JP2017093321A - Bacillus subtilis mutants and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Bacillus subtilis mutant strains capable of high yield production of PGA and technology for producing PGA using the same.SOLUTION: The invention provides a mutant strain of Bacillus subtilis characterized in that one or more regions selected from the group consisting of 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, prophage7 region, (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region is deleted, abrB gene or a corresponding gene is constructed to be expressed constantly, kinA gene is deleted or inactivated, and at least one of alsS gene and alsD gene is deleted or inactivated.SELECTED DRAWING: None

Description

  The present invention relates to a novel Bacillus subtilis mutant and use.

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

  Poly-γ-glutamic acid (hereinafter also referred to as “PGA”) is a high molecular compound in which a carboxyl group at the γ-position and an amino group at the α-position of glutamic acid are bonded by a peptide bond, and Bacillus subtilis var. Natto), Bacillus subtilis var. Some bacteria belonging to the genus Bacillus such as chungkookjang, Bacillus licheniformis, Bacillus megaterium, Bacillus anthracis, Bacillus halodurans, etc., and are known to produce Natalba aehypti. In recent years, PGA has attracted attention as a useful polymer material due to various properties such as water retention, hydrophilicity, thickening, biodegradability and the like, and improvement of its productivity has become one of the important issues.

  In recent years, with respect to Bacillus subtilis, about 4100 kinds of disrupted strains of genes present in the Bacillus subtilis genome have been comprehensively studied, and it has been clarified that 271 genes are essential for growth (Non-Patent Documents). 1) Various mutant strains in which genes unnecessary for growth have been deleted have been created. Moreover, mutant strains excellent in productivity of various enzymes have been found by comprehensive analysis of mutant strains in which large genomic regions or genes have been deleted. MGB874 strain, which is one of the Bacillus subtilis mutants obtained in this study, is Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) derived from a mutant strain with a total of 874 kb deleted, but it is reported that the secretory productivity of the protein is significantly improved compared to the wild-type 168 strain (Patent Document 1). Recently, a method for efficiently producing the above PGA using a Bacillus subtilis strain obtained by deleting a large region of the genome has been reported (Patent Document 2).

On the other hand, Bacillus subtilis AbrB plays an important role in the control of the expression of various genes involved in spore formation, competence, or nutrient acquisition in the transitional phase and stationary phase during the transition from the logarithmic growth phase to the stationary phase. A transcription factor that fulfills (Non-Patent Documents 2 and 3), for example, a skf operon (skfA-H) containing a skf gene encoding a killing factor as a gene under the control of AbrB is negatively regulated Has been reported (Non-Patent Document 4). Moreover, it has been reported that the productivity of amylase and protease is improved in a Bacillus subtilis strain in which the abrB gene is deleted (Patent Document 3).
However, no Bacillus subtilis mutant strain in which AbrB is constantly expressed has been reported.

On the other hand, Bacillus subtilis KinA exists in the cytoplasm, and is a multi-component regulatory histidine kinase involved in the early stage of sporulation. It is said that phosphorylation is transferred to Spo0F through its own phosphorylation to generate Spo0F to P. ing. In the KinA mutant, the spore-forming ability is reduced to 1 to 30% compared to the wild type (Non-patent Document 5). Moreover, it has been recognized that alkaline cellulase secretion productivity is improved in the KinA mutant (Patent Document 4).
Bacillus subtilis AlsS and AlsD are acetolactate synthase and acetolactate decarboxylase, respectively, and both are known as enzyme groups involved in acetoin synthesis.
However, there is no known report that examines how KinA, AlsS, and AlsD affect PGA production in Bacillus subtilis.

JP 2007-130013 A JP 2013-252115 A JP 2007-74934 A JP 2006-296268 A

K. Kobayashi et al., Proc. Natl. Acad. Sci. USA., 100, 4678-4683, 2003 M. A. Strauch, et al., Mol. Microbiol., 1993, 7: 337-342 Z. E. V. Phillips, et al., Cell Mol. Life Sci., 2002, 59: 392-402 M. Fujita, et al., J. Bacteriol., 2005, 187: 1357-1368 Japanese Bacteriological Journal, 1996, 51: 789-801

  The present invention relates to providing a mutant strain of Bacillus subtilis capable of producing PGA at a high yield and a PGA production technique using the same.

  The present inventor has advanced development of a microorganism strain capable of efficiently producing PGA. As a result, Bacillus subtilis Marburg No. 168 (B. subtilis strain 168) is a Bacillus subtilis transcription factor that suppresses genes specifically expressed in the transitional phase and stationary phase in a Bacillus subtilis mutant strain lacking a large region of the 168 genome. In addition to the constant expression of AbrB, the Bacillus subtilis mutant strain that has been deleted or inactivated in combination with the kinA gene and the acetoin synthesis gene group has an excellent PGA production ability. We found that it can be manufactured efficiently.

That is, the present invention relates to the following (1) to (5).
(1) a Bacillus subtilis mutant,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region Having one or more selected regions of the deleted genome;
The gene is constructed so that the abrB gene or a gene equivalent thereto is constitutively expressed, the kinA gene is deleted or inactivated, and at least one gene selected from the group consisting of the alsS gene and the alsD gene is missing. Bacillus subtilis mutant strain that has been inactivated or inactivated.
(2) In the Bacillus subtilis mutant strain of (1), a poly-γ-glutamate synthase gene, or a poly-γ-glutamate synthase gene and a poly-γ-glutamate degrading enzyme gene are enhanced or introduced so that they can be expressed. Bacillus subtilis.
(3) A method for producing a Bacillus subtilis mutant,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region In a Bacillus subtilis mutant strain having a genome in which one or more selected regions are deleted, the abrB gene or a gene equivalent thereto is constitutively expressed. A method comprising a step of constructing a gene, a step of deleting or inactivating a kinA gene, and a step of deleting or inactivating at least one gene selected from the group consisting of an alsS gene and an alsD gene.
(4) A method for producing recombinant Bacillus subtilis,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region In a Bacillus subtilis mutant strain having a genome in which one or more selected regions are deleted, the abrB gene or a gene equivalent thereto is constitutively expressed. A step of constructing a gene, a step of deleting or inactivating the kinA gene, a step of deleting or inactivating at least one gene selected from the group consisting of an alsS gene and an alsD gene, and poly-γ-glutamic acid synthesis A method comprising the step of enhancing or introducing an enzyme gene, or a poly-γ-glutamate synthase gene and a poly-γ-glutamate degrading enzyme gene in an expressible manner.
(5) A method for producing poly-γ-glutamic acid, comprising culturing the recombinant Bacillus subtilis of (2) and recovering poly-γ-glutamic acid from the culture.

  According to the present invention, a Bacillus subtilis mutant strain having high PGA production ability is provided. Efficient production of PGA can be realized by using the Bacillus subtilis mutant strain of the present invention.

The schematic diagram which shows an example of the method of deleting a predetermined area | region from the genome of Bacillus subtilis. The schematic diagram which shows the procedure which removes a foreign drug resistance gene from a Bacillus subtilis genome. The schematic diagram which shows the procedure of the marker free deletion method using a mazF gene fusion cassette. The figure of β-galactosidase activity measurement.

  The name of each gene and genomic region of Bacillus subtilis described in this specification is publicly available on the Internet ([bacillus.genome.ad.jp/], January 2006 in Japan Functional Analysis Network for Bacillus subtilis (BSORF DB)). It is described based on Bacillus subtilis genome data updated on the 18th).

  In this specification, the identity of an amino acid sequence and a nucleotide sequence is calculated by the Lipman-Pearson method (Lipman, DJ., Pearson. WR .: Science, 1985, 227: 1435-1441). Specifically, genetic information processing software Genetyx-Win Ver. 11 (software development) using the homology analysis (Search homology) program, the unit size to compare (ktup) is set to 2 and calculated.

  In this specification, unless otherwise defined, “one or several” used for amino acid or nucleotide deletion, substitution, addition or insertion in an amino acid sequence or nucleotide sequence means, for example, 1 to 18 amino acid sequences. , Preferably 1-9, more preferably 1-4, still more preferably 1-2, nucleotide sequence 1-56, preferably 1-28, more preferably 1-14, Preferably it may be 1-7. In the present specification, “addition” of amino acids or nucleotides includes addition of one or several amino acids or nucleotides to one and both ends of the sequence.

  In the present specification, unless otherwise defined, “stringent conditions” for hybridization are conditions that allow identification of a gene having a nucleotide sequence having a sequence identity of about 80% or more, preferably about 90% or more. It is. “Stringent conditions” include those described in Molecular Cloning-A Laboratory Manual THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001). Those skilled in the art of hybridization can appropriately create stringent conditions by adjusting the salt concentration, temperature, etc. of the hybridization solution according to the nucleotide sequence, concentration, length, etc. of the probe. For example, the “stringent condition” is preferably 5 × SSC, 70 ° C. or higher, more preferably 5 × SSC, 85 ° C. or higher, or 6 × SSC, 60 ° C. as hybridization conditions. The above is preferable, 6 × SSC, 65 ° C. or more is more preferable, and the cleaning condition is preferably 1 × SSC, 60 ° C. or more, and more preferably 1 × SSC, 73 ° C. or more. The combination of the SSC and the temperature condition is merely an example, and those skilled in the art can realize appropriate stringency by appropriately combining the above or other factors that determine the stringency of hybridization.

  In the present specification, upstream and downstream of a gene or region indicate a region continuing on the 5 'side and 3' side of the gene or region regarded as a target, respectively. Unless otherwise defined, upstream and downstream of a gene are not limited to the upstream region from the translation start point or transcription start point of the gene and the downstream region from the stop codon.

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

In this specification, “operably linking” a target gene and a control region means that the control region and the target gene are combined on DNA so that the function of controlling gene expression by the control region acts on the target gene. It means arranging. Examples of the method of operably linking the target gene and the control region include a method of linking the target gene downstream of the control region.
Further, in this specification, a Bacillus subtilis mutant strain into which a gene related to PGA synthesis or a gene related to PGA degradation, which is a target gene, is referred to as recombinant Bacillus subtilis, and the target gene is not expressed, but the target gene is expressed. Bacillus subtilis improved as a host with excellent ability is called Bacillus subtilis mutant.

In the present specification, “transcription factor” (“transcription control factor”) means a protein that binds to a transcription initiation control region on DNA or the like and promotes or suppresses the transcription process from DNA to RNA.
In the present specification, the term “transition phase” means the transition from the logarithmic growth phase or the exponential growth phase to the stationary phase or the stationary phase in the growth of bacteria.

[1. Construction of Bacillus subtilis mutants]
The Bacillus subtilis mutant strain of the present invention has a genome in which a large region on the genome of the Bacillus subtilis wild strain is deleted, and is constructed so that the abrB gene or a gene corresponding thereto is constantly expressed, and A Bacillus subtilis mutant strain in which the kinA gene is deleted or inactivated, and at least one gene selected from the group consisting of the alsS gene and the alsD gene is deleted or inactivated.
The Bacillus subtilis mutant strain of the present invention has a genetic modification that constitutively expresses the abrB gene or a gene corresponding to the Bacillus subtilis mutant strain in which a large region of the genome is deleted, and a kinA gene deleted or not. It can be produced by adding a modification that activates and a modification that deletes or inactivates at least one gene selected from the group consisting of the alsS gene and the alsD gene.

[1-1. Bacillus subtilis mutant lacking genomic region]
A Bacillus subtilis mutant lacking a large genomic region, which is a parent strain of the Bacillus subtilis mutant of the present invention, is a wild strain of Bacillus subtilis (for example, Bacillus subtilis Marburg No. 168; hereinafter, 168 strains of Bacillus subtilis). Compared to the genome of 168 strain or wild strain), a large region in the genome is deleted. That is, in the genome of the Bacillus subtilis mutant strain, in the genome of the Bacillus subtilis wild strain, the profile6 (yoaV-yobO) region, the profile1 (ybbU-ybdE) region, the profile4 (yjcM-yjdJ) region, PBSX (ykdA-xlyA) ) Region, profile5 (ynxB-dut) region, profile3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region , Profile2 (ydcL-ydeJ) region, ydcL-ydeK-ydhU region, yzB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeK-yesX region, pd One or more of the regions selected from the group consisting of -rocR region, ycxB-sipU region, profile7 (yrkM-yraK or yrkS-yraK) region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region are deleted. doing.
“Region deletion” as used herein refers to deletion of a region sandwiched by the above-described gene at both ends, including the genes at both ends.

  Preferably, the Bacillus subtilis mutant is a prophage6 (yoaV-yobO) region, a prophage1 (ybbU-ybdE) region, a prophage4 (yjcM-yjdJ) region, or a PBSX (ykdA-xlyA) region in the genome of a Bacillus subtilis wild strain. , Profile5 (ynxB-dut) region, profile3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA2) region (YdcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-locR region Bacillus subtilis MGB874 strain lacking all of the region selected from the group consisting of the YcxB-sipU region, the profile7 (yrkS-yraK) region, the sbo-ywhH region, the yybP-yyaJ region and the yncM-fosB region (specially No. 2007-130013), and in the genome of Bacillus subtilis wild strain, the profile6 (yoaV-yobO) region, the profile1 (ybbU-ybdE) region, the profile4 (yjcM-yjdJ) region, the PBSX (ykdA-xlyA) region, profile5 (ynxB-dut) region, profile3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps ( PSE-ppsA) region, prophage2 (ydcL-ydeJ) region, prophage7 (yrkM-yraK) OA105 strain lacking all region selected from the group consisting of area and the like.

The OA105 strain includes a profile6 (yoaV-yobO) region, a profile1 (ybbU-ybdE) region, a profile4 (yjcM-yjdJ) region, a PBSX (ykdA-xlyA) region, and a property5 (described in JP2007-130013A). ynxB-dut) region, profile3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region, profile2 (ydcL) ydeJ) is prepared by further deleting the profile7 (yrkM-yraK) region with respect to the strain lacking the region (MGB469 strain).
The skin (spoIVCB-spoIIIC) region and the profile7 (yrkS-yraK) region, which are deletion regions of the MGB874 strain described in the present specification, are adjacent regions and are described in JP-A-2007-130013. This is the same region as the SKIN-Pro7 (spoIVCB-yraK) region.
The MGB469 strain and MGB874 strain can be sold by the National Institute of Genetics NBRP (National BioResource Project) (http://www.shigen.nig.ac.jp/bsub/kaoListAction.do).

  A Bacillus subtilis mutant strain in which the large region of the genome is deleted can be prepared by deleting the above-described genomic region from any Bacillus subtilis strain such as 168 strain, for example. The target genomic region to be deleted can be determined, for example, by comparing the genome of any Bacillus subtilis strain with the published genome of Bacillus subtilis 168 strain. The complete nucleotide sequence and genome information of Bacillus subtilis 168 strain can be obtained from the above-mentioned BSORF DB or GenBank: AL009126.2 ([www.ncbi.nlm.nih.gov/nuccore/38680335]). Those skilled in the art can determine the target genomic region to be deleted based on the genomic information of Bacillus subtilis 168 obtained from these information sources. Here, the genomic region to be deleted is one or several (for example, 1 to 100, preferably 1 to 50) of the nucleotide sequence of the aforementioned genomic region in the publicly available Bacillus subtilis 168 strain. More preferably 1 to 30, more preferably 1 to 10, and even more preferably 1 to 5 nucleotides including natural or artificially induced deletions, substitutions, insertions, additions, etc. It can be a genomic region having a sequence. Alternatively, the genomic region to be deleted is preferably 80% or more identical, even more preferably 90% or more identical, and still more preferably 95%, in the nucleotide sequence with respect to the aforementioned genomic region in the publicly available Bacillus subtilis 168 strain. % Or more of the genome region.

  Alternatively, the genomic region to be deleted can be expressed as a region sandwiched between a pair of oligonucleotide sets shown in Table 1. The method for deleting the region described in Table 1 from the Bacillus subtilis genome is not particularly limited. For example, the region is prepared by SOE-PCR (splicing by overlap extension PCR: Gene, 1989, 77: 61-68) or the like. A double crossover method using the deleted DNA fragment. The procedure for preparing a mutant strain in which a predetermined genomic region has been deleted from a Bacillus subtilis wild strain by this method is described in detail in JP-A-2007-130013.

  FIG. 1 shows an outline of procedures for preparing a deletion DNA fragment by the SOE-PCR method and deleting the target region by the double crossover method using the deletion DNA fragment. First, by the SOE-PCR method, a fragment corresponding to an about 0.1 to 3 kb region adjacent to the upstream of the target region to be deleted (referred to as an upstream fragment) and an about 0.1 to 3 kb adjacent to the downstream in the same manner A DNA fragment in which fragments corresponding to regions (referred to as downstream fragments) are linked is prepared. Preferably, in order to confirm the deletion of the target region, a DNA fragment in which a marker gene fragment such as a drug resistance gene is further linked between the upstream fragment and the downstream fragment is prepared.

  First, by the first PCR, an upstream fragment A and a downstream fragment B of the region to be deleted and, if necessary, three fragments of a marker gene fragment (in FIG. 1, chloramphenicol resistance gene fragment Cm) are prepared. To do. In PCR amplification of the upstream fragment and the downstream fragment, a primer added with a sequence of terminal 10 to 30 nucleotides of the fragment to be ligated later is used. For example, when linking the upstream fragment A, the drug resistance marker gene fragment Cm, and the downstream fragment B in this order, the upstream side of the drug resistance marker gene fragment Cm is attached to the 5 ′ end of the primer that binds to the downstream end of the upstream fragment A. A sequence corresponding to 10 to 30 nucleotides is added (FIG. 1, primer DR1), and at the 5 ′ end of the primer that binds to the upstream end of the downstream fragment B, to the downstream 10 to 30 nucleotides of the drug resistance marker gene fragment Cm. The corresponding sequence is added (FIG. 1, primer DF2). When the upstream fragment A and the downstream fragment B are amplified by PCR using the primer set designed in this way, a region corresponding to the upstream side of the drug resistance marker gene fragment Cm is present on the downstream side of the amplified upstream fragment A ′. Thus, a region corresponding to the downstream side of the drug resistance marker gene fragment Cm is added to the upstream side of the amplified downstream fragment B ′.

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

  A deletion DNA fragment obtained by the above-described method or the like is inserted into a plasmid using an ordinary restriction enzyme and DNA ligase to construct a deletion introduction plasmid. Alternatively, after preparing a deletion DNA fragment in which the upstream fragment and the downstream fragment are directly linked, the deletion DNA fragment is inserted into a plasmid containing a drug resistance marker gene, so that the drug is added to the upstream fragment and the downstream fragment. A plasmid for introducing a deletion having a resistance marker gene fragment can be constructed.

  The deletion-introducing plasmid constructed by the above procedure is introduced into Bacillus subtilis for which the genomic region is to be deleted by a conventional technique such as competent cell transformation. The introduction of the plasmid causes double crossover homologous recombination between the upstream and downstream fragments on the plasmid and the region homologous to those of the Bacillus subtilis genome, and the region to be deleted becomes a drug resistance marker gene. Replaced transformants are obtained (FIG. 1). The transformant can be selected using the expression of a marker gene such as a drug resistance gene present in the deletion DNA fragment as an indicator. For example, a bacterium that has been transformed with a chloramphenicol resistance gene fragment is cultured in a medium containing an antibiotic (such as chloramphenicol), and the grown colonies are recovered, so that the target region is deleted. A transformant substituted 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 can be confirmed that the target region has been deleted.

  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 as shown in FIG. 2 can be used (Japanese Patent Laid-Open No. 2009-254350). In this method, first, a DNA fragment (donor DNA) for the first homologous recombination is prepared. Although it does not specifically limit as a preparation method, The above-mentioned SOE-PCR method etc. are mentioned. Examples of donor DNA include an about 0.1 to 3 kb fragment (upstream fragment) corresponding to a region adjacent to the upstream of the marker gene region to be removed (ie, the deleted region) and a region adjacent to the downstream as well. A DNA fragment in which an approximately 0.1 to 3 kb fragment (downstream fragment) corresponding to is bound to a fragment in the downstream region of the marker gene to be removed can be used. Preferably, a DNA fragment in which a second marker gene or the like serving as an index for homologous recombination is inserted between the downstream fragment and the downstream region fragment of the first marker gene to be removed (see FIG. 2). reference).

  Next, the prepared donor DNA is introduced into the transformant by a conventional technique such as competent cell transformation, and corresponds to the upstream fragment and the downstream region of the first marker gene on the transformant genome. Homologous recombination occurs between the regions (first homologous recombination). A transformant in which the desired homologous recombination has occurred can be selected using the expression of the second marker gene inserted into the donor DNA as an indicator. In the genomic DNA of a transformant in which the first homologous recombination has occurred appropriately, an upstream fragment, a downstream fragment, and, if necessary, a second marker gene, a first marker gene downstream region, and a downstream fragment are arranged in order. (See FIG. 2). In genomic DNA having such an arrangement, homologous recombination can occur spontaneously between the two downstream fragments (intragenomic homologous recombination). By this intragenomic homologous recombination, the region located between the two downstream fragments is deleted, whereby the first marker gene is removed from the transformant genome.

  As a means for selecting a transformant in which homologous recombination has occurred in the genome, when the first marker gene is a drug resistance gene, a method of selecting a bacterium having no drug resistance can be mentioned. Penicillin antibiotics act bactericidally on proliferating cells but not non-proliferating cells. Therefore, if a bacterium is cultured in the presence of a drug and a penicillin antibiotic, a drug non-resistant bacterium that does not grow in the presence of the drug can be selectively concentrated (Methods in Molecular Genetics, Cold Spring Harbor Labs, 1970). . Another means is a method of introducing a lethal gene. For example, if a lethal gene such as the chpA (mazF) gene is introduced into the bacterium as the second marker, a bacterium that has not undergone homologous recombination within the genome will die due to the action of the lethal gene. A transformant that has undergone the treatment can be selected. By extracting genomic DNA from the selected strain and performing PCR using this as a template, it can be confirmed that the target region has been deleted (see JP 2009-254350 A).

  As described above, a Bacillus subtilis mutant strain lacking a predetermined region on the genome can be prepared. Furthermore, by repeating this procedure, a Bacillus subtilis mutant strain in which a part or all of the above-described genomic region is deleted can be produced.

[1-2. Constant expression of abrB gene]
In the Bacillus subtilis mutant strain of the present invention, the gene is constructed so that the abrB gene or a gene corresponding thereto is constantly expressed.
In Bacillus subtilis, the abrB gene is a gene that encodes the transcription factor AbrB, and is expressed in the logarithmic growth phase and suppressed in the transition phase and during the stationary phase. This transcription factor AbrB directly or indirectly suppresses the expression of a large number of genes related to sporulation, competence, or nutrient source acquisition in the logarithmic growth phase. In the transition phase and the stationary phase, the expression suppression by AbrB is released, and the expression of the above-mentioned many genes is induced.
The Bacillus subtilis abrB gene is specified as gene number: BG10204, and encodes a protein AbrB consisting of the nucleotide sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2.

  Examples of the gene corresponding to the abrB gene include abrB genes of microorganisms belonging to the genus Bacillus other than Bacillus subtilis. Examples of microorganisms other than Bacillus subtilis include, but are not limited to, microorganisms belonging to the genus Bacillus such as Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus anthracis, Bacillus cereus, and the like. For example, for a gene corresponding to abrB, genomic DNA is extracted from the above-mentioned microorganism, and Southern hybridization is performed under stringent conditions using a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or a fragment thereof as a probe. Can be identified. Further, for example, a gene corresponding to abrB in the microorganism can be identified by comparing the nucleotide sequence of the genome of the microorganism with the nucleotide sequence represented by SEQ ID NO: 1 based on known genome sequence information.

  For example, the abrB gene (SEQ ID NO: 10, 11) of Bacillus amyloliquefaciens DSM7 strain, the abrB gene (SEQ ID NO: 12, 13) of Bacillus licheniformis ATCC14580 strain, the abrB gene of Bacillus megaterium DMS319 strain, the abrB gene of Bacillus megasium DMS319 strain, The sequence identity of the 168 strain with the abrB gene is 91%, 88% and 80% in the nucleotide sequence, respectively, and 98%, 94% and 85% in the amino acid sequence encoded by them. These are exemplified as genes corresponding to the abrB gene.

Accordingly, the abrB gene or a gene corresponding thereto includes the following:
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(B) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 1 Preferably, it comprises a nucleotide sequence having an identity of 98% or more, more preferably 99% or more, and encodes a protein having an activity as a transcription factor of a gene induced in the transition phase and the stationary phase, similar to AbrB A polynucleotide;
(C) As a transcription factor for a gene that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 and is induced in the transition phase and stationary phase, similar to AbrB A polynucleotide encoding a protein having the activity of:
(D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) a transcription factor of a gene consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in the transition phase and stationary phase similar to AbrB A polynucleotide encoding a protein having activity as:
(F) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 2. Preferably, it comprises an amino acid sequence having an identity of 98% or more, more preferably 99% or more, and encodes a protein having an activity as a transcription factor of a gene induced in the transition phase and the stationary phase, similar to AbrB Polynucleotide.

  In the present invention, constructing a gene so that the abrB gene or a gene corresponding thereto is constantly expressed means constructing the gene so that the abrB gene or a gene corresponding thereto is always expressed while the microorganism is alive, That is, it means that the gene is constructed so that the gene is expressed even in the stationary phase after the logarithmic growth phase, which is not normally expressed. Examples of gene construction in which the abrB gene or a gene corresponding thereto is constitutively expressed include, for example, a transcription initiation control region located upstream of the abrB gene or a gene corresponding thereto, or a transcription initiation control region and a ribosome binding site, or Inactivating the binding site of the inhibitory regulator in the region from the transcription initiation control region to the translation initiation control region can be mentioned. Here, “suppressive regulator” means a protein that binds to a specific base sequence involved in the control of transcription on a gene and controls transcription in a suppressive manner. As an example of the transcription initiation control region of the abrB gene, the transcription initiation control region and the ribosome binding site, or the region from the transcription initiation control region to the translation initiation control region, DNA comprising the base sequence shown in SEQ ID NO: 9, Alternatively, DNA having a base sequence homologous to the sequence and having a function as a transcription initiation control region, a transcription initiation control region and a ribosome binding site, or a region from the transcription initiation control region to the translation initiation control region can be mentioned. As a base sequence homologous to the base sequence shown in SEQ ID NO: 9, for example, a base sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the base sequence shown in SEQ ID NO: 9 Or a base sequence consisting of a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 9.

In the control region, examples of the binding site of the inhibitory regulator include, for example, a region consisting of the 20th to 49th bases or a region corresponding to the base sequence shown in SEQ ID NO: 9, from the 73th to 89th bases. Or a region corresponding thereto.
Here, the region corresponding to the region consisting of the 20th to 49th bases or the region consisting of the 73th to 89th bases in the base sequence shown in SEQ ID NO: 9 consists of the base sequence shown in SEQ ID NO: 9 described above. A DNA comprising a base sequence homologous to DNA and having a function as a transcription initiation control region, a transcription initiation control region and a ribosome binding site, or a region from the transcription initiation control region to the translation initiation control region. And a region that corresponds to the region when compared with a base sequence that functions as a binding site for an inhibitory regulatory factor.
Means for inactivating the site include mutagenesis of one or more nucleotides on the nucleotide sequence of the site, substitution or insertion of another nucleotide sequence into the nucleotide sequence, or part of the sequence of the site Alternatively, all of them can be deleted. Here, the term “part” includes a partial sequence consisting of 1 to 40, preferably 5 to 30, or 10 to 17 bases. Among these, preferably, in the base sequence shown in SEQ ID NO: 9, a region consisting of the 20th to 49th bases or a region corresponding thereto, and / or a region consisting of the 73th to 89th bases or a region corresponding thereto Is preferably deleted, more preferably the region consisting of the 73rd to 89th bases or the region corresponding thereto is deleted.
Specific methods for the above-described mutagenesis and nucleotide sequence substitution or insertion include ultraviolet irradiation, site-specific mutagenesis, and the above [1-1. Examples include the SOE-PCR method and the homologous recombination method described in “Genomic region-deficient Bacillus subtilis mutant strain”.

  Further, as another means for constructing a gene that constantly expresses the abrB gene or a gene corresponding thereto, the abrB gene on the genome or a gene corresponding thereto can be added to a control region capable of enhancing the expression of the gene in a stationary phase. Examples include operably linking the (strong expression control region), and introducing the abrB gene operably linked to the strong expression control region or a gene corresponding thereto into the intracellular genome or plasmid. Examples of such a strong expression control region include a Bacillus subtilis rplK gene, a fus gene or an atpI gene, or a transcription initiation control region located upstream of a gene corresponding to these genes or a transcription initiation control region and a ribosome binding site. .

[1-3. Deletion or inactivation of kinA gene]
In the Bacillus subtilis mutant strain of the present invention, the kinA gene or a gene corresponding thereto is deleted or inactivated. In Bacillus subtilis mutants constructed so that the abrB gene or a gene corresponding thereto is constantly expressed, the lysis phenomenon in the medium is suppressed by deletion or inactivation of the kinA gene or a gene corresponding thereto. It was found.
The kinA gene is identified as gene number: BG10204 (SEQ ID NO: 3), and the protein encoded by the gene is a multi-component sensor histidine kinase involved in the early stage of sporulation. of stimulation).

Examples of the kinA gene include the following.
(G) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3;
(H) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 3 A polynucleotide encoding a protein consisting of a nucleotide sequence having preferably 98% or more, more preferably 99% or more identity, and having activity as a sensor histidine kinase of a multi-component regulatory system involved in the early stage of sporulation;
(I) a protein that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 and has activity as a histidine kinase of a multi-component regulatory system involved in the early stage of sporulation The encoding polynucleotide;
(J) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(K) The activity as a histidine kinase of a multi-component control system which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 4 and which is involved in the early stage of sporulation A polynucleotide encoding a protein having;
(L) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 4 A polynucleotide encoding a protein comprising an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having an activity as a histidine kinase of a multicomponent regulatory system involved in the early stage of sporulation.

[1-4. Deletion or inactivation of alsS gene and alsD gene]
In the Bacillus subtilis mutant of the present invention, at least one gene selected from the group consisting of the alsS gene and the alsD gene is deleted or inactivated.
Here, the alsS gene and the alsD gene are Bacillus subtilis genes involved in acetoin synthesis. The alsS gene and the alsD gene form one operon as an acetoin synthesis gene group (alsSD). In the present invention, it is preferable that both the alsS gene and the alsD gene are deleted or inactivated.

  Table 2 below shows the registration numbers of the alsS gene and the alsD gene in the BSORF DB and the encoded proteins.

Examples of the alsS gene include the following.
(M) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5;
(N) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 5 A polynucleotide encoding a protein consisting of a nucleotide sequence preferably having 98% or more identity, more preferably 99% or more identity, and having α-acetolactic acid synthase activity;
(O) a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 and encodes a protein having α-acetolactic acid synthase activity;
(P) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;
(Q) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 6 and encoding a protein having α-acetolactic acid synthase activity;
(R) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 6 A polynucleotide encoding a protein comprising an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having α-acetolactic acid synthase activity.

Moreover, the following are mentioned as an alsD gene.
(S) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7;
(T) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 7 A polynucleotide comprising a nucleotide sequence preferably having 98% or more identity, more preferably 99% or more identity, and encoding a protein having α-acetolactic acid decarboxylase activity;
(U) a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 7 and encodes a protein having α-acetolactic acid decarboxylase activity;
(V) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8;
(W) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 8, and encoding a protein having α-acetolactic acid decarboxylase activity;
(X) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 8 A polynucleotide encoding a protein consisting of an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having α-acetolactic acid decarboxylase activity.

  Means for deleting or inactivating the gene include mutagenesis for one or more nucleotides on the nucleotide sequence of the gene, substitution or insertion of another nucleotide sequence for the nucleotide sequence, or sequence of the gene Deletion of a part or all of. Examples of means for introducing a mutation that inhibits transcription of the gene include mutagenesis in the promoter region of the gene, and inactivation of the promoter by substitution or insertion with another nucleotide sequence. Specific methods for the above-described mutagenesis and nucleotide sequence substitution or insertion include ultraviolet irradiation, site-specific mutagenesis, and the above [1-1. Examples include the SOE-PCR method and the homologous recombination method described in “Genomic region-deficient Bacillus subtilis mutant strain”. The position or nucleotide sequence of the gene to be deleted or inactivated on the Bacillus subtilis genome can be confirmed by the above-described BSORF DB.

[2. Construction of recombinant Bacillus subtilis
The recombinant Bacillus subtilis of the present invention is constructed such that the above-mentioned abrB gene or a gene corresponding thereto is constitutively expressed, the kinA gene is deleted or inactivated, and comprises an alsS gene and an alsD gene. At least one gene selected from the group is deleted or inactivated, and a target PGA synthase gene, or a PGA synthase gene and a PGA degrading enzyme gene (hereinafter referred to as “target gene”) can be expressed. It is preferable that it is strengthened or introduced as described above.

[2-1. PGA synthase gene]
Examples of the PGA synthase gene include a Bacillus subtilis gene pgsB (SEQ ID NO: 16), pgsC (SEQ ID NO: 18), pgsA (SEQ ID NO: 20) and pgsE (SEQ ID NO: 22), and a group consisting of genes corresponding thereto. And at least one selected gene, preferably a combination comprising at least a Bacillus subtilis pgsB gene or an equivalent gene thereof, and a Bacillus subtilis pgsC gene or an equivalent gene thereof, more preferably a Bacillus subtilis pgsB gene or an equivalent thereof. Combination of equivalent gene and Bacillus subtilis pgsC gene or its equivalent gene, Bacillus subtilis pgsB gene or its equivalent gene, Bacillus subtilis pgsC gene or its equivalent gene, Bacillus subtilis pgsA gene or its equivalent gene, Bacillus subtilis pgsE Combination with a gene or its equivalent gene It is not.

Examples of the Bacillus subtilis pgsB gene or a gene equivalent thereto include the following:
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 16;
(B) at least 62%, for example, 62% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably the nucleotide sequence shown in SEQ ID NO: 16 Is 85% or more, 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, more preferably 99% or more. A polynucleotide encoding a protein consisting of a nucleotide sequence and having an amide ligase activity;
(C) a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16 and encodes a protein having amide ligase activity;
(D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 17;
(E) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 17 and having amide ligase activity;
(F) at least 55%, for example, 55% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably the amino acid sequence shown in SEQ ID NO: 17 Is 80% or more, more preferably 85% or more, 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, more preferably A polynucleotide encoding a protein comprising an amino acid sequence having 99% or more identity and having an amide ligase activity.

Examples of the Bacillus subtilis pgsC gene or a gene corresponding thereto include the following:
(A ′) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 18;
(B ′) at least 59%, for example, 59% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more than the nucleotide sequence shown in SEQ ID NO: 18 Preferably 80% or more, more preferably 85% or more, 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, more preferably A polynucleotide encoding a protein comprising a nucleotide sequence having an identity of 99% or more and having a function of generating PGA in the presence of a protein encoded by the Bacillus subtilis pgsB gene or a gene corresponding thereto;
(C ′) in the presence of a protein that hybridizes under stringent conditions to the complementary strand of the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 18 and that is encoded by the Bacillus subtilis pgsB gene or a gene corresponding thereto. A polynucleotide encoding a protein having a function of generating PGA;
(D ′) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 19;
(E ′) Presence of a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 19 and encoded by the Bacillus subtilis pgsB gene or a gene corresponding thereto. A polynucleotide encoding a protein having the function of generating PGA below;
(F ′) at least 51%, for example, 51% or more, preferably 55% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more than the amino acid sequence shown in SEQ ID NO: 19. Preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably Encodes a protein having an amino acid sequence having an identity of 98% or more, more preferably 99% or more and having a function of generating PGA in the presence of the protein encoded by the Bacillus subtilis pgsB gene or a gene corresponding thereto. Polynucleotide.

Examples of the Bacillus subtilis pgsA gene or a gene equivalent thereto include the following:
(A ″) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 20;
(B ″) at least 48%, for example, 48% or more, preferably 50% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more, with the nucleotide sequence shown in SEQ ID NO: 20, More preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more Preferably, PGA is generated in the presence of the nucleotide sequence having identity of 98% or more, more preferably 99% or more, and in the presence of the Bacillus subtilis pgsB gene or its equivalent gene and the Bacillus subtilis pgsC gene or its equivalent gene. A polynucleotide encoding a protein having the function of:
(C ″) hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 20, and the Bacillus subtilis pgsB gene or its equivalent gene and the Bacillus subtilis pgsC gene or its A polynucleotide encoding a protein having a function of generating PGA in the presence of a corresponding gene;
(D ″) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 21;
(E ″) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 21, and the Bacillus subtilis pgsB gene or its equivalent gene and the Bacillus subtilis pgsC gene Or a polynucleotide encoding a protein having a function of generating PGA in the presence of an equivalent gene;
(F ″) at least 30%, for example, 30% or more, preferably 50% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more, with the amino acid sequence shown in SEQ ID NO: 21; More preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more Preferably, PGA is produced in the presence of the amino acid sequence having an identity of 98% or more, more preferably 99% or more, and in the presence of the Bacillus subtilis pgsB gene or its equivalent gene and the Bacillus subtilis pgsC gene or its equivalent gene. A polynucleotide encoding a protein having a function.

Examples of the Bacillus subtilis pgsE gene or a gene equivalent thereto include the following:
(A ′ ″) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 22;
(B ′ ″) at least 51%, for example 51% or more, preferably 55% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more with the nucleotide sequence shown in SEQ ID NO: 22 , More preferably 75% or more, more preferably 80% or more, more preferably 85% or more, 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, more preferably 99% or more of the nucleotide sequence, and the Bacillus subtilis pgsB gene or its equivalent gene, the Bacillus subtilis pgsC gene or its equivalent gene, and the Bacillus subtilis pgsA A polynucleotide encoding a protein having a function of generating PGA in the presence of a gene or an equivalent gene thereof Plastid;
(C ′ ″) hybridizing under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 22, and the Bacillus subtilis pgsB gene or its equivalent gene, and the Bacillus subtilis pgsC gene Or a polynucleotide encoding a protein having a function of generating PGA in the presence of the Bacillus subtilis pgsA gene or an equivalent gene thereof;
(D ′ ″) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 23;
(E ′ ″) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 23, and the Bacillus subtilis pgsB gene or its equivalent gene, and the Bacillus subtilis a polynucleotide encoding a protein having a function of generating PGA in the presence of the pgsC gene or its equivalent gene and the Bacillus subtilis pgsA gene or its equivalent gene;
(F ″ ′) at least 35%, for example, 35% or more, preferably 50% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more with the amino acid sequence shown in SEQ ID NO: 23 , More preferably 75% or more, more preferably 80% or more, more preferably 85% or more, 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, more preferably 99% or more of the amino acid sequence, and the Bacillus subtilis pgsB gene or its equivalent gene, the Bacillus subtilis pgsC gene or its equivalent gene, and the Bacillus subtilis pgsA gene Alternatively, a polynucleotide encoding a protein having a function of generating PGA in the presence of an equivalent gene.

In the above, “amide ligase activity” of a protein can be measured by detecting the production of PGA in a reaction solution in which glutamic acid and ATP or GTP are used as a substrate and manganese chloride is added thereto (J. Bacteriol). , 2002, 184: 337-343). The “amide ligase activity” of the protein encoded by the Bacillus subtilis pgsB gene or its equivalent gene refers to the introduction of the pgsB gene or its equivalent gene into Bacillus subtilis or its mutant strain together with the pgsC gene or its equivalent gene. In this case, it may be the ability to cause production of PGA and secretion outside the cell in the strain.
Evaluation of PGA production ability in a predetermined microorganism is, for example, a method of visually observing a translucent viscous substance formed around a colony when cultured on a PGA production agar medium containing glutamic acid or a salt thereof; SDS-PAGE (Biosci. Biotechnol. Biochem., 1996, 60: 255-258); Method for quantification using an amino acid analyzer after acid hydrolysis (Biosci. Biotechnol. Biochem., 1997, 61: 1684-) 1687); Quantification method for purification from culture broth and determination of dry weight (Biochem. Biophys. Res. Commun., 1999, 263: 6-12); Quantification / qualitative analysis by HPLC (high performance liquid chromatography) using a gel filtration column Law Biosci.Biotechnol.Biochem, 1993,57:. 1212-1213) can be carried out by, for example.

  Further, examples of genes corresponding to genes of Bacillus subtilis pgsB, pgsC, pgsA, and pgsE include genes involved in PGA synthesis of microorganisms belonging to the genus Bacillus other than Bacillus subtilis or other genera. Microorganisms other than Bacillus subtilis may be PGA-producing microorganisms or non-PGA-producing microorganisms, for example, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus megathium, Bacillus anthracis, etc. Examples include, but are not limited to, Naturalba aegyptica, Hydra, Oceanobacillus iheyensis, and the like. For example, a gene corresponding to Bacillus subtilis pgsB, pgsC, pgsA or pgsE is obtained by extracting genomic DNA from the above-mentioned microorganism and probing a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 16, 18, 20 or 22 or a fragment thereof. Can be identified by performing Southern hybridization. Further, for example, by comparing the nucleotide sequence of the microorganism genome with the nucleotide sequence represented by SEQ ID NO: 16, 18, 20, or 22 based on known genome sequence information, Bacillus subtilis pgsB, pgsC, A gene corresponding to pgsA or pgsE can be identified.

  For example, Bacillus sp. PgsB gene of KSM-366 strain (FERM BP-6262), pgsB gene of Bacillus licheniformis ATCC14580 strain, pwsB gene of Bacillus amyloliquefaciens strain 16 The sexes are 99.9%, 78%, 81% and 62% in the nucleotide sequence, respectively, and 99.7%, 89%, 93% and 55% in the amino acid sequence encoded by them, respectively. These are exemplified as genes corresponding to the Bacillus subtilis pgsB gene, and specifically, it has been shown that they can be used in PGA production by Bacillus subtilis (JP 2010-213664 A).

  For example, Bacillus sp. The pgsC gene of KSM-366 strain (FERM BP-6262), the pgsC gene of Bacillus licheniformis ATCC14580 strain, the ywtA gene of Bacillus amyloliquefaciens FZB42 strain, and the Bacillus strain HB8 Sex is 100%, 78%, 83% and 59% in the nucleotide sequence, respectively, and 100%, 89%, 93% and 51% in the amino acid sequence encoded by them, respectively. These are exemplified as genes corresponding to the Bacillus subtilis pgsC gene, and specifically, it has been shown that they can be used in PGA production by Bacillus subtilis (JP 2010-213664 A).

  For example, Bacillus sp. The pgsA gene of KSM-366 strain (FERM BP-6262), the pgsA gene of Bacillus licheniformis ATCC14580 strain, the ywtB gene of Bacillus amyloliquefaciens strain FZB42 strain 16 and the Oceanbacillus strain 17 The sexes are 100%, 68%, 75% and 48%, respectively, in the nucleotide sequence and 100%, 67%, 78% and 30%, respectively, in their encoded amino acid sequence. These are exemplified as a gene corresponding to the Bacillus subtilis pgsA gene (Japanese Patent Laid-Open No. 2010-213664).

  Further, for example, the pgsE gene of Bacillus licheniformis ATCC 14580 strain, the pgsE gene of Bacillus amyloliquefaciens FZB42 strain, and the capE gene of Bacillus anthracis H9401 strain are pgs of the E. coli strain 168, % And 51%, and 47%, 72% and 35%, respectively, in their encoded amino acid sequences. These are exemplified as genes corresponding to the Bacillus subtilis pgsE gene.

  Specific examples of genes of microorganisms belonging to the genus Bacillus or other genera corresponding to the Bacillus subtilis gene pgsBCAE include the genes shown in Table 3 below.

  In a preferred embodiment, the PGA synthase genes that are forcibly enhanced or introduced in the Bacillus subtilis mutant strain of the present invention are the above (a) to (f), (a ′) to (f ′), (a ″). ) To (f ″) and (a ′ ″) to (f ′ ″), at least one selected from the group consisting of genes, preferably at least the above (a) to (f) And a combination of at least one selected from the group consisting of the genes represented by (a ′) to (f ′) and at least one selected from the group consisting of the genes represented by (a ′) to (f ′). Also preferably, the gene to be enhanced or introduced so that it can be expressed is at least one selected from the group consisting of the genes represented by (a), (a ′), (a ″) and (a ′ ″) above. More preferably, a combination of at least the gene represented by (a) and the gene represented by (a ′) is included. More preferably, the gene to be enhanced or introduced so that it can be expressed is a combination of the genes represented by (a) and (a ′) above, or (a), (a ′), (a ″) above. And a combination of genes represented by (a ′ ″).

  In another preferred embodiment, PGA synthase genes that can be expressed or enhanced in the Bacillus subtilis mutant strain of the present invention include, for example, SEQ ID NOs: 16, 18, 20, 22 and 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and at least one of the genes consisting of the sequence shown in FIG. A gene consisting of at least one of the sequences shown in any of Nos. 16, 26, 32, 40, 48 and 54 and a sequence shown in any of SEQ ID Nos. 18, 28, 34, 42, 50 and 56 A combination with at least one of the above. More preferably, the gene to be enhanced or introduced so that expression is possible is at least one of the genes consisting of the sequences shown in any of SEQ ID NOs: 16, 26, 32, 40, 48 and 54, and SEQ ID NOs: 18, 28, At least one of the genes consisting of the sequence shown in any of 34, 42, 50 and 56 and at least one of the genes consisting of the sequence shown in any of SEQ ID NOs: 20, 30, 36, 44, 52 and 58 And at least one of the genes consisting of the sequence shown in any one of SEQ ID NOs: 22, 38, 46 and 60.

[2-2. Gene encoding PGA degrading enzyme]
In the recombinant Bacillus subtilis in which the PGA synthase gene is enhanced or introduced so that it can be expressed, it is preferable that the gene encoding the PGA degrading enzyme is further enhanced or introduced so that it can be expressed. When microorganisms produce PGA at a high level, the viscosity of the medium increases, and as a result, the survival of microorganisms and the productivity of PGA may be adversely affected. For example, the Bacillus subtilis pgdS gene or a gene corresponding thereto is strengthened or introduced so that it can be expressed, thereby producing a low molecular weight PGA. The problem can be solved.
Therefore, examples of the gene encoding PGA degrading enzyme include the Bacillus subtilis pgdS gene (SEQ ID NO: 24) or a gene corresponding thereto.

  Examples of the gene corresponding to the Bacillus subtilis pgdS gene include genes involved in PGA degradation of microorganisms belonging to the genus Bacillus other than Bacillus subtilis or other genera. A gene corresponding to the Bacillus subtilis pgdS gene of a microorganism other than Bacillus subtilis can be searched or identified based on the nucleotide sequence shown in SEQ ID NO: 24 in the same procedure as described above for the pgsBCAE gene. Specific examples of Bacillus microbial genes corresponding to the Bacillus subtilis pgdS gene include the pgdS gene (SEQ ID NO: 62) of Bacillus licheniformis ATCC 14580 strain and the pgdS gene (SEQ ID NO: 64) of Bacillus amyloliquefaciens FZB42 strain.

Examples of the Bacillus subtilis pgdS gene or a gene equivalent thereto include the following:
(G) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 24;
(H) at least 62%, for example, 62% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably, the nucleotide sequence shown in SEQ ID NO: 24 Is 85% or more, 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, more preferably 99% or more. A polynucleotide encoding a protein consisting of a nucleotide sequence and having PGA-degrading activity;
(I) a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 24 and encodes a protein having PGA degrading activity;
(J) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 25;
(K) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 25 and encoding a protein having PGA degrading activity;
(L) at least 57%, for example, 57% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably the amino acid sequence shown in SEQ ID NO: 25 Is 80% or more, more preferably 85% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably A polynucleotide encoding a protein having an amino acid sequence having an identity of 97% or more, more preferably 98% or more, more preferably 99% or more and having PGA degrading activity.

  In a preferred embodiment, the gene encoding a PGA-degrading enzyme that is fortified or introduced so that it can be expressed in the recombinant Bacillus subtilis of the present invention is selected from the group consisting of the genes represented by (g) to (l) above. And preferably the gene represented by (g) above.

  In another preferred embodiment, the gene encoding Bacillus subtilis PGA degrading enzyme is at least one of the genes consisting of the sequence shown in any of SEQ ID NOs: 24, 62 and 64.

[2-3. Reinforcement or introduction of genes related to PGA production]

Enhancing or introducing a gene related to PGA production (target gene) so that it can be expressed means that in the mutant strain of Bacillus subtilis of the present invention, the target gene is placed in a state where it can be expressed at least. It does not matter whether it is expressed in the Bacillus subtilis mutant of the present invention.
Such expression enhancement or introduction of the target gene can be achieved by replacing the control region sequence of the target gene (eg, pgsBCAE gene) on the genome of the parent strain with a strong control region, or operably linked to the strong control region. A fragment containing the target gene can be introduced into the genome of the parent strain or into a plasmid. Alternatively, it can also be made by modifying a parent strain so that a plurality of target genes can be expressed.
An example of a procedure for enhancing or introducing a target gene so that it can be expressed is described below.

(1) A DNA fragment is constructed in which a target gene is operably linked to a control region and is further linked to a homologous region with the parental genome. For example, from the upstream, a fragment in which a control region (eg, rrnO operon control region) and a target gene (eg, pgsBCAE gene) are arranged in this order is prepared, and a homologous region with a part of the parental genome is linked to the fragment. To prepare a DNA fragment. Subsequently, when this DNA fragment is introduced into the parent strain, the DNA fragment is inserted into the parent strain genome, and the parent strain can be transformed. The resulting transformant has an increased expression level of the target gene compared to the strain before transformation.

(2) Construct a vector containing the gene of interest operably linked to the control region. For example, a vector containing DNA arranged in the order of a control region (for example, a control region of a cellulase gene of KSM-S237) and a target gene (for example, a pgsBC gene) is prepared from upstream. Subsequently, the parent strain can be transformed by introducing this vector into the parent strain. The resulting transformant has an increased expression level of the target gene compared to the strain before transformation.

  Here, the control region that can be used for the expression of the target gene preferably has a function of increasing the expression of the downstream target gene in the host, and more preferably, the downstream target gene is constitutively expressed or highly expressed. This is a control area having a function to be performed. Also preferred is a control region (strong control region) that can enhance expression of the target gene as compared to the wild type control region of the target gene.

  Examples of control regions that can be used for expression of a gene encoding a PGA synthase gene and a PGA-degrading enzyme include a control region of an α-amylase gene derived from a bacterium belonging to the genus Bacillus, a control region of a protease gene, an rrnO operon control region, tufA Gene regulatory region, aprE gene regulatory region, spoVG gene regulatory region, Bacillus sp. Examples include a cellulase gene control region of KSM-S237 strain, a staphylococcus aureus-derived kanamycin resistance gene control region, a chloramphenicol resistance gene control region, and the like (all of which are described in JP-A-2009-089708).

  Preferably, Bacillus sp. A 0.4 to 1.0 kb region upstream of the translation start point of the cellulase gene, which is the control region of the cellulase gene of the KSM-S237 strain (SEQ ID NO: 66), and 80% or more, preferably 90% or more in the region and nucleotide sequence More preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and gene expression equivalent to that region. 80% or more, preferably 90% or more, more preferably 95% or more of the base sequence having a control function, the upstream 0.2-1.0 kb region (SEQ ID NO: 166) of the 16S rRNA of the rrnO operon, and the region and nucleotide sequence More preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably A nucleotide sequence having 9% identity or more and having a gene expression control function equivalent to that of the region, a region of 1 kb from the upstream of the translation start point of the tufA gene (SEQ ID NO: 167), and the nucleotide sequence of the region 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, more preferably 99% or more And a base sequence having a gene expression control function equivalent to that of the region.

  More preferably, for the PGA synthase gene (for example, the pgsBC gene or its equivalent gene), the control region of the KSM-S237 cellulase gene is the entire Bacillus subtilis poly-γ-glutamate synthase gene group (for example, the pgsBCAE gene). Alternatively, the rrnO operon regulatory region is used for the corresponding gene group), and the tufA gene regulatory region is used for the gene encoding Bacillus subtilis poly-γ-glutamate degrading enzyme or its corresponding gene.

  The vector used for introducing the target gene or control region into the parent strain may be a vector generally used for transformation of a plasmid or the like. The type of vector can be selected as appropriate, but it is preferably a vector (for example, a plasmid) capable of self-replication in the parent strain, and more preferably a multicopy vector. Furthermore, the copy number of the plasmid may be 2 or more and 100 or less, preferably 2 or more and 50 or less, more preferably 2 or more and 30 or less, relative to the genome (chromosome) of the parent strain. Examples of preferable plasmids include pT181, pC194, pUB110, pE194, pSN2, pHY300PLK, and the like.

  Insertion of a gene of interest or a control region into a vector can be performed according to a conventional method in the art. For example, the target gene and control region fragments may be amplified by PCR or the like, and these fragments may be inserted into a vector such as a plasmid by a restriction enzyme method or the like and ligated. Alternatively, a fragment in which a target gene and a regulatory region fragment are linked in advance may be prepared and inserted into a vector such as a plasmid. At that time, on the vector, the control region fragment and the target gene fragment are ligated in this order from the upstream.

  When the PGA synthase gene is a combination of the pgsB gene or its corresponding gene and the pgsC gene or its corresponding gene, the control region fragment, the pgsB gene or its corresponding gene fragment from the upstream on the DNA fragment or vector to be introduced into the parent strain Then, it is preferable that the pgsC gene or its corresponding gene fragment is linked in this order.

  DNA fragments or vectors can be introduced into the parent strain by, for example, 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).

  According to the above procedure, the recombinant Bacillus subtilis of the present invention can be prepared. In the finally obtained microorganism, the recombinant Bacillus subtilis is a deletion of a large region of the genome, the abrB gene or the Gene construction in which the corresponding gene is constitutively expressed, deletion or inactivation of the kinA gene, and deletion or inactivation of at least one gene selected from the group consisting of the alsS gene and the alsD gene The order of the respective gene modification / introduction steps is not particularly limited. For example, for a Bacillus subtilis mutant strain in which a large region of the genome is deleted, a predetermined gene is modified by the above-described procedure, and then the predetermined gene is deleted or inactivated by the above-described procedure. Alternatively, for a Bacillus subtilis mutant strain in which a large region of the genome described above is deleted, a predetermined gene is deleted or inactivated by the procedure described above, and then the predetermined gene is modified by the procedure described above. May be manufactured. Preferably, the abrB gene or a gene corresponding thereto is constructed in a gene-deleted strain in which a large region of the genome is deleted, and the kinA gene is deleted or inactivated. A Bacillus subtilis mutant can be used as a parent strain, and at least one gene selected from the group consisting of the alsS gene and the alsD gene can be deleted or inactivated.

[3. Production of PGA]
The recombinant Bacillus subtilis of the present invention thus obtained is compared to the recombinant Bacillus subtilis with enhanced expression of the PGA synthase gene, compared to the Bacillus subtilis mutant lacking a large region of the parent genome. It has a much higher PGA production capacity.
Therefore, by culturing the recombinant Bacillus subtilis of the present invention, PGA can be efficiently produced outside the bacteria.
In the method for producing PGA of the present invention, first, the recombinant Bacillus subtilis of the present invention is cultured in an appropriate medium, and PGA produced outside the cells is recovered from the medium. Medium includes carbon sources such as glycerol, glucose, fructose, maltose, xylose, arabinose, various organic acids, various amino acids, polypeptone, tryptone, nitrogen sources such as ammonium sulfate and urea, inorganic salts such as sodium salts, and other necessary nutrients A medium having a composition containing a source, a trace metal salt and the like can be used. The medium may be a synthetic medium or a natural medium.

  In the production of PGA, it is not always necessary to add glutamic acid to the medium. However, in order to further improve the productivity of PGA, the amount of glutamic acid required for the growth of the recombinant Bacillus subtilis of the present invention is minimal. The recombinant Bacillus subtilis can be cultured in a medium to which glutamic acid is added. Glutamic acid is preferably added to the medium as sodium glutamate, and its concentration (in terms of glutamic acid) is preferably, for example, 0.005 to 600 g / L, more preferably 0.05 to 500 g / L in the medium. More preferably, it may be 0.1-400 g / L, More preferably, it may be 0.5-300 g / L. The glutamic acid concentration in the medium is preferably 0.005 g / L or more from the viewpoint of improving the productivity of PGA. On the other hand, sodium glutamate and other medium components in the medium due to exceeding the saturated solubility of sodium glutamate From the viewpoint of avoiding precipitation, the glutamic acid concentration is preferably 600 g / L or less.

As the culture conditions in this case, the optimum temperature is preferably 10 to 40 ° C, more preferably 30 to 40 ° C. The optimum pH is preferably pH 4-8, more preferably 5-8. Moreover, the culture days are preferably 1 to 10 days, more preferably 1 to 5 days after the inoculation of bacteria.
When recovering the PGA accumulated in the medium, it is necessary to separate it from the cells that produced the PGA. Examples of means for separating PGA include centrifugation, ultrafiltration membrane, electrodialysis, a method of recovering as a precipitate by maintaining pH near the isoelectric point of PGA, and a combination of the above methods.

The following are further disclosed herein as exemplary embodiments of the invention. However, the present invention is not limited to these embodiments.
<1> a Bacillus subtilis mutant,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region Having one or more selected regions of the deleted genome;
The gene is constructed so that the abrB gene or a gene equivalent thereto is constitutively expressed, the kinA gene is deleted or inactivated, and at least one gene selected from the group consisting of the alsS gene and the alsD gene is missing. Bacillus subtilis mutant strain that has been inactivated or inactivated.
<2> profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA- YurT region, cgeE-ypmQ region, yek-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region <1> Bacillus subtilis mutant.
<3> The gene construction that constantly expresses the abrB gene or a gene corresponding thereto is a transcription inhibitory region of the abrB gene or a gene corresponding thereto, or a repression property in a transcription initiation control region and a ribosome binding site <1> or <2> a Bacillus subtilis mutant strain produced by inactivating a binding site of a regulatory factor.
<4> The transcription initiation control region of the abrB gene or a gene corresponding thereto, the transcription initiation control region and the ribosome binding site, or the binding site of the inhibitory regulatory factor in the region from the transcription initiation control region to the translation initiation control region <3> Bacillus subtilis which is a region consisting of the 20th to 49th bases or a region corresponding thereto, or a region consisting of the 73th to 89th bases or a region corresponding thereto, in the base sequence shown in SEQ ID NO: 9 Mutant strain.
<5> The Bacillus subtilis mutant according to <4>, wherein the region consisting of the 73rd to 89th bases or the region corresponding thereto is deleted from the base sequence represented by SEQ ID NO: 9.
<6> The Bacillus subtilis mutant strain according to any one of <1> to <5>, wherein the abrB gene of Bacillus subtilis or a gene corresponding thereto is selected from the group consisting of the following (a) to (f).
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(B) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 1 Poly which encodes a protein comprising a nucleotide sequence having preferably 98% or more, more preferably 99% or more identity, and having the activity as a transcription factor of a gene induced in the transition phase and stationary phase similar to AbrB nucleotide;
(C) As a transcription factor for a gene that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and is induced in the transition phase and stationary phase similar to AbrB A polynucleotide encoding a protein having activity;
(D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) a transcription factor of a gene consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in the transition phase and stationary phase similar to AbrB A polynucleotide encoding a protein having activity as:
(F) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 2. Poly which encodes a protein comprising an amino acid sequence having an identity of preferably 98% or more, more preferably 99% or more, and having activity as a transcription factor of a gene induced in the transition phase and stationary phase similar to AbrB nucleotide.
<7> The Bacillus subtilis mutant strain according to any one of <1> to <6>, wherein the kinA gene is selected from the group consisting of the following (g) to (l).
(G) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3;
(H) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 3 A polynucleotide encoding a protein consisting of a nucleotide sequence having preferably 98% or more, more preferably 99% or more identity, and having activity as a sensor histidine kinase of a multi-component regulatory system involved in the early stage of sporulation;
(I) a protein that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 and has activity as a histidine kinase of a multi-component regulatory system involved in the early stage of sporulation The encoding polynucleotide;
(J) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(K) The activity as a histidine kinase of a multi-component control system which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 4 and which is involved in the early stage of sporulation A polynucleotide encoding a protein having;
(L) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 4 A polynucleotide encoding a protein comprising an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having an activity as a histidine kinase of a multicomponent regulatory system involved in the early stage of sporulation.
<8> The Bacillus subtilis mutant strain according to any one of <1> to <7>, wherein the alsS gene is selected from the group consisting of the following (m) to (r).
(M) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5;
(N) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 5 A polynucleotide encoding a protein consisting of a nucleotide sequence preferably having 98% or more identity, more preferably 99% or more identity, and having α-acetolactic acid synthase activity;
(O) a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 and encodes a protein having α-acetolactic acid synthase activity;
(P) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;
(Q) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 6 and encoding a protein having α-acetolactic acid synthase activity;
(R) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 6 A polynucleotide encoding a protein comprising an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having α-acetolactic acid synthase activity.
<9> The Bacillus subtilis mutant strain according to any one of <1> to <8>, wherein the alsD gene is selected from the group consisting of the following (s) to (x).
(S) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7;
(T) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 7 A polynucleotide comprising a nucleotide sequence preferably having 98% or more identity, more preferably 99% or more identity, and encoding a protein having α-acetolactic acid decarboxylase activity;
(U) a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 7 and encodes a protein having α-acetolactic acid decarboxylase activity;
(V) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8;
(W) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 8, and encoding a protein having α-acetolactic acid decarboxylase activity;
(X) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 8 A polynucleotide encoding a protein consisting of an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having α-acetolactic acid decarboxylase activity.
<10> The Bacillus subtilis mutant strain according to any one of <1> to <9>, wherein both the alsS gene and the alsD gene are deleted or inactivated.
<11> In the Bacillus subtilis mutant strain of any one of <1> to <10>, a poly-γ-glutamate synthase gene, or a poly-γ-glutamate synthase gene and a poly-γ-glutamate degrading enzyme gene can be expressed Recombinant Bacillus subtilis enhanced or introduced into
<12> The gene encoding poly-γ-glutamate synthase is at least one gene selected from the group consisting of a Bacillus subtilis pgsB gene, a pgsC gene, a pgsA gene, a pgsE gene, and a gene corresponding thereto. <11> The recombinant Bacillus subtilis according to <11>, wherein the poly-γ-glutamic acid degrading enzyme gene is a Bacillus subtilis pgdS gene or a gene corresponding thereto.
<13> The poly-γ-glutamate synthase gene and the poly-γ-glutamate degrading enzyme gene are Bacillus subtilis pgsB gene, pgsC gene, pgsA gene, pgsE gene and pgdS gene, or genes corresponding thereto <12 > Recombinant Bacillus subtilis.
<14> A method for producing a Bacillus subtilis mutant,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region In a Bacillus subtilis mutant strain having a genome in which one or more selected regions are deleted, the abrB gene or a gene equivalent thereto is constitutively expressed. A method comprising a step of constructing a gene, a step of deleting or inactivating a kinA gene, and a step of deleting or inactivating at least one gene selected from the group consisting of an alsS gene and an alsD gene.
<15> A method for producing recombinant Bacillus subtilis,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region In a Bacillus subtilis mutant strain having a genome in which one or more selected regions are deleted, the abrB gene or a gene equivalent thereto is constitutively expressed. A step of constructing a gene, a step of deleting or inactivating the kinA gene, a step of deleting or inactivating at least one gene selected from the group consisting of an alsS gene and an alsD gene, and poly-γ-glutamic acid synthesis A method comprising the step of enhancing or introducing an enzyme gene, or a poly-γ-glutamate synthase gene and a poly-γ-glutamate degrading enzyme gene in an expressible manner.
<16> A method for producing poly-γ-glutamic acid, comprising a step of culturing the recombinant Bacillus subtilis of any one of <11> to <13> and recovering poly-γ-glutamic acid from the culture.

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

Example 1 Construction of Strain (1-1) Preparation of Tryptophan Requirement Release Strain Tryptophan Requirement in a Bacillus subtilis Mutant (MGB874 Strain; Japanese Patent Application Laid-Open No. 2007-130013) in which a large region of the Bacillus subtilis wild type genome is deleted Was released. The Bacillus subtilis 168 strain, which is the original strain of the Bacillus subtilis mutant MGB874, possesses trpC2 as a genotype. In other words, strain 168 is required for tryptophan synthesis due to the trpC gene mutation encoding indole-3-glycerol phosphate synthase involved in tryptophan synthesis. Also, it is known that the Bacillus subtilis ATCC6051T strain is tryptophan non-requirement (Albertini, AM & Galizzi, A. Microbiology, 1999, 145 (Pt12): 3319-3320). When the trpC genes of Bacillus subtilis 168 and ATCC6051T were compared, it was found that nucleotides ATT from 328 to 330 were not present in Bacillus subtilis 168. Thus, PCR was performed by using the ATCC6051T strain genome as a template and using the trpC_F1 and trpC_R1 primers listed in Table 4 to construct a PCR product. Using this PCR product, the Bacillus subtilis mutant strain MGB874 was transformed by the competent method (J. Bacteriol., 1960, 81: 741-746), and tryptophan-free SMA agar medium (0.2% ammonium sulfate, 1% .4% dipotassium hydrogen phosphate, 0.6% potassium dihydrogen phosphate, 0.1% trisodium citrate dihydrate, 0.5% glucose, 0.035% magnesium sulfate heptahydrate, Colonies that grew on 1.5% agar) were isolated as transformants. Thus, a Bacillus subtilis mutant MGB874T strain not requiring tryptophan was obtained.

(1-2) Construction of Bacillus subtilis mutant strain (168_abrB * strain) into which the abrB gene constant expression mutation was introduced The abrB gene constant expression mutation was introduced. The abrB gene upstream region fragment (A) was amplified by PCR using the genomic DNA of Bacillus subtilis wild strain 168 as a template and using primers Dspo0Abox-FF and Dspo0Abox-FR shown in Table 5. In addition, the abrB gene upstream region fragment (B) was amplified by PCR using the primers Dspo0Abox-PF and Dspo0Abox-PR shown in Table 5. Further, using the primers Dspo0Abox-BF and Dspo0Abox-BR shown in Table 5, the abrB gene upstream region / midstream region fragment (C) was amplified by PCR. Next, using the plasmid pDLT3 (Morimoto T. et al., Microbiology, 2002, 148: 3539-3552) carrying the chloramphenicol resistance gene as a template, using the primers rPCR-CmF2 and rPCR-CmR2 described in Table 5, PCR amplification was performed, and the chloramphenicol resistance gene fragment (D) was PCR amplified.

  Next, the obtained fragment (A) and fragment (D) were combined by the SOE-PCR method using primers Dspo0Abox-FF and rPCR-CmR2 described in Table 5, and fragment (B) and fragment (C) were displayed. The primers Dspo0Abox-PF and Dspo0Abox-BR described in 5 were used for binding by the SOE-PCR method. Finally, fragment (A + D) and fragment (B + C) were ligated by SOE-PCR method using primers Dspo0Abox-FF and Dspo0Abox-BR described in Table 5 to obtain the final PCR product (A + D + B + C). The resulting final PCR product was used to transform 168 strains of Bacillus subtilis wild strain to obtain a Bacillus subtilis mutant strain 168_abrB * _Cm.

  Next, a drug resistance gene replacement plasmid pCm :: Em (Lei Y. et al., J. Bacteriol., 2013, 195: 1697-1705) was transformed with a Bacillus subtilis mutant 168_abrB * _Cm strain, A Bacillus subtilis mutant 168_abrB * in which the chloramphenicol resistance gene was replaced with the erythromycin resistance gene was constructed.

  It was confirmed that the target mutation was introduced at a predetermined position on the genome by PCR using the obtained Bacillus subtilis mutant 168_abrB * strain genome and subsequent sequencing by the Sanger method.

(1-3) Construction of a Bacillus subtilis mutant strain (168_ΔkinA strain) lacking the kinA gene A mutated mutation of the kinA gene was introduced. Using the genomic DNA of Bacillus subtilis wild strain 168 as a template and using the primers kinA-uF1 and kinA-uR1 shown in Table 5, the fragment (A) in the upstream region of the kinA gene was amplified by PCR. In addition, a fragment (B) in the downstream region of the kinA gene was amplified by PCR using the primers kinA-dF1 and kinA-dR1 listed in Table 5. Next, PCR amplification was performed using the plasmid pAPNCK (Yoshimura M. et al., BMC Microbiology, 2007, 7:69) carrying the kanamycin resistance gene as a template and using the primers rPCR-KmF and rPCR-KmR described in Table 5. The kanamycin resistance gene fragment (C) was PCR amplified.

  Next, fragment (A), fragment (C) and fragment (B) were combined in this order using primers kinA-uF1 and kinA-dR1 listed in Table 5 by the SOE-PCR method, and the final PCR product ( A + C + B) was obtained. The obtained final PCR product was used to transform 168 strains of Bacillus subtilis wild strain to obtain Bacillus subtilis mutant 168_ΔkinA strain.

  It was confirmed that the target gene was deleted at a predetermined position on the genome by PCR using the genome of the obtained Bacillus subtilis mutant 168_ΔkinA strain and subsequent sequencing by the Sanger method.

(1-4) Introduction of constitutive expression mutation of abrB gene and construction of Bacillus subtilis mutant strain lacking kinA gene (168_abrB * ΔkinA strain) Competent method (J. Bacteriol., 1960, 81: 741-746) was transformed into a Bacillus subtilis mutant 168_ΔkinA strain, and a colony grown on an LB agar medium containing erythromycin was constructed as a Bacillus subtilis mutant 168_abrB * ΔkinA strain.

  Using the PCR method using the obtained Bacillus subtilis modified genome, it was confirmed that the target mutation was introduced and deleted at a predetermined position on the genome.

(1-5) Construction of Bacillus subtilis mutant strain (MGB874T_PGA strain) with enhanced expression of PGA synthase gene group and gene encoding PGA degrading enzyme First, PGA synthase gene group originally possessed by Bacillus subtilis on the genome Deletion mutagenesis of genes encoding (pgsBCAE) and PGA degrading enzyme was performed. This mutant strain was constructed by a marker-free deletion method using a cassette in which the IPTG regulatory region and a free mRNA-cleaving ribonuclease mazF gene were fused (Morimoto, T. et al., In Strain Engineering, pp 345-358. Springer).

  Using the genomic DNA extracted from the Bacillus subtilis mutant strain MGB874T constructed in Example (1-1) as a template, the fragments (A) were amplified using the primers pgs-DF1 and pgs-DR1 described in Table 6 as fragments (B ) Primers pgs-DF2 and pgs-DR2 described in Table 6 were used for amplification, and primers pgs-IF and pgs-IR described in Table 6 were used for fragment (C) amplification. Furthermore, Bacillus subtilis mutant TOM310 (Morimoto, T. et al., In Strain Engineering, pp 345-358. Springer) was used as a template, and primers pAPNC-F and chpA-R described in Table 6 were used to resist spectinomycin. The mazF cassette fragment (D) containing the gene was amplified by PCR. Next, SOE-PCR was performed using the primers pgs-DF1 and pgs-IR described in Table 6 so that the obtained fragment (A), fragment (B), fragment (D) and fragment (C) were in this order. The final DNA fragment was obtained by ligation by the method. The constructed final PCR product was transformed with the Bacillus subtilis mutant MGB874T strain. By PCR using the genome of the obtained transformant (B. subtilis MGB874T_ΔpgsBCAEΔpgdS strain) and subsequent sequencing by the Sanger method, it was confirmed that each gene introduced with mutation on the genome had a deletion. The PGA synthase gene group (pgsBCAE) constructed by the marker-free deletion method using the mazF gene cassette in this example and the gene deletion method encoding the PGA degrading enzyme are shown in FIG.

  Next, a gene encoding PGA degrading enzyme was introduced. Using the genomic DNA of the Bacillus subtilis mutant MGB874T strain as a template and the primers tufA-1f and tufA-1r shown in Table 6, the fragment (A) in the downstream region of the tufA gene was amplified by PCR. In addition, a fragment (B) in the downstream region of the tufA gene was amplified by PCR using the primers tufA-2f and tufA-2r shown in Table 5. Subsequently, the fragment (C) of the pgdS gene ORF was amplified by PCR using the genomic DNA of the Bacillus subtilis mutant strain MGB874T as a template and using the primers BSpgdS-F and BSpgdS-R described in Table 6. Next, using the plasmid pAPNC213 (Morimoto T. et al., Microbiology, 2002, 148: 3539-3552) carrying the spectinomycin resistance gene as a template and using the primers spc-F and spc-R described in Table 6, Amplification was performed, and a spectinomycin resistance gene fragment (D) was PCR amplified.

  Next, SOE-PCR was performed using the primers tufA-1f and tufA-2r described in Table 6 so that the obtained fragment (A), fragment (C), fragment (D) and fragment (B) were in this order. The final PCR product was obtained by ligation by the method. The obtained final PCR product was used to transform with the aforementioned Bacillus subtilis MGB874T_ΔpgsBCAEΔpgdS strain to obtain a Bacillus subtilis mutant MGB874T_ΔpgsBCAEΔpgdS_PtufA-pgdS strain.

  The obtained Bacillus subtilis mutant MGB874T_ΔpgsBCAEΔpgdS_PtufA-pgdS strain was subjected to PCR using the genome followed by sequencing by the Sanger method to confirm that the target gene was introduced at a predetermined position on the genome.

  Subsequently, the PGA synthase gene group was introduced. Using the genomic DNA of the Bacillus subtilis mutant MGB874T strain as a template, the amyE upstream region fragment (A) was amplified by PCR using the primer amyE-UF described in Table 6 and the primer amyE-UR described in Table 6. A mutant strain obtained by transforming the wild strain 168 of Bacillus subtilis with pDLT3 (Morimoto T. et al., Microbiology, 2002, 148: 3539-3552) as a template, primer cat-F described in Table 6 and primer amyE described in Table 6 Using -DR, a fragment (B) in the downstream region of amyE containing the chloramphenicol resistance gene was amplified by PCR. Next, using the genomic DNA of Bacillus subtilis mutant strain RIK356 (Natori Y. et al., J. Bacteriol., 2009, 191: 4555-4561) as a template, primers amyE / PrrnO-F and PrrnO / bs described in Table 6 were used. The fragment (C) of the control region of the rrnO operon was amplified by PCR using -pgsR. Next, PCR amplification was performed using the genomic DNA of the Bacillus subtilis mutant MGB874T strain as a template and using the primers bs-pgsF and bs-pgs / catR described in Table 6, and the fragment (D) of the pgsBCAE gene group was PCR amplified. .

  Next, SOE-PCR was performed using the primers amyE-UF and amyE-DR described in Table 6 so that the obtained fragment (A), fragment (C), fragment (D) and fragment (B) were in this order. The final PCR product was obtained by ligation by the method. The resulting final PCR product was used to transform with MGB874T_ΔpgsBCAEΔpgdS_PtufA-pgdS strain to obtain a Bacillus subtilis mutant MGB874T_PGA strain.

  Using the PCR method using the genome of the obtained Bacillus subtilis mutant MGB874T_PGA strain, it was confirmed that the target gene group was introduced at a predetermined position on the genome.

(1-6) Bacillus subtilis mutant strain (MGB874T_PGA_abrB * ΔkinA strain) in which the abrB gene constitutive expression mutation was introduced, the kinA gene was deleted, and the expression of genes encoding PGA synthase gene group and PGA degrading enzyme was enhanced First, the Bacillus subtilis mutant MGB874T_PGA strain was transformed by the competent method (J. Bacteriol., 1960, 81: 741-746) using the genomic DNA of the Bacillus subtilis mutant 168_abrB * ΔkinA strain, and erythromycin was transformed. The colonies grown on the LB agar medium containing Bacillus subtilis were constructed as the Bacillus subtilis mutant MGB874T_PGA_abrB * strain and the colonies grown on the LB agar medium containing kanamycin as the Bacillus subtilis mutant MGB874T_PGA_ΔkinA strain.

  Next, the Bacillus subtilis mutant MGB874T_PGA_ΔkinA strain was transformed by the competent method (J. Bacteriol., 1960, 81: 741-746) using the genomic DNA of the Bacillus subtilis mutant MGB874T_PGA_abrB * strain, and LB containing erythromycin was obtained. A colony grown on the agar medium was constructed as a Bacillus subtilis mutant MGB874T_PGA_abrB * ΔkinA strain.

  Using the PCR method using the obtained genome of Bacillus subtilis, it was confirmed that the target gene group was introduced and deleted at a predetermined position on the genome.

(1-7) A hay that has been introduced with a constitutive expression mutation of the abrB gene, the kinA gene is deleted, the alsSD gene group is further deleted, and the expression of genes encoding the PGA synthase gene group and the PGA degrading enzyme is enhanced. Construction of fungal mutant (MGB874T_PGA_abrB * ΔkinAΔalsSD strain)

  First, using the Bacillus subtilis MGB874T_ΔpgsBCAEΔpgdS strain as a host, the primers alsSD-DF1, alsSD-DR1, alsSD-DF2, alsSD-DR2, Deletion of the alsSD gene group was performed using alsSD-IF, alsSD-IR, pAPNC-F and chpA-R.

  Next, by the same method as in (1-5), a mutation that enhances the expression of a gene encoding a PGA synthase gene group and a PGA degrading enzyme was introduced on the genome to construct a Bacillus subtilis mutant strain MGB874T_PGA_ΔalsSD.

  Finally, in the same manner as in (1-6), the abrB gene constitutive expression mutation was introduced, the kinA gene was deleted, the acetoin synthetic gene group was deleted, and the PGA synthase gene group and PGA degrading enzyme were A Bacillus subtilis mutant MGB874T_PGA_abrB * ΔkinAΔalsSD strain in which expression of the encoded gene was enhanced was constructed.

  Using the PCR method using the obtained genome of Bacillus subtilis, it was confirmed that the target gene group was introduced and deleted at a predetermined position on the genome.

Example 2 PGA Productivity Evaluation The Bacillus subtilis mutant obtained in Example 1 was treated with 2% glucose MYC pre-auto medium (2% glucose, 1.8% ammonium chloride, 0.15% dipotassium hydrogen phosphate, 0. 035% magnesium sulfate heptahydrate, 0.005% manganese sulfate pentahydrate, 50 mM MOPS (monophorinopropane sulfonic acid) buffer (pH 7.0), other trace metals, 0.05% yeast extract, 0 The cells were cultured for 24 hours at 30 ° C. with 0.02% casamino acid, 2% agar, and the obtained colonies were treated with 30 mL of 5% glucose M modified medium (5% glucose, 1.2% ammonium chloride, 0.15%). Dipotassium hydrogen phosphate, 0.035% magnesium sulfate heptahydrate, 0.005% manganese sulfate pentahydrate, 50 mM MOPS (monophorinopropanesulfonic acid) buffer Inoculated to an agent (pH 7.0, other trace metals) so that the OD600 is 0.02 to 0.1, and cultured with shaking at 30 ° C. for 12 to 16 hours. 5% glucose M modified medium (5% glucose, 1.2% ammonium chloride, 0.15% dipotassium hydrogen phosphate, 0.035% magnesium sulfate heptahydrate, 0.005% manganese sulfate pentahydrate, 50 mM MOPS (monophorinopropanesulfonic acid) buffer (pH 7.0), other trace metals, 4.0% calcium carbonate) was inoculated so that OD600 would be 0.05, and at 37 ° C. and 200 rpm for 1 day. Shaking culture was performed for 2 days. After completion of the culture, the PGA production amount was measured under the analysis conditions shown in the following measurement examples. The reached PGA production amount of each strain is shown as a relative value when the reached PGA production amount in the MGB874T_PGA strain is 100%.

  The results are shown in Table 8. A Bacillus subtilis mutant MGB874T_PGA_abrB * ΔkinAΔalsSD strain in which the abrB gene is constitutively expressed and the kinA gene is deleted and the alsSD gene group is further deleted is a Bacillus subtilis mutant MGB874T_PGA, a Bacillus subtilis mutant MGB874T_PGA_abrB * ΔkinA mutant Compared with the MGB874T_PGA_ΔalsSD strain, the reached PGA production amount was the highest.

<Quantitative analysis and molecular weight analysis of PGA>
The culture solution sample after completion of the culture is subjected to centrifugation at 14,800 rpm for 30 minutes at room temperature (Hitachi Koki, himacCF15RX). And quantitative analysis and molecular weight analysis. The HPLC apparatus used was a liquid feed pump (Shimadzu, LC-9A), an autosampler (Hitachi Measuring Instruments, AS-2000), a column oven (Shimadzu, CTO-6A), a UV detector (Shimadzu, SPD-10A), A degasser (GL Science, DG660B) and a chromatographic data analyzer (Hitachi Instrumentation, D-2500) were connected and used. The analytical columns were two types of gel filtration columns for hydrophilic polymer TSKgel G6000PWXL (7.8 mm ID × 30 cm, exclusion limit> 5 × 10 ⁷ ) and TSKgel G4000PWXL (7.8 mm I) with different exclusion limits. D. × 30 cm, exclusion limit 1 × 10 ⁶ ), and guard column TSK guardcolumn PWXL (6.0 mm ID × 4.0 cm) was connected immediately before these analytical columns. The eluent used was 0.1 M sodium sulfate, the flow rate was 1.0 mL / min, the column temperature was 50 ° C., and the detection wavelength of the elution peak was 210 nm. The sample injection amount was 50 μL per analysis using a sample loop (rheodyne). Samples to be subjected to HPLC analysis were prepared by filtering with MULTI SCREEN MNHV45 (manufactured by MILLIPORE, 0.45 μm Durapore membrane) as a pretreatment for removing insoluble matters. For the concentration test, a calibration curve was prepared using PGA (Meiji Food Material) with a molecular weight of 800,000.

Reference example Confirmation of abrB gene constitutive expression In order to confirm that the abrB gene is constitutively expressed by the abrB gene constitutive expression mutation, a mutant strain in which a lacZ gene and a promoter region are introduced immediately before is constructed, and β-galactosidase is constructed. The assay was performed.
(1-1) Construction of Bacillus subtilis mutant strain (OC-014 strain) into which the lacZ gene was introduced First, the lacZ gene was introduced into the Bacillus subtilis amyE gene region. Bacillus subtilis wild strain 168 was transformed with pDL2 plasmid (Fukuchi K. et al., Microbiology, 2000, 146: 1573-1583), and it has lacZ gene and chloramphenicol resistance gene in the amyE gene region A Bacillus subtilis mutant OC-014 strain was constructed.

(1-2) Construction of a Bacillus subtilis mutant strain (OA-021 strain and OA-022 strain) into which a wild-type abrB promoter region or abrB gene constitutive expression mutant abrB promoter region and a lacZ gene are introduced

  Using the genomic DNA of the Bacillus subtilis mutant OC-014 strain as a template and using the primers lacZ-FF and lacZ-FR listed in Table 9, the fragment (A) of the lacZ upstream region including the amyE gene upstream region was amplified by PCR. . Further, using the primers lacZ-BF and lacZ-BR shown in Table 9, a fragment (B) of the lacZ gene midstream region containing the SD sequence of the lacZ gene was amplified by PCR. Further, using the genomic DNA of Bacillus subtilis wild strain 168 as a template and using the primers abrBP-lacZ-F and abrBP-lacZ-R described in Table 9, the fragment (C) of the wild type abrB promoter region was amplified by PCR. . Next, using the plasmid pJL62 carrying the spectinomycin resistance gene (LeDeaux and Grossman, J. Bacteriol., 1995, 177: 166-175) as a template and using the primers rPCR-specF and rPCR-specR described in Table 9, Amplification was performed, and a spectinomycin resistance gene fragment (D) was PCR amplified.

  Next, the obtained fragment (A) and fragment (D) were bound by the OE-PCR method using primers lacZ-FF and rPCR-specF described in Table 9, and fragment (C) and fragment (B) were displayed. The primers abrBP-lacZ-F and lacZ-BR described in 9 were used for binding by the OE-PCR method. Finally, the fragment (A + D) and the fragment (C + B) were ligated by the OE-PCR method using the primers lacZ-FF and lacZ-BR described in Table 9 to obtain the final PCR product (A + D + C + B). The resulting final PCR product is used to transform the Bacillus subtilis mutant OC-014 strain to obtain a chloramphenicol sensitive and spectinomycin resistant transformant, and the wild type abrB promoter region and the lacZ gene are introduced. B. subtilis mutant OA-021 was obtained.

  Next, using the genomic DNA of the Bacillus subtilis mutant 168_abrB * _Cm as a template and using the primers abrBP-lacZ-F and abrBP-lacZ-R described in Table 9, a fragment of the abrB gene constitutive expression mutant abrB promoter region ( E) was amplified by PCR. The final PCR product (A + D + E + B) was obtained in the same manner as described above. The resulting final PCR product is used to transform Bacillus subtilis mutant OC-014 strain to obtain a chloramphenicol sensitive and spectinomycin resistant transformant, and an abrB gene constitutive expression mutant abrB promoter region and A Bacillus subtilis mutant OA-022 introduced with the lacZ gene was obtained.

  It was confirmed that the target mutation was introduced at a predetermined position on the genome by PCR using the genome of each obtained Bacillus subtilis mutant strain and subsequent sequencing by the Sanger method.

(1-3) β-galactosidase assay The Bacillus subtilis mutant OA-021 and Bacillus subtilis mutant OA-022 constructed in (1-2) were streaked on an LB agar medium containing spectinomycin to obtain The obtained colonies were inoculated into S7 (50) medium (Jacks K., et al., J. Bacteriol., 1989, 171: 4121-4129) and cultured at 37 ° C. with shaking. After cultivation, the cells were collected by centrifugation, and the activity of β-galactosider was measured using the obtained cells.

  As a result, in the case of the Bacillus subtilis mutant OA-021 strain having the wild type abrB promoter region, transcriptional repression is observed when entering the stationary phase, but the Bacillus subtilis mutant strain OA- having the abrB gene constitutive expression mutant abrB promoter region is observed. The 022 strain showed high transcriptional activity even in the stationary phase (FIG. 4). From the above, it was suggested that the abrB gene is constantly expressed in the abrB gene constitutive expression mutant.

<Measurement of β-galactosidase activity>
β-galactosidase activity was measured by suspending the collected cells in 1 mL of Z buffer (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 10 mM KCl, 1 mM MgSO ₄ .7H ₂ O and 1 mM DTT). 10 μL of sample solution was mixed with 0.63 mL of Z buffer. Next, 0.16 mL of lysozyme / DNase I solution (2.5 mg / mL lysozyme and 0.05 mg / mL DNase I) dissolved in Z buffer was added to the obtained mixed solution, and the mixture was kept at 37 ° C. for 5 minutes. Next, 8 μL of 10% Trition-X100 was added, stirred and allowed to stand on ice. The prepared final sample solution was incubated at 30 ° C. for 3 minutes, and 0.2 mL of ONPG solution (4.0 mg / mL o-Nitrophenyl-BD-galactoside (ONPG)) dissolved in Z buffer was added and mixed, and yellowed. Keep warm until colored. Finally, 0.4 mL of Na ₂ CO ₃ solution was added to stop the reaction, the absorbance at 420 nm (OD 420 nm) was measured, and the activity value calculated from the following formula (1) was determined.

Formula (1)
Miller unit = (1000 × OD420nm) / (T × V × OD600 unit of cell)

OD600 unit of cell: Turbidity at the time of collection (OD600 nm)
T: Reaction time (min)
V: Volume of culture solution used for activity measurement (mL)

Claims (16)

Bacillus subtilis mutant strain,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region Having one or more selected regions of the deleted genome;
The gene is constructed so that the abrB gene or a gene equivalent thereto is constitutively expressed, the kinA gene is deleted or inactivated, and at least one gene selected from the group consisting of the alsS gene and the alsD gene is missing. Bacillus subtilis mutant strain that has been inactivated or inactivated.   profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region a genome having a deleted cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region, The Bacillus subtilis mutant according to claim 1.   The gene construction that constitutively expresses the abrB gene or a gene corresponding thereto is a transcription initiation control region of the abrB gene or a gene equivalent thereto, or a transcription initiation control region and a ribosome binding site, or a transcription initiation control region The Bacillus subtilis mutant according to claim 1 or 2, which is to inactivate a binding site of an inhibitory regulatory factor in a region ranging from a translation initiation control region to a translation initiation control region.   The transcription initiation control region of the abrB gene or a gene corresponding thereto, the transcription initiation control region and the ribosome binding site, or the binding site of the inhibitory regulatory factor in the region from the transcription initiation control region to the translation initiation control region is SEQ ID NO: The Bacillus subtilis according to claim 3, which is a region consisting of the 20th to 49th bases in the base sequence shown in Fig. 9 or a region corresponding thereto, and / or a region consisting of the 73rd to 89th bases or a region corresponding thereto. Mutant strain.   Gene construction in which the abrB gene or the gene corresponding thereto is constantly expressed is to delete the region consisting of the 73-89th bases in the base sequence represented by SEQ ID NO: 9 or the region corresponding thereto. The Bacillus subtilis mutant according to claim 4. The Bacillus subtilis mutant according to any one of claims 1 to 5, wherein the abrB gene or a gene corresponding thereto is selected from the group consisting of the following (a) to (f).
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
(B) a protein comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and having activity as a transcription factor of a gene induced in the transition phase and stationary phase similar to AbrB A polynucleotide to:
(C) As a transcription factor for a gene that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and is induced in the transition phase and stationary phase similar to AbrB A polynucleotide encoding a protein having activity;
(D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) a transcription factor of a gene consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in the transition phase and stationary phase similar to AbrB A polynucleotide encoding a protein having activity as:
(F) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having activity as a transcription factor of a gene induced in the transition phase and stationary phase similar to AbrB Polynucleotide. The Bacillus subtilis mutant according to any one of claims 1 to 6, wherein the kinA gene is selected from the group consisting of the following (g) to (l).
(G) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3;
(H) a polynucleotide encoding a protein comprising the nucleotide sequence shown in SEQ ID NO: 3 and having a nucleotide sequence having 90% or more identity and having an activity as a histidine kinase of a multicomponent regulatory system involved in the early stage of sporulation;
(I) a protein that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 and has activity as a histidine kinase of a multi-component regulatory system involved in the early stage of sporulation The encoding polynucleotide;
(J) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4;
(K) The activity as a histidine kinase of a multi-component control system which consists of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 4 and which is involved in the early stage of sporulation A polynucleotide encoding a protein having;
(L) A polynucleotide encoding a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4 and having activity as a histidine kinase of a multicomponent regulatory system involved in the early stage of sporulation. The Bacillus subtilis mutant according to any one of claims 1 to 7, wherein the alsS gene is selected from the group consisting of the following (m) to (r).
(M) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5;
(N) a polynucleotide comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 5 and encoding a protein having α-acetolactic acid synthase activity;
(O) a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 and encodes a protein having α-acetolactic acid synthase activity;
(P) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;
(Q) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 6 and encoding a protein having α-acetolactic acid synthase activity;
(R) A polynucleotide encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 6 and having an amino acid sequence of 90% or more and having α-acetolactic acid synthase activity. The Bacillus subtilis mutant according to any one of claims 1 to 8, wherein the alsD gene is selected from the group consisting of the following (s) to (x).
(S) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7;
(T) a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 7 and having a nucleotide sequence of 90% or more and encoding a protein having α-acetolactic acid decarboxylase activity;
(U) a polynucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 7 and encodes a protein having α-acetolactic acid decarboxylase activity;
(V) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8;
(W) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 8, and encoding a protein having α-acetolactic acid decarboxylase activity;
(X) A polynucleotide encoding a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 8 and having α-acetolactic acid decarboxylase activity.   The Bacillus subtilis mutant according to any one of claims 1 to 9, wherein both the alsS gene and the alsD gene are deleted or inactivated.   The Bacillus subtilis mutant according to any one of claims 1 to 10, wherein the poly-γ-glutamate synthase gene, or the poly-γ-glutamate synthase gene and the poly-γ-glutamate degrading enzyme gene are enhanced to be expressed. Or the introduced recombinant Bacillus subtilis.   The gene encoding poly-γ-glutamate synthase is at least one gene selected from the group consisting of the Bacillus subtilis pgsB gene, the pgsC gene, the pgsA gene and the pgsE gene, and a gene corresponding thereto; The recombinant Bacillus subtilis according to claim 11, wherein the γ-glutamic acid degrading enzyme gene is a Bacillus subtilis pgdS gene or a gene corresponding thereto.   13. The poly-γ-glutamate synthase gene and the poly-γ-glutamate degrading enzyme gene are the Bacillus subtilis pgsB gene, pgsC gene, pgsA gene, pgsE gene and pgdS gene, or genes corresponding thereto. Recombinant Bacillus subtilis. A method for producing a Bacillus subtilis mutant,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region In a Bacillus subtilis mutant strain having a genome in which one or more selected regions are deleted, the AbrB gene or a gene corresponding thereto is constantly expressed. A method comprising a step of constructing a gene, a step of deleting or inactivating a kinA gene, and a step of deleting or inactivating at least one gene selected from the group consisting of an alsS gene and an alsD gene. A method for producing recombinant Bacillus subtilis,
profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, profile7 region (yrkM-yraK or yrkS-yraK), sbo-ywhH region, yybP-yyaJ region and yncM-fosB region In a Bacillus subtilis mutant strain having a genome in which one or more selected regions are deleted, the abrB gene or a gene equivalent thereto is constitutively expressed. A step of constructing a gene, a step of deleting or inactivating the kinA gene, a step of deleting or inactivating at least one gene selected from the group consisting of an alsS gene and an alsD gene, and poly-γ-glutamic acid synthesis A method comprising the step of enhancing or introducing an enzyme gene, or a poly-γ-glutamate synthase gene and a poly-γ-glutamate degrading enzyme gene in an expressible manner.   A method for producing poly-γ-glutamic acid comprising culturing the recombinant Bacillus subtilis according to any one of claims 10 to 13 and recovering poly-γ-glutamic acid from the culture.