JP2014161284A - Mutant microorganism and useful substance production method using the same - Google Patents

Abstract

Provided is a microbial method capable of efficiently producing secreted useful substances.
A mutant Bacillus bacterium in which EndoA is inactivated, a gene encoding a target gene product is introduced, and a secretory signal peptide region is linked upstream of the gene.
[Selection figure] None

Description

  The present invention relates to a mutant microorganism having a high substance-producing ability and a method for producing a useful substance using the microorganism.

  Industrial production of useful substances by microorganisms is carried out in a wide range of fields such as daily necessities such as foods, pharmaceuticals, detergents, cosmetics, and various chemical products. There are various types of useful substances produced by microorganisms such as foods such as alcoholic beverages, miso and soy sauce, amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, proteins, and the like. One important issue in industrial production of useful substances using microorganisms is improvement of productivity.

  In the past, useful substance-producing bacteria have been developed by genetic techniques such as mutation. Furthermore, in recent years, the genome information of industrially useful microorganisms has been decoded, and useful substance-producing bacteria have been developed by genetic engineering techniques based on the decoded genome information. Bacillus subtilis Marburg No. 168 is one of useful microorganisms whose genome information has been disclosed so far (Non-patent Document 1).

  For example, it is known that high cellulase productivity can be obtained by transforming a plasmid derived from pAMα1 into which a cellulase gene derived from Bacillus sp. KSM64 is introduced in Bacillus subtilis (Patent Document 1).

  Intracellular protein or peptide synthesis takes place in the ribosome. Processing and degradation of rRNA constituting the ribosome are controlled by RNase, which is a kind of endoribonuclease. In Escherichia coli, RNaseG encoded by the rng gene performs 5'-end processing of 16S rRNA, and RNaseE encoded by the rne gene is involved in 5SRNA maturation (Patent Document 2). Furthermore, Patent Document 2 discloses an expression vector comprising a sequence in which a gene encoding a target protein is linked to a 5′-untranslated region of a gene encoding a polypeptide overexpressed in an E. coli strain deficient in RNaseG. A method for improving the productivity of the target protein by introducing mRNA is disclosed. Regarding Bacillus subtilis, it is suggested that rRNA is specifically degraded by RNase that depends on the sigH gene in the early stage of sporulation (Non-patent Document 2).

  EndoA, a member of the RNase family, is an enzyme that is toxic to Bacillus subtilis when overexpressed. EndoA in Bacillus subtilis is considered to correspond to the MazF / PemK family, which is an endoribonuclease that inactivates mRNA in E. coli. EndoA is encoded only by the ydcE gene and its activity is suppressed by the ydcD gene (Non-patent Document 3). The authors of Non-Patent Document 3 proposed that the gene encoding EndoA be the ndoA gene.

JP 2001-69986 A Japanese Patent Laid-Open No. 2002-238576

Nature, 1997, 390: 249-256 6th Annual Meeting of the Genomic Microbiology (2012) p85, 2P-28 Molecular Microbiology, 2005, 56 (5): 1139-1148

  The present invention relates to a mutant microorganism having a high ability to produce a useful substance, and a method for producing a useful substance using the microorganism.

  In Bacillus subtilis, 70S ribosomes that actively synthesize proteins are formed in the logarithmic growth phase, but in the sporulation phase, dimer ribosomes in which two 70S ribosomes are combined are formed and rRNA degradation occurs. It is known. In the Bacillus subtilis strain lacking the ndoA gene, dimer ribosomes are formed at the early stage of sporulation, but rRNA degradation is suppressed. Therefore, RNase encoded by ndoA is involved in rRNA degradation. (The 84th Annual Meeting of the Genetics Society of Japan, Proceedings [Published: August 31, 2012]) The present inventors have also found that an ndoA gene-deficient Bacillus subtilis mutant has a high ability to produce a protein or peptide. Based on these findings, the present inventors have found that EndoA-inactivated Bacillus strains can efficiently secrete and produce useful substances such as proteins and polypeptides, and have completed the present invention. .

That is, in the present invention, EndA is inactivated and a vector containing a gene encoding a heterologous protein, peptide or polypeptide is introduced, or a gene encoding a heterologous protein, peptide or polypeptide is introduced into the genome. And a mutant Bacillus bacterium, wherein a secretory signal peptide region is linked upstream of the gene encoding the heterologous protein, peptide or polypeptide.
The present invention also provides a method for producing a protein, peptide or polypeptide, comprising culturing the mutant Bacillus bacterium.

  The mutant Bacillus bacterium of the present invention can produce highly useful substances such as proteins and polypeptides. By using the mutant genus Bacillus, a useful substance can be produced efficiently. Furthermore, according to the bacterium belonging to the genus Bacillus of the present invention, the produced useful substance is secreted to the outside of the cell body, so that more efficient useful substance production can be achieved.

The schematic diagram of the preparation of the DNA fragment for gene deletion by SOE-PCR, and the procedure of the target gene deletion using the said DNA fragment.

  In the present specification, the “amino acid residue” means 20 kinds of amino acid residues constituting a protein, alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L) ), Lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W) , Tyrosine (Tyr or Y) and valine (Val or V).

  In the present specification, the “base” relating to the nucleotide sequence refers to a base that usually constitutes DNA or RNA, adenine (A), thymine (T) or uracil (U), guanine (G), and cytosine (C). Point to.

  As used herein, “identity” between amino acid sequences or nucleotide sequences means that two amino acid sequences or nucleotide sequences are identical in both sequences when they are aligned (aligned) for maximum identity. The ratio (%) of the number of amino acid residues or bases to the total number of amino acid residues or the total number of bases. More specifically, the “identity” between amino acid sequences or nucleotide sequences is calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)), and genetic information processing software Genetyx-Win ( Using the homology analysis program of Ver.5.1.1 (software development), the unit size to compare (ktup) is set to 2 for the calculation.

  In the present specification, unless otherwise limited, “upstream” of a gene refers to a region continuing 5 ′ of the start codon of the gene of interest in each operation step, whereas “downstream” of a gene It refers to the region that continues 3 'of the stop codon of the gene of interest in the manipulation process. In the present specification, “upstream” and “downstream” of a gene do not refer to a region based on the position from the replication start point unless otherwise limited.

  In the present specification, EndoA can be a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. In addition, EndoA in the present specification can include a polypeptide that is an endoribonuclease having an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having rRNA degrading activity. The amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 is at least 80%, preferably at least 85%, more preferably at least 90%, still more preferable with the amino acid sequence shown in SEQ ID NO: 2. Includes amino acid sequences having at least 95% identity, even more preferably at least 98%, still more preferably at least 98%, even more preferably at least 99%.

  The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 represents Bacillus subtilis endoribonuclease EndoA represented by Genbank: NP_388347. Bacillus subtilis EndoA is a protein encoded by the Bacillus subtilis ndoA gene (Genbank: AL009126 REGION: 518943..519290) consisting of the nucleotide sequence shown in SEQ ID NO: 1. It has been suggested that Bacillus subtilis EndoA has rRNA degradation activity in the early stage of sporulation (Non-patent Document 2). In Bacillus subtilis, 70S ribosomes that actively synthesize proteins are formed in the logarithmic growth phase, but in the sporulation phase, dimer ribosomes in which two 70S ribosomes are combined are formed and rRNA degradation occurs. . Therefore, when Bacillus subtilis is cultured under normal conditions, the activity of ribosomes decreases at the later stage of culture when the bacteria enter the spore formation stage, and the protein productivity of the bacteria decreases. However, inactivation of fungal EndoA suppresses ribosome degradation, so that the ribosome activity is maintained even in the later stage of culture, and the protein production ability of the fungus can be maintained. As a result, the productivity of the bacteria such as proteins and polypeptides can be improved by inactivating the bacterial EndoA.

  Therefore, the mutant Bacillus genus bacterium of the present invention is a mutated Bacillus bacterium having improved substance production ability due to the inactivation of EndoA. In one embodiment, the mutant Bacillus bacterium of the present invention may include a Bacillus bacterium mutant in which EndoA is inactivated by a natural mutation. In another embodiment, the mutant Bacillus bacterium of the present invention may include a Bacillus bacterium mutant in which EndoA is inactivated by artificial modification. Preferably, the mutant Bacillus bacterium of the present invention is a Bacillus bacterium mutant in which EndoA is inactivated by artificial modification.

  Examples of the Bacillus genus that is the parent strain of the mutant Bacillus bacterium of the present invention include bacteria belonging to the genus Bacillus, such as Bacillus subtilis, Bacillus cereus, Bacillus coagulans, Bacillus clausii, Bacillus circulans, Bacillus licheniformis, Paenibacillus sp. Etc. When the mutant Bacillus bacterium of the present invention is produced by artificial modification, as a parent strain, the whole genome information of a wild strain has been clarified, genetic engineering and genome engineering techniques have been established, and protein It is preferable to use a wild strain (168 strain) of Bacillus subtilis and a mutant thereof from the viewpoint of having the ability to secrete and produce the bacterium outside the cell. The mutant Bacillus genus bacterium of the present invention can be produced by modifying the parent strain so as to inactivate EndoA.

  The Bacillus subtilis wild strain (also referred to herein as Bacillus subtilis 168 or simply 168) is a Bacillus subtilis strain known as Bacillus subtilis Marburg No.168. Bacillus subtilis 168 strain is usually used as a wild strain in the production of a recombinant protein. In addition, the entire base sequence and gene of Bacillus subtilis 168 have been reported and published on the Internet (Non-patent Document 1, BSORF: Bacillus subtilis Genome Database [http://bacillus.genome.jp/], and GenBank: AL009126.2 [http://www.ncbi.nlm.nih.gov/nuccore/38680335]). Those skilled in the art can perform various genetic manipulations based on the genome information of Bacillus subtilis 168 strain obtained from these information sources.

  In the mutant Bacillus bacterium of the present invention, for example, the expression level of EndoA is 50% or less, preferably 40% or less, more preferably 30% or less, still more preferably 20% or less, even more preferably, the expression level of the wild strain. May be a mutant Bacillus bacterium that is reduced to 10% or less, more preferably 5% or less. The expression level of EndoA can be measured by a commonly used protein expression quantification method such as, but not limited to, quantitative PCR, colorimetric quantification, fluorescence method, Western blotting, ELISA, radioimmunoassay and the like.

  For mutants of the genus Bacillus, in which EndoA is inactivated by natural mutation, search for mutants in which EndoA expression level is reduced or completely inhibited by the screening method using EndoA expression level as an indicator. Can be obtained. As such a mutant strain, the gene encoding EndoA is partially or completely deleted, or the gene encoding EndoA or its regulatory region has a mutation, so that EndoA is not expressed, or Examples include strains in which the polypeptide expressed from the gene does not have the desired EndoA activity (the activity of an endoribonuclease that degrades rRNA). The expression level of EndoA can be measured by the usual protein expression quantitative method described above.

  A Bacillus bacterium mutant in which EndoA has been inactivated by artificial modification can be produced by any means known in the art as a method for suppressing the expression of a target protein by genetic engineering. Typically, a method of deleting or inactivating a gene encoding EndoA in a cell of the Bacillus bacterium, a method of inactivating mRNA encoding EndoA in the cell, and encoding EndoA in the cell Include a method for suppressing translation of mRNA into EndoA and a method for inactivating translated EndoA, including a method for deleting or inactivating a gene encoding EndoA, and encoding EndoA A method of inactivating mRNA is preferred.

  As a gene encoding EndoA, a Bacillus subtilis ndoA gene represented by (Genbank: AL009126 REGION: 518943..519290); a gene consisting of a nucleotide sequence represented by SEQ ID NO: 1; a nucleotide sequence represented by SEQ ID NO: 1 and 80% A gene comprising a nucleotide sequence having the above identity and encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2; and a nucleotide sequence having 80% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 And a gene encoding a polypeptide which is an endoribonuclease having an rRNA degrading activity, comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2. The nucleotide sequence having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 is at least 80%, preferably at least 85%, more preferably at least 90%, still more preferably the nucleotide sequence represented by SEQ ID NO: 1. Includes nucleotide sequences having at least 95% identity, even more preferably at least 98%, still more preferably at least 98%, even more preferably at least 99%. The amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 is as described above. Examples of mRNA encoding EndoA include mRNA transcribed from the above-mentioned gene encoding EndoA.

  Examples of a method for inactivating mRNA encoding EndoA in cells include target-specific mRNA inhibition using siRNA. The basic procedure of siRNA is well known to those skilled in the art (see, for example, JP-T-2007-512808), and reagents or kits for siRNA are commercially available. A person skilled in the art can prepare a desired target-specific siRNA and inhibit the target mRNA based on known literature and manuals of commercially available products.

  As a method for deleting or inactivating a gene encoding EndoA in a cell, part or all of the DNA sequence of a gene encoding EndoA (hereinafter referred to as a target gene) is removed from the genome, or other DNA Examples thereof include a method of replacing the sequence, a method of inserting another DNA fragment into the DNA sequence in the target gene, a method of mutating the transcription or translation initiation region of the target gene, and the like. Preferably, part or all of the DNA sequence of the target gene is deleted or inactivated. More specific examples include a method for specifically deleting or inactivating a target gene in a cell, and a random deletion or inactivating mutation in a gene in the cell, and then encoding the target gene. And a method for selecting a cell having a desired mutation by conducting evaluation of protein productivity and gene analysis.

  Examples of a method for specifically deleting or inactivating a target gene include a method by homologous recombination. More specifically, a DNA fragment of a target gene having another fragment inserted therein, a DNA fragment of a target gene inactivated by mutation such as base substitution or base insertion, or upstream and downstream of the target gene as shown in FIG. A DNA fragment containing only the downstream region is constructed, and the DNA fragment is cloned into an appropriate plasmid vector to prepare a circular recombinant plasmid. When this plasmid is incorporated into a cell having a target gene, homologous recombination occurs between the DNA fragment in the incorporated plasmid and the corresponding target gene region on the genome, so the target gene sequence is deleted. Or inactivated by splitting or replacing with another sequence.

  For example, there have already been several reports on methods for specifically deleting or inactivating genes on the genome of Bacillus subtilis cells by homologous recombination (for example, Molecular Genetics and Genomics, 1990, 223: 268). -272). A person skilled in the art can obtain a Bacillus bacterium having a target gene deleted or inactivated based on these methods.

  As a method for randomly deleting or inactivating a gene in a cell, a method for causing homologous recombination similar to the above method using a DNA fragment obtained by randomly cloning a gene, γ-rays, etc. Examples include a method of inducing mutation by irradiation. By confirming the genomic sequence of cells having random mutations obtained by these methods, cells in which the gene encoding EndoA has been deleted or inactivated can be selected. Alternatively, cells in which EndoA is inactivated can be selected using the EndoA expression level measured by the above-described normal protein expression quantification method as an index.

  Hereinafter, as an example of a method for specifically deleting or inactivating a target gene, DNA for deletion prepared by SOE (splicing by overlap extension) -PCR method (Gene, 1989, 77: 61-68) The deletion method by the double crossing method using fragments will be described. Regarding the method, the descriptions in JP2007-130013A, JP2011-160686A, and the like can be referred to. However, the gene deletion or inactivation method usable in the present invention is not limited to this.

  For the deletion DNA fragment, for example, a drug resistance marker gene fragment is inserted between the approximately 0.1 to 3 kb fragment adjacent to the upstream of the gene to be deleted and the approximately 0.1 to 3 kb fragment adjacent to the downstream as well. Fragment. Although it does not specifically limit as a drug resistance marker gene, For example, a kanamycin resistance gene, a spectinomycin resistance gene, a chloramphenicol resistance gene, an erythromycin resistance gene, a tetracycline resistance gene etc. are mentioned.

  First, an upstream fragment and a downstream fragment of a deletion target gene and three fragments of a drug resistance marker gene fragment are prepared by the first PCR. For the amplification of the upstream fragment, primers designed so that the upstream side (for example, 10 to 30 bases) sequence of the drug resistance marker gene is added to the downstream end of the upstream region of the deletion target gene, For amplification, a primer designed so that the downstream side (for example, 10 to 30 bases) sequence of the drug resistance marker gene is added to the upstream end of the downstream region of the deletion target gene is used. By this first PCR, the upstream fragment (A) in which the upstream sequence of the drug resistance marker gene is added to the downstream end of the upstream region of the deletion target gene, and the drug resistance marker at the upstream end of the downstream region of the deletion target gene Three fragments of the downstream fragment (B) to which the downstream sequence of the gene is added and the drug resistance marker gene fragment (C) are prepared (FIG. 1).

  Next, using the three types of PCR fragments prepared in the first PCR as templates, the second PCR is performed using the upstream primer of the upstream fragment and the downstream primer of the downstream fragment. In this reaction, annealing occurs between the drug resistance marker gene sequence added to the downstream end of the upstream fragment and the upstream end of the downstream fragment and the drug resistance marker gene fragment, and as a result of PCR amplification, between the upstream fragment and the downstream fragment. In addition, a DNA fragment (D) into which a drug resistance marker gene is inserted can be obtained (FIG. 1). The obtained DNA fragment is used for subsequent homologous recombination as a DNA fragment for gene deletion. The first and second PCRs are performed using commercially available enzyme kits for PCR and the like (PCR Protocols. Current Methods and Applications, Edited by BA White, Humana Press pp251, 1993, Gene, 1989, 77:61). -68) It can be performed under the normal conditions shown in the above.

  Alternatively, the three fragments prepared by the first PCR are ligated on an appropriate plasmid vector in the order of upstream fragment (A), drug resistance marker gene fragment (C), and downstream fragment (B), and cloned. Then, a DNA fragment for gene deletion can be obtained by cutting out the target fragment from the obtained plasmid vector with a restriction enzyme.

  The obtained DNA fragment for gene deletion is introduced into cells having the gene to be deleted by a conventional method such as a competent method. When gene recombination occurs between the introduced DNA fragment for gene deletion and the upstream and downstream homologous regions of the corresponding deletion target gene in the cell, the deletion target gene is replaced with a drug resistance gene. Or a cell in which a drug resistance gene is inserted into the gene to be deleted is generated (FIG. 1E). Cells that have undergone recombination are selected using the substituted or inserted drug resistance marker as an index. For example, recombinant Bacillus subtilis deficient in the target gene can be obtained by culturing cells introduced with the DNA fragment for gene deletion on an agar medium containing a drug and isolating growing colonies. Furthermore, it can be confirmed by PCR method using the genome as a template that the target gene is deleted. By the above procedure, a cell in which the gene encoding the target EndoA is specifically deleted or inactivated can be obtained.

  The mutant Bacillus genus bacterium of the present invention can be produced by introducing a gene encoding a target gene product into a Bacillus bacterium in which EndoA is inactivated obtained by the procedure as described above. The target gene product encoded by the gene to be introduced is not particularly limited in type, but is preferably a protein, peptide or polypeptide, preferably contractile protein, transport protein, signal protein, biological defense protein, receptor protein, structural protein, Examples include functional proteins such as gene regulatory proteins, enzymes, and storage proteins, peptides, and polypeptides. The protein, peptide or polypeptide may be of a kind that is naturally expressed by the Bacillus bacterium into which the gene is introduced, but preferably, the kind of protein that the Bacillus bacterium is not naturally expressed. Is a heterologous protein, peptide or polypeptide.

  In a preferred embodiment, the target gene product includes, for example, proteins, peptides, or polypeptides such as industrially useful enzymes and physiologically active factors used in the fields of foods, pharmaceuticals, cosmetics, detergents, fibers, medical tests, etc. Is mentioned. The industrially useful enzymes include oxidoreductase (oxidoreductase), transferase (transferase), hydrolase (hydrolase), eliminase (lyase), isomerase (isomerase), and synthetic enzyme (ligase / synthetase). ) And the like, preferably hydrolyzing enzymes such as glycoside hydrolase, α-amylase and protease.

  Examples of the glycoside hydrolase include enzymes belonging to glycoside hydrolase family 8 (GH family 8), preferably in the classification of polysaccharide hydrolases (Biochem. J., 1991, 280: 309-316). Cellulases belonging to family 5 are mentioned, and cellulases derived from Bacillus bacteria are more preferred. More specific examples include alkaline cellulase (SEQ ID NO: 4) derived from Bacillus genus bacteria KSM-S237 strain (FERM BP-7875); alkaline cellulase (sequence) derived from Bacillus genus bacteria KSM-64 strain (FERM BP-2886) No. 6); Alkaline cellulase derived from Bacillus genus bacteria KSM-N257 strain (FERM P-17473) (JP 2002-85078, GenBank accession no. AB059267); Cellulolytic xylanase derived from Bacillus circulans (GenBank accession no. Paenibacillus sp. Y412MC10-derived lichenase (GenBank accession no. YP_003240630); Paenibacillus sp. HGF5-derived glycosyl hydrolase family 8 (GenBank accession no. ZP_08283397); Paenibacillus vortex V453-derived lichenase (GenBank accession no. Paenibacillus lactis 154-derived lichenase (GenBank accession no. ZP_09002324); and the amino acid sequences of these glycoside hydrolases and more than 80% preferred Alternatively, a glycoside hydrolase consisting of an amino acid sequence having 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more.

  Examples of α-amylase include microorganism-derived α-amylase, and liquefied amylase derived from bacteria belonging to the genus Bacillus is preferred. More specific examples include alkaline amylase derived from Bacillus genus KSM-K38 strain (FERM BP-6946) (Japanese Patent Laid-Open No. 2002-112792, GenBank accession no. AB051102), and the amino acid sequence of the amylase and 80% or more. An amylase comprising an amino acid sequence having preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more.

  Examples of the protease include microorganism-derived proteases, and serine proteases, metal proteases, and the like derived from bacteria belonging to the genus Bacillus are preferable. As a more specific example, an alkaline protease derived from Bacillus clausii strain KSM K-16 (FERM BP-3376) (JP-A-5-308967, GenBank accession no. YP_174261); Bacillus genus bacteria KSM-KP43 Strain (FERM BP-6532) -derived alkaline protease (GenBank accession no. AB051423); protease KP9860 [from Bacillus sp. KSM-KP9860 (FERM BP-6534), WO99 / 18218, GenBank accession no. AB046403]; protease E-1 [Derived from Bacillus No. D-6 (FERM P-1592), JP-A-49-71191, GenBank accession no. AB046402]; Protease Ya [Derived from FERM BP-1029, JP-A-61- No. 280268, GenBank accession no. AB046404]; Protease SD521 [derived from Bacillus SD521 (FERM P-11162), JP-A-3-191781, GenBank accession no. AB046405]; Protease A-1 [derived from NCIB12289, WO88 / 01293 , Gen Bank accession no. AB046406]; Protease A-2 [derived from NCIB12513, WO98 / 56927]; Protease 9865 [Derived from Bacillus SP KSM-9865 (FERM P-18566), GenBank accession no. AB084155]; and amino acids of these proteases Examples include a protease comprising an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more.

  Furthermore, in the mutant Bacillus bacterium of the present invention, a polynucleotide encoding a secretory signal peptide (hereinafter also referred to as secretory signal peptide region) is linked upstream of the gene encoding the introduced target gene product. . In other words, the gene encoding the target gene product introduced into the mutant Bacillus bacterium of the present invention is located downstream of the secretory signal peptide region. By such linkage with the secretory signal peptide region, when the target gene product is expressed in the mutant Bacillus bacterium of the present invention, it is then secreted outside the cell. That is, if the target gene product is a protein, peptide or polypeptide, it becomes a secretory protein, peptide or polypeptide. In the bacterium belonging to the genus Bacillus of the present invention, the gene encoding the target gene product and the secretory signal peptide region upstream of the gene are secreted outside the cell body by the secretory signal peptide so that both can be expressed. As long as they are connected in such a manner, there is no limitation on the configuration and arrangement.

  In the mutant Bacillus bacterium of the present invention, the secretory signal peptide region linked upstream of the gene encoding the target gene product is expressed in the mutant Bacillus bacterium, and the target gene product is put outside the cell body. The polynucleotide is not particularly limited as long as it is a polynucleotide encoding a secretory signal peptide having a function of secreting. The secretory signal peptide region may be naturally present in the genome of the mutant Bacillus bacterium or may be heterologous. In addition, the secretory signal peptide region is an original secretory signal peptide of a protein, peptide, or polypeptide that is a target gene product (that is, secretion related to the control of secretion of the target gene product in an organism from which the target gene product is derived). Signal peptide) or a secretory signal peptide region of a different protein, peptide or polypeptide. Preferably, the secretory signal peptide region is a polynucleotide encoding the original secretory signal peptide of the protein, peptide, or polypeptide that is the target gene product.

  Examples of the secretory signal peptide region include a secretory signal peptide region of the alkaline cellulase gene derived from the Bacillus sp. KSM-S237 strain (FERM BP-7875) (a nucleotide sequence represented by nucleotide numbers 573 to 662 of SEQ ID NO: 3). Nucleotide), a secretory signal peptide region of the alkaline cellulase gene derived from Bacillus sp. KSM-64 strain (FERM BP-2886) (a polynucleotide comprising the nucleotide sequence represented by base numbers 610 to 696 of SEQ ID NO: 5), and Bacillus no. Examples include a secretion signal peptide region of the protease E-1 gene derived from the D-6 strain (FERM P-1592) (polynucleotide comprising the nucleotide sequence represented by base numbers 1 to 90 of SEQ ID NO: 17). In the examples described below, the secretory signal peptide regions derived from the Bacillus sp. KSM-S237 or Bacillus No. D-6 are the alkaline cellulase gene derived from the KSM-S237 strain and the protease E-1 gene derived from the D-6 strain, respectively. And secrete the alkaline cellulase and protease. Furthermore, it is known that the secretion signal peptide of alkaline cellulase derived from the KSM-S237 strain and KSM-64 strain functions also for secretion of proteins other than the alkaline cellulase, for example, alkaline amylase derived from Bacillus sp. KSM-K38. (For example, JP 2002-112792 A).

  More preferably, in addition to the above-mentioned secretory signal peptide region, in addition to the above-mentioned secretory signal peptide region, a control region involved in transcription, translation or secretion of the gene is introduced upstream of the gene encoding the target gene product introduced into the mutant Bacillus bacterium of the present invention. That is, at least one region selected from the group consisting of a transcription initiation control region including a promoter and a transcription initiation site and a translation initiation region including a ribosome binding site and an initiation codon is operably linked. More preferably, three regions consisting of the transcription initiation control region, translation initiation control region, and secretory signal peptide region are operably linked upstream of the gene encoding the target gene product. Preferably, the transcription initiation control region and the translation initiation control region include a region corresponding to an upstream region of 0.6 to 1 kb in length starting from the initiation codon of the cellulase gene or protease gene of the genus Bacillus, Examples of the secretory signal peptide region include the secretory signal peptide region derived from the cellulase gene or protease gene of the Bacillus bacterium exemplified above.

  Preferred examples of the three regions comprising the transcription initiation regulatory region, translation initiation region and secretory signal peptide region include cellulase genes derived from KSM-S237 strain (FERM BP-7875) or KSM-64 strain (FERM BP-2886). Examples include a combination of a transcription initiation control region, a translation initiation region, and a secretory signal peptide region. More preferred examples include a polynucleotide comprising the nucleotide sequence represented by base numbers 1 to 662 of SEQ ID NO: 3; a polynucleotide comprising the nucleotide sequence represented by base numbers 1 to 696 of SEQ ID NO: 5; Either, at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 98%, even more preferably at least at least 90% in nucleotide sequence Mention may be made of polynucleotides having 99% identity and retaining functions related to gene transcription, translation and secretion.

  Another preferred example of the three regions comprising the transcription initiation regulatory region, translation initiation region and secretory signal peptide region is cellulase derived from KSM-S237 strain (FERM BP-7875) or KSM-64 strain (FERM BP-2886). Examples include a combination of a transcription initiation control region and a translation initiation region of a gene and a secretion signal peptide region of a protease E-1 gene derived from a Bacillus No. D-6 strain (FERM P-1592). As a more preferred example, a combination of a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 1 to 572 of SEQ ID NO: 3 and a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 1 to 90 of SEQ ID NO: 17; A combination of a polynucleotide consisting of a nucleotide sequence represented by nucleotide numbers 1 to 609 of 5 and a polynucleotide consisting of a nucleotide sequence represented by nucleotide numbers 1 to 90 of SEQ ID NO: 17; and any of the combinations of the above polynucleotides , At least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 98%, even more preferably less in nucleotide sequence Both have a 99% identity, and transcription of the gene, can be exemplified a polynucleotide retains the function related to the translation and secretion.

  In the present specification, “operably linked” means that a gene encoding a target gene product and a control region related to transcription, translation or secretion of the gene are under the control of the control region. It is linked so that a gene can be expressed.

  The mutant Bacillus bacterium of the present invention can be prepared by introducing a vector containing a gene encoding the target gene product (hereinafter also referred to as target gene) into the Bacillus bacterium in which EndoA is inactivated. it can. In a preferred procedure, first, a vector in which the secretory signal peptide region is linked upstream of the target gene is constructed. For example, a DNA fragment in which a secretory signal peptide region is linked upstream of the target gene is inserted into an appropriate plasmid vector. Alternatively, the DNA fragment of the target gene and the DNA fragment of the secretory signal peptide region are inserted into an appropriate plasmid vector, and the secretory signal peptide region is placed upstream of the target gene. Next, the resulting vector is incorporated into the bacterium belonging to the genus Bacillus using a general transformation method, thereby encoding the target gene product in which EndoA is inactivated and the secretion signal peptide region is linked upstream. The bacterium belonging to the genus Bacillus of the present invention into which a vector containing the gene to be introduced is introduced can be produced. Preferably, further, an expression vector is used as the vector, or the gene of interest is efficiently ligated in bacteria by operably linking the target gene and the above-described control region involved in transcription or translation of the gene. It can be expressed well. Procedures for generating expression vectors and introducing vectors into bacteria are well known to those skilled in the art. For example, the target gene DNA can be inserted into the vector by cleaving the vector with a restriction enzyme and adding the target gene DNA constructed so as to have a restriction enzyme cleavage sequence at the end thereof (restriction enzyme method) . Although it does not specifically limit as a vector which can be utilized, pHY300PLK (Takara Bio Inc.) etc. are mentioned as a preferable example. What is necessary is just to introduce | transduce the produced expression vector containing the target gene DNA into a Bacillus genus bacteria by normal methods, such as a competent method.

  Alternatively, the mutant Bacillus bacterium of the present invention can be prepared by directly introducing a gene (target gene) encoding a target gene product into the genome of the Bacillus bacterium in which EndoA is inactivated. In a preferred procedure, first, a secretory signal peptide region is ligated upstream of the target gene, and a DNA fragment to which an appropriate homologous region of the genome of the bacterium belonging to the genus Bacillus is introduced is bound. To introduce. When homologous recombination occurs between the DNA fragment and the Bacillus bacterial genome, EndoA is inactivated and a gene encoding a target gene product with a secretory signal peptide region linked upstream is introduced into the genome. Thus, the mutant Bacillus bacterium of the present invention can be produced. In another preferred procedure, the mutant Bacillus bacterium of the present invention can be produced by incorporating a DNA fragment containing a target gene downstream of an appropriate secretory signal peptide region in the genome of the Bacillus bacterium. Preferably, further, on the DNA fragment containing the target gene, the target gene is operably linked downstream of the control region involved in transcription and translation of the gene described above, and the DNA fragment is incorporated into the bacterial genome. Thus, the target gene can be efficiently expressed in bacteria. Alternatively, DNA containing an appropriate region on the bacterial genome and the target gene so that the target gene in the DNA fragment integrated into the bacterial genome is operably linked downstream of the control region involved in transcription or translation on the genome. By recombination with the fragments, the target gene can be efficiently expressed in bacteria. The procedure for homologous recombination for introducing a DNA fragment containing the target gene is the same as the procedure for homologous recombination for gene deletion described above.

  Alternatively, the mutant Bacillus bacterium of the present invention can be prepared by inactivating EndoA in a Bacillus bacterium into which a gene encoding the target gene product has been introduced. However, the mutant Bacillus genus bacterium of the present invention can be produced more simply by the method of introducing a vector encoding the target gene product into the Bacillus bacterium in which EndoA is inactivated.

  By the above procedure, the mutant Bacillus bacterium of the present invention into which EndoA is inactivated and a gene encoding the target gene product is introduced can be produced. When the Bacillus bacterium is cultured, the target gene product encoded by the introduced gene is highly expressed, so that the target gene product can be efficiently secreted and produced.

  Production of the target gene product according to the present invention involves inoculating the mutant Bacillus genus bacterium of the present invention into a medium containing an assimilable carbon source, a nitrogen source, and other essential components, and cultivating it by a normal microbial culture method. The target gene product can be recovered from the culture. The composition of the medium used for the culture and the culture conditions can be appropriately selected by those skilled in the art according to the type of microorganism used, the type of target gene product, and the like. Usually, it is preferable to culture bacteria under aerobic conditions such as shaking culture by liquid culture and aeration and agitation culture.

  The target gene product may be extracted from the cells after completion of the culture, but the target gene product is produced and secreted by a mutant Bacillus bacterium having the ability to secrete the gene product out of the cell. It is preferable to recover the target gene product secreted therein. In a preferred embodiment, the target gene product is expressed in the mutant bacterium of the genus Bacillus of the present invention, and then secreted outside the cells by the action of a secretory signal peptide. Therefore, the target gene product can be easily recovered without destroying the fungus body, and the fungus body can be reused after the target gene product is recovered.

  In recovering the target gene product from the cells or culture, the target gene product may be extracted or separated from the supernatant or cells obtained by centrifuging the culture according to a conventional method. If necessary, it is preferable to further purify the separated target gene product by appropriately combining ammonium sulfate precipitation or chromatography.

  The present invention also includes the following compositions, manufacturing methods, uses or methods as exemplary embodiments. However, the present invention is not limited to these embodiments.

<1> EndA is inactivated and a vector containing a gene encoding the target gene product is introduced, or a gene encoding the target gene product is introduced into the genome and encodes the target gene product A mutant Bacillus bacterium, wherein a secretory signal peptide region is linked upstream of the gene to be

<2> The mutant Bacillus bacterium according to <1>, wherein the inactivation of EndA is preferably a deletion or inactivation of a gene encoding EndoA.

<3> The mutant Bacillus bacterium according to <2>, wherein the gene encoding EndoA is preferably an ndoA gene.

<4> The ndoA gene is preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 80% of the nucleotide sequence represented by SEQ ID NO: 1. 98%, still more preferably at least 98%, more preferably at least 99% of the nucleotide sequence having identity and preferably with the amino acid sequence shown in SEQ ID NO: 2 at least 80%, more preferably at least 85% More preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, even more preferably at least 98%, more preferably at least 99% amino acid sequences comprising rRNA degradation activity A gene encoding a polypeptide which is an endoribonuclease having, <3>, wherein the mutant Bacillus bacterium.

<5> Preferably, the mutant Bacillus bacterium according to any one of <1> to <4>, wherein the secretory signal peptide region is derived from a cellulase gene or a protease gene of a Bacillus bacterium.

<6> Preferably, the secretory signal peptide region is an alkaline cellulase secretory signal peptide region derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) or a protease E derived from Bacillus No. D-6 (FERM P-1592) The mutant Bacillus bacterium according to any one of <1> to <5>, which is a -1 secretion signal peptide region.

<7> The mutant Bacillus bacterium according to any one of <1> to <6>, wherein the Bacillus bacterium is preferably Bacillus subtilis.

<8> Preferably, the mutant Bacillus bacterium according to any one of <1> to <7>, wherein a gene encoding the target gene product is incorporated in a vector.

<9> The target gene product is preferably a protein, peptide or polypeptide, more preferably a heterologous protein, peptide or polypeptide, <1> to <8> the mutant Bacillus according to any one of <1> Genus bacteria.

<10> The mutant Bacillus bacterium according to any one of <1> to <9>, wherein the target gene product is preferably a glycoside hydrolase or a protease.

<11> Preferably, the glycoside hydrolase is derived from an alkaline cellulase (SEQ ID NO: 4) derived from Bacillus genus KSM-S237 (FERM BP-7875); from Bacillus genus KSM-64 (FERM BP-2886). Alkaline cellulase (SEQ ID NO: 6); Alkaline cellulase derived from Bacillus genus KSM-N257 strain (FERM P-17473) (JP 2002-85078, GenBank accession no. AB059267); Cellulolytic xylanase derived from Bacillus circulans (GenBank paenibacillus sp. Y412MC10-derived lichenase (GenBank accession no. YP_003240630); Paenibacillus sp. HGF5-derived glycosyl hydrolase family 8 (GenBank accession no. ZP_08283397); Paenibacillus vortex V453-derived lichenase (GenBank accession no. AAP22946); ZP_07897964); Paenibacillus lactis 154-derived lichenase (GenBank accession no. ZP_09002324); or the amino acids of these glycoside hydrolases Glycoside hydrous consisting of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, still more preferably 99% or more identity with the nonacid sequence The mutant Bacillus bacterium according to <10>, which is a lyase.

<12> Preferably, the protease is an alkaline protease derived from Bacillus clausii KSM K-16 strain (FERM BP-3376) (JP-A-5-308967, GenBank accession no. YP_174261); KSM-KP43 strain (FERM BP-6532) -derived alkaline protease (GenBank accession no. AB051423); Protease KP9860 [Bacillus sp. KSM-KP9860 (FERM BP-6534) -derived, WO99 / 18218, GenBank accession no. AB046403]; Protease E-1 [Derived from Bacillus No. D-6 (FERM P-1592), JP-A-49-71191, GenBank accession no. AB046402]; Protease Ya [Derived from Bacillus SP Y (FERM BP-1029), JP Sho 61-280268, GenBank accession no. AB046404]; Protease SD521 [Derived from Bacillus SD521 (FERM P-11162), JP-A-3-191781, GenBank accession no. AB046405]; Protease A-1 [NCIB12289 Origin, WO88 / 01293, GenBank accession no. AB046406]; Protease A-2 [derived from NCIB12513, WO98 / 56927]; Protease 9865 [Derived from Bacillus SP KSM-9865 (FERM P-18566), GenBank accession no. AB084155]; or An amino acid sequence having an identity of preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more, with the amino acid sequence of these proteases The mutant Bacillus bacterium according to <10>, which is a protease comprising

<13> A method for producing a target gene product, comprising culturing the mutant Bacillus bacterium according to any one of <1> to <12>.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

  Table 1 shows the names, base sequences, and sequence numbers of the PCR primers used in this example. The underlined portion in the base sequence shown in Table 1 indicates a restriction enzyme recognition sequence.

Production Example 1 Production of modified Bacillus subtilis lacking the ndoA gene (1) Production of gene fragment for gene deletion Using the genomic DNA extracted from Bacillus subtilis 168 strain as a template, NDOA-F1 (SEQ ID NO: 7) shown in Table 1 And the upstream region of the ndoA gene on the genome was amplified by PCR using the primer set of NDOA-R1 (SEQ ID NO: 8) (fragment (A)). Further, using the genomic DNA as a template, the downstream region of the ndoA gene in the genome was amplified by PCR using the primer set of NDOA-F2 (SEQ ID NO: 9) and NDOA-R2 (SEQ ID NO: 10) (fragment (B )). The DNA of plasmid pDG1727 (Guerout-Fleury et al., Gene., 1995, 167: 335-336; available from Bacillus Genetic Stock Center [http://www.bgsc.org/]) is used as a template, and Using the SPC-F (SEQ ID NO: 11) and SPC-R (SEQ ID NO: 12) primer sets shown in 1, a fragment (C) containing a spectinomycin resistance gene (Spc) was prepared.

  Next, the obtained DNA fragments (A), (B) and (C) were mixed to form a template, and SOE using the primer sets of NDOA-F1 (SEQ ID NO: 7) and NDOA-R2 (SEQ ID NO: 10). -The 3 fragments were combined by PCR in the order of (A)-(C)-(B) to prepare a DNA fragment for gene deletion (D).

(2) Production of competent cells Bacillus subtilis 168 strain was treated with CI medium (0.20% by mass ammonium sulfate, 1.40% by mass dipotassium hydrogen phosphate, 0.60% by mass potassium dihydrogen phosphate, 0.10% by mass). Trisodium citrate dihydrate, 0.50% by mass glucose, 0.03% by mass Amicase (manufactured by SIGMA), 5 mM magnesium chloride, 50 μg / mL tryptophan) has a growth degree (OD600) value at 37 ° C. The culture was shaken until it reached about 1. After shaking culture, a part (0.5 mL) of the culture solution is collected, and only the cells are collected by centrifugation, and then 1 mL of CII medium (0.20 mass% ammonium sulfate, 1.40 mass% hydrogen phosphate). Dipotassium, 0.60 mass% potassium dihydrogen phosphate, 0.10 mass% trisodium citrate dihydrate, 0.50 mass% glucose, 0.015 mass% Amicase (manufactured by SIGMA), 5 mM magnesium chloride A competent cell suspension of Bacillus subtilis strain was prepared by resuspending the cells in 5 μg / mL tryptophan).

(3) Transformation of Bacillus subtilis 168 strain To 100 μL of the competent cell suspension prepared in (2) above, the final concentration of the DNA fragment (D) obtained in (1) above is 15 μg / mL or more. After addition and shaking culture at 37 ° C. for 90 minutes, 100 μL of LB liquid medium (1% by mass tryptone, 0.50% by mass yeast extract, 0.50% by mass NaCl) was added, followed by incubation at 37 ° C. for 1 hour. Thereafter, 10 μL or 100 μL of the culture solution was added to LB agar medium (1% by mass tryptone, 0.50% by mass yeast extract, 0.50% by mass NaCl, 1.5% by mass agar) containing spectinomycin (100 μg / mL). Smeared. After stationary culture at 37 ° C., the grown colonies were isolated as transformants. Genomic DNA of the obtained transformant was extracted, and PCR and sequencing were performed using this as a template, and it was confirmed that the target transformant was obtained by replacing the ndoA gene with a spectinomycin resistance gene. The obtained transformant was obtained as a Bacillus subtilis mutant lacking the ndoA gene (RIK1910 strain) and used in the following procedure.

Production Example 2 Construction of Cellulase Gene Introduction Vector Using a genomic DNA prepared from Bacillus sp. KSM-S237 strain (FERM BP-7875) as a template, a primer set of 237UB1 primer (SEQ ID NO: 13) and 237DB1 primer (SEQ ID NO: 14) A fragment of the alkaline cellulase gene (see JP 2000-210081, SEQ ID NO: 3) containing the upstream promoter region and secretory signal peptide region derived from the KSM-S237 strain was amplified by polymerase chain reaction (PCR). The amplified DNA fragment was treated with BamHI restriction enzyme, and the fragment was inserted into the BamHI restriction enzyme cleavage point of shuttle vector pHY300PLK to prepare recombinant plasmid pHY-S237. On this plasmid, the promoter region and secretory signal peptide region of KSM-S237 are linked upstream of the gene encoding KSM-S237 alkaline cellulase.

Production Example 3 Transformation of Bacillus subtilis by introducing cellulase gene introduction vector Bacillus subtilis 168 strain (wild type) and Bacillus subtilis mutant strain (RIK1910 strain) produced in Production Example 1 were produced in Production Example 2. The recombinant plasmid pHY-S237 was introduced by a protoplast transformation method (Mol. Gen. Genet., 1979, 168: 111-115) to obtain a transformant.

Example 1 Cellulase Productivity Evaluation The transformant obtained in Production Example 3 was used in 5 mL of LB medium (1% by mass tryptone, 0.5% by mass yeast extract, 1% by mass NaCl, 15 ppm tetracycline) at 30 ° C. overnight. Incubated with shaking. 0.4 mL of this culture solution was each 2 × L-maltose medium (2% by mass tryptone, 1% by mass yeast extract, 1% by mass NaCl, 7.5% by mass maltose, 7.5 ppm manganese sulfate 4-5 hydrate) 20 mL was inoculated and cultured with shaking at 30 ° C. for 3 days (N = 3).

  After culturing, the cellulase activity of the culture supernatant from which the cells were removed by centrifugation was measured by the following procedure, and the amount of cellulase secreted and produced outside the cells was determined. 50 μL of 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation) was added to 50 μL of the culture supernatant sample appropriately diluted with 1 / 7.5 M phosphate buffer (pH 7.4, Wako Pure Chemical Industries). The mixture was mixed and reacted at 30 ° C., and the amount of p-nitrophenol liberated by the reaction was measured as the absorbance at 420 nm (OD 420 nm). The amount of cellulase in the sample was quantified based on the change in absorbance. The enzyme activity that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U, and the cellulase production amount was calculated from the specific activity. The results are shown in Table 2. The cellulase production amount in Table 2 represents a relative value with respect to the production amount of Bacillus subtilis 168 strain.

  As is clear from the results in Table 2, cellulase productivity was improved in the ndoA gene-deficient strain (RIK1910 strain) compared to the control 168 strain (wild type). From these results, it was found that Bacillus subtilis in which the gene encoding EndoA was deleted or inactivated has high protein productivity and is suitable for production of useful substances.

Production Example 4 Construction of Protease Gene Introduction Vector (1) Construction of Plasmid pHY-SP64 Primer SP64-F (EcoRI) (SEQ ID NO :) using genomic DNA prepared from Bacillus sp. KSM-64 strain (FERM P-10482) as a template 15) and a primer set of primer SP64-R (BamHI) (SEQ ID NO: 16), and the Endo-1,4-beta-glucanase gene (Genbank) containing the upstream promoter region derived from the Bacillus sp. KSM-64 strain A DNA fragment of ACCESSION No. M84963, SEQ ID NO: 5) was prepared. An appropriate amount of this DNA fragment and pHY300PLK (manufactured by Takara Bio Inc.) were mixed, and after double digestion with restriction enzymes EcoRI and BamHI, ligation was performed and the plasmid was purified by ethanol precipitation. This was introduced into Bacillus sp. KSM-9865 strain (FERM P-18566) by electroporation and transformed, and skim milk-containing alkaline LB agar medium (1% by mass bactotryptone, 0.5% by mass yeast extract, 1 mass% sodium chloride, 1 mass% skim milk, 1.5 mass% agar, 0.05 mass% sodium carbonate, and 15 ppm tetracycline). Several days later, colonies generated on the agar medium were isolated as transformants, and the plasmid was extracted. The promoter sequence inserted in the multicloning site was analyzed to confirm that no unintended mutation due to PCR error was introduced, and this was designated as a recombinant plasmid pHY-SP64.

(2) Construction of plasmid pHY-SP64-E-1 The base sequence of the gene encoding protease E-1 (Bacillus No. D-6 (FERM P-1592) (Japanese Patent Publication No. 56-4236, GenBank accession no. DNA containing AB046402, SEQ ID NO: 17 (having a Bam HI site at the 5 ′ end upstream of the gene and an Xba I site at the 3 ′ end downstream of the gene) was co-digested with Bam HI and Xba I. Endo-1,4-beta-glucanase gene by ligation reaction using Ligation High (Toyobo Co., Ltd.) after mixing with the plasmid pHY-SP64 constructed in (1) above and digested with restriction enzymes BamHI and XbaI. A plasmid containing the gene encoding protease E-1 was constructed downstream, and the ligation product was purified by ethanol precipitation and introduced into the Bacillus sp. KSM-9865 strain (FERM P-18566) by electroporation. Then transform and skim A milk-containing alkaline LB agar medium was smeared, and colonies that appeared on the agar medium a few days later were selected for transformants into which a protease gene had been introduced based on the presence or absence of skim milk melting spots, and plasmid DNA was extracted from these transformants. It was confirmed that the gene encoding protease E-1 was correctly inserted, and the resulting plasmid was designated as pHY-SP64-E-1, on which the gene encoding protease E-1 was identified. The secretion signal peptide region of protease E-1 is linked upstream.

Production Example 5 Transformation of Bacillus subtilis by introducing a vector for introducing a protease gene The recombinant plasmid pHY produced in Production Example 4 was added to each of the Bacillus subtilis 168 strain and the Bacillus subtilis mutant strain produced in Production Example 1 (RIK1910 strain). -SP64-E-1 was introduced by a protoplast transformation method (Mol. Gen. Genet., 1979, 168: 111-115) to obtain a transformant.

Example 2 Evaluation of Alkaline Protease Productivity The transformant obtained in Production Example 5 was treated with 5 mL of LB medium (1% by mass tryptone, 0.5% by mass yeast extract, 1% by mass NaCl, 15 ppm tetracycline) overnight 30 Cultured with shaking at 0 ° C. 0.4 mL of this culture solution was each 2 × L-maltose medium (2% by mass tryptone, 1% by mass yeast extract, 1% by mass NaCl, 7.5% by mass maltose, 7.5 ppm manganese sulfate 4-5 hydrate) 20 mL was inoculated and cultured with shaking at 30 ° C. for 3 days (N = 3).

  After the culture, the alkaline protease activity of the culture supernatant excluding the bacterial cells was measured by the following procedure, and the amount of alkaline protease secreted and produced outside the bacterial cells was determined. Take 0.9 ml of 1/15 M phosphate buffer (pH 7.4), 0.05 ml of 40 mM Glt-Ala-Ala-Pro-Leu-p-nitroanilide / dimethylsulfoxide solution in a test tube, and incubate at 30 ° C. for 5 minutes. did. To this was added 0.05 ml of enzyme solution (culture supernatant) and reacted at 30 ° C. for 10 minutes. Then, 2.0 ml of 5% (w / v) aqueous citric acid solution was added to stop the reaction, and the spectrophotometer Was used to measure the absorbance at 420 nm. Based on the change in absorbance, the amount of alkaline protease in the sample was quantified. The enzyme activity for producing 1 μmol of p-nitroaniline per minute was defined as 1 U, and the amount of alkaline protease produced was calculated from the specific activity. The results are shown in Table 3. The production amount of alkaline protease in Table 3 represents a relative value with respect to the production amount of Bacillus subtilis 168 strain.

  As is clear from the results in Table 3, the productivity of alkaline protease was improved in the ndoA gene-deficient strain (RIK1910 strain) compared to the control 168 strain (wild type). This result shows that Bacillus subtilis in which the gene encoding EndoA has been deleted or inactivated is suitable for the production of various types of proteins.

Claims (7)

  A vector comprising a gene in which EndA is inactivated and a heterologous protein, peptide or polypeptide is introduced, or a gene encoding a heterologous protein, peptide or polypeptide is introduced into the genome, A mutant Bacillus bacterium, wherein a secretory signal peptide region is linked upstream of a gene encoding a heterologous protein, peptide or polypeptide.   The mutant Bacillus bacterium according to claim 1, wherein the inactivation of EndA is deletion or inactivation of a gene encoding EndoA.   The mutant Bacillus bacterium according to claim 2, wherein the gene encoding EndoA is an ndoA gene.   The ndoA gene is composed of a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and is composed of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2. The mutant Bacillus bacterium according to claim 3, which is a gene encoding a polypeptide which is an endoribonuclease having degrading activity.   The mutant Bacillus bacterium according to any one of claims 1 to 4, wherein the Bacillus bacterium is Bacillus subtilis.   The mutant Bacillus bacterium according to any one of claims 1 to 5, wherein the heterologous protein, peptide or polypeptide is a glycoside hydrolase or a protease.   A method for producing a protein, peptide, or polypeptide, comprising culturing the mutant Bacillus bacterium according to any one of claims 1 to 6.