CN111560342B - Recombinant bacillus subtilis for synthesizing MK-4 and construction method and application thereof - Google Patents
Recombinant bacillus subtilis for synthesizing MK-4 and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant Bacillus subtilis for synthesizing MK-4 and a construction method and application thereof, wherein the recombinant Bacillus subtilis168 for synthesizing MK-4 is used as a host, menA, menG and crtE genes from blue algae Synechocystis sp.PCC 6803 are integrated, and hepT is knocked out. And dxs, dxr and ispD-ispF genes are integrated on the chromosome of the recombinant strain, and the expression of MEP pathway key genes dxs, dxr and ispD-ispF is enhanced. The MK-4 synthesized by the recombinant bacillus subtilis has the extracellular accumulation amount of 78mg/L, and lays a foundation for further metabolic engineering modification of the bacillus subtilis to produce MK-4.
Description
Technical Field
The invention relates to recombinant bacillus subtilis for synthesizing MK-4 and a construction method and application thereof, belonging to the technical field of metabolic engineering.
Background
Menaquinones are a class of compounds that are widely found in microorganisms and play important roles in sporulation, oxidative phosphorylation, and electron transfer. Currently, there are 14 MK-n where n is the number of isoprene units in the side chain. The length of this tail varies from microorganism to microorganism. In humans, menaquinones are known as vitamin K2; they are important cofactors in the posttranslational conversion of glutamic acid residues of vitamin K-dependent proteins. The Menaquinones have important functions in the aspects of blood coagulation, osteoporosis prevention, diabetes prevention, cardiovascular calcification alleviation, inflammation inhibition and the like. Therefore, vitamin K2 is widely used as a pharmaceutical therapy and dietary supplement in the food, health and pharmaceutical industries.
Chemical synthesis of MK-4 is challenging because the all-trans configuration of MK-4 is biologically active. Microbial production of MK-4 has the advantage of selective production of the all-trans isomer; therefore, researchers have used wild-type Bacillus subtilis natto and Flavobacterium sp.238-7-K3-15 strains to produce MK-4. Bacillus subtilis is Generally Recognized As Safe (GRAS), has a fast growth rate, good genetic characteristics, synthetic biological tools and a specialized library.
Therefore, we chose Bacillus subtilis as a platform microorganism for rational route engineering to increase MK-4 production. Bacillus subtilis168 has considerable potential for the production of MK-4 in metabolic engineering. Therefore, how to utilize Bacillus subtilis to synthesize MK-4 through metabolic engineering and fermentation becomes a problem which needs to be solved by the technical personnel in the field.
Disclosure of Invention
In order to solve the technical problems, the invention provides the recombinant bacillus subtilis for synthesizing MK-4 and the construction method and the application thereof, and the constructed recombinant bacillus subtilis can efficiently synthesize MK-4.
The first purpose of the invention is to provide a recombinant bacillus subtilis for synthesizing MK-4, wherein the recombinant bacillus subtilis takes bacillus subtilis as a host, integrates and expresses menA, menG and crtE genes derived from Synechocystis sp.PCC 6803 on a genome, and knocks out a hepT gene; and enhancing the expression of one or more of MEP pathway genes dxs, dxr or ispD-ispF on the genome.
Further, the amino acid sequence expressed by the menA gene is shown as SEQ ID NO. 1; the amino acid sequence expressed by the menG gene is shown as SEQ ID NO. 2; the amino acid sequence of crtE gene expression is shown in SEQ ID NO. 3.
Furthermore, the Gene ID of the hepT Gene is 939002.
Furthermore, the amino acid sequence expressed by the dxs gene is shown in SEQ ID NO. 4; the amino acid sequence expressed by the dxr gene is shown as SEQ ID NO. 5; the amino acid sequence of ispD-ispF gene expression is shown in SEQ ID NO. 6.
Further, the bacillus subtilis is bacillus subtilis 168.
The second purpose of the invention is to provide a construction method of the recombinant bacillus subtilis, which comprises the following steps:
(1) Construction of a plasmid containing a homologous arm of the dacA gene, the menA gene and P by fusion PCR 43 A promoter, a recombinant fragment of a bleomycin resistance gene sequence; transforming the recombinant fragment into Bacillus subtilis168 to obtain a menA gene-containing strain;
(2) Construction of a plasmid containing the homologous arm of the ganA gene, the menG gene, P by fusion PCR 43 A recombinant fragment of a promoter, chloramphenicol resistance gene sequence; transforming the recombinant fragment into the menA gene-containing strain in the step (1) to obtain a recombinant strain BY01;
(3) Construction of a plasmid containing the SacB Gene homology arm, the crtE Gene, and P by fusion PCR 43 A promoter, a recombinant fragment of a spectinomycin resistance gene sequence; transforming the recombinant fragment into a recombinant strain BY01 to obtain a recombinant strain BY02;
(4) Constructing a recombinant fragment containing upstream and downstream homology arms of the hepT gene and a bleomycin resistance gene sequence by fusion PCR; transforming the recombinant fragment into a recombinant strain BY02 to obtain a recombinant strain BY03;
(5) Construction of upstream and downstream homology arms containing terminator, dxs Gene, P by fusion PCR 43 A recombinant fragment of a promoter, a chloramphenicol resistance gene sequence;
(6) Construction of upstream and downstream homology arms containing terminator, dxr gene, P by fusion PCR 43 A promoter, a recombinant fragment of a spectinomycin resistance gene sequence;
(7) Construction of a Gene construct containing upstream and downstream homology arms of terminator, ispD-sipF, P by fusion PCR 43 A promoter, a recombinant fragment of a bleomycin resistance gene sequence;
(8) And (3) transforming one or more of the recombinant fragments obtained in the steps (5), (6) and (7) into a recombinant strain BY03, and carrying out the recombinant Bacillus subtilis.
The third purpose of the invention is to provide the application of the recombinant bacillus subtilis in synthesizing MK-4.
Further, the application specifically comprises the following steps: inoculating the recombinant bacillus subtilis into a seed culture medium, culturing at 35-38 ℃ and 200-250 rpm for 10-12 h to obtain a seed solution, inoculating the seed solution into a fermentation culture medium, culturing at 38-42 ℃ and 200-250 rpm for 6-8 days, and separating the fermentation broth to obtain MK-4.
Further, the seed culture medium comprises: 8-12 g/L peptone, 4-6 g/L yeast extract and 8-12 g/L NaCl.
Further, the fermentation culture medium consists of 4-6% (w/v) glucose and 0.05-0.07% (w/v) KH 2 PO 4 4-6% (w/v) soybean peptone and 4-6% (w/v) glycerol.
The invention has the beneficial effects that:
the invention constructs a recombinant Bacillus subtilis for synthesizing MK-4, which takes Bacillus subtilis168 as a host, integrates menA, menG and crtE genes derived from blue-green algae Synechocystis sp.PCC 6803, and knocks out hepT. And dxs, dxr and ispD-ispF genes are integrated on the chromosome of the recombinant strain, and the expression of MEP pathway key genes dxs, dxr and ispD-ispF is enhanced. The MK-4 synthesized by the recombinant bacillus subtilis has the extracellular accumulation amount of 78mg/L, and lays a foundation for further metabolic engineering modification of the bacillus subtilis to produce MK-4.
Drawings
FIG. 1 shows the major metabolic pathways in B.subtilis associated with MK-4 biosynthesis.
FIG. 2 is a diagram showing enhancement of Bacillus subtilis MK-4 biosynthesis by the menadione synthetic pathway; (a) menA and menG from Synechocystis sp.pcc 6803 were overexpressed on the b.subtilis genome, yielding the BY01 strain; the crtE gene from Synechocystis sp.pcc 6803 was integrated into the BY01 gene to generate BY02 strain; (b) shake flask fermentation results of BY01 strain; (c) growth curves of BY01, BY02 strains; (d) shake flask fermentation result of BY02 strain.
FIG. 3 knock-out of hepT gene increases MK-4 biosynthesis at B.subtilis; (a) knocking out hepT gene in BY02 strain; (b) BY03 shake flask fermentation result.
FIG. 4B enhancement of the MEP pathway in the subtilis metabolic pathway increases MK-4 production; (a) representation of overexpression on the BY03 genome; (b) Shake flask fermentation results of BY03, BY04, BY05, BY06, BY07, BY08, BY09, and BY10 strains.
Detailed Description
The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can carry out the present invention, but the embodiments are not to be construed as limiting the present invention.
Extraction and HPLC detection of MK-4: adding a mixture of isopropanol and n-hexane (1. The supernatant was collected, MK-4 was dissolved in this phase, frozen in a freezer at-80 ℃ to remove the lipid crystals, the filtrate was collected and assayed for MK-4 content by HPLC. MK-4 production by HPLC: using an Agilent ZORBAX eclipseXDB-C18 separation column (5 μm, 250X 4.6 mm), the temperature was measured at 40 ℃ and the mobile phase was purified using methanol: dichloromethane (9, v/v), flow rate of 1mL/min, detection wavelength 254nm, sample size 10 u L.
Example 1: construction of recombinant fragment containing menA gene
Upstream homology arm primers with sequences of P1 and P2 are respectively designed by taking a Bacillus subtilis168 genome as a template according to a dacA Gene sequence (Gene ID: 940000) published on NCBI:
P1:TCATTGAGTTCCCGAACTCATCTAAAG
P2:GAAATTGTTATCCGCTCTCGTACGACCTCCGTATTTCATTTTCTTCA TCT
a plasmid containing a promoter and a bleomycin resistance gene is used as a template, and homologous arm primers with sequences of P3 and P4 are respectively designed:
P3:CGGAGGTCGTACGAGAGCGGATAACAATTTCACACAGGAAAC
P4:GCGGGCTAGATTCTGTCATGTGTACATTCCTCTCTTACCTATAATGG T
taking the menA gene as a template, designing homologous arm primers with sequences of P5 and P6 respectively:
P5:GTAAGAGAGGAATGTACACATGACAGAATCTAGCCCGCTGGCTCC
P6:CCATCAGAGCTCTTTCAATTGATTATCCAAGTCCAGCCCATCCATAT CC
taking a bacillus subtilis genome as a template, respectively designing downstream homology arm primers with sequences of P7 and P8:
P7:GGCTGGACTTGGATAATCAATTGAAAGAGCTCTGATGGATGTTAG G
P8:TGACAACTCAGCGATTAATTTGTAATCAGTAAAG。
the gene fragment amplified by using the above primer. And (3) carrying out column recovery after gel running verification on the left homologous arm of the dacA gene, the right homologous arm of the dacA gene, the ZP43 fragment and the menA gene fragment. Fusing the left homologous arm of the dacA gene, the right homologous arm of the dacA gene, the ZP43 fragment and the menA gene fragment by a fusion PCR technology. The first round of PCR conditions were: equimolar amounts of DNA recovered on the column, total amount greater than 1000ng, primer star enzyme amount 25. Mu.L, plus ddH 2 O constant volume is 50 mu L, PCR conditions are 55 ℃ and 11 cycles. Second round PCR conditions: and (3) taking the PCR product as a template, taking sequences shown by P1 and P8 as upstream and downstream primers respectively, obtaining a recombinant fragment according to conventional PCR setting conditions, and transferring the recombinant fragment into Bacillus subtilis168 to obtain a menA gene-containing strain.
Example 2: construction of recombinant fragment containing menG Gene
Upstream homology arm primers with sequences P9 and P10 were designed based on ganA Gene sequence published at NCBI (Gene ID: 936313) using Bacillus subtilis genome as template:
P9:TAAATTGACAATGCAGTCCAGCCAATATTC
P10:GTGAAATTGTTATCCGCTCATTCTCCTCCTTGTTCTCTTAGCCCTT
a plasmid containing a promoter and a chloramphenicol resistance gene is used as a template, and homologous arm primers with sequences of P11 and P12 are respectively designed:
P11:GCTAAGAGAACAAGGAGGAGAATGAGCGGATAACAATTTCACAC AGGAAA
P12:AGCAGGCTATTTGACATGTGTACATTCCTCTCTTACCTATAATGGT ACCG
taking the menG gene as a template, designing homologous arm primers with sequences of P13 and P14 respectively:
P13:AGGTAAGAGAGGAATGTACACATGTCAAATAGCCTGCTTACACA ACCGAC
P14:GCGGAGCATCAGCTTATTTTTCTGCGACCAGCACTCCCATT
using a bacillus subtilis genome as a template, respectively designing homologous arm primers with sequences of P15 and P16:
P15:GGTCGCAGAAAAATAAGCTGATGCTCCGCTCGATATGGGCGGA
P16:ATTTCCATGCCCATCGCCATCCTCAGC。
the gene fragment amplified by using the above primer. And (3) running glue on the left homologous arm of the ganA gene, the right homologous arm of the ganA gene, the CP43 fragment and the menG gene fragment to verify the correctness, and then carrying out column recovery. And fusing the left homologous arm of the ganA gene, the right homologous arm of the ganA gene, the CP43 fragment and the menG gene fragment by a fusion PCR technology. The first round of PCR conditions were: equimolar amounts of DNA recovered on the column, total amount greater than 1000ng, primer star enzyme amount 25. Mu.L, plus ddH 2 O constant volume is 50 mu L, PCR conditions are 55 ℃ and 11 cycles. Second round PCR conditions: and (3) taking the PCR product as a template, taking sequences shown BY P9 and P16 as upstream and downstream primers respectively, obtaining a recombinant fragment according to conventional PCR setting conditions, and transferring the recombinant fragment into a menA gene-containing strain to obtain a recombinant strain BY01.
Example 3: construction of recombinant fragment containing crtE gene
Using Bacillus subtilis genome as template, designing homology arm primers with sequences P17 and P18 according to sacB Gene sequence published on NCBI (Gene ID: 936413):
P17:GCTGTACATCCAGCCTTCATTCGGTTCA
P18:GTGAAATTGTTATCCGCTCCGTTCATGTCTCCTTTTTTATGTACTGT
a plasmid containing a promoter and a spectinomycin resistance gene is used as a template, and homologous arm primers with sequences of P19 and P20 are respectively designed:
P19:AAAAGGAGACATGAACGGAGCGGATAACAATTTCACACAGGAA AC
P20:GTCTGTTGAGCCACCATGTGTACATTCCTCTCTTACCTATAATGGT A
using crtE gene as a template, designing homology arm primers with sequences of P21 and P22 respectively:
P21:AAGAGAGGAATGTACACATGGTGGCTCAACAGACAAGAACAGA TT
P22:CATTTTCTTTTGCGTTTTTAATATTTGCGCGCCACGATATATTCC
using a bacillus subtilis genome as a template, respectively designing homologous arm primers with sequences of P23 and P24:
P23:TCGTGGCGCGCAAATATTAAAAACGCAAAAGAAAATGCCGATAT CCTA
P24:CCGCTTTTCTAATGGATCTGTGCTTTTG。
the gene fragment amplified by using the above primer. And (3) carrying out column recovery after the gel running verification of the left homologous arm of the sacB gene, the right homologous arm of the sacB gene, the SP43 fragment and the crtE gene fragment is correct. And fusing the left homologous arm of the sacB gene, the right homologous arm of the sacB gene, the SP43 fragment and the crtE gene fragment by a fusion PCR technology. The first round of PCR conditions were: equimolar amounts of DNA recovered on the column, total amount greater than 1000ng, primer star enzyme amount 25. Mu.L, plus ddH 2 O constant volume is 50 mu L, PCR conditions are 55 ℃ and 11 cycles. Second round PCR conditions: taking the PCR product as a template, taking sequences shown BY P17 and P24 as upstream and downstream primers respectively, obtaining a recombinant fragment according to conventional PCR setting conditions, and transferring the recombinant fragment into a strain BY01 to obtain a recombinant strain BY02.
Example 4: construction of recombinant knockout fragment hepT
Using Bacillus subtilis genome as template, designing homology arm primers with sequences of P25 and P26 according to hepT Gene (Gene ID: 939002) published on NCBI and adjacent Gene sequences:
P25:TGATCGTCATTCTGTCCAATGTTTCATT
P26:CCTGTGTGAAATTGTTATCCGCTCTTTTTGTAGATATTAAGAAATT TCTCCGCCCC
plasmid containing promoter and bleomycin resistance gene is used as template, and homology arm primers with sequences of P27 and P28 are respectively designed:
P27:AATATCTACAAAAAGAGCGGATAACAATTTCACACAGGAAACA
P28:GCATATCGGATGGAAATGAGTGTACATTCCTCTCTTACCTATAATG GT
using a bacillus subtilis genome as a template, respectively designing homologous arm primers with sequences of P29 and P30:
P29:GGTAAGAGAGGAATGTACACTCATTTCCATCCGATATGCGTGGCA
P30:ATTCCATCGTGTGGCGGAACACTTCAACCTC。
the gene fragment amplified by using the above primer. And (3) running glue on the left homologous arm of the hepT gene, the right homologous arm of the hepT gene and the ZP43 fragment to verify the correctness, and then carrying out column recovery. The left homology arm of hepT gene, the right homology arm of hepT gene and ZP43 fragment are fused by fusion PCR technology. The first round of PCR conditions were: equimolar amounts of DNA recovered in the column, in a total amount of more than 1000ng, primer star enzyme 25. Mu.L, plus ddH 2 O constant volume is 50 mu L, PCR conditions are 55 ℃ and 11 cycles. Second round PCR conditions: taking the PCR product as a template, taking sequences shown BY P25 and P30 as upstream and downstream primers respectively, obtaining a recombinant fragment according to conventional PCR setting conditions, and transferring the recombinant fragment into a strain BY02 to obtain a recombinant strain BY03.
Example 5: construction of recombinant fragment containing dxs Gene
Using a bacillus subtilis genome as a template, and respectively designing homology arm primers with sequences of P31 and P32 according to a bacillus subtilis genome sequence:
P31:CGTCTCTTCATTGGAGTTGTCATCCATAAT
P32:CTGTGTGAAATTGTTATCCGCTCTTATCTTGGCGAATAGGACGCCA TGGA
taking plasmids containing promoters and chloramphenicol resistance genes as templates, designing homologous arm primers with sequences of P33 and P34 respectively:
P33:ATTCGCCAAGATAAGAGCGGATAACAATTTCACACAGGAAACA
P34:GGTCCTGTATTGATAAAAGATCCATGTGTACATTCCTCTCTTACCT ATAATGGTACCG
using dxs gene as a template, designing homology arm primers with sequences of P35 and P36 respectively:
P35:AGGTAAGAGAGGAATGTACACATGGATCTTTTATCAATACAGGAC CCGTCG
P36:GGCAGTCTGACAAGTTATTCTGCAATAGTCATGATCCAATTCCTTT GTGTGTCTTTGGTG
using a bacillus subtilis genome as a template, respectively designing homologous arm primers with sequences of P37 and P38:
P37:AATTGGATCATGACTATTGCAGAATAACTTGTCAGACTGCCGGGA AATCCCGGCAGTCTTTTTTCCATTAAAACACGGCTTATAGATATTTGACGG CTGCGACAACG
P38:GTCGTGGACATCGCCATGAAAAAATTCAGCA。
the gene fragment amplified by using the above primer. And (3) running gel on the left homologous arm, the right homologous arm, the CP43 fragment and the dxs gene fragment of the insertion site to verify the correctness, and then performing column recovery. Fusing the homologous arm on the left side of the insertion site, the homologous arm on the right side of the insertion site, the CP43 fragment and the dxs gene fragment by a fusion PCR technology. The first round of PCR conditions were: equimolar amounts of DNA recovered on the column, the total amount being greater than 1000ng, 25. Mu.L of primer star enzyme, plus ddH 2 O constant volume is 50 mu L, PCR conditions are 55 ℃ and 11 cycles. Second round PCR conditions: and (3) taking the PCR product as a template, taking sequences shown BY P31 and P38 as upstream and downstream primers respectively, obtaining a recombinant fragment according to conventional PCR setting conditions, and transferring the recombinant fragment into a strain BY03 to obtain a recombinant strain BY04.
Example 6: construction of recombinant fragment containing dxr gene
Respectively designing homology arm primers with sequences of P39 and P40 by taking a bacillus subtilis genome as a template according to a bacillus subtilis genome sequence:
P39:GCTGATTGCTTGCGCCAGTGACGGTATCGC
P40:CTGTGTGAAATTGTTATCCGCTCCTACCACACAAACAGGCCGACA ATCGCA
a plasmid containing a promoter and a spectinomycin resistance gene is used as a template, and homologous arm primers with sequences of P41 and P42 are respectively designed:
P41:TGTTTGTGTGGTAGGAGCGGATAACAATTTCACACAGGAAACAG
P42:CCTGTTGCTCCTAAAAGACAAATATTTTTCATGTGTACATTCCTCT CTTACCTATAATGGTACCG
using dxr gene as template, designing homologous arm primers with sequences of P43 and P44 respectively:
P43:GAGAGGAATGTACACATGAAAAATATTTGTCTTTTAGGAGCAACA GGAT
P44:AAAAAAGACTGCCGGGATTTCCCGGCAGTCTGACAAGTTATTCT GCAATAGTTATGTGAGTATTGAATTGACGTATCCCCG
taking a bacillus subtilis genome as a template, respectively designing homology arm primers with sequences of P45 and P46:
P45:GCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCTTAT TGATAAATAGAGGCTGGCACCTGC
P46:CGCCAAAGCGTATCCGCAGCAAAAGCTG。
the gene fragment amplified by using the above primer. And (4) running glue on the left homologous arm, the right homologous arm, the SP43 fragment and the dxr gene fragment of the insertion site to verify the correctness, and then performing column recovery. Fusing the homologous arm on the left side of the insertion site, the homologous arm on the right side of the insertion site, the SP43 fragment and the dxr gene fragment by a fusion PCR technology. The first round of PCR conditions were: equimolar amounts of DNA recovered on the column, the total amount being greater than 1000ng, 25. Mu.L of primer star enzyme, plus ddH 2 O constant volume is 50 mu L, PCR conditions are 55 ℃ and 11 cycles. Second round PCR conditions: and (3) taking the PCR product as a template, taking sequences shown BY P39 and P46 as upstream and downstream primers respectively, obtaining a recombinant fragment according to conventional PCR setting conditions, and transferring the recombinant fragment into a strain BY03 to obtain a recombinant strain BY05.
Example 7: construction of recombinant fragment containing ispD-ispF Gene
Taking a bacillus subtilis genome as a template, and respectively designing homology arm primers with sequences of P47 and P48 according to the sequence of the bacillus subtilis genome:
P47:TTCATAGACATTTGAACACAAGCTGAAATTATCATACG
P48:TGTTTCCTGTGTGAAATTGTTATCCGCTCTTATATGCGCCCAGTGA GCCTTTGCAA
the plasmid containing the promoter and the bleomycin resistance gene is used as a template, and homologous arm primers with sequences of P49 and P50 are respectively designed:
P49:GCGCATATAAGAGCGGATAACAATTTCACACAGGAAACAG
P50:CTGCAGGAATCACCACATCATAACTCATGTGTACATTCCTCTCTTA CCTATAATGGT
using ispD-ispF gene as a template, designing homology arm primers with sequences of P51 and P52 respectively:
P51:GGAATGTACACATGAGTTATGATGTGGTGATTCCTGCAG
P52:CCGGGATTTCCCGGCAGTCTGACAAGTTATTCTGCAATAGTTAGC CTTTTTGTATCAGTACTGTCGCCTGA
using a bacillus subtilis genome as a template, respectively designing homologous arm primers with sequences of P53 and P54:
P53:TTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAA CACGGCTTATGATTCTCGTTCAGACAAAAGCTCT
P54:AAGTATTTTAGGGATAAAGTATATAAGACAGTTATTCCGC。
the gene fragment amplified by using the above primer. And (3) running glue on the left homologous arm, the right homologous arm, the ZP43 fragment and the ispD-ispF gene fragment of the insertion site to verify the correctness, and then carrying out column recovery. The homologous arm on the left side of the insertion site, the homologous arm on the right side of the insertion site, the ZP43 fragment and the ispD-ispF gene fragment are fused by a fusion PCR technology. The first round of PCR conditions were: equimolar amounts of DNA recovered on the column, total amount greater than 1000ng, primer star enzyme amount 25. Mu.L, plus ddH 2 O constant volume is 50 mu L, PCR conditions are 55 ℃, and 11 cycles are carried out. Second round PCR conditions: and (3) taking the PCR product as a template, taking sequences shown BY P47 and P54 as upstream and downstream primers respectively, obtaining a recombinant fragment according to the conventional PCR setting conditions, and transferring the recombinant fragment into a strain BY03 to obtain a recombinant strain BY06.
Example 8:
the dxs-containing recombinant fragment (obtained in example 5) was transferred to the BY05 strain (obtained in example 6) to obtain a recombinant strain BY07. The recombinant fragment containing ispD-ispF (example 7) was transferred into BY04 strain (example 5) to obtain recombinant strain BY08. The recombinant fragment containing ispD-ispF (example 7) was transferred into BY05 strain (example 6) to obtain recombinant strain BY09. The dxs recombinant fragment (example 5) was transferred into BY09 strain to obtain recombinant strain BY10.
Example 9: production of MK-4 by horizontal fermentation in shake flask
The recombinant bacillus subtilis synthesizes MK-4. All strains were subjected to genetic experiments in liquid LB medium (10.0 g peptone, 5.0g yeast extract, 10.0g NaCl per liter) at 37 ℃ with a rotation speed of 220rpm. The fermentation medium consists of 5% (w/v) glucose and 0.06% (w/v) KH 2 PO4, 5% (w/v) Soy peptone and 5% (w/v) glycerol. The medium was sterilized at 121 ℃ for 20 minutes. All strains were cultured in 2mL LB at 37 ℃ with shaking at 220rpm for 11h. Seeds (2 mL) were inoculated into a 250mL shake flask containing 20mL of fermentation medium and incubated at 40 ℃ at 220rpm for 7 days.
The menA and menG genes from Synechocystis sp.PCC 6803 were integrated into the B.subtilis 168 genome to obtain strain BY01 with a yield of 6.1 + -0.15 mg/L (FIG. 2 b). However, in Bacillus subtilis BY01, crtE was integrated into the sacB site of the chromosome of strain BY01, resulting in 8.1. + -. 0.2mg/L production BY strain BY02 (FIG. 2 d). As shown in FIG. 2c, the expression of crtE gene had less effect on cell growth than BY01. Notably, the MK-4 content in the pellet was almost the same as in the supernatant (FIG. 2 d). The enzyme encoded by HepT plays an important role in catalyzing the conversion of FPP to HDP, which competes with the MK-4 synthetic pathway. By knocking out the hepT gene (FIG. 3 a), MK-4 production was increased 3.8 fold to 31.53 + -0.95 mg/L (FIG. 3 b). These results indicate that overexpression of key enzymes in the synthetic pathway and deletion of enzymes in the competing branched pathway in the biosynthetic pathway can significantly increase the production of MK-4. Deletion of the hepT gene reduces consumption of FPP, which can be used to synthesize GGPP, a precursor of MK-4.
BY over-expressing dxs gene in the strain BY03, the MK-4 yield of the strain BY04 is increased BY 106.3 percent and reaches 65 +/-1.8 mg/L. The BY05 strain, which overexpresses dxr in strain BY03, showed an increase in MK-4 production of 58.2. + -. 1.2mg/L, which was 84.7% (FIG. 4 b). The yield of strain BY06 overexpressing ispD-ispF gene in strain BY03 reached 55.5. + -. 1.1mg/L, which is 76.1% greater than BY03 (FIG. 4 b).
Combined overexpression of the dxs, dxr, ispD-ispF genes further promoted MK-4 synthesis (FIG. 4 a). When both genes are expressed simultaneously. As shown in FIG. 4b, MK-4 production increased to 66. + -. 1.8mg/L (BY 07, dxs and dxr gene overexpression), 72. + -. 1.4mg/L (BY 08, dxs and ispD-ispF gene overexpression), 63. + -. 1.1mg/L (BY 09, dxr and ispD-ispF gene overexpression). Furthermore, when 3 genes were simultaneously overexpressed (FIG. 4 b), the production of MK-4 increased to 78. + -. 1.6mg/L (overexpression of the BY10, dxs, dxr and ispD-ispF genes). Thus, overexpression of the dxs, dxr, and ispD-ispF genes promotes MK-4 synthesis. These results indicate that insufficient supply of IPP is indeed the rate-limiting step in MK-4 synthesis, and that overexpression of enzymes in the MEP pathway contributes to increased MK-4 production.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
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Claims (8)
1. The recombinant bacillus subtilis for synthesizing MK-4 is characterized in that the recombinant bacillus subtilis takes bacillus subtilis as a host, and the integrated expression of the bacillus subtilis on a genome comes fromSynechocystis sp. PCC 6803IsmenA、menG、crtEGene and knock outhepTA gene; and enhanced expression of MEP pathway genes on the genomedxs、dxrOrispD- ispFOne or more of (a);
saidmenAThe amino acid sequence of the gene code is shown in SEQ ID NO. 1; menGthe amino acid sequence of the gene code is shown as SEQ ID NO. 2;crtEthe amino acid sequence of the gene code is shown in SEQ ID NO. 3;
saiddxsThe amino acid sequence of the gene code is shown in SEQ ID NO. 4;dxrthe amino acid sequence of the gene code is shown as SEQ ID NO. 5;ispD-ispFthe amino acid sequence of the gene code is shown in SEQ ID NO. 6.
2. According to the claimThe recombinant Bacillus subtilis according to claim 1, wherein the recombinant Bacillus subtilis ishepTGene ID is 939002.
3. The recombinant Bacillus subtilis of claim 1, wherein the Bacillus subtilis is Bacillus subtilis 168.
4. A construction method of the recombinant Bacillus subtilis of any one of claims 1 to 3, which is characterized by comprising the following steps:
(1) Obtaining a fusion PCR construct comprisingdacAA gene homology arm,menAGene, P 43 A promoter, a recombinant fragment of a bleomycin resistance gene sequence; transformation of the recombinant fragmentBacillus subtilis168, obtaining a mixture containingmenAA genetic strain;
(2) Obtaining a fusion PCR construct comprisingganAA gene homology arm,menGGene, P 43 A recombinant fragment of a promoter, a chloramphenicol resistance gene sequence; transforming the recombinant fragment into the recombinant fragment of step (1) containingmenAGene strain to obtain recombinant strain BY01;
(3) Obtaining a fusion PCR construct comprisingsacBA gene homologous arm,crtEGene, P 43 A promoter, a recombinant fragment of a spectinomycin resistance gene sequence; transforming the recombinant fragment into a recombinant strain BY01 to obtain a recombinant strain BY02;
(4) Obtaining a fusion PCR construct comprisinghepTThe upstream and downstream homology arms of the gene and the recombinant fragment of the bleomycin resistance gene sequence; transforming the recombinant fragment into a recombinant strain BY02 to obtain a recombinant strain BY03;
(5) Obtaining fusion PCR construction including upstream and downstream homologous arms of terminator,dxsGene, P 43 A recombinant fragment of a promoter, a chloramphenicol resistance gene sequence;
(6) Obtaining a fusion PCR construct comprising upstream and downstream homology arms of a terminator,dxrGene, P 43 A promoter, a recombinant fragment of a spectinomycin resistance gene sequence;
(7) Obtaining fusion PCR construction including upstream and downstream homologous arms of terminator,ispD-sipFGene, P 43 A promoter, a recombinant fragment of a bleomycin resistance gene sequence;
(8) And (3) transforming one or more of the recombinant fragments obtained in the steps (5), (6) and (7) into a recombinant strain BY03 to obtain the recombinant bacillus subtilis.
5. Use of the recombinant Bacillus subtilis of any one of claims 1 to 3 in the synthesis of MK-4.
6. The application according to claim 5, characterized in that it comprises in particular the following steps: inoculating the recombinant bacillus subtilis into a seed culture medium, culturing at 35-38 ℃ and 200-250rpm for 10-12h to obtain a seed solution, inoculating the seed solution into a fermentation culture medium, culturing at 38-42 ℃ and 200-250rpm for 6-8 days, and separating the fermentation liquor to obtain MK-4.
7. The use of claim 6, wherein said seed medium comprises: 8 to 12g/L peptone, 4 to 6g/L yeast extract and 8 to 12g/L NaCl.
8. The use of claim 6, wherein the fermentation medium comprises: 4 to 6% (w/v) glucose and 0.05 to 0.07% (w/v) KH 2 PO 4 4 to 6% (w/v) soybean peptone and 4 to 6% (w/v) glycerin.
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