JP6527669B2 - 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 capable of producing dipicolinic acid, 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 masking agent, an antioxidant, etc. for industrial use. It is reported that it can be used.

  Dipicolinic acid is known to be synthesized from 2,6-lutidine by an oxidation reaction. That is, it is known that the compound 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 ratio and selectivity of the reaction are insufficient, so 2,6-lutidine as a raw material and alkylpyridine such as 6-methyl-2-pyridinecarboxylic acid as a reaction intermediate are reaction solutions. It remains inside, and these are mixed as impurities in the product 2,6-pyridinedicarboxylic acid (dipicolinic acid). That is, in the conventional dipicolinic acid synthesis method, it was difficult to produce dipicolinic acid of high purity.

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

  Also, methods for producing dipicolinic acid using genetically engineered microorganisms are known. Patent Document 4 discloses a method for producing dipicolinic acid using genetically modified lysine-producing bacteria. Patent Document 5 discloses a method for producing dipicolinic acid using Bacillus or Clostridium microorganisms, 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 obtained by deleting a large region of the genome is excellent in productivity of enzymes such as protease. Patent Document 7 discloses an efficient method for producing L-glutamic acid by enhancing the pyruvate carboxylase activity in cells of coryneform bacteria having L-glutamic acid-producing ability. However, these mutant microorganisms are strains developed for the production of enzymes or L-glutamic acid.

JP 2004-275075 A German Patent No. 2300056 U.S. Pat. No. 3,334,021 JP, 2002-371063, A Japanese Patent Application Publication No. 2008-048732 Japanese Patent Application Publication No. 2007-130013 JP 2000-201692 A

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

  The present inventors promoted development of a microorganism strain capable of efficiently producing DPA. As a result, the present inventors have enhanced expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto in a Bacillus subtilis mutant in which a large region of the genome has been deleted, and Bacillus subtilis alpha-acetolactate synthase The inventors have found that the obtained Bacillus subtilis mutant strain has a remarkably high DPA producing ability by deleting or inactivating any one or more of the gene alsS and the Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD. Completed.

That is, the present invention provides in one aspect:
A Bacillus subtilis mutant,
prophage6 area, prophage1 area, prophage4 area, PBSX area, prophage5 area, prophage area, spb area, pks area, skin area, pps area, propagation2 area, ydcL-ydeK-ydhU area, yisB-yitD area, yunA-yurT area, One or more regions selected from the group consisting of cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region Have a deleted genome,
The expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced, and at least one selected from the group consisting of the Bacillus subtilis alpha-acetolactate synthase gene alsS and the Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD. The gene has been deleted or inactivated
Bacillus subtilis mutant strain.

In another aspect, the present invention provides:
A method for producing a Bacillus subtilis mutant, which comprises:
prophage6 area, prophage1 area, prophage4 area, PBSX area, prophage5 area, prophage area, spb area, pks area, skin area, pps area, propagation2 area, ydcL-ydeK-ydhU area, yisB-yitD area, yunA-yurT area, One or more regions selected from the group consisting of cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region Enhance the expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto in a Bacillus subtilis mutant strain having a genome deleted from
Deleting or inactivating at least one gene selected from the group consisting of Bacillus subtilis alpha-acetolactate synthase gene alsS and Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD;
How, 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 high DPA producing ability. By using the Bacillus subtilis mutant strain of the present invention, DPA or a salt thereof can be efficiently produced.

The schematic diagram which shows an example of the method of deleting a predetermined | prescribed area | region on 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 one Embodiment regarding the introduction procedure and introduction | transduction position of the dipicolinate synthase gene to a Bacillus subtilis mutant strain. The schematic diagram which shows the procedure which deletes alsS gene and alsD gene from a Bacillus subtilis mutant strain by the marker free deletion method using a mazF gene fusion cassette.

(1. Definition)
The name of each gene and genome region of Bacillus subtilis described in the present specification is published on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) ([bacillus. Genome. Ad. Jp /], January 2006. Based on the B. subtilis genome data (updated on the 18th).

  In the present specification, the identity of amino acid sequences and nucleotide sequences is calculated by the Lipman-Pearson method (Lipman, DJ., Pearson. WR .: Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing analysis with Unit size to compare (ktup) of 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

  In the present specification, unless otherwise defined, “one or several” used for deletion, substitution, addition or insertion of an amino acid or nucleotide in an amino acid sequence or nucleotide sequence is, for example, 1 to 12, preferably It may be 1 to 8, more preferably 1 to 4. Also, as used herein, "addition" of an amino acid or nucleotide includes the addition of one or several amino acids or nucleotides at one and both ends of the sequence.

  As used herein, “stringent conditions” for hybridization are conditions that allow for the identification of genes having a nucleotide sequence of about 80% or more, or about 90% or more in sequence identity. As the "stringent conditions", conditions described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001) can be 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. If an example is shown, as said "stringent conditions", as hybridization conditions, 5 * SSC, 70 degreeC or more is preferable, 5 * SSC, 85 degreeC or more is more preferable, As washing conditions, 1 * SSC 60 ° C. or higher is preferable, 1 × SSC, 73 ° C. or higher is more preferable. The combination of the above-mentioned SSC and temperature conditions is an exemplification, and one of ordinary skill in the art can realize appropriate stringency by appropriately combining the above or other factors that determine the stringency of hybridization.

  As used herein, upstream and downstream of a gene refers to a region that continues 5 'and 3' of a gene or region that is taken as an object, respectively. Unless defined otherwise, upstream and downstream of a gene is not limited to regions upstream and downstream from the translation initiation point of the gene.

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

  In the present specification, the expression enhancement of a target gene is to increase the expression level of the target gene as compared to that before modification. As a 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 control region) capable of enhancing the expression of the gene as compared to the wild type The target gene placed under the control of the expression control region may be introduced into the genome of the cell or into a plasmid, or modified to make a plurality of target genes expressible in the cell. As a modification that allows multiple target genes to be expressed in cells, it is necessary to further introduce the target genes in the genome or in the plasmid in the cells in which the target genes can be expressed, as well as the control region as necessary. Alternatively, modifications to increase the number of target genes expressed in cells can be mentioned by introducing a plurality of target genes into the genome of cells or into a plasmid, if necessary, together with control regions.

  In the present specification, the term “place the target gene under control of the control region” means that the control region and the target gene are on the DNA such that the function of controlling the expression of the gene by the control region acts on the target gene It says to arrange. As a method of disposing a target gene under the control of a control region, there is a method of linking the target gene downstream of the control of the control region.

  In the present specification, dipicolinate synthase activity is an activity catalyzing a synthesis reaction of dipicolinic acid from dihydrodipicolinic acid shown in the following formula (1). Further, in the present specification, the alpha-acetolactate synthase activity and the alpha-acetolactate dehydrogenase activity are activities for catalyzing a conversion reaction from phosphoenolpyruvate to acetoin shown in the following formula (2).

(2. Production of Bacillus subtilis Mutant of the Present Invention)
(2-1. Genome 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 Bacillus subtilis wild strain has been deleted, expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced, and Bacillus subtilis. Bacillus subtilis mutant strain in which at least one gene selected from the group consisting of Bacillus alpha-acetolactate synthase gene (alsS gene) and Bacillus subtilis alpha-acetolactate dehydrogenase gene (alsD gene) is deleted or inactivated It is. Preferably, the Bacillus subtilis mutant strain of the present invention comprises a modification that enhances expression of a Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto with respect to a Bacillus subtilis mutant strain in which a large region of the genome is deleted, and a Bacillus subtilis strain. It is produced by adding a modification which deletes or inactivates at least one gene selected from Bacillus als S gene and B. subtilis als D gene.

  The Bacillus subtilis mutant in which the large region of the genome has been 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 a wild strain). In comparison to, the large region in the genome has a deleted genome. That is, in the genome of the Bacillus subtilis mutant strain, the prophage 6 (yoaV-yobO) region, the prophage 1 (ybbU-ybdE) region, the prophage 4 (yjcM-yjdJ) region, and the PBSX (ykdA-xlyA) region in the genome of the Bacillus subtilis wild strain Region, prophage 5 (ynxB-dut) region, prophage 3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region , Prophage 2 (ydcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pd Deletion of one or more regions selected from the group consisting of -rocR region, ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region . Preferably, the Bacillus subtilis mutant strain has a prophage 6 (yoaV-yobO) region, a prophage 1 (ybbU-ybdE) region, a prophage 4 (yjcM-yjdJ) region, a PBSX (ykdA-xlyA) region in the genome of a Bacillus subtilis wild strain. , Prophage 5 (ynxB-dut) region, prophage 3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region, prophage2 (YdcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region Lacks ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) region, sbo-ywhH region, all yybP-yyaJ region and yncM-fosB region. As an example of such a Bacillus subtilis mutant strain, Bacillus subtilis MGB874 strain described in Japanese Patent Application Laid-Open No. 2007-130013 can be mentioned.

  The Bacillus subtilis mutant 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 any Bacillus subtilis strain with the genome of the published Bacillus subtilis 168 strain. The complete nucleotide sequence and genomic information of B. subtilis strain 168 can be obtained from BSORF DB described above, or from GenBank: AL009126.2 ([www. Ncbi. Nlm. Nih. Gov / nuccore / 38680335]). One skilled in the art can determine the genomic region of interest to be deleted based on the genomic information of Bacillus subtilis 168 strain obtained from these sources. Here, the genomic region to be deleted is one or several (eg, 1 to 100, preferably 1 to 50) relative to the nucleotide sequence of the above-mentioned genomic region in Bacillus subtilis 168 strain that has been published. More preferably, nucleotides containing mutations such as natural or artificially caused deletions, substitutions, insertions, additions, etc. in 1 to 30, more preferably 1 to 10, even more preferably 1 to 5) nucleotides It may be a genomic region having a sequence. Alternatively, the genomic region to be deleted is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more identical in nucleotide sequence to the above-mentioned genomic region in the published Bacillus subtilis 168 strain. % Or more may be identical genomic regions.

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

  An outline of the procedure for preparation of a DNA fragment for deletion by SOE-PCR method and deletion of a target region by double crossover method using the DNA fragment for deletion is shown in FIG. First, by SOE-PCR, 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 as well 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 linked between the upstream fragment and the downstream fragment is prepared.

  First, three fragments of the upstream fragment A and downstream fragment B of the region to be deleted and, if necessary, the marker gene fragment (in FIG. 1, chloramphenicol resistance gene fragment Cm) are prepared by the first PCR. Do. In PCR amplification of the upstream fragment and the downstream fragment, a primer to which a terminal 10 to 30 nucleotide sequence 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 linked in this order, the 5 'end of the primer linked to the downstream end of the upstream fragment A is upstream of the drug resistance marker gene fragment Cm. A sequence corresponding to 10 to 30 nucleotides is added (FIG. 1, primer DR1), and 10 'to 30 nucleotides downstream of the drug resistance marker gene fragment Cm at the 5' end of the primer bound to the upstream end of downstream fragment B 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 downstream of the amplified upstream fragment A ′. As a result, 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 in the first PCR are mixed to form a template, and a primer that binds to the upstream side of the upstream fragment (FIG. 1, primer DF1) A second round of PCR is performed using a pair of primers consisting of and a primer that binds downstream of the downstream fragment (Figure 1, primer DR2). By the second PCR, it is possible to amplify a 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.

  The DNA fragment for deletion obtained by the above-mentioned method and the like is inserted into a plasmid using a conventional restriction enzyme and DNA ligase to construct a plasmid for introduction of deletion. 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, thereby adding the drug in addition to the upstream fragment and the downstream fragment. A plasmid for deletion can be constructed which has a resistance marker gene fragment.

  The plasmid for deletion introduction constructed in the above-mentioned 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 results in a double crossover homologous recombination between the upstream fragment and the downstream fragment on the plasmid and the region homologous to them in the Bacillus subtilis genome, and the region to be deleted is the drug resistance marker gene. Transformed transformants are obtained (Figure 1). For selection of transformants, expression of a marker gene such as a drug resistance gene present in the DNA fragment for deletion may be used as an index. For example, a bacterium transformed with a chloramphenicol resistance gene fragment is cultured in a medium containing an antibiotic (eg, chloramphenicol), and a grown colony is recovered to delete the target region. A transformant in which the chloramphenicol resistance gene has been replaced can be obtained. Furthermore, by extracting genomic DNA of a transformant and performing PCR using this as a template, it can be confirmed that the target region is deleted.

  Next, the above-mentioned marker gene inserted into genomic DNA is removed from the obtained transformant. Although it does not specifically limit as the procedure of removal, The two-step homologous-recombination method as shown in FIG. 2 can be used (Unexamined-Japanese-Patent No. 2009-254350). In the method, first, a DNA fragment (donor DNA) for the first homologous recombination is prepared. Although it does not specifically limit as a method of preparation, SOE-PCR method etc. which were mentioned above etc. are mentioned. As donor DNA, for example, an about 0.1 to 3 kb fragment (upstream fragment) corresponding to the region adjacent upstream of the marker gene region to be removed (ie, the deleted region) and the region adjacent downstream similarly A DNA fragment in which a fragment to which an about 0.1 to 3 kb fragment (downstream fragment) corresponding to is linked and a fragment of the marker gene downstream region to be removed can be used. 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 (FIG. 2). reference).

  Then, the prepared donor DNA is introduced into the transformant by a conventional method such as the competent cell transformation method, which corresponds to the upstream fragment and the first marker gene downstream region on the transformant genome. Homologous recombination occurs with the region (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 has appropriately occurred, the upstream fragment, the downstream fragment, the second marker gene as required, the first marker gene downstream region, and the downstream fragment are arranged in order (See Figure 2). In genomic DNA having such an arrangement, homologous recombination can occur spontaneously between the two downstream fragments (in-genome 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.

  As a means for selecting a transformant which has undergone homologous recombination in the genome, when the first marker gene is a drug resistant gene, a method of selecting a non-drug resistant bacterium can be mentioned. Penicillin antibiotics act bactericidally on proliferating cells but not on non-proliferating cells. Therefore, culture of bacteria in the presence of drug and penicillin antibiotics can selectively concentrate drug-resistant bacteria that do not grow in the presence of drug (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 chpA gene is introduced into the bacterium as the second marker, the bacterium in which homologous recombination in the genome did not occur is killed by the action of the lethal gene, so that homologous recombination occurs in the genome. Transformants 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 is deleted (Japanese Patent Laid-Open No. 2009-254350).

  As described above, a Bacillus subtilis mutant 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 the above-mentioned genomic regions are deleted can be prepared.

2-2. Dipicolinate Synthase Gene Expression Enhancement
Next, in the Bacillus subtilis mutant strain in which the predetermined region has been deleted, expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced.

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

  Dipicolinate synthase is composed of subunit A and subunit B, and a gene encoding each subunit is present. 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 B. 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.

  The gene corresponding to the Bacillus subtilis dipicolinate synthase gene is at least 80%, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably to the Bacillus subtilis spoVFA gene. 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 with Bacillus subtilis dipicolinate synthase subunit B to catalyze the above-described dipicolinate synthesis reaction Polynucleotides encoding proteins that exert the activity of

  Alternatively, the gene corresponding to the Bacillus subtilis dipicolinate synthase gene is at least 80%, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more with the B. subtilis spoVFB gene. , More preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and the above-mentioned dipicolinic acid synthesis reaction which works together with Bacillus subtilis dipicolinate synthase subunit A. And polynucleotides encoding proteins that exert catalyzing activity.

  Furthermore, examples of the gene corresponding to the Bacillus subtilis dipicolinate synthase gene include genes encoding dipicolinate synthase subunit A or B derived from microorganisms 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 There may be mentioned (SEQ ID NO: 9) and spoVFB (SEQ ID NO: 10), as well as 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 or any two or more of the above-mentioned Bacillus subtilis dipicolinate synthase genes or genes corresponding thereto may be enhanced in expression. Preferably, a gene encoding subunit A of Bacillus subtilis dipicolinate synthase described above or a gene corresponding thereto is combined with a gene encoding subunit B of Bacillus subtilis dipicolinate synthase described above or a gene corresponding thereto The expression should be enhanced. More preferably, expression may be enhanced by combining the subunit A of the Bacillus subtilis dipicolinate synthase described above and the gene encoding the subunit B.

Preferred examples of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto which is enhanced in expression in the Bacillus subtilis mutant strain of the present invention are selected from the group consisting of the genes described in (i) and (ii) below: There is at least one.
(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 set forth 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 of the nucleotide sequence shown in SEQ ID NO: 1 A poly encoding a protein comprising a nucleotide sequence having preferably 98% or more, more preferably 99% or more identity and exhibiting dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii) nucleotide;
(C) Dipicolinate synthase activity which hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and in the presence of the protein encoded by the gene described in (ii) A polynucleotide encoding a protein that exerts
(D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) 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 dipicoline in the presence of the protein encoded by the gene described in (ii) A polynucleotide encoding a protein that exerts acid synthase activity;
(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 the amino acid sequence shown in SEQ ID NO: 2 And a polynucleotide encoding a protein that exhibits dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii), consisting of an amino acid sequence having an identity of 99% or more;
(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 set forth 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 of the nucleotide sequence shown in SEQ ID NO: 3 Preferably, a poly comprising a nucleotide sequence having an identity of 98% or more, more preferably 99% or more, and encoding a protein that exerts dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) nucleotide;
(L) Hybridization under stringent conditions with the complementary strand of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 and dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) A polynucleotide encoding a protein that exerts
(M) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4;
(N) 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 dipicolin in the presence of the protein encoded by the gene described in (i) A polynucleotide encoding a protein that exerts acid synthase activity;
(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 the amino acid sequence shown in SEQ ID NO: 4 A polynucleotide comprising an amino acid sequence having an identity of 99% or more, and encoding a protein that exerts 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 gene of (i) (a) and at least one selected from the group consisting of the genes of (ii) (j) to (o) above. Or the combination of at least one selected from the group consisting of the genes of (i) (a) to (f) above and the gene of (ii) (j) above is enhanced. More preferably, in the Bacillus subtilis mutant strain of the present invention, the combination of the gene of (i) (a) and the gene of (ii) (j) is enhanced.

  As another preferable example of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto which is enhanced in expression in the Bacillus subtilis mutant strain of the present invention, the above mentioned spoVFA gene and spoVFB gene of Bacillus subtilis, spoVFA gene and spoVFB gene of Bacillus licheniformis And at least one member selected from the group consisting of the genes spoVFA and spoVFB of Bacillus clausii, the spoVFA and spoVFB genes of Clostridium stercorarium, and the spoVFA and spoVFB genes of Bacillus amyloliquefaciens, among which the Bacillus subtilis spoVFA and More preferably, at least one selected from the group consisting of Bacillus subtilis spoVFB genes.

  As still another preferred example of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto that is enhanced in expression in the Bacillus subtilis mutant strain of the present invention, the Bacillus spoVFA gene, the Bacillus licheniformis spoVFA gene, and the Bacillus clausii spoVFA gene And at least one member selected from the group consisting of Clostridium stercorarium spoVFA gene, and Bacillus amyloliquefaciens spoVFA gene, Bacillus subtilis spoVFB gene, Bacillus licheniformis spoVFB gene, Bacillus clausii spoVFB gene, Clostridium stercorarium spoVF gene, and Bacillus sp. The combination with at least one selected from the group consisting of the spoVFB gene of amyloliquefaciens is mentioned, and the combination of the Bacillus subtilis spoVFA gene and the Bacillus subtilis spoVFB gene is Ri preferred.

  As a means for enhancing the expression of the above-mentioned dipicolinate synthase gene or a gene corresponding thereto, for example, a control region (strong control region) capable of enhancing the expression of the gene of interest on the genome as compared to a wild type gene. Placing under control, and adding the gene of interest placed under the control of a control region, preferably a strong control region, to the dipicolinate synthase gene inherently possessed by the host, is introduced into the genome or plasmid in the cell To do, etc.

  For example, the Bacillus subtilis mutant strain of the present invention strongly expresses the control region sequence of the target dipicolinate synthase gene on its genome, using as a host the Bacillus subtilis mutant strain in which one or more, preferably all of the above-mentioned genomic regions are deleted. It can be produced by substituting control regions. Alternatively, the Bacillus subtilis mutant strain of the present invention is prepared by introducing a target dipicolinate synthase gene or a gene corresponding thereto placed under the control of the strong expression control region into the genome or plasmid of the host. Can. Alternatively, the expression of the target gene can be enhanced by modifying a plurality of target dipicolinate synthase genes or genes corresponding thereto in the host.

  An example of a detailed procedure for enhancing dipicolinate synthase gene expression is described below. First, the control region and the above-described dipicolinate synthase gene can be constructed as a DNA fragment which is arranged such that the gene is expressed under the control of the control region, and which is further linked to a region homologous to the host genome. For example, a fragment in which the regulatory region (for example, the spoVG gene regulatory region described later), the dipicolinate synthase subunit A gene, and the dipicolinate synthase subunit B gene are arranged in order from upstream is prepared, and the fragment is further added to the host A region of homology with a part of the genome is ligated to prepare a DNA fragment. Then, this DNA fragment is introduced into the above-described host, whereby the DNA fragment can be inserted into the host genome to transform the host. As a transformation method, an appropriate and general method can be adopted according to Bacillus subtilis as a host. The resulting transformant carries a large number of dipicolinate synthase genes as compared to the host before transformation, so the expression level of the genes is increased.

  Examples of control regions that can be used to enhance the expression of the dipicolinate synthase gene in the present invention include the control region of the α-amylase gene derived from Bacillus bacteria, the control region of the protease gene, the gene encoding ribosomal protein (rplS gene, etc. Control region of aprE gene, or a control region of spoVG gene; Bacillus sp. A control region of a cellulase gene of KSM-S237 strain; a control region of a kanamycin resistance gene derived from Staphylococcus aureus, a control region of a chloramphenicol resistance gene and the like (all refer to JP2009-089708A) There is no particular limitation. A control region capable of enhancing expression of the target gene as compared to the wild-type control region of the target gene is preferred.

(2-3. Deletion or inactivation of alsS gene or alsD gene)
In the Bacillus subtilis mutant strain of the present invention, at least one gene selected from the group consisting of Bacillus subtilis alsS gene and Bacillus subtilis alsD gene is deleted or inactivated. In a preferred embodiment, in the Bacillus subtilis mutant strain of the present invention, the Bacillus subtilis alsS gene and the Bacillus subtilis alsD gene are deleted or inactivated.

  In the present specification, the alsS gene and the alsD gene are genes encoding alpha-acetolactate synthase and alpha-acetolactate dehydrogenase, respectively, which have an activity of causing the chemical reaction of the following formula (2) to proceed.

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

  As a means for deleting or inactivating the gene, mutation of one or more nucleotides on the nucleotide sequence of the gene, or substitution or insertion of another nucleotide sequence to the nucleotide sequence, or sequence of the gene Deletion of part or all of Examples of means for introducing a mutation that inhibits transcription of the gene include mutagenesis of the promoter region of the gene, and inactivation of the promoter by substitution or insertion with another nucleotide sequence. Specific methods for the above-mentioned mutagenesis and substitution or insertion of nucleotide sequences are explained by ultraviolet irradiation, site-directed mutagenesis, and the above (2-1. Genomic region deletion Bacillus subtilis mutant strain). SOE-PCR method, homologous recombination method, etc. can be mentioned. The position on the Bacillus subtilis genome and the nucleotide sequence of the gene to be deleted or inactivated can be confirmed by the above-described BSORF DB.

(3. DPA production using the Bacillus subtilis mutant strain of the present invention)
The Bacillus subtilis mutant strain of the present invention has high DPA producing ability. The Bacillus subtilis mutant strain of the present invention has a genome in which a large region of the genome has been deleted, and in comparison with a Bacillus subtilis mutant strain in which expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced. And DPA production ability is improved (see Example 2 below). By culturing the Bacillus subtilis mutant strain of the present invention, DPA is efficiently produced outside the cells. Therefore, another aspect of the present invention relates to a method for producing DPA or a salt thereof using the Bacillus subtilis mutant strain of the present invention described above.

  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. There is no restriction | limiting in particular in the method to culture | cultivate, The culture | cultivation method of normal microorganisms can be used. That is, the medium to be used is a normal medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic components. As a carbon source, glucose, lactose, galactose, fructose and starch and cellulose hydrolysates of cellulose, saccharides such as molasses, alcohols such as glycerol, ethanol and sorbitol, 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 organic nitrogen sources, organic nitrogen such as soybean hydrolysate, ammonia gas, ammonia water, urea, glutamic acid, lysine, glycine, alanine, methionine, aspartic acid, arginine acid etc Can be used. As organic trace nutrient sources, it is desirable to contain required substances such as vitamins and amino acids or, if necessary, yeast extract, corn steep liquor and the like. In addition to these, it is desirable to add potassium phosphate, magnesium sulfate, calcium chloride, sodium chloride, copper ion, iron ion, manganese ion and the like as needed. In some cases, an antifoaming agent or the like is also added.

  The pH of the culture medium may be adjusted to a range where Bacillus subtilis can grow, preferably 6.0 to 8.0, and adjustment of the pH is carried out by using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, etc. You may 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 if necessary, aeration or agitation may be added.

  As described above, by culturing the Bacillus subtilis mutant strain of the present invention, DPA is produced in the culture supernatant. Alternatively, it is 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 produce DPA more efficiently by coexisting a substance added to the above-mentioned culture medium as an energy source.

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

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

  The following compositions, methods of manufacture, or uses are further disclosed herein as exemplary embodiments of the present invention. However, the present invention is not limited to these embodiments.

<1> Bacillus subtilis mutant strain
prophage6 area, prophage1 area, prophage4 area, PBSX area, prophage5 area, prophage area, spb area, pks area, skin area, pps area, propagation2 area, ydcL-ydeK-ydhU area, yisB-yitD area, yunA-yurT area, One or more regions selected from the group consisting of cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region Have a deleted genome,
The expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is enhanced, and
At least one gene selected from the group consisting of Bacillus subtilis alpha-acetolactate synthase gene alsS and Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD is deleted or inactivated.
Bacillus subtilis mutant strain.

<2> A method for producing a Bacillus subtilis mutant, which comprises:
prophage6 area, prophage1 area, prophage4 area, PBSX area, prophage5 area, prophage area, spb area, pks area, skin area, pps area, propagation2 area, ydcL-ydeK-ydhU area, yisB-yitD area, yunA-yurT area, One or more regions selected from the group consisting of cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region Enhance the expression of the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto in a Bacillus subtilis mutant strain having a genome deleted from
Deleting or inactivating at least one gene selected from the group consisting of Bacillus subtilis alpha-acetolactate synthase gene alsS and Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD;
How, including.

<3> In any one of the above <1> to <2>, preferably, the Bacillus subtilis mutant strain is a prophage 6 area, a prophage 1 area, a prophage 4 area, a PBSX area, a prophage 5 area, a prophage 3 area, an spb area, a pks area , Skin region, pps region, prophage 2 region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 It has a genome in which the region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region are deleted.

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

<5> In any one of <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: At least one more selected:
(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 set forth in SEQ ID NO: 1;
(B) A protein consisting of a nucleotide sequence having 80% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 and coding for a protein that exerts dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii) Polynucleotides to be
(C) Dipicolinate synthase activity which hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and in the presence of the protein encoded by the gene described in (ii) A polynucleotide encoding a protein that exerts
(D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) 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 dipicoline in the presence of the protein encoded by the gene described in (ii) A polynucleotide encoding a protein that exerts acid synthase activity;
(F) A protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and coding for a protein that exerts dipicolinate synthase activity in the presence of the protein encoded by the gene described in (ii) Polynucleotides to be
(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 set forth in SEQ ID NO: 3;
(K) A protein consisting of a nucleotide sequence having 80% or more identity to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein that exerts dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) Polynucleotides to be
(L) Hybridization under stringent conditions with the complementary strand of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 and dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) A polynucleotide encoding a protein that exerts
(M) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4;
(N) 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 dipicolin in the presence of the protein encoded by the gene described in (i) A polynucleotide encoding a protein that exerts acid synthase activity;
(O) A protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and coding for a protein that exerts dipicolinate synthase activity in the presence of the protein encoded by the gene described in (i) Polynucleotides.

<6> In the above <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 gene of (i) above and at least one selected from the group consisting of the gene of (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 genes of (i) (a) to (f) and the gene of (ii) (j) above; or
The combination of the gene of said (i) (a), and the gene of said (ii) (j).

<7> In any one of <1> to <6>, the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is:
Preferably, it comprises the spoVFA gene and spoVFB gene of Bacillus subtilis, the spoVFA gene and spoVFB gene of Bacillus licheniformis, the spoVFA gene and spoVFB gene of Bacillus clausii, the spoVFA gene and spoVF gene of Clostridium stercorarium, and the spoVFA gene and spoVF gene of Bacillus amyloliquefaciens 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 <7>, the Bacillus subtilis dipicolinate synthase gene or a gene corresponding thereto is:
Preferably, at least one selected from the group consisting of the spoVFA gene of Bacillus subtilis, the spoVFA gene of Bacillus licheniformis, the spoVFA gene of Bacillus clausii, the spoVFA gene of Clostridium stercorarium, and the spoVFA gene of Bacillus amyloliquefaciens A combination of at least one selected from the group consisting of the spoVFB gene of Bacillus licheniformis, the spoVFB gene of Bacillus clausii, the spoVFB gene of Clostridium stercorarium, and the spoVFB gene of Bacillus amyloliquefaciens;
More preferably, it is a combination of B. subtilis spoVFA gene and B. subtilis spoVFB gene.

<9> In any one of the above <1> to <8>, preferably, the Bacillus subtilis alpha-acetolactate synthase gene alsS and the Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD are deleted or inactivated. Be done.

<10> In any one of the above <1> to <9>, preferably, the Bacillus subtilis alpha-acetolactate synthase gene alsS consists of the nucleotide sequence shown in SEQ ID NO: 75, and the Bacillus subtilis alpha-acetolactate dehydrogenase The gene alsD consists of the nucleotide sequence shown in SEQ ID NO: 76.

<11> In any one of the above <1> to <10>, preferably, enhancement of 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 in a plasmid:
A control region of an α-amylase gene derived from Bacillus bacteria, a control region of a protease gene, a control region of a gene encoding a ribosomal protein (such as rplS gene), a control region of aprE gene, or a control region of spoVG gene; A control region of a cellulase gene of KSM-S237 strain; and a control region of a kanamycin resistance gene derived from Staphylococcus aureus or a control region of a chloramphenicol resistance gene.

The manufacturing method of dipicolinic acid or its salt using the Bacillus subtilis mutant strain as described in any one of <12> said <1> and <3>-<11>.

The manufacturing method of dipicolinic acid or its salt using the Bacillus subtilis mutant manufactured by the method as described in any one of <13> said <2>-<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 using 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 auxotrophy release strain Tryptophan in Bacillus subtilis mutant strain (MGB 874 strain; JP 2007-130013A) in which a large region of Bacillus subtilis wild strain genome has been deleted Canceled the requirement. Bacillus subtilis 168 strain, which is a parent strain of Bacillus subtilis mutant strain MGB 874, has trpC2 as a genotype. Thus, strain 168 is a tryptophan synthesis requirement for the trpC gene mutation encoding indole-3-glycerol phosphate synthase involved in tryptophan synthesis. In addition, Bacillus subtilis ATCC 6051 strain is known to be non-tryptophan-dependent (Albertini, AM & Galizzi, A. Microbiology, 1999, 145 (Pt12): 3319-3320). When the trpC genes of Bacillus subtilis 168 strain and ATCC 6051 T strain were compared, it was found that nucleotide ATT from 328th to 330th nucleotide does not exist in Bacillus subtilis 168 strain. Therefore, PCR was performed using the ATCC 6051 T strain genome as a template and the primers trpC_F1 (SEQ ID NO: 13) and trpC_R1 (SEQ ID NO: 14) listed in Table 3 to construct a PCR product. Bacillus subtilis mutant strain MGB 874 was transformed by the competent method using this PCR product, and SMA agar medium supplemented with tryptophan (0.2% ammonium sulfate, 1.4% dipotassium hydrogen phosphate, 0.6% phosphorus) Transformants grown on potassium dihydrogen acid, 0.1% trisodium citrate dihydrate, 0.5% glucose, 0.035% magnesium sulfate heptahydrate, 1.5% agar) As separated. Thus, the tryptophan non-requiring MGB 874 T strain was obtained.

(1-2) Construction of Bacillus subtilis mutant strain (MGB874T_PspoVG-spoVFAB strain) in which the expression of the dipicolinate synthase gene is enhanced: the expression of the dipicolinate synthase gene is enhanced using the strain MGB874T constructed in the above (1-1) as a host A Bacillus subtilis mutant (MGB874T_PspoVG-spoVFAB strain) was constructed. Fragment (A) was amplified by PCR using genomic DNA extracted from B. subtilis strain 168 as a template and the primers spoVFA-F1 (SEQ ID NO: 15) and spoVFA-R1 (SEQ ID NO: 16) described in Table 3. This fragment (A) is a region adjacent to the upstream of the start codon of the spoVFA gene (SEQ ID NO: 1). Moreover, 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) described in Table 3. This fragment (B) is a region downstream from the start codon of the spoVFA gene. Furthermore, a fragment (C) containing the spoVG control region sequence was amplified by PCR using the same genomic DNA as a template and the primers spoVGpro-F (SEQ ID NO: 19) and spoVGpro-R (SEQ ID NO: 20) described in Table 3 . Furthermore, using the plasmid pDLT3 (Morimoto, T. et al., Microbiology, 2002, Pt 11: 3539-3552) as a template, the primers Cm-F (SEQ ID NO: 21) and Cm-R (SEQ ID NO: 22) described in Table 3 were used. Using this, a fragment (D) containing the chloramphenicol resistance gene and its control region was amplified by PCR.

  Next, the resulting fragments (A), (D), (C) and (B) were sequenced in this order using the primers spoVFA-F1 (SEQ ID NO: 15) and spoVFA-R2 (see Table 3). It joined by SOE-PCR method using SEQ ID NO: 18) to obtain a DNA fragment of about 2.2 kb. Using the obtained DNA fragment, transformation of Bacillus subtilis strain MGB 874 T was performed by the competent method (J. Bacteriol., 1960, 81: 741-746), and grown on LB agar medium containing chloramphenicol. The colony was isolated 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 having enhanced expression of the spoVFAB gene, which has on its genome both the native spoVFAB gene and the introduced spoVFAB gene under the control of PspoVG.

  Between the spoVFA control region and the spoVFA gene on the genome of the MGB874T_PspoVG-spoVFAB strain constructed 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. The procedure for introducing the dipicolinate synthase gene into the Bacillus subtilis mutant obtained in this Example and the introduction position are shown in FIG.

(1-3) Construction of a Bacillus subtilis mutant (MGB 874 T_PspoVG-spoVFAB_ΔalsSD strain) in which the alpha-acetolactate synthase gene alsS and the alpha-acetolactate dehydrogenase gene alsD are deleted

  Using the MGB874T_PspoVG-spoVFAB strain constructed in (1-2) above as a host, a Bacillus subtilis mutant strain (MGB874T_PspoVG-spoVFAB_ΔalsSD strain) in which the alsSD gene group was deleted was constructed. This mutant was constructed by a marker-free deletion method using a cassette in which the IPTG regulatory region and the mazF gene, which is a free mRNA-cleaved ribonuclease, were fused (Morimoto, T. et al., In Strain Engineering, pp 345-358. Springer).

  First, using the genomic DNA extracted from Bacillus subtilis mutant strain MGB 874 T as a template, and the primers alsSD-DF1 (SEQ ID NO: 23) and alsSD-DR1 (SEQ ID NO: 24) described in Table 3, the start codon of alsSD gene cluster Fragment (A), which is the upstream flanking region, was amplified by PCR. Also, using the same genomic DNA as a template and the primers alsSD-DF2 (SEQ ID NO: 25) and alsSD-DR2 (SEQ ID NO: 26) described in Table 3, fragment (B) which is a region downstream of the stop codon of the alsD gene It amplified by PCR. Furthermore, using the same genomic DNA as a template and the primers alsSD-IF (SEQ ID NO: 27) and alsSD-IR (SEQ ID NO: 28) described in Table 3, a fragment (C) which is a homologous region within the alsSD gene group ORF It amplified by PCR. Furthermore, using the Bacillus subtilis mutant TOM310 (Morimoto, T. et al., In Strain Engineering, pp 345-358. Springer) as a template, the primers pAPNC-F (SEQ ID NO: 29) described in Table 3 and chpA-R (SEQ ID NO: 30, the mazF cassette fragment (D) containing the spectinomycin resistance gene was amplified by PCR.

  Next, the obtained fragment (A), fragment (B), fragment (D) and fragment (C) are sequenced in this order with primers alsSD-DF1 (SEQ ID NO: 23) and alsSD-IR (listed in Table 3). The final DNA fragment was obtained by SOE-PCR method using SEQ ID NO: 28). Using the obtained DNA fragment, transformation of Bacillus subtilis MGB874T_PspoVG-spoVFAB strain is carried out according to the competent method (J. Bacteriol., 1960, 81: 741-746), and IPTG does not contain LB agar containing spectinomycin. The 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 resulting mutant is a mutant from 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 MGB 874 T_PspoVG-spoVFAB_ΔalsSD strain is a Bacillus subtilis mutant having a mutation in which the alsD gene stop codon region is deleted from the alsS gene start codon on the genome. A method for deleting the alsSD gene group constructed by the marker-free deletion method using the mazF gene cassette in this example is shown in FIG.

  By 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), the alsSD gene cluster on the genome is obtained. It confirmed that the deletion had arisen.

(Example 2) DPA productivity evaluation The MGB874T_PspoVG-spoVFAB strain constructed in Example 1 and the MGB874T_PspoVG-spoVFAB_ΔalsSD strain were treated with 50 mL of a synthetic medium (5.0% glucose, 1.8% ammonium chloride, 0.15% phosphorus) Dibasic acid hydrogen, 0.035% magnesium sulfate heptahydrate, 0.005% manganese sulfate pentahydrate, 50 mM MOPS (monophorinopropane sulfonic acid) buffer (pH 7.0), other trace metals, 4 Shake culture with 0% calcium carbonate, 0.6% ammonium chloride at 37 ° C. for 8 hours, obtain a culture broth, and use synthetic medium (5.0% glucose, 0.6% ammonium chloride, 0.15) % Dibasic hydrogen phosphate, 0.035% magnesium sulfate heptahydrate, 0.005% sulfuric acid Inoculate 50 mL of Ngangan pentahydrate, 50 mM MOPS (monophoropropane sulfonic acid) buffer (pH 7.0), other trace metals, 4.0% calcium carbonate, and shake culture at 37 ° C, 200 rpm for 2 days Did.
After completion of the culture, the DPA production amount was measured under the analysis conditions shown in the following reference example, and the relative value was determined when the production amount of the MGB874T_PspoVG-spoVFAB strain was 100%.

  The Bacillus subtilis mutant strain MGB874T_PspoVG-spoVFAB_ΔalsSD strain in which the alsS gene and the alsD gene were deleted had a significantly higher DPA production amount as compared with the MGB874T_PspoVG-spoVFAB strain, which is the strain before the alsSD gene deletion (Table 4).

(Reference Example) Quantification of DPA The culture solution sample after completion of culture is subjected to centrifugation (Hitachi Koki, himacCF15 RX) for 30 minutes at 14,800 rpm at room temperature, and the obtained sample supernatant after centrifugation For the DPA contained, quantification and molecular weight analysis were performed by HPLC method. The HPLC apparatus includes a liquid transfer pump (Shimadzu, LC-9A), an autosampler (Hitachi measuring instrument, AS-2000), a column oven (Shimadzu, CTO-6A), a UV detector (Shimadzu, SPD-10A), and degassing. What connected the apparatus (GL science, DG660B) and the chromatography data analyzer (Hitachi measuring instrument, D-2500) was used. The analytical column used was High Performance Carbohydrate Column 60 (Å) 4 μm 4.6 × 250 mm HPLC Column (Aminopropylmethylsilyl bonded amorphous silica) (Waters). As an eluent, a solution in which an aqueous solution of 20 mM EDTA in water, adjusted to pH 3.4 with concentrated phosphoric acid, and an acetonitrile solution were mixed 1: 1 was used. Measurement conditions were such that the detection wavelength was 270 nm and the flow rate was 1 mL / min. The sample to be subjected to HPLC analysis was pretreated by filter filtration with MULTI SCREEN MNHV 45 (manufactured by MILLIPORE, 0.45 μm Durapore membrane) to remove insolubles. The concentration test was performed based on a calibration curve prepared using 2,6-pyridinedicarboxylic acid: DPA, 99% (SIGMA-ALDRICH).

Claims (7)

prophage6 area, prophage1 area, prophage4 area, PBSX area, prophage5 area, prophage area, spb area, pks area, skin area, pps area, propagation2 area, ydcL-ydeK-ydhU area, yisB-yitD area, yunA-yurT area, A Bacillus subtilis strain having a genome in which all of the cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region are deleted. A mutant strain,
The expression of the Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto, and the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto is enhanced, and
The Bacillus subtilis alpha-acetolactate synthase gene alsS and the Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD have been deleted or inactivated.
The Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto is
The following (a) , (b), (d) to (f):
(A) a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1;
(B) Dipicoline consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity ;
( D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) 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 the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts dipicolinate synthase activity in the presence of a protein encoded by
(F) Dipicoline consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity;
At least one selected from the group consisting of
The Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto is
The following (j) , (k), (m) to (o):
(J) a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3;
(K) Dipicoline consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 3 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity ;
( M) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4;
(N) 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 consisting of Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts dipicolinate synthase activity in the presence of a protein encoded by
(O) Dipicoline consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity;
At least one selected from the group consisting of
Bacillus subtilis mutant strain.   The Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto is a Bacillus subtilis spoVFA gene, and the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto is a Bacillus subtilis spoVFB gene. The Bacillus subtilis mutant according to claim 1.   The expression of the B. subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto, and the expression of the B. subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto are under the control of the control region of the spoVG gene. The Bacillus subtilis mutant according to claim 1 or 2, wherein the Bacillus subtilis mutant is reinforced by being arranged to be expressed. A method for producing Bacillus subtilis,
prophage6 area, prophage1 area, prophage4 area, PBSX area, prophage5 area, prophage area, spb area, pks area, skin area, pps area, propagation2 area, ydcL-ydeK-ydhU area, yisB-yitD area, yunA-yurT area, A Bacillus subtilis strain having a genome in which all of the cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region are deleted. In the mutant,
To enhance expression of the Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto, and the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto,
To delete or inactivate the Bacillus subtilis alpha-acetolactate synthase gene alsS and the Bacillus subtilis alpha-acetolactate dehydrogenase gene alsD,
Including
The Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto is
The following (a) , (b), (d) to (f):
(A) a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1;
(B) Dipicoline consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity ;
( D) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) 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 the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts dipicolinate synthase activity in the presence of a protein encoded by
(F) Dipicoline consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity;
At least one selected from the group consisting of
The Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto is described in the following (j) , (k), (m) to (o):
(J) a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3;
(K) Dipicoline consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 3 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity ;
( M) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4;
(N) 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 consisting of Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts dipicolinate synthase activity in the presence of a protein encoded by
(O) Dipicoline consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and in the presence of a protein encoded by the Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto A polynucleotide encoding a protein that exerts acid synthase activity;
At least one selected from the group consisting of
Method.   The Bacillus subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto is a Bacillus subtilis spoVFA gene, and the Bacillus subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto is a Bacillus subtilis spoVFB gene. The method of claim 4.   The expression of the B. subtilis dipicolinate synthase subunit A gene or a gene corresponding thereto, and the expression of the B. subtilis dipicolinate synthase subunit B gene or a gene corresponding thereto are under the control of the control region of the spoVG gene. 6. The method of claim 4 or 5, wherein the gene is enhanced by being arranged to be expressed.   The manufacturing method of dipicolinic acid or its salt using the Bacillus subtilis mutant strain of any one of Claims 1-3.