JP5601783B2 - Method for adjusting the molecular weight of poly-gamma-glutamic acid - Google Patents

Description

  The present invention relates to a method for adjusting the molecular weight of poly-gamma-glutamic acid using a recombinant microorganism.

Poly-gamma-glutamic acid is a polymer compound in which a carboxyl group at the γ-position and an amino group at the α-position of glutamic acid are bonded by a peptide bond. 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. (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 (Non-patent Document 4). Among these PGA synthase systems PgsBCA, PgsB (also called YwsC) is known to be involved in the amide ligase reaction using the γ-position carboxy group in glutamic acid (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, pgsB gene and pgsC gene are essential for PGA production, and pgsA gene is necessary to obtain the maximum PGA productivity. 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. In Patent Document 8, 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.

  On the other hand, microorganisms originally have a wide variety of genes to cope with various environmental changes in nature. From recent results of genome decoding, cell proliferation / division, metabolism, nucleic acid synthesis, information transmission It has been clarified that there are many gene groups with unknown functions. On the other hand, when microorganisms are used for the production of industrial useful substances, abundantly supplied limited medium components provide an environment where they can survive stably. Therefore, they are useful among the above-mentioned genes encoded on the genome. It is considered that there are genes that are unnecessary or harmful to the production of substances.

  Therefore, attempts have been made to improve the productivity of useful substances (for example, heterologous proteins) by deleting or inactivating genes that are considered to be unnecessary or harmful (Patent Documents 7 and 7). 8), there are many reports on the productivity of proteins produced in a ribosome-dependent manner, and PGA and other poly-amino acids produced in a ribosome-independent manner are genetically modified into microbial strains used as hosts. There are few reports on attempts to efficiently produce PGA by mutating using technology, specifically by deleting or inactivating specific genes.

  From the above, the technology necessary for efficiently producing PGA of various molecular weights according to the purpose has not been disclosed, and as a PGA production method so far, it has been actively applied to microbial strains used as hosts. No attempt has been made to efficiently produce PGA with various molecular weights adjusted according to the purpose, using a host microorganism in which a specific gene has been deleted or inactivated. .

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

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-A-2005-348641 JP 2007-130013 A

  The present invention relates to a method for adjusting the molecular weight of PGA using a recombinant microorganism, a method for producing PGA using the molecular weight adjustment method, and a recombinant microorganism.

In light of the above-described circumstances, the present inventors have intensively studied methods for adjusting the molecular weight of PGA, and as a result, have introduced all of the pgsB gene, pgsC gene and pgsA gene involved in PGA synthesis into the host microorganism. contrary to conventional wisdom that say it is possible to produce PGA, and gene corresponding to pgsB gene or the gene, the resulting specific genes genes corresponding to pgsC gene or the gene deleted or inactivated host By using a recombinant microorganism obtained by introducing into a microorganism and without introducing the pgsA gene in Bacillus subtilis or a gene corresponding to that gene, PGA having a different molecular weight can be obtained, that is, the molecular weight of PGA is adjusted. As a result, the present inventors have found the knowledge that it can be performed and have completed the present invention.
Here, “adjustment of the molecular weight of PGA” means that the PGA produced by the microorganism after introduction is polymerized or reduced in molecular weight compared to the molecular weight of PGA produced by the microorganism before introduction by the gene introduction of the present invention. And adjusting its molecular weight. Here, the molecular weight and the weight average molecular weight are synonymous.

  That is, the present invention relates to the following 1) to 5). 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 microorganisms, the ackA gene, acoA gene, acoR gene, ahrC gene, argC gene, argD gene, argF gene, argJ gene, asnO gene, carA gene, codY gene, comP in Bacillus subtilis Gene, comQ gene, comX gene, dhaS gene, gapB gene, glcK gene, glcP gene, gltB gene, licR gene, oppA-F gene ( oppABCDF gene operon), phrA gene, phrE gene, phrI gene, phrK gene, proB gene , RocA gene, rocF gene, sigE gene, sigF gene, sigG gene, sigH gene, sigK gene ( spoIVCB to spoIIIC gene), skfA gene, ygaC gene, yqfN gene, yrzL gene, yuzB gene, ywpG gene and these genes A host microorganism obtained by deleting or inactivating one or more genes selected from genes involved in PGA synthesis. Among gene group, introduced a gene corresponding to pgsB gene or the gene in B. subtilis, a gene corresponding to pgsC gene or the gene in B. subtilis, and, corresponding to the pgsA gene or the gene in B. subtilis A method for adjusting the molecular weight of PGA, wherein the gene to be transferred is not introduced into a host microorganism.

2) Delete or inactivate one or more genes selected from ackA gene, acoR gene, argC gene, argD gene, phrE gene, proB gene, rocF gene, ywpG gene and genes corresponding to these genes in Bacillus subtilis. The method for adjusting the molecular weight of PGA according to (1) above, wherein the PGA is polymerized.

3) acoA gene, ahrC gene, argF gene, argJ gene, carA gene, codY gene, comP gene, comQ gene, gapB gene, glcP gene, glpP gene, gltB gene, licR gene, oppA-F gene ( oppABCDF ) in Bacillus subtilis Gene operon), phrI gene, phrK gene, sigE gene, sigF gene, sigG gene, sigH gene, ygaC gene, yqfN gene, yrzL gene, yuzB gene and one or more genes selected from these genes are deleted Alternatively, the method of adjusting the molecular weight of PGA according to (1) above, wherein the molecular weight of PGA is reduced by inactivation.

4) A method for adjusting the molecular weight of PGA, characterized by using the above-mentioned recombinant microorganism.
5) A method for producing PGA, wherein the produced PGA is obtained using the molecular weight adjustment method for PGA.

  If the molecular weight adjusting method for PGA according to the present invention is used, PGA having various molecular weights can be obtained according to the purpose.

It is the construction flow of the vector for PGA recombinant production for demonstrating an example of the cloning process of the gene group involved in the synthesis | combination of PGA. It is a flow for demonstrating the target gene deletion procedure by the double crossing method using the SOE-PCR fragment in manufacture example 6. It is the construction flow of the vector for PGA recombinant production for demonstrating the cloning process of the gene group involved in the synthesis | combination of PGA in manufacture example 2 and 3. FIG.

  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 name of each gene and gene region of Bacillus subtilis described in this specification has been reported as a result of an international consortium [Kunst, F., et al .: Nature, 390, 249-256 (1997)], JAFAN: Japan It is described based on the Bacillus subtilis genome data published on the Internet (http://bacillus.genome.ad.jp/, updated on January 18, 2006) on the Functional Analysis Network for Bacillus subtilis (BSORF DB).

The recombinant microorganism according to the present invention corresponds to a pgsA gene in Bacillus subtilis or a gene of interest among host genes obtained by deleting or inactivating a specific gene described later, among genes involved in PGA synthesis. Except genes, pgsB gene in Bacillus subtilis or a gene corresponding to the gene and a pgsC gene in Bacillus subtilis or a gene corresponding to the gene are obtained by introducing into a host microorganism and have PGA production ability It is.

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 shown in SEQ ID NO: 1, but is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, or 1 or 2 in the amino acid sequence shown in SEQ ID NO: 2. It is meant to include a gene encoding a protein having an amide ligase activity which consists of an amino acid sequence in which several amino acids are deleted, substituted, added or inserted, and glutamic acid as a substrate.
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.

In the present invention, the pgsB gene is 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more with respect to the amino acid sequence shown in 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 of an amino acid sequence having an amide ligase activity Is meant to include the gene encoding.

Further, in the present invention, the pgsB gene is 60% or more, preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 90% or more with respect to the base sequence shown in SEQ ID NO: 1. 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 has an amide ligase activity. It means to include a gene encoding a protein.
Here, the amide ligase activity can be measured by confirming the formation of PGA in the reaction solution when glutamic acid and ATP or GTP are used as a substrate and manganese chloride is added to the substrate as a reaction solution. [Urushibata, Y., et al .: J. Bacteriol., 184, pp.337-343 (2002)].

Further, in the present invention, the pgsB gene is meant to include a gene that hybridizes under stringent conditions to a complementary strand of the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having amide ligase activity.
Here, “stringent conditions” include, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell, Cold Spring Harbor Laboratory Press (2001)]. For example, 6 × SSC (1 × SSC composition: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. And the conditions of constant temperature for 8 to 16 hours for hybridization. The “stringent conditions” described later have the same meaning.

The above-mentioned amide ligase activity means the ability to produce PGA outside the cell when introduced into a host microorganism together with the pgsC gene. The evaluation of PGA production ability in a given microorganism is performed by, for example, SDS-PAGE, a method of visually observing a translucent viscous substance formed around a colony when cultured on a PGA production agar medium containing glutamic acid or a salt thereof. Method for detecting PGA [Yamaguchi, F., et al .: Biosci. Biotechnol. Biochem. 60, pp. 255-258 (1996)], or a method of quantification using an amino acid analyzer after acid hydrolysis [Ogawa, Y., et al .: Biosci. Biotechnol. Biochem., 61, pp. 1684-1687 (1997)], a quantitative method for purifying the culture liquid and determining the dry weight [Ashiuchi, M., et al .: Biochem. Biophys. Res. Commun., 263: pp. 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, pp. 1212-1213 (1993)].

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.
In the present invention, the pgsC gene is not limited to the gene consisting of the nucleotide sequence shown in SEQ ID NO: 3, but is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, or one or several in the amino acid sequence shown in SEQ ID NO: 4. It means that it includes a gene encoding a protein having an amino acid sequence in which a single amino acid is deleted, substituted, added or inserted, and having the function of generating PGA in the presence of the aforementioned PgsB protein.
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.
In the present invention, the pgsC gene is 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more with respect to the amino acid sequence shown in SEQ ID NO: 4. More preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more, and has a function of generating PGA in the presence of PgsB protein. It means to include a gene encoding a protein.

In the present invention, the pgsC gene is 55% or more, preferably 60% or more, more preferably 70% or more, more preferably 75% or more, and more preferably 80% or more with respect to the base sequence shown in SEQ ID NO: 3. 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 from a nucleotide sequence having identity. Thus, it means to include a gene encoding a protein having a function of generating PGA in the presence of the aforementioned PgsB protein.

Furthermore, in the present invention, the pgsC gene refers to a protein that has a function of hybridizing to a complementary strand of the base sequence shown in SEQ ID NO: 3 under stringent conditions and generating PGA in the presence of the aforementioned PgsB protein. The meaning includes the gene to be encoded. Here, “stringent conditions” uses the same definition as above.

Also, the functions of PgsC proteins such generates a PGA in the presence of PgsB proteins described above, it refers to the ability to produce PGA when was expressed pgsC gene with pgsB gene in a host microorganism. This is not production PGA in the host microorganism to express pgsB gene alone when expressed the pgsC gene with pgsB gene, based on the knowledge of the present inventors have found such PGA is produced Yes.

Incidentally, The nucleotide sequence of the pgsA gene is shown in SEQ ID NO: 5 shows the amino acid sequence of the protein encoded by the pgsA gene is shown in SEQ ID NO: 6.
As mentioned above, the function of the protein encoded by the pgsA gene has not been clarified, but it has been suggested that it is a protein involved in the extracellular export of PGA, and is considered essential for PGA production. ing. On the other hand, as will be described later, the recombinant microorganism according to the present invention has a feature that it is excellent in the productivity of PGA even though the pgsA gene is not introduced.

In the present invention, the pgsA gene is not limited to the gene consisting of the nucleotide sequence shown in SEQ ID NO: 5, but is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6, or one or several in the amino acid sequence shown in SEQ ID NO: 6. The term includes a gene encoding a protein that is composed of an amino acid sequence in which a single amino acid is deleted, substituted, added, or inserted, and is presumed to have PGA synthesis activity in the presence of PgsB and PgsC.
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 the present invention, the pgsA gene is 30% or more, preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, relative to the amino acid sequence shown in 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, and an amino acid sequence having identity with PgsB and PgsC. And a gene encoding a protein presumed to have PGA synthesis activity.

In the present invention, the pgsA gene refers to 45% or more, preferably 50% or more, more preferably 60% or more, more preferably 65% or more, more preferably 70% or more with respect to the base sequence shown in SEQ ID NO: 5. , More preferably 75% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, More preferably, it means that it includes a base sequence having 99% or more identity and includes a gene encoding a protein presumed to have PGA synthesis activity in the presence of PgsB and PgsC. Here, “identity of base sequences” has the same definition as described above.
Furthermore, in the present invention, the pgsA gene is a protein that is hybridized under stringent conditions to the complementary strand of the base sequence shown in SEQ ID NO: 5, and is presumed to have PGA synthesis activity in the presence of PgsB and PgsC. Is meant to include the gene encoding. Here, the same definition as above is used for “stringent conditions”.

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 licheniformis , Bacillus megaterium, Bacillus anthracis, and Bacillus halodurans, Oceanobacillus iheyensis, Natrialba aegyptiaca , there may be mentioned Hydra like, pgsB gene from these microorganisms , A gene corresponding to the pgsC gene, or a 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 70% or more, more preferably 75% or more, more preferably 80% or more, more than the amino acid sequence of a gene having the same function as the pgsB gene of Bacillus subtilis described above Preferably 85%, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more and less than 100% identity, or with the base sequence of the pgsB gene In the nucleotide sequence, 60% or more, preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably Other microorganisms (preferably Bacillus bacteria and Oceanobacillus bacteria, more preferably Bacillus bacteria, having an identity of 97% or more, more preferably 98% or more, more preferably 99% or more and less than 100% ) Is considered to be a gene corresponding to the pgsB gene.
More specifically, pgsB gene and Bacillus licheniformis ATCC 14580 strain of Bacillus subtilis 168 strain, identity with the pgsB gene from Bacillus amyloliquefacience FZB42 strain and Oceanobacillus iheyensis HTE831 strain in the base sequence, respectively 79%, 82% and 62% And their amino acid sequences are 90%, 93% and 55%, respectively, which are considered to be corresponding genes.

50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more, more than the amino acid sequence of a gene having the same function as the pgsC gene of Bacillus subtilis described above Preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more and less than 100% identity, or 55% or more in the base sequence of the pgsC gene, Preferably 60% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably Other microorganisms (preferably Bacillus bacteria and Oceanobacillus bacteria, more preferably Bacillus bacteria, having an identity of 97% or more, more preferably 98% or more, more preferably 99% or more and less than 100% ) Gene is considered to be a gene corresponding to the pgsC gene.
More specifically, the identity between the pgsC gene of Bacillus subtilis 168 and the pgsB gene derived from Bacillus licheniformis ATCC14580 , Bacillus amyloliquefacience FZB42 and Oceanobacillus iheyensis HTE831 is 78%, 84% and 59%, respectively, in the nucleotide sequence. And their amino acid sequences are 90%, 94% and 51%, respectively, which are considered to be corresponding genes.

30% or more, preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more than the amino acid sequence of a gene having a function equivalent to the pgsA gene of Bacillus subtilis described above 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 45% or more in the base sequence of the pgsA gene, 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 90% or more, more preferably 95% or more, more preferably Is 96% or more, more preferably 97% or more, 98% or more, more preferably 99% or more and less than 100% identity, other microorganisms (preferably Bacillus bacteria and Oceanobacillus bacteria, more preferably Bac The gene derived from the genus illus ) is considered to be a gene corresponding to the pgsA gene.
More specifically, the identity between the pgsA gene of Bacillus subtilis 168 strain and the pgsB gene from Bacillus licheniformis ATCC14580 strain, Bacillus amyloliquefacience FZB42 strain and Oceanobacillus iheyensis HTE831 strain is 69%, 75% and 49%, respectively, in the nucleotide sequence. And their amino acid sequences are 67%, 79% and 30%, respectively, which are considered to be corresponding genes.

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, as a gene corresponding to the pgsB gene, one to several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31 or each amino acid sequence. Examples thereof include a gene (for example, SEQ ID NO: 12, 18, 24, or 30) that encodes a protein having an amino acid sequence and having an amide ligase activity using glutamic acid as a substrate.
The gene corresponding to the pgsC gene includes 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 amino acid sequence. And a gene encoding a protein having a function of generating PGA in the presence of the above-described PgsB protein (for example, SEQ ID NO: 14, 20, 26, or 32).
The gene corresponding to the pgsA gene is an amino acid sequence represented by SEQ ID NO: 17, 23, 29 or 35 or an amino acid sequence in which 1 to several amino acids are deleted, substituted, added or inserted in each amino acid sequence. Thus, a gene encoding a protein presumed to have PGA synthesis activity in the presence of PgsB and PgsC (for example, SEQ ID NO: 16, 22, 28 or 34) can be mentioned.

The recombinant microorganism according to the present invention lacks one or more genes selected from the specific genes described later and the genes corresponding to the pgsB gene or pgsB homologous gene and the pgsC gene or pgsC homologous gene. Alternatively, it is produced by introduction into an inactivated host microorganism.
One or more genes selected from the following specific genes on the genome of the parent microorganism into which the pgsBC gene has been introduced and genes corresponding to these genes may be deleted or inactivated, or the specific genes on the genome may be deleted. Recombinant microorganisms may be produced by introducing the pgsBC gene into a parental microorganism in which one or more genes selected from the above genes have been deleted or inactivated, and the timing of gene introduction, inactivation or inactivation Is not particularly limited.

The recombinant microorganism according to the present invention is selected from the above-mentioned specific genes and genes corresponding to these genes, the gene corresponding to the pgsB gene or pgsB gene and the gene corresponding to the pgsC gene or pgsC gene described above. It is produced by introducing a gene of more than one species into a deleted or inactivated host microorganism.
In addition, one or more genes selected from the following specific genes on the genome of the parent microorganism into which the pgsBC gene has been introduced and genes corresponding to these genes may be deleted or inactivated, or A recombinant microorganism may be produced by introducing the pgsBC gene into a parental microorganism in which one or more genes selected from a specific gene have been deleted or inactivated. Introduction of the target gene or inactivation of the gene・ The time of inactivation is not particularly limited.

Here, the parent microorganism for constructing the host microorganism of the present invention is specifically Bacillus amyloliquefaciens , Bacillus pumilus , Bacillus licheniformis , Bacillus megaterium , Bacillus anthracis , Bacillus halodurans , Bacillus subtilis having wild-type PGA-producing ability. and Bacillus (Bacillus) bacteria etc., Clostridium imparted with ability of producing PGA by the gene recombination (Clostridium) bacteria, Corynebacterium (Corynebacterium) bacteria, E. coli (Escherichia coli) or a yeast, and the like. 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. Furthermore, since whole genome information has been clarified, genetic engineering and genome engineering techniques have been established, and it has been developed as a microbial strain that is recognized as safe and excellent as a host microorganism, Bacillus subtilis in particular. Is preferred.

Further, the selected host microorganism may be a microorganism that originally has PGA-producing ability, or may be a microorganism that does not inherently have PGA-producing ability. That is, a microorganism having the pgsBCA gene on the chromosome (genome) and having the ability to produce PGA, a microorganism having the pgsBCA gene on the chromosome but not having the ability to produce PGA, and the pgsBCA gene Any of the microorganisms that are not. When the above-mentioned pgsB gene or a gene corresponding to the pgsB gene and a gene corresponding to the pgsC gene or pgsC gene are introduced into a microorganism having PGA-producing ability, the PGA-producing ability is enhanced and / or PGA has a high molecular weight. It will become. On the other hand, when the aforementioned pgsB gene or the gene corresponding to the pgsB gene and the gene corresponding to the pgsC gene or the pgsC gene are introduced into a microorganism not having the PGA producing ability, the PGA producing ability is imparted.

As a host microorganism, a transcription initiation control region and / or translation having a function in a microorganism as described later, upstream of the pgsBCA gene in Bacillus subtilis possessed on the chromosome (genome) or a gene corresponding to the gene. A microorganism having the ability to produce PGA can also be used by introducing an initiation control region.

In the present invention, ackA gene of Bacillus subtilis to be inactivated or inactivated, acoA gene, acoR gene, ahrC gene, argC gene, argD gene, argF gene, argJ gene, asnO gene, carA gene, codY gene, comP gene, comQ gene, comX gene, dhaS gene, gapB gene, glcK gene, glcP gene, glpP gene, gltB gene, licR gene, oppA-F gene ( oppABCDF gene operon), phrA gene, phrE gene, phrI gene, phrK Gene, proB gene, rocA gene, rocF gene, sigE gene, sigF gene, sigG gene, sigH gene, sigK gene ( spoIVCB to spoIIIC gene), skfA gene, ygaC gene, yqfN gene, yrzL gene, yuzB gene, ywpG gene AckA, AcoA, AcoR, AhrC, ArgC, ArgD, ArgF, ArgJ, AsnO, CarA, CodY, ComP, ComQ, ComX, DhaS, GapB, GlcK, GlcP, GlpP, GltB, LicR, OppABCDF, It is a gene encoding PhrA, PhrE, PhrI, PhrK, ProB, RocA, RocF, SigE, SigF, SigG, SigH, SigK, SkfA, YgaC, YqfN, YrzL, YuzB, YwpG.

Among the above genes, ackA gene, acoA gene, acoB gene, argC gene, argD gene, argE gene, argF gene, argG gene, argJ gene, asnH gene, asnO gene, carA gene, dhaS gene, gapB gene, glcK gene, glcP gene, GlpP gene, gltA gene, gltB gene, proA gene, proB gene, rocA gene, rocF gene, AnsZ gene, Acor gene, AHRC gene, cody gene, GltR gene, LICR gene sugar, organic acids, amino acid metabolism Among the above genes, the acoR gene, ahrC gene, codY gene, gltR gene, and licR gene are gene groups involved in the control of expression of genes involved in metabolism. The comP gene, comQ gene, comX gene, oppA-F gene ( oppABCDF gene operon), phrA gene, phrE gene, phrG gene, phrI gene, and phrK gene are gene groups involved in information transmission between cells. Furthermore, the sigE gene, the sigF gene, the sigG gene, the sigH gene, the sigK gene ( spoIVCB to spoIIIC gene), and the skfA gene are genes involved in sporulation. Furthermore, the ygaC gene, yhaJ gene, yqfN gene, yuzB gene, ywpG gene, yjbM gene, and yrzL gene were functions unknown genes in the aforementioned database. Among these function unknown genes, the yjbM gene is a GTP pilot gene. It encodes a protein having phosphokinase activity [Nanamiya, H., et al .: Mol. Microbiol., 67, 291-304 (2008)], and the yrzL gene encodes a protein having tRNA methyltransferase activity. [Roovers, M., et al .: Nucleic Acids Res., 36, 3252-3262 (2008)] has been reported recently. The protein encoded by the above gene has various functions described in Table 2 below.

Further, it has substantially the same function as each gene of Bacillus subtilis shown in Table 2, preferably 70% or more, preferably 80% or more, more preferably 90% or more, more preferably in each gene and base sequence. Derived from other microorganisms (preferably Bacillus bacteria) having an identity of 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more Genes are considered to be genes corresponding to these genes, and are included in the genes to be deleted or inactivated in the present invention.

These deletion or inactivation genes may be deletion or inactivation of each of the above genes alone, or a combination of two or more.
One or more selected from ackA gene, acoR gene, argC gene, argD gene, phrE gene, proB gene, rocF gene, ywpG gene and genes corresponding to these genes in terms of the ability to polymerize PGA It is preferable to delete or inactivate the gene of the parent microorganism when the PGA is polymerized to 150% or more than when the wild strain is used as the host microorganism, and the parent microorganism ackA gene, argC gene, acoR gene and It is preferable to use a host microorganism obtained by deleting or inactivating one or more genes selected from genes corresponding to these genes.
In addition, since the PGA can be made smaller , the parent microorganism's acoA gene, ahrC gene, argF gene, argJ gene, carA gene, codY gene, comP gene, comQ gene, gapB gene, glcP gene, glpP gene, gltB gene, licR Gene, oppA-F gene ( oppABCDF gene operon), phrI gene, phrK gene, sigE gene, sigF gene, sigG gene, sigH gene, ygaC gene, yqfN gene, yrzL gene, yuzB gene and genes corresponding to these genes It is preferable to delete or inactivate one or more genes, and when reducing the molecular weight of PGA to 70% or less than when a wild strain is used as a host microorganism, the sigH gene of the parent microorganism, the codY gene, It is preferable to use a host microorganism obtained by deleting or inactivating one or more genes selected from the glpP gene and genes corresponding to these genes.
In addition, the deletion or inactivation of a gene includes the insertion or insertion of a base into the gene in addition to the substitution / deletion of all or a part of the base in the gene.

Moreover, in order to further increase the productivity, further deletion or inactivation of genes other than the specific gene may be performed.
For example, a better effect can be expected by using, as a host, a mutant strain in which a gene encoding a PGA-degrading enzyme (PGA-degrading enzyme gene) is deleted or inactivated. As a PGA-degrading enzyme gene in Bacillus subtilis, the ggt gene and the like are known [Kimura, K., et al .: Microbiology, 150, pp. 4115-4123 (2004)]. The nucleotide sequence of the ggt gene is shown in SEQ ID NO: 10 shows the amino acid sequence of the PGA degrading enzyme encoded by ggt gene in SEQ ID NO: 11. In addition, when a microorganism other than Bacillus subtilis is used as a host, it is preferable to use a mutant strain in which the ggt gene or a gene corresponding to the gene is deleted or inactivated.
In the present invention, the ggt gene is not limited to the gene consisting of the base sequence shown in SEQ ID NO: 10, but a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 11, or one or several in the amino acid sequence shown in SEQ ID NO: 11. It is meant to include a gene encoding a protein having a PGA degrading activity, consisting of an amino acid sequence deleted, substituted, added or inserted. Here, the term “several” is, for example, preferably 2 to 6, more preferably 2 to 3.
In the present invention, the ggt gene is 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, with respect to the base sequence shown in SEQ ID NO: 10. , Most preferably 98% or more, more preferably 99% or more of the base sequence having the identity, and means to include a gene encoding a protein 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: 11 and encodes a protein having PGA degradation activity. Here, the same definition as above is used for “stringent conditions”.
Further, it has the same function as the Bacillus subtilis ggt gene, preferably 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably in the nucleotide sequence and nucleotide sequence of the gene. A gene derived from another microorganism (preferably Bacillus bacterium) having 98% or more identity is considered to be a gene corresponding to the ggt gene.

  Deletion or inactivation of the gene of the present invention can be performed by a method such as inserting another DNA fragment into each gene or giving a mutation to the transcription / translation initiation region of the gene. For this, it is more desirable to physically delete the target gene.

  As a procedure for deletion or inactivation of the above-mentioned gene, in addition to a method of systematically deleting or inactivating the gene (target gene), random gene deletion or inactivation mutation is given. Thereafter, a method for evaluating protein productivity and performing gene analysis by an appropriate method may be mentioned.

  As a method for deleting or inactivating a target gene or a homologous gene thereof, a conventionally known method can be appropriately used. For example, a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of each specific gene into an appropriate plasmid (vector) is incorporated into the parent microorganism cell, and homologous in a partial region of the target gene. Recombination can disrupt and inactivate target genes on the parental microbial genome [Leenhouts, K., et al .: Mol. Gen. Genet., 253, pp. 217-224 (1996)].

  In particular, when Bacillus subtilis is used as a parent microorganism for constructing the microorganism of the present invention, there have already been several reports on methods for deleting or inactivating a target gene by homologous recombination [Itaya, M. , Tanaka, T .: Mol. Gen. Genet., 223, pp. 268-272 (1990)], by repeating such a method, 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 parent microorganism with ultraviolet rays, gamma 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 target structural gene, it is related to the introduction of the same mutation into the control region such as the promoter region or the expression control of the target gene. The same effect can be expected by introducing a similar mutation into a gene. Furthermore, as a more efficient technique, a linear DNA fragment that includes upstream and downstream regions of the target gene but does not include the target gene is obtained by SOE (splicing by overlap extension) -PCR method [Horton, RM. , Et al .: Gene, 77, pp. 61-68 (1989)], which is incorporated into the parent microbial cell and crossed twice at two locations outside the target gene on the parent microbial genome. It is also possible to carry out a technique of deleting a target gene on the genome by causing homologous recombination (see FIG. 2).

In addition, the recombinant microorganism according to the present invention has a positive expression in which the introduced pgsB gene or a gene corresponding to pgsB and a gene corresponding to pgsC or pgsC have a function in the host cell upstream thereof. It is preferable that the transcription initiation control region and the translation initiation control region to be controlled are combined. The control region is more preferably one capable of expressing the linked downstream gene and enhancing the expression of the gene in the host cell, particularly one that constitutively expresses and / or highly expresses the downstream gene. preferable. Examples of such control regions include control regions of α-amylase genes derived from bacteria belonging to the genus Bacillus , protease gene control regions, aprE gene control regions, spoVG gene control regions, rapA gene control regions, Bacillus sp. Examples include, but are not limited to, the cellulase gene control region of KSM-S237 strain, the kanamycin resistance gene control region derived from Staphylococcus aureus , the control region of the chloramphenicol resistance gene.

Bacillus sp. The base sequence of the control region derived from the 0.4 to 1.0 kb region upstream of the translation start point of the cellulase gene, which is the cellulase gene control region of the KSM-S237 strain, is shown in SEQ ID NO: 7. In addition, the control region is not limited to the base sequence shown in SEQ ID NO: 7, and for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably the base sequence shown in SEQ ID NO: 7. It means a base sequence having an identity of 96% or more, more preferably 97% or more, most preferably 98% or more, more preferably 99% or more and having a function equivalent to that of the control region.

The base sequence of the control region of the rapA gene (BG10652) derived from Bacillus subtilis is shown in SEQ ID NO: 8. In addition, the control region is not limited to the base sequence shown in SEQ ID NO: 8, and for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably the base sequence shown in SEQ ID NO: 8. Means a base sequence having an identity of 96% or more, more preferably 97% or more, most preferably 98% or more, more preferably 99% or more and having a function equivalent to that of the control region.

Furthermore, SEQ ID NO: 9 shows the base sequence of the control region of the spoVG gene (BG10112) derived from Bacillus subtilis. The control region is not limited to the base sequence shown in SEQ ID NO: 9, and for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 96% with respect to the base sequence shown in SEQ ID NO: 9. % Or more, more preferably 97% or more, most preferably 98% or more, more preferably 99% or more, and a base sequence having a function equivalent to that of the control region.

When introducing the aforementioned pgsB gene or a gene corresponding to pgsB and a gene corresponding to pgsC or pgsC into a host microorganism, a normal 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, 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). In addition, for transformation using an expression vector, conventional techniques such as the protoplast method [Chamg, S., Cohen, SH .: Mol. Gen. Genet., 168, pp. 111-115 (1979)] and competent cell method [Young, FE., Spizizen, J .: J. Bacteriol., 86, pp. 392-400 (1963)] Can do.

As shown in the examples below, conditions for introducing a recombinant plasmid (pHY-P_S237 / pgsBC) having the cellulase gene control region of Bacillus sp. KSM-S237 strain upstream of the Bacillus subtilis pgsBC gene into Bacillus subtilis strains When the molecular weight of PGA obtained under this control condition is 100%, the ackA gene, argC gene, phrE gene, argD gene, rocA gene, rocF gene, proB gene, skfA gene, ywpG of the Bacillus subtilis strain A Bacillus subtilis pgsBC gene having a cellulase gene control region of the aforementioned Bacillus sp. KSM-S237 strain or a mutant of Bacillus subtilis having one or more genes selected from genes and genes corresponding to these genes deleted or inactivated By introducing a gene corresponding to the gene, PGA having a molecular weight of 105 to 240% is obtained. On the other hand, ahrC gene, argF gene, argJ gene, asnO gene, codY gene, comP gene, comQ gene, comX gene, gapB gene, glcK gene, glcP gene, glpP gene, gltB gene, licR gene, oppA-F of Bacillus subtilis strain 1 selected from genes ( oppABCDF gene operon), phrI gene, phrK gene, sigE gene, sigH gene, sigK gene ( spoIVCB to spoIIIC gene), ygaC gene, yqfN gene, yrzL gene, yuzB gene and genes corresponding to these genes When a Bacillus subtilis mutant strain in which the above genes are deleted or inactivated is used, PGA having a molecular weight reduced to 60 to 95% is obtained.

In addition, the control conditions were the conditions in which the recombinant plasmid (pHY-P_spoVG / pgsBC) having the control region of the Bacillus subtilis spoVG gene described above was introduced upstream of the Bacillus subtilis pgsBC gene. When the molecular weight is 100%, one or more genes selected from the ackA gene, acoR gene, asnO gene, phrA gene, sigK gene ( spoIVCB to spoIIIC gene) of Bacillus subtilis and genes corresponding to these genes are deleted or By introducing the same pgsBC gene into an inactivated Bacillus subtilis mutant, PGA having a molecular weight of 110 to 330% can be obtained. On the other hand, the acoA gene, ahrC gene, carA gene, codY gene, comX gene, dhaS gene, gapB gene, glpP gene, glcK gene, rocA gene, sigE gene, sigF gene, sigG gene, skfA gene and these genes of Bacillus subtilis strain When a similar pgsBC gene is introduced into a Bacillus subtilis mutant strain in which one or more genes selected from the corresponding genes have been deleted or inactivated, PGA having a molecular weight reduced to 40 to 95% can be obtained.

Further, as a control condition the conditions introduced recombinant plasmid (pHY-P_rapA / pgsBC) in B. subtilis strain having a control region upstream of the Bacillus subtilis pgsBC gene of B. subtilis rapA gene described above, the PGA obtained in this control conditions One or more selected from ackA gene, comX gene, dhaS gene, glcK gene, rocA gene, sigK gene ( spoIVCB to spoIIIC gene), skfA gene and genes corresponding to these genes when the molecular weight is 100% By introducing the same pgsBC gene into a Bacillus subtilis mutant strain in which this gene has been deleted or inactivated, PGA having a molecular weight of 110 to 240% can be obtained. On the other hand, similar to Bacillus subtilis mutant strains in which one or more genes selected from the carA gene, codY gene, gapB gene, glpP gene, phrA gene, sigH gene and genes corresponding to these genes are deleted or inactivated. When the pgsBC gene is introduced, PGA having a molecular weight reduced to 70 to 95% can be obtained.

In addition, the control condition was the condition in which a recombinant plasmid (pHY-P_S237 / pgsBC) having the control region of the cellulase gene of Bacillus sp. KSM-S237 described above upstream of the Bacillus subtilis pgsBC gene was introduced into the Bacillus subtilis strain. When the molecular weight of PGA obtained under the conditions is 100%, Bacillus subtilis mutant strains in which one or more genes selected from the sigH gene of the Bacillus subtilis strain and a gene corresponding to the gene have been deleted or inactivated are added to the aforementioned Bacillus. sp. from pgsBC gene Bacillus amyloliquefaciens NBRC3022 strain having a control region of a cellulase gene of KSM-S237 strain, Bacillus licheniformis ATCC9945a line derived pgsBC gene, by introducing the derived pgsBC gene Bacillus licheniformis AHU1371 strain derived pgsBC gene or Oceanobacillus iheyensis HTE831 strain Since a PGA having a molecular weight reduced to 15 to 60% can be obtained, a gene corresponding to the Bacillus subtilis pgsBC gene as described above is also used. The effect is obtained.

That is, the ackA gene, acoA gene, acoR gene, ahrC gene, argC gene, argD gene, argF gene, argJ gene, asnO gene, carA gene, codY gene, comP gene, comQ gene, comX gene, dhaS gene in Bacillus subtilis , gapB gene, glcK gene, glcP gene, glpP gene, gltB gene, licR gene, oppA-F gene ( oppABCDF gene operon), phrA gene, phrE gene, phrI gene, phrK gene, proB gene, rocA gene, rocF gene, sigE One or more genes selected from genes, sigF genes, sigG genes, sigH genes, sigK genes ( spoIVCB to spoIIIC genes), skfA genes, yqga genes, yqfN genes, yrzL genes, yuzB genes, ywpG genes and genes corresponding to these genes A Bacillus subtilis mutant strain with the gene deleted or inactivated is used as the host microorganism, and the host microorganism is transformed into Bacillus subtilis. That pgsA gene or without the introduction of gene corresponding to the gene, and the gene corresponding to pgsBC gene or the gene of Bacillus subtilis is introduced into the host microorganism, to adjust the molecular weight of PGA, various in accordance with the intended Efficient production of molecular weight PGA.

  By using the recombinant microorganism according to the present invention described above, PGAs having various molecular weights can be efficiently produced according to the purpose. The produced PGA can be used for various applications such as cosmetics, pharmaceuticals, foods, water purification agents, water retention materials, thickeners and the like. In particular, in the recombinant microorganism according to the present invention, it is possible to significantly reduce the production cost of PGA used in the above-mentioned applications because superior productivity can be obtained as compared with the conventional PGA production method by genetic recombination technology. Can do.

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

  From the viewpoint of improving 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 growth of the recombinant microorganism to be cultured. Specifically, it is preferable to add sodium glutamate to the medium, but the concentration (in terms of glutamic acid) is, for example, in the medium, preferably 0.005 to 600 g / L, more preferably 0.05 to 500 g / L, more preferably 0.1 to 400 g / L, more preferably 0.5 to 300 g / L. Since glutamic acid in the medium is consumed for the growth of recombinant microorganisms, the glutamic acid concentration is preferably 0.005 g / L or more from the viewpoint of improving the productivity of PGA, and glutamic acid in the medium due to exceeding the saturated solubility of sodium glutamate In view of avoiding precipitation of sodium and other medium components, a glutamic acid concentration of 600 g / L or less is preferable.

  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 recovering the PGA accumulated in the medium, it is necessary to remove the microbial cells that produced the PGA. For this, a method by centrifugation, a method using an ultrafiltration membrane, an electrodialysis method, Examples include a method of recovering as a precipitate by maintaining the pH near the isoelectric point of PGA, and a combination of the above methods is also possible.

  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. Tables 3 and 4 collectively show the names of the primers used in this Example, their base sequences, and SEQ ID NOs. In the base sequence shown in Table 3, the underlined portion indicates the added restriction enzyme recognition sequence.

[Production Example 1] Construction of vector (1)
In this production example, a method for cloning genes involved in PGA synthesis was shown (see FIG. 1).
First, Bacillus sp. Using the chromosomal DNA prepared from KSM-366 strain (FERM BP-6262) as a template, using the primer set of P-S237 / BSpgsBatg FW (SEQ ID NO: 36) and HindIII_BSpgsBCA RV (SEQ ID NO: 37) shown in Table 3 Using a primer set of pgsBCA gene 2.9 kb DNA fragment (BCA) and P-S237 / BSpgsB atg FW (SEQ ID NO: 36) and HindIII_BSpgsBC RV (SEQ ID NO: 38), a pgsBC gene 1.7 kb DNA fragment (BC) Each was prepared. Bacillus sp. The nucleotide sequence of the pgsB gene of KSM-366 strain is SEQ ID NO: 12, the amino acid sequence of the protein encoded by the pgsB gene is SEQ ID NO: 13, the nucleotide sequence of the pgsC gene is SEQ ID NO: 14, and the protein encoded by the pgsC gene the amino acid sequence in SEQ ID NO: 15, in SEQ ID NO: 16 the nucleotide sequence of the pgsA gene, the amino acid sequence of the protein the pgsA gene is encoded by SEQ ID NO: 17. Furthermore, Bacillus sp. Using chromosomal DNA prepared from the KSM-S237 strain (FERM BP-7875) as a template, and using the primer set of Cm_S237 FW (SEQ ID NO: 39) and P_S237-RV (SEQ ID NO: 40) shown in Table 3, KSM-S237 A cellulase gene derived from a strain (see Japanese Patent Application Laid-Open No. 2000-210081) A 0.6 kb DNA fragment (P_S237) was prepared.

Next, a 3.5 kb DNA fragment obtained by fragmenting (P_S237) and (BCA) into one fragment by SOE-PCR using the fragments (P_S237) and (BCA) as a template and a primer set of Cm_S237 FW and HindIII_BSpgsBCA RV (P_BCA), 2.3 kb obtained by similarly fragmenting (P_S237) and (BC) by SOE-PCR using the fragments (P_S237) and (BC) as templates and using a primer set of Cm_S237 FW and HindIII_BSpgsBC RV DNA fragment (P_BC) was obtained. Subsequently, the DNA fragments (P_BCA) and (P_BC) were treated with restriction enzymes Bam HI and Hind III, and the plasmid vectors pHY300PLK (Takara) and DNA Ligation kit Ver. Ligation reaction (conjugation) was performed using 2 (Takara).

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-ALDRICH) Transformants were used. 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, to the DNA fragment of interest to the Bam HI and Hin dIII restriction enzyme recognition sites on the plasmid vector (P_BC) is inserted by electrophoresis It was confirmed.

  In this production example, each of the recombinant plasmids in which insertion of the DNA fragment (P_BC) was confirmed was designated as a PGA recombinant production vector pHY-P_S237 / pgsBC. 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 (2)
In this production example, a method for cloning genes involved in PGA synthesis was shown (see FIG. 3).
First, using a chromosomal DNA prepared from Bacillus subtilis Marburg No.168 (Bacillus subtilis 168) as a template, primer sets of P_spoVG FW2 (SEQ ID NO: 41) and P_spoVG / Cm R (SEQ ID NO: 42) shown in Table 3 were used. Plasmid pC194 [for example, Horinouchi, S., Weisblum, B .: J., about 0.6 kb upstream of the spoVG gene ORF. Bacteriol., 150, pp. 815-825 (1982)] An upstream 0.9 kb DNA fragment (spoVG-UP) for inserting a chloramphenicol resistance gene (Cm ^r ) derived from Bacteriol. Similarly, a downstream 0.6 kb DNA fragment (spoVG-DW) was prepared using the primer set of P_spoVG / Cm F (SEQ ID NO: 43) and P_spoVG RV (SEQ ID NO: 44) shown in Table 3 (note that In this case, the primer for P_spoVG RV was designed to change the start codon gtg of the spoVG gene ORF to atg). In addition, using a plasmid pC194 as a template and a primer set of comp_Cm FW (SEQ ID NO: 45) and comp_Cm RV (SEQ ID NO: 46) shown in Table 3, a chloramphenicol resistance gene 0.9 kb DNA fragment (comp_Cm) was obtained. Prepared.

  Next, using the obtained fragments (spoVG-UP), (spoVG-DW) and (comp_Cm) as templates, using the primer set of P_spoVGFW2 and P_spoVG RV, one fragment of the above 3 fragments by SOE-PCR method A 2.4 kb DNA fragment (Cm-P_spoVG) was obtained. Furthermore, LB agar medium (LBCm) containing 10 ppm of chloramphenicol (SIGMA-ALDRICH) was transformed by the competent cell method (described above) using the DNA fragment thus obtained. Colonies that grew on the agar medium were isolated as transformants.

Next, the genomic DNA extracted from the obtained transformant was used as a template, and PCR using a primer set of comp_Cm FW and P_spoVG RV shown in Table 3 was carried out. The 1.5 consisting of a chloramphenicol resistance gene and a spoVG gene promoter region. A kb DNA fragment (P-spoVG-Cm2) was prepared. Furthermore, by using a primer set of comp_P-spoVG R / BSpgsB atg FW (SEQ ID NO: 47) and pgsC FW (SEQ ID NO: 48), PCR using genomic DNA prepared from Bacillus subtilis 168 strain as a template results in a 1.2 kb pgsB gene. A DNA fragment (pgsB-UP) upstream of the pgsB gene ORF was prepared from a DNA fragment (pgsB1) using a primer set of pgsB / CmF (SEQ ID NO: 49) and pgsB RV (SEQ ID NO: 50). Furthermore, using the obtained 3 fragments of (pgsB1), (P-spoVG-Cm2) and (pgsB-UP) as a template, 3.6 kb DNA by SOE-PCR using a primer set of pgsC FW and pgsB RV Fragments were prepared.

Subsequently, Bacillus subtilis 168 strain was transformed by the competent cell method using the thus obtained DNA fragment, and colonies grown on the LBCm agar medium were isolated as transformants. Genomic DNA extracted from the transformant obtained as a template, using a primer set of HindIII_BSpgsBC RV and BamHI_PspoVG FW (SEQ ID NO: 51), the 2.3kb with Bacillus subtilis spoVG gene promoter upstream of the Bacillus subtilis pgsBC gene DNA A fragment (P_spoVG / pgsBC) was prepared. Further, the obtained DNA fragment was cloned into a plasmid vector by the same procedure as in Production Example 1, and the obtained vector for recombinant PGA production was designated as pHY-P_spoVG / pgsBC.

[Production Example 3] Construction of vector (3)
In this production example, a method for cloning genes involved in PGA synthesis was shown as in Production Example 2 (see FIG. 3).
Using chromosomal DNA prepared from Bacillus subtilis 168 as a template, using the rapA FW (SEQ ID NO: 52) and P_rapA / Cm R (SEQ ID NO: 53) primer sets shown in Table 3, about 0.5 kb upstream from the rapA gene ORF An upstream 0.5 kb DNA fragment (rapA-UP) for insertion of the plasmid pC194-derived chloramphenicol resistance gene was prepared. Similarly, a downstream 0.5 kb DNA fragment (rapA-DW) was prepared using the primer set of P_rapA / CmF (SEQ ID NO: 54) and P_rapA RV (SEQ ID NO: 55) shown in Table 3 (note that At this time, the primer for P_rapA RV was designed to change the start codon ttg of the rapA gene ORF to atg).

  Next, using the obtained fragments (rapA-UP) and (rapA-DW) and the DNA fragment (comp_Cm) prepared in Production Example 2 as a template, using a primer set of rapA FW and P_rapA RV, SOE- A 1.9 kb DNA fragment (Cm-P_rapA) in which the above three fragments were fragmented by PCR was obtained. Further, the Bacillus subtilis strain 168 was transformed by the competent cell method using the thus obtained DNA fragment, and colonies grown on the LBCm agar medium were isolated as transformants.

Next, using genomic DNA extracted from the obtained transformant as a template, PCR using the comp_CmFW and P_rapA RV primer sets shown in Table 3 was carried out to perform a PCR using a chloramphenicol resistance gene and a rapA gene promoter region. A kb DNA fragment (P-rapA-Cm2) was prepared. In addition, a 1.2 kb DNA fragment of pgsB gene (pgsB2) by PCR using genomic DNA prepared from 168 strains of Bacillus subtilis using primer set of comp_P-rapA R / BSpgsB atg FW (SEQ ID NO: 56) and pgsC FW Was prepared. Furthermore, using the obtained (pgsB2), (P-rapA-Cm2) and 3 fragments of the DNA fragment (pgsB-UP) prepared in Production Example 2 as a template, using a primer set of pgsC FW and pgsB RV A 3.5 kb DNA fragment was prepared by SOE-PCR.

Subsequently, Bacillus subtilis 168 strain was transformed by a competent cell method using a DNA fragment obtained by the same procedure as in Production Example 2. Genomic DNA is prepared from the obtained transformant, and has a Bacillus subtilis rapA gene promoter upstream of the Bacillus subtilis pgsBC gene using the genomic DNA as a template and a primer set of HindIII_BSpgsBC RV and BamHI_PrapA FW (SEQ ID NO: 57). A 2.2 kb DNA fragment (P_rapA / pgsBC) was prepared. Further, the obtained DNA fragment was cloned into a plasmid vector in the same manner as in Production Example 1, and the obtained recombinant plasmid was designated as a PGA recombinant production vector pHY-P_rapA / pgsBC.

[Production Example 4] Construction of vector (4)
In this production example, a method for cloning a gene group corresponding to the Bacillus subtilis pgsBC gene involved in PGA synthesis was shown.
Recombinant plasmid pHY-S237, in which an alkaline cellulase gene DNA fragment (approximately 3.1 kb) derived from Bacillus genus KSM-S237 strain (FERM BP-7785) was inserted at the Bam HI restriction enzyme breakpoint of plasmid vector pHY300PLK (Takara) JP 2007-130013 A] is treated with restriction enzyme Xba I, and this linearized plasmid DNA is used as a template, and P_S237-RV (SEQ ID NO: 40) and INFU_pHY-HindIII FW (SEQ ID NO: 40) shown in Table 3 are used. 58), a 5.5 kb DNA fragment (pHY-P_S237) comprising a cellulase gene promoter region derived from the KSM-S237 strain and a plasmid vector pHY300PLK was prepared. Subsequently, using chromosomal DNA prepared from Bacillus amyloliquefaciens NBRC3022 as a template, INFU_P-S237 RV / BAL pgsBatg FW (SEQ ID NO: 59) and INFU_pHY-HindIII / BAL pgsC RV (SEQ ID NO: 62) shown in Table 3 were used. A 1.7 kb DNA fragment (BALpgsBC-3022) derived from Bacillus amyloliquefaciens pgsBC gene was prepared using the primer set. Furthermore, a binding reaction was carried out using In-Fusion PCR Cloning Kit (Takara) using the above DNA fragments in combination (pHY-P_S237 and BALpgsBC-3022) as substrates. Escherichia coli (HB101 strain) was transformed by the competent cell method using the obtained binding sample, and colonies that appeared on the LBTc agar medium were used as transformants. The obtained transformant was grown again on an LBTc agar medium, and a recombinant plasmid was prepared from the resulting cell body using a high-purity plasmid isolation kit (Roche Diagnostics). The resulting plasmid was restriction enzyme treatment with restriction enzymes Bam HI and Hin dIII, to confirm that the DNA fragment of interest on the plasmid vector (BALpgsBC-3022) is inserted by electrophoresis, the The recombinant plasmid obtained in the production example was used as a PGA recombinant production vector (pHY-P_S237 / BALpgsBC-3022). The design of primer sequences and the experimental conditions used in this production example were carried out according to the protocol attached to the kit.

[Production Example 5] Construction of vector (5)
In this Production Example, a method for cloning a gene group corresponding to the Bacillus subtilis pgsBC gene involved in PGA synthesis as in Production Example 5 was shown.
Using chromosomal DNA prepared from Bacillus licheniformis ATCC9945a strain and AHU1371 strain as templates, INFU_P-S237 RV / BLi pgsBatg FW (SEQ ID NO: 60) and INFU_pHY-HindIII / BAL pgsC RV (SEQ ID NO: 63) shown in Table 3 were used. INFU_P-S237 RV shown in Table 3 using a primer set and a 1.7 kb DNA fragment of the pgsBC gene derived from Bacillus licheniformis (BLipgsBC-9945, BLipgsBC-1371) and chromosomal DNA prepared from Oceanobacillus iheyensis HTE831 strain as a template A 1.7 kb DNA fragment (OBipgsBC-831) of the pgsBC gene derived from Oceanobacillus iheyensis was prepared using a primer set of / OBi pgsB atg FW (SEQ ID NO: 61) and INFU_pHY-HindIII / OBi pgsC RV (SEQ ID NO: 64). Subsequently, the DNA fragment (pHY-P_S237) obtained in the above Production Example 4 and the above DNA fragment were combined (pHY-P_S237 and BLipgsBC-9945, pHY-P_S237 and BLipgsBC-1371, pHY-P_S237 and OBipgsBC-831). ), A binding reaction was performed using In-Fusion PCR Cloning Kit (Takara). Escherichia coli (HB101 strain) was transformed by the competent cell method using the obtained binding sample, and a recombinant plasmid was prepared by the same procedure as in Production Example 4 described above. It was confirmed that the target DNA fragment (BLipgsBC-9945, BLipgsBC-1371, OBipgsBC-831) was inserted into the obtained plasmid, and the recombinant plasmid obtained in this Production Example was used as a vector for PGA recombination production. (PHY-P_S237 / BLipgsBC-9945, pHY-P_S237 / BLipgsBC-1371, pHY-P_S237 / OBipgsBC-831).

[Production Example 6] Production of modified Bacillus subtilis (1)
In this production example, a method for producing a Bacillus subtilis mutant strain for introducing the vectors obtained in Production Examples 1 to 3 was shown (see FIG. 2).
Using the genomic DNA prepared from Bacillus subtilis 168 as a template, and using the primer set of ackA-FW (SEQ ID NO: 71) and ackA / Cm-R (SEQ ID NO: 72) shown in Table 4, the ackA gene on the genome Adjacent to the upstream of the ackA gene on the genome using a primer set of 1.0 kb DNA fragment (ackA-UP) adjacent to the downstream, ackA / Cm-F (SEQ ID NO: 73) and ackA-RV (SEQ ID NO: 74) A 1.0 kb DNA fragment (ackA-DW) was prepared. A 0.9 kb DNA fragment (Cm ^r ) containing the chloramphenicol resistance gene using the plasmid pC194-derived template as a primer set of CmFW (SEQ ID NO: 69) and CmRV (SEQ ID NO: 70) shown in Table 4 Was prepared.

Next, the obtained DNA fragments (ackA-UP), (ackA-DW) and (Cm ^r ) were mixed and used as a template, and SOE-PCR using a primer set of ackA-FW and acoA-RV was performed. The three fragments were combined in the order of (ackA-UP)-(Cm ^r )-(ackA-DW) to obtain a 2.9 kb DNA fragment for gene deletion. Next, using this DNA fragment for gene deletion, Bacillus subtilis 168 strain was transformed by the competent cell method, and colonies grown on the LBCm agar medium were isolated as transformants. Furthermore, genomic DNA was extracted from the obtained transformant, and the desired Bacillus subtilis mutant strain (hereinafter referred to as ΔackA) in which the ackA gene was replaced with the chloramphenicol resistance gene (fragment Cm ^r ) by PCR using this as a template. It was confirmed that it was a stock). Similarly, other gene-deficient strains were prepared in the same manner as shown in Table 4 with primer sets of gene name-FW and gene name / Cm-R, and primers of gene name / Cm-F and gene name-RV. The set was used for the preparation, and the resulting Bacillus subtilis mutant strain was given the symbol “Δ” to the name of the deleted gene to give the mutant strain name.

[Production Example 7] Transformation by introduction of plasmid In this production example, a transformation method for Bacillus subtilis strains and their mutants as hosts was shown. The wild-type Bacillus subtilis Marburg No.168 strain and the Bacillus subtilis mutant produced in Production Example 6 were compared with the PGA recombinant production vector (pHY-P_S237 / pgsBC) obtained in Production Examples 1-5. , PHY-P_spoVG / pgsBC, pHY-P_rapA / pgsBC pHY-P_S237 / BALpgsBC-3022, pHY-P_S237 / BLipgsBC-9945, pHY-P_S237 / BLipgsBC-1371 and pHY-P_S237 / OBipgsBC-831) I did it. The plasmid DNA was introduced by supplying 20 to 100 ng of the plasmid DNA to 10 ⁵ to 10 ⁶ protoplasts prepared from the wild strain and mutant by lysozyme treatment. Colonies that grew on DM3 protoplast regeneration medium (DM3 medium) supplemented with 30 ppm tetracycline-hydrochloride were selected as target Bacillus subtilis transformants into which the plasmid was introduced.

[Example 1] Productivity evaluation (1)
Using the Bacillus subtilis 168 strain as a control, the transformants obtained by introducing pHY-P_S237 / pgsBC prepared in Production Example 7 were each inoculated on an LBTc agar medium and subjected to stationary culture at 30 ° C. overnight. . After this, the Bacillus subtilis transformant grown on this LBTc agar medium was modified 2xL / Maltose + MSG medium (2.0% tryptone, 1.0% yeast extract, 1.0% sodium chloride, 7.5% maltose, 8.0% sodium glutamate monohydrate , 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline-hydrochloride) and left to stand as a seed culture solution. This seed culture is inoculated into 1% (v / v) of a new modified 2xL / Maltose + MSG medium and reciprocally shaken at 37 ° C, 120rpm for 3 days (TB-20R-3F, Takasaki) Scientific equipment). After the evaluation culture, the PGA molecular weight was measured under the analysis conditions shown in the measurement examples described later, and the relative values are shown in Table 5.

  As shown in Table 5, the molecular weights of PGA produced in Bacillus subtilis 168 strain and Bacillus subtilis mutant strain into which pHY-P_S237 / pgsBC had been introduced were compared. When the molecular weight of PGA of Bacillus subtilis strain 168 introduced with pHY-P_S237 / pgsBC is set to 100, the molecular weight of PGA of each mutant strain of Bacillus subtilis introduced with pHY-P_S237 / pgsBC changes greatly, and the molecular weight of PGA is adjusted. It became clear that it was possible.

[Example 2] Productivity evaluation (2)
Using Bacillus subtilis 168 strain as a control, the transformants obtained by introducing pHY-P_spoVG / pgsBC prepared in Production Example 7 were inoculated on LBTc agar medium, followed by overnight culture at 30 ° C. . After this, the Bacillus subtilis transformant grown on this LBTc agar medium was modified 2xL / Maltose + MSG medium (2.0% tryptone, 1.0% yeast extract, 1.0% sodium chloride, 7.5% maltose, 8.0% sodium glutamate monohydrate , 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline-hydrochloride) and left to stand as a seed culture solution. This seed culture is inoculated into 1% (v / v) of a new modified 2xL / Maltose + MSG medium and reciprocally shaken at 37 ° C, 120rpm for 3 days (TB-20R-3F, Takasaki) Scientific equipment). After completion of the evaluation culture, the PGA molecular weight was measured under the analysis conditions shown in the measurement examples described later. The evaluation results are shown in Table 6.

  As shown in Table 6, the molecular weights of PGA produced in Bacillus subtilis 168 strain and Bacillus subtilis mutant strain into which pHY-P_spoVG / pgsBC had been introduced were compared. When the molecular weight of PGA of Bacillus subtilis strain 168 introduced with pHY-P_spoVG / pgsBC is set to 100, the molecular weight of PGA of each mutant strain of Bacillus subtilis introduced with pHY-P_spoVG / pgsBC changes greatly, and the molecular weight of PGA is adjusted. It became clear that it was possible.

[Example 3] Productivity evaluation (3)
Using the Bacillus subtilis 168 strain as a control, the transformants obtained by introducing pHY-P_rapA / pgsBC prepared in Production Example 7 were inoculated on an LBTc agar medium, followed by stationary culture at 30 ° C. overnight. . After this, the Bacillus subtilis transformant grown on this LBTc agar medium was modified 2xL / Maltose + MSG medium (2.0% tryptone, 1.0% yeast extract, 1.0% sodium chloride, 7.5% maltose, 8.0% sodium glutamate monohydrate , 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline-hydrochloride) and left to stand as a seed culture solution. This seed culture is inoculated into 1% (v / v) of a new modified 2xL / Maltose + MSG medium and reciprocally shaken at 37 ° C, 120rpm for 3 days (TB-20R-3F, Takasaki) Scientific equipment). After completion of the evaluation culture, the PGA molecular weight was measured under the analysis conditions shown in the measurement examples described later. The evaluation results are shown in Table 7.

  As shown in Table 7, the molecular weights of PGA produced in Bacillus subtilis 168 and Bacillus subtilis mutants into which pHY-P_rapA / pgsBC had been introduced were compared. When the molecular weight of PGA of Bacillus subtilis strain 168 introduced with pHY-P_rapA / pgsBC is set to 100, the molecular weight of PGA of each mutant strain of Bacillus subtilis introduced with pHY-P_rapA / pgsBC changes greatly, and the molecular weight of PGA is adjusted. It became clear that it was possible.

[Example 4] Productivity evaluation (4)
PHY-P_S237 / pgsBC, pHY-P_S237 / BALpgsBC-3022, pHY-P_S237 / BLipgsBC-9945, pHY-P_S237 / BLipgsBC-1371 or pHY-P_S237 / OBipgsBC- prepared in Production Example 7 using Bacillus subtilis 168 strain as a control Transformants obtained by introducing 831 were each inoculated on LBTc agar medium, and statically cultured at 30 ° C. overnight. After this, the Bacillus subtilis transformant grown on this LBTc agar medium was modified 2xL / Maltose + MSG medium (2.0% tryptone, 1.0% yeast extract, 1.0% sodium chloride, 7.5% maltose, 8.0% sodium glutamate monohydrate , 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline-hydrochloride) and left to stand as a seed culture solution. This seed culture is inoculated into 1% (v / v) of a new modified 2xL / Maltose + MSG medium and reciprocally shaken at 37 ° C, 120rpm for 3 days (TB-20R-3F, Takasaki) Scientific equipment). After completion of the evaluation culture, the PGA molecular weight was measured under the analysis conditions shown in the measurement examples described later, and the evaluation results are shown in Table 7.

  As shown in Table 8, Bacillus subtilis strain 168 introduced with pHY-P_S237 / pgsBC and pHY-P_S237 / BALpgsBC-3022, pHY-P_S237 / BLipgsBC-9945, pHY-P_S237 / BLipgsBC-1371 or pHY-P_S237 / OBipgsBC The molecular weight of PGA produced in the Bacillus subtilis mutant strain (ΔsigH strain) introduced with -831 was compared. pHY-P_S237 / BALpgsBC-3022, pHY-P_S237 / BLipgsBC-9945, pHY-P_S237 / BLipgsBC-1371 or pHY-P_S237 when the molecular weight of PGA of Bacillus subtilis strain 168 introduced with pHY-P_S237 / pgsBC is 100 The molecular weight of PGA of each Bacillus subtilis mutant strain into which / OBipgsBC-831 was introduced was greatly changed (lower molecular weight), and it became clear that the PGA molecular weight could be adjusted.

[Measurement example] Quantification of PGA and molecular weight measurement method The culture solution sample after completion of the evaluation culture shown in Examples 1 to 4 was subjected to centrifugation (trade name himacCF15RX, Hitachi Koki) at 14,800 rpm for 30 minutes at room temperature. The amount of recombinant PGA produced in the culture supernatant obtained by centrifugation was measured. Detection of PGA in the supernatant sample was performed according to the method of Yamaguchi et al. (Described above). After subjecting the sample to agarose gel electrophoresis, the gel was stained with methylene blue, and recombinant PGA production was performed by the presence or absence of a PGA-derived staining substance. It was confirmed. Furthermore, HPLC analysis using TSKGel G4000PWXL and TSKGel G6000PWXL gel filtration columns (trade name, Tosoh) was performed on samples in which recombinant PGA was detected. 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. In addition, polyglutamic acids with different molecular weights obtained using a pullulan (Shodex STANDRD P-82, trade name, Showa Denko) in advance for molecular weight tests [Wako Pure Chemical Industries (162-21411, 162-21401) , SIGMA-ALDRICH (P-4886, P-4761), Meiji Food Material (molecular weight 880,000)].

Claims (7)

In a method for producing poly-gamma-glutamic acid using a microorganism, the ackA gene, acoA gene, acoR gene, ahrC gene, argC gene, argD gene, argF gene, argJ gene, asnO gene, carA gene, codY gene in Bacillus subtilis , ComP gene, comQ gene, comX gene, dhaS gene, gapB gene, glcK gene, glcP gene, glpP gene, gltB gene, licR gene, oppA-F gene (oppABCDF gene operon), phrA gene, phrE gene, phrI gene, phrK gene, proB gene, rocA gene, rocF gene, sigE gene, sigF gene, sigG gene, sigH gene, sigK gene (spoIVCB to spoIIIC gene), skfA gene, ygaC gene, yqfN gene, yrzL gene, yuzB gene, ywpG gene and have these genes substantially the same function, and selected from a gene having these genes and keep nucleotide sequence identity of 90% or more A host microorganism obtained by deleting or inactivating the above genes, among the genes involved in poly-gamma-glutamic acid synthesis, the pgsB gene in Bacillus subtilis or a gene corresponding to the gene, and introducing the gene corresponding to pgsC gene or the gene, and the pgsA gene in B. subtilis, characterized in that not introduced into the host microorganism, poly - gamma - a molecular weight adjustment method of glutamate,
The host microorganism is a Bacillus bacterium,
pgsB gene or a gene corresponding to the gene, the gene corresponding to the pgsC gene or the gene is a gene shown in the following respectively (1) to (4), molecular weight adjustment method.
(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 represented by SEQ ID NO: 2 and having an amide ligase activity
(2) Gene corresponding to pgsB gene:
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: 13, 19, 25 or 31
(3) pgsC gene: a gene encoding the following protein (d), (e) or (f)
(D) a protein comprising the amino acid sequence shown in SEQ ID NO: 4
(E) 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: 4, and has a function of generating poly-gamma-glutamic acid in the presence of PgsB protein. protein
(F) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having a function of generating PGA in the presence of a PgsB protein
(4) Gene corresponding to pgsC gene:
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: 15, 21, 27 or 33, and produces poly-gamma-glutamic acid in the presence of PgsB protein. Genes encoding functional proteins AckA gene, acoR gene, argC gene, argD gene, phrE gene, proB gene, rocF gene, ywpG gene, and these genes in Bacillus subtilis have substantially the same functions, and 90% or more of these genes and nucleotide sequences The poly-gamma glutamic acid is polymerized using a host microorganism obtained by deleting or inactivating one or more genes selected from genes having the same identity. -Method for adjusting the molecular weight of gamma-glutamic acid. AcoA gene, ahrC gene, argF gene, argJ gene, carA gene, codY gene, comP gene, comQ gene, gapB gene, glcP gene, glpP gene, gltB gene, licR gene, oppA-F gene (oppABCDF gene) in Bacillus subtilis Operon), phrI gene, phrK gene, sigE gene, sigF gene, sigG gene, sigH gene, ygaC gene, yqfN gene, yrzL gene, yuzB gene and the same function as these genes, and these genes and bases Poly-gamma glutamic acid is reduced in molecular weight using a host microorganism obtained by deleting or inactivating one or more genes selected from genes having 90% or more identity in the sequence The method for adjusting the molecular weight of poly-gamma-glutamic acid according to claim 1. The pgsB gene or a gene corresponding to the gene and the pgsC gene or a gene corresponding to the gene are incorporated into a plasmid (vector) capable of self-replication in the host cell of the microorganism . 3 according to any one of poly - gamma - molecular weight adjustment method of glutamate. pgsB gene or a gene corresponding to the gene and pgsC gene or the gene corresponding to the gene, upstream thereof, any of claims 1 to 4 having a transcription initiation control region and / or translation initiation control region having a function in a microorganism A method for adjusting the molecular weight of poly-gamma-glutamic acid according to claim 1. The method for adjusting the molecular weight of poly-gamma-glutamic acid according to any one of claims 1 to 5, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis. A method for producing poly-gamma-glutamic acid, wherein the produced poly-gamma-glutamic acid is obtained using the molecular weight adjustment method for poly-gamma-glutamic acid according to any one of claims 1 to 6 .