JP6694696B2 - Bacillus subtilis mutant strain and method for producing dipicolinic acid using the same - Google Patents

Description

  The present invention relates to a Bacillus subtilis mutant having dipicolinic acid-producing ability, and a method for producing dipicolinic acid or a salt thereof using the Bacillus subtilis mutant.

  Dipicolinic acid (2,6-pyridinedicarboxylic acid, DPA) is a natural product and is known as a highly biodegradable chelating agent, and is used as a detergent composition, a metal hiding agent, an antioxidant, etc. for industrial use. It is reported to be available.

  Dipicolinic acid is known to be synthesized from 2,6-lutidine by an oxidation reaction. That is, it is known that it can be synthesized by a method using a hypohalite as an oxidizing agent in the presence of a nickel compound, an electrolytic oxidation method, an air oxidation method, or the like. However, in these methods, the conversion rate and selectivity of the reaction are insufficient, so that the raw material 2,6-lutidine or the reaction intermediate alkylpyridine such as 6-methyl-2-pyridinecarboxylic acid is used as the reaction liquid. Remain inside, and these are mixed as impurities in the product 2,6-pyridinedicarboxylic acid (dipicolinic acid). That is, it has been difficult to produce high-purity dipicolinic acid by the conventional dipicolinic acid synthesis method.

  On the other hand, according to the method for producing dipicolinic acid using a microorganism, it is expected that dipicolinic acid can be produced in high purity without containing alkylpyridine. As a method for producing dipicolinic acid using a microorganism, a fermentation method (Patent Documents 1 and 2) using a Bacillus genus, which is a spore-forming microorganism, and a mold method (Patent Document 3) )It has been known.

  Further, a method for producing dipicolinic acid using a genetically modified microorganism is known. Patent Document 4 discloses a method for producing dipicolinic acid using a recombinant lysine-producing bacterium. Patent Document 5 discloses a method for producing dipicolinic acid using a microorganism belonging to the genus Bacillus or Clostridium in which the expression pattern of a gene involved in the dipicolinic acid synthesis pathway is modified.

  Patent Document 6 describes that a Bacillus subtilis mutant strain obtained by deleting a large region of the genome is excellent in productivity of enzymes such as protease. However, this mutant microorganism is a strain developed for enzyme production.

JP 2004-275075 A German Patent No. 2300056 U.S. Pat. No. 3,333,021 JP-A-2002-371063 JP, 2008-048732, A JP, 2007-130013, A

  The present invention provides a Bacillus subtilis mutant strain capable of highly producing dipicolinic acid (2,6-pyridinedicarboxylic acid, also referred to as DPA in the following specification), a method for producing the Bacillus subtilis mutant strain, and the Bacillus subtilis. The present invention relates to a method for producing DPA or a salt thereof using a bacterial mutant strain.

  The present inventors proceeded with the development of a microbial strain capable of efficiently producing DPA. As a result, in the Bacillus subtilis mutant strain in which the large region of the genome was deleted, enhancing the expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto, both the gene relating to acetoin synthesis and the gene relating to acetic acid synthesis were used together. It was found that the Bacillus subtilis mutant strain obtained by deletion or inactivation has a remarkably high DPA-producing ability, and completed the present invention.

That is, the present invention provides, in one aspect, the following:
A Bacillus subtilis mutant strain,
Prophage 6 region, Prophage 1 region, Prophage 4 region, PBSX region, Prophage 5 region, Prophage 3 region, Spb region, PKs region, Skin region, pps region, Prophage 2 region, ydcL-ydeK-ydhUy region, DysL, DyBy, DysBy, DysY, DysBy, DysBy, DysBy. One or more regions selected from the group consisting of the cgeE-ypmQ region, the yeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yayaJ region, and the ncM-fosB region. Has a deleted genome,
Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced in expression, and at least one gene selected from the group consisting of alsS gene and alsD gene is deleted or inactivated, and ackA gene and A Bacillus subtilis mutant strain in which at least one gene selected from the group consisting of pta genes is deleted or inactivated.

In another aspect, the invention provides:
A method for producing a Bacillus subtilis mutant strain,
Prophage 6 region, Prophage 1 region, Prophage 4 region, PBSX region, Prophage 5 region, Prophage 3 region, Spb region, PKs region, Skin region, pps region, Prophage 2 region, ydcL-ydeK-ydhUy region, DysL, DyBy, DysBy, DysY, DysBy, DysBy, DysBy. One or more regions selected from the group consisting of the cgeE-ypmQ region, the yeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yayaJ region, and the ncM-fosB region. Enhances the expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto in a Bacillus subtilis mutant strain having a deleted genome, and
deleting or inactivating at least one gene selected from the group consisting of alsS gene and alsD gene, and deleting or inactivating at least one gene selected from the group consisting of ackA gene and pta gene, A method, including:

  In another aspect, the present invention provides a method for producing dipicolinic acid or a salt thereof using the Bacillus subtilis mutant strain.

  The Bacillus subtilis mutant strain of the present invention has a high DPA producing ability. By using the Bacillus subtilis mutant strain of the present invention, it becomes possible to efficiently produce DPA or a salt thereof.

FIG. 3 is a schematic view showing an example of a method for deleting a predetermined region from the genome of Bacillus subtilis. FIG. 3 is a schematic diagram showing a procedure for removing a foreign drug resistance gene from the Bacillus subtilis genome. The schematic diagram which shows one Embodiment regarding the introduction procedure and the introduction position of a dipicolinate synthase gene to a Bacillus subtilis mutant strain. FIG. 3 is a schematic diagram showing a procedure for deleting a specific gene from a Bacillus subtilis mutant strain by a marker-free deletion method using a mazF gene fusion cassette.

The names of the respective genes and genomic regions of Bacillus subtilis described in the present specification are JAFAN: Japan.
It is described based on the Bacillus subtilis genomic data published on the Internet ([bacillus.genome.ad.jp/], updated on January 18, 2006) in the Functional Analysis Network for Bacillus subtilis (BSORF DB).

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

In the present specification, unless otherwise defined, the “several” of “one or several” used for deletion, substitution, addition or insertion of an amino acid or nucleotide in an amino acid sequence or nucleotide sequence means, for example, The maximum integer of 10% or less of the total number of amino acid residues or the total number of nucleotides of the amino acid sequence or the nucleotide sequence of It also means a maximum integer of 3% or less, more preferably a maximum integer of 2% or less, even more preferably a maximum integer of 1% or less. In the present specification, "addition" of amino acids or nucleotides includes addition of one or several amino acids or nucleotides at one end and both ends of the sequence.

In the present specification, “stringent conditions” regarding hybridization are conditions that allow confirmation of a gene having a nucleotide sequence with a sequence identity of about 80% or more or about 90% or more. "Stringent conditions" include Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold.
The conditions described in Spring Harbor Laboratory Press, 2001) are mentioned. 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. As an example, the above “stringent condition” means that the hybridization condition is preferably 5 × SSC, 70 ° C. or higher, more preferably 5 × SSC, 85 ° C. or higher, and the washing condition is 1 × SSC. , 60 ° C. or higher, preferably 1 × SSC, more preferably 73 ° C. or higher. The combination of the above SSC and temperature conditions is an exemplification, and a person skilled in the art can realize an appropriate stringency by appropriately combining the above or other elements that determine the stringency of hybridization.

  In the present specification, the terms “upstream” and “downstream” of a gene or region refer to regions following the 5 ′ side and the 3 ′ side of the gene or region captured as a target, respectively.

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

  In the present specification, enhancing the expression of a target gene means increasing the expression level of the target gene as compared with before modification. As means for increasing the expression level of the target gene, for example, placing the target gene on the genome under the control of a control region (strong expression control region) capable of enhancing the expression of the gene as compared with the wild type, Examples include introduction of a gene of interest placed under the control of a strong expression control region into the genome of a cell or a plasmid, or modification for allowing a plurality of genes of interest to be expressed in a cell. As a modification for allowing a plurality of target genes to be expressed in a cell, a target gene may be further introduced, together with a control region, into a genome or a plasmid in a cell in which the target genes are expressed. Alternatively, a plurality of target genes may be introduced together with a control region, if necessary, into the genome of a cell or into a plasmid to modify the number of target genes expressed in the cell.

  In the present specification, “arranging a target gene under the control of a control region” refers to a control region and a target gene on DNA so that the function of controlling the expression of the gene by the control region acts on the target gene. It means placing. Examples of the method of placing the target gene under the control of the control region include a method of ligating the target gene downstream of the control of the control region.

(1. Production of Bacillus subtilis mutant strain)
(1-1. Genomic deletion Bacillus subtilis mutant strain)
The Bacillus subtilis mutant strain of the present invention has a genome in which a large region on the genome of a Bacillus subtilis wild strain is deleted, expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced, and alsS At least one gene selected from the group consisting of a gene and an asD gene is deleted or inactivated, and at least one gene selected from the group consisting of an ackA gene and a pta gene is deleted or inactivated It is a Bacillus subtilis mutant strain. Preferably, the Bacillus subtilis mutant strain of the present invention is a modification to enhance the expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto, with respect to the Bacillus subtilis mutant strain in which the large region of the genome is deleted, and A modification that deletes or inactivates at least one gene selected from the gene and the alsD gene, and a modification that deletes or inactivates at least one gene selected from the group consisting of the ackA gene and the pta gene It is made by adding.

  The Bacillus subtilis mutant strain in which the large region of the genome is deleted is the genome of Bacillus subtilis wild strain (Bacillus subtilis Marburg No. 168; hereinafter referred to as Bacillus subtilis 168 strain or simply 168 strain, or wild strain). Compared to, it has a genome in which a large region in its genome has been 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 (AppsEpsEp). , Profile2 (ydcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeK-yesX region, pdp-rocR region, ycxB-SipU region, ycxB-SipU region. spoIVCB-yraK) region, sbo-ywhH region, yybP-yayaJ region, and yncM-fosB region are deleted. The region means a region of a gene sandwiched between specific genes, and the region is deleted means that the entire region sandwiched between specific genes is deleted.

  The Bacillus subtilis mutant strain is preferably, in the genome of a Bacillus subtilis wild strain, a phase6 (yoaV-yobO) region, a profile1 (ybbU-ybdE) region, a profile4 (yjcM-yjdJ) region, a 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 (AppsEpsEp). , Profile2 (ydcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeK-yesX region, pdp-rocR region, ycxB-SipU region, ycxB-SipU region. spoIVCB-yraK) region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region all lacking MGB874 strain (Japanese Patent Laid-Open No. 2007-130013) and the genome of Bacillus subtilis wild strain, Prophage6 (yoaV-yobO) region, prophase1 (ybbU-ybdE) region, propage4 (yjcM-yjdJ) region, PBSX (ykdA-xlyA) region, prophase5 (ynxB-dyt) 3, progage5 (ynxB-dut) region, prog. yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region, profile2 (ydcL-ydeJ) region, and a region selected from prophage7 (yrkM-yraK) region. The OA105 strain lacking all of the regions described above is more preferable, and the MGB874 strain is more preferable.

  The Bacillus subtilis mutant strain can be prepared, for example, by deleting the above-mentioned genomic region from any Bacillus subtilis strain such as 168 strain. The genomic region of interest to be deleted can be determined, for example, by comparing the genome of the arbitrary Bacillus subtilis strain with the genome of the published Bacillus subtilis strain 168. Information on the entire nucleotide sequence and genome of Bacillus subtilis strain 168 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 genomic region of interest to be deleted based on the genomic information of Bacillus subtilis strain 168 obtained from these sources. Here, the genomic region to be deleted is one or several (for example, 1 to 100, preferably 1 to 50, with respect to the nucleotide sequence of the above-mentioned genomic region in the published Bacillus subtilis 168 strain. (More preferably 1 to 30, even more preferably 1 to 10 and even more preferably 1 to 5) nucleotides containing mutations such as deletions, substitutions, insertions and additions caused by natural or artificial conditions. It can be a genomic region having a sequence. Alternatively, the genomic region to be deleted is preferably 80% or more identical in nucleotide sequence, more preferably 90% or more identical, still more preferably 95% to the above-mentioned genomic region of the published Bacillus subtilis strain 168. Genomic regions that are at least% identical may be present.

  Alternatively, the genomic region to be deleted can be expressed as a region sandwiched by a pair of oligonucleotide sets shown in Table 1. The method for deleting the region shown in Table 1 from the Bacillus subtilis genome is not particularly limited, but prepared by, for example, the SOE-PCR method (splicing by overlap extension PCR: Gene, 1989, 77: 61-68). A double-crossing method using a DNA fragment for deletion can be mentioned. The procedure for producing a mutant strain in which a predetermined genomic region has been deleted from a Bacillus subtilis wild strain by the method is described in detail in Japanese Patent Laid-Open No. 2007-130013, and the outline will be described below.

  An outline of the procedure 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 is shown in FIG. First, by the SOE-PCR method, a fragment (referred to as an upstream fragment) corresponding to a region of about 0.1 to 3 kb adjacent to the upstream of the target region to be deleted, and a fragment of about 0.1 to 3 kb adjacent to the same downstream. A DNA fragment in which a fragment corresponding to the region (referred to as a downstream fragment) is ligated 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 ligated between the upstream fragment and the downstream fragment is prepared.

  First, three fragments of an upstream fragment A and a downstream fragment B of the region to be deleted, and a marker gene fragment (in Fig. 1, chloramphenicol resistance gene fragment Cm) are prepared by the first PCR. To do. In PCR amplification of the upstream fragment and the downstream fragment, a primer to which a sequence of 10 to 30 nucleotides at the end of the fragment to be ligated later is added is used. For example, when the upstream fragment A, the drug resistance marker gene fragment Cm, and the downstream fragment B are ligated in this order, the 5 ′ end of the primer that binds to the downstream end of the upstream fragment A has the upstream side of the drug resistance marker gene fragment Cm. A sequence corresponding to 10 to 30 nucleotides was added (FIG. 1, primer DR1), and the 5 ′ end of the primer binding to the upstream end of the downstream fragment B was added to the downstream 10 to 30 nucleotides of the drug resistance marker gene fragment Cm. Add the corresponding sequence (Figure 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 ′. As a result, the 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 ′ prepared by the first PCR, the drug resistance marker gene fragment Cm, and the downstream fragment B ′ were mixed and used as a template, and the primer was bound to the upstream side of the upstream fragment (FIG. 1, primer DF1). And a pair of primers consisting of a primer (FIG. 1, primer DR2) that binds to the downstream side of the downstream fragment is used to perform the second PCR. By the second PCR, the deletion DNA fragment D in which the upstream fragment A, the drug resistance marker gene fragment Cm, and the downstream fragment B are linked in this order can be amplified.

  The deletion DNA fragment obtained by the method described above is inserted into a plasmid using a usual restriction enzyme and DNA ligase to construct a deletion-introducing plasmid. Alternatively, by preparing a deletion DNA fragment in which an upstream fragment and a downstream fragment are directly ligated, and then inserting the deletion DNA fragment into a plasmid containing a drug resistance marker gene, the deletion fragment is added to the upstream fragment and the downstream fragment. A deletion-introducing plasmid having a resistance marker gene fragment can be constructed.

  The deletion-introducing plasmid constructed by the above procedure is introduced into Bacillus subtilis in which the genomic region is to be deleted by a conventional method such as the competent cell transformation method. The introduction of the plasmid causes double crossover homologous recombination between the upstream fragment and the downstream fragment on the plasmid and the region homologous to those of the Bacillus subtilis genome, and the region to be deleted becomes the drug resistance marker gene. A displaced transformant is obtained (Fig. 1). The transformant may be selected by using the expression of a marker gene such as a drug resistance gene present in the DNA fragment for deletion as an index. For example, by culturing a bacterium transformed with a chloramphenicol resistance gene fragment in a medium containing an antibiotic (chloramphenicol etc.) and recovering the grown colonies, the target region is deleted. It is possible to obtain a transformant in which the chloramphenicol resistance gene is replaced. 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 is 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 (JP-A-2009-254350). In this method, first, a DNA fragment (donor DNA) for the first homologous recombination is prepared. The method of preparation is not particularly limited, and examples thereof include the SOE-PCR method described above. Examples of the donor DNA include, for example, a fragment of about 0.1 to 3 kb (upstream fragment) corresponding to a region adjacent to the upstream of a marker gene region to be removed (that is, a deleted region) and a region adjacent to the same downstream. It is possible to use a DNA fragment in which a fragment to which about 0.1 to 3 kb fragment (downstream fragment) corresponding to is linked to a fragment of the marker gene downstream region to be removed is ligated. Preferably, a DNA fragment in which a second marker gene or the like serving as an indicator of homologous recombination is inserted between the downstream fragment and the first marker gene downstream region fragment to be removed is used (see FIG. 2). reference).

  Then, the prepared donor DNA is introduced into the transformant by a usual method such as the competent cell transformation method, and corresponds to the upstream fragment and the first marker gene downstream region on the transformant genome. Homologous recombination with the region is caused (first homologous recombination). The transformant in which the desired homologous recombination has occurred can be selected using the expression of the second marker gene inserted in the donor DNA as an index. In the genomic DNA of the transformant in which the first homologous recombination was appropriately generated, the upstream fragment, the downstream fragment, the second marker gene, the first marker gene downstream region, and the downstream fragment were arranged in order. (See FIG. 2). In the genomic DNA having such an arrangement, homologous recombination can occur spontaneously between the above two downstream fragments (intragenomic homologous recombination). By this homologous recombination within the genome, the first marker gene is removed from the transformant genome by deleting the region located between the two downstream fragments.

  Examples of means for selecting a transformant that has undergone homologous recombination within the genome include a method of selecting a bacterium that does not have drug resistance when the first marker gene is a drug resistance gene. Penicillin antibiotics act bactericidally on proliferating cells but not on non-proliferating cells. Therefore, by culturing the bacterium in the presence of the drug and the penicillin antibiotic, drug-non-resistant bacteria that do not grow in the presence of the drug can be selectively concentrated (Methods in Molecular Genetics, Cold Spring Harbor Labs, 1970). .. Another means is to introduce a lethal gene. For example, if a lethal gene such as the chpA gene is introduced into the bacterium as the second marker, the bacterium that did not undergo homologous recombination within the genome will die due to the action of the lethal gene. Transformants can be selected. It is possible to confirm that the target region is deleted by extracting genomic DNA from the selected strain and performing PCR using this as a template (Japanese Patent Laid-Open No. 2009-254350).

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

(1-2. Enhanced dipicolinate synthase gene expression)
Then, in the Bacillus subtilis mutant strain lacking the predetermined region, the expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced.

  In the present specification, the dipicolinate synthase gene is a gene encoding dipicolinate synthase having an activity of promoting the chemical reaction of the following formula (1).

  Dipicolinate synthase is composed of subunit A and subunit B, and there are genes encoding each subunit. Examples of the Bacillus subtilis dipicolinate synthase gene include spoVFA encoding dipicolinate synthase subunit A and spoVFB encoding dipicolinate synthase subunit B. The nucleotide sequence of Bacillus subtilis spoVFA gene and the amino acid sequence of dipicolinate synthase subunit A encoded by the spoVFA gene are shown in SEQ ID NOS: 1 and 2, respectively. The nucleotide sequence of Bacillus subtilis spoVFB gene and the amino acid sequence of dipicolinate synthase subunit B encoded by the spoVFB gene are shown in SEQ ID NOS: 3 and 4, respectively.

  As a gene corresponding to the Bacillus subtilis dipicolinate synthase gene, the Bacillus subtilis spoVFA gene and at least 80%, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more Preferably, it comprises a nucleotide sequence having an identity of 97% or more, more preferably 98% or more, more preferably 99% or more, and works together with Bacillus subtilis dipicolinate synthase subunit B to catalyze the above-described dipicolinic acid synthesis reaction. A polynucleotide encoding a protein that exerts an activity (dipicolinate synthase activity) that activates

  Alternatively, as the gene corresponding to the Bacillus subtilis dipicolinate synthase gene, at least 80%, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more with the Bacillus subtilis spoVFB gene. , More preferably 97% or more, more preferably 98% or more, more preferably 99% or more of a nucleotide sequence having an identity, and working together with Bacillus subtilis dipicolinate synthase subunit A, the dipicolinic acid synthesis reaction described above. Examples include a polynucleotide encoding a protein that exerts an activity that catalyzes (dipicolinate synthase activity).

  Furthermore, examples of the gene corresponding to the Bacillus subtilis dipicolinate synthase gene include a gene encoding a dipicolinate synthase subunit A or B derived from a microorganism other than Bacillus subtilis. Examples of such genes include spoVFA (SEQ ID NO: 5) and spoVFB (SEQ ID NO: 6) from Bacillus licheniformis, spoVFA (SEQ ID NO: 7) and spoVFB (SEQ ID NO: 8) from Bacillus clausii, spoVFA from Clostridium stercorarium. (SEQ ID NO: 9) and spoVFB (SEQ ID NO: 10), and spoVFA (SEQ ID NO: 11) and spoVFB (SEQ ID NO: 12) derived from Bacillus amyloliquefaciens.

  In the Bacillus subtilis mutant strain of the present invention, any one of the above-mentioned Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto, or any two or more, may be enhanced in expression. Preferably, a gene encoding subunit A of Bacillus subtilis dipicolinate synthase or a gene corresponding thereto and a gene encoding subunit B of Bacillus subtilis dipicolinate synthase or a gene corresponding thereto are combined. It is good that the expression is enhanced. More preferably, the expression is enhanced by combining the genes encoding subunit A and subunit B of Bacillus subtilis dipicolinate synthase described above.

Preferred examples of the Bacillus subtilis dipicolinate synthase gene whose expression is enhanced in the Bacillus subtilis mutant strain of the present invention or a gene corresponding thereto are selected from the group consisting of the genes described in (i) and (ii) below. At least one is mentioned.
(I) At least one Bacillus subtilis dipicolinate synthase subunit A gene selected from the group consisting of the following (a) to (f) or a gene corresponding thereto:
(A) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) at least 80%, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more with the nucleotide sequence shown in SEQ ID NO: 1; Poly, which encodes a protein having a nucleotide sequence having an identity of preferably 98% or more, more preferably 99% or more, and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene according to (ii) nucleotide;
(C) Dipicolinate synthase activity in the presence of a protein encoded by the gene described in (ii), which hybridizes to a complementary strand of a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions A polynucleotide encoding a protein that exerts
(D) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 2;
(E) Dipicoline in the presence of the protein encoded by the gene described in (ii), 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: 2 A polynucleotide encoding a protein exhibiting acid synthase activity, wherein several is 59, preferably 44, more preferably 29, more preferably 14, more preferably 5, and more preferably 2. Individual; and
(F) At least 80%, preferably 80% or more, more preferably 85%, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably with the amino acid sequence shown in SEQ ID NO: 2. Is a polynucleotide consisting of an amino acid sequence having 99% or more identity, and encoding a protein exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene according to (ii);
(Ii) at least one Bacillus subtilis dipicolinate synthase subunit B gene selected from the group consisting of the following (j) to (o) or a gene corresponding thereto:
(J) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3;
(K) at least 80%, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, with the nucleotide sequence shown in SEQ ID NO: 3; Poly, which encodes a protein having a nucleotide sequence having an identity of preferably 98% or more, more preferably 99% or more, and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) nucleotide;
(L) Dipicolinic acid synthase activity in the presence of a protein encoded by the gene described in (i), which hybridizes to a complementary strand of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 under stringent conditions A polynucleotide encoding a protein that exerts
(M) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 4;
(N) Dipicoline in the presence of the protein encoded by the gene described in (i), 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. A polynucleotide encoding a protein exhibiting acid synthase activity, wherein several is 40, preferably 30, more preferably 20, more preferably 10, more preferably 4, more preferably 2. Individual; and
(O) At least 80%, preferably 80% or more, more preferably 85%, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably with the amino acid sequence shown in SEQ ID NO: 4. Is a polynucleotide consisting of an amino acid sequence having 99% or more identity, and encoding a protein exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i).

  More preferably, in the Bacillus subtilis mutant strain of the present invention, expression is enhanced by combining the genes described in (i) and (ii) above. More preferably, in the Bacillus subtilis mutant strain of the present invention, a combination of the above-mentioned (i) (a) gene with at least one selected from the group consisting of the above-mentioned (ii) (j) to (o) genes Alternatively, the expression of the combination of at least one selected from the group consisting of the above-mentioned genes (i) (a) to (f) and the above-mentioned gene (ii) (j) is enhanced. In addition, preferably, in the Bacillus subtilis mutant strain of the present invention, the expression of the combination of the gene of (i) (a) and the gene of (ii) (j) is enhanced.

  Another preferable example of the Bacillus subtilis dipicolinate synthase gene or the gene corresponding thereto, the expression of which is enhanced in the Bacillus subtilis mutant strain of the present invention, includes the above-mentioned Bacillus subtilis spoVFA gene and spoVFB gene, Bacillus licheniformis spoVFA gene and spoVFB gene. Gene, at least one selected from the group consisting of Bacillus clausii spoVFA gene and spoVFB gene, Clostridium stercorarium spoVFA gene and spoVFB gene, and Bacillus amyloliquefaciens spoVFA gene and spoVFB gene. Among them, Bacillus subtilis spoVFA gene More preferably, at least one selected from the group consisting of Bacillus subtilis spoVFB gene.

  Still another preferable example of the Bacillus subtilis dipicolinate synthase gene whose expression is enhanced in the Bacillus subtilis mutant strain of the present invention or a gene corresponding thereto is, as a Bacillus subtilis spoVFA gene, a Bacillus licheniformis spoVFA gene, or a Bacillus clausii spoVFA gene. , The Clostridium stercorarium spoVFA gene and the Bacillus amyloliquefaciens spoVFA gene, and the Bacillus subtilis spoVFB gene, the Bacillus licheniformis spoVFB gene, the Bacillus clausii spoVFBsp gene, and the Clostridium spoVFA gene, and the Clostridium Clostridium gene. Examples thereof include a combination with at least one selected from the group consisting of amyloliquefaciens spoVFB genes, and among these, a combination of Bacillus subtilis spoVFA gene and Bacillus subtilis spoVFB gene is more preferable.

  As means for enhancing the expression of the dipicolinate synthase gene or a gene corresponding thereto, for example, the target gene on the genome, a control region capable of enhancing the expression of the gene compared to the wild type (strong expression control region) Of the target gene, which is placed under the control of a control region, preferably a strong expression control region, in addition to the dipicolinate synthase gene originally possessed by the host, Introduced into, and so on.

  For example, the Bacillus subtilis mutant strain of the present invention uses a Bacillus subtilis mutant strain having one or more, preferably all deletions of the above-mentioned genomic region as a host, and strongly expresses the control region sequence of the target dipicolinate synthase gene on the genome. It can be produced by substituting the control region. Alternatively, the Bacillus subtilis mutant strain of the present invention is prepared by introducing the dipicolinate synthase gene of interest placed under the control of a strong expression control region or a gene corresponding thereto into the genome of the host or into a plasmid. You can Alternatively, the expression of the target gene can be enhanced by modifying a plurality of target dipicolinate synthase genes or genes corresponding thereto to be operably present in the host.

  An example of a detailed procedure for enhancing dipicolinate synthase gene expression is described below. First, the control region and the dipicolinate synthase gene are arranged so that the gene is expressed under the control of the control region, and can be constructed as a DNA fragment bound to a region homologous to the host genome. For example, a fragment in which a regulatory region (for example, a spoVG gene regulatory region described later), a dipicolinic acid synthase subunit A gene, and a dipicolinic acid synthase subunit B gene are arranged in this order from the upstream, and the fragment is used as a host A DNA fragment is prepared by ligating the homologous region with a part of the genome. Then, when this DNA fragment is introduced into the above-mentioned host, the DNA fragment is inserted into the host genome and the host can be transformed. As a transformation method, an appropriate and general method can be adopted depending on the host Bacillus subtilis. Since the obtained transformant possesses a large number of dipicolinate synthase genes as compared with the host before transformation, the expression level of the genes is increased.

  Examples of the control region that can be used for enhancing the expression of the dipicolinate synthase gene in the present invention include a control region of an α-amylase gene derived from Bacillus bacterium, a control region of a protease gene, a gene encoding a ribosomal protein (rplS gene, etc. ) Control region, aprE gene control region, or spoVG gene control region; Bacillus sp. The control region of the cellulase gene of the KSM-S237 strain; the control region of the kanamycin resistance gene derived from Staphylococcus aureus, the control region of the chloramphenicol resistance gene, etc. (both refer to JP-A-2009-089708) are exemplified. However, it is not particularly limited. A control region capable of enhancing the expression of the target gene is preferable as compared to the wild-type control region of the target gene.

(1-3. Deletion or inactivation of alsS gene or alsD gene, and deletion or inactivation of ackA gene or pta gene)
In the Bacillus subtilis mutant strain 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. In a preferred embodiment, in the Bacillus subtilis mutant strain of the present invention, the alsS gene and the alsD gene are deleted or inactivated.

  As used herein, the alsS gene is a gene encoding alpha-acetolactate synthase, and the alsD gene is a gene encoding alpha-acetolactate dehydrogenase. Both alpha-acetolactate synthase (AlsS) and alpha-acetolactate dehydrogenase (AlsD) are genes involved in acetoin synthesis, and are responsible for the synthesis reaction of acetoin represented by the following formula (2).

  In Bacillus subtilis, the alsS gene and the alsD gene form one operon as a group of alsSD genes. Table 2 below shows the accession numbers in BSORF DB of the genes that can be deleted or inactivated in the Bacillus subtilis mutant strain of the present invention and the encoded proteins. Those skilled in the art can identify the gene in the parent strain based on the information in the BSORF DB.

In the Bacillus subtilis mutant strain of the present invention, in addition to deletion or inactivation of at least one gene selected from the group consisting of the alsS gene and the alsD gene, at least one gene selected from the ackA gene and the pta gene is further selected. The gene has been deleted or inactivated. In a preferred embodiment, in the Bacillus subtilis mutant strain of the present invention, the ackA gene and the pta gene are deleted or inactivated.
As used herein, the ackA gene is a gene encoding acetate kinase, and the pta gene is a gene encoding phosphate acetyltransferase (Table 3).
Acetate kinase (AckA) and phosphate acetyltransferase (Pta) are both enzymes involved in acetic acid synthesis, AckA is a reaction that produces ATP and acetic acid from ADP and acetylphosphate, and Pta is acetyl. It is responsible for the reaction that produces CoA and acetyl phosphate from CoA and phosphoric acid.

  As a means for deleting or inactivating a gene, a mutation is introduced to one or more nucleotides on the nucleotide sequence of the gene, or a substitution or insertion of another nucleotide sequence to the nucleotide sequence, or a sequence of the gene Some or all of them may be deleted. Means for introducing a mutation that inhibits transcription of a gene include inactivating the promoter by introducing a mutation into the promoter region of the gene or by substitution or insertion with another nucleotide sequence. Specific methods for introducing the above-mentioned mutation and for replacing or inserting the nucleotide sequence include UV irradiation, site-specific mutation introduction, and the above (1-1. Genomic region deletion Bacillus subtilis mutant strain). The SOE-PCR method and the homologous recombination method described above can be mentioned. The position of the gene to be deleted or inactivated on the Bacillus subtilis genome and the nucleotide sequence can be confirmed by the above-mentioned BSORF DB.

(2. DPA production using Bacillus subtilis mutant strain)
The Bacillus subtilis mutant strain of the present invention in which both the acetoin synthetic gene and the gene involved in acetic acid synthesis are deleted or inactivated has an extremely high DPA-producing ability, as shown in Examples below. That is, the Bacillus subtilis mutant strains in which the acetoin synthesis gene and the gene involved in acetic acid synthesis are individually deleted or inactivated have an improved DPA-producing ability. In the Bacillus subtilis mutant strain that is deleted or inactivated at the same time, its DPA-producing ability synergistically improves far beyond the additive effect (see Example 2).
Therefore, by culturing the Bacillus subtilis mutant strain described above, DPA can be efficiently produced outside the cell, and another aspect of the present invention is the production of DPA or a salt thereof using the Bacillus subtilis mutant strain described above. It relates to the method.

  In the method for producing DPA or a salt thereof of the present invention, first, the Bacillus subtilis mutant strain of the present invention is cultured. The method of culturing is not particularly limited, and an ordinary method for culturing a microorganism can be used. That is, the medium used is a normal medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic components. As the carbon source, glucose, lactose, galactose, fructose, starch and cellulose hydrolysates, sugars such as molasses, alcohols such as glycerol, ethanol and sorbitol, and organic acids such as fumaric acid, citric acid and succinic acid are used. be able to. Inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate as nitrogen sources, organic nitrogen such as soybean hydrolyzate, ammonia gas, ammonia water, urea, glutamic acid, lysine, glycine, alanine, methionine, aspartic acid, arginine, etc. Can be used. It is desirable to contain required substances such as vitamins and amino acids, or if necessary, yeast extract, corn steep liquor, etc., as an organic trace nutrient source. In addition to these, it is desirable to add potassium phosphate, magnesium sulfate, calcium chloride, sodium chloride, copper ions, iron ions, manganese ions, and the like, if necessary. In some cases, a defoaming agent or the like is also added.

  The pH of the medium may be adjusted to a range in which Bacillus subtilis can grow, preferably 6.0 to 8.0. The pH may be adjusted with an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. You can use it. Culturing is carried out at 15 to 42 ° C, preferably 28 to 37 ° C for 6 to 96 hours, preferably 2 to 4 days, and aeration and stirring may be added if necessary.

  As described above, by culturing the Bacillus subtilis mutant strain of the present invention, DPA is produced in the culture supernatant. Alternatively, it is also possible to produce DPA in the reaction supernatant by suspending the microorganism cultured as described above in an aqueous solution containing aspartic acid and / or pyruvic acid. At this time, it is possible to more efficiently produce DPA by coexisting with a substance that is added to the medium as an energy source.

  The DPA produced is then recovered from the culture. Collection of DPA can be performed according to a known method. For example, isolation of DPA from the above culture supernatant or reaction supernatant is carried out by a known method, such as adsorption / desorption with an ion exchange resin, solid-liquid separation by crystallization, removal of impurities by membrane treatment, or a combination thereof. It can be carried out. By this method, crystals or precipitate of dipicolinic acid can be easily obtained.

  A salt of DPA can be obtained by adding an alkali in an amount suitable for the purpose to the DPA-containing solution obtained by the above-mentioned ion exchange resin treatment or the like. As a specific example, dipicolinic acid sodium salt can be obtained by adding NaOH.

  The following compositions and methods of manufacture 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 strain,
Prophage 6 region, Prophage 1 region, Prophage 4 region, PBSX region, Prophage 5 region, Prophage 3 region, Spb region, PKs region, Skin region, pps region, Prophage 2 region, ydcL-ydeK-ydhUy region, DysL, DyBy, DysBy, DysY, DysBy, DysBy, DysBy. One or more regions selected from the group consisting of the cgeE-ypmQ region, the yeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yayaJ region, and the ncM-fosB region. Has a deleted genome,
Bacillus subtilis dipicolinate synthase gene or the expression of a gene corresponding thereto is enhanced, and,
at least one gene selected from the group consisting of alsS gene and alsD gene is deleted or inactivated, and at least one gene selected from the group consisting of ackA gene and pta gene is deleted or inactivated Bacillus subtilis mutant strain.

<2> A method for producing a Bacillus subtilis mutant strain,
Prophage 6 region, Prophage 1 region, Prophage 4 region, PBSX region, Prophage 5 region, Prophage 3 region, Spb region, PKs region, Skin region, pps region, Prophage 2 region, ydcL-ydeK-ydhUy region, DysL, DyBy, DysBy, DysY, DysBy, DysBy, DysBy. One or more regions selected from the group consisting of the cgeE-ypmQ region, the yeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yayaJ region, and the ncM-fosB region. Enhances the expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto in a Bacillus subtilis mutant strain having a deleted genome, and
at least one gene selected from the group consisting of alsS gene and alsD gene is deleted or inactivated, and at least one gene selected from the group consisting of ackA gene and pta gene is deleted or inactivated. A method, including:

<3> In any one of the above items <1> to <2>, preferably, the Bacillus subtilis mutant strain is a profile6 region, a profile1 region, a profile4 region, a PBSX region, a profile5 region, a profile3 region, a spb region, and a PKS region. , Skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeK-yesX region, pdp-roCR region, ycxB-sipU7 region, SKU region. The region, the sbo-ywhH region, the yybP-yayaJ region and the ncM-fosB region are deleted.

<4> In any one of the above items <1> to <3>, each region is preferably a region sandwiched by the oligonucleotide sets shown in Table 1.

<5> In any one of the above items <1> to <4>, preferably, the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is a group consisting of the genes described in (i) and (ii) below. Is at least one selected from:
(I) At least one Bacillus subtilis dipicolinate synthase subunit A gene selected from the group consisting of the following (a) to (f) or a gene corresponding thereto:
(A) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) 80% 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 with the nucleotide sequence shown in SEQ ID NO: 1. More preferably, a polynucleotide comprising a nucleotide sequence having 99% or more identity and encoding a protein exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene according to (ii);
(C) Dipicolinate synthase activity in the presence of a protein encoded by the gene described in (ii), which hybridizes to a complementary strand of a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions A polynucleotide encoding a protein that exerts
(D) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 2;
(E) Dipicoline in the presence of the protein encoded by the gene described in (ii), 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: 2 A polynucleotide encoding a protein exhibiting acid synthase activity, wherein several is 59, preferably 44, more preferably 29, more preferably 14, more preferably 5, and more preferably 2. Individual;
(F) 80% 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 with the amino acid sequence shown in SEQ ID NO: 2; More preferably, a polynucleotide encoding a protein consisting of an amino acid sequence having 99% or more identity and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene according to (ii);
(Ii) at least one Bacillus subtilis dipicolinate synthase subunit B gene selected from the group consisting of the following (j) to (o) or a gene corresponding thereto:
(J) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3;
(K) 80% 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 with the nucleotide sequence shown in SEQ ID NO: 3. More preferably, a polynucleotide comprising a nucleotide sequence having 99% or higher identity and encoding a protein exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene according to (i);
(L) Dipicolinic acid synthase activity in the presence of a protein encoded by the gene described in (i), which hybridizes to a complementary strand of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 under stringent conditions A polynucleotide encoding a protein that exerts
(M) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 4;
(N) Dipicoline in the presence of the protein encoded by the gene described in (i), 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. A polynucleotide encoding a protein exhibiting acid synthase activity, wherein several is 40, preferably 30, more preferably 20, more preferably 10, more preferably 4, more preferably 2. Individual;
(O) 80% 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 with the amino acid sequence shown in SEQ ID NO: 4. More preferably, a polynucleotide encoding a protein having an amino acid sequence having 99% or higher identity and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i).

<6> In the above item <5>, preferably, the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is the following gene:
A combination of at least one selected from the group consisting of the genes described in (i) above and at least one selected from the group consisting of the genes described in (ii) above;
A combination of the gene of (i) (a) and at least one selected from the group consisting of the genes of (ii) (j) to (o);
A combination of at least one selected from the group consisting of the above-mentioned (i) (a) to (f) genes with the above-mentioned (ii) (j) genes; or
A combination of the gene of (i) (a) and the gene of (ii) (j).

<7> In any one of <1> to <6> above, the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is:
Preferably, Bacillus subtilis spoVFA gene and spoVFB gene, Bacillus licheniformis spoVFA gene and spoVFB gene, Bacillus clausii spoVFA gene and spoVFB gene, Clostridium stercorarium spoVFA gene and spoVFB gene, and Vacillus amylo are Vacillus amylo. Is at least one selected from the group;
More preferably, it is at least one selected from the group consisting of Bacillus subtilis spoVFA gene and Bacillus subtilis spoVFB gene.

<8> In the above item <7>, the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is:
Preferably, at least one selected from the group consisting of Bacillus subtilis spoVFA gene, Bacillus licheniformis spoVFA gene, Bacillus clausii spoVFA gene, Clostridium stercorarium spoVFA gene, and Bacillus amyloliquefaciens spoVFA gene; Bacillus licheniformis spoVFB gene, Bacillus clausii spoVFB gene, Clostridium stercorarium spoVFB gene, and at least one selected from the group consisting of Bacillus amyloliquefaciens spoVFB gene;
More preferably, it is a combination with the B. subtilis spoVFA gene and the B. subtilis spoVFB gene.

<9> In any one of the above items <1> to <8>, preferably, the arsS gene and alsD gene are deleted or inactivated, and the ackA gene and pta gene are deleted or inactivated. Has been done.

<10> In any one of the above items <1> to <9>, preferably, the alsS gene comprises the nucleotide sequence represented by SEQ ID NO: 89, the alsD gene comprises the nucleotide sequence represented by SEQ ID NO: 90, and the ackA gene comprises It consists of the nucleotide sequence shown in SEQ ID NO: 91, and the pta gene consists of the nucleotide sequence shown in SEQ ID NO: 92.

<11> In any one of the above items <1> to <10>, preferably, the enhanced expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is a control region selected from the group consisting of: Introduction of the gene under control into the genome or into a plasmid:
Control region of α-amylase gene, control region of protease gene, control region of gene encoding ribosomal protein (rplS gene etc.), control region of aprE gene, or control region of spoVG gene derived from Bacillus bacterium; Bacillus sp. Control region of cellulase gene of KSM-S237 strain; and control region of kanamycin resistance gene or chloramphenicol resistance gene derived from Staphylococcus aureus.

<12> A method for producing dipicolinic acid or a salt thereof, which uses the Bacillus subtilis mutant strain according to any one of <1> and <3> to <11>.

<13> A method for producing dipicolinic acid or a salt thereof using the Bacillus subtilis mutant strain produced by the method according to any one of <2> to <11>.

<14> The method according to <12> or <13>, which comprises culturing the Bacillus subtilis mutant strain and recovering dipicolinic acid from the culture.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to the following examples.

(Example 1) Construction of strain (1-1) Preparation of tryptophan requirement-eliminating strain Tryptophan in Bacillus subtilis mutant strain (MGB874 strain; JP 2007-130013 A) in which large region of Bacillus subtilis wild strain genome was deleted The requirement is released. The Bacillus subtilis 168 strain, which is the original strain of the Bacillus subtilis mutant strain MGB874, has trpC2 as a genotype. That is, strain 168 is auxotrophic for tryptophan due to the trpC gene mutation encoding indole-3-glycerol phosphate synthase involved in tryptophan synthesis. The Bacillus subtilis ATCC6051T strain is known to be non-tryptophan-requiring (Albertini, AM & Galizzi, A. Microbiology, 1999, 145 (Pt12): 3319-3320). When the trpC genes of the Bacillus subtilis 168 strain and the ATCC 6051T strain were compared, it was found that the nucleotides ATTs 328 to 330 were not present in the Bacillus subtilis 168 strain. Therefore, PCR was carried out using the ATCC6051T strain genome as a template and the primers trpC_F1 (SEQ ID NO: 13) and trpC_R1 (SEQ ID NO: 14) described in Table 1 to construct a PCR product. Using this PCR product, a Bacillus subtilis mutant strain MGB874 was transformed by the competent method, and tryptophan-free SMA agar medium (0.2% ammonium sulfate, 1.4% dipotassium hydrogenphosphate, 0.6% phosphorus) was added. Transformed colonies grown on potassium dihydrogen acid, 0.1% trisodium citrate dihydrate, 0.5% glucose, 0.035% magnesium sulfate heptahydrate, 1.5% agar) As separated. Thus, a tryptophan non-auxotrophic MGB874T strain was obtained.

(1-2) Construction of Bacillus subtilis mutant strain (MGB874T_PspoVG-spoVFAB strain) having enhanced expression of dipicolinate synthase gene Using the MGB874T strain constructed in (1-1) as a host, the expression of dipicolinate synthase gene was enhanced. A Bacillus subtilis mutant strain (MGB874T_PspoVG-spoVFAB strain) was constructed. Using the genomic DNA extracted from Bacillus subtilis strain 168 as a template and using the primers spoVFA-F1 (SEQ ID NO: 15) and spoVFA-R1 (SEQ ID NO: 16) shown in Table 1, the fragment (A) was amplified by PCR. This fragment (A) is a region adjacent to the start codon of the spoVFA gene (SEQ ID NO: 1) upstream. The fragment (B) was amplified by PCR using the same genomic DNA as a template and the primers spoVFA-F2 (SEQ ID NO: 17) and spoVFA-R2 (SEQ ID NO: 18) shown in Table 1. This fragment (B) is a region downstream from the start codon of the spoVFA gene. Furthermore, using the same genomic DNA as a template and using the primers spoVGpro-F (SEQ ID NO: 19) and spoVGpro-R (SEQ ID NO: 20) shown in Table 1, a fragment (C) containing the spoVG control region sequence was amplified by PCR. .. Furthermore, using plasmid pDLT3 (Morimoto, T. et al., Microbiology, 2002, Pt 11: 3539-3552) as a template, primers Cm-F (SEQ ID NO: 21) and Cm-R (SEQ ID NO: 22) shown in Table 1 were used. The fragment (D) containing the chloramphenicol resistance gene and its control region was amplified by PCR.
Next, the obtained fragment (A), fragment (D), fragment (C) and fragment (B) are arranged in this order in order of the primers spoVFA-F1 (SEQ ID NO: 15) and spoVFA-R2 ( SEQUENCE ID NO: 18) was used for ligation by the SOE-PCR method to obtain a DNA fragment of about 2.2 kb. The obtained DNA fragment was used to transform the Bacillus subtilis MGB874T strain by the competent method (J. Bacteriol., 1960, 81: 741-746) and grown on LB agar medium containing chloramphenicol. The colony was separated as a transformant (hereinafter, the separated transformant is referred to as MGB874T_PspoVG-spoVFAB strain). The MGB874T_PspoVG-spoVFAB strain is a Bacillus subtilis mutant strain having enhanced expression of the spoVFABB gene, which has both the native spoVFAB gene and the introduced spoVFAB gene under PspoVG control on the genome.
Between the spoVFA regulatory region and the spoVFA gene on the genome of the constructed MGB874T_PspoVG-spoVFAB strain by PCR and subsequent sequencing by the Sanger method (Proc. Natl. Acad. Sci. USA, 1982, 74: 5463-5467). It was confirmed that the chloramphenicol resistance gene and the spoVG gene control region were inserted. FIG. 3 shows the procedure for introducing the dipicolinate synthase gene into the Bacillus subtilis mutant strain obtained in this Example, and the introduction position.

(1-3) Construction of Bacillus subtilis mutant strain (MGB874T_PspoVG-spoVFAB_ΔalsSD strain) in which the alsS gene and the alsD gene are deleted, the alsSD gene group is deleted using the MGB874T_PspoVG-spoVFABB strain constructed in (1-2) above as a host. The Bacillus subtilis mutant strain (MGB874T_PspoVG-spoVFAB_ΔalsSD strain) was constructed. This mutant strain was constructed by a marker-free deletion method using a cassette in which an IPTG control region and a mazF gene, which is a free mRNA-cleaving ribonuclease, were fused (Morimoto, T. et al., In Strain Engineering, pp345-358. Springer).
First, using the genomic DNA extracted from Bacillus subtilis mutant strain MGB874T as a template and using primers alsSD-DF1 (SEQ ID NO: 23) and alsSD-DR1 (SEQ ID NO: 24) shown in Table 1, the start codon of the alsSD gene group A fragment (A), which is a region adjacent to the upstream, was amplified by PCR. In addition, using the same genomic DNA as a template and using the primers alsSD-DF2 (SEQ ID NO: 25) and alsSD-DR2 (SEQ ID NO: 26) shown in Table 1, a fragment (B) that is a region downstream of the stop codon of the alsD gene was obtained. It was amplified by PCR. Furthermore, using the same genomic DNA as a template and using the primers alsSD-IF (SEQ ID NO: 27) and alsSD-IR (SEQ ID NO: 28) shown in Table 1, a fragment (C) that is a homologous region in the arsSD gene group ORF is obtained. It was amplified by PCR. Furthermore, using the Bacillus subtilis mutant strain TOM310 (Morimoto, T. et al., In Strain Engineering, pp345-358. Springer) as a template, the primers pAPNC-F (SEQ ID NO: 29) and chpA-R (SEQ ID NO :) shown in Table 1 were used. 30) was used to amplify the mazF cassette fragment (D) containing the spectinomycin resistance gene by PCR.
Next, the obtained fragment (A), fragment (B), fragment (D) and fragment (C) are arranged in this order in the order of primer alsSD-DF1 (SEQ ID NO: 23) and alsSD-IR ( The final DNA fragment was obtained by ligation by SOE-PCR method using SEQ ID NO: 28). The obtained DNA fragment was used to transform the Bacillus subtilis MGB874T_PspoVG-spoVFAB strain by the competent method (J. Bacteriol., 1960, 81: 741-746), and LB agar containing spectinomycin without IPTG was transformed. Colonies grown on the medium were isolated as transformants.
Finally, in order to remove the mazF gene cassette, the cells were cultured on LB agar medium containing IPTG and grown colonies were selected. The obtained mutant strain becomes a mutant strain in which the mazF cassette has been removed by intracellular homologous recombination in the fragment (B) (hereinafter referred to as MGB874T_PspoVG-spoVFAB_ΔalsSD strain). The MGB874T_PspoVG-spoVFAB_ΔalsSD strain is a Bacillus subtilis mutant strain having a mutation in which the alsD gene stop codon region is deleted from the alsS gene start codon on the genome and further the drug selection marker is removed. FIG. 4 shows the method for deleting the alsSD gene group constructed by the marker-free deletion method using the mazF gene cassette in this example.
PCR using the genome of the obtained transformant MGB874T_PspoVG-spoVFAB_ΔalsSD strain and subsequent sequencing by the Sanger method (Proc. Natl. Acad. Sci. USA, 1982, 74: 5463) of the alsSD gene group on the genome. It was confirmed that the deletion had occurred.

(1-4) Construction of Bacillus subtilis mutant strain lacking ackA gene and pta gene (MGB874T_PspoVG-spoVFAB_ΔackAΔpta strain) Using the MGB874T_PspoVG-spoVFAB strain constructed in (1-2) above as a host, (1-3) above A mutant strain (MGB874T_PspoVG-spoVFAB_ΔackAΔpta strain) in which both the ackA gene and the pta gene were deleted was constructed using the same method as described in (1) above.
First, the ackA gene was deleted. When the ackA gene was deleted, the primers ackA-DF1 (SEQ ID NO: 31) and ackA-DR1 (SEQ ID NO: 32) described in Table 1 were used for amplification of the fragment (A), and the primers described in Table 4 were used for amplification of the fragment (B). ackA-DF2 (SEQ ID NO: 33) and ackA-DR2 (SEQ ID NO: 34), and the fragment (C) amplification was performed using the primers ackA-IF (SEQ ID NO: 35) and ackA-IR (SEQ ID NO: 36) described in Table 4. Used respectively.
Subsequently, the pta gene was deleted. When pta gene was deleted, primers pta-DF1 (SEQ ID NO: 37) and pta-DR1 (SEQ ID NO: 38) shown in Table 1 were used for amplification of the fragment (A), and primers of Table 4 were used for amplification of the fragment (B). pta-DF2 (SEQ ID NO: 39) and pta-DR2 (SEQ ID NO: 40), and the fragment (C) amplification was performed using the primers pta-IF (SEQ ID NO: 41) and pta-IR (SEQ ID NO: 42) described in Table 4. Used respectively.
Finally, the MGB874T_PspoVG-spoVFAB_ΔackAΔpta strain was constructed. It was confirmed by PCR using the genome of the obtained transformant MGB874T_PspoVG-spoVFAB_ΔackAΔpta strain and subsequent sequencing by the Sanger method that a deletion has occurred in each gene into which a mutation was introduced on the genome.

(1-5) Construction of Bacillus subtilis mutant strain (MGB874T_PspoVG-spoVFFAB_ΔalsSDΔackAΔpta strain) in which alsS gene and alsD gene, ackA gene and pta gene are deleted, MGB874T_PspoVG-sposVFAB_Δ is used as the MGB874T_PspoVG-spoVSDB host, which was constructed in (1-3) above. The MGB874T_PspoVG-spoVFAB_ΔalsSDΔackAΔpta strain was constructed using a method similar to the method described in (1-4) above. It was confirmed by PCR using the genome of the obtained transformant MGB874T_PspoVG-spoVFAB_ΔalsSDΔackAΔpta strain and subsequent sequencing by the Sanger method that a deletion has occurred in each gene on which the mutation was introduced on the genome.

(Example 2) DPA and MGB874T_PspoVG-spoVFAB strain constructed in productivity Evaluation Example 1, MGB874T_PspoVG-spoVFAB_ΔalsSD strain, the MGB874T_PspoVG-spoVFAB_ΔackAΔpta strain and MGB874T_PspoVG-spoVFAB_ΔalsSDΔackAΔpta strain, synthetic medium (5.0% glucose 5 mL, 0 0.6% by mass ammonium chloride, 0.15% by mass dipotassium hydrogen phosphate, 0.035% by mass magnesium sulfate heptahydrate, 0.005% by mass manganese sulfate pentahydrate, 50 mM MOPS (monoholinopropane sulfone) Acid) buffer (pH 7.0) and other trace metals) at 30 ° C. with shaking overnight, and the resulting culture solution is used as a synthetic medium (5.0% by mass glucose, 0.6% by mass ammonium chloride). 0.15% by mass dipotassium hydrogen phosphate, 0.035% by mass magnesium sulfate heptahydrate, 0.005% by mass manganese sulphate pentahydrate, 50 mM MOPS (monoholinopropanesulfonic acid) buffer (pH 7. 0), other trace metals, 4.0 mass% calcium carbonate) (30 mL) were inoculated, and shake culture was performed at 37 ° C. and 200 rpm for 2 days.
After the culture was completed, the DPA production amount was measured under the analysis conditions shown below, and the relative value was calculated when the production amount of the MGB874T_PspoVG-spoVFAB strain was 100%.
The Bacillus subtilis mutant strain MGB874T_PspoVG-spoVFABB_ΔalsSD strain in which the acetoin synthesis genes arsS gene and alsD gene are deleted, and the Bacillus subtilis mutant strain MGB874T_PspoVG-spoAFAt_Δac, which is a gene involved in acetic acid synthesis, lacking the ackA gene and the pta gene are , Compared with MGB874T_PspoVG-spoVFAB strain which is a strain before gene deletion involving acetoin synthesis gene and acetic acid synthesis, DPA production increased up to 142% and 110% respectively, but involved in acetoin synthesis gene and acetic acid synthesis In the Bacillus subtilis mutant strain MGB874T_PspoVG-spoVFAB_ΔalsSDΔackAΔpta strain in which the gene was simultaneously deleted, the DPA production amount was further increased to 184%, and a high synergistic effect was confirmed (Table 5).

Quantification of DPA The culture solution sample after completion of culture was subjected to centrifugation (Hitachi Koki, himacCF15RX) at 14,800 rpm for 30 minutes at room temperature to obtain DPA contained in the obtained sample supernatant after centrifugation. Quantitation was performed by the HPLC method. The HPLC device is a liquid-sending pump (Shimadzu, LC-9A), auto sampler (Hitachi measuring instrument, AS-2000), column oven (Shimadzu, CTO-6A), UV detector (Shimadzu, SPD-10A), degassing. An instrument (GL Science, DG660B) and a chromatographic data analyzer (Hitachi Instrument Co., Ltd., D-2500) connected to each other were used. As the analytical column, a High Performance Carbohydrate Column 60 (Å) 4 μm 4.6 × 250 mm HPLC Column (Aminopropoxymethyl bonded amorphous silicon) (Waters) was used. As the eluent, a 1: 1 mixture of an aqueous solution containing 20 mM EDTA in water with concentrated phosphoric acid adjusted to pH 3.4 and an acetonitrile solution was used. The measurement conditions were a detection wavelength of 270 nm and a flow rate of 1 mL / min. The sample to be subjected to HPLC analysis was pretreated by filter filtration with MULTI SCREEN MNHV45 (manufactured by MILLIPORE, 0.45 μm Durapore membrane) to remove insoluble matter. The concentration test was performed based on a calibration curve prepared using 2,6-pyridinedical boxlic acid: DPA, 99% (SIGMA-ALDRICH).

Claims (7)

A Bacillus subtilis mutant strain,
Prophage 6 region, Prophage 1 region, Prophage 4 region, PBSX region, Prophage 5 region, Prophage 3 region, Spb region, PKs region, Skin region, pps region, Prophage 2 region, ydcL-ydeK-ydhUy region, DysL, DyBy, DysBy, DysY, DysBy, DysBy, DysBy. It has a genome in which the cgeE-ypmQ region, theyeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yayaJ region, and the ncM-fosB region are deleted,
Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced in expression, and at least one gene selected from the group consisting of alsS gene and alsD gene is deleted or inactivated, and ackA gene and at least one gene selected from the group consisting of pta genes is deleted or inactivated,
A Bacillus subtilis mutant strain, wherein the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is at least one selected from the group consisting of the genes described in (i) and (ii) below:
(I) the following (a) ~ (b), at least one gene selected from the group consisting of (d) ~ (f):
(A) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) encodes a protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii) A polynucleotide that:
(D) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 2;
(E) Dipicoline in the presence of the protein encoded by the gene described in (ii), 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: 2 A polynucleotide encoding a protein exhibiting acid synthase activity;
(F) encodes a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii) A polynucleotide that:
(Ii) below (j) ~ (k), at least one gene selected from the group consisting of (m) ~ (o):
(J) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3;
(K) encodes a protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 3 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) A polynucleotide that:
(M) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 4;
(N) Dipicoline in the presence of the protein encoded by the gene described in (i), 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. A polynucleotide encoding a protein exhibiting acid synthase activity;
(O) codes for a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) A polynucleotide.   The Bacillus subtilis mutant strain according to claim 1, wherein the alsS gene and the alsD gene are deleted or inactivated, and the ackA gene and the pta gene are deleted or inactivated.   The Bacillus subtilis mutant strain according to claim 1 or 2, wherein the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is at least one selected from the group consisting of Bacillus subtilis spoVFA gene and Bacillus subtilis spoVFB gene. A method for producing a Bacillus subtilis mutant strain,
Prophage 6 region, Prophage 1 region, Prophage 4 region, PBSX region, Prophage 5 region, Prophage 3 region, Spb region, PKs region, Skin region, pps region, Prophage 2 region, ydcL-ydeK-ydhUy region, DysL, DyBy, DysBy, DysY, DysBy, DysBy, DysBy. Bacillus subtilis mutant strain having a genome in which the cgeE-ypmQ region, the yeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yyaJ region, and the yncM-fosB region are deleted. At
Enhance the expression of Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto, and
Deletion or inactivation of at least one gene selected from the group consisting of alsS gene and alsD gene, and deletion or inactivation of at least one gene selected from the group consisting of ackA gene and pta gene Including,
The method wherein the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is at least one selected from the group consisting of the genes described in (i) and (ii) below:
(I) the following (a) ~ (b), at least one gene selected from the group consisting of (d) ~ (f):
(A) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) encodes a protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii) A polynucleotide that:
(D) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 2;
(E) Dipicoline in the presence of the protein encoded by the gene described in (ii), 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: 2 A polynucleotide encoding a protein exhibiting acid synthase activity;
(F) encodes a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii) A polynucleotide that:
(Ii) below (j) ~ (k), at least one gene selected from the group consisting of (m) ~ (o):
(J) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3;
(K) encodes a protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 3 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) A polynucleotide that:
(M) a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 4;
(N) Dipicoline in the presence of the protein encoded by the gene described in (i), 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. A polynucleotide encoding a protein exhibiting acid synthase activity;
(O) codes for a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) A polynucleotide.   The method according to claim 4, wherein the alsS gene and the alsD gene are deleted or inactivated, and the ackA gene and the pta gene are deleted or inactivated.   The method according to claim 4, wherein the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is at least one selected from the group consisting of Bacillus subtilis spoVFA gene and Bacillus subtilis spoVFB gene. ..   A method for producing dipicolinic acid or a salt thereof using the Bacillus subtilis mutant strain according to any one of claims 1 to 3.