CN110157749B - Method for synthesizing MK-7 by using bacillus subtilis group response regulation and control system - Google Patents

Abstract

The invention discloses a method for synthesizing MK-7 by using a bacillus subtilis group response regulation and control system, belonging to the field of genetic engineering. The invention carries out truncation mutation on the phosphokinase KinAAnd use of a constitutive promoter P_vegExpression, enabling the cell to produce a spore signal in a nutrient rich medium. The Spo0A system which is regulated by the response of the Phr60-Rap60 population is obtained by applying the method, and the high-efficiency synthesis of MK-7 is realized by dynamically regulating the synthesis way of MK-7, without additional inducer, and the dynamic balance between the high-efficiency synthesis of a target product and the growth of cells is realized. The yields of MK-7 produced by shake flask fermentation of finally obtained Bacillus subtilis recombinant bacteria BS19 and BS20 are respectively as high as 170mg/L and 360mg/L, which are 17.89 times and 37.89 times of Bacillus subtilis 168.

Description

Method for synthesizing MK-7 by using bacillus subtilis group response regulation and control system

Technical Field

The invention relates to a method for synthesizing MK-7 by applying a bacillus subtilis group response regulation and control system, belonging to the field of genetic engineering.

Background

Heptaene menadione (MK-7) is an important fat-soluble vitamin, which is used as a cofactor of gamma-glutamate carboxylase and plays an important role in regulating calcium distribution, promoting skeletal development to reverse osteoporosis, protecting blood vessels, and preventing atherosclerosis and cardiovascular disease response. More attention has been paid to heptamenadione due to its better affinity for the human body and longer half-life.

Bacillus subtilis is commonly used in the production of food enzyme preparations and important nutritional chemicals, and its products are certified by the FDA as "produced regulated as safe" (GRAS) grade. Therefore, the construction of the recombinant bacillus subtilis by using a metabolic engineering means is an effective way for efficiently synthesizing MK-7. However, the MK-7 synthesis pathway is very complex, leading to a major precursor phosphoenolpyruvate PEP flowing to the TCA cycle, which limits chorismate synthesis, and in addition, isopentenyl pyrophosphate is also an important substance for cell wall synthesis, which inhibits cell growth if knocked out; the MEP pathway intermediates 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBPP) and dimethylallyl Diphosphate (DMAPP) are both cytotoxic. How to adjust the supply of metabolic flux of Bacillus subtilis and increase the synthesis of MK-7 is a problem worth in-depth discussion.

Professor of Hanai.T, Kyushu university adopts LuxR population response regulation and control system from Vibrio fischeri (Vibrio fischeri) to realize automatic switching of carbon flow from a central metabolic pathway to an isopropanol pathway in Escherichia coli. The group of professor prather. k, academy of science and technology of ma kenchu, utilized the Esa system from bacterial blight of maize (Pantoea stewartii) to integrate the binding site of EsaR into the promoter region of the e.coli phosphofructokinase (pfkA) gene, inhibiting the transcription of pfkA gene and increasing the yield of the product inositol by 5.5 times. However, these studies use heterogeneous group response systems for regulation, which increases the burden of cell metabolism and weakens the system stability, and if the cell's own group response system is used to dynamically regulate the metabolic network, no additional burden is imposed on the cell metabolism. There have been no studies using dynamic regulation of population response in the study of menatetrenone.

Disclosure of Invention

In order to solve the existing technical problems, the invention constructs a population response dynamic regulation system of Phr60-Rap60-Spo0A and transforms P_spoiiaAnd P_abrbThe promoter is used for obtaining a promoter library with different transcriptional activities under the control of Spo0A-P, and key genes of HepS/T, ispH, pyk and uppS in a heptanenaphthoquinone synthetic pathway are dynamically controlled by the obtained promoter, the yields of MK-7 produced by shake-flask fermentation of finally obtained Bacillus subtilis recombinant bacteria BS19 and BS20 are respectively as high as 170mg/L and 360mg/L which are respectively 17.89 times and 37.89 times of Bacillus subtilis168, the recombinant bacteria BS20 is fermented in a fermentation tank for 3 days, and the yield of MK-7 can reach 230 mg/L.

The first object of the present invention is to provide a method for synthesizing MK-7 using a Bacillus subtilis population response regulatory system, which comprises the following modification on the chromosome of Bacillus subtilis:

(1) carrying out truncation mutation on the gene of the histidine kinase KinA, carrying out constitutive expression, and knocking out the gene of the histidine kinase KinB;

(2) will contain P_hagThe Rap60 gene of the promoter is integrated on a chromosome, and two copies of the expression frame of the signal molecule Phr60 are integrated on the chromosome at the downstream of the Rap60 gene;

(3) using P_abrB(cs-1)The promoter replaces the promoters of the pyruvate kinase gene and the undecenyl pyrophosphate synthetase gene; the P is_abrB(cs-1)The sequence of the promoter is shown in SEQ ID NO. 14.

In one embodiment, the genebank ID of the histidine kinase KinA in step (1) is 939230; the truncation mutation refers to the knocking out of A PAS-A region of A signal conduction part of A histidine kinase KinA gene, and the sequence of the knocked-out histidine kinase KinA gene kinA-deltA PAS-A is shown in SEQ ID NO. 9; the genebank ID of the histidine kinase KinB is 937167.

In one embodiment, said P in step (2)_hagThe promoter sequence is shown as SEQ ID NO. 16; the genebank ID of the transcription factor Rap60 is 1115983; the sequence of the expression cassette of the signal molecule Phr60 is shown in SEQ ID NO. 17.

In one embodiment, the pyruvate kinase gene pyk in step (3) has genebank ID 936596; the genebank ID of the undecenyl pyrophosphate synthetase gene uppS is 939640.

In one embodiment, the Bacillus subtilis is Bacillus subtilis 168.

In one embodiment, the Bacillus subtilis is Bacillus subtilis 168P₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrCP_menadanA, in Bacillus subtilis168On the basis of the promoter P₄₃Natural promoters for replacing menF and menB genes; by P_hbsThe promoter replaces the natural promoter of the menE gene; by containing P₄₃The promoter entC gene replaces the dhbB gene; by P_hbsThe promoter replaces the native promoter of the tkt gene; by containing P₄₃Integrating the ppsA gene of the promoter into a bacillus subtilis chromosome, and knocking out the ptsG gene; will aroG^fbrGenes and promoters P_hbsIntegration into chromosome after fusion; by P₄₃The promoter replaces the native promoter of the aroK gene; by P_hbsThe promoter replaces the native promoter of the ispA gene; by P₄₃The promoter replaces the native promoter of the hepS/T gene; mixing the kdpG gene with promoter P_hbsIntegration into chromosome after fusion; by P₄₃The promoter replaces the natural promoters of dxr gene, dxs gene and fni gene; the open reading frame of menA is integrated and expressed at ganA, thrC and dacA sites of the bacillus subtilis genome, and the finally obtained strain is named as BS 17.

In one embodiment, the genebank ID of the menF is 937190; the genebank ID of menB is 937195; the P is_hbsThe sequence of the promoter is shown as SEQ ID NO. 5; the genbank ID of menE is 937132; the entC gene is derived from e.coli K12, genebank ID is 945511; the genebank ID of the dhbB is 936582; the genebank ID of the tkt gene is 937377; the ppsA is derived from E.coli K12, and the genebank ID of the ppsA is 946209; the genebank ID of the ptsG is 939255; the aroG^fbrThe sequence of the gene is shown as SEQ ID NO. 6; the genebank ID of the aroA is 937853; the genebank ID of the aroK is 938343; the hepS/T, genebank ID is 938998; the genebank ID of the kdPG is 33073472; the genebank ID of dxr is 939636; the genebank ID of dxs is 938609; the genebank ID of the fni is 938985; the sequence of the menA open reading frame is shown as SEQ ID NO. 8; the genebank ID of ganA is 936313; the genebank ID of thrC is 936660; the genebank ID of dacA is 940000.

In one embodiment, the method further comprises using P_{spoiia(cs-1,3)}Promoter expression of heptameric dimerAllyl diphosphate synthase gene and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene; the P is_{spoiia(cs-1,3)}The sequence of the promoter is shown in SEQ ID NO. 12.

In one embodiment, the heptapoly (diallyl-diphosphate) synthase gene HepS/T has genebank ID of 938998; the genebank ID of the 4-hydroxy-3-methylbut-2-enyldiphosphate reductase gene ispH is 937900.

The second purpose of the invention is to provide a recombinant bacterium for producing heptaene menadione, which is characterized in that the chromosome of bacillus subtilis is modified as follows:

(1) carrying out truncation mutation on the gene of the histidine kinase KinA, carrying out constitutive expression, and knocking out the gene of the histidine kinase KinB;

(2) will contain P_hagThe Rap60 gene of the promoter is integrated on a chromosome, and two copies of the expression frame of the signal molecule Phr60 are integrated on the chromosome at the downstream of the Rap60 gene;

(3) using P_abrB(cs-1)The promoter replaces the promoters of the pyruvate kinase gene and the undecenyl pyrophosphate synthetase gene; the P is_abrB(cs-1)The sequence of the promoter is shown in SEQ ID NO. 14.

In one embodiment, the recombinant bacterium is further modified on the basis of BS17 as follows: carrying out truncation mutation on the gene of the histidine kinase KinA and constitutive expression to knock out the gene of the histidine kinase KinB; will contain P_hagThe Rap60 gene of the promoter is integrated on a chromosome, and two copies of the expression frame of the signal molecule Phr60 are integrated on the chromosome at the downstream of the Rap60 gene; finally constructing to obtain a strain Bacillus subtilis168, P₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrC P_mena-menA::dacA P_veg-kinA-ΔPAS-AΔkinB P_hag-Rap60(P_native-Phr60)₂Hag, which is named BS 18.

In one embodiment, the recombinant bacterium is further modified on the basis of BS18 as follows: using P_abrB(cs-1)The promoter replaces the promoters of pyruvate kinase (pyk, genebank ID: 936596) gene and undecenyl pyrophosphate synthetase (uppS, genebank ID: 939640) gene to obtain strain Bacillus subtilis168, P₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroKP_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrC P_mena-menA::dacA P_veg-kinA-ΔPAS-AΔkinB P_hag-Rap60(P_native-Phr60)₂::hagP_abrB(cs-1)-pyk::pyk P_abrB(cs-1) -uppS:: uppS, which is named BS 19.

In one embodiment, the recombinant bacterium is further modified on the basis of BS19 as follows: using P_{spoiia(cs-1,3)}The promoter expresses heptadiallyl diphosphate synthase (HepS/T, genebank ID: 938998) gene and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH, genebank ID: 937900) gene to finally obtain strain Bacillus subtilis168, P₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fniP_mena-menA::ganA P_mena-menA::thrC P_mena-menA::dacA P_veg-kinA-ΔPAS-AΔkinB P_hag-Rap60(P_native-Phr60)₂::hag P_abrB(cs-1)-pyk::pyk P_abrB(cs-1)-uppS::uppS P_{spoiia(cs-1,3)}-ispH::ispH P_{spoiia(cs-1,3)}HepS/T, which is named BS 20.

The third purpose of the invention is to provide a method for producing heptaene menadione, which is to use the recombinant bacterium for fermentation production.

The invention has the beneficial effects that:

(1) by truncation mutation of phosphokinase KinA and use of constitutive promoter P_vegExpression, enabling the cell to produce a spore signal in a nutrient rich medium.

(2) The Spo0A system which is regulated by the response of the Phr60-Rap60 population is obtained by applying the method, and the high-efficiency synthesis of MK-7 is realized by dynamically regulating the MK-7 synthesis pathway.

(3) No extra inducer is needed, and the dynamic balance between the high-efficiency synthesis of the target product and the cell growth is realized.

(4) When the strain obtained by the invention is added with sucrose, the yield of MK-7 is obviously improved.

(5) The yields of MK-7 produced by shake flask fermentation of finally obtained Bacillus subtilis recombinant bacteria BS19 and BS20 are respectively 170mg/L and 360mg/L, which are 17.89 times and 37.89 times of Bacillus subtilis 168. And the recombinant strain BS20 is fermented in a fermentation tank for 3 days, and the yield of MK-7 can reach 230 mg/L.

Biological material

Bacillus subtilis168 of the present invention was purchased from American type culture Collection, and the accession number thereof was ATCC No. 27370.

TABLE 1 genotype of the strains

TABLE 2 sequence listing

Drawings

FIG. 1: phosphokinase KinA structural schematic diagram and truncation mutation.

FIG. 2: the influence of different ratios of the signal molecules Phr60-Rap60 on the Spo0A regulation capability; p_spoiia-GFP: strain BS168-P_spoiia-GFP; rap 60: strain BS17-2-Rap 60-GFP; phr60-Rap 60: the strain BS17-2-Phr60-Rap 60-GFP; 2 Phr60-Rap 60: strain BS17-2- (Phr60) 2-Rap 60-GFP.

FIG. 3: the number of Spo0A binding sites and the change of the sequence of the Spo0A binding sites on the regulation and control ability of the promoter.

FIG. 4: effect of cell concentration on Spo0A regulatory ability.

FIG. 5: phr60-Rap60-Spo0A dynamically regulates the MK-7 synthetic pathway.

FIG. 6: the level of transcription of key genes.

FIG. 7: and (3) carrying out shake flask fermentation on the recombinant bacteria to produce MK-7.

FIG. 8: and fermenting in a recombinant bacterium fermentation tank to produce MK-7.

Detailed Description

Extraction and HPLC detection of MK-7: adding 4 times volume of isopropanol and n-hexane mixture (1:2V/V) into the fermentation liquid, vortex shaking for 30min, filtering to obtain extractive solution, and centrifuging at 8000r/min for 15 min. The supernatant was collected, whereupon MK-7 was dissolved in this phase, frozen in a freezer at-80 ℃ to remove the lipid crystals, the filtrate was collected and assayed for MK-7 content by HPLC.

MK-7 production by HPLC: using an Agilent ZORBAX eclipseXDB-C18 separation column (5 μm, 250X 4.6mm), the temperature was measured at 40 ℃ and the mobile phase was purified using methanol: dichloromethane (9:1, v/v), flow rate 1mL/min, detection wavelength 254nm, sample size 10 uL.

Example 1: construction of recombinant bacterium BS17

(1) Construction of recombinant bacterium BS1

By constitutive promoter P₄₃Replacement of the native promoter of menF on the chromosome of bacillus subtilis to enhance menF isochorismate synthase (a)menF, genebank ID: 937190) expression of the gene. Using a marker-free genetic modification strategy, see the article (Yan, x., Yu, h. -j., Hong, q., Li, s.p.,2008.Cre/lox system and PCR-basedgenome engineering in Bacillus subtilis, appl Environ micro b.74,5556-5562), the specific construction procedure is as follows:

(a) cloning of genes

① Bacillus subtilis168 genome as template, and primer menF_upFOR and menF_upREV amplification to obtain upstream homology arm sequence menF of menF gene_up(the sequence is shown as SEQ ID NO. 1).

② artificially synthesizing a lox71-zeo-lox66 box (the sequence is shown as SEQ ID NO. 2) containing bleomycin gene.

③ Bacillus subtilis168 genome as template, primer P₄₃For and P₄₃Rev amplification to obtain P₄₃A promoter sequence (the sequence is shown as SEQ ID NO. 3).

And fourthly, amplifying by using a bacillus subtilis168 genome as a template and adopting primers menF.FOR and menF.REV to obtain a menF gene segment (the sequence is shown as SEQ ID NO. 4).

(b) Obtaining fusion fragments

Subjecting the four fragments obtained in step (a) to a reaction_upLox71-zeo-lox66 box, P₄₃Carrying out overlap extension PCR on the promoter sequence and the menF gene segment, wherein the PCR conditions are as follows: pre-denaturation at 98 ℃ for 5min, then denaturation at 98 ℃ for 10s and annealing at 55 ℃ for 5s and 72 ℃ for 2min, carrying out 30 cycles in total, cutting gel and recovering the segment with the correct size to obtain the fusion gene segment menF_up-lox71-zeo-lox66-P₄₃-menF。

(c) Homologous recombination

Transforming the fusion fragment obtained in the step (b) into a wild type strain Bacillus subtilis168 competent cell. Because the upstream sequence of the menF existing in the fusion fragment is homologous with the upstream sequence gene of the menF on the chromosome 168 of the bacillus subtilis, the menF gene existing in the fusion fragment is homologous with the menF gene on the chromosome 168 of the bacillus subtilis, and the bleomycin resistance genes zeo and P in the fusion fragment are homologous through homologous recombination₄₃Promoter replacement of subtillisThe menF gene native promoter on chromosome 168 of bacillus sp. The method comprises the following specific steps:

electrically transforming the fusion fragment constructed in the step (b) into competent cells of the bacillus subtilis168, wherein the addition amount of the fusion fragment is 100-300ng, and the electrical transformation conditions are as follows: the voltage is 2.5kV, the electric shock time is 5ms, the mixture is revived at 37 ℃ for 5h and coated with a bleomycin resistant LB plate with the final concentration of 10 mu g/mL, and the mixture is subjected to anaerobic culture at 37 ℃ for 48 h. The positive bleomycin resistance is transformed successfully by bacillus subtilis.

② selecting single colony growing on the plate, performing colony PCR verification with primers BS1YZ. FOR and BS1YZ.REV, replacing, amplifying to obtain fragment with length of 1350 bp., and sequencing to obtain lox71-zeo-lox66-P₄₃The fusion gene successfully replaces the menF gene natural promoter on the chromosome 168 of Bacillus subtilis. Finally knocking out the bleomycin resistance gene zeo through a Cre/lox recombination system, and obtaining a strain Bacillus subtilis168, P after verification is correct₄₃menF, named BS 1.

TABLE 3 primer sequence Listing

(2) Construction of recombinant bacterium BS2

Based on the strain BS1 obtained in step (1), P was used in a similar manner to that of step (1)₄₃The promoter replaces the natural promoter of the dihydroxynaphthalene formate synthetase (menB, genebank ID: 937195) gene on the chromosome of the Bacillus subtilis168, and the strain Bacillus subtilis168, P is obtained after the verification is correct₄₃-menF P₄₃menB, named BS 2.

(3) Construction of recombinant bacterium BS3

Based on the strain BS2 obtained in step (2), P was used in a similar manner to that of step (1)_hbsThe promoter (the sequence is shown as SEQ ID NO. 5) replaces the natural promoter of the O-succinylbenzoic acid-CoA ligase (menE, genebank ID: 937132) gene in the Bacillus subtilis, and the strain Bacillus subtilis168, P is obtained after the verification is correct₄₃-menF P₄₃-menB P_hbsmenE, which was named BS 3.

(4) Construction of recombinant bacterium BS4

On the basis of the strain BS3 obtained in step (3), a strain containing P₄₃The isochorismate synthase (entC, genebank ID: 945511) gene of the promoter derived from E.coli K12 replaces the isochorismate (dhbB, genebank ID: 936582) gene on the chromosome of Bacillus subtilis. The specific construction process is as follows:

(a) obtaining fusion fragments

Respectively amplifying to obtain dhbB gene upstream homologous arm sequences by taking bacillus subtilis168 genome as a template_updhbB downstream homology arm sequence dhbB_down、P₄₃A promoter sequence; artificially synthesizing a lox71-zeo-lox66 box sequence (the sequence is shown as SEQ ID NO. 2); taking E.coli K12 genome as template, amplifying to obtain entC gene sequence, and then separating five segments dhbB_upLox71-zeo-lox66 box, P₄₃Promoter sequence, entC gene sequence, dhbB_downOverlapping extension PCR is carried out on the gene segment to obtain a fusion gene segment dhbB_up-lox71-zeo-lox66-P₄₃-entC-dhbB_down。

(b) Homologous recombination

Transforming the fusion fragment obtained in the step (a) into a BS3 competent cell, knocking out a bleomycin resistance gene zeo in a strain through a Cre/lox recombination system to obtain a strain Bacillus subtilis168, P₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entC Δ dhbB, which is named BS 4.

(5) Construction of recombinant bacterium BS5

Based on the strain BS4 obtained in step (4), P was used in a similar manner to that of step (1)_hbsThe promoter replaces the natural promoter of the transketolase (tkt, genebank ID: 937377) gene on the Bacillus subtilis chromosome, and the strain Bacillus subtilis168, P is obtained after the verification is correct₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbBP_hbs-tkt, which is named BS 5.

(6) Construction of recombinant bacterium BS6

On the basis of the strain BS5 obtained in step (5), the strain contains P₄₃The phosphoenolpyruvate synthase (ppsA, genebank ID: 946209) gene of the promoter derived from E.coli K12 was integrated between the gene of yjeA (genebank ID: 936440) and the gene of yjfA (genebank ID: 939830) on the chromosome of Bacillus subtilis, and the glucose phosphate transporter (ptsG, genebank ID: 939255) gene on the chromosome was knocked out. The specific construction process is as follows:

(a) obtaining fusion fragments

Taking E.coli K12 genome as a template, amplifying to obtain ppsA gene, taking Bacillus subtilis168 genome as a template, respectively amplifying to obtain yjeA gene, yjfA gene and P₄₃A promoter sequence; artificially synthesizing a lox71-zeo-lox66 box sequence (the sequence is shown as SEQ ID NO. 2). Then five fragments, yjeA, lox71-zeo-lox66 box, P₄₃The PCR was performed by overlap extension of ppsA and yjfA to obtain the fusion gene fragment yjeA-lox71-zeo-lox66-P₄₃-ppsA-yjfA。

Respectively amplifying ptsG upstream homology arms ptsG by using genome of bacillus subtilis168 as template_upAnd downstream homology arm ptsG_downTo ptsG_up、lox71-zeo-lox66、ptsG_downPerforming overlap extension PCR to obtain fusion gene fragment ptsG_up-lox71-zeo-lox66-ptsG_down。

(b) Homologous recombination

Fusing the fusion fragment yjeA-lox71-zeo-lox66-P obtained in the step (a)₄₃-ppsA-yjfA and ptsG_up-lox71-zeo-lox66-ptsG_downAll are transformed into BS5 competent cells, then a bleomycin resistance gene zeo in a strain is knocked out through a Cre/lox recombination system, and the strain Bacillus subtilis 168P is obtained after verification is correct₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃ppsA. DELTA. ptsG, which was named BS 6.

(7) Construction of recombinant bacterium BS7

Artificially synthesized aroG is added on the basis of the strain BS6 obtained in the step (6)^fbrGene (sequence shown in SEQ ID NO. 6) and promoterSeed P_hbs(the sequence is shown as SEQ ID NO. 5) is fused and integrated between a stress protein (ytxj, genebank ID: 937308) gene and a 3-deoxy-D-arabinose-heptasulfonate 7-phosphate synthetase enzyme (aroA, genebank ID: 937853) gene on a bacillus subtilis chromosome, and the specific construction process is as follows:

(a) obtaining fusion fragments

Artificially synthesized aroG^fbrGene (sequence shown in SEQ ID NO. 6); respectively amplifying ytxj gene, aroA gene and P by using Bacillus subtilis168 genome as template_hbsA promoter sequence. Then five fragments ytxj, lox71-zeo-lox66, P_hbs、aroG^fbrPerforming overlap extension PCR on aroA to obtain a fusion gene fragment ytxj-lox71-zeo-lox66-P_hbs-aroG^fbr-aroA。

(b) Homologous recombination

Transforming the fusion fragment obtained in the step (a) into a BS6 competent cell, knocking out a bleomycin resistance gene zeo in a strain through a Cre/lox recombination system, and obtaining a strain Bacillus subtilis 168P after verification is correct₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrIt is named BS 7.

(8) Construction of recombinant bacterium BS8

On the basis of the strain BS7 obtained in step (7), P was used in a similar manner to that in step (1)₄₃The promoter replaces the natural promoter of shikimate kinase (aroK, genebank ID: 938343) gene on the Bacillus subtilis chromosome, and the strain Bacillus subtilis 168P is obtained after the verification is correct₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbBP_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃aroK, named BS 8.

(9) Construction of recombinant bacterium BS9

On the basis of the strain BS8 obtained in step (8), P was used in a similar manner to that of step (1)_hbsPromoter replacement of farnesyl on Bacillus subtilis chromosomeThe natural promoter of the diphosphonate synthase (ispA, genebank ID: 938652) gene is verified to be correct to obtain the strain Bacillus subtilis 168P₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbsispA, which was named BS 9.

(10) Construction of recombinant bacterium BS10

On the basis of the strain BS9 obtained in step (9), P was used in a similar manner to that in step (1)₄₃The promoter replaces the natural promoter of heptadiallyl diphosphate synthase (hepS/T, genebank ID: 938998) gene on the Bacillus subtilis chromosome, and the strain Bacillus subtilis 168P is obtained after verification₄₃-menF P₄₃-menB P_hbs-menEP₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/T, named BS 10.

(11) Construction of recombinant bacterium BS11

On the basis of the strain BS10 obtained in the step (10), 2-dehydro-3-deoxy-phosphogluconate aldolase (kdpG, genebank ID: 33073472) gene derived from Zymomonas mobilis (Zymomonas mobilis) and promoter P_hbsAfter fusion, the gene is integrated between urokinase (yclG, genebank ID: 938292) gene and spore germination response protein (gerkA, genebank ID: 938285) gene on the chromosome of the bacillus subtilis, and the specific construction process is as follows:

(a) obtaining fusion fragments

Synthesizing a kdpG gene by using a Zymomonas mobilis (Zymomonas mobilis) genome as a template; respectively amplifying to obtain yclG gene and P gene by using Bacillus subtilis168 genome as template_hbsA promoter sequence and a gerkA gene; artificially synthesizing a lox71-zeo-lox66 box sequence (the sequence is shown as SEQ ID NO. 2). The five fragments yclG, lox71-zeo-lox66 box, P were then inserted_hbsOverlapping extension PCR is carried out on the promoter sequence, the kdpG and the gerkA to obtain a fusion gene fragment yclG-lox71-zeo-lox66-P_hbs-kdpG-gerkA

(b) Homologous recombination

Transforming the fusion fragment obtained in the step (a) into a BS10 competent cell, knocking out a bleomycin resistance gene zeo in a strain through a Cre/lox recombination system, and obtaining a strain Bacillus subtilis 168P after verification is correct₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbr::lox72P₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbskdpG, named BS 11.

(12) Construction of recombinant bacterium BS12

On the basis of the strain BS11 obtained in step (11), P was used in a similar manner to that in step (1)₄₃The promoter replaces the natural promoter of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (dxr, genebank ID: 939636) gene on Bacillus subtilis chromosome, and strain Bacillus subtilis 168P is obtained after verification is correct₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃Dxr, which is named BS 12.

(13) Construction of recombinant bacterium BS13

On the basis of the strain BS12 obtained in step (12), P was used in a similar manner to that of step (1)₄₃The promoter replaces the natural promoter of the 1-deoxyxylulose-5-phosphate synthase (dxs, genebank ID: 938609) gene in the Bacillus subtilis168, and the strain Bacillus subtilis 168P is obtained after the verification is correct₄₃-menF P₄₃-menB P_hbs-menEP₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/TP_hbs-kdpG P₄₃-dxr P₄₃Dxs, which is named BS 13.

(14) Construction of recombinant bacterium BS14

In the step of(13) Based on the obtained strain BS13, P was used in a similar manner to that in step (1)₄₃The promoter replaces the natural promoter of the gene in isopentenyl pyrophosphate isomerase (fni, genebank ID: 938985) on the Bacillus subtilis chromosome, and the strain Bacillus subtilis 168P is obtained after the verification is correct₄₃-menF P₄₃-menB P_hbs-menEP₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/TP_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃Fni, named BS 14.

(15) Construction of recombinant bacterium BS15

Inserting menA reading frame into ganA site of Bacillus subtilis genome based on the strain BS14 obtained in step (14). ganA (genebank ID: 936313) expresses arabinogalactan type I oligomer exohydrolases. The specific construction process is as follows:

(a) cloning of genes

① the upstream homologous arm sequence ganA of ganA gene is obtained by using Bacillus subtilis168 genome as template and adopting primers mena-up and mena-up_up。

② artificially synthesizing a lox71-zeo-lox66 box (the sequence is shown as SEQ ID NO. 2) containing bleomycin gene.

③ using Bacillus subtilis168 genome as template, amplifying open reading frame of 1, 4-dihydroxy-2-naphthoic acid heptapentenyl transferase (menA) by using primers menA-orf.FOR and menA-orf.REV, detecting the amplified gene fragment with agarose gel to 1537bp, cutting the gel and recovering the fragment with correct size to obtain the nAme reading frame sequence P with nucleotide sequence as SEQ ID NO.8_mena-menA。

④ the downstream homologous arm sequence ganA of ganA gene is obtained by taking Bacillus subtilis168 genome as a template and adopting primers menA-down and menA-down_down。

(b) Obtaining fusion fragments

Subjecting the four pieces obtained in step (a) toSegment ganA_upLox71-zeo-lox66 box, P_mena-menA、ganA_downThe gene segment is subjected to overlap extension PCR to obtain a fusion gene segment ganA_up-lox71-zeo-lox66-P_mena-menA-ganA_down。

(c) Homologous recombination

Transforming the fusion fragment obtained in the step (b) into BS14 competent cells, coating the competent cells on a bleomycin-resistant LB plate, selecting single colonies growing on the plate, carrying out colony PCR verification and sequencing. Finally knocking out the bleomycin resistance gene zeo in the strain through a Cre/lox recombination system to finally obtain the strain Bacillus subtilis 168P₄₃-menFP₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbr::lox72P₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_menaganA, which is named as BS 15.

TABLE 4 primer sequence Listing

(16) Construction of recombinant bacterium BS16

On the basis of the strain BS15 obtained in step (15), the reading frame of menA is inserted into the thrC site of the Bacillus subtilis genome by a method similar to the step (15). thrC (genebank ID: 936660) translationally expresses threonine synthase. Obtaining a strain Bacillus subtilis 168P after verification₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbr::lox72P₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_menathrC, which is named as BS 16.

(17) Construction of recombinant bacterium BS17

On the basis of the strain BS16 obtained in step (16), the insertion of the menA reading frame into the dacA site of the Bacillus subtilis genome was performed in a manner similar to that in step (15). dacA (genebank ID: 940000) expresses alanine carboxypeptidase. Obtaining a strain Bacillus subtilis 168P after verification₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbr::lox72P₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrC P_menamenA, dacA, which is named BS 17.

Example 2: truncation mutation of histidine kinase KinA and expression using constitutive promoter

Histidine kinases, KinA and KinB, can phosphorylate themselves only under carbon starvation, then, DNA binding protein Spo0A is phosphorylated and activated, and phosphorylated Spo0A is bound with a promoter, so that the corresponding gene expression is up-regulated or down-regulated, and the ability of regulating the gene expression is exerted. However, efficient synthesis of the desired product requires a continuous supply of available carbon sources. Thus, KinA was obtained by truncating the histidine kinase and using the constitutive promoter P_vegThe mutation method for expressing truncated phosphokinase KinA and truncated PAS-A domain as shown in FIG. 1 enables the continuous expression of histidine phosphokinase KinA.

(1) Obtaining fusion fragments

Respectively amplifying to obtain upstream homology arm sequence kinA of histidine kinase (kinA, genebank ID: 939230) by using Bacillus subtilis168 genome as template_upTruncated histidine kinase gene fragments kinA-deltA PAS-A (the sequence is shown as SEQ ID NO. 9) and P_vegA promoter sequence (shown as SEQ ID NO. 10), artificially synthesizing a lox71-zeo-lox66 box sequence (shown as SEQ ID NO. 2), and then separating four fragments of kinA_upLox71-zeo-lox66 box, P_vegThe promoter sequence and the kinA-deltA PAS-A gene segment are subjected to overlap extension PCR to obtain A fusion gene segment kinA_up-lox71-zeo-lox66-P_veg-kinA-ΔPAS-A。

② homologous recombination

Transforming the fusion fragment obtained in the step ① into the BS17 competent cell obtained in the example 1, coating the cell on a bleomycin resistant LB plate, selecting a single colony growing on the plate, carrying out colony PCR verification and sequencing, finally knocking out the bleomycin resistant gene zeo in the strain through a Cre/lox recombination system to obtain a strain Bacillus subtilis168, P₄₃-menF P₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbr::lox72P₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrC P_mena-menA::dacA P_veg-kinA- Δ PAS-A, named BS 17-1.

Example 3: knockout of histidine kinase KinB

In order to reduce the influence of other histidine phosphokinases on the regulatory system, another major phosphokinase KinB (kinB, genebank ID: 937167) was knocked out to ensure the uniqueness of the regulatory system. On the basis of the recombinant bacterium BS17-1 obtained in example 2, the phosphokinase gene kinB on the chromosome of Bacillus subtilis is knocked out.

(1) Obtaining fusion fragments

Respectively amplifying to obtain kinB by taking bacillus subtilis168 genome as a template_up、kinB_downArtificially synthesizing lox71-zeo-lox66 box sequence, and then dividing three fragments into kinB_upLox71-zeo-lox66 box, kinB_downPerforming overlap extension PCR to obtain fusion gene fragment kinB_up-lox71-zeo-lox66-kinB_down。

② homologous recombination

Transforming the fusion fragment obtained in the step ① into a BS17-1 competent cell, coating the fusion fragment on a bleomycin-resistant LB plate, selecting a single colony growing on the plate, carrying out colony PCR verification and sequencing, and finally knocking out the bacterial strain by a Cre/lox recombination systemThe bleomycin resistance gene zeo finally obtains a strain Bacillus subtilis168, P₄₃-menFP₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbr::lox72P₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrC P_mena-menA::dacA P_veg-kinA- Δ PAS-A Δ kinB, which was named BS 17-2.

Example 4: construction of population response dynamic regulation system of Phr60-Rap60-Spo0A

The phosphorylation level of Spo0A is regulated by histidine kinase KinA, and in the strain BS17-2 obtained in example 3, KinA is subjected to truncation mutation and constitutive expression, and another main phosphokinase KinB is knocked out, so that Spo0A can be subjected to phosphorylation modification under the condition of rich nutrition, and thus the function is achieved. However, the Phr-Rap group response regulation and control system in the bacillus subtilis can only control the phosphorylation level of Spo0A-P, and cannot regulate the phosphorylation degree of KinA, so that KinA has phosphorylation capacity in the early stage of cell growth, which is not beneficial to dynamic regulation and synthesis of menatetrenone, and the solution is that the phosphorylation of KinA can be inhibited in the early stage of cell growth and can be released along with the growth of cells. It is reported that (DOI: 10.1111/mmi.12939) Phr60-Rap60 can inhibit not only the phosphorylation level of Spo0A, but also the phosphorylation level of KinA. Therefore, a Phr60-Rap60 population response system was introduced into the cells. And the proportion relation between the introduced Phr60 and Rap60 is determined through experiments, and the specific process is as follows:

(1) recombinant vector pHT01-P_spoiiaConstruction of GFP

To verify the effect of Phr60-Rap60 on Spo0A-P phosphorylation, P regulated by Spo0A-P was used_spoiiaThe promoter is fused with Green Fluorescent Protein (GFP), and the regulation and control capacity of Spo0A-P is characterized through the change of the fluorescent value.

① primer P_spoiiaFor and P_spoiiaRev expansionPromoter P_spoiia(the sequence is shown as SEQ ID NO. 11), a green fluorescent protein GFP gene (the sequence is shown as SEQ ID NO.15) is obtained by utilizing primers GFP. for and GFP. rev amplification, the two fragments are connected to a pHT01 plasmid through a Gibson multi-fragment assembly technology, and finally, a recombinant vector pHT01-P is obtained_spoiia-GFP。

TABLE 5 primer sequence Listing

(2) On the basis of the recombinant strain BS17-2 obtained in example 3, a heterologous transcription factor Rap60(genebank ID: 1115983) was inserted into the chromosome of Bacillus subtilis.

Obtaining of fusion fragment

Respectively amplifying to obtain an upstream homologous arm sequence Rap60 of Rap60 gene by taking bacillus subtilis168 genome as a template_upRap60 gene fragment and P_hagA promoter sequence (shown as SEQ ID NO. 16), artificially synthesizing a lox71-zeo-lox66 box sequence (shown as SEQ ID NO. 2), and then combining four fragments of Rap60_upLox71-zeo-lox66 box, P_hagOverlapping extension PCR is carried out on the promoter sequence and the Rap60 gene segment to obtain a fusion gene segment Rap60_up-lox71-zeo-lox66-P_hag-Rap60。

② homologous recombination

Transforming the fusion fragment obtained in the step ① into a BS17-2 competent cell, coating the fusion fragment on a bleomycin resistant LB plate, selecting a single colony growing on the plate, carrying out colony PCR verification and sequencing, knocking out a bleomycin resistant gene zeo in a strain through a Cre/lox recombination system, and finally obtaining a strain Bacillus subtilis168, P₄₃-menFP₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbr::lox72P₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::lacA P_mena-menA::thrC P_mena-menA::dacA P_veg-kinA-ΔPAS-AΔkinB P_hag-Rap60, named BS17-2-Rap 60.

(3) On the basis of the recombinant bacteria BS17-2-Rap60 obtained in the step (2), an expression box (shown as SEQ ID NO. 17) of a signal molecule Phr60 with one copy and two copies is not inserted, inserted and inserted into the downstream of a bacillus subtilis chromosome Rap60 gene, and the genebank ID of the Phr60 is 1115984, so that three bacteria BS17-2-Rap60, BS17-2-Phr60-Rap60, BS17-2- (Phr60) × 2-Rap60 are obtained.

(4) The recombinant plasmid pHT01-P constructed in the step (1)_spoiiaRespectively transforming the wild type Bacillus subtilis168 and the three strains obtained in the step (3) by GFP to obtain four strains, which are respectively named as BS168-P_spoiia-GFP，BS17-2-Rap60-P_spoiia-GFP，BS17-2-Phr60-Rap60-P_spoiia-GFP，BS17-2-(Phr60)*2-Rap60-P_spoiia-GFP。

(5) Measurement of GFP fluorescence value

Culturing the 4 strains of bacteria constructed in the step (4) by using an LB culture medium, detecting the change of a fluorescent value by using a 96-well plate through a microplate reader every hour, wherein the fluorescent protein excitation wavelength is as follows: 395nm and the emission wavelength is 509 nm. As shown in FIG. 2, the fluorescence values of the strains expressing only Rap60 are very weak, indicating that Spo0A cannot activate promoter P_spoiiaThe fluorescence intensity of the cells expressing two Phr60 is obviously higher than that of the cells expressing one copy, which shows that the higher the concentration of the signal molecule is, the higher the degree of activating Spo0A phosphorylation is, and the more the promoter expression can be activated. Therefore, the strain BS17-2- (Phr60) × 2-Rap60 is used as the starting strain of the next experiment and is named as BS18, the genotype of which is Bacillus subtilis168, P₄₃-menF P₄₃-menB P_hbs-menEP₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/TP_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrC P_mena-menA::dacAP_veg-kinA-ΔPAS-AΔkinB P_hag-Rap60(P_native-Phr60)₂Hag, used in the subsequent experiments.

Example 5: relationship between Spo0A-P regulatory gene expression and cell concentration

As the invention is based on the relation between the cell concentration and the gene expression intensity, in order to determine the influence of the cell concentration on Spo0A-P, the relation between the constructed population response Phr60-Rap60-Spo0A and the cell growth is detected, and P is used on pHT01 plasmid_spoiiaAnd P_abrbThe promoter expresses GFP protein, and recombinant plasmids pHT01-P are respectively constructed_spoiiaGFP and pHT01-P_abrbGFP, then the recombinant plasmid was transferred into the strain BS18 obtained in example 4, and the change in fluorescence value with cell growth was examined, as shown in FIG. 4, from which it can be seen that the transcription ability of the promoter was controlled by Spo0A-P when the cells grew to around 1.433.

Example 6: obtaining of mutant promoters

(1) Obtaining of Up-regulated promoters

In order to obtain a promoter with different regulatory abilities under the Phr60-Rap60, a promoter P up-regulated by Spo0A-P_spoiia(the sequence is shown as SEQ ID NO. 11) four Spo0A binding sites are respectively mutated to obtain 3 promoters P with different transcription capacities_spoiia(cs-3)、P_spoiia(OA-3)、P_{spoiia(cs-1，3)}These promoters were fused to the GFP gene (sequence shown in SEQ ID NO.15), and the transcriptional capacities of the different promoters were characterized using the change in fluorescence.

The results show that: post-mutation P_{spoiia(cs-1，3)}The transcription ability of the promoter (the sequence is shown as SEQ ID NO. 12) is improved by about 7 times than the original ability, so that the promoter P is added_{spoiia(cs-1，3)}Used for enhancing the expression of key genes in the synthesis pathway of heptaene menadione.

(2) Obtaining of Down regulated promoters

Promoter P to be downregulated by Spo0A-P_abrbThe Spo0A-P binding site on the sequence shown in SEQ ID NO.13 is subjected to conservative sequence mutation to obtain 3 promoters P with different transcription capacities_abrB(OA-2)、P_abrB(cs-1)、P_abrB(cs-2)And will beThese promoters were fused with the GFP gene (sequence shown in SEQ ID NO.15), and the transcriptional capacities of the different promoters were characterized using the change in fluorescence.

The results show that: as shown in FIG. 3, P_abrB(cs-1)(the sequence is shown as SEQ ID NO. 14) the transcription capacity of the promoter is only 18 percent of the original transcription capacity, so that the promoter P_abrB(cs-1)The method is used for inhibiting the expression of key genes of a heptaene menadione competition pathway.

Example 7: construction of recombinant bacterium BS19

To increase MK-7 synthesis, P was used on the basis of recombinant strain BS18_abrB(cs-1)The recombinant strain BS19 was obtained by in situ replacement of the pyruvate kinase (pyk, genebank ID: 936596) gene and the undecenyl pyrophosphate synthetase (uppS, genebank ID: 939640) gene promoter with promoters that gradually limited the flux of carbon to competing pathways as the cells grew. The final transcript levels of the relevant genes are shown in FIG. 6. The specific construction process is as follows:

(1) cloning of genes

① respectively amplifying to obtain pyk gene upstream homologous arm sequences pyk by using Bacillus subtilis168 genome as template_upPyk gene fragment, artificially synthesized P_abrB(cs-1)A promoter sequence (shown as SEQ ID NO. 14) and a lox71-zeo-lox66 box sequence (shown as SEQ ID NO. 2), and then four fragments pyk_upLox71-zeo-lox66 box, P_abrB(cs-1)Overlapping extension PCR is carried out on the promoter sequence and the pyk gene segment to obtain a fusion gene segment pyk_up-lox71-zeo-lox66-P_abrB(cs-1)-pyk。

② respectively amplifying to obtain the upstream homologous arm sequences uppS of uppS gene by taking bacillus subtilis168 genome as a template_upuppS gene fragment, artificially synthesized P_abrB(cs-1)A promoter sequence (shown as SEQ ID NO. 14) and a lox71-zeo-lox66 box sequence (shown as SEQ ID NO. 2), and then four fragments of uppS_upLox71-zeo-lox66 box, P_abrB(cs-1)Overlapping extension PCR is carried out on the promoter sequence and the uppS gene segment to obtain a fusion gene segment uppS_up-lox71-zeo-lox66-P_abrB(cs-1)-uppS。

(2) Homologous recombination

And (2) transforming the fusion fragment obtained in the step (1) into a BS18 competent cell, coating the competent cell on a bleomycin-resistant LB plate, selecting a single colony growing on the plate, carrying out colony PCR verification and sequencing. Finally knocking out the bleomycin resistance gene zeo in the strain through a Cre/lox recombination system to finally obtain the strain Bacillus subtilis168, P₄₃-menFP₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::lacA P_mena-menA::thrCP_mena-menA::dacA P_veg-kinA-ΔPAS-AΔkinB P_hag-Rap60(P_native-Phr60)₂::hag P_abrB(cs-1)-pyk::pyk P_abrB(cs-1)-uppS:: uppS, which is named BS 19.

Example 8: construction of recombinant bacterium BS20

Using a method similar to that of example 7, the recombinant bacterium BS19 obtained in example 7 was supplemented with P obtained in example 6_{spoiia(cs-1,3)}The promoter expresses heptadiallyldiphosphate synthase (HepS/T, genebank ID: 938998) gene and 4-hydroxy-3-methylbut-2-enyldiphosphate reductase (ispH, genebank ID: 937900) gene, resulting in recombinant strain BS20, and allows carbon flux to flow to MK-7 synthesis pathway as the cells grow. The final transcript levels of the relevant genes are shown in FIG. 6.

(1) Obtaining fusion fragments

Respectively amplifying to obtain an upstream homologous arm sequence HepS/T of the HepS/T gene by taking a bacillus subtilis168 genome as a template_upA HepS/T gene fragment, a lox71-zeo-lox66 box sequence (the sequence is shown as SEQ ID NO. 2), and then four fragments of HepS/T_upLox71-zeo-lox66 box, P_{spoiia(cs-1,3)}Performing overlap extension PCR on the promoter sequence and the HepS/T gene segment to obtain a fusion gene segment HepS/T_up-lox71-zeo-lox66-P_{spoiia(cs-1,3)}-HepS/T。

Respectively amplifying to obtain the upstream homologous arm sequences of the ispH genes by taking the bacillus subtilis168 genome as a template_upispH gene fragment, lox71-zeo-lox66 box sequence (shown as SEQ ID NO. 2), and then the four fragments are subjected to ispH_upLox71-zeo-lox66 box, P_{spoiia(cs-1,3)}Carrying out overlap extension PCR on the promoter sequence and the ispH gene segment to obtain a fusion gene segment ispH_up-lox71-zeo-lox66-P_{spoiia(cs-1,3)}-ispH。

(2) Homologous recombination

And (2) transforming the fusion fragment obtained in the step (1) into a BS19 competent cell, coating the competent cell on a bleomycin-resistant LB plate, selecting a single colony growing on the plate, carrying out colony PCR verification and sequencing. Finally knocking out the bleomycin resistance gene zeo in the strain through a Cre/lox recombination system to finally obtain the strain Bacillus subtilis168, P₄₃-menFP₄₃-menB P_hbs-menE P₄₃-entCΔdhbB P_hbs-tkt P₄₃-ppsAΔptsG P_hbs-aroG^fbrP₄₃-aroK P_hbs-ispA P₄₃-hepS/T P_hbs-kdpG P₄₃-dxr P₄₃-dxs P₄₃-fni P_mena-menA::ganA P_mena-menA::thrCP_mena-menA::dacA P_veg-kinA-ΔPAS-AΔkinB P_hag-Rap60(P_native-Phr60)₂::hag P_abrB(cs-1)-pyk::pyk P_abrB(cs-1)-uppS::uppS P_{spoiia(cs-1,3)}-ispH::ispH P_{spoiia(cs-1,3)}HepS/T, which is named BS 20.

Example 9: fermentation of the Strain

The seed culture medium formula (mass percent): tryptone 1%, yeast extract 0.5%, sodium chloride 1%.

The formula (mass percent) of the shake flask fermentation medium is as follows: 3-8% of soybean peptone, 3-8% of glucose, 3-8% of sucrose and KH₂PO₃0.06％。

The fermentation medium formula of the fermentation tank (mass percent): soybean peptone 3-8%, glucose 3-8%, caneSugar 3-8%, KH₂PO₃0.06％。

(1) Preparing a seed solution:

the recombinant bacteria BS 1-BS 17, BS19 and BS20 constructed in examples 1, 7 and 8 were inoculated into seed culture media, and cultured at 37 ℃ and 220rpm for 12 hours to obtain a Bacillus subtilis seed solution.

(2) Shake flask fermentation culture

Transferring the seed solution obtained in the step (1) into a 250mL shake flask with the inoculation amount of 5%, wherein the liquid loading amount is 15 mL. The fermentation conditions were 40 ℃ and the fermentation conditions were 220rpm for 6 days, and the fermentation results are shown in FIG. 7, in which the yield of BS19 reached 170mg/L after 6 days of fermentation, the yield of BS20 reached 360mg/L after 6 days of fermentation, and the MK-7 yield was 37.89 times that of Bacillus subtilis 168.

(3) Fermentation culture

Transferring the seed liquid obtained in the step (1) into a fermentation medium with 15% of inoculation amount, fermenting the recombinant strain BS20 by using a 15-L fermentation tank under the fermentation condition of 40 ℃, the tank pressure of 0.05Mpa, the ventilation amount of 10vvm, stirring at 500rpm, and controlling the concentration of glucose at 5g/L by feeding glucose. The fermentation result is shown in FIG. 8, the yield reaches 230mg/L, and the pH of the fermentation liquid at the later stage of fermentation is alkaline.

TABLE 6 fermentation production of MK-7 by recombinant strains

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> method for synthesizing MK-7 by using Bacillus subtilis group response regulation and control system

<160>37

<170>PatentIn version 3.3

<210>1

<211>846

<212>DNA

<213>Bacillus subtilis 168

<400>1

gcattcatcg tcatatcatc atagctgagc ggagggtttt ccatatcctg cggcgtttgt 60

gagttcccat atttttcgac atcctgatag gaatcctcac tgtcatatgg cgctctgatt 120

tcttcatcat catcaaattc aaattgcccg aatggtgttt cttcttcaat cggacggtct 180

ttggaaacca cgtcctgtga cgaatactcc gcgagggttg tggcagtcgg aagcgcttca 240

agtcgttcga aagggatttc ctttccgctg acttcacaaa tgccatacgt accgttttct 300

atcgccttca atgaatgctc aatgtcccga aggtgctctc tctcatgcaa gtctagagcg 360

atgtctttct cacgctcgta aagttctgtc gcctgatcgc cgggatggtt gtcgtatgcc 420

gaaagctcac cccacgaatc ataaggaaag gctgagttaa gctgaaaatg atcattgtct 480

ttgaaacggt ttaagatatc ttttttcgtt tgttccagtt cattttttaa atgctgaagc 540

tgttctttcg taagcaatgt gatcgcctcg tttctctgtg atgcatacct ttagtatgaa 600

cagatcgcct gagaactttc ataaatggcg ggtggaggaa tataggaggt tttcctttta 660

tggtaagcgg atacaacctt tgctatcagt ggagaaagaa atttaagctt tgtttctttt 720

tcatttctga aattaggttt ataataggta aggcaggcca tttggactgc atgatctgtg 780

tttgacacaa aggagacaca ggtgtatggt attaaaagca ttttaaggat tatggaggaa 840

tgtctc 846

<210>2

<211>588

<212>DNA

<213> Artificial Synthesis

<400>2

gagcggataa caatttcaca caggaaacag ctatgaccat gattacgaat tcgagctcgg 60

tacccgggga tcctctagag ataccgttcg tatagcatac attatacgaa gttatcttga 120

tatggctttt tatatgtgtt actctacata cagaaaggag gaactaaaca tggccaagtt 180

gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 240

cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 300

cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 360

ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 420

gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 480

ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 540

ctgaataact tcgtatagca tacattatac gaacggtaaa tcgtcgac 588

<210>3

<211>300

<212>DNA

<213>Bacillus subtilis 168

<400>3

tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60

taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120

tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180

aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240

gcgattatgt aaaatataaa gtgatagcgg taccattata ggtaagagag gaatgtacac 300

<210>4

<211>847

<212>DNA

<213>Bacillus subtilis 168

<400>4

atggtgacaa cggtgcagcg tacgttccga aaggaagttc tacatgcatt acataaagcc 60

aaagaagtca accatgctgt cttaataagc tattcgagac aaatcgagtc tcttgaccct 120

ctatcatttt tcaattacgg agcaaaaaaa tatacaggca atcgattttt ttggtcagat 180

cctgaaagtg aattgacaat agtcggtctt ggcaaagaag cggttttcca gacaaatcaa 240

aaaaacagcg agcggtatcg tgaggttttt gaacaatggg agcgctttaa aaagacggct 300

tttcatattt atgaagaaga aaagctgcag cattctgcag tgggacctgt gttattcgga 360

ggattttctt ttgacccttg cgaagaaaga ggttcacaat gggaccattt ctcggaaggg 420

gatttctttg tgcctgcgct tatgctgacg atgactgctg aaggcccgtt cttaacagtt 480

aacagatggg taagcggagg agaagacgca gaagctgttt tagaaggctt aaaagctttt 540

gcggcggaat ttatggttcc cgatttcaag caagaagatc aggctgtgat tgcagcagcc 600

gaagagctgg ataaggatga ttggctgaaa gcaatcgaaa cggccacaag ccaaattaaa 660

gagaaacaat atgataaggt tgttcttgcc cgagagctgc tgctcacgtt tgacggtcca 720

atccaaattg aaccggtgct taaaacgctt ctggacgatc agcagacaag ctatgttttt 780

gcaattgaac aagaaggcaa aacctttgtc ggcgcgtctc cggaaagact gatcaaaaga 840

gacggcg 847

<210>5

<211>328

<212>DNA

<213>Bacillus subtilis 168

<400>5

cttaataatg gaaaaggatc aaggaatagg atgaaaaaag gaaaaaaagg aatattcgtt 60

cggtaaatca ccttaaatcc ttgacgagca agggattgac gctttaaaat gcttgatatg 120

gctttttata tgtgttactc tacatacaga aattcttcac tttgttggac aaacattcct 180

cagagtgcag tttttcttaa aaagccgttt aattgtcttt ctcttacttg ctctcatttt 240

tttctgagac aggtttagaa tcagactgaa ctgtgaagaa atgataataa acgaactgaa 300

tgtatccttt tgggaggagg tgaaaggc 328

<210>6

<211>1053

<212>DNA

<213> Artificial Synthesis

<400>6

atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60

gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120

aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180

tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240

gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300

acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360

gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420

gcaggtgagt ttctcgatat gatcaccctg caatatctcg ctgacctgat gagctggggc 480

gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540

tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600

aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660

gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720

tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780

caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840

gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900

gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960

ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020

ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053

<210>7

<211>936

<212>DNA

<213>Bacillus subtilis 168

<400>7

atgaaccaaa caaataaggg tgagggtcag acagcgccgc aaaaagaaag catggggcag 60

atcctttggc agttaacccg tcctcatacg ttaaccgcat cgtttgtgcc tgtgctgctc 120

ggaaccgttt tggcgatgtt ttatgtgaag gttgatctgc tgctgttttt ggctatgctg 180

ttttcttgcc tatggatcca gatcgcgacg aacttattta atgaatacta tgattttaaa 240

cgcggattag atacagcaga atcagtcgga atcggagggg ccattgtacg ccacggaatg 300

aagcctaaaa cgattttgca attagctctt gcctcatacg ggattgccat tttgctcggt 360

gtctatattt gtgccagcag cagctggtgg cttgcgctga tcggccttgt cggcatggcg 420

atcggctacc tgtatacagg cgggccgctg ccgattgcgt acacgccgtt cggtgaatta 480

ttctcaggca tttgcatggg ttcggtgttt gtgctgattt cgtttttcat tcagacagat 540

aagatcaata tgcagagcat tttgatttcc atcccgattg cgattcttgt cggcgcgatt 600

aacctgtcga acaacattcg tgatattgaa gaggacaaaa aaggcggacg caaaacattg 660

gcgattttga tggggcataa gggagctgtt actctgttag ctgcgtcgtt tgccgtcgct 720

tatatctggg ttgtcggctt ggttattacc ggtgccgcaa gcccatggct gtttgtcgtc 780

tttttgagcg tgcctaagcc ggttcaggca gtgaagggct tcgtccagaa cgaaatgccg 840

atgaatatga ttgtcgcaat gaaatcaaca gcccaaacaa atacattttt cggattcctg 900

ctctcgatcg gattattgat cagctatttc cgataa 936

<210>8

<211>1537

<212>DNA

<213>Bacillus subtilis 168

<400>8

cttgacagcg ggtttttcat acagctgttt cattaattgc agcatatcaa aaattccctt 60

cccgtttttt cgacaatcac atgattattt gacgatcaga tgctcaaaaa gtttcatttt 120

ttccctaatc tccctcaaat taatatatcg gcgtatgttt ttgaaaatga atctttatac 180

ctgcaagggc aagaatggaa tgtttcgagc tgattcataa taaaggaaaa gatacctgga 240

gtcaaaagat ctttttaact tttaaaaagt gtaatgtact taaaaaaatt tttctgtacc 300

tgttcgttca agtgaaagca gaaacctaat gaaatcaggg cgagaagcgt ataataagaa 360

agaaaacgcc ttcttgcggt atcttgtgaa ataagggaga agtcattatg atagataagg 420

aagcaaaagg agttttttat taagctgccg gctgaaaaaa agtaagcctt ttaatagaaa 480

ggaagaggaa gatgaaccaa acaaataagg gtgagggtca gacagcgccg caaaaagaaa 540

gcatggggca gatcctttgg cagttaaccc gtcctcatac gttaaccgca tcgtttgtgc 600

ctgtgctgct cggaaccgtt ttggcgatgt tttatgtgaa ggttgatctg ctgctgtttt 660

tggctatgct gttttcttgc ctatggatcc agatcgcgac gaacttattt aatgaatact 720

atgattttaa acgcggatta gatacagcag aatcagtcgg aatcggaggg gccattgtac 780

gccacggaat gaagcctaaa acgattttgc aattagctct tgcctcatac gggattgcca 840

ttttgctcgg tgtctatatt tgtgccagca gcagctggtg gcttgcgctg atcggccttg 900

tcggcatggc gatcggctac ctgtatacag gcgggccgct gccgattgcg tacacgccgt 960

tcggtgaatt attctcaggc atttgcatgg gttcggtgtt tgtgctgatt tcgtttttca 1020

ttcagacaga taagatcaat atgcagagca ttttgatttc catcccgatt gcgattcttg 1080

tcggcgcgat taacctgtcg aacaacattc gtgatattga agaggacaaa aaaggcggac 1140

gcaaaacatt ggcgattttg atggggcata agggagctgt tactctgtta gctgcgtcgt 1200

ttgccgtcgc ttatatctgg gttgtcggct tggttattac cggtgccgca agcccatggc 1260

tgtttgtcgt ctttttgagc gtgcctaagc cggttcaggc agtgaagggc ttcgtccaga 1320

acgaaatgcc gatgaatatg attgtcgcaa tgaaatcaac agcccaaaca aatacatttt 1380

tcggattcct gctctcgatc ggattattga tcagctattt ccgataataa aaaagaccgc 1440

tcgtttcatg cggtcttttt ttgttacaat cgaccgcatt ttgtaaaaaa attcatagaa 1500

ccttgcagca gacagggacg tctagtacat ggacagc 1537

<210>9

<211>1490

<212>DNA

<213> Artificial Synthesis

<400>9

tggaacagga tgaagaagaa acaggccatc aatccctaaa ctgcgaaaaa catgaaatcg 60

aacctgcaag cccggaatcg actacatata taacggatga ttatgaacgg ttggttgaaa 120

atctcccgag tccgctatgc atcagtgtca aaggcaagat cgtctatgta aacagcgcga 180

tgctttcaat gctgggagcc aaaagcaagg atgctattat tggtaaatcg tcctatgaat 240

ttattgaaga agaatatcat gatatcgtga aaaacaggat tatacgaatg caaaaaggaa 300

tggaagtcgg aatgattgaa cagacgtgga aaaggcttga tggcacacct gttcatttag 360

aagtgaaagc atccccgacc gtctacaaaa accagcaggc tgagctgctg ctgctgatcg 420

atatctcttc aaggaaaaaa ttccaaacca tcctgcaaaa aagccgtgaa cgatatcagc 480

tgctgattca aaattccatt gataccattg cggtgattca caatggaaaa tgggtattta 540

tgaatgaatc gggaatttcc ctgtttgaag cggctacata tgaggattta attggcaaaa 600

acatatacga tcagctgcat ccttgcgatc acgaggatgt aaaagagaga atccaaaaca 660

ttgccgagca aaaaacagaa tctgaaattg tcaagcaatc ctggttcacc tttcagaaca 720

gggtcatcta tacggagatg gtctgcattc cgacgacctt ttttggtgaa gcggccgtcc 780

aggtcattct tcgggacatc tcagagagaa aacaaacaga agaattgatg ctgaaatcgg 840

aaaaattatc aatcgcaggg cagctcgcgg cgggaatcgc ccatgagatc cgcaaccctc 900

ttacagcgat caaaggattt ttacagctga tgaaaccgac aatggaaggc aacgaacatt 960

actttgatat tgtgttttct gaactcagcc gtatcgaatt aatactcagt gaactgctca 1020

tgctggcgaa acctcagcaa aatgctgtca aagaatattt gaacttgaaa aaattaattg 1080

gtgaggtttc agccctgtta gaaacgcagg cgaatttaaa tggcattttt atcagaacaa 1140

gttatgaaaa agacagcatt tatataaacg gggatcaaaa ccaattaaag caggtattca 1200

ttaatttaat caaaaatgca gttgaatcaa tgcctgatgg gggaacagta gacattatca 1260

taaccgaaga tgagcattct gttcatgtta ctgtcaaaga cgaaggggaa ggtatacctg 1320

aaaaggtact aaaccggatt ggagagccat ttttaacaac aaaagaaaaa ggtacggggc 1380

ttggattaat ggtgacattt aatatcattg aaaaccatca gggagttata catgtggaca 1440

gccatcctga aaaaggcaca gcgtttaaaa tttcatttcc aaaaaaataa 1490

<210>10

<211>320

<212>DNA

<213>Bacillus subtilis 168

<400>10

tatgggaagt gctccgtaat acgctgacaa gagagaaagg gcttggaggt attgaaacaa 60

gaggagttct gagaattggt atgccttata agtccaatta acagttgaaa acctgcatag 120

gagagctatg cgggtttttt attttacata atgatacata atttaccgaa acttgcggaa 180

cataattgag gaatcataga attttgtcaa aataatttta ttgacaacgt cttattaacg 240

ttgatataat ttaaatttta tttgacaaaa atgggctcgt gttgtacaat aaatgtagtg 300

aaaggaggtg aaatgtacac 320

<210>11

<211>574

<212>DNA

<213>Bacillus subtilis 168

<400>11

ctggtgtaga cggcgtaaaa acaggctata caggcgaagc gaaatattgt ctgactgctt 60

cggctaaaaa aggaaacatg cgggccatag cggttgtatt cggagcgagc acgcctaaag 120

aaagaaacgc gcaagtgaca aaaatgcttg acttcgcctt tagccaatat gaaacgcatc 180

ctttatataa acgaaatcaa acagtagcaa aagtaaaggt caaaaaaggg aaacaaaaat 240

ttatcgaact cactacatct gagccgattt caatattgac gaaaaaaggc gaggatatga 300

acgatgtgaa aaaagaaatc aagatgaagg acaatattag tgctccgatt caaaaaggcc 360

aagagcttgg cactcttgtt ctgaaaaagg atggagaagt actcgctgaa agtcctgttg 420

ctgcaaaaga agatatgaag aaagccgggt ttatcacatt cttaaagcgg acgatgggag 480

actggacaaa atttaagtaa ttatgaccac tagtttgtga aggaattcat tcatcgaaac 540

actcattatc cgatcatatc aaggaggaat gagc 574

<210>12

<211>603

<212>DNA

<213> Artificial Synthesis

<400>12

ctggtgtaga cggcgtaaaa acaggctata caggcgaagc gaaatattgt ctgactgctt 60

cggctaaaaa aggaaacatg cgggccatag cggttgtatt cggagcgagc acgcctaaag 120

aaagaaacgc gcaagtgaca aaaatgcttg acttcgcctt tagccaatat gaaacgcatc 180

ctttatataa acgaaatcaa acagtagcaa aagtaaaggt caaaaaaggg aaacaaaaat 240

ttatcgaact cactacatct gagccgattt caatattgac gaaaaaaggc gaggatatga 300

acgatgtgaa aaaagaaatc aagatgaagg acaatattag tgctccgatt caaaaaggcc 360

aagagcttgg cactcttgtt ctgaaaaagg atggagaagt actcgctgaa agtcctgttg 420

ctgcaaaaga agatatgaag aaagccgggt ttatcacatt cttaaagcgg acgatgggag 480

actggacaaa atttaagtaa ttatttgtcg aaaatgacca ctagttttgt cacggtgaag 540

gaattcattc tttgtcgaaa aatcgaaaca ctcattatcc gatcatatca aggaggaatg 600

agc 603

<210>13

<211>580

<212>DNA

<213>Bacillus subtilis 168

<400>13

catctcctgc gactgtcgta tatgcatggc cgatatgtaa ttttccgctc ggataataaa 60

tcggtgttgt aatgtaaaat gtattgtttt cttgcggcat cttgaaacct cctaatcaga 120

atgtcaaaaa gctcccgccc aaaggacgag agcggcgata agtgcaaatc atatatgaat 180

attatttata tttatccttt atctatgata ccatcttagc ctttaagatc aagaatatat 240

acatacgccc tgaaaaagaa taattaaaat taaaaatctt taatgaaaat aaaaaaagtc 300

tgttcgtgtc gaattttgtc gtatataatc aaatgaaata aaaaactttg gttcgaaagt 360

cttgatttaa aaggattttt agtaggataa tagctttatt aaatatttat aaaatgctgt 420

tatttcggta gtttccaaga cattactgac tataagaact aattcttaca atcaatagta 480

aacaaaatga ttgacgatta ttggaaacct tgttatgcta tgaaggtaag gattttaaga 540

aaaatataat ttaaacaaat aagtatctct tgggaggaga 580

<210>14

<211>601

<212>DNA

<213> Artificial Synthesis

<400>14

catctcctgc gactgtcgta tatgcatggc cgatatgtaa ttttccgctc ggataataaa 60

tcggtgttgt aatgtaaaat gtattgtttt cttgcggcat cttgaaacct cctaatcaga 120

atgtcaaaaa gctcccgccc aaaggacgag agcggcgata agtgcaaatc atatatgaat 180

attatttata tttatccttt atctatgata ccatcttagc ctttaagatc aagaatatat 240

acatacgccc tgaaaaagaa taattaaaat taaaaatctt taatgaaaat aaaaaaagtc 300

tgttcgtgtc gaattttgtc gtatataatc aaatgaaata aaaaactttg gttcgaaagt 360

cttgatttaa aaggattttt agtaggataa tagctttatt aaatatttat aaaatgctgt 420

tatttcggta gtttccaaga cattactgac tataagaact aattcttaca atcaatagta 480

aacaaaatga ttgacgatta ttggaaacct tgttatgcta tgaaggtaag gattttttgt 540

cgaaaataat ttgtcgaaag aaaaatataa tttaaacaaa taagtatctc ttgggaggag 600

a 601

<210>15

<211>717

<212>DNA

<213> Artificial Synthesis

<400>15

atgggtaagg gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaagctgc ctgttccttg gccaacactt 180

gtcactactc ttacttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg 240

cacgacttct tcaagagcgc catgcctgag ggatacgtgc aggagaggac catcttcttc 300

aaggacgacg ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc 360

aacagaatcg agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag 420

ttggaataca actacaactc ccacaacgta tacatcatgg cagacaaaca aaagaatgga 480

atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660

cttgagtttg taacagctgc tgggattaca catggcatgg atgaactgta caaataa 717

<210>16

<211>564

<212>DNA

<213> Artificial Synthesis

<400>16

aactgctgaa cttttggata tcgataatat tcaagacgta gaagtcatga caatattgac 60

tatggcagag ccatttgaaa agtctactgc gaatttattg gctcccatta ttgtgaatcg 120

caagaacatg atggctaagc aagtcgtttt acacgactcc tcatatacga caaagcatcc 180

gattggagga gaatcatgct agttttatcg cggaaaataa acgaagcgat tcaaataggt 240

gctgatattg aagtaaaagt gattgcggtt gaaggggatc aagtgaagct tggaattgac 300

gccccaaagc atattgatat tcacaggaaa gaaatttact tgaccattca ggaagaaaat 360

aaccgtgcag cagcgttatc cagcgatgtg atctccgcat tatcctcaca aaaaaagtga 420

ggattttttt atttttgtat taacaaaatc agagacaatc cgatattaat gatgtagccg 480

ggaggaggcg caaaagactc agccagttac aaaataaggg cacaaggacg tgccttaaca 540

acatattcag ggaggaacaa aaca 564

<210>17

<211>396

<212>DNA

<213>Bacillus subtilis 168

<400>17

gctttttcgg aaaagcaatg gaacaggcgg aagaatttaa cgattctctg tttcaagatc 60

tgcttaacgt tctgaaagcc ctgtttatcg aaacaggctc aagacagaaa gtgatgaacg 120

ctcttgaagc cttacgcaca ggccaaggat acccgtactt tgaagaactt gcactgatcg 180

ctgccgaatt ttatacaatg gataaacgca tggaagattc aatctacttt tacaacgaaa 240

tggtttgtgc gcaaagacag atccaacgcg gagattttct gtatgaagtt taaaggctta 300

ttttctgctg tcctgattgt ttcactgctt gtgggcgcag gatactcatt tgttcatcat 360

gatgaagttt cagtggcaag cagaaatgcg acataa 396

<210>18

<211>30

<212>DNA

<213> Artificial Synthesis

<400>18

gcattcatcg tcatatcatc atagctgagc 30

<210>19

<211>59

<212>DNA

<213> Artificial Synthesis

<400>19

tgtgaaattg ttatccgctc gagacattcc tccataatcc ttaaaatgct tttaatacc 59

<210>20

<211>27

<212>DNA

<213> Artificial Synthesis

<400>20

tgataggtgg tatgttttcg cttgaac 27

<210>21

<211>28

<212>DNA

<213> Artificial Synthesis

<400>21

gtgtacattc ctctcttacc tataatgg 28

<210>22

<211>53

<212>DNA

<213> Artificial Synthesis

<400>22

tttaaggatt atggaggaat gtctcgagcg gataacaatt tcacacagga aac 53

<210>23

<211>30

<212>DNA

<213> Artificial Synthesis

<400>23

aatcatacct accacaatat catgctcaag 30

<210>24

<211>22

<212>DNA

<213> Artificial Synthesis

<400>24

ttccatatcc tgcggcgttt gt 22

<210>25

<211>25

<212>DNA

<213> Artificial Synthesis

<400>25

acgaagttat tcagtcctgc tcctc 25

<210>26

<211>25

<212>DNA

<213> Artificial Synthesis

<400>26

cttgacagcg ggtttttcat acagc 25

<210>27

<211>23

<212>DNA

<213> Artificial Synthesis

<400>27

gctgtccatg tactagacgt ccc 23

<210>28

<211>19

<212>DNA

<213> Artificial Synthesis

<400>28

actggcttgg acggtggtt 19

<210>29

<211>47

<212>DNA

<213> Artificial Synthesis

<400>29

gtatgaaaaa cccgctgtca agtcctcctt gttctcttag cccttcg 47

<210>30

<211>39

<212>DNA

<213> Artificial Synthesis

<400>30

ctgggaaaac cctggcggct gatgctccgc tcgatatgg 39

<210>31

<211>20

<212>DNA

<213> Artificial Synthesis

<400>31

cgccaatcgc tgacctgaag 20

<210>32

<211>24

<212>DNA

<213> Artificial Synthesis

<400>32

ctggtgtaga cggcgtaaaa acag 24

<210>33

<211>32

<212>DNA

<213> Artificial Synthesis

<400>33

gctcattcct ccttgatatg atcggataat ga 32

<210>34

<211>41

<212>DNA

<213> Artificial Synthesis

<400>34

tatcaaggag gaatgagcat gggtaaggga gaagaacttt t 41

<210>35

<211>42

<212>DNA

<213> Artificial Synthesis

<400>35

ccaagcttct gcagttattt gtacagttca tccatgccat gt 42

<210>36

<211>36

<212>DNA

<213> Artificial Synthesis

<400>36

ctgtacaaat aactgcagaa gcttggcgta atcatg 36

<210>37

<211>44

<212>DNA

<213> Artificial Synthesis

<400>37

gccgtctaca ccagttgctc aaaaaaatct cggtcagatg ttac 44

Claims (10)

1. A method for synthesizing MK-7 by using a Bacillus subtilis group response regulation and control system is characterized in that the following modification is carried out on the chromosome of the Bacillus subtilis:

(1) carrying out truncation mutation on the gene of the histidine kinase KinA, carrying out constitutive expression, and knocking out the gene of the histidine kinase KinB; the truncation mutation refers to the knocking out of A PAS-A region of A signal conduction part of A histidine kinase KinA gene, and the sequence of the knocked-out histidine kinase KinA gene kinA-deltA PAS-A is shown in SEQ ID NO. 9;

(2) will contain P_hagThe Rap60 gene of the promoter is integrated on a chromosome, and two copies of the expression frame of the signal molecule Phr60 are integrated on the chromosome at the downstream of the Rap60 gene;

(3) using P_abrB(cs-1)The promoter replaces the promoters of the pyruvate kinase gene and the undecenyl pyrophosphate synthetase gene; the P is_abrB(cs-1)The sequence of the promoter is shown in SEQ ID NO. 14.

2. The method according to claim 1, wherein the genebank ID of the histidine kinase KinA in step (1) is 939230; the genebank ID of the histidine kinase KinB is 937167.

3. The method of claim 1, wherein said P in step (2)_hagThe promoter sequence is shown in SEQ ID NO. 16; the genebank ID of the transcription factor Rap60 is 1115983; the sequence of the expression cassette of the signal molecule Phr60 is shown in SEQ ID NO. 17.

4. The method according to claim 1, wherein the pyruvate kinase gene pyk in step (3) has a genebank ID of 936596; the genebank ID of the undecenyl pyrophosphate synthetase gene uppS is 939640.

5. The method of claim 1, further comprising using P_{spoiia(cs-1,3)}The promoter expresses hepta-diallyl diphosphate synthase gene and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene; the P is_{spoiia(cs-1,3)}The sequence of the promoter is shown in SEQ ID NO. 12.

6. A recombinant bacterium for producing heptaene menadione is characterized in that the chromosome of Bacillus subtilis is modified as follows:

(1) carrying out truncation mutation on the gene of the histidine kinase KinA, carrying out constitutive expression, and knocking out the gene of the histidine kinase KinB; the truncation mutation refers to the knocking out of A PAS-A region of A signal conduction part of A histidine kinase KinA gene, and the sequence of the knocked-out histidine kinase KinA gene kinA-deltA PAS-A is shown in SEQ ID NO. 9;

(2) will contain P_hagThe Rap60 gene of the promoter is integrated on a chromosome, and two copies of the expression frame of the signal molecule Phr60 are integrated on the chromosome at the downstream of the Rap60 gene;

(3) using P_abrB(cs-1)The promoter replaces the promoters of the pyruvate kinase gene and the undecenyl pyrophosphate synthetase gene; the P is_abrB(cs-1)The sequence of the promoter is shown in SEQ ID NO. 14.

7. The recombinant bacterium according to claim 6, wherein the genebank ID of the histidine kinase KinA in step (1) is 939230; the genebank ID of the histidine kinase KinB is 937167.

8. The recombinant bacterium according to claim 6, wherein P in step (2) is_hagThe promoter sequence is shown in SEQ ID NO. 16; the genebank ID of the transcription factor Rap60 is 1115983; the expression cassette of the signal molecule Phr60The sequence is shown as SEQ ID NO. 17.

9. The recombinant bacterium according to claim 6, wherein the genebank ID of the pyruvate kinase gene pyk in step (3) is 936596; the genebank ID of the undecenyl pyrophosphate synthetase gene uppS is 939640.

10. The recombinant bacterium according to claim 6, wherein the recombinant bacterium is modified as follows based on claim 6: using P_{spoiia(cs-1,3)}The promoter expresses hepta-diallyl diphosphate synthase gene and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene; the P is_{spoiia(cs-1,3)}The sequence of the promoter is shown in SEQ ID NO. 12.

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