JP5432635B2 - Method for producing vitamin K2 - Google Patents

Description

  The present invention relates to a method for producing vitamin K2 using a mutant strain of Bacillus subtilis.

  Vitamin K is known as a factor necessary for blood coagulation, and since this vitamin deficiency causes a decrease in blood coagulation ability, it is a kind of fat-soluble vitamin also called anti-hemorrhagic vitamin. In recent years, vitamin K has a bone formation promoting action and a bone resorption suppressing action, and it has been clarified that administration of vitamin K increases bone density, and is expected as a prophylactic or therapeutic drug for osteoporosis.

Vitamin K exists in nature in two forms, vitamin K1 and vitamin K2. Vitamin K1 (phylloquinone) is contained in green plants, and group K2 (menaquinone MK) is contained in animals including intestinal bacteria and some bacteria.
In the vitamin K2 group, homologues of menaquinone-1 to 14 (MK-1 to 14) (1 to 14 represent the number of isoprenoid residues) are known depending on the side chain length. Of these, menaquinone-7 (also simply referred to as “MK-7”) is a typical substance of vitamin K2.

  Vitamin K2 is very difficult to isolate because it exists in nature only in trace amounts in natto and has a short half-life. Therefore, it is attempted to produce vitamin K2 in large quantities using microorganisms such as natto bacteria. For example, a method for producing vitamin K by inoculating fermented natto bacteria in soybean soup or tofu cake (for example, a patent) Reference 1), a method of producing vitamin K2 using Bacillus subtilis mutant GN13 / 72 (Patent Document 2) and menadion-resistant mutant (Non-Patent Document 1), culture of microorganisms belonging to the genus Flavobacterium A method of collecting vitamin K2 from the liquid (for example, Patent Document 3) has been reported. However, its productivity is still not sufficient.

  On the other hand, in the industrial production of useful substances by microorganisms, improvement of productivity is one of the important issues, and breeding of producing bacteria by genetic techniques such as mutation has been performed as the technique. Particularly recently, with the development of microbial genetics and biotechnology, more efficient breeding of production bacteria using genetic recombination techniques has been carried out. Furthermore, in response to the rapid development of genome analysis technology in recent years, attempts have been made to decode genome information of target microorganisms and actively apply them to industry.

  Regarding Bacillus subtilis, about 4100 types of disrupted strains in the Bacillus subtilis genome have been extensively studied, and it has been pointed out that 271 genes are essential for growth (Non-patent Document 2). Has succeeded in comprehensively analyzing gene-disrupted strains derived from Bacillus subtilis, deleting large amounts of the Bacillus subtilis genome, and finding mutant strains excellent in productivity of various enzymes (Patent Document 4). ).

  However, attempts to produce vitamin K2 using a genome-deficient strain of Bacillus subtilis have not been performed so far.

JP 10-295393 A JP 2007-181461 A Japanese Patent Publication No. 7-28748 JP 2007-130013 A

Journal of Industrial Microbiology & Biotechnology (2001) 26, 115-120 K. Kobayashi et al., Proc. Natl. Acad. Sci. USA., 100, 4678-4683, 2003

  The present invention relates to providing a method for efficiently producing vitamin K2 using a genome-deficient strain of Bacillus subtilis.

  The present inventors examined a method for producing vitamin K2 by culturing Bacillus subtilis having the ability to produce vitamin K2, and when using a Bacillus subtilis mutant MGB874 strain lacking a large region of the Bacillus subtilis genome. It was found that vitamin K2 (MK-7) can be efficiently produced.

  That is, the present invention relates to a method for producing vitamin K2, which comprises culturing a Bacillus subtilis mutant MGB874 strain and collecting vitamin K2 from the culture.

  According to the present invention, vitamin K2 can be efficiently produced, and more efficient production of vitamin K2 useful as an osteoporosis preventive / therapeutic agent can be realized.

It is a schematic diagram for demonstrating an example of the method of deleting a predetermined area | region from the genome of Bacillus subtilis. It is a schematic diagram for demonstrating the procedure which produces 168 (DELTA) upp strain from Bacillus subtilis 168 strain. It is a schematic diagram for demonstrating the procedure which produces recombinant plasmid pBRcatupp formed by inserting a cat-upp cassette DNA fragment. FIG. 3 is a schematic diagram for explaining a procedure for preparing a Δ-deleted target region :: cat-upp strain. It is a schematic diagram for demonstrating the procedure which produces (DELTA) deletion target area | region strain | stump | stock. FIG. 3 is a schematic diagram for explaining a procedure for producing a Δ deletion target region :: tet strain. It is a schematic diagram for demonstrating the procedure of deleting the deletion target area | region in a predetermined mutant using a pBRcatupp (DELTA) deletion target area | region. It is a schematic diagram for demonstrating the preparation process of Bacillus subtilis MGB874 strain.

  In the present invention, vitamin K2 means vitamin K2 group, that is, menaquinone-1 to 14 (MK-1 to 14), preferably MK-7.

In the present invention, the Bacillus subtilis mutant MGB874 strain used is Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168) as a wild strain, and a large region of its genome, ie, the prophage6 (yoaV-yobO) region, prophage1 ( ybbU-ybdE) region, prophage4 (yjcM-yjdJ) region, PBSX (ykdA-xlyA) region, prophage5 (ynxB-dut) region, prophage3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA- ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region, prophage2 (ydcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, YeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region are deleted (the above patent) Reference 4).
The Bacillus subtilis Marburg 168 strain, a Bacillus subtilis Marburg 168 parent strain, became a tryptophan-requiring mutant by X-irradiation by Paul Burkholder and Norman Giles of Yale University [Burkholder, PR, and NH Giles. Am J. Bot., 34, 345-348 (1947)].

The Bacillus subtilis mutant strain MGB874 can be produced by deleting the above-mentioned deletion region from the Bacillus subtilis genome. Examples of a method for deleting such a region include the following method shown in FIG. Can be applied.
That is, by the method using the two-step single crossover method using the deletion plasmid inserted with the deletion DNA fragment prepared by the so-called SOE-PCR method (Gene, 77, 61 (1989)), The deletion region is deleted from the Bacillus subtilis genome. The DNA fragment for deletion used in this method is a DNA fragment (downstream fragment) in which about 0.1 to 3 kb fragment adjacent to the upstream of the deletion target region (referred to as upstream fragment) and about 0.1 to 3 kb fragment adjacent to the downstream are bound. Are called DNA fragments. A DNA fragment in which a drug resistance marker gene fragment such as a chloramphenicol resistance gene is bound downstream or upstream of the DNA fragment can also be used.

  First, by the first PCR, an upstream fragment and a downstream fragment of a deletion target gene, and, if necessary, three fragments of a drug resistance marker gene fragment are prepared. At this time, a primer to which a sequence of 10 to 30 base pairs at the end of the DNA fragment to be bound is added is designed. For example, when the upstream fragment and the downstream fragment are joined in this order, a sequence corresponding to 10 to 30 bases upstream of the downstream fragment is added to the 5 ′ end of the primer located at the downstream end of the upstream fragment (annealing). In addition, a sequence corresponding to 10 to 30 bases downstream of the upstream fragment is added to the 5 ′ end of the primer located at the upstream end of the downstream fragment (annealing). When the upstream fragment and the downstream fragment are amplified using the primer set designed in this way, a region corresponding to the upstream side of the downstream fragment is added to the downstream side of the amplified upstream fragment. A region corresponding to the downstream side of the upstream fragment is added to the upstream side of the fragment.

  Next, the upstream fragment and the downstream fragment prepared in the first round are mixed to form a template, and a pair consisting of a primer located on the upstream side of the upstream fragment (annealed) and a primer located on the downstream side of the downstream fragment (annealed) Perform a second PCR using these primers. By this second PCR, a deletion DNA fragment in which the upstream fragment and the downstream fragment are combined in this order can be amplified.

  When the drug resistance marker gene fragment is linked to the deletion DNA fragment, the drug resistance marker gene fragment is amplified so that a region corresponding to the downstream side of the downstream fragment is added in the first PCR. Further, in the second PCR, a pair of primers consisting of a primer located upstream of the upstream fragment (annealing) and a primer located downstream of the drug resistance marker gene fragment (annealed) are used. As a result, the deletion DNA fragment in which the upstream fragment, the downstream fragment, and the drug resistance marker gene fragment are combined in this order can be amplified.

  Further, after amplifying the deletion DNA fragment obtained by joining the upstream fragment and the downstream fragment in this order by the second PCR, by inserting the deletion DNA fragment into the plasmid containing the drug resistance marker gene, the upstream fragment, A deletion DNA fragment having the downstream fragment and the drug resistance marker gene fragment in this order may be prepared.

  Furthermore, the DNA fragment for deletion obtained by the above-mentioned method is inserted into plasmid DNA that cannot be easily amplified in a host bacterium using normal restriction enzymes and DNA ligase, or plasmid DNA that can be easily removed, such as a temperature-sensitive plasmid. Thus, a plasmid for introducing a deletion is constructed. Examples of plasmid DNA that is not amplified in the host bacterium include, but are not limited to, pUC18, pUC118, pBR322, etc. when Bacillus subtilis is used as the host.

  Next, transformation of the host bacterium with the deletion plasmid is performed by the competent cell transformation method (J. Bacteriol. 93, 1925 (1967)), etc., and homology on the genome with the upstream or downstream fragment inserted into the plasmid. A transformant in which the deletion plasmid is fused into the host bacterial genomic DNA is obtained by homologous recombination between the regions. For selection of transformants, drug resistance by a marker gene such as a chloramphenicol resistance gene in a plasmid for deletion introduction may be used as an index.

  In the genome of the transformant thus obtained, there are duplicates of the upstream and downstream sequences of the drug resistance gene on the genome to be deleted that are derived from the host fungus genome and the deletion plasmid. ing. In this upstream region or downstream region, by causing homologous recombination in the genome in a region different from the region homologous recombination when obtaining the transformant, drug resistance on the genome together with the region derived from the deletion plasmid Deletion of the target gene to be deleted occurs, such as a gene. As a method for causing homologous recombination in the genome, for example, a method of inducing competence (J. Bacteriol. 93, 1925 (1967)) can be mentioned, but it can be induced spontaneously even during culturing in a normal medium. Homologous recombination occurs. Strains that have undergone homologous recombination within the genome as intended can be selected from strains that have become drug-sensitive because the drug resistance gene is lost at the same time and the drug resistance is lost. Genomic DNA may be extracted from these strains and the deletion of the target gene may be confirmed by PCR or the like.

  When selecting a target deletion strain, it is difficult to directly select a strain that has changed from drug resistance to sensitivity, and homologous recombination in the genome is considered to occur at a low frequency of about 10-4 or less. Therefore, in order to efficiently obtain the target deletion strain, it is desirable to devise measures such as increasing the ratio of drug-sensitive strains. As a method for concentrating drug-sensitive strains, for example, a method of concentration utilizing methods that penicillin antibiotics such as ampicillin act on bactericidal cells while not acting on non-proliferating cells (Methods in Molecular Genetics). Cold Spring Harbor Labs, (1970)). In the case of concentration using ampicillin or the like, it is effective for deletion of a resistance gene for a drug that acts bacteriostatically on host cells such as tetracycline and chloramphenicol. In an appropriate medium containing an appropriate amount of such a bacteriostatic agent, a resistant strain carrying the drug resistance gene can grow, and a sensitive strain lacking the drug resistance gene does not grow or die. Under such conditions, when a suitable concentration of penicillin antibiotics such as ampicillin is added and cultured, resistant strains to be grown are killed, while sensitive strains are not affected by ampicillin or the like, resulting in The proportion of sensitive strains will increase. To efficiently select sensitive strains by smearing and culturing such a concentrated culture solution on an appropriate agar medium, and confirming the presence or absence of resistance to the marker drug of the colonies that appeared by replica method etc. Is possible.

  As described above, a Bacillus subtilis mutant strain having a genome structure in which a predetermined region on the genome is deleted alone can be produced. Furthermore, a Bacillus subtilis mutant strain having a genomic structure lacking a plurality of regions can be produced by a so-called LP (lysis of protoplasts) transformation method. LP transformation methods are described in `` T. Akamatsu and J. Sekiguchi, `` Archives of Microbiology '', 1987, 146, p.353-357 '' and `` T. Akamatsu and H. Taguchi, `` Bioscience, Biotechnology, and Biochemistry ”, 2001, Vol. 65, No. 4, p. 823-829”. That is, in the LP transformation method, a protoplast having a cell wall lysed is provided as a donor DNA to a competent cell of a recipient strain. The added protoplast is considered to be destroyed by osmotic shock, and the donor DNA released into the culture medium is taken up into the competent cell of the recipient strain. In addition, according to the LP transformation method, DNA damage to be introduced is greatly reduced as compared with a general transformation method.

By applying this LP transformation method, a Bacillus subtilis mutant strain having a genome structure in which a plurality of regions are deleted can be newly produced from a Bacillus subtilis mutant strain having a genome structure deleted alone. Specifically, first, a Bacillus subtilis mutant strain having a genomic structure lacking a specific region (referred to as a first deletion region) is protoplasted, and a different region (second deletion region) is deleted. And co-existing with competent cells of Bacillus subtilis mutants having the genomic structure. Thereby forming a pair of interchain exchange structures between genomic DNA having a first deletion region (donor DNA) and genomic DNA having a second deletion region (host DNA); Become. This set of interstrand exchange structures occurs at a position sandwiching the first deletion region in the donor DNA, whereby the first deletion region in the donor DNA is introduced into the host DNA. Thus, by applying the LP transformation method, a Bacillus subtilis mutant strain having a genomic structure in which the first deletion region and the second deletion region are deleted can be produced. By applying this method, a Bacillus subtilis mutant strain having a genomic structure in which a plurality of regions are deleted can be produced unless a gene essential for growth is deleted.
A specific method for producing the Bacillus subtilis mutant MGB874 strain is shown in Reference Examples below.

The medium used for culture of Bacillus subtilis of the present invention contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by Bacillus subtilis, and can be a natural medium if it can efficiently culture transformants. Any synthetic medium may be used.
For example, as a carbon source, monosaccharides such as glucose and fructose, disaccharides such as sucrose and maltose, polysaccharides such as soluble starch, etc., nitrogen sources as yeast extract, fish meat extract, corn steep liquor (CSL), various animals and plants A known medium containing a component obtained by degrading the above protein with an acid or an enzyme or various amino acids can be used.
The inorganic salt is not particularly limited as long as it can produce vitamin K2 satisfactorily. Specifically, phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc, 1 type (s) or 2 or more types selected from hydrochloride, sulfate, acetate, etc. can be used.

  Specific examples of the medium that can be used for culturing Bacillus subtilis of the present invention include the following LB medium, 2 × L-maltose medium, glucose broth medium, gravy medium, 2SY medium (P2002-238569A), TM medium ( P2002-238569A), MMG medium (Appl. Microbiol. Biotechnol., 62, 369-373) and the like.

The culture can be carried out in the same manner as a conventionally known method, and the culture conditions at that time are appropriately selected depending on the composition of the medium and the culture method, and particularly if the strain can proliferate and produce vitamin K2 efficiently. Not limited.
For example, the culture temperature is usually 20 to 40 ° C., preferably 25 to 35 ° C., and the culture time is not particularly limited, but is 24 to 120 hours, preferably 48 to 72 hours. May be.

  Moreover, the pH of the culture medium suitable for culture is usually 6.0 to 9.5, preferably 6.5 to 8.5. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

  As shown in Examples below, when the Bacillus subtilis mutant MGB874 strain is cultured under the above conditions, vitamin K2 (MK-7) is contained in the cells and / or in the medium in a short period of about 48 hours. Since it is accumulated, the culture is terminated when the production amount reaches the maximum, and vitamin K2 can be isolated and purified from the culture by a conventional method and collected.

  Vitamin K2 is isolated and purified by, for example, extraction using an organic solvent such as methanol, ethanol, acetone or chloroform or a mixed solvent of these with water, column chromatography using silica gel, alumina, Sephadex, etc. It can be performed by using a technique such as chromatography alone or in combination.

Example 1 Production of Vitamin K2 Bacillus subtilis strain 168 and MGB874 strain {(Biotech. Appl. Biochem. 46: 169-178 2007) (DNA research 15, 73-81 2008) (Japanese Patent Laid-Open No. 2007-130013)} In each case, shake culture in 5 mL of LB medium at 30 ° C overnight, and then add 0.6 mL of this culture to 30 mL of 2 × L maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose monohydrate) And 7.5 ppm manganese sulfate 4-5 hydrate), followed by shaking culture at 30 ° C. for 2 days. The supernatant was removed from the culture broth by centrifugation, and the resulting cells were ground with water and methanol and filtered. The filtrate is appropriately diluted with 2-propanol and analyzed by high performance liquid chromatography (reduction column-fluorescence spectrophotometer). The amount of MK-7 produced per culture and the amount of MK-7 produced per cell Was measured. The results are shown in Table 8.

  The Bacillus subtilis mutant MGB874 was prepared from Bacillus subtilis Marburg No.168 (Bacillus subtilis 168 strain) by the method described in Patent Document 4.

  From Table 8, the production amount of the Bacillus subtilis mutant strain MGB874 was significantly increased compared to 168 strains after 2 days of culture.

Reference Example Preparation of MGB874 strain (1) Preparation of upp gene deletion strain (including cat-upp cassette) As shown in Fig. 2, genomic DNA extracted from Bacillus subtilis 168 strain was used as a template and upp-AFW and upp- Using the ARV and upp-BFW and upp-BRV primer sets, a 1.0 kb fragment adjacent to the upstream of the upp gene (BG 13408; uracil phosphoribosyl-transferase) on the genome (a1) and adjacent downstream The 1.0 kb fragment (b1) was amplified by PCR. In addition, a 1.2 kb fragment (c1) containing an erythromycin resistance gene was prepared by PCR using plasmid pMutinT3 (Microbiology, 144, 3097, 1998) as a template and a set of Erm-FW and Erm-RV primers. In the PCR reaction, the reaction system was 20 μL, and LATaq polymerase (manufactured by Takara Bio Inc.) was used according to the attached protocol. For (a1) and (b1), 50 ng of the genome of Bacillus subtilis 168 strain, which is a template DNA, In the above, 1 ng of plasmid DNA, 200 nM each of the above primers, 200 μM each of dATP, dTTP, dCTP, and dGTP, LATaq 0.2 U, and the attached 10 × buffer solution were mixed so as to be 1 ×. For the amplification reaction, a PCR system (GeneAmp9700 manufactured by Applied Biosystem) was used. After heat denaturation at 95 ° C. for 5 minutes, constant temperature was maintained at 95 ° C. for 15 seconds, then 55 ° C. for 30 seconds, and then 72 ° C. for 60 seconds This was repeated 25 times, and finally the extension was completed at 72 ° C. for 30 seconds. The three PCR amplification fragments (a1), (b1) and (c1) thus obtained were purified with Centricon (Millipore), mixed with 0.5 μL of each, and further added with primer upp- AFW and upp-BRV were added, and the constant temperature at 72 ° C. of the above PCR conditions was changed to 3 minutes, and SOE-PCR was performed. As a result of this PCR, a 3.2 kb DNA fragment (d1) was obtained in which the three fragments were combined in the order of (a1), (c1) and (b1). Using this DNA fragment, 168 strains of Bacillus subtilis were transformed by a competent method (J. Bacteeriol., 81, 741, 1960). That is, 168 strains of Bacillus subtilis were treated with SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.02% casamino acid ( Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan) at 37 ° C. until the degree of growth (OD600) is about 1. After shaking culture, a portion of the culture broth was added to 9 times the amount of SPII medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% Inoculated with glucose, 0.01% casamino acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / ml tryptophan), and further cultured with shaking until the growth degree (OD600) is about 0.4. 168 strain competent cells were prepared. Next, 5 μL of the solution containing the above DNA fragment (d1) (the reaction solution of the above SOE-PCR) was added to 100 μL of the prepared competent cell suspension (culture medium in SPII medium), and after shaking culture at 37 ° C. for 1 hour, The whole amount was smeared on LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing 0.5 g / mL erythromycin. After static culture at 37 ° C., the grown colonies were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR using this as a template that the upp gene was deleted from the genome and replaced with an erythromycin resistance gene. This strain is hereinafter referred to as 168Δupp. In addition, transformation by the competent method described later was performed in the same manner as described above except that the DNA to be used and the agar medium for selection of transformants were appropriately changed.

(2) Construction of cat-upp cassette DNA fragment As shown in FIG. 3, plasmid pSM5022 whose transcription has been confirmed in Bacillus subtilis to ensure that the upp gene and the chloramphenicol resistance gene (cat) are transcribed. (Mol. Microbiol. 6, 309, 1992) The upp gene was ligated downstream of the cat gene. That is, a 1.3-kb DNA fragment containing cat was amplified by PCR using pSM5022 as a template using a primer set of cat-Fw and cat-Rv. Further, PCR was performed using primers upp-Fw and upp-RV with the 168 strain genome as a template to obtain a 1.1-kb DNA fragment containing the upp gene. Next, these two fragments were purified and then combined by SOE-PCR to prepare a 2.4 kb ca-upp cassette DNA fragment (C) in which the upp gene was bound downstream of the cat gene. Further, this fragment was inserted into the ClaI cleavage site of plasmid pBR322 to obtain a recombinant plasmid pBRcatupp.

(3) Preparation of Pro1 region-deficient strain (including cat-upp cassette fragment) As shown in FIG. 4, using Pro1-AFW and Pro1-ARV, Pro1-BFW and Pro1-BRV primer sets, 168 Using the strain genome as a template, a 0.6 kb fragment adjacent to the upstream of the Pro1 region (A) and a 0.3 kb fragment adjacent to the downstream (B) were prepared by PCR. In FIG. 4, the Pro1 region is described as “deletion target region”.

Next, SOE-PCR using primers Pro1-AFW and Pro1-BRV was performed using the obtained PCR amplified fragments (A) and (B) and the above-described cat-upp cassette fragment (C) as a template to obtain 3 fragments. They were combined in the order of (A), (C) and (B). The obtained DNA fragment (D) was used to transform the 168Δupp strain shown in Example 2 by a competent method, and a transformant capable of growing on an LB agar medium containing 10 ppm of chloramphenicol was isolated. . It was confirmed by PCR that the Pro1 region was deleted from the genome and replaced with a cat-upp cassette DNA fragment by PCR. Furthermore, Cg + glucose agar medium (7% dipotassium hydrogen phosphate, 3% potassium dihydrogen phosphate, 0.5% sodium citrate) to which this transformant was added with various concentrations of 5FU (manufactured by Sigma-Aldrich) 1% ammonium sulfate, 0.1% magnesium sulfate, 0.05% glutamic acid, 0.5% glucose, 10 ng / mL L-tryptophan, 0.55 μg / mL calcium chloride, 0.17 μg / mL zinc chloride, 43 ng / mL Culture was performed on copper chloride dihydrate, 60 ng / mL cobalt chloride hexahydrate, 60 ng / mL sodium molybdate (IV) dihydrate, 1.5% agar), but 0.5 μg / mL No growth was observed in the medium supplemented with 5FU. On the other hand, growth of the 168Δupp strain, the parent strain of the transformant, was also observed in a medium supplemented with 5 μg / mL of 5FU under the same conditions. From the above results, it was estimated that the upp gene introduced into the transformant was expressed by transcription from the cat gene promoter and became sensitive to 5FU. The strain thus obtained was designated as ΔPro1 :: cat-upp strain. In FIG. 4, the ΔPro1 :: cat-upp strain is described as a Δ deletion target region :: cat-upp strain.

(4) Deletion of cat-upp cassette fragment (C) from Pro1 region-deficient strain As shown in FIG. 5, using ΔPro1 :: cat-upp genome as a template, Pro1-AFW, Pro1-ERV and Pro1-FFW And Pro1-BRV primer set to amplify a 0.6 kb fragment (E) adjacent to the upstream of the cat-upp cassette fragment (C) and a 0.3 kb fragment (F) adjacent to the downstream, A 0.9 kb fragment (G) in which both fragments were bound was prepared by SOE-PCR using Pro1-AFW and Pro1-BRV primer sets using both of the obtained DNA fragments as templates. Using this fragment (G), the above-mentioned ΔPro1 :: cat-upp strain was transformed by a competent method to obtain a strain capable of growing on a Cg + glucose agar medium supplemented with 1 μg / mL / 5FU. In the obtained strain, chloramphenicol sensitivity was confirmed, and it was confirmed that the Pro1 region on the genome and the cat-upp cassette DNA fragment were deleted, and this was designated as ΔPro1 strain. In FIG. 5, ΔPro1 :: cat-upp strain and ΔPro1 strain are described as “Δ deletion target region :: cat-upp strain” and “Δ deletion target region strain”, respectively.
A ΔPro7 :: cat-upp strain was prepared by the method described in FIG. 4 in accordance with the above-described procedure for preparing the ΔPro1 strain. Table 9 below shows primer sets used for amplifying fragment (A) to fragment (G) in the step of producing the strain.

(5) Construction of MGB07 strain Next, a strain in which a plurality of regions were deleted (multiple deletion strain) was constructed using ΔPro7 strain (also referred to as MGB01 strain). First, a double deletion strain of Pro7 region and Pro6 region was constructed as follows. Specifically, ΔPro7 strain was transformed by competent method using the genomic DNA of ΔPro6 :: cat-upp strain in which Pro6 region was replaced with cat-upp cassette fragment, and the LB agar medium containing 10 ppm of chloramphenicol was used. Growing colonies were isolated as transformants. Next, the obtained chloramphenicol resistant transformant was transformed with ΔPro6 strain genomic DNA by the competent method and capable of growing on a Cg + glucose agar medium supplemented with 1 μg / mL of 5FU. Acquired shares. In the obtained strain, chloramphenicol sensitivity was confirmed, and both the Pro6 region and the Pro7 region were deleted, and a double deletion strain containing no cat-upp cassette fragment was isolated. This strain was designated as MGB02 strain.

  By repeating the same operation, an MGB03 strain in which the Pro7 region, the Pro6 region, and the Pro1 region were sequentially deleted was constructed. By repeating the same operation, the MGB04 strain in which the Pro7 region, Pro6 region, Pro1 region and Pro4 region were sequentially deleted was constructed. By repeating the same operation, the MGB05 strain in which the Pro7 region, the Pro6 region, the Pro1 region, the Pro4 region and the PBSX region were sequentially deleted was constructed. By repeating the same operation, the MGB06 strain in which the Pro7 region, the Pro6 region, the Pro1 region, the Pro4 region, the PBSX region, and the Pro5 region were sequentially deleted was constructed. By repeating the same operation, the MGB07 strain in which the Pro7 region, Pro6 region, Pro1 region, Pro4 region, PBSX region, Pro5 region and Pro3 region were sequentially deleted was constructed.

(6) Construction of SPβ region-deficient strain Next, an example of construction of a strain lacking the SPβ region will be described. As shown in FIG. 6, using a primer set of spB-AFW and spB-ARV2 and spB-BFW2 and spB-BRV, using a 168 strain genome as a template, a 0.6 kb adjoining upstream of the SPβ region by PCR. Fragment (H) and a downstream adjacent 0.3 kb fragment (I) were prepared. In FIG. 6, SPβ is described as “deletion target region”.

Further, a tetracycline resistance gene region fragment (J) was amplified using a primer set of tet-FW and tet-RV. Next, SOE-PCR using primers spB-AFW and spB-BRV was performed using the obtained PCR amplified fragments (H) (I) (J) as templates, and the three fragments were in the order of (H) (J) (I). It was made to combine. Using the obtained DNA fragment (K), the above-described 168Δupp strain was transformed by a competent method to isolate a transformant capable of growing on an LB agar medium containing 15 ppm tetracycline. The resulting transformant was confirmed by PCR to have the SPβ region deleted from the genome and replaced with a tetracycline resistance gene fragment, and this was designated as ΔSPβ :: tet strain. In FIG. 6, the ΔSPβ :: tet strain is described as “Δ deletion target region :: tet strain”.
Similarly, a strain in which each region described above was deleted alone was prepared. The prepared strain is referred to as “Δ deletion target region :: tet strain” in the same manner as the ΔSPβ :: tet strain.

(7) Deletion of SPβ region from MGB07 strain ΔPro7 strain was transformed with the genomic DNA of ΔSPβ :: tet strain prepared above to obtain a tetracycline resistant strain MGB07ΔSPβ :: tet strain. On the other hand, the tetracycline resistance gene fragment was removed from the genome as follows.

  As shown in FIG. 7, using the primer sets of spB-AFW and spB-ERV, and spB-FFW and spB-BRV, a 0.6 kb fragment adjacent to the upstream of the SPβ region by PCR using the 168 strain genome as a template (L) and a downstream adjacent 0.3 kb fragment (M) were prepared. In FIG. 7, SPβ is described as “deletion target region”.

  Next, using the obtained PCR amplified fragment (L) (M) as a template, SOE-PCR was performed using primers spB-AFW and spB-BRV to bind the two fragments in the order of (L) (M). . The obtained DNA fragment (N) was inserted into the above-described sacI-KpnI restriction enzyme site of pBRcatupp (blunted after cleavage) to construct a plasmid pBRcatuppΔSPβ for deletion of the tetracycline resistance gene fragment. In FIG. 7, pBRcatuppΔSPβ is described as “pBRcatuppΔ deletion target region”.

  By transforming the MGB07ΔSPβ :: tet strain with the constructed pBRcatuppΔSPβ, single crossover recombination occurred between the region upstream or downstream of SPβ on the plasmid and the region upstream or downstream of SPβ on the genome. A plasmid was introduced into the genome to obtain a strain MGB07ΔSPβ (pBR) strain exhibiting chloramphenicol resistance.

  The obtained transformant MGB07ΔSPβ (pBR) was inoculated into 50 mL of LB medium (500 mL Sakaguchi flask) containing 1.5 μg / mL of tetracycline so that OD600 = 0.3, and cultured at 37 ° C. with shaking for 1 hour. Ampicillin 15 mg (300 μg / mL) was added to the eye, and then the culture was continued while adding ampicillin 15 mg every 2 hours. After 8.5 hours of culture, the culture was washed with 2% sodium chloride aqueous solution, and the drug-free LB agar medium Smeared on. Among the grown colonies, those that became chloramphenicol sensitive as the plasmid region dropped out were selected.

  Using the genomic DNA of the selected strain as a template, PCR was performed to confirm that the SPβ region and the tetracycline resistance gene fragment were deleted, and the MGB08 strain was obtained.

(8) Deletion of pks region from MGB08 strain and restoration of Pro5 region Deletion of pks region from MGB08 strain prepared above was performed using the Δpks :: tet strain according to the method described above (FIG. 7). went. A strain in which the pks region was deleted from the MGB08 strain was named MGB09 strain. When the genomic DNA of the obtained MGB09 strain was confirmed by PCR, the pks region was deleted, but the presence of a sequence within the Pro5 region that was located close to the pks region on the genome was confirmed, and the Pro5 region was I found out that I have returned. When the MGB08 strain was transformed with the Δpks :: tet genomic DNA, the upstream region of the pks region on the Δpks :: tet strain genome was introduced simultaneously with the introduction of the tetracycline resistance gene accompanying the deletion of the pks region. And the region downstream of the Pro5 region were considered to be due to the occurrence of homologous recombination between the corresponding region on the genome of the MGB08 strain.

(9) Deletion of SKIN region from MGB09 strain and restoration of Pro7 region Deletion of SKIN region from MGB09 strain prepared above was performed using the ΔSKIN :: tet strain according to the method described above (FIG. 7). went. A strain in which the SKIN region was deleted from MGB09 strain was designated as MGB10 strain. When the genomic DNA of the obtained MGB10 strain was confirmed by PCR, the SKIN region was deleted, but the presence of a sequence within the Pro7 region that was located close to the SKIN region and the genome was confirmed, and the Pro7 region was I found out that I have returned. When the MGB09 strain was transformed using the ΔSKIN :: tet genomic DNA, the upstream region of the SKIN region on the ΔSKIN :: tet strain genome was introduced simultaneously with the introduction of the tetracycline resistance gene accompanying the deletion of the SKIN region. And the region downstream of the Pro7 region were considered to be due to the occurrence of homologous recombination between the corresponding region on the genome of the MGB09 strain.

(10) Deletion of pps region from MGB10 strain Deletion of the pps region from the MGB10 strain prepared above was performed using the Δpps :: tet strain in accordance with the method described above (FIG. 7). A strain in which the pps region was deleted from MGB10 strain was designated as MGB11 strain. When the obtained genomic DNA of the MGB11 strain was confirmed by PCR, the pps region was deleted without restoring other regions.

(11) Deletion of Pro2 region from MGB11 strain Deletion of the Pro2 region from the MGB11 strain prepared above was performed using the ΔPro2 :: tet strain in accordance with the method described above (FIG. 7). A strain in which the Pro2 region was deleted from the MGB11 strain was named MGB12 strain. When the obtained genomic DNA of the MGB12 strain was confirmed by PCR, the Pro2 region was deleted without restoring other regions.

(12) Deletion of Pro5 region from MGB12 strain
In order to delete again the Pro5 region that was restored when the MGB09 strain was prepared, the Pro5 region was deleted from the MGB12 strain prepared above using the ΔPro5 :: tet strain according to the method described above (FIG. 7). I went. A strain in which the Pro5 region was deleted from MGB12 strain was designated as MGB11d strain. When the obtained genomic DNA of the MGB11d strain was confirmed by PCR, the Pro5 region was deleted without restoring other regions.

(13) Construction of MGB874 stock
The MGB874 strain was prepared as follows from the MGB11d strain prepared as described above (see FIG. 8). That is, according to the method described above (FIG. 7), deletion of the NED0302 region (ydcL-ydeK-ydhU region) from the MGB11d strain was performed using the ΔNED0302 :: tet strain (ΔydcL-ydeK-ydhU :: tet strain). went. A strain in which the NED0302 region (ydcL-ydeK-ydhU region) was deleted from the MGB11d strain was named MGB533 strain. The obtained MGB533 strain has a genomic structure in which the NED0302 region (ydcL-ydeK-ydhU region) is deleted in addition to the deletion region in the MGB11d strain.

Subsequently, the NED0803 region (yisB-yitD region), NED3200 region (yunA-yurT region), NED1902 region (cgeE-ypmQ region), and NED0501 region (yeeK-yesX region) were sequentially deleted to construct a mutant strain. The constructed mutant strain was named MGB625 strain.
Furthermore, a strain in which the NED40002 region (pdp-rocR region) was deleted from the MGB625 strain was designated as the MGB723 strain.

  Thereafter, the NED02021 region (ycxB-sipU region), SKIN-Pro7 region, and NED3701 region (sbo-ywhH region) were deleted in this order to construct a mutant strain. The constructed mutant strain was designated as MGB834 strain.

  Next, according to the method described above (FIG. 7), deletion of the NED4100 region (yybP-yyaJ region) from the constructed MGB834 strain was performed using the ΔNED4100 :: tet strain (ΔyybP-yyaJ :: tet strain). went. A strain in which the NED4100 region (yybP-yyaJ region) was deleted from the MGB834 strain was named MGB860 strain.

  Thereafter, the NED1602 region (yncM-fosB region) was deleted in order to construct a mutant strain. The constructed mutant strains were named MGB874 strain, respectively.

Table 10 below shows the primer sets used when amplifying fragment (H) to fragment (N) in the step of preparing each strain.
Moreover, regarding the various primers used in the reference examples, the correspondence between the primer name, the base sequence, and the sequence number is shown in Tables 11-13.

Claims (1)

  A method for producing vitamin K2, comprising culturing a Bacillus subtilis mutant strain MGB874 and collecting vitamin K2 from the culture.