JP5498651B2 - Method for producing dipicolinic acid or a salt thereof - Google Patents

Description

  The present invention relates to a method for producing dipicolinic acid or a salt thereof using a microorganism.

  Dipicolinic acid (2,6-pyridinedicarboxylic acid) is a natural product and is known as a highly biodegradable chelating agent. For industrial use, it is a detergent composition (Patent Document 1) and a metal masking agent (Patent Document). 2) It has been reported that it can be used as an antioxidant (Patent Documents 3 and 4).

  Dipicolinic acid is known to be synthesized from 2,6-lutidine by an oxidation reaction. That is, a method using hypohalite as an oxidizing agent in the presence of a nickel compound (Patent Document 5), a manufacturing method by oxidation using ozone (Patent Documents 6 and 7), an electrolytic oxidation method (Patent Document 8) An air oxidation method (Non-patent Document 1) is disclosed. However, in these methods, since the conversion and selectivity of the reaction are insufficient, alkylpyridine such as 2,6-lutidine as a raw material and 6-methyl-2-pyridinecarboxylic acid as a reaction intermediate is used as a reaction solution. They remain in the product and are mixed as impurities in the product 2,6-pyridinedicarboxylic acid (dipicolinic acid). That is, these methods have a problem that it is difficult to produce high-purity dipicolinic acid.

On the other hand, according to the method for producing dipicolinic acid using microorganisms, it is expected that dipicolinic acid can be produced with high purity without containing alkylpyridine. As a method for producing dipicolinic acid using a microorganism to a process for the production by fermentation using Bacillus (Bacillus) genus is a microorganism to form a spore production method using (Patent Documents 9 and 10) and mold (Patent Document 11 ). However, these methods have a problem that the industrially usable production amount cannot be achieved.

  Moreover, as a manufacturing method of dipicolinic acid by a fermentation method, the manufacturing method (patent document 12) using gene recombinant lysine producing microbe is mentioned. However, since this method requires expensive diaminopimelic acid for growth, there is a problem that dipicolinic acid cannot be produced at low cost.

Japanese translations of PCT publication No. 2002-532616, US Pat. No. 5,536,625 JP-A-5-158195 JP-A-6-166621 JP-A-3-163002 JP-A-11-322716 Japanese Patent Laid-Open No. 11-343483 SU943236A1 EP253439B1 JP 2004-275075 A DE2300056 publication US Pat. No. 3,334,021 JP 2002-371063 A Synthesis Commun. (1992), 22,2691-6

  The present invention solves the problems in the conventional method for producing dipicolinic acid as described above, can produce dipicolinic acid or a salt thereof with excellent productivity and low cost, and industrial production of dipicolinic acid. An object of the present invention is to provide a method for producing dipicolinic acid or a salt thereof applicable to the above.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have modified the expression pattern of a gene involved in the dipicolinic acid synthesis pathway using a microorganism having spore-forming ability or having lost spore-forming ability as a host. As a result, it was found that dipicolinic acid can be produced in a high yield without using a special medium that is a main factor for increasing the cost, and the present invention has been completed.

That is, the present invention includes the following.
(1) culturing a microorganism having a spore-forming ability or having lost a spore-forming ability and having a dipicolinate synthase gene transcribed under the control of a promoter that works before the spor coat protein deposition period in the spore formation stage And a step of recovering dipicolinic acid from the culture supernatant and / or aqueous reaction solution of the microorganism, and a method for producing dipicolinic acid or a salt thereof.

  The method for producing dipicolinic acid or a salt thereof according to the present invention may further include a step of purifying the recovered dipicolinic acid.

In the method for producing dipicolinic acid or a salt thereof according to the present invention, the spore coat protein deposition period can be a period corresponding to the V-stage of Bacillus subtilis spore formation. In dipicolinic acid or a salt thereof according to the present invention, it is preferable as the above-mentioned microorganism is a microorganism belonging to Bacillus (Bacillus) genus or Clostridium (Clostridium) genus. In the method for producing dipicolinic acid or a salt thereof according to the present invention, a microorganism belonging to the genus Bacillus is particularly preferable as the microorganism. Here it is more preferable examples of the microorganisms belonging to the Bacillus (Bacillus) genus Bacillus subtilis (Bacillus subtilis) and Bacillus megaterium (Bacillus megaterium).

  In the method for producing dipicolinic acid or a salt thereof according to the present invention, a σA-dependent promoter or a σH-dependent promoter can be used as the promoter. Further, in the method for producing dipicolinic acid or a salt thereof according to the present invention, an S237 promoter or a spoVG promoter can be used as the promoter.

Moreover, this invention includes the following.
(2) A microorganism having a spore-forming ability or having lost the spore-forming ability and having a dipicolinate synthase gene transcribed under the control of a promoter that works before the spore coat protein deposition period in the spore formation period.

In the microorganism according to the present invention, the spore coat protein deposition period may be a period corresponding to the V-stage of Bacillus subtilis spore formation. Further, as the microorganism of the present invention is preferably a microorganism belonging to Bacillus (Bacillus) genus or Clostridium (Clostridium) genus. It is particularly preferred examples of the microorganisms according to the present invention is a microorganism belonging to Bacillus (Bacillus) genus. Here it is more preferable examples of the microorganisms belonging to the Bacillus (Bacillus) genus Bacillus subtilis (Bacillus subtilis) and Bacillus megaterium (Bacillus megaterium).

  In the microorganism according to the present invention, a σA-dependent promoter or a σH-dependent promoter can be used as the promoter. In the microorganism according to the present invention, S237 promoter or spoVG promoter can be used as the promoter.

  According to the present invention, dipicolinic acid or a salt thereof can be produced with excellent productivity and low cost. That is, according to the method for producing dipicolinic acid or a salt thereof according to the present invention, dipicolinic acid or a salt thereof that can be used for a highly biodegradable detergent composition, a metal masking agent, an antioxidant, or the like is provided in a large amount and at a low cost. be able to. In addition, according to the microorganism of the present invention, dipicolinic acid that can be used for a highly biodegradable detergent composition, a metal masking agent, an antioxidant, and the like can be provided in large quantities and at low cost.

Hereinafter, the present invention will be described in more detail.
In the method for producing dipicolinic acid or a salt thereof according to the present invention, first, a microorganism having a spore-forming ability or having lost the spore-forming ability, which is under the control of a promoter that works before the spor coat protein deposition stage in the spore-forming stage. A microorganism having a dipicolinate synthase gene to be transcribed is prepared.

Here, the microorganism having the ability to form spores means a microorganism capable of forming endospores in response to depletion of nutrients contained in the medium and various environmental stresses. The microorganism having a spore forming ability, for example, a Bacillus (Bacillus) genus or Clostridium (Clostridium) microorganism belonging to the genus.

Examples of the microorganisms belonging to the genus Bacillus, specifically, Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus stearotheromophilus, Bacillus coagulans, Bacillus megaterium, Bacillus halodurans, Bacillus brevis (Brevibacillus brevis), Bacillus pumilus, Bacillus alcalophilus, Examples include Bacilus amylolyticus, Bacillus polymyxa (Paenibacillus polymyxa), Bacillus sphaericus, Bacillus firmus, Bacillus clausii, Bacillus macerans and the like. Examples of microorganisms belonging to the genus Clostridium include Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, and the like. In addition, Bacillus brevis may be classified as a Brevibacillus genus and described as Brevibacillus brevis, and Bacillus polymyxa may be classified as a Paenibacillus genus and described as Paenibacillus polymyxa. However, in this specification, Bacillus brevis and Bacillus polymyxa are both defined as bacteria belonging to the genus Bacillus. That is, in the present invention, a microorganism belonging to the genus Bacillus is interpreted as including a microorganism described as Brevibacillus brevis and a microorganism described as Paenibacillus polymyxa.

In particular, in this method, it is preferable to use a microorganism belonging to the genus Bacillus having a spore-forming ability, and it is preferable to use Bacillus subtilis or Bacillus megaterium. Examples of Bacillus subtilis include Bacillus subtilis 168, Marburg, natto, and ISW1214. As Bacillus megaterium, ATCC14581 strain preserve | saved in the American Type Culture Collection can be used.

  In addition, a microorganism that has lost the ability to sporulate is to induce a mutation in the microorganism having the ability to form a spore, for example, or to functionally delete a part of a gene group involved in sporulation. It means a mutant microorganism that has lost its sporulation ability. In particular, in this method, a Bacillus subtilis mutant strain that has lost its sporulation ability by inducing a mutation in Bacillus subtilis, or a part of a gene related to sporulation (spo gene) is deleted, resulting in a sporulation ability. It is preferable to use a Bacillus subtilis mutant strain that has lost the amount.

  On the other hand, in the present method, the above-described microorganism has a dipicolinate synthase gene that is transcribed under the control of a promoter that works before the spore coat protein deposition phase in the spore formation phase.

  Here, the spore coat protein deposition period in the spore formation period means a period called the V stage in the spore formation process of Bacillus subtilis. The spore formation process of Bacillus subtilis begins in phase 0 when cell division by vegetative growth stops, phase II in which the diaphragm is formed, phase III in which the forapore is formed on the other side of the diaphragm, and the outer periphery of the forepore Phase IV in which the cortex layer is formed, and phase V in which spore coat protein is deposited on the outer periphery of the cortex layer are included. In the 0th phase of Bacillus subtilis sporulation, RNA polymerases σA and σH control gene transcription. In phase II, σF controls transcription on one side separated by a diaphragm, and σE controls transcription on the other side. In stage III, σG controls transcription within the forespore, and σE controls transcription outside the forespore. Furthermore, in the IV stage, σG controls transcription inside the spore covered with the spore coat layer, and σK controls transcription outside the spore. Furthermore, in the V stage, σG controls transcription inside the spores covered with the spore coat, and σK controls transcription outside the spores.

  Therefore, in the case of Bacillus subtilis, promoters that function before the spor coat protein deposition phase in the sporulation phase mean σA factor-dependent promoter, σH factor-dependent promoter, σE factor-dependent promoter, and σF factor-dependent promoter It will be. In particular, it is more preferable to use a promoter that acts constitutively. Also, use a σB factor-dependent promoter that is expressed in response to environmental stress or an ECF (extracytoplasmic function) σ factor-dependent promoter (FEMS Microbiol Lett. (2003) 14; 220 (1): 155-60.) You can also.

  The σA factor-dependent promoter is not particularly limited, but includes Bacillus subtilis phage SP01 promoter (Proc. Natl. Acd. Sci. USA. (1984) 81: 439-443.), veg gene, amyE (amylase) gene, aprE (Subtilisin) gene and S237 (S237 cellulase, JP 2000-210081) promoter. The σH factor-dependent promoter is not particularly limited, and examples include promoters of citG gene, spoVS gene and spoVG (Proc. Natl. Acd. Sci. USA. (1986) 83: 9438-9442.) gene. The σE factor-dependent promoter is not particularly limited, and examples include promoters of cotE gene and spoIVA gene. In this method, the promoter of the spoVG gene of Bacillus subtilis is particularly desirable.

  Here, the promoter of a predetermined gene is defined as a region that exists upstream of the coding region in the gene and has a function of controlling the transcription of the gene by the interaction of RNA polymerase. More specifically, the promoter of a predetermined gene means a region of about 200 to 600 bases upstream of the coding region in the gene.

  More specifically, SEQ ID NO: 1 shows the base sequence of the spoVG gene promoter, which is a σH factor-dependent promoter preferably used in the present method. In this method, the polynucleotide comprises a nucleotide sequence in which one or several bases are substituted, deleted, added or inserted in the nucleotide sequence shown in SEQ ID NO: 1, and functions as a σH factor-dependent promoter. Polynucleotides can also be used as promoters. Here, “functions as a σH factor-dependent promoter” means that the transcription is specifically regulated by the σH factor which is an RNA polymerase. This specificity can be evaluated by binding a reporter gene downstream of the polynucleotide to be evaluated and observing the expression of the reporter gene in the presence and absence of the σH factor.

  In addition, SEQ ID NO: 2 shows the base sequence of the S237 promoter, which is a σA factor-dependent promoter preferably used in this method. In this method, the polynucleotide comprises a nucleotide sequence in which one or several bases are substituted, deleted, added or inserted in the nucleotide sequence shown in SEQ ID NO: 2, and functions as a σA factor-dependent promoter. Polynucleotides can also be used as promoters. Here, “functions as a σA factor-dependent promoter” means that the transcription is specifically regulated by the σA factor which is an RNA polymerase. This specificity can be evaluated by binding a reporter gene downstream of the polynucleotide to be evaluated and observing the expression of the reporter gene in the presence and absence of the σA factor.

  Similarly, SEQ ID NO: 3 shows the base sequence of the cotE gene promoter, which is a σE factor-dependent promoter preferably used in this method.

  In addition, even if it is microorganisms other than Bacillus subtilis, for example, a microorganism of the genus Clostridium, spores are formed through a process similar to the spore formation process of Bacillus subtilis. That is, even for microorganisms other than Bacillus subtilis, the spor coat deposition period is included in the spore formation process. Therefore, even a microorganism other than Bacillus subtilis can uniquely identify and isolate a promoter that works before the spoacoat deposition period. For example, in microorganisms belonging to the genus Clostridium, a ptb gene promoter (SEQ ID NO: 4) is known as a promoter corresponding to a σA-dependent promoter. In addition, the spoVG gene promoter (SEQ ID NO: 5) is known as a promoter corresponding to the σH-dependent promoter in Clostridium microorganisms. Furthermore, the flgE gene promoter (SEQ ID NO: 6) is known as a promoter corresponding to the σE-dependent promoter in Clostridium microorganisms (Journal of Bacteriology (2005), 187, 7103-7118 and Nucleic Acids Research (2004). ), 32, 6, 1973-1981).

  On the other hand, in the present method, the dipicolinate synthase gene whose transcription is controlled by the above-described promoter is a gene encoding dipicolinate synthase having an activity to promote the following chemical reaction.

  The dipicolinate synthase gene in Bacillus subtilis is composed of spoVFA encoding dipicolinate synthase subunit A and spoVFB encoding dipicolinate synthase subunit B. The nucleotide sequence of the spoVFA gene and the amino acid of dipicolinate synthase subunit A encoded by the spoVFA gene are shown in SEQ ID NOs: 7 and 8, respectively. The nucleotide sequence of the spoVFB gene and the amino acid sequence of dipicolinate synthase subunit B encoded by the spoVFB gene are shown in SEQ ID NOs: 9 and 10, respectively. In wild-type Bacillus subtilis, spoVFA and spoVFB are arranged in this order to form an operon, and both are transcriptionally controlled by a σK factor-dependent promoter. Therefore, in the wild-type Bacillus subtilis, the spoVFA gene and the spoVFB gene are expressed in the V stage in the Bacillus subtilis spore formation process, and dipicolinic acid accumulates in the endospore.

  Moreover, in this method, it is not limited to the dipicolinate synthase gene derived from Bacillus subtilis, The dipicolinate synthase gene derived from microorganisms other than Bacillus subtilis can also be used. Dipicolinate synthase genes include spoVFA (SEQ ID NO: 11), spoVFB (SEQ ID NO: 12) derived from Bacillus licheniformis, spoVFA (SEQ ID NO: 13), spoVFB (SEQ ID NO: 14) derived from Bacillus clausii, CtheDRAFT_2170 derived from Clostridium thermocellum (sequence SEQ ID NO: 14) No. 15), CtheDRAFT_2171 (SEQ ID NO: 16) and the like. In this method, any microorganism-derived dipicolinate synthase gene described above can be used.

  The promoter and dipicolinate synthase gene, which function before the spor coat protein deposition phase in the spore formation phase, are arranged so that the gene is expressed under the control of the promoter and binds the homologous region with the host genome. Thus, the above-mentioned microorganism can be transformed using this DNA fragment. As a transformation method, an appropriate and general method can be adopted depending on the microorganism as a host. For example, when Bacillus subtilis is used as a host microorganism, the above-mentioned promoter (for example, spoVG gene promoter) is arranged upstream of spoVFA, and a DNA fragment is prepared by binding a homologous region with a part of the Bacillus subtilis genome. Using the obtained DNA fragment, for example, Bacillus subtilis 168 strain is transformed.

  A PCR method or the like can be used for gene amplification. For example, Pyrobest DNA polymerase (Takara Bio) can be used for the PCR reaction, and the reaction conditions may be performed according to the instructions.

  In addition, the promoter and dipicolinate synthase gene that act before the spor coat protein deposition phase in the sporulation phase described above were incorporated into a desired vector so that the gene was expressed under the control of the promoter, and obtained. The above-mentioned microorganism can be transformed with a recombinant vector according to a conventional method.

  Furthermore, a recombinant microorganism capable of highly expressing the above-described dipicolinate synthase gene can be obtained by transforming the recombinant microorganism transformed with the above-described DNA fragment again with the above-described recombinant vector. it can. In the transformed recombinant microorganism, the dipicolinate synthase gene is transcribed under the control of a promoter that works before the spore coat protein deposition phase in the spore formation phase. Therefore, dipicolinic acid can be produced in the microorganism by culturing (growing) the obtained recombinant microorganism.

  There is no particular limitation on the method for growing the microorganism, and a normal method for culturing 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 other organic components as required. As the carbon source, glucose, lactose, galactose, fructose, starch and cellulose hydrolysates, sugars 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 the nitrogen source, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia, urea, glutamic acid, lysine, glycine, alanine, methionine, aspartic acid, arginic acid, etc. Can be used. As organic trace nutrient sources, it is desirable to contain required substances such as vitamins and amino acids, or yeast extract, corn steep liquor and the like as necessary. 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 as necessary. In some cases, an antifoaming agent or the like is also added.

  Further, it is appropriate to adjust the pH of the medium to 6.0 to 8.0, and the pH may be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Culturing is carried out at 15 to 45 ° C., preferably 25 to 45 ° C., for 6 to 96 hours, more preferably 24 to 72 hours, and aeration or agitation may be added if necessary. In the case of recombinant Bacillus subtilis, it is preferable to adjust the pH of the medium to a range where the Bacillus subtilis used can grow, for example, pH 6.0 to 8.0. The culture conditions may be 15 to 42 ° C., preferably 28 to 37 ° C. for 2 to 4 days with shaking or aeration and agitation.

  As described above, by culturing (growing) the recombinant microorganism, dipicolinic acid can be produced in the culture supernatant, and dipicolinic acid can be recovered by a known method. In addition, it is also possible to produce dipicolinic acid in the reaction supernatant by suspending the microorganisms grown as described above in an aqueous solution containing aspartic acid and / or pyruvic acid. At this time, dipicolinic acid can be produced more efficiently by coexisting a substance that is added to the medium as an energy source.

  Isolating and collecting dipicolinic acid from the culture supernatant or reaction supernatant of the recombinant microorganism can be carried out by combining known methods. For example, dipicolinic acid crystals or precipitates can be easily obtained by combining adsorption / desorption with an ion exchange resin, solid-liquid separation by crystallization, and removal of impurities by membrane treatment.

  Here, a salt of dipicolinic acid can be obtained by adding an amount of alkali according to the purpose to a solution containing dipicolinic acid obtained by treatment with an ion exchange resin or the like. Specifically, dipicolinic acid sodium salt can be obtained by adding NaOH.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
[Example 1]
(1-1)
Primers of PsVFA FW (5'-CCCTTCCGGCAATATGCC-3 ') (SEQ ID NO: 17) and PsVFA / Cm RV (5'-ATGGGTGCTTTAGTTGAAGATCTAAACGTTCACCTTC-3') (SEQ ID NO: 18) using genomic DNA extracted from Bacillus subtilis 168 strain as a template A 0.6 kb fragment (A) was amplified by PCR. This 0.6 kb fragment (A) is a 0.6 kb region adjacent to the start codon of the spoVFA gene (SEQ ID NO: 1). In addition, using the genomic DNA as a template, and using the primers of spoVG / spoVFA FW (5′-GGTGGTGAACTACTATGTTAACCGGATTGAAAATTGC-3 ′) (SEQ ID NO: 19) and spoVFA RV (5′-GATGCGCTGAACTTCTTGC-3 ′) (SEQ ID NO: 20) The kb fragment (B) was amplified by PCR. This 0.6 kb fragment (B) is a 0.6 kb region downstream from the start codon of the spoVFA gene. Furthermore, using the genomic DNA as a template, spoVG using primers Cm / spoVG FW (5'-CTGCCCCGTTAGTTGAAGGTTAGTCGAGATCGAAGTTA-3 ') (SEQ ID NO: 21) and spoVG RV (5'-CATAGTAGTTCACCACC-3') (SEQ ID NO: 22). A 0.2 kb fragment (C) containing the promoter sequence was amplified by PCR. Furthermore, using plasmid pC194 (source: Staphylococcus aureus origin) as a template, Cat F (5'-TCTTCAACTAAAGCACCCAT-3 ') (SEQ ID NO: 23) and Cm RV (5'-CTTCAACTAACGGGGCAG-3') (SEQ ID NO: 24) A 0.9 kb fragment (D) containing a chloramphenicol resistance gene was amplified by PCR using primers.

  Next, PsVFA FW2 (5′-CCCCATAATGGAGCAGGC-3 ′) (SEQ ID NO: 25) and spoVFA are arranged so that the obtained fragment (A), fragment (D), fragment (C) and fragment (B) are in this order. Using a primer of RV2 (5'-GCGCGGCAAATGTACGG-3 ') (SEQ ID NO: 26), it was ligated by SOE-PCR (splicing by overlap extension PCR: Gene, 77, 61, 1989) to obtain a 2.2 kb DNA fragment It was. The resulting DNA fragment was used to transform Bacillus subtilis 168 strain by competent method (J. Bacteriol., 81, 741, 1960), and colonies grown on LB agar medium containing chloramphenicol It was isolated as a transformant (hereinafter referred to as 168VGspoVF strain).

  The spoVFA promoter on the genome was obtained by PCR using the genome of the obtained transformant 168VGspoVF and subsequent sequencing by the Sanger method (Proc. Natl. Acad. Sci. USA, 74: 5463 (1982)). It was confirmed that the chloramphenicol resistance gene and the spoVG gene promoter were inserted between the and the spoVFA genes.

(1-2)
On the other hand, S237UB1 (5′-GCAGGATCCGATTTGCCGATGCAACAGGCTTATATTTAG-3 ′) (SEQ ID NO: 27) using a recombinant plasmid pHYEglS containing a cellulase gene derived from Bacillus sp. KSM-S237 strain (source: JP 2000-210081) as a template. A 0.6 kb fragment (E) containing the S237 promoter was amplified by PCR using primers of S237sVFABd1 (5′-CGGTTAACATATTACCTCCTAAATATTTTTAAAG-3 ′) (SEQ ID NO: 28). Also, using the genomic DNA extracted from Bacillus subtilis 168 as a template, dipicoline using S237sVFABu2 (5'-AGGAGGTAATATGTTAACCGGATTGAAAATTGC-3 ') (SEQ ID NO: 29), S237sVFABd2 (5'-GCTTCTAGAATTAGTCATTTCCCTGATAATTCTCAAC-3') (SEQ ID NO: 30) A 1.5 kb fragment (F) containing the acid synthase structural gene was amplified by PCR.

  Next, the obtained fragment (E) and fragment (F) were combined in this order by SOE-PCR using S237UB1 and S237sVFABd2 to obtain a 2.1 kb DNA fragment. The obtained DNA fragment was inserted into EcoRI and XbaI restriction enzyme cleavage points of shuttle vector pHY300PLK to construct a recombinant plasmid pH237sVFAB. It was confirmed that the target promoter and gene were inserted by Sanger sequencing for the plasmid pH237sVFAB.

  Next, using the genomic DNA extracted from the 168VGspoVF strain prepared in (1-1) above as a template, and using primers of PsVFA FW and Cm RV, a 0.6 kb DNA fragment adjacent to the upstream of the start codon of the spoVFA gene and A 1.5 kb fragment (G) containing the chloramphenicol resistance gene was amplified by PCR. In addition, using plasmid pH237sVFAB as a template, Cm / PS237FW (5'-CTGCCCCGTTAGTTGAAGGATTTGCCGATGCAACAGG-3 ') (SEQ ID NO: 31) and spoVFA RV primer, 0.6 kb DNA fragment downstream from the start codon of S237 promoter sequence and spoVFA A 1.1 kb fragment (H) containing was amplified by PCR. The obtained fragments (G) and (H) were combined in this order by the SOE-PCR method using PsVFA FW2 and spoVFA RV2 primers to obtain a 2.5 kb DNA fragment. The obtained DNA fragment was used to transform Bacillus subtilis 168 strain by competent method, and colonies grown on LB agar medium containing chloramphenicol were isolated as transformants (hereinafter referred to as 168S237spoVF strain). Called).

  It was confirmed that the chloramphenicol resistance gene and the S237 promoter were inserted between the spoVFA promoter and spoVFA gene on the genome by PCR using the genome of the obtained transformant 168S237spoVF strain and subsequent sequencing. confirmed.

(1-3)
The 168VGspoVF strain obtained in (1-1) above and the 168S237spoVF strain obtained in (1-2) above were each 20 mL of 2% (w / v) yeast extract (Difco) and 4% corn steep liquor. (Oriental Yeast Co., Ltd.), 0.05% magnesium sulfate heptahydrate, 0.001% manganese sulfate, 0.20% L-tryptophan, 0.50% lysine hydrochloride, 4.0% sodium aspartate, 0.15% monopotassium phosphate, 0.35% phosphoric acid Using a medium containing 2 potassium and 14% maltose monohydrate, shaking culture was performed at 30 ° C. for 72 hours. After culturing, the cells were removed by centrifugation, the amount of dipicolinic acid in the culture supernatant was measured by HPLC, and the amount of dipicolinic acid secreted and produced outside the cells by culturing was determined. For HPLC analysis, a High Performance Carbohydrate Column 60 (60) 4μm 4.6 × 250mm HPLC Column {Aminopropylmethylsilyl bonded amorphos silica} (Waters) was used as the column, and water containing 20 mM EDTA was used as the eluent with concentrated phosphoric acid. A solution prepared by mixing an aqueous solution adjusted to pH 3.4 with an acetonitrile solution in a ratio of 1: 1 was used. The measurement conditions were a detection wavelength of 270 nm and a flow rate of 1 ml / min. The measurement results are shown in Table 1.

  As shown in Table 1, both 168VGspoVF strain and 168S237spoVF strain showed significantly higher production of dipicolinic acid than the wild strain (168 strain). From this result, the dipicolinate synthase gene operon {spoVFA, spoVFB, asd (SEQ ID NO: 32), dapG (SEQ ID NO: 33), dapA (SEQ ID NO: 33) under the control of the promoter that works before the spore coat protein deposition phase in the sporulation phase. 34)} was transcribed, and it was revealed that dipicolinic acid can be produced in microorganisms having spore-forming ability regardless of whether they are in a specific stage in the spore formation process.

  In addition, the method of this example achieves a very excellent productivity as compared to the productivity of dipicolinic acid by a conventionally known fermentation method, and expensive additives such as diaminopimelic acid are used. I don't need it. From this, it can be concluded that the production method of dipicolinic acid or a salt thereof excellent in productivity and cost can be newly established by this example.

[Example 2]
The recombinant plasmid pH237sVFAB obtained in Example 1 (1-2) was introduced into the gene recombinant strain 168VGspoVF obtained in Example 1 (1-1) by the protoplast method. The obtained recombinant strain was designated as 168VGspoVF-AB strain.

  168VGspoVF-AB 20mL 2% (w / v) yeast extract (Difco), 0.20% corn steep liquor (Oriental Yeast), 0.05% magnesium sulfate heptahydrate, 2% urea, 0.20% L- Shaking culture at 30 ° C for 72 hours in a medium containing tryptophan, 0.50% lysine hydrochloride, 4.0% sodium aspartate, 0.15% monopotassium phosphate, 0.35% dipotassium phosphate and 10% maltose monohydrate Went. After culturing, the cells were removed by centrifugation, the amount of dipicolinic acid in the culture supernatant was measured by HPLC, and the amount of dipicolinic acid secreted and produced outside the cells by culturing was determined. For HPLC analysis, a High Performance Carbohydrate Column 60 Column 4μm 4.6 × 250mm HPLC Column {Aminopropylmethylsilyl bonded amorphos silica} (Waters) was used as the column, and water containing 20 mM EDTA as the eluent was added with concentrated phosphoric acid to pH 3.4. A solution prepared by mixing 1: 1 an aqueous solution 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 measurement results are shown in Table 2.

  As shown in Table 2, in the 168VGspoVF-AB strain, about twice as much dipicolinic acid secretion production was observed as compared with the 168VGspoVF strain obtained in Example 1 (1-1). According to this example, by combining the high expression of dipicolinate synthase in the early stage of culture and the high expression of dipicolinate synthesizing operon in the middle to late stage of the culture when the lysine biosynthesis system is reduced, the productivity of dipicolinate can be further improved. It became clear that we could do it.

Example 3
The recombinant plasmid pH237sVFAB obtained in Example 1 (1-2) was introduced into Bacillus megaterium ATCC 14581 strain (hereinafter referred to as 14581 strain) by the protoplast method. The obtained recombinant strain was designated as 14581-AB strain.

  14581 strain, 14581-AB strain 20mL 2% (w / v) yeast extract (Difco), 4% corn steep liquor (Oriental Yeast), 0.05% magnesium sulfate heptahydrate, 0.001% manganese sulfate, Using a medium containing 0.20% L-tryptophan, 0.50% lysine hydrochloride, 4.0% sodium aspartate, 0.15% monopotassium phosphate, 0.35% dipotassium phosphate and 14% maltose monohydrate at 30 ° C The shaking culture was performed for 72 hours. After culturing, the cells were removed by centrifugation, the amount of dipicolinic acid in the culture supernatant was measured by HPLC, and the amount of dipicolinic acid secreted and produced outside the cells by culturing was determined. For HPLC analysis, a High Performance Carbohydrate Column 60 Column 4μm 4.6 × 250mm HPLC Column {Aminopropylmethylsilyl bonded amorphos silica} (Waters) was used as the column, and water containing 20 mM EDTA as the eluent was added with concentrated phosphoric acid to pH 3.4. A solution prepared by mixing 1: 1 an aqueous solution and an acetonitrile solution was used. The measurement conditions were a detection wavelength of 270 nm and a flow rate of 1 ml / min. Table 3 shows the measurement results.

  As shown in Table 3, significantly higher dipicolinic acid secretory production was observed in the 14581-AB strain compared to the original 14581 strain.

Claims (11)

A microorganism belonging to the genus Bacillus having spore-forming ability or having lost spore-forming ability, which is a dipicolinate synthase gene transcribed under the control of a promoter that works before the spor coat protein deposition stage in the spore formation stage. Culturing a microorganism having a medium containing no diaminopimelic acid;
See containing and recovering the dipicolinic acid from the culture supernatant and / or the reaction solution of the microorganism,
The method for producing dipicolinic acid or a salt thereof , wherein the promoter in the microorganism belonging to the genus Bacillus that has lost the spore-forming ability is a σA-dependent promoter or a σH-dependent promoter .   The method for producing dipicolinic acid or a salt thereof according to claim 1, further comprising a step of purifying the recovered dipicolinic acid.   The method for producing dipicolinic acid or a salt thereof according to claim 1 or 2, wherein the spore coat protein deposition period is a period corresponding to the V stage of Bacillus subtilis spore formation. The Bacillus (Bacillus) microorganism is Bacillus subtilis (Bacillus subtilis) which belongs to the genus or Bacillus megaterium (Bacillus megaterium) a is claim 1 dipicolinic acid or a salt thereof as claimed. The dipicolinic acid according to any one of claims 1 to 4 , wherein the promoter in the microorganism belonging to the genus Bacillus having spore-forming ability is a σA-dependent promoter or a σH-dependent promoter. Method for producing salt. The method for producing dipicolinic acid or a salt thereof according to any one of claims 1 to 5 , wherein the promoter is S237 promoter or spoVG promoter. A microorganism belonging to the genus Bacillus having spore-forming ability or having lost spore-forming ability, which is a dipicolinate synthase gene transcribed under the control of a promoter that works before the spor coat protein deposition stage in the spore formation stage. Yes, and
The microorganism, wherein the promoter in the microorganism belonging to the genus Bacillus that has lost the spore-forming ability is a σA-dependent promoter or a σH-dependent promoter . The microorganism according to claim 7, wherein the spoa coat protein deposition period is a period corresponding to the V stage of Bacillus subtilis spore formation. The microorganism according to claim 7 , wherein the microorganism belonging to the genus Bacillus is Bacillus subtilis or Bacillus megaterium. The microorganism according to any one of claims 7 to 9 , wherein the promoter in the microorganism belonging to the genus Bacillus having spore-forming ability is a σA-dependent promoter or a σH-dependent promoter. The microorganism according to any one of claims 7 to 10 , wherein the promoter is S237 promoter or spoVG promoter.