JP5599153B2 - Process for producing poly-gamma-glutamic acid - Google Patents

Description

The present invention relates to a method for improving the productivity of poly-gamma-glutamic acid by a microorganism in which the pgsBCA gene is expressed, a recombinant microorganism having improved productivity of poly-gamma-glutamic acid, and poly-gamma-glutamic acid using the microorganism. It relates to a manufacturing method.

Poly-gamma-glutamic acid is a polymer compound in which a carboxy group at the γ-position and an amino group at the α-position of glutamic acid are bonded by a peptide bond. Poly-gamma-glutamic acid may also be referred to as γ-polyglutamic acid. Poly-gamma-glutamic acid is known as a viscous substance produced by Bacillus subtilis var. Natto and has recently attracted attention as a new polymer material due to various properties. In the following description, poly-gamma-glutamic acid is abbreviated as PGA.

Microorganisms that produce PGA include some Bacillus bacteria including natto and related species ( Bacillus subtilis var. Chungkookjang , Bacillus licheniformis , Bacillus megaterium , Bacillus anthracis , Bacillus halodurans ), Natrialba aegyptiaca , Hydra, etc. (See Non-Patent Document 1). Among the above microorganisms, Bacillus natto having PGA productivity is classified as a subspecies of Bacillus subtilis that does not produce PGA in terms of taxonomy, but pgsB encodes PGA synthase in many Bacillus bacteria including Bacillus natto. PgsC and pgsA genes are present downstream of the same transcription initiation regulatory region and translation initiation regulatory region, and have a cluster structure (operon) (for example, Non-patent Documents 3, 6 and 8). Furthermore, it is speculated that the PGA production control mechanism of Bacillus natto is subjected to complicated control involving many intracellular control factors together with the mechanism for acquiring Bacillus subtilis transformation ability (for example, Non-patent Document 9 and 10). Although it is possible to increase the productivity of PGA by breeding microbial species that produce PGA in the wild type as described above, optimization for obtaining these stable productivity requires complex nutrition of wild strains. It is judged that this is not an efficient PGA production method because it requires a great deal of labor, such as eliminating requirements and releasing the strict control that the PGA synthesis system receives. In addition, PGA synthesis systems that have a cluster structure are expected to be regulated by the same control system in the cell, so methods such as mutation breeding strictly regulate the expression level of individual gene functions. It seems difficult. For this reason, it is considered that a high production method using genetic recombination technology is being studied as an efficient method to replace wild-type breeding. As an example of PGA production using genetic recombination techniques known so far, about 9 g / L / 5 days in a recombinant Bacillus subtilis strain ( Bacillus subtilis ISW1214) introduced with a plasmid (non-patent literature) 2), about 4 g / L / 1.5 days (see Non-Patent Document 7) or 2.5 mg / 40 mg-dried cells (see Patent Document 1 and Non-Patent Document 6) in recombinant Escherichia coli introduced with a plasmid. It is disclosed that productivity can be obtained. However, it is determined that these disclosed production methods do not achieve sufficient productivity. Furthermore, in the production method using genetic recombination technology as described above, productivity can be improved by expressing one selected from a group of proteins (PgsBCA) involved in PGA synthesis alone or in combination. No report has been made.

  PGA has been shown to be produced in a ribosome-independent manner by a membrane-bound enzyme system called PgsBCA in Bacillus subtilis (see Non-Patent Document 4). Among these PGA synthase systems PgsBCA, PgsB (also known as YwsC) is known to be involved in the amide ligase reaction that acts on the γ-position carboxyl group in glutamic acid (see Non-Patent Documents 3 and 5). ). On the other hand, according to Non-Patent Document 3, it is speculated that PgsC (also called YwtA) is a membrane-bound protein and is involved in the discharge of PGA out of the cell. Coexisting with PgsB and involved in glutamate binding reactions, the function of PgsC is currently unclear. In Non-Patent Documents 4 and 5, PgsA (also referred to as YwtB) is presumed to be involved in PGA discharge, but its function has not been clarified so far.

Further, Non-Patent Document 2, pgsB gene on chromosome Bacillus subtilis deficient in pgsC gene and pgsA gene, pgsB gene, expressed singly or in combination of a plurality of one gene selected from the pgsC gene and pgsA gene vector And the results of studying PGA productivity are disclosed. According to Non-Patent Document 2, it is concluded that PGA cannot be produced unless all of the pgsB gene, pgsC gene and pgsA gene are expressed. Non-patent document 3 describes that a pgsB gene (also referred to as ywsC gene) -deficient strain, a pgsC gene (also referred to as ywtA gene) -deficient strain, and a pgsA gene ( ywtB gene and (Also known as) Deficient strains are constructed and the PGA productivity in each defective strain is verified. Among them, according to Non-Patent Document 3, in natto bacteria having PGA production ability, the pgsB gene and the pgsC gene are essential for PGA production, and the pgsA gene is necessary for obtaining the maximum high PGA production. It is inferred that there is.

Furthermore, in Patent Document 1, by transforming Escherichia coli using a vector into which the pgsB gene, pgsC gene and pgsA gene have been introduced in this order, the ability to produce PGA in Escherichia coli not originally capable of producing PGA. However, according to Non-Patent Document 6, production of PGA is observed under the condition where the pgsBCA gene is introduced in recombinant Escherichia coli, but PGA is not produced under the condition where the pgsBC gene is introduced. It is specified. From the above, PgsBCA is essential for PGA production, and it is not generally considered that PgsA is not required.

  Patent Documents 2 to 5 disclose methods for producing PGA using PGA-producing microorganisms such as Bacillus natto. According to these, it is said that PGA having a molecular weight of several thousands to 2 million can be produced by cultivating Bacillus natto or the like in various nutrient media. Patent Document 6 discloses that PGA having a molecular weight of 3 million or more can be produced by applying external stress to Bacillus natto.

On the other hand, Non-Patent Document 11 and Patent Documents 7 and 8 disclose that PGA with high optical purity can be produced by microorganisms. In Patent Document 7, it is shown that PGA having an L-glutamic acid content of 90% or more can be produced in about 10 g / L / 5 days using a Bacillus megaterium strain. It is disclosed that a mutant of ( Natrialba aegyptiaca ) can produce poly-gamma-L-glutamic acid having a molecular weight of 1.3 million or more in about 5 g / L / 6 days.

  From the above, in the PGA production method using genetic recombination known so far, sufficient productivity has not been obtained (see Non-Patent Documents 2, 6, 7 and Patent Document 1), Furthermore, by using a genetic engineering technique, expression of one selected from a group of proteins involved in PGA synthesis (PgsBCA), alone or in combination, improves PGA productivity, molecular weight adjustment and L- No attempt to adjust body ratio has been reported.

Ashiuchi, M., et al .: Appl. Microbiol. Biotechnol., 59, 9-14 (2002) Ashiuchi, M., et al .: Biosci. Biotechnol. Biochem., 70, 1794-1797 (2006) Urushibata, Y., et al .: J. Bacteriol., 184, 337-343 (2002) Tsujiuchi et al .: Future Materials, 3,44-50 (2003) Ashiuchi, M., et al .: Eur. J. Biochem., 268, 5321-5328 (2001) Ashiuchi, M., et al .: Biochem. Biophys. Res. Commun., 263, 6-12 (1999) Jiang, H., et al .: Biotechnol. Lett., 28, 1241-1246 (2006) Presecan, E., et al .: Microbiology, 143, 3313-3328 (1997) Itaya, M., et al .: Biosci. Biotechnol. Biochem., 63, 2034-2037 (1999) Imanaka Tadayuki et al .: Development of microbe utilization, Chapter 1, Section 4, 657-663 (2002) Shimizu, K., et al .: Appl. Environ. Microbiol., 73, 2378-2

JP 2001-17182 A Japanese Patent Publication No.43-24472 Japanese Patent Laid-Open No. 1-174397 JP-A-3-47087 Japanese Patent No. 3081901 JP 2006-42617 A JP 2007-228957 A JP 2007-314434 A

  The present invention relates to a method for improving PGA productivity using a recombinant microorganism, a recombinant microorganism having improved PGA productivity, and a method for producing PGA using the microorganism.

As a result of intensive studies on a method for increasing the production of PGA using a microorganism, the present inventor has identified a pgsBCA gene, which is a gene group involved in PGA synthesis, in the PgsBCA gene group. It has been found that the productivity of PGA is improved by introducing these genes alone or in combination.

That is, the present invention relates to the following (1) to (4). In the present invention, the corresponding gene means a gene encoding a protein having a function equivalent to that of a protein having a specific function encoded by a target gene.
(1) In the method of producing PGA using a microorganism, the pgsBCA gene in Bacillus subtilis or a gene corresponding to the gene expressed is expressed in the pgsB gene in Bacillus subtilis or the gene corresponding to the gene and the pgsA gene or The gene corresponding to the gene, the pgsC gene in Bacillus subtilis, the gene corresponding to the gene and the pgsA gene or the gene corresponding to the gene, the pgsB gene in Bacillus subtilis or the gene corresponding to the gene, or pgsC in Bacillus subtilis A method for improving the productivity of PGA, which comprises introducing a gene or a gene corresponding to the gene.

(2) The pgsBCA gene in Bacillus subtilis or the gene corresponding to the gene is expressed in the pgsB gene in Bacillus subtilis or the gene corresponding to the gene and the pgsA gene or the gene corresponding to the gene, or in Bacillus subtilis a pgsC gene or a gene corresponding to the gene and a pgsA gene or a gene corresponding to the gene, a pgsB gene in Bacillus subtilis or a gene corresponding to the gene, or a pgsC gene in Bacillus subtilis or a gene corresponding to the gene Microorganisms obtained by

  (3) A method for producing PGA using a microorganism as described above, wherein the recombinant microorganism is used to improve the productivity of PGA.

  (4) A method for producing PGA, wherein the recombinant microorganism is cultured and the produced PGA is obtained.

    According to the method for improving the productivity of PGA according to the present invention, the PGA productivity of microorganisms can be improved. By using the recombinant microorganism according to the present invention, PGA can be produced with excellent productivity. According to the method for producing PGA according to the present invention, PGA can be produced with excellent productivity.

It shows the cloning method of genes involved in PGA synthesis. The preparation of a DNA fragment for gene deletion by SOE-PCR and a method for deleting a target gene (substitution with a drug resistance gene) using the DNA fragment are schematically shown. The method for producing a Bacillus subtilis mutant (1cp-P_S237 / pgsBCAcm) is schematically shown. The method for producing a Bacillus subtilis mutant (1cp-P_rapA / pgsBCAcm) is schematically shown.

  In the present invention, the identity of the amino acid sequence and the base sequence is calculated by the Lipman-Pearson method [Lipman, DJ., Pearson. WR .: Science, 227, 1435-1441 (1985)]. Specifically, it is calculated by performing an analysis with a 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 invention, upstream / downstream of a gene is not a position from the translation start point, but upstream refers to a region continuing on the 5 ′ side of a gene or region regarded as a target, while downstream refers to a target. The region following the 3 ′ side of the gene or region being shown is indicated.

  In the present specification, the transcription initiation control region is a region including a promoter and a transcription start site, and the translation initiation control region is a Shine-Dalgarno (SD) sequence that forms a ribosome binding site together with the initiation codon [Shine, J., Dalgarno, L.:Proc. Natl. Acad. Sci. USA., 71, 1342-1346 (1974)].

    The names of the genes and gene regions of Bacillus subtilis described in this specification are reported as a result of genome decoding of an international consortium [Kunst, F., et al .: Nature, 390, 249-256, (1997)] , JAFAN: Based on Bacillus subtilis genome data published online on the Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http://bacillus.genome.ad.jp/, updated on January 18, 2006). ing.

Here, the gene group involved in PGA synthesis is also called pgsB gene (BG12531: ywsC gene or capB gene), pgsC gene (BG12532: ywtA gene or capC gene) in Bacillus subtilis. ) And pgsA gene (BG12533: also called ywtB gene or capA gene). The Bacillus subtilis Marburg No.168 strain widely used as a host microorganism for genetic recombination has these pgsB gene, pgsC gene and pgsA gene on the chromosome, but does not have the ability to produce PGA. Similarly, Bacillus subtilis var. Natto has these pgsB gene, pgsC gene and pgsA gene on the chromosome (genome), but has the ability to produce PGA.

The nucleotide sequence of the pgsB gene is shown in SEQ ID NO: 1 shows the amino acid sequence of PgsB protein encoded by pgsB gene is shown in SEQ ID NO: 2.

In the present invention, the pgsB gene is not limited to the gene consisting of the base sequence represented by SEQ ID NO: 1, and includes the following (a) to (e).

(A) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
(B) A gene encoding a protein having an amide ligase activity, comprising 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.

Here, in the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted is, for example, 1 to 12, preferably 1 to 8, more preferably Includes an amino acid sequence in which 1 to 4 amino acids are deleted, substituted or added, and the addition includes addition of one to several amino acids at both ends.
A protein having amide ligase activity is an enzyme that synthesizes PGA by polymerizing glutamic acid due to the involvement of a membrane-bound enzyme system involved in PGA synthesis, and the amide ligase activity includes glutamic acid and ATP or GTP. When the substrate and the enzyme and manganese chloride added thereto are used as a reaction solution, it can be measured by confirming the production of PGA in the reaction solution [Urushibata, Y., et al .: J. Bacteriol., 184, 337-343 (2002)].

(C) 50% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, with the amino acid sequence represented by SEQ ID NO: 2. More preferably 85%, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more amino acid sequence having an amide ligase activity. The gene to encode.
(D) 60% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, with the base sequence represented by SEQ ID NO: 1. More preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more. A gene encoding a protein having amide ligase activity.

  (E) A gene that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a protein having amide ligase activity.

  Here, examples of “stringent conditions” include conditions described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)], and 6 × SSC (1 × SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. for 8-16 Examples include conditions of constant temperature for hybridization. The “stringent conditions” described later have the same meaning.

The nucleotide sequence of the pgsC gene is shown in SEQ ID NO: 3 shows the amino acid sequence of PgsC protein encoded by pgsC gene is shown in SEQ ID NO: 4.

The pgsC gene in the present invention is not limited to the gene consisting of the base sequence shown in SEQ ID NO: 3, and includes the following (f) to (j).
(F) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4.
(G) The amino acid sequence shown in SEQ ID NO: 4 comprises an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted, and encodes a protein having a function of generating PGA in the presence of a PgsB protein. gene.

Here, in the amino acid sequence represented by SEQ ID NO: 4, in the amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted, for example, 1 to 12, preferably 1 to 8, more preferably Includes an amino acid sequence in which 1 to 4 amino acids are deleted, substituted or added, and the addition includes addition of one to several amino acids at both ends.
Also, the functions of PgsC proteins such as "generating a PGA in the presence of PgsB protein" described above refers to the ability to produce PGA extracellularly when expressed the pgsC gene with pgsB gene in a host microorganism To do. This is because, in the host microorganism to express pgsB gene alone PGA is not produced, when expressed the pgsC gene with pgsB gene, PGA is based on the present inventors have found the knowledge such produced .

(H) 50% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, with the amino acid sequence represented by SEQ ID NO: 4, More preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more, and an amino acid sequence having the identity of PgsB protein. A gene that encodes a protein having the function of generating PGA.
(I) 55% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, with the base sequence represented by SEQ ID NO: 3; More preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more. A gene that encodes a protein having a function to generate PGA in the presence of a PgsB protein, comprising a base sequence having sex.
(J) A gene encoding a protein that hybridizes with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 under stringent conditions and has a function of generating PGA in the presence of a PgsB protein.

The nucleotide sequence of the pgsA gene is shown in SEQ ID NO: 5 shows the amino acid sequence of PgsA protein encoded by pgsA gene in SEQ ID NO: 6.

The pgsA gene in the present invention is not limited to the gene consisting of the base sequence shown in SEQ ID NO: 5, and includes the following (k) to (o).
(K) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6.
(L) It consists of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 6, and encodes a protein having the ability to synthesize PGA in the presence of PgsB and PgsC. gene.

Here, in the amino acid sequence represented by SEQ ID NO: 6, in the amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted, for example, 1 to 12, preferably 1 to 8, more preferably Includes an amino acid sequence in which 1 to 4 amino acids are deleted, substituted or added, and the addition includes addition of one to several amino acids at both ends.
In addition, the above-mentioned “protein having the ability to synthesize PGA in the presence of PgsB and PgsC proteins” is a protein that has the ability to synthesize PGA in the presence of PgsB and PgsC proteins, and is involved in the extracellular discharge of PGA. However, to date, its function has not been clarified [Ashiuchi, M., et al .: Eur. J. Biochem., 268, 5321-5328 (2001)].

(M) 30% or more, preferably 50% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, with the amino acid sequence represented by SEQ ID NO: 6 More preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more amino acid sequence having the identity of PgsB and PgsC protein A gene encoding a protein capable of synthesizing PGA in the presence of coexistence
(N) 45% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, with the base sequence represented by SEQ ID NO: 5, More preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more. A gene that encodes a protein having a PGA synthesis ability in the presence of PgsB and PgsC.
(O) a gene that hybridizes with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 under stringent conditions and encodes a protein having PGA synthesis ability in the presence of PgsB and PgsC proteins .

In addition, the genes to be introduced into the host microorganism are not limited to the pgsB gene, pgsC gene and pgsA gene derived from Bacillus subtilis as described above. For example, genes belonging to PGA synthesis-related genes derived from microorganisms other than Bacillus subtilis Of these, a gene corresponding to the pgsB gene, a gene corresponding to the pgsC gene, and a gene corresponding to the pgsA gene may be used.

A gene corresponding to the pgsB gene, a gene corresponding to the pgsC gene, or a gene corresponding to the pgsA gene can be isolated / identified from a microorganism known to produce PGA according to a conventional method. The microorganism having ability of producing PGA, in addition to Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus , Bacillus licheniformis, Bacillus megaterium, Bacillus anthracis, Bacillus halodurans, Natrialba aegyptiaca, and the like can be exemplified Hydra, the pgsB gene from these microorganisms The corresponding gene, the gene corresponding to the pgsC gene or the gene corresponding to the pgsA gene can be isolated / identified. As a method for isolating / identifying the above gene, for example, Southern hybridization is performed using genomic DNA extracted from the above-mentioned microorganism and a polynucleotide comprising the base sequence shown in SEQ ID NO: 1, 3 or 5 as a probe. The gene corresponding to the pgsB gene, the gene corresponding to the pgsC gene or the gene corresponding to the pgsA gene can be isolated. Furthermore, due to the rapid progress of genome decoding in recent years, even if it is a non-PGA microorganism, it corresponds to the pgsC gene, a gene corresponding to the Bacillus subtilis pgsB gene defined as described above from Oceanobacillus iheyensis etc. Or a gene corresponding to the pgsA gene can be isolated / identified.

50% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more than the amino acid sequence of a gene having a function equivalent to the aforementioned pgsB gene of Bacillus subtilis Preferably 80% or more, more preferably 85%, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more, less than 100% identity, or In the base sequence and base sequence of the pgsB gene, 60% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably Is 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and less than 100% identity. Microorganisms ( Mashiku the genus Bacillus bacteria and Oceanobacillus bacteria, more preferably Bacillus bacteria) derived from the gene are believed gene corresponding to pgsB gene.

50% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more than the amino acid sequence of a gene having a function equivalent to the aforementioned pgsC gene of Bacillus subtilis Preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more and less than 100%, or In the base sequence and base sequence of the pgsC gene, 55% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably Is 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more and less than 100%. Same Having, other microorganisms (preferably bacteria of the genus Bacillus and Oceanobacillus bacteria, more preferably Bacillus bacteria) derived from the gene are believed gene corresponding to pgsC gene.

30% or more, preferably 50% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more, and more, the amino acid sequence of a gene having a function equivalent to the pgsA gene of Bacillus subtilis described above Preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more and less than 100% identity, or In the base sequence and base sequence of the pgsA gene, 45% or more, preferably 60% or more, more preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably Is 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more and less than 100%. Same Having, other microorganisms (preferably bacteria of the genus Bacillus and Oceanobacillus bacteria, more preferably Bacillus bacteria) derived from the gene are believed gene corresponding to pgsA gene.

Suitable genes corresponding to these genes include genes encoding proteins consisting of the amino acid sequences shown in Table 1 and genes consisting of base sequences.
Specifically, the gene corresponding to the pgsB gene includes the amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31, or an amino acid in which 1 to several amino acids are deleted, substituted, added or inserted in each sequence. Examples thereof include genes (for example, SEQ ID NO: 12, 18, 24 or 30) encoding a protein having a sequence and having amide ligase activity.
The gene corresponding to the pgsC gene consists of the amino acid sequence shown in SEQ ID NO: 15, 21, 27 or 33, or an amino acid sequence in which 1 to several amino acids are deleted, substituted, added or inserted in each sequence. And a gene encoding a protein having a function of generating PGA in the presence of PgsB protein (for example, SEQ ID NO: 14, 20, 26, or 32).
The gene corresponding to the pgsA gene consists of the amino acid sequence shown in SEQ ID NO: 17, 23, 29 or 35, or an amino acid sequence in which several amino acids are deleted, substituted, added or inserted in each sequence, and PgsB And a gene encoding a protein having the ability to synthesize PGA in the presence of PgsC (for example, SEQ ID NO: 16, 22, 28, or 34).

To be introduced into a host microorganism, the gene corresponding to pgsC gene or the gene in a gene and pgsA gene or genes corresponding to the gene, or Bacillus subtilis corresponding to pgsB gene or the gene in B. subtilis and pgsA gene or the gene Or a pgsB gene in Bacillus subtilis or a gene corresponding to the gene, or a pgsC gene in Bacillus subtilis or a gene corresponding to the gene constitutively expresses a downstream gene linked upstream of the gene. It is preferable that a transcription initiation control region and / or a translation initiation control region capable of increasing the expression of in the host cell are combined.
Such control regions, Bacillus sp. Upstream 0.4~1.0kb region of a cellulase gene of KSM-S237 strain (control region), the control region of the rapA gene of Bacillus bacteria, such as the control region of the spoVG gene from Bacillus genus bacteria Although illustrated, it is not particularly limited.
The base sequence of the control region derived from the cellulase gene of Bacillus sp. KSM-S237 described above is represented by base numbers 1 to 572 of SEQ ID NO: 7, and the control region of the base sequence of the rapA gene of the genus Bacillus is In SEQ ID NO: 8, the control region of the base sequence of the spoVG gene derived from the genus Bacillus is represented by SEQ ID NO: 9, and each of these base sequences is 90% or more, preferably 95% or more, more preferably 96% or more, More preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and a region consisting of a base sequence having an equivalent function is also included. Here, the same definition as above is used for the identity of the base sequence.
When introducing the gene into a host microorganism, a conventionally known plasmid (vector) can be used. Moreover, a plasmid can be suitably selected according to the kind of host microorganism for gene transfer. When Bacillus subtilis is used as a host, for example, pT181, pC194, pUB110, pE194, pSN2, pHY300PLK and the like can be exemplified. The selected plasmid is preferably a plasmid capable of self-replication in the host cell, and more preferably the plasmid is multicopy.
Furthermore, since it is expected that such a fragment will exhibit a sufficient effect on the production of the target protein by introducing one copy per cell, when introduced by a plasmid, some plasmids may be dropped during production culture. However, the copy number of the plasmid may be 2 or more and 100 or less, preferably 2 or more and 50 or less, more preferably 2 or more and 30 or less, relative to the host genome (chromosome). I need it. For transformation using an expression vector, conventionally known methods such as the protoplast method [Chamg, S., Cohen, SH .: Mol. Gen. Genet., 168, 111-115 (1979)] and competent methods are used. The cell method [Young, FE., Spizizen, J .: J. Bacteriol., 86, 392-400 (1965)] can be applied.

The host microorganism used in the present invention is not particularly limited as long as it is a microorganism that expresses the pgsBCA gene in Bacillus subtilis or a gene corresponding to the gene, and produces PGA. But it ’s okay. That is, a wild-type microorganism having the pgsBCA gene on the chromosome (genome) and having PGA-producing ability, or a mutant type having the pgsBCA gene on the chromosome (genome) and having enhanced PGA-producing ability. Any of microorganisms and mutant microorganisms to which the pgsBCA gene is introduced onto the chromosome and PGA production ability is imparted may be used.
Wherein said host microorganism is not particularly limited, specifically, Bacillus amyloliquefaciens having ability of producing PGA in the wild-type, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus anthracis, Bacillus halodurans, Bacillus such as Bacillus subtilis (Bacillus) Examples include bacteria belonging to the genus, Clostridium genus bacteria that have been given the ability to produce PGA by genetic recombination, bacteria belonging to the genus Corynebacterium , Escherichia coli, and yeast. Among them, the whole genome information is revealed, genetic engineering, that genome engineering technology has been established, from the point that has been developed as a safe and and microbial strains accepted excellent as a host microorganism, among bacteria of the genus Bacillus However, Bacillus subtilis is particularly preferable.

In a host microorganism that has a pgsBCA gene on the genome or a gene corresponding to the gene but does not express PGA-producing ability, a method for imparting PGA-producing ability to the host microorganism is to use pgsBCA in Bacillus subtilis on the genome. It can be obtained by introducing a transcription initiation control region and / or a translation initiation control region (hereinafter also simply referred to as “control region”) having a function in the microorganism upstream of the gene or a gene corresponding to the gene. it can. In addition, it is desirable that the transcription initiation control region and / or the translation initiation control region having a function in the microorganism of the present invention are bound in an appropriate form, and the translation initiation region is bound downstream of the transcription initiation control region. It is preferable that The control region is more preferably one that constitutively expresses the bound downstream gene and enhances the expression of the gene in the host cell. If a host microorganism for constructing the microorganism of the present invention using Bacillus subtilis, such control as the region Bacillus sp. Regulatory region of a cellulase gene of KSM-S237 strain, the control regions of rapA gene derived from Bacillus bacteria, the Examples include the control region of spoVG gene.

In order to introduce the control region into the upstream region of the gene corresponding to the pgsBCA gene or the gene on the genome, not only the control region is inserted into the upstream region of the gene corresponding to the pgsBCA gene or the gene, A part or all of the control region may be replaced. Regarding the insertion / substitution of the control region, there has been a report on a method of deleting or inactivating a target gene by homologous recombination when Bacillus subtilis is used as a host microorganism for constructing the microorganism of the present invention [Itaya, M , Tanaka, T .: Mol. Gen. Genet., 223, 268-272 (1990)], the target host microorganism can be obtained using such a method.
For example, first, a drug resistance gene fragment containing the original translation initiation control region and / or the transcription initiation control region of the pgsBCA gene is prepared upstream of the DNA fragment containing such a control region, and the structural gene region of the pgsBCA gene is further downstream. A DNA fragment containing a part or all of is bound by a known method such as SOE (splicing by overlap extension) -PCR [Horton, RM., Et al .: Gene, 77, 61-68 (1989)]. In this way, the drug resistance gene fragment containing the original control region of the pgsBCA gene and its upstream region, the target control region fragment, and the DNA fragment containing a part or all of the structural gene region of the pgsBCA gene are combined in this order. DNA fragment is obtained.
Next, by incorporating such a DNA fragment into a host microorganism cell by a known method, the original transcription initiation control region of the pgsBCA gene in the host microorganism genome and its upstream region, and the structural gene region of the pgsBCA gene , Double crossover homologous recombination occurs. As a result, the transformant in which the original control region is replaced with the target control region can be isolated using the drug resistance gene as an index. As a result, the control region introduced upstream of the pgsBCA gene on the genome is genetically stable.

Also, if on the genome does not have a gene corresponding to pgsBCA gene or the gene in B. subtilis, the pgsBCA gene or the gene of Bacillus subtilis which control regions are combined in the proper form having the functions in the host microorganism The corresponding gene may be introduced at a position on the genome of any host microorganism using the same method as described above. For example, first, a control region that enables expression of a gene linked downstream, a DNA fragment containing a part or all of the structural gene region of the pgsBCA gene downstream of this control region, and further a drug resistance gene is added to SOE- Binding by PCR method. Next, DNA fragments in the upstream and downstream regions of the position where the above DNA fragment is introduced on an arbitrary genome are prepared, including the drug resistance gene prepared by the SOE-PCR method described above, and the control region bound upstream. the DNA fragment consisting of a part or all of the structural gene region of the pgsBCA gene, (upstream region) - (including the drug resistance gene, consisting of some or all of the structural gene region of the pgsBCA gene control region linked to an upstream DNA The fragments are bound by the SOE-PCR method in the order of (fragment)-(downstream region). Thereafter, the obtained DNA fragment is incorporated into the host microbial cell by a known method, so that the homologous pair of the two crosses in the upstream region and the downstream region at any position on the host microbial genome is obtained. A change occurs. As a result, a transformant in which a part or all of the structural gene region of the pgsBCA gene having a control region bound to the target genomic region is introduced can be isolated using drug resistance as an index. As a result, the pgsBCA gene in Bacillus subtilis or a gene corresponding to the gene can be genetically stably held at an arbitrary position on the genome.

As the host microorganism to be selected, either a wild-type microorganism or a mutant strain into which a predetermined mutation has been introduced can be used. Further, among the mutant strains, a better effect can be expected by using as a host a mutant strain in which a gene encoding PGA-degrading enzyme (PGA-degrading enzyme gene) is deleted. The ggt gene is known as a PGA-degrading enzyme gene in Bacillus subtilis [Kimura, K., et al .: Microbiology, 150, pp4115-4123 (2004)].
The base sequence of the Ggt gene of Bacillus subtilis is shown in SEQ ID NO: 10, and the amino acid sequence of PGA-degrading enzyme GGT encoded by the ggt gene is shown in SEQ ID NO: 11. In addition, when a microorganism other than Bacillus subtilis is used as the host microorganism, it is preferable to use a mutant strain in which a gene corresponding to the ggt gene is deleted.
It has the same function as the B. subtilis ggt gene, and / or the base sequence and base sequence of this gene is 90% or more, preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably Genes derived from other microorganisms (preferably Bacillus bacteria) having identity of 98% or more, more preferably 99% or more are considered to be genes corresponding to these genes, and are deleted or inactivated in the present invention. Included in genes.

In the present invention, the ggt gene is not limited to the gene consisting of the nucleotide sequence shown in SEQ ID NO: 10, but 90% or more, preferably 95% or more, more preferably 96% or more, relative to the nucleotide sequence shown in SEQ ID NO: 10. More preferably 97% or more, more preferably 98% or more, and more preferably 99% or more, and includes a gene encoding a protein having a PGA degrading activity. Here, “identity of base sequences” has the same definition as described above.
The ggt gene includes an amino acid sequence having PGA resolution in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 11. Here, the “amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted” includes, for example, 1 to 12, preferably 1 to 8, more preferably 1 to 4 amino acids. A deleted, substituted or added amino acid sequence is included, and the addition includes the addition of one to several amino acids at both ends.
Further, the ggt gene is 90% or more, preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99%, with the amino acid sequence represented by SEQ ID NO: 11. It is meant to include a gene encoding a protein having an amino acid sequence having% or more identity and having PGA degradation activity.

Furthermore, in the present invention, the ggt gene is meant to include a gene that hybridizes under stringent conditions to a complementary strand of the base sequence shown in SEQ ID NO: 10 and encodes a protein having PGA degradation activity.
Here, the same definition as above is used for “stringent conditions”. As a method for deleting the ggt gene or a gene corresponding to the gene, a conventionally known method can be appropriately used. For example, a circular recombination plasmid obtained by cloning a DNA fragment containing a part of the ggt gene into an appropriate plasmid (vector) is taken into the host microorganism cell, and homologous recombination in a partial region of the target gene Can disrupt and inactivate target genes on the host microbial genome [Leenhouts, K., et al .: Mol. Gen. Genet., 253, 217-224 (1996)] .

In particular, when Bacillus subtilis is used as a host microorganism for constructing the microorganism of the present invention, there are already several reports on the method for deleting or inactivating a target gene by homologous recombination, as described above, By repeating these methods, the target host microorganism can be obtained. In addition, a random gene inactivation method can be carried out by using a mutagen or by irradiating the host microorganism with ultraviolet rays, γ rays or the like.
In this case, in addition to the introduction of mutations such as base substitution, base insertion, or partial deletion into the ggt structural gene, to the genes related to the same mutation introduction into the control region such as the promoter region or the expression control of the ggt gene. The same effect can be expected by introducing the same mutation.
Furthermore, not more as an efficient approach to construct upstream of ggt gene, by a linear DNA fragment including downstream region does not include the ggt gene aforementioned SOE-PCR method, which incorporated into a host microorganism cell It is also possible to carry out a technique of deleting the ggt gene on the genome by causing double crossover homologous recombination at two locations outside the ggt gene on the host microorganism genome (see FIG. 2).

As shown in the examples below, the host microorganism imparted with ability of producing PGA, pgsB in gene and pgsA gene or genes corresponding to the gene or Bacillus subtilis, corresponding to pgsB gene or the gene in B. subtilis genes Or a gene corresponding to the gene, a pgsC gene in Bacillus subtilis, a gene corresponding to the gene and a pgsA gene or a gene corresponding to the gene, a pgsC gene in Bacillus subtilis or a gene corresponding to the gene, or pgsB in Bacillus subtilis By introducing a gene or a gene corresponding to the gene, the productivity of PGA can be improved compared to the host microorganism, the production cost can be reduced, and if the obtained recombinant microorganism is used, PGA can be industrialized. Can be manufactured efficiently.

In particular, when a PGA was produced using a Bacillus subtilis strain in which the cellulase gene control region of Bacillus sp. KSM-S237 was inserted into the control region upstream of the pgsBCA gene, the microorganism was used as a host microorganism in Bacillus subtilis. by introducing the gene corresponding to the gene and pgsA gene or the gene corresponding to the pgsB gene or the gene from 110 to 150% the productivity of PGA controls productivity to the relative value to 100% Can be improved. Further, in the above same host microorganism, by introducing the gene corresponding to pgsC gene or the gene in a gene and pgsA gene or genes corresponding to the gene, or Bacillus subtilis corresponding to pgsC gene or the gene in B. subtilis The PGA productivity can be improved to 110-130% with respect to the relative value where the control productivity is 100%. Above all, in view of improving productivity, it is preferable to introduce a gene corresponding to the gene and pgsA gene or the gene corresponding to the pgsB gene or the gene in B. subtilis.

Further, as a control when to produce PGA using Bacillus strains were inserted control region of rapA gene derived from Bacillus bacteria in the control region of the pgsBCA gene upstream, heritage pgsB in B. subtilis the microorganism as a host microorganism Den By introducing a child or a gene corresponding to the gene and a pgsA gene or a gene corresponding to the gene, the productivity of PGA is improved to 110 to 500% with respect to a relative value where the productivity of the control is 100%. be able to. Moreover, by introducing the pgsB gene in Bacillus subtilis or a gene corresponding to the gene, the productivity of PGA can be improved to 110 to 1,500% with respect to the relative value where the productivity of the control is 100%.

  Therefore, if the present invention is used, the production cost of PGA used for various purposes can be greatly reduced. Since the production cost of PGA can be reduced in this way, it is also useful for industrial production of PGA. Furthermore, the PGA produced using the present invention can be used in various applications such as cosmetics, pharmaceuticals, foods, water purification agents, water retention materials, thickeners and the like.

  When producing PGA using the recombinant microorganism of the present invention, first, the recombinant microorganism is cultured in an appropriate medium, and PGA produced outside the cells is obtained from the medium. Medium includes starch, maltooligosaccharide, maltose, glucose, fructose, xylose, arabinose, glycerol, carbon sources such as various organic acids, various amino acids, nitrogen sources such as polypeptone, tryptone, ammonium sulfate and urea, and inorganic salts such as sodium salts In addition, a medium having a composition containing necessary nutrients, trace metal salts, and the like can be used. The medium may be a synthetic medium or a natural medium.

  In particular, in order to improve the productivity of PGA, it is preferable to use a medium to which an excess amount of glutamic acid is added than the amount of glutamic acid required for the growth of the recombinant microorganism to be cultured. Specifically, the sodium salt (sodium glutamate) is preferably added to the medium, but the concentration is, for example, 0.005 to 600 g / L, preferably 0.05 to 500 g / L, more preferably 0.1 to 400 g / L, Most preferably, it is 0.5 to 300 g / L.

  As culture conditions, the optimum temperature is preferably 10 to 40 ° C, more preferably 30 to 40 ° C. The optimum pH is preferably pH 4-8, more preferably 5-8. Moreover, the culture days are preferably 1 to 10 days, more preferably 2 to 5 days after inoculation with the inoculum. The culture is not particularly limited, and examples include shaking culture, agitation culture, aeration culture, and stationary culture.

  When obtaining PGA accumulated in the culture medium, it is necessary to remove the cells of the host microorganism that produced PGA. This includes centrifugation, ultrafiltration membranes, and electrodialysis. And a method of obtaining the precipitate as a precipitate by maintaining the pH near the isoelectric point of PGA, and a combination of the above methods is also possible.

  In addition, the evaluation of PGA production ability in microorganisms is performed by, for example, the method using the above-mentioned amide ligase activity, and the semitransparent viscous substance formed around the colony when cultured on a PGA production agar medium containing glutamic acid or a salt thereof. The method of observation by HPLC, the method of detecting PGA by SDS-PAGE [Yamaguchi, F., et al .: Biosci. Biotechnol. Biochem. 60, 255-258 (1996)], or using an amino acid analyzer after acid hydrolysis [Ogawa, Y., et al .: Biosci. Biotechnol. Biochem., 61, 1684-1687 (1997)], quantification method for purifying from culture broth and determining dry weight [Ashiuchi, M., et al .: Biochem. Biophys. Res. Commun., 263: 6-12 (1999)], quantitative / qualitative method by HPLC (high performance liquid chromatography) using a gel filtration column [Kubota, H., et al .: Biosci Biotechnol. Biochem., 57, 1212-1213 (1993)].

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

[Production Example 1] Cloning of PGA synthesis-related genes (1)
In this production example, a method for cloning genes involved in PGA synthesis was shown (see FIG. 1). Primer set of P-S237 / BSpgsB atg FW (SEQ ID NO: 38) and HindIII_BSpgsBCA RV (SEQ ID NO: 43) shown in Table 2 using chromosomal DNA prepared from Bacillus genus KSM-366 strain (FERM BP-6262) as a template Was used to prepare a 2.9 kb pgsBCA gene DNA fragment (BCA) (see FIG. 1). Similarly, using a primer set of P-S237_BSpgsC FW (SEQ ID NO: 40) and HindIII_BSpgsBCA RV (SEQ ID NO: 43), a 1.7 kb pgsCA gene DNA fragment (CA), P-S237 / BSpgsB atg FW (SEQ ID NO: 38) and HindIII_BSpgsB 1.2 kb pgsB gene DNA fragment (B) using the primer set of RV (SEQ ID NO: 45), 0.5 kb pgsC gene DNA using the primer set of P-S237_BSpgsC FW (SEQ ID NO: 40) and HindIII_BSpgsBC RV (SEQ ID NO: 44) Fragment (C) was prepared. In addition, using a primer set of P-S237 / BSpgsB atg FW (SEQ ID NO: 38) and pgsC FW (SEQ ID NO: 42), a 1.2 kb pgsB gene DNA fragment (b), comp_pgsA / pgsC RV (SEQ ID NO: 41) and HindIII_BSpgsBCA RV A 1.2 kb pgsA gene DNA fragment (a) was prepared in advance using the primer set of (SEQ ID NO: 43), and the SOE-PCR method using the primer set of SEQ ID NOs: 38 and 43 using these fragments (b and a) as templates. To prepare a 2.4 kb DNA fragment (BA) of the pgsBA gene. Furthermore, using a chromosomal DNA prepared from Bacillus genus KSM-S237 strain (FERM BP-7875) as a template, Cm_S237 FW (SEQ ID NO: 36) and P-S237 RV (SEQ ID NO: 37) primer sets shown in Table 2 were used. Using this, a 0.6 kb promoter region DNA fragment (P_S237) of the S237 cellulase gene (Japanese Patent Laid-Open No. 2000-210081) derived from Bacillus genus KSM-S237 strain (FERM BP-7875) was prepared. Next, using the obtained fragments (P_S237) and (BCA) and the primer sets of SEQ ID NOs: 36 and 43, 3.5 kb DNA obtained by fragmenting fragments (P_S237) and (BCA) into one fragment by SOE-PCR method A fragment (P_BCA) was obtained (see FIG. 1). Similarly, using the primer sets of fragments (P_S237) and (BA) and SEQ ID NOs: 36 and 43, using the 3.0 kb DNA fragment (P_BA), fragments (P_S237) and (CA), and the primer sets of SEQ ID NOs: 36 and 43 Using a primer set of 2.3 kb DNA fragment (P_CA), fragment (P_S237) and (B) and SEQ ID NOs: 36 and 45, 1.8 kb DNA fragment (P_B), fragment (P_S237) and (C) and SEQ ID NO: 36 and A 1.1 kb DNA fragment (P_C) was obtained using 44 primer sets. The PCR reaction was performed using GeneAmp 9700 (PE Applied Biosystems) and TaKaRa LA Taq (Takara) according to the protocol attached to the kit.

[Production Example 2] Construction of vector (1)
The DNA fragment prepared in Production Example 1 was digested with restriction enzymes Bam HI and Hin dIII, respectively, the same restriction enzyme treatment alms plasmid vector pHY300PLK (Takara) and DNA Ligation kit Ver.2 the (Takara) A ligation reaction (conjugation) was performed. Using the above ligation sample, E. coli (HB101 strain) was transformed by competent cell method, and colonies that appeared on LB agar medium (LBTc agar medium) supplemented with 15 ppm of tetracycline-hydrochloride (SIGMA) were transformed. It was. The obtained transformant was grown again on the LBTc agar medium, and a recombinant plasmid was prepared from the resulting cell using a high-purity plasmid isolation kit (Roche Diagnostics). These were restriction enzyme treatment with restriction enzymes Bam HI and Hin dIII, the multiple cloning site on the plasmid vector by electrophoresis, DNA fragments of interest as shown in Production Example 1 (P_BCA), (P_BA) , ( It was confirmed that (P_CA), (P_B), and (P_C) were inserted. In this production example, the recombinant plasmid that confirmed the insertion of the DNA fragment (P_BCA) was used as the PGA recombinant production vector pHY-P_S237 / pgsBCA, and the recombinant plasmid that was confirmed to be inserted into the DNA fragment (P_BA) was produced by the PGA recombinant production. Recombinant plasmid with confirmed insertion of vector for pHY-P_S237 / pgsBA, DNA fragment (P_CA) Recombinant plasmid with confirmed vector for PGA recombination production pHY-P_S237 / pgsCA, DNA fragment (P_B) PGA recombinant production vector pHY-P_S237 / pgsB, and a recombinant plasmid in which insertion of a DNA fragment (P_C) could be confirmed was designated as PGA recombinant production vector pHY-P_S237 / pgsC.

[Production Example 3] Production of modified Bacillus subtilis (1)
In this production example, a method for producing a Bacillus subtilis mutant for introducing the vector obtained in Production Example 2 was shown (see FIG. 3).
Using the pHY-P_S237 / pgsBCA prepared in Production Example 2 as a template and using the HindIII_BSpgsB RV (SEQ ID NO: 45) and TET4-1 F (SEQ ID NO: 46) primer sets shown in Table 2, the tetracycline resistance gene on the plasmid A 3.3 kb pgsB gene fragment (tet-P_B) linked to the S237 cellulase promoter sequence containing (Tet ^r ) was prepared. In addition, using a chromosomal DNA prepared from Bacillus subtilis Marburg No.168 (Bacillus subtilis 168) as a template, using a primer set of tet4-1_comp / pgsB FW (SEQ ID NO: 47) and pgsB RV (SEQ ID NO: 48), A 1.0 kb upstream DNA fragment (pgsB-UP) of the pgsBCA gene related to PGA synthesis on the Bacillus subtilis genome was prepared. Using these fragments (tet-P_B, pgsB-UP) as a template and the primer sets of SEQ ID NOs: 45 and 48, DNA fragments (tet-P_S237) for upstream promoter replacement of the pgsBCA gene operon on the Bacillus subtilis genome by SOE-PCR method ) Subsequently, 168 strains of Bacillus subtilis were transformed by the competent cell method using the obtained DNA fragment (tet-P_S237), and colonies grown on the LBTc agar medium were isolated as transformants. Furthermore, it is a Bacillus subtilis mutant strain (1cp-P_S237 / pgsBCAtet) in which the S237 cellulase promoter is introduced upstream of the pgsBCA gene operon by extracting genomic DNA from the obtained transformant and using this as a template. confirmed. Subsequently, the S237 cellulase promoter region 0.6 kb DNA fragment prepared in Production Example 1 (P_S237), the pC194 plasmid derived from Staphylococcus aureus [Horinouchi, S., Weisblum, B., J. Bacteriol., 150, 815 (1982) ], A chloramphenicol resistance gene 0.9 kb DNA fragment (Cm ^r ) prepared with a primer set of P-S237F_comp / Cm FW (SEQ ID NO: 49) and pC194Cm RV (SEQ ID NO: 50), Bacillus subtilis 168 strain A kbsB RV (SEQ ID NO: 48) and pgsB / CmF (SEQ ID NO: 51) primer set was used as a template to prepare a 1.0 kb DNA fragment (pgsB-UP2) containing the upstream promoter of the pgsBCA gene operon. Prepared. Furthermore, using these DNA fragments (P_S237, Cm, pgsB-UP2) as a template, P-S237 RV (SEQ ID NO: 37) and pgsB RV (SEQ ID NO: 48) primer sets were used to make a single fragment by SOE-PCR. A 2.5 kb DNA fragment was obtained. The resulting DNA fragment was used to transform a Bacillus subtilis mutant (1cp-P_S237 / pgsBCAtet) by a competent cell method, and on an LB agar medium (LBCm agar medium) supplemented with 10 ppm chloramphenicol (SIGMA) ) Was used as a transformant, and the obtained transformant was a Bacillus subtilis mutant (1cp-P_S237 / pgsBCAcm).

[Production Example 4] Production of modified Bacillus subtilis (2)
In this production example, a method for producing a Bacillus subtilis mutant for introducing the vector obtained in Production Example 2 was shown (see FIG. 4). Structural gene of rapA gene (BG10652) on Bacillus subtilis genome using chromosomal DNA prepared from Bacillus subtilis 168 as a template and using primer set of rapA FW (SEQ ID NO: 52) and P_rapA / Cm R (SEQ ID NO: 53) (ORF) 0.5 kb upstream DNA fragment (rapA-UP) for inserting a chloramphenicol resistance gene 0.5 kb upstream, as well as P_rapA / Cm F (SEQ ID NO: 54) and P_rapA RV (SEQ ID NO: 55) ) Was used to prepare a downstream 0.5 kb DNA fragment (rapA-DW) for insertion of the chloramphenicol resistance gene (in this case, the primer of SEQ ID NO: 55 was the ORF of the rapA gene). Designed to change the start codon ttg to atg). A chloramphenicol resistance gene 0.9 kb DNA fragment (Cm2) was prepared using plasmid pC194 as a template and a primer set of Cm_comp FW (SEQ ID NO: 56) and Cm_comp RV (SEQ ID NO: 57). Furthermore, using these DNA fragments (rapA-UP, Cm2, and rapA-DW) as templates, a primer set of SEQ ID NOS: 52 and 55 was used to obtain a 1.9 kb DNA fragment that was fragmented by the SOE-PCR method. Using the obtained DNA fragment, Bacillus subtilis 168 strain was transformed by the competent cell method, colonies grown on LBCm agar medium were used as transformants, and the obtained transformants were transformed into Bacillus subtilis mutant strains ( P-rapA / Cm). Subsequently, using the chromosomal DNA prepared from the Bacillus subtilis mutant strain (P-rapA / Cm) as a template, using a primer set of P_rapA RV (SEQ ID NO: 55) and pC194Cm FW (SEQ ID NO: 58), Lambphenicol resistance gene and 0.5 kb on Bacillus subtilis genome A 1.4 kb DNA fragment (Cm-P_rapA) consisting of a promoter region (P_rapA) upstream of the rapA gene was prepared. In addition, chromosomal DNA prepared from Bacillus subtilis 168 strain was used as a template, and the pgsBCA gene on the Bacillus subtilis genome was used using a primer set of pgsC FW (SEQ ID NO: 42) and comp_P-rapA R / BSpgsBatg FW (SEQ ID NO: 60). Using a primer set of 1.2 kb DNA fragment (pgsBatg), pgsB RV (SEQ ID NO: 48) and CmFW_comp / pgsBF (SEQ ID NO: 59) from the ORF start codon atg to the downstream, 1.0 pgsBCA gene upstream 1.0 on the Bacillus subtilis genome A kb DNA fragment (pgsB-UP3) was prepared. Furthermore, using these DNA fragments (Cm-P_rapA, pgsBatg, pgsB-UP3) as a template, a primer set of SEQ ID NOS: 42 and 48 was used to obtain a 3.6 kb DNA fragment that was fragmented by the SOE-PCR method. Using the obtained DNA fragment, Bacillus subtilis 168 strain was transformed by the competent cell method, colonies grown on LBCm agar medium were used as transformants, and the obtained transformants were transformed into Bacillus subtilis mutant strains ( 1cp-P_rapA / pgsBCAcm).

[Production Example 5] Transformation by introduction of plasmid In this production example, a method for transforming Bacillus subtilis and its modified strain was shown.
The Bacillus subtilis mutant strain produced in Production Example 3 (1cp-P_S237 / pgsBCAcm) and the Bacillus subtilis mutant strain produced in Production Example 4 (1cp-P_rapA / pgsBCAcm) are used as a source strain for gene transfer, and are used as controls for recombinant production. Plasmid before introduction of recombinant DNA fragment (pHY300PLK) and PGA synthesis-related gene expression vectors prepared in Production Example 2 (pHY-P_S237 / pgsBCA, pHY-P_S237 / pgsBA, pHY-P_S237 / pgsCA, pHY-P_S237 / pgsB, pHY -P_S237 / pgsC) and transformed by the protoplast method. Colonies that grew on DM3 protoplast regeneration medium (DM3 medium) supplemented with 30 ppm tetracycline-hydrochloride were selected as the desired Bacillus subtilis transformants into which the plasmid was introduced.

[Example 1] Productivity evaluation-1
Of the Bacillus subtilis transformants obtained in Production Example 5, 1cp-P_S237 / pgsBCAcm strain was used as the original strain for gene transfer, pHY300 PLK was used as a control, pHY-P_S237 / pgsBCA, pHY-P_S237 / pgsBA, pHY-P_S237. A transformant introduced with / pgsB was inoculated on an LBTc agar medium, and statically cultured at 30 ° C. overnight. Thereafter, the transformant grown on the LBTc agar medium was transformed into 0.8 to 1.6 mL of a modified 2 × L / Maltose + MSG medium (hereinafter referred to as 2 × L / Mal + MSG medium; 2.0) in a presterilized 2.0 mL microcentrifuge tube. Stir / suspend in 1% tryptone, 1.0% yeast extract, 1.0% sodium chloride, 8.0% sodium glutamate monohydrate, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline-hydrochloride) The supernatant after standing still was used as a seed culture solution. This seed culture is inoculated to a new 2xL / Mal + MSG medium at 1% (v / v), and reciprocal shaking culture at 37 ° C, 120 rpm for 3 days (TB-20R-3F, Takasaki Scientific Instruments) ).
After completion of the evaluation culture, the productivity and molecular weight of PGA were measured under the analysis conditions shown in the measurement examples described later, and the productivity evaluation results are shown in Table 3. As shown in Table 3, it is clear that the productivity of PGA is improved in the pHY-P_S237 / pgsBA, pHY-P_S237 / pgsCA and pHY-P_S237 / pgsC-introduced strains as compared to the control pHY300PLK vector-introduced strains. It became. In addition, the effect was remarkable compared with the pHY-P_S237 / pgsBCA-introduced strain. The molecular weights of PGA produced by pHY-P_S237 / pgsBA, pHY-P_S237 / pgsCA and pHY-P_S237 / pgsC-introduced strains (1 day of culture) were 8,000,000-15,000,000 PGA and 6,000,000-9,000,000 PGA, respectively.

[Example 2] Productivity evaluation-2
Among the Bacillus subtilis transformants obtained in Production Example 5, 1cp-P_rapA / pgsBCAcm strain was used as the original strain for gene transfer, pHY300 PLK was used as a control, pHY-P_S237 / pgsBCA, pHY-P_S237 / pgsBA, pHY-P_S237. The transformant introduced with / pgsB was subjected to the same productivity evaluation as in Example 1 described above.
After completion of the evaluation culture, analysis was performed under the conditions shown in the measurement examples in the same manner as in Example 1, and the productivity evaluation results are shown in Table 4. As shown in Table 4, it was revealed that the productivity of PGA was improved in the pHY-P_S237 / pgsBA and pHY-P_S237 / pgsB-introduced strains compared to the control-introduced strain of pHY300PLK vector. In addition, the effect was remarkable compared with the pHY-P_S237 / pgsBCA-introduced strain. The molecular weights of PGA produced by pHY-P_S237 / pgsBA and pHY-P_S237 / pgsB-introduced strains (1 day of culture) were 800,000 to 8,000,000 PGA and 5,000,000 to 9,000,000 PGA, respectively.

[Measurement example] Quantification of PGA and molecular weight measurement method After completion of the evaluation culture shown in Examples 1 and 2, the culture solution sample was centrifuged at 14,800 rpm at room temperature for 30 minutes (himacCF15RX, Hitachi Koki) and centrifuged. The amount of recombinant PGA produced in the culture supernatant obtained by the separation was measured. HPLC analysis using TSKGel G4000PWXL and TSKGel G6000PWXL gel filtration columns (Tosoh) was performed. The analysis conditions were such that 0.1 M sodium sulfate was used as the eluent, the flow rate was 1.0 mL / min, the column temperature was 50 ° C., and the UV detection wavelength was 210 nm. For the concentration test, a calibration curve was prepared using PGA (Meiji Food Material) with a molecular weight of 800,000. Furthermore, polyglutamic acid with different molecular weights (Wako Pure Chemical Industries (162-21411, 162-21401), SIGMA-ALDRICH) whose weight average molecular weight was determined in advance using pullulan Shodex STANDRD P-82 (Showa Denko) for molecular weight test. (P-4886, P-4761), Meiji Food Material (molecular weight 880,000)].

If the primers of SEQ ID NO: 37 and SEQ ID NO: 61-71 are used according to the method described in the above production example, Bacillus subtilis mutant strains (1cp-P_S237 / pgsBCAcm strain and 1cp-P_rapA / pgsBCAcm strain) are used as hosts. A Bacillus subtilis transformant into which a gene corresponding to the pgsBC gene in Bacillus subtilis has been introduced can be obtained.

Claims (8)

Using a microorganism poly - gamma - in the method of producing glutamic acid, the microorganism pgsBCA gene in B. subtilis is expressed, pgsB gene in Bacillus subtilis 及 beauty pgsA gene alone, or pgsC gene及 beauty in B. subtilis pgsA gene alone, or only the pgsB gene in Bacillus subtilis, or poly and introducing only pgsC gene in B. subtilis - gamma - a method of improving the glutamate productivity,
The microorganism is a bacterium belonging to the genus Bacillus,
A method in which the pgsB gene, the pgsC gene, and the pgsA gene are genes represented by the following (1) to (3), respectively.
(1) pgsB gene: a gene encoding the following protein (a), (b) or (c)
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(B) a protein having an amide ligase activity, comprising 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
(C) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having an amide ligase activity
(2) pgsC gene: a gene encoding the following protein (f), (g) or (h)
(F) a protein comprising the amino acid sequence shown in SEQ ID NO: 4
(G) a protein comprising 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 having a function of generating PGA in the presence of a PgsB protein
(H) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4 and having a function of generating PGA in the presence of a PgsB protein
(3) pgsA gene: a gene encoding the following protein (k), (l) or (m)
(K) a protein comprising the amino acid sequence shown in SEQ ID NO: 6
(L) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 6, and having the ability to synthesize PGA in the presence of PgsB and PgsC proteins
(M) A protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and having the ability to synthesize PGA in the presence of PgsB and PgsC A microorganism pgsBCA gene in B. subtilis is expressed, pgsB gene in Bacillus subtilis 及 beauty pgsA gene alone, or pgsC gene in B. subtilis 及 beauty pgsA gene alone, or pgsB gene in Bacillus subtilis only, or Bacillus subtilis A recombinant microorganism obtained by introducing only the pgsC gene in
The microorganism is a bacterium belonging to the genus Bacillus,
A recombinant microorganism in which the pgsB gene, the pgsC gene, and the pgsA gene are genes represented by the following (1) to (3), respectively.
(1) pgsB gene: a gene encoding the following protein (a), (b) or (c)
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(B) a protein having an amide ligase activity, comprising 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
(C) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having an amide ligase activity
(2) pgsC gene: a gene encoding the following protein (f), (g) or (h)
(F) a protein comprising the amino acid sequence shown in SEQ ID NO: 4
(G) a protein comprising 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 having a function of generating PGA in the presence of a PgsB protein
(H) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4 and having a function of generating PGA in the presence of a PgsB protein
(3) pgsA gene: a gene encoding the following protein (k), (l) or (m)
(K) a protein comprising the amino acid sequence shown in SEQ ID NO: 6
(L) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 6, and having the ability to synthesize PGA in the presence of PgsB and PgsC proteins
(M) A protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and having the ability to synthesize PGA in the presence of PgsB and PgsC The recombinant microorganism according to claim 2 , wherein the introduced gene is incorporated into a plasmid (vector) capable of self-replication in the host cell of the microorganism. The recombinant microorganism according to claim 2 or 3 , wherein the introduced gene has a transcription initiation control region and / or a translation initiation control region having a function in the microorganism upstream thereof. The recombinant microorganism according to any one of claims 2 to 4 , wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis. A method for improving the productivity of poly-gamma-glutamic acid, comprising improving the productivity of poly-gamma-glutamic acid using the recombinant microorganism according to any one of claims 2 to 5 . In the method for producing poly-gamma-glutamic acid using a microorganism, the recombinant microorganism according to any one of claims 2 to 5 is used to improve the productivity of poly-gamma-glutamic acid. A method for producing poly-gamma-glutamic acid. A method for producing poly-gamma-glutamic acid, comprising culturing the recombinant microorganism according to any one of claims 2 to 5 and obtaining the produced poly-gamma-glutamic acid.