdpC gene is deleted or inactivated. Host Bacillus subtilis strain. 2) Recombinant Bacillus subtilis for producing Cry protein, wherein a gene encoding Cry protein is introduced into the host Bacillus subtilis strain described in 1) in a manner that enables expression. 3)2) IBaCC (Inactivated Bacillus with Cytosolic Crystals), which is an inactivated recombinant Bacillus subtilis cell containing Cry protein. 4) A method for producing a host Bacillus subtilis strain for producing Cry protein, 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 In a Bacillus subtilis mutant strain having a genome in which at least one region selected from the group consisting of the yncM-fosB region is deleted, the following are performed: 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, sdpB, and sdpC genes. Methods that include... 5) A method for producing recombinant Bacillus subtilis for the production of Cry protein, In a Bacillus subtilis mutant having a genome in which at least one region selected from the group consisting of the 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 region is deleted, deleting or inactivating all of the genes epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX; deleting or inactivating the sigF gene; deleting or inactivating the lytC gene; deleting or inactivating at least one gene selected from the group consisting of the sdpA gene, sdpB gene, and sdpC gene; and introducing a gene encoding a Cry protein in an expressible manner A method comprising the above. A method for producing a Cry protein or a culture containing the same in a bacterium, comprising culturing the recombinant Bacillus subtilis according to 6)2). A method for producing an IBaCC containing a Cry protein, comprising culturing the recombinant Bacillus subtilis according to 7)2) and treating the cultured cells with a bactericide after culturing.

Advantages of the Invention

[0012] According to the present invention, there are provided a host Bacillus subtilis strain for producing a recombinant Bacillus subtilis suitable for producing an IBaCC containing a Cry protein and a recombinant Bacillus subtilis in which a gene encoding a Cry protein is introduced in an expressible manner into the host Bacillus subtilis strain. By using the recombinant Bacillus subtilis, an IBaCC containing a Cry protein or the Cry protein can be efficiently produced.

Brief Description of the Drawings

[0013] [Figure 1]It schematically shows a method for preparing a DNA fragment for gene deletion by SOE-PCR and deleting (and substituting with a drug resistance gene) a target gene (region) using the DNA fragment. [Figure 2] It schematically shows a method for deleting (and substituting with a drug resistance gene) a target gene using a plasmid for gene deletion. [Figure 3] It schematically shows a method for constructing a Cry protein (Cry5B) expression plasmid. [Figure 4] It is a schematic diagram showing the procedure of a marker-free deletion method using the mazF cassette. [Figure 5] It schematically shows a method for introducing a cry gene (cry5B gene) into the genome of Bacillus subtilis without a marker.

Mode for Carrying Out the Invention

[0014] The names of each gene and genomic region of Bacillus subtilis described in this specification are described based on the Bacillus subtilis genomic data publicly available on the Internet ([www.ncbi.nlm.nih.gov / nuccore / 38680335]) in GenBank: AL009126.2.

[0015] In this specification, 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 homology analysis (Search homology) program of the genetic information processing software GENETYX Ver.12 with Unit size to compare (ktup) set to 2.

[0016] In this specification, unless otherwise defined, "one or several" as used with respect to deletions, substitutions, additions or insertions 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 still more preferably 1 to 3, in an amino acid sequence, and 1 to 180, preferably 1 to 90, more preferably 1 to 30, even more preferably 1 to 15, and still more preferably 1 to 9, in a nucleotide sequence. Also in this specification, "addition" of amino acids or nucleotides includes the addition of one or several amino acids or nucleotides to one end and both ends of a sequence.

[0017] In this specification, "stringent conditions" for hybridization refer to the conditions described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001), which may include conditions in which a solution containing 6×SSC (composition of 1×SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhart, and 100 mg / mL herring sperm DNA is incubated with the probe at 65°C for 8 to 16 hours to allow hybridization.

[0018] In this specification, upstream and downstream of a gene or region refer to the regions immediately following the 5' and 3' ends of the gene or region under consideration, respectively. Unless otherwise defined, upstream and downstream of a gene are not limited to the region upstream from the translation start site or transcription start site and the region downstream from the stop codon.

[0019] In this specification, the regulatory region of a gene is a region that has the function of controlling the intracellular expression of downstream genes, and preferably has the function of constitutively expressing or over-expressing downstream genes. Specifically, it can be defined as a region located upstream of the coding region of the gene that has the function of controlling the transcription of the gene through interaction with RNA polymerase. Preferably, the regulatory region of a gene in this specification refers to a region of about 200 to 600 nucleotides upstream of the coding region of the gene. The regulatory region includes the transcription initiation regulatory region and / or the translation initiation regulatory region of the gene, or the region from the transcription initiation regulatory region to the translation initiation regulatory region. The transcription initiation regulatory region is a region that includes the promoter and the transcription start site, and the translation initiation regulatory region is a region corresponding to the Shine-Dalgarno (SD) sequence that forms a ribosome binding site together with the start codon (Shine, J., Dalgarno, L., Proc. Natl. Acad. Sci. USA., 1974, 71:1342-1346).

[0020] In this specification, "operably linked" a gene encoding the Cry protein (target gene) and a regulatory region means arranging the regulatory region and the target gene on the DNA such that the regulatory function of the regulatory region acts on the target gene. One method for operably linking a target gene and a regulatory region is to link the target gene downstream of the regulatory region.

[0021] In this invention, Cry protein refers to a crystalline insecticidal protein produced by Bacillus thuringiensis. An insecticidal protein is a protein that causes death or prevents normal growth of invertebrates that are inoculated with it. In this invention, IBaCC (Inactivated Bacillus with Cytosolic Crystals) refers to an inactivated Bacillus subtilis cell containing crystalline protein, particularly Cry protein, in its cytoplasm.

[0022] [1. Host Bacillus subtilis strain for producing Cry protein] As shown in the reference examples and examples below, recombinant Bacillus subtilis was created by introducing a gene encoding Cry protein into a Bacillus subtilis mutant lacking nine protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX), the spore formation-related gene (sigF), and the lysis-related gene (lytC) as a host. This recombinant Bacillus subtilis was cultured to produce Cry protein within the cell, and then the cultured recombinant Bacillus subtilis cells were inactivated to attempt to produce IBaCC. While the productivity of Cry protein was good, the efficiency of IBaCC production was low. Next, a similar study was conducted using a Bacillus subtilis mutant lacking not only the nine protease genes, the sigF gene, and the lytC gene, but also a large region of the genome unnecessary for survival, proliferation, and protein production. While the productivity of Cry protein was good and the efficiency of IBaCC production improved, further improvement in IBaCC production efficiency was required considering practical production. Therefore, when we conducted similar studies using a Bacillus subtilis mutant strain with a large region of deletion in its genome, and in addition to the deletions of the nine protease genes mentioned above, the sigF gene, and the lytC gene, we also deleted the sdpABC gene, which is associated with cannibalism, the production of Cry protein was good, and the efficiency of IBaCC production was significantly improved. Thus, such a Bacillus subtilis mutant strain is useful as a host Bacillus subtilis strain for producing Cry protein.

[0023] The host Bacillus subtilis strain of the present invention is a host Bacillus subtilis strain for producing Cry protein, having a genome in which a large region of the genome of the wild-type Bacillus subtilis is deleted, with all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE and aprX genes deleted or inactivated, the sigF gene deleted or inactivated, the lytC gene deleted or inactivated, and at least one gene selected from the group consisting of the sdpA, sdpB and sdpC genes deleted or inactivated. Here, "host Bacillus subtilis strain for producing Cry protein" refers to a Bacillus subtilis mutant strain capable of producing Cry protein by introducing a gene encoding Cry protein so that it can be expressed. The host Bacillus subtilis strain of the present invention can be produced, for example, by modifying a Bacillus subtilis mutant strain in which a large region of the genome is deleted, by deleting or inactivating all of the epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX genes, by deleting or inactivating the sigF gene, by deleting or inactivating the lytC gene, and by deleting or inactivating at least one gene selected from the group consisting of the sdpA, sdpB, and sdpC genes.

[0024] [1-1. Bacillus subtilis mutant strains with genomic region deletions] A Bacillus subtilis mutant strain having a large region of genome deletion that can serve as the parent strain of the host Bacillus subtilis strain of the present invention has a genome in which a large region of the genome is deleted compared to the genome of the wild-type Bacillus subtilis strain (for example, Bacillus subtilis Marburg No. 168; hereinafter referred to herein as Bacillus subtilis strain 168 or simply strain 168, or wild-type strain), and an example of such a mutant strain is the one described in Japanese Patent No. 4955358.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 the sE-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(yrkM-yraK, or yrkS-yraK) region, sbo-ywhH region, yybP-yyaJ region, and yncM-fosB region. Examples include Bacillus subtilis mutant strains lacking specific regions, preferably the prophage6(yoaV-yobO) region, prophage1(ybbU-ybdE) region, prophage4(yjcM-yjdJ) region, PBSX(ykdA-xlyA) region, prophage5(ynxB-dut) region, prophage3(ydiM-ydjC) region, spb(yodU-ypqP) region, pks(pksA-ymaC) region, and skin(spoIVCB-spoIIIC) region. One example is the Bacillus subtilis strain MGB874, which lacks all of the following regions: pps(ppsE-ppsA), prophage2(ydcL-ydeJ), ydcL-ydeK-ydhU, yisB-yitD, yunA-yurT, cgeE-ypmQ, yeeK-yesX, pdp-rocR, ycxB-sipU, prophage7(yrkS-yraK), sbo-ywhH, yybP-yyaJ, and yncM-fosB. This MGB874 strain is available for download from the National Institute of Genetics' NBRP (National BioResource Project) (http: / / www.shigen.nig.ac.jp / bsub / kaoListAction.do). The term "regional deletion" as used here refers to the deletion of the region flanked by the genes described above, including the genes at both ends.

[0025] A Bacillus subtilis mutant strain lacking the aforementioned large genomic region can be created, for example, by deleting the aforementioned genomic region from any Bacillus subtilis strain, such as strain 168 (NBRC 111470). The genomic region to be deleted can be determined, for example, by comparing the genomic sequence of the arbitrary Bacillus subtilis strain with the publicly available genomic sequence of Bacillus subtilis strain 168. The complete nucleotide sequence and genomic information of Bacillus subtilis strain 168 can be obtained from GenBank:AL009126.2 ([www.ncbi.nlm.nih.gov / nuccore / 38680335]). Those skilled in the art can determine the genomic region to be deleted based on the genomic information of Bacillus subtilis strain 168 obtained from these sources. Here, the genomic region to be deleted may be a genomic region having a nucleotide sequence that includes mutations such as naturally or artificially caused deletions, substitutions, insertions, or additions of one or more nucleotides (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) in the nucleotide sequence of the aforementioned genomic region in the publicly available 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 aforementioned genomic region in the publicly available Bacillus subtilis strain 168.

[0026] Alternatively, the genomic region to be deleted can be represented as a region sandwiched between a pair of oligonucleotide sets shown in Table 1. While not particularly limited, a method for deleting the region described in Table 1 from the Bacillus subtilis genome is employed. For example, a double crossover method using deletion DNA fragments prepared by SOE-PCR (splicing by overlap extension PCR: Gene, 1989, 77:61-68) is used. The procedure for producing a mutant strain with a predetermined genomic region deleted from a wild-type Bacillus subtilis strain using this method is described in detail in Japanese Patent Publication No. 2007-130013, but an overview is provided below.

[0027] [Table 1]

[0028] Figure 1 shows an overview of the procedure for preparing deletion DNA (SOE-PCR) fragments by SOE-PCR and deleting the target region using the deletion DNA fragments by double crossover. First, a DNA fragment is prepared by SOE-PCR by concatenating a fragment corresponding to an approximately 0.1-3kb region adjacent to the upstream of the target region to be deleted (referred to as the upstream fragment) and a fragment corresponding to an approximately 0.1-3kb region adjacent to the downstream of the target region (referred to as the downstream fragment). Preferably, in order to confirm the deletion of the target region, a DNA fragment is further prepared by concatenating a marker gene fragment, such as a drug resistance gene, between the upstream and downstream fragments.

[0029] First, three fragments are prepared by the first PCR: the upstream and downstream fragments of the region to be deleted, and, if necessary, a marker gene fragment (in Figure 1, the drug resistance gene fragment). When PCR amplifying the upstream and downstream fragments, primers are used that have the sequence of the last 10-30 nucleotides of the fragment to be later ligated added to them. For example, when ligating the upstream fragment, drug resistance gene fragment, and downstream fragment in this order, the 5' end of the primer that binds to the downstream end of the upstream fragment has a sequence corresponding to the upstream 10-30 nucleotides of the drug resistance gene fragment added to it, and the 5' end of the primer that binds to the upstream end of the downstream fragment has a sequence corresponding to the downstream 10-30 nucleotides of the drug resistance gene fragment added to it. When the upstream and downstream fragments are amplified by PCR using this primer set, the region corresponding to the upstream side of the drug resistance gene fragment is added to the downstream side of the amplified upstream fragment, and the region corresponding to the downstream side of the drug resistance gene fragment is added to the upstream side of the amplified downstream fragment.

[0030] Next, the upstream fragment, drug resistance gene fragment, and downstream fragment prepared in the first PCR are mixed to form a template, and a second PCR is performed using a pair of primers consisting of a primer that binds to the upstream side of the upstream fragment and a primer that binds to the downstream side of the downstream fragment. This second PCR allows for the amplification of a deletion DNA fragment in which the upstream fragment, drug resistance gene fragment, and downstream fragment are joined in that order.

[0031] The deletion DNA fragment obtained by the methods described above is inserted into a plasmid using a standard restriction enzyme and DNA ligase to construct a deletion-introducing plasmid. Alternatively, a deletion DNA fragment can be prepared by directly ligating the upstream and downstream fragments, and then inserting this deletion DNA fragment into a plasmid containing a drug resistance gene to construct a deletion-introducing plasmid that has the drug resistance gene fragment in addition to the upstream and downstream fragments.

[0032] The deletion plasmid constructed using the above procedure is introduced into Bacillus subtilis (Bacillus subtilis) in which the genomic region to be deleted is to be deleted using a standard method such as competent cell transformation. Upon introduction of the plasmid, double crossover homologous recombination occurs between the upstream and downstream fragments on the plasmid and homologous regions in the Bacillus subtilis genome, resulting in a transformant in which the region to be deleted is replaced with an antibiotic resistance gene (Figure 1). The selection of transformants can be performed using the expression of marker genes, such as antibiotic resistance genes, present in the deletion DNA fragment as an indicator. For example, by culturing bacteria transformed with a chloramphenicol resistance gene fragment in a medium containing an antibiotic (such as chloramphenicol) and collecting the grown colonies, transformants in which the target region is deleted and replaced with a chloramphenicol resistance gene can be obtained. Furthermore, by extracting the genomic DNA of the transformant and performing PCR using this as a template, it is possible to confirm that the target region has been deleted.

[0033] Next, the marker gene inserted into the genomic DNA is removed from the obtained transformant. The removal procedure is not particularly limited, but a two-step homologous recombination method can be used (Japanese Patent Publication No. 2009-254350). In this method, first, a DNA fragment for the first homologous recombination (donor DNA) is prepared. The preparation method is not particularly limited, but the SOE-PCR method described above can be used. As the donor DNA, for example, a DNA fragment can be used in which a fragment of about 0.1 to 3 kb corresponding to a region adjacent upstream of the marker gene region to be removed (i.e., the deleted region) (upstream fragment) and a fragment of about 0.1 to 3 kb corresponding to a region adjacent downstream (downstream fragment) are linked together, and a fragment of the downstream region of the marker gene to be removed is linked together. Preferably, a DNA fragment is used in which a second marker gene, which serves as an indicator of homologous recombination, is inserted between the downstream fragment and the downstream region fragment of the first marker gene to be removed.

[0034] Next, the prepared donor DNA is introduced into the transformant using a conventional method such as competent cell transformation, and homologous recombination is induced between the upstream fragment and the region corresponding to the downstream region of the first marker gene on the transformant genome (first homologous recombination). Transformants in which the desired homologous recombination has occurred can be selected based on the expression of the second marker gene inserted into the donor DNA. In the genomic DNA of a transformant in which the first homologous recombination has occurred appropriately, the upstream fragment, the downstream fragment, the second marker gene if necessary, the downstream region of the first marker gene, and the downstream fragment are arranged in that order. In genomic DNA with such an arrangement, homologous recombination can occur spontaneously between the two downstream fragments (intragenomic homologous recombination). This intragenomic homologous recombination removes the first marker gene from the transformant genome by deleting the region located between the two downstream fragments.

[0035] One method for selecting transformants that have undergone homologous recombination within the genome is to select bacteria that do not have drug resistance, if the first marker gene is a drug resistance gene. Penicillin antibiotics have a bactericidal effect on proliferating cells but do not affect non-proliferating cells. Therefore, by culturing bacteria in the presence of a drug and a penicillin antibiotic, drug-resistant bacteria that do not proliferate in the presence of the drug can be selectively enriched (Methods in Molecular Genetics, Cold Spring Harbor Labs, 1970). Another method is to introduce 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 did not undergo homologous recombination within the genome will die due to the action of the lethal gene, thus allowing for the selection of transformants that have undergone homologous recombination within the genome. Genomic DNA can be extracted from the selected bacterial strain and used as a template for PCR to confirm that the target region has been deleted (see Japanese Patent Publication No. 2009-254350).

[0036] As described above, a Bacillus subtilis mutant strain lacking a predetermined region of the genome can be produced. Furthermore, by repeating this procedure, a Bacillus subtilis mutant strain lacking some or all of the aforementioned genomic regions can be produced.

[0037] [1-2. Deletion or inactivation of various genes] In the host Bacillus subtilis strain of the present invention, in addition to the deletion of a large region of the genome as 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 that encode extracellular proteases of Bacillus subtilis. The sigF gene is a gene that encodes a foraspore-specific σF factor in Bacillus subtilis. The lytC(cwlB) gene is the gene that encodes the major cell wall-lytic enzyme (N-acetylmuramoyl-L-alanine amidase) of Bacillus subtilis. Furthermore, 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 preferable that all of the sdpA, sdpB, and sdpC genes are deleted or inactivated.

[0038] Furthermore, in the host Bacillus subtilis strain of the present invention, in addition to the deletion or inactivation of the above-mentioned gene, the deletion or inactivation of the spoIIE gene is preferable in terms of improving the productivity of the Cry protein. Here, the spoIIE gene is a gene that activates the foraspore-specific σF factor in Bacillus subtilis.

[0039] Table 2 below shows the gene names, gene numbers, and functional descriptions of the encoded proteins of the above-mentioned genes. 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 / .

[0040] [Table 2]

[0041] The genes listed in Table 2 may, for example, be genes having the same function as the genes, consisting of nucleotide sequences in which one or more nucleotides are deleted, substituted, or added to the nucleotide sequence of the gene. 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 sequences with each of the genes 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.

[0042] Means of deleting or inactivating the above-mentioned gene include introducing a mutation into 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 of introducing a mutation that inhibits the transcription of the above-mentioned gene include introducing a mutation into the promoter region of the gene, or inactivating the promoter by substituting or inserting another nucleotide sequence. Specific methods for introducing the above-mentioned mutation or substitution or insertion of nucleotide sequences include ultraviolet irradiation, site-directed mutagenesis, and SOE-PCR and homologous recombination methods as described in [1-1. Bacillus subtilis mutant strains with genomic region deletion] above. The location and nucleotide sequence of the gene to be deleted or inactivated on the Bacillus subtilis genome can be confirmed 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 / .

[0043] The host Bacillus subtilis strain of the present invention can be produced by the above procedure, but the host Bacillus subtilis strain only needs to achieve the following in the final microorganism: deletion of a large region of the genome as described above, 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, sdpB, and sdpC genes, and the order of each gene modification step is not particularly limited.

[0044] [2. Recombinant Bacillus subtilis for Cry protein production] The recombinant Bacillus subtilis for producing the Cry protein of the present invention is a recombinant Bacillus subtilis strain in which the gene encoding the target Cry protein (hereinafter referred to as the "target gene") has been introduced into the host Bacillus subtilis strain described above so as to be expressible. The recombinant Bacillus subtilis for producing the Cry protein in which the target gene has been introduced so as to be expressible includes Bacillus subtilis strains that originally express the target gene but have undergone the genetic modification related to the present invention. The recombinant Bacillus subtilis should, in the final microorganism, achieve the deletion of a large region of the genome as described above, 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, deletion or inactivation of at least one gene selected from the group consisting of the sdpA, sdpB, and sdpC genes, and introduction that enables the expression of the target gene. The order of each gene modification / introduction step is not particularly important. Preferably, the target gene can be produced by introducing it into a parent strain of Bacillus subtilis in which, in addition to a genome deletion strain in which a large region of the genome is deleted, 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.

[0045] [2-1. Cry Protein] Cry proteins are classified into classes from Cry1 to Cry75 based on their primary structure (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)). Each class is further subdivided into subclasses based on the degree of sequence similarity. For example, there are more than 100 proteins belonging to the Cry1 class. The major Cry proteins are shown in Tables 3-1 to 3-3 below. The access numbers shown in these tables are GenBank Accession No.

[0046] [Table 3-1]

[0047] [Table 3-2]

[0048] [Table 3-3]

[0049] The Cry Protein of the present invention is preferably 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, or Cry21Aa Protein; more preferably Cry1A Protein, Cry1Ca Protein, Cry3A Protein, Cry11Aa Protein, Cry21Aa Protein, Cry5B Protein, Cry4Aa Protein, Cry4Ba Protein, or Cry11Aa Protein; and even more preferably Cry5B Protein.

[0050] 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 shown in SEQ ID NO: 2, and an example of the nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO: 1.

[0051] The Cry1A and Cry1C proteins are known as insecticidal toxins effective against lepidopteran and diptera (Palma L. et al. Toxins 6, 3296-3325 (2014)). An example of the amino acid sequence of Cry1A is shown in SEQ ID NO: 231, and an example of the nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO: 230. An example of the amino acid sequence of Cry1Ca is shown in SEQ ID NO: 235, and an example of the nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO: 234.

[0052] 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 shown in SEQ ID NO: 204, and an example of the nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO: 203.

[0053] The Cry4Aa, Cry4Ba, and Cry11Aa proteins 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 shown in SEQ ID NO: 206, and an example of the nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO: 205. An example of the amino acid sequence of Cry4Ba is shown in SEQ ID NO: 208, and an example of the nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO: 207. An example of the amino acid sequence of Cry11Aa is shown in SEQ ID NO: 210, and an example of the nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO: 209.

[0054] 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 sequence shown in SEQ ID NO: 212, and an example of the nucleotide sequence of the gene encoding this protein is the sequence shown in SEQ ID NO: 211. An example of the amino acid sequence of Cry21Aa is the sequence shown in SEQ ID NO: 215, and an example of the nucleotide sequence of the gene encoding this protein is the sequence shown in SEQ ID NO: 214.

[0055] It is well known that among such natural proteins, there exist mutant proteins with one to several amino acid mutations due to differences in ecotype, or the existence of very similar isozymes. Therefore, the Cry protein of the present invention includes not only the Cry protein shown in Table 3, but also variants of the Cry protein in which one to several amino acid residues are added or substituted, or one to several amino acid residues are deleted, and which have similar insecticidal activity.

[0056] Furthermore, it is known that most Cry proteins have toxins composed of three domains: domain I, which contributes to membrane perforation; domain II, which is involved in the interaction between the toxin and the receptor; and domain III, which is involved in receptor binding and pore formation. In some cases, they also form a protoxin structure consisting of domains IV, V, VI, and VII (Palma L. et al. Toxins 6, 3296-3325 (2014)). In addition to the generally known mutations, a domain swap method is known that focuses on the similarity of domain structures between Cry proteins in order to change the properties of Cry proteins. Domain swap methods are known to those skilled in the art and are carried out by methods such as inducing homologous recombination using the homology of DNA between Cry proteins, linking Cry proteins that have been cut with restriction enzymes, or exchanging corresponding domains or loop regions connecting domains of two or more Cry proteins. Examples have been reported of domain swaps between Cry1Aa and Cry1Ac resulting in up to a 37-fold increase in activity, between Cry1Ca and Cry1Ac resulting in up to a 172-fold increase, and between Cry11A and Cry11B resulting in up to a 6-fold increase (Vilchez, S. Toxins 12, 600 (2020)). Therefore, the Cry protein of the present invention also includes domain swap proteins of the Cry protein or a variant of the Cry protein shown in Table 3, that is, domain swap proteins in which at least one domain of the Cry protein or a variant of the Cry protein shown in Table 3 is swapped with the domain of another Cry protein or a variant of the Cry protein shown in Table 3, and which have insecticidal activity. As such a domain swap protein of the Cry protein, a protein in which insecticidal activity is improved compared to the parent protein is preferred.

[0057] For example, Cry5B protein, Cry1A protein, Cry1Ca protein, Cry3A protein, Cry4Aa protein, Cry4Ba protein, Cry11Aa protein, Cry14Ab protein, and Cry21Aa protein include (A) to (D) below. (A) Proteins consisting of the amino acid sequences shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235 (B) Proteins having insecticidal activity, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235. (C) A protein having insecticidal activity, comprising 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 with the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231 or 235. (D) A domain swap protein in which at least one domain of any of the proteins (A) to (C) is swapped with a domain of any other protein (A) to (C), and which has insecticidal activity.

[0058] 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 derived DNA, recombinant DNA, or chemosynthetic DNA, and may also be any of a genomic DNA clone or a cDNA clone.

[0059] The cry genes of the present invention typically refer to the cry genes shown in Table 3 above. However, 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 existence of very similar isozymes. In addition, so-called domain-swapped proteins, in which the domains of cry proteins are swapped, are also known. Therefore, the cry genes of the present invention are not limited to the genes shown in Table 3, but encompass all genes encoding the above-mentioned cry proteins.

[0060] For example, the cry5B gene, cry1A gene, cry1Ca gene, cry3A gene, cry4Aa gene, cry4Ba gene, cry11Aa gene, cry14Ab gene, and cry21Aa gene include (a) to (h) below. (a) Polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1, 203, 205, 207, 209, 211, 214, 230, or 234 (b) A polynucleotide encoding a protein having insecticidal activity, comprising a nucleotide sequence having 80% or more identity, 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 with the nucleotide sequence shown in SEQ ID NOs: 1, 203, 205, 207, 209, 211, 214, 230 or 234. (c) A polynucleotide encoding a protein that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1, 203, 205, 207, 209, 211, 214, 230, or 234, and which has insecticidal activity. (d) A polynucleotide comprising a nucleotide sequence obtained by swapping a nucleotide sequence encoding at least one domain in any of the polynucleotides of (a) to (c) with a nucleotide sequence encoding a domain of another polynucleotide of any of the other polynucleotides of (a) to (c), and which encodes a protein having insecticidal activity. (e) Polynucleotides encoding a protein consisting of the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235 (f) Polynucleotides encoding a protein having insecticidal activity, which consist of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235. (g) A polynucleotide encoding a protein having insecticidal activity, comprising 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 with the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231 or 235. A polynucleotide encoding a protein having insecticidal activity, comprising the amino acid sequence of a domain-swapped protein obtained by swapping at least one domain of any of the proteins described in (h)(e) to (g) with a domain of any other protein described in (e) to (g).

[0061] In the above, the "insecticidal activity" of a protein can be evaluated using the mortality rate when the protein is brought into contact with a suitable insect as an indicator. For example, using a 24-well flat-bottom plate, 10 five-day-old Aedes albopictus larvae, 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 it at 25°C for 24 hours, the mortality rate can be calculated based on the number of dead 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). For example, using a 48-well flat-bottom plate, 20 μL of 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 incubation at 20°C for 3 days, the insecticidal activity of the Cry protein against nematodes can be evaluated using the degree of inhibition of nematode growth in each well as an indicator (Patent No. 7218090).

[0062] [2-2. Expression-enabled introduction of genes encoding the Cry protein] Introducing a gene encoding the Cry protein (target gene) in a way that enables its expression means that the target gene is at least expressible 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. Such expressible introduction of a target gene can be achieved, for example, by introducing the target gene, operably linked to a regulatory region, preferably a strongly regulatory region, into the genome or plasmid of a host Bacillus subtilis strain. Alternatively, it can be achieved by modifying the host Bacillus subtilis strain to enable the expression of multiple target genes. An example of a procedure for introducing a target gene so that it can be expressed is described below.

[0063] (1) A DNA fragment is constructed in which the target gene is operably linked to a regulatory region, and further linked to a fragment corresponding to a region adjacent to the target gene introduction site upstream of the host Bacillus subtilis strain genome (referred to as the upstream fragment) and a fragment corresponding to a region adjacent to it downstream (referred to as the downstream fragment). For example, a fragment is prepared in which the regulatory region (e.g., the regulatory region of the cellulase gene of KSM-S237) and the target gene (e.g., the cry5B gene) are arranged in that order from upstream, and then the upstream fragment is linked upstream and the downstream fragment is linked downstream to prepare a DNA fragment. The DNA fragment may appropriately contain marker genes such as drug resistance genes. Next, when this DNA fragment is introduced into the host Bacillus subtilis strain, the DNA fragment is inserted into the host Bacillus subtilis strain genome, and the host Bacillus subtilis strain can be transformed. In the resulting transformant, the target gene is expressed.

[0064] In procedure (1), by simultaneously introducing marker genes such as drug resistance genes into the genome of the host Bacillus subtilis strain, the desired transformant can be easily selected using the expression of the marker gene as an indicator. It is preferable that the marker gene be removed from the obtained transformant in order to prevent the propagation of the marker gene. The procedure for removing the marker gene is not particularly limited and can include the two-step homologous recombination method or the method of introducing a lethal gene as described in [1-1. Bacillus subtilis mutant strain with genomic region deletion] above (see, for example, Figure 5). In the finally obtained transformant, the marker gene is not included and the target gene is expressed.

[0065] (2) Construct a vector containing a regulatory region and a target gene operably linked to it. For example, prepare a vector containing DNA arranged in the order of a regulatory region (e.g., the regulatory region of the KSM-S237 cellulase gene) followed by the target gene (e.g., the cry5 gene) from upstream. The vector may optionally contain marker genes such as drug resistance genes. Then, by introducing this vector into a host Bacillus subtilis strain, the host Bacillus subtilis strain can be transformed. In the resulting transformant, the target gene is expressed.

[0066] Here, the regulatory region that can be used for the expression of the target gene is preferably a regulatory region that has the function of increasing the expression of the downstream target gene in the host, and more preferably a regulatory region that has the function of constitutively expressing or highly expressing the downstream target gene. Also preferably, it is a regulatory region (strong regulatory region) that can enhance the expression of the target gene compared to the wild-type regulatory region of the target gene.

[0067] Examples of regulatory regions that can be used for the expression of genes encoding the Cry protein include the regulatory region of the α-amylase gene, the regulatory region of the protease gene, the rrnO operon regulatory region, the tufA gene regulatory region, the aprE gene regulatory region, the spoVG gene regulatory region, the cellulase gene regulatory region of Bacillus sp. KSM-S237 strain, the kanamycin resistance gene regulatory region of Staphylococcus aureus, and the chloramphenicol resistance gene regulatory region (see Japanese Patent Publication No. 2009-089708 for all of these).

[0068] Preferably, the regulatory region of the cellulase gene of Bacillus sp. KSM-S237 strain is the 0.4-1.0 kb region upstream of the translation start site of the cellulase gene (SEQ ID NO: 202), and a base sequence that has 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 with the said region in terms of nucleotide sequence, and has a gene expression regulatory function equivalent to that of the said region.

[0069] The target gene or regulatory region can be introduced into the host Bacillus subtilis strain by newly introducing it into the intracellular genome or plasmid, and the vector used can be any vector commonly used for transformation, such as a plasmid. The type of vector can be selected as appropriate, but it is preferable that it be a self-replicating vector (e.g., a plasmid) within the host Bacillus subtilis strain, and more preferably a multi-copy vector. Furthermore, the copy number of the plasmid is 2 to 100 copies, preferably 2 to 50 copies, and more preferably 2 to 30 copies, relative to the genome (chromosome) of the host Bacillus subtilis strain. Examples of preferred plasmids include, for example, pT181, pC194, pUB110, pE194, pSN2, and pHY300PLK.

[0070] Insertion of the target gene or regulatory region into a vector can be carried out according to the usual methods in the field. For example, fragments of the target gene and regulatory region can be amplified by PCR or the like, and these fragments can be inserted into a vector such as a plasmid by restriction enzyme assay or the like and then ligated. Alternatively, a fragment in which the target gene and regulatory region fragments are pre-ligated can be prepared and inserted into a vector such as a plasmid. In this case, the regulatory region fragment and the target gene fragment should be ligated in that order from upstream to downstream on the vector.

[0071] The introduction of DNA fragments or vectors into host Bacillus subtilis strains can be carried out according to conventional methods 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).

[0072] [3. Production of Cry protein or cultures containing it] The recombinant Bacillus subtilis obtained in this way has good Cry protein production ability. Therefore, by culturing the recombinant Bacillus subtilis of this invention in a nutrient medium, Cry protein can be expressed (produced) within the bacterial cells. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of Bacillus subtilis (transformed organism). Examples of carbon sources include glucose, dextran, soluble starch, sucrose, methanol, etc. Examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. In addition, 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.)) may be included if desired. Culturing is carried out by methods known in the art. Culture conditions, such as temperature, aeration and stirring conditions, pH of the culture medium, and culture time, are appropriately selected so that the Cry protein of the present invention is produced in large quantities.

[0073] The culture obtained by the above culture containing the Cry protein of the present invention is obtained by collecting cells by operations such as centrifugation and filtration, and suspending them in a suitable buffer (for example, a buffer such as Tris buffer, phosphate buffer, HEPES buffer, or MES buffer with a concentration of about 10M to 100mM (a pH range of 5.0 to 9.0 is desirable)) or water. Furthermore, the cells can also be destroyed by appropriately combining known cell disruption methods, such as lysozyme, freeze-thaw, sonication, French press, or bead disruption, and the Cry protein can be recovered by centrifugation.

[0074] The recovered Cry protein can be purified as appropriate using methods such as sucrose density gradient spectroscopy, recrystallization, ion exchange chromatography, gel filtration, hydrophobic chromatography, isoelectric focusing chromatography, or affinity columns with polyclonal antibodies against Cry protein as ligands.

[0075] [4. Manufacturing of IBaCC] In the culture of recombinant Bacillus subtilis of the present invention, the recombinant Bacillus subtilis contains the produced Cry protein in the cytoplasm in the form of crystals (Bacillus with Cytosolic Crystals; BaCC). Therefore, when the cultured recombinant Bacillus subtilis cells of the present invention are treated with a fungicide, inactivated Bacillus subtilis cells containing 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 the wild-type Bacillus subtilis 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, at least one gene selected from the group consisting of the sdpA, sdpB and sdpC genes is deleted or inactivated, and a gene encoding the Cry protein is introduced in an expressible manner, and contains the Cry protein.

[0076] While not particularly limited, disinfectants that can also be used in food are preferred, such as terpenes. Terpenes can be used individually or as plant-derived essential oils containing terpenes as the main component. Examples of terpenes include carvacrol, thymol, eugenol, geraniol, and citral, with carvacrol being particularly preferred. Terpenes can be used individually or in combination of two or more.

[0077] The amount of fungicide used can be appropriately set depending on the type of fungicide and the amount of bacterial cells. For example, in the case of carvacrol, 0.125 to 50 mg / mL is preferred, and 0.25 to 2.0 mg / mL is more preferred, relative to bacterial cells suspended in OD600 < 250.

[0078] The treatment of the recombinant Bacillus subtilis cells of the present invention with a fungicide after cultivation is carried out by bringing the cells and the fungicide into contact at a predetermined temperature and for a predetermined time. The contact temperature is preferably 1 to 40°C, and more preferably 4 to 30°C. The contact time is preferably 10 to 300 minutes, and more preferably 15 to 60 minutes. Contact can be carried out, for example, by suspending the recombinant Bacillus subtilis cells of the present invention, after cultivation, in a suitable buffer solution, and then adding a bactericide to the suspension.

[0079] It is desirable to wash away the disinfectant after treatment. For example, it is preferable to collect the recombinant Bacillus subtilis cells of the present invention after treatment by operations such as centrifugation or filtration, and then wash and suspend them with a suitable buffer (for example, a buffer such as Tris buffer, phosphate buffer, HEPES buffer, or MES buffer with a concentration of about 10M to 100mM (a pH range of 5.0 to 9.0 is desirable)) or water.

[0080] The efficiency of IBaCC production using recombinant Bacillus subtilis according to the present invention can be evaluated by observing the resulting processed product under a microscope. Specifically, the IBaCC rate (number of IBaCCs / (number of IBaCCs + number of free crystals) × 100) (%), which represents the ratio of the number of IBaCCs to the number of free crystals of Cry protein, can be calculated and used as an indicator for evaluation. The IBaCC rate using recombinant Bacillus subtilis of the present invention is higher than that of recombinant Bacillus subtilis identical to the recombinant Bacillus subtilis of the present invention, except that the nine protease genes, the sigF gene, and the lytC gene are deleted, in other words, the prophage6(yoaV-yobO) region to yncM-fosB region of the genome is not deleted, and the sdpA gene, sdpB gene, and sdpC gene are not deleted or inactivated. Alternatively, the IBaCC rate using recombinant Bacillus subtilis identical to the recombinant Bacillus subtilis of the present invention is higher than that of recombinant Bacillus subtilis identical to the recombinant Bacillus subtilis of the present invention, except that the prophage6(yoaV-yobO) region to yncM-fosB region of the genome, the nine protease genes, and the sigF gene are deleted, in other words, the lytC gene, sdpA gene, sdpB gene, and sdpC gene are not deleted or inactivated. The IBaCC rate of recombinant Bacillus subtilis of the present invention is preferably 125% or higher, more preferably 150% or higher, and even more preferably 175% or higher, compared to the IBaCC rate of recombinant Bacillus subtilis of the present invention, which is the same as the IBaCC rate of recombinant Bacillus subtilis of the present invention except that the lytC gene, sdpA gene, sdpB gene, and sdpC gene are not deleted or inactivated, with the IBaCC rate of recombinant Bacillus subtilis of the present invention being set as the reference IBaCC rate (100%).

[0081] Since the bacterial cells are killed in IBaCC, IBaCC is highly stable in production, storage, and use, and is also advantageous for stable passage through the stomach, making it useful as a pharmaceutical active ingredient.

[0082] The Cry protein produced by the recombinant Bacillus subtilis of the present invention, the culture containing the Cry protein, or the IBaCC containing the Cry protein (hereinafter referred to as the IBaCC, etc. of the present invention) can serve as an insecticide having insecticidal activity depending on the type of Cry protein, and can also be used to manufacture insecticides. Furthermore, IBaCC and the like of the present invention can be used for anthelmintic purposes. Here, such use may be administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic use. Note that "non-therapeutic" is a concept that does not include medical procedures, that is, a concept that does not include methods of surgery, treatment, or diagnosis of humans, and more specifically, a concept that does not include methods of surgery, treatment, or diagnosis performed on humans by a physician or a person under the direction of a physician.

[0083] The IBaCC and the like of the present invention can themselves be pharmaceuticals or quasi-drugs for anthelmintic purposes, or they can be materials or formulations used in combination with such pharmaceuticals or quasi-drugs.

[0084] When the anthelmintic agent of the present invention is used as a pharmaceutical product (including quasi-drugs), the pharmaceutical product can be administered in any form of administration. Examples of administration forms include oral administration in the form of tablets, capsules, granules, powders, syrups, etc., or parenteral administration in the form of injections, suppositories, inhalants, transdermal agents, topical agents, etc., but oral administration is preferred. Such various dosage forms of pharmaceutical preparations can be prepared by appropriately combining IBaCC of the present invention with other pharmaceutically acceptable excipients, binders, bulking agents, disintegrants, diluents, thickeners, emulsifiers, lubricants, dispersants, coating agents, surfactants, coating agents, osmotic pressure regulators, buffers, pH adjusters, preservatives, stabilizers, antioxidants, colorants, flavoring agents, deodorizing agents, fragrances, etc.

[0085] The amount of IBaCC, etc., contained in the above-mentioned pharmaceuticals (including quasi-drugs) varies depending on the target of anthelmintics, the recipient, and the route of administration, and is therefore not particularly limited and can be appropriately selected over a wide range of areas.

[0086] The dosage and administration schedule of the anthelmintic of the present invention may be appropriately determined by those skilled in the art according to the target species, weight, sex, age, condition, or other factors.

[0087] The anthelmintic agent of the present invention can be administered to both humans and non-human animals. Examples of non-human animals include non-human mammals, such as great apes, other primates, mice, rats, horses, cattle, pigs, sheep, dogs, cattle, hamsters, and companion animals. Preferably, the anthelmintic agent of the present invention is administered to humans who need it.

[0088] Exemplary embodiments of the present invention are further disclosed herein. However, the present invention is not limited to these embodiments. <1> A host Bacillus subtilis strain for producing Cry protein, 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 The genome has a deletion in at least one region selected from the group consisting of the P-yyaJ region and the yncM-fosB region, 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, sdpB gene and sdpC gene is deleted or inactivated. Host Bacillus subtilis strain. <2> Furthermore, the spoIIE gene is deleted or inactivated. <1> The host Bacillus subtilis strain described. <3> 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- Has a genome in which the 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 region are deleted, <1> or <2> The host Bacillus subtilis strain described. <4> The sdpA, sdpB, and sdpC genes are deleted or inactivated. <1> ~ <3> A host Bacillus subtilis strain as described in any one of the items.

[0089] <5> Recombinant Bacillus subtilis for producing Cry protein, <1> ~ <4> Recombinant Bacillus subtilis, wherein a host Bacillus subtilis strain described in any one of the items is modified to enable expression of a gene encoding the Cry protein. <6> The Cry Protein is selected from one of the following: Cry5B, Cry1A, Cry1Ca, Cry3A, Cry4Aa, Cry4Ba, Cry11Aa, Cry14Ab, and Cry21Aa. <5> Recombinant Bacillus subtilis as described. <7> The Cryptin Protein is Cry5B. <5> or <6> Recombinant Bacillus subtilis as described. <8> The Cry Protein is either Cry1A or Cry1Ca. <5> or <6> Recombinant Bacillus subtilis as described. <9> The Cry Protein is Cry3A. <5> or <6> Recombinant Bacillus subtilis as described. <10> The Cry Protein is Cry4Aa, Cry4Ba, or Cry11Aa. <5> or <6> Recombinant Bacillus subtilis as described. <11> The Cry protein is as follows (A)~(D): (A) Proteins consisting of the amino acid sequences shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235; (B) Proteins having insecticidal activity, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235; (C) A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence IDs 2, 204, 206, 208, 210, 212, 215, 231, or 235, and that has insecticidal activity; (D) A domain swap protein in which at least one domain of any of the proteins (A) to (C) is swapped with a domain of any other protein (A) to (C), and which has insecticidal activity. It is one of the following, <5> or <6> Recombinant Bacillus subtilis as described.

[0090] <12> <5> ~ <11> IBaCC (Inactivated Bacillus with Cytosolic Crystals), which is an inactivated recombinant Bacillus subtilis cell containing Cry protein, as described in any one of the items. <13> <12> An insecticide containing the IBaCC listed as an active ingredient. <14> <12> Use of IBaCC as described for anthelmintic purposes. <15> <12> Use of IBaCC described above for the manufacture of anthelmintics. <16> <12> A method of anthelmintics comprising administering the IBaCC described herein to a subject requiring it.

[0091] <17> A method for producing a host Bacillus subtilis strain for the production of Cry protein, 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 In a Bacillus subtilis mutant strain having a genome in which at least one region selected from the group consisting of the yncM-fosB region is deleted, the following are performed: 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, sdpB gene and sdpC gene. Methods that include... <18> A method for producing recombinant Bacillus subtilis for the production of Cry protein, 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, Consists of 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 region In a Bacillus subtilis mutant strain having a genome in which at least one region selected from the group is deleted, the following actions are performed: 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; deletion or inactivation of at least one gene selected from the group consisting of the sdpA, sdpB, and sdpC genes; and introduction of a gene encoding the Cry protein to enable expression. Methods that include... <19> Furthermore, this includes deleting or inactivating the spoIIE gene. <17> or <18> Method of description. <20> Bacillus subtilis mutant strain has 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, y Has a genome in which the isB-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 region are deleted; <17> ~ <19> The method described in any one of the items. <21> This includes deleting or inactivating the sdpA gene, the sdpB gene, and the sdpC gene. <17> ~ <20> The method described in any one of the items. <22> The Cry Protein is selected from one of the following: Cry5B, Cry1A, Cry1Ca, Cry3A, Cry4Aa, Cry4Ba, Cry11Aa, Cry14Ab, and Cry21Aa. <17> ~ <21> The method described in any one of the items. <23> The Cryptin Protein is Cry5B. <17> ~ <22> The method described in any one of the items. <24> The Cry Protein is either Cry1A or Cry1Ca. <17> ~ <22> The method described in any one of the items. <25> The Cry Protein is Cry3A. <17> ~ <22> The method described in any one of the items. <26> The Cry Protein is Cry4Aa, Cry4Ba, or Cry11Aa. <17> ~ <22> The method described in any one of the items. <27> The Cry protein is as follows (A)~(D): (A) Proteins consisting of the amino acid sequences shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235; (B) Proteins having insecticidal activity, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235; (C) A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence IDs 2, 204, 206, 208, 210, 212, 215, 231, or 235, and that has insecticidal activity; (D) A domain swap protein in which at least one domain of any of the proteins (A) to (C) is swapped with a domain of any other protein (A) to (C), and which has insecticidal activity. It is one of the following, <17> ~ <22> The method described in any one of the items.

[0092] <28> <5> ~ <11> A method for producing intracellular Cry protein or a culture containing the same, comprising culturing recombinant Bacillus subtilis as described in any one of the items. <29> <5> ~ <11> A method for producing IBaCC containing Cry protein, comprising culturing recombinant Bacillus subtilis as described in any one of the items, and treating the cultured bacterial cells with a fungicide. <30> The fungicide is a terpene, preferably carvacrol. <29> Method of description. <31> <29> or <30> IBaCC containing Cry protein obtained by the method described. [Examples]

[0093] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0094] In the following examples, polymerase chain reaction (PCR) for DNA fragment amplification was performed using a GeneAmp PCR System (Applied Biosystems) with PrimeSTAR Max DNA polymerase (Takara Bio Inc.) and accompanying reagents. The PCR reaction mixture 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, bringing the total reaction volume to 50 μL. The PCR reaction conditions consisted of 30 cycles of three temperature changes: 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 5-50 seconds (adjusted according to the target amplification product, with a guideline of 5 seconds per 1 kb), followed by a reaction at 72°C for 5 minutes.

[0095] Furthermore, in the following reference examples and embodiments, the upstream and downstream of a gene do not refer to positions from the replication start site. Instead, upstream refers to the region following the 5' end of the start codon of the target gene in each operation / process, while downstream refers to the region following the 3' end of the stop codon of the target gene in each operation / process.

[0096] Transformation of Bacillus subtilis was performed using the competent cell method (J. Bacteriol. 93, 1925 (1967)). Specifically, Bacillus subtilis strains were cultured with shaking at 37°C in SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.02% casamino acid (Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / mL tryptophan; % is (w / v)%) until the growth rate (OD600) was approximately 1. After shaking culture, a portion of the culture medium was used. Competent cells of the Bacillus subtilis strain were prepared by inoculating the strain into nine times the 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 acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / mL tryptophan), and then culturing with shaking until the growth rate (OD600) reached approximately 0.4.

[0097] Next, 5 μL of a solution containing various DNA fragments (such as the reaction solution for SOE-PCR) was added to 100 μL of the prepared competent cell suspension (culture medium in SPII medium). After shaking and culturing at 37°C for 1 hour, the entire volume was spread onto LB agar medium containing appropriate agents (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar). After static culture at 37°C, the grown colonies were isolated as transformants. The genomes of the obtained transformants were extracted, and PCR was performed using these genomes as templates to confirm that the desired genome structure had been modified.

[0098] The genes encoding the target protein or polypeptide were introduced into host microorganisms by one 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)).

[0099] For culturing recombinant microorganisms for protein production, we used LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), 2×YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl), 2×L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate tetra-pentahydrate), 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).

[0100] Example 1: Construction of a plasmid for EPR gene deletion Using genomic DNA extracted from 168 strains of Bacillus subtilis as a template, 0.6kb fragments (A) adjacent to the upstream and 0.5kb fragments (B) adjacent to the downstream of the epr gene on the genome were prepared using the eprfw1 and eprUPr, and eprDNf and eprrv-rep primer sets shown in Table 4-1. Separately, a 1.2kb fragment (C) was prepared by ligating the promoter region of the repU gene from plasmid pUB110 (Plasmid 15,93 (1986)) (Nucleic Acids Res. 17,4410 (1989)) upstream of the chloramphenicol resistance gene from plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)). Next, the three obtained fragments (A), (B), and (C) were mixed to form a template, and SOE-PCR was performed using the primers eprfw2 and Cmrv2 from Table 4-1 to ligate the three fragments in the order of (A), (B), and (C), obtaining a 2.2kb DNA fragment (Figure 2). The ends of this DNA fragment were blunted and 5'-phosphorylated, and inserted into the SmaI restriction enzyme site of plasmid pUC118 (Methods Enzymol. 153,3 (1987)) to construct the epr gene deletion plasmid pUC118-CmrΔepr. The 1.2kb fragment (C) mentioned above was prepared by mixing a 0.4kb fragment (D) containing the repU gene promoter region, which was prepared using the primer set of repUfw and repUr-Cm (Table 4-1) and plasmid pUB110 as a template, with a 0.8kb fragment (E) containing the chloramphenicol resistance gene, which was prepared using the primer set of CmUf-rep and Cmrv1 (Table 4-1) and plasmid pC194 as a template, and performing SOE-PCR using the primers repUfw and Cmrv1 shown in Table 4-1.

[0101] [Table 4-1]

[0102] [Table 4-2]

[0103] Reference Example 2: Construction of an epr gene deletion strain using a deletion plasmid The epr gene deletion plasmid pUC118-CmrΔepr, constructed in Reference Example 1, was introduced into 168 strains of Bacillus subtilis using the competent cell transformation method (J. Bacteriol. 93, 1925 (1967)). Transformed strains, fused with genomic DNA via single crossover homologous recombination between the upstream or downstream regions of the epr gene, were obtained using chloramphenicol resistance as an indicator. The obtained transformant strains were inoculated into LB medium and cultured at 37°C for 2 hours. Competence induction was then performed again to induce intragenomic homologous recombination between the overlapping upstream or downstream regions of the epr gene on the genome. As shown in Figure 2, when homologous recombination occurred in a different region than during plasmid introduction, the epr gene was deleted due to the loss of the chloramphenicol resistance gene and the pUC118 vector region derived from the plasmid. Next, to increase the proportion of chloramphenicol-sensitive strains, ampicillin enrichment was performed as follows. The culture medium after competent cell induction was inoculated into 1 mL of LB medium containing 5 ppm chloramphenicol and 100 ppm ampicillin sodium at a final concentration, so that the turbidity (OD600) at 600 nm was 0.003. After incubation at 37°C for 5 hours, 10 μL of 10,000 ppm ampicillin sodium aqueous solution was added, and incubation was continued for a further 3 hours. After incubation, the bacterial cells were centrifuged and washed with 2% sodium chloride aqueous solution, then suspended in 1 mL of 2% sodium chloride aqueous solution, and 100 μL of the suspension was spread onto LB agar medium. After incubation at 37°C for approximately 15 hours, strains that became chloramphenicol-sensitive due to plasmid region deletion were selected from among the grown strains. Using the genomic DNA of the selected strains as a template, PCR was performed using the primers eprfw2 and eprrv-rep shown in Table 4-1 to confirm epr gene deletion, and epr gene deletion strains were obtained.

[0104] Reference Example 3: Construction of protease gene octavo deletion strains and protease gene nepta deletion strains For the epr gene deletion strain, the wprA gene was deleted in the same manner as the epr gene deletion. Specifically, a plasmid for wprA gene deletion, pUC118-CmrΔwprA, was constructed in the same manner as in Reference Example 1. By introducing the constructed plasmid into genomic DNA and subsequently deleting the wprA gene by homologous recombination within the genome, a double deletion strain of both the epr and wprA genes was obtained. By repeating the same procedure, the mpr, nprB, bpr, nprE, vpr, and aprE genes were sequentially deleted, and an octuplet deletion strain of proteases with eight different protease genes was constructed and named the Δ8prt strain. Further repeating the same procedure resulted in the construction of a nnuplet deletion strain of proteases with the aprX gene deleted, and it was named the Δ9prt strain. Tables 4-1 and 4-2 show the sequences of the primers used for each deletion, and Table 5 shows the correspondence between each primer and the primer used for the epr gene deletion shown in Reference Example 1.

[0105] [Table 5]

[0106] Reference Example 4: Deletion of the lytC(cwlB) gene, PBSX gene group, 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 sets for cwlB gene deletion shown in Tables 4-2 and 5, and the Δ9B strain was constructed, which lacked nine protease genes and the cwlB gene. Next, the PBSX gene group was deleted. In this invention, the PBSX gene group refers to the 38 genes from the xlyB gene to the spoIISA gene that are continuously present on the Bacillus subtilis genome (Table 6). The PBSX gene group was deleted from the Δ9B strain using the primer sets for PBSX gene deletion shown in Tables 4-2 and 5, and the Δ9BP strain was constructed. Furthermore, the cwlF gene was deleted from the Δ9B strain using the primer sets for cwlF gene deletion shown in Tables 4-2 and 5, and the Δ9BF strain was constructed. Finally, the cwlF gene was deleted from the Δ9BP strain using the primer sets for cwlF gene deletion shown in Tables 4-2 and 5, and the Δ9BPF strain was constructed.

[0107] [Table 6]

[0108] Reference example 5: Deletion of the spoIIIC gene from the Δ9BPF strain Based on Figure 1, the method of deletion of the spoIIIC gene in the genome by a drug resistance gene will be explained. The spoIIIC gene, along with the spoIVCB gene, encodes the sigma factor SigK, which functions specifically during the spore formation stage of Bacillus subtilis.

[0109] Using genomic DNA extracted from 168 strains of Bacillus subtilis as a template, a 1.0 kb fragment (A) adjacent to the upstream of the spoIIIC gene in the genome was amplified by PCR using the spoIIIC-FW and spoIIIC / Em-R primer sets shown in Table 4-2. Furthermore, using the same 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 using the spoIIIC / Em-F and spoIIIC-RV primer sets.

[0110] Furthermore, using plasmid pMUTIN4 (Microbiology. 144, 3097 (1998)) as a template, a 1.3 kb erythromycin (Em) resistance gene region (C) was prepared by PCR using the emf2 and emr2 primer sets shown in Table 4-2.

[0111] Next, as shown in Figure 1, the three obtained fragments—1.0kb fragment (A), 1.0kb fragment (B), and Cm resistance gene region (C)—were mixed and used as a template. Using the spoIIIC-FW2 and spoIIIC-RV2 primer sets shown in Table 4-2, a 3.3kb DNA fragment (D) was obtained by SOE-PCR, in which the three fragments were contained in the order of 1.0kb fragment (A), Em resistance gene region (C), and 1.0kb fragment (B).

[0112] Furthermore, the Δ9BPF strain was transformed using the obtained DNA fragment (D) by the competent cell transformation method. After transformation, colonies grown on LB agar medium containing erythromycin (1 μg / mL) and lincomycin (25 μg / mL) were isolated as transformants.

[0113] The genomic DNA of the obtained transformants was extracted, and PCR confirmed that the spoIIIC gene was deleted and replaced with an Em resistance gene. In this way, the Δ9BPFC strain was constructed.

[0114] Reference example 6: Deletion of the sigD gene from the Δ9BPFC strain Based on Figure 1, the method of deleting the sigD gene in the genome using a drug resistance gene will be explained. The sigD gene is a gene that encodes the sigma factor SigD, which controls the expression of genes involved in cell wall lysis, flagellar formation, and chemotaxis in Bacillus subtilis.

[0115] Using genomic DNA extracted from 168 strains of Bacillus subtilis as a template, a 1.0 kb fragment (A) adjacent to the upstream of the sigD gene in the genome was amplified by PCR using the sigD-FW and sigD / Cm-R primer sets shown in Table 7. Furthermore, using the same 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 using the sigD / Cm-F and sigD-RV primer sets.

[0116] Furthermore, using plasmid DNA pC194 as a template, a 0.85 kb chloramphenicol (Cm) resistance gene region (C) was prepared by PCR using the catf and catr primer sets shown in Table 7.

[0117] Next, as shown in Figure 1, the three obtained fragments—1.0 kb fragment (A), 1.0 kb fragment (B), and Cm resistance gene region (C)—were mixed and used as a template. Using the sigD-FW2 and sigD-RV2 primer sets shown in Table 7, a 2.8 kb DNA fragment (D) was obtained by SOE-PCR, in which the three fragments were contained in the order of 1.0 kb fragment (A), Cm resistance gene region (C), and 1.0 kb fragment (B).

[0118] Furthermore, the Δ9BPFC strain was transformed using the obtained DNA fragment (D) by the competent cell transformation method. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.

[0119] The genomic DNA of the obtained transformants was extracted, and PCR confirmed that the sigD gene was deleted and replaced with a Cm resistance gene. In this way, the Δ9BPFCD strain (Δ9lyt strain) was constructed.

[0120] Reference example 7: Deletion of the sigF gene Based on Figure 1, the method of deletion of the sigF gene in the genome by a drug resistance gene will be explained. The sigF gene is a gene that encodes the sigma factor SigF, which controls the expression of genes involved in spore formation in Bacillus subtilis.

[0121] Using genomic DNA extracted from 168 strains of Bacillus subtilis as a template, a 1kb fragment (A) adjacent to the upstream of the sigF gene in the genome was amplified by PCR using the sigFfw and sigF-cat-r primer sets shown in Table 7. Furthermore, using the same genomic DNA as a template, a 1kb fragment (B) adjacent to the downstream of the sigF gene in the genome was amplified by PCR using the sigF-cat-f and sigFrv primer sets.

[0122] Furthermore, using plasmid DNA pC194 as a template, a 1kb Cm resistance gene region (C) was prepared by PCR using the catf and catr primer sets shown in Table 7.

[0123] Next, the three fragments obtained—fragment (A), fragment (B), and the Cm resistance gene region (C)—were mixed and used as a template. Using the sigFfw2 and sigFrv2 primer sets shown in Table 7, a DNA fragment (D) containing the three fragments in the order of (A), (C), and (B) was obtained by SOE-PCR (Figure 1).

[0124] Furthermore, the Bacillus subtilis mutant strain Δ9prt, constructed in Reference Example 3, was transformed using the obtained DNA fragment (D) by the competent cell transformation method. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.

[0125] The genomic DNA of the obtained transformants was extracted, and PCR confirmed that the sigF gene was deleted and replaced with a Cm resistance gene. In this manner, the Δ9F strain was constructed. By repeating the same procedure thereafter, 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 were transformed to construct Δ9BF, Δ9BPF, Δ9BPFF, Δ9BPFCF, and Δ9BPFCDF strains, respectively, which lack sigF.

[0126] [Table 7]

[0127] Reference Example 8: Synthesis of artificial genes 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). 3738 bp of the synthesized gene was cloned into the KpnI and HindIII sites of pUC57 to obtain pUC57-cry5B.

[0128] Reference Example 9: Construction of an 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 create the cry5B gene expression plasmid (Figure 3). The construction method followed the protocol of the In-Fusion® HD EcoDry® Cloning Kit (Clontech).

[0129] Using the vect+pF and vect+tR primer sets shown in Table 8, a vector containing the S237 promoter and terminator region was amplified using pHYS237 DNA as a template. Next, the cry5B gene insert was amplified by PCR using the cry5BpF and cry5BtR primer sets with pUC57-cry5B DNA as a template. Subsequently, the vector and insert were ligated using the In-Fusion HD EcoDry Cloning Kit (Clontech) and transformed into E. coli HB101 competent cells (Takara Bio). Transformants selected for tetracycline resistance were confirmed by colony PCR and named pPscry5B (Figure 3). The extracted plasmids were further confirmed by PCR, and the plasmid digestion patterns were examined using restriction enzymes EcoRI, SpeI, and XbaI.

[0130] [Table 8]

[0131] The constructed plasmids were sequenced. A sequencing template was prepared by PCR, and the 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 then sequenced using the 10 primers SEQ-P1 to SEQ-P10 shown in Table 8. The analysis revealed no mutations in any of the plasmids, confirming that the plasmids were constructed as designed.

[0132] Reference Example 10: Plasmid introduction into Bacillus subtilis host and culture 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 at 30°C with shaking at 250 rpm for 4 days. 0.2 mL of the culture medium was centrifuged at 15000 rpm at 4°C to separate the culture supernatant from the cells. The cell pellet was washed with 1×PBS, suspended in 1 mL of 1×PBS, and 2 mg / mL of lysozyme was added. The cells were incubated at 37°C for 1 hour. Subsequently, cells were disrupted using a Biorupter by sonication for 30 seconds x 20 times, then centrifuged at 15000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the precipitate was suspended in 1 mL of 1×PBS to obtain the cell lysate.

[0133] Reference Example 11: Quantification of Cry5B protein Protein quantification was performed by SDS-polyacrylamide electrophoresis (SDS-PAGE). Equal volumes of 10 μL of prepared cell disruption solution and Laemmli Sample Buffer (BIO-RAD) containing 100 mM DTT were mixed, heated at 100°C for 10 minutes, and cooled on ice for 3 minutes. An appropriate amount was then applied to Mini-Protean TGX gel StainFree 7.5% (BIO-RAD) and electrophoresis was performed at 200 V for 35 minutes. After completion, the gel was washed with deionized water for 5 minutes, and the protein bands were analyzed using ChemiDoc™ MP Imaging System (BIO-RAD). A calibration curve was created using BSA as the standard protein, and the amount of Cry5B protein was calculated by multiplying by a coefficient. Because the fluorescence intensity of this method changes depending on the Trp content of the protein, the coefficient was determined by comparing it with the Coomassie staining method. The Cry5B productivity of each strain is shown in Table 9. While the productivity of the Δ9BF strain for Cry5B was equivalent to that of the Δ9F strain, strains from Δ9BPF onward, in which PBSX was removed, showed decreased productivity (Table 9).

[0134] [Table 9]

[0135] Reference Example 12: IBaCC Rate Confirmation IBaCC (Inactivated Bacillus with Cytosolic Crystals) refers to cells that have been inactivated to contain crystalline proteins within the bacterial cell body. IBaCC was prepared based on International Publication No. 2017 / 123946. A cell pellet was collected from 20 mL of culture medium by centrifugation (8000 rpm, 15 minutes) and suspended in 1× PBS buffer to an OD600 < 250. 1 mg / g (w / w) of carvacrol (Carvacrol > 98%, Sigma) was added and shaken at room temperature for 15 minutes (to wet the entire inner surface of the container). Subsequently, IBaCC was recovered by centrifugation (8000 rpm, 15 minutes), the pellet was washed three times with 20 mL of 1× PBS buffer or ultrapure water, and then resuspended in 20 mL of 1× PBS buffer for use in SDS-PAGE analysis or microscopic observation. If not used immediately, it was stored frozen at -80°C. The IBaCC percentage (%) was calculated by microscopic examination, measuring the number of IBaCCs and free crystals within the field of view (100 or more), and then using the formula: (Number of IBaCCs) / (Number of IBaCCs + Number of free crystals) × 100.

[0136] The Δ9BF strain showed productivity comparable to the Δ9F strain, but its IBaCC rate was low at 50%. Considering that subsequent strains showed a significant decrease in Cry5B productivity, we decided to use the MGB874 strain to investigate how to improve its IBaCC rate.

[0137] Example 1: Constructing the 874dpr7 (874P) strain Using a Bacillus subtilis mutant strain (MGB874 strain; Japanese Patent Publication No. 4955358) in which a large region of the Bacillus subtilis wild-type genome was deleted, an extracellular protease-deficient strain was constructed. Excluding wprA and aprX, which had already been deleted during the large region deletion, the deletion of seven protease genes, epr, mpr, nprB, bpr, nprE, vpr, and aprE, was performed in the same manner as in Reference Examples 2 and 3, and the 874dpr7 (874P) strain was constructed.

[0138] Example 2: Constructing the 874PΔsigF (874PF) strain The 874P strain constructed in Example 1 was modified by deleting the sigF gene involved in spore formation using the same method as in Reference Example 7 to construct the 874PΔsigF(874PF) strain.

[0139] Example 3: Constructing the 874PΔsigFΔlytC (874PF1) strain The lysogenesis-related gene lytC was deleted from the 874PF strain constructed in Example 2. This mutant strain was constructed using a marker-free deletion method that fused the IPTG regulatory region with the free mRNA-cleaving ribonuclease mazF gene, 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), the region adjacent to the upstream codon of the lytC gene, was amplified by PCR using the primers lytC-DF1 and lytC-DR1 listed in Table 7. Then, using the same genomic DNA as a template, fragment (B), the region downstream of the stop codon of the lytC gene, was amplified by PCR using the primers lytC-DF2 and lytC-DR2 listed in Table 7. Furthermore, using the same genomic DNA as a template, a fragment (C) homologous to the ORF of the lytC gene was amplified by PCR using the primers lytC-DF and lytC-DR listed in Table 7. In addition, using the Bacillus subtilis mutant strain TOM31 (Genes Genet Syst. 2009, 84(4):315-8) as a template, a mazF cassette (mazF-lacI-spc) fragment (D) containing the spectinomycin resistance gene (spc) was amplified by PCR using the primers casf and casur listed in Table 7.

[0140] Next, the obtained fragments (A), (B), (D), and (C) were joined in that order using the primers lytC-DF1 and lytC-DR listed in Table 7 by SOE-PCR to obtain the final DNA fragment. Using the obtained DNA fragment, Bacillus subtilis 874PF strain was transformed by the competent method, and colonies grown on LB agar medium containing spectinomycin but not IPTG were isolated as transformants (lytC::mazF cassette).

[0141] Finally, to remove the mazF cassette, the cells were cultured on LB agar medium containing IPTG, and the resulting colonies were selected. The resulting mutant strain was mutant strain 874PF1, in which the mazF cassette was removed by intracellular homologous recombination in fragment (B). This mutant strain has a deletion of the stop codon region from the start codon of the lytC gene on the genome, and furthermore, the drug selection marker has been removed. PCR and sequencing analysis of the genome of the obtained mutant strain 874PF1 confirmed the introduction of the cassette and the deletion of the lytC gene on the genome.

[0142] Example 4: Construction of the 874PΔsigFΔlytCΔsdp (874PFL) strain and the 874PΔsigFΔlytCΔsdpΔspoIIE (874PFLE) strain Using the same method as in the construction of the 874PF1 strain in Example 3, the sdpABC gene group was deleted to construct the 874PF2 strain (874PFΔlytCΔsdpABC). Furthermore, using a similar method, the Cm marker introduced during the deletion of the sigF gene was removed from the 874PF2 strain, thereby constructing a markerless 874PFL strain. Next, the spoIIE gene was deleted from the 874PFL strain using the same method as in Example 3 to construct the 874PFLE strain. The correspondence between the primers used to construct the 874PF2, 874PFL, and 874PFLE strains and the primers used to construct the 874PF1 strain is shown in Table 10.

[0143] [Table 10]

[0144] Example 5: Plasmid introduction into MGB874 mutant strain and culture of the resulting transformant Plasmid pPscry5B, constructed in Reference Example 9, was introduced into the 874PF, 874PF1, 874PFL, and 874PFLE strains constructed in Examples 2-4 by protoplast transformation. The resulting transformants were cultured in 2xL / mal medium at 30°C with shaking at 250 rpm for 4 days. 0.2 mL of the culture medium was centrifuged at 15000 rpm at 4°C to separate the culture supernatant from the cells. The cell pellet was washed with 1×PBS, suspended in 1 mL of 1×PBS, and 2 mg / mL of lysozyme was added. The cells were incubated at 37°C for 1 hour. Subsequently, the cells were lysed using a Biorupter by sonication for 30 seconds x 20 times, then centrifuged at 15000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the precipitate was suspended in 1 mL of 1×PBS to obtain the cell lysate.

[0145] Example 6: Determination of Cry5B protein Protein quantification was performed by SDS-polyacrylamide electrophoresis (SDS-PAGE). Equal volumes of 10 μL of prepared cell disruption solution and Laemmli Sample Buffer (BIO-RAD) containing 100 mM DTT were mixed, heated at 100°C for 10 minutes, and cooled on ice for 3 minutes. An appropriate amount was then applied to MiniProtean TGX gel StainFree 7.5% (BIO-RAD) and electrophoresis was performed at 200 V for 35 minutes. After completion, the gel was washed with deionized water for 5 minutes, and the protein bands were analyzed using the ChemiDoc MP Imaging System (BIO-RAD). A calibration curve was created using BSA as the standard protein, and the amount of Cry5B protein was calculated by multiplying by a coefficient. Cry5B protein productivity is shown in Table 11.

[0146] [Table 11]

[0147] Example 7: Confirmation of IBaCC rate Table 11 shows the results of examining the IBaCC rates of strains 874PF, 874PF1, and 874PFL using a method similar to that of Reference Example 12. It was confirmed that strain 874PFL, which lacked lysis-related genes (lytC, sdpABC), maintained high Cry5B productivity and also showed a significant improvement in the IBaCC rate.

[0148] Example 8: Synthesis of artificial genes Eurofins artificially synthesized a sequence (SEQ ID NO: 134) consisting of the chloramphenicol resistance gene (cat) and a promoter expressible in Bacillus subtilis. The 1930 bp portion of the synthesized gene was inserted into a pUC plasmid to obtain pUC-CatP2.

[0149] Example 9: Genome transfer of cry5B using Cm markers The first copy of the cry5B genome was inserted between the yliA and hutP genes. Based on Figure 5, the method of introducing cry5B simultaneously with the drug resistance gene cat will be explained. Using genomic DNA extracted from Bacillus subtilis strain MGB874 as a template, a 1.0 kb fragment (upstream) adjacent to the insertion site in the genome was amplified by PCR using the yxiAfw2 and In3-cry5B-R primer sets shown in Table 12. Furthermore, using the same genomic DNA as a template, a 1.0 kb fragment (downstream) adjacent to the insertion site in the genome was amplified by PCR using the In3-cry5B-F and hutPrv2 primer sets.

[0150] Furthermore, using the plasmid DNA pUC-CatP2 as a template, a 1.9kb chloramphenicol resistance gene region (cat) and promoter region (cat-promoter) were prepared by PCR using the Ter-recA-F and pro-cry5B-rv primer sets shown in Table 12.

[0151] Furthermore, using the plasmid DNA pUC57-cry5B as a template, a 3.7kb cry5B gene region (cry5B) was prepared by PCR using the cry5BaF and Ter-TS43-R primer sets shown in Table 12.

[0152] Next, as shown in Figure 5, the four fragments obtained—a 1.0kb fragment (upstream), a 1.0kb fragment (downstream), a 1.9kb fragment (cat-promoter), and the cry5B gene region (cry5B)—were mixed and used as a template. Using the SOE-PCR method with the yxiAfw1 and hutPrv1 primer sets shown in Table 12, a 7.6kb cry5B introduction PCR fragment was obtained in which the four fragments contained, in the order of 1.0kb fragment (upstream), 1.9kb fragment (cat-promoter), 3.7kb cry5B gene region (cry5B), and 1.0kb fragment (B).

[0153] Furthermore, using the PCR fragment for cry5B introduction obtained by the competent cell transformation method, the 874PFL strain obtained in Example 4 was transformed. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.

[0154] The genomic DNA of the obtained transformants was extracted, and PCR confirmed the insertion of cat-promoter-cry5B between the yliA and hutP genes. In this manner, the 874PFL-cry5B1c strain was constructed. Similarly, the 874PFLE-cry5B1c strain was constructed from the 874PFLE strain.

[0155] [Table 12]

[0156] Example 10: Deletion of the Cm marker in the 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 Bacillus subtilis strain MGB874 as a template, a 1.0 kb fragment (upstream) adjacent to the cry5B insertion site in the genome was amplified by PCR using the yxiAfw2 and In3-Psp64-R primer sets shown in Table 12.

[0157] Next, using the Bacillus subtilis mutant strain TOM310 (Genes Genet Syst. 2009, 84(4):315-8) as a template, a 2.8kb mazF cassette (mazF-lacI-spc) fragment containing the spectinomycin resistance gene (spc) was amplified by PCR using the casf and casr primers listed in Table 12.

[0158] Furthermore, using plasmid DNA pUC-CatP2 as a template, a 1.1kb promoter fragment was amplified by PCR using the Psp64-fw and sp64rv primer sets shown in Table 12. Additionally, a 1kb chloramphenicol resistance gene region (cat) was prepared by PCR using the cas-catr and catf primer sets shown in Table 12.

[0159] Next, as shown in Figure 5, the four fragments obtained—a 1.0kb fragment (upstream), a 1.1kb promoter fragment, a 2.8kb mazF cassette fragment, and a 1kb chloramphenicol resistance gene region (cat)—were mixed and used as a template. Using the SOE-PCR method with the yxiAfw1 and catf primer sets shown in Table 12, a 5.9kb cat deletion PCR fragment was obtained in which the four fragments contained, in the order of 1.0kb fragment (upstream), 1.1kb fragment (promoter), 2.8kb mazF cassette, and 1.0kb chloramphenicol resistance gene region (cat).

[0160] Furthermore, the 874PFL-cry5B1c strain obtained in Example 9 was transformed using the PCR fragment for cat deletion obtained by the competent cell transformation method. 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 was inserted in the predetermined position in these Spc-resistant transformants. Next, these transformants were cultured overnight in LB liquid medium, and colonies grown on LB agar medium containing IPTG (0.1 mM) were isolated as marker-free transformants.

[0161] The genomic DNA of the obtained marker-free transformants was extracted, and the deletion of the cat gene region was confirmed by PCR. In this manner, the 874PFL-cry5B1 ​​strain was constructed. Similarly, the 874PFLE-cry5B1 ​​strain was constructed by deleting the cat gene from the 874PFLE-cry5B1c strain.

[0162] Example 11: Genome transfer of copy 2 cry5B using Cm marker Using the 874PFL-cry5B1 ​​and 874PFLE-cry5B1 ​​strains constructed in Example 10 as hosts, the 874PFL-cry5B2 and 874PFLE-cry5B2 strains were constructed by inserting a second copy of cry5B between the yweA gene and the spsL gene using the same procedure as in Examples 9 and 10. The correspondence between the primers used to construct 874PFL-cry5B2 (874PFLE-cry5B2) and the primers used to construct 874PFL-cry5B1 ​​(874PFLE-cry5B1) is shown in Table 13.

[0163] [Table 13]

[0164] Example 12: Culture of cry5B genome-transformed strain The 874PFL-cry5B1, 874PFLE-cry5B1, 874PFL-cry5B2, and 874PFLE-cry5B2 strains constructed in Examples 10 and 11 were cultured in 2x L / mal medium for 4 days at 30°C with shaking at 250 rpm. 0.2 mL of the culture medium was centrifuged at 15000 rpm at 4°C to separate the culture supernatant from the cells. The cell pellet was washed with 1×PBS, then suspended in 1 mL of 1×PBS, 2 mg / mL of lysozyme was added, and the cells were incubated at 37°C for 1 hour. Subsequently, the cells were lysed using a Biorupter by sonication for 30 seconds x 20 times, then centrifuged at 15000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the precipitate was suspended in 1 mL of 1×PBS to obtain the cell lysate.

[0165] Example 13: Determination of Cry5B protein Protein quantification was performed by SDS-polyacrylamide electrophoresis (SDS-PAGE). Equal volumes of 10 μL of prepared cell disruption solution and Laemmli Sample Buffer (BIO-RAD) containing 100 mM DTT were mixed, heated at 100°C for 10 minutes, and cooled on ice for 3 minutes. An appropriate amount was then applied to Mini-Protean TGX gel StainFree 7.5% (BIO-RAD) and electrophoresis was performed at 200 V for 35 minutes. After completion, the gel was washed with deionized water for 5 minutes, and the protein bands were analyzed using the ChemiDoc MP Imaging System (BIO-RAD). A calibration curve was created using BSA as the standard protein, and the amount of Cry5B protein was calculated by multiplying by a coefficient. The Cry5B protein productivity of the plasmid expression system is shown in Table 14, and the Cry5B protein productivity of the genome expression system is shown in Table 15. From these results, a significant improvement in productivity was confirmed by spoIIE deletion in both the plasmid expression system and the genome expression system.

[0166] [Table 14]

[0167] [Table 15]

[0168] Example 14: Confirmation of IBaCC rate Table 16 shows the results of confirming the IBaCC rate of strains 874PFLE-cry5B1 ​​and 874PFLE-cry5B2 using a method similar to that of Reference Example 12. It was confirmed that in strains lacking lysis-related genes (lytC, sdpABC) and the spoIIE gene, Cry5B productivity improved and the IBaCC rate remained high.

[0169] [Table 16]

[0170] Example 15: Acquisition of the Cry gene The 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), derived from Bacillus thuringiensis, 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), and 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. PCR reactions were performed using artificially synthesized gene fragments as templates to obtain the cry3A fragment using the primer combination of SEQ ID NO: 216 and SEQ ID NO: 217, the cry4Aa fragment using the primer combination of SEQ ID NO: 218 and SEQ ID NO: 219, the cry4Ba fragment using the primer combination of SEQ ID NO: 220 and SEQ ID NO: 221, the cry11Aa fragment using the primer combination of SEQ ID NO: 222 and SEQ ID NO: 223, the cry14Ab fragment using the primer combination of SEQ ID NO: 224 and SEQ ID NO: 225, and the cry21Aa fragment using the primer combination of SEQ ID NO: 226 and SEQ ID NO: 227. Furthermore, PCR reactions were performed using Basilex wettable powder (SDS Biotech Co., Ltd.) as a template. Using the primer combination of SEQ ID NO: 228 and SEQ ID NO: 229, a cry1A fragment (SEQ ID NO: 230, amino acid sequence: SEQ ID NO: 231, GenBank: WP_259384207.1 with 99% agreement) was obtained, and using the primer combination of SEQ ID NO: 232 and SEQ ID NO: 233, a cry1Ca fragment (SEQ ID NO: 234, amino acid sequence: SEQ ID NO: 235, GenBank: QEU48942.1 with 99% agreement) was obtained.

[0171] Example 16: Construction of an expression plasmid PCR was performed using plasmid pPscry5B as a template, and the vector fragment was amplified using the primer combination of SEQ ID NO: 236 and SEQ ID NO: 237. The resulting vector was then ligated with the cry1A, cry1Ca, cry3A, cry4Aa, cry4Ba, cry11Aa, cry14Ab, and cry21Aa fragments, respectively, using the In-Fusion HD Cloning Kit (Clontech), and expression plasmids for each cry gene were obtained via cloning in E. coli.

[0172] Example 17: Plasmid introduction into Bacillus subtilis host and cultivation of the resulting transformants In the same manner as in Example 5, the expression plasmids of each constructed cry gene were introduced into the 874PΔsigF (874PF) strain, the 874PΔsigFΔlytCΔsdp (874PFL) strain, and the 874PΔsigFΔlytCΔsdpΔspoIIE (874PFLE) strain, and the resulting transformants were cultured to obtain cell lysates.

[0173] Example 18: Determination of Cry protein Each Cry protein was quantified in the same manner as in Reference Example 11 and Example 6 for the quantification of Cry5B protein. The Cry protein productivity of each strain is shown in Table 17. As a result, improved Cry protein productivity was confirmed by spoIIE deletion in all strains expressing the Cry gene (Table 17).

[0174] [Table 17]

[0175] Example 19: Confirmation of IBaCC rate In the same manner as in Reference Example 12, IBaCC was prepared for each strain and the IBaCC rate was calculated. As a result, it was confirmed that the IBaCC rate was maintained higher in the 874PFL strain, which lacked lysis-related genes (lytC, sdpABC) than the 874PF strain, and in the 874PFLE strain, which further lacked the spoIIE gene, than in the 874PF strain (Table 18).

[0176] Table 18

Claims

1. A host Bacillus subtilis strain for producing Cry protein, 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 The genome has a deletion in at least one region selected from the group consisting of the P-yyaJ region and the yncM-fosB region, and 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, sdpB gene and sdpC gene is deleted or inactivated. Host Bacillus subtilis strain.

2. Furthermore, the host Bacillus subtilis strain according to claim 1, wherein the spoIIE gene is deleted or inactivated.

3. Recombinant Bacillus subtilis for producing Cry protein, wherein a gene encoding Cry protein is introduced into a host Bacillus subtilis strain according to claim 1 or 2 in an expressible manner.

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

5. The Cry protein is as follows (A) to (D): (A) Proteins consisting of the amino acid sequences shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235; (B) Proteins having insecticidal activity, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231 or 235; (C) A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NOs: 2, 204, 206, 208, 210, 212, 215, 231, or 235, and which has insecticidal activity; (D) A domain swap protein in which at least one domain of any of the proteins (A) to (C) is swapped with a domain of any other protein (A) to (C), and which has insecticidal activity. Recombinant Bacillus subtilis according to claim 3, which is one of the following selected from:

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

7. A method for producing a host Bacillus subtilis strain for the production of Cry protein, 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 In a Bacillus subtilis mutant strain having a genome in which at least one region selected from the group consisting of the yncM-fosB region is deleted, the following are performed: 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, sdpB, and sdpC genes. Methods that include...

8. A method for producing recombinant Bacillus subtilis for the production of Cry protein, 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, Consists of 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 region In a Bacillus subtilis mutant strain having a genome in which at least one region selected from the group is deleted, the following actions are performed: 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; deletion or inactivation of at least one gene selected from the group consisting of the sdpA, sdpB, and sdpC genes; and introduction of a gene encoding the Cry protein to enable expression. Methods that include...

9. A method for producing a Cry protein within a bacterial cell or a culture containing the same, comprising culturing the recombinant Bacillus subtilis described in claim 3.

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

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