CN110157749A - Using the method for bacillus subtilis group response regulator control system synthesis MK-7 - Google Patents

Using the method for bacillus subtilis group response regulator control system synthesis MK-7 Download PDF

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CN110157749A
CN110157749A CN201910490611.1A CN201910490611A CN110157749A CN 110157749 A CN110157749 A CN 110157749A CN 201910490611 A CN201910490611 A CN 201910490611A CN 110157749 A CN110157749 A CN 110157749A
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刘龙
崔世修
吕雪琴
李江华
堵国成
陈坚
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Jiangnan University
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Abstract

The invention discloses the methods of application bacillus subtilis group response regulator control system synthesis MK-7, belong to field of genetic engineering.The present invention is by phosphokinase KinA truncated mutant, and with constitutive promoter PvegExpression, enables cell to generate gemma signal in the case where culture medium is full of nutrition.The Spo0A system that regulation is responded by Phr60-Rap60 group is obtained using the method for the present invention, pass through the dynamic regulation to MK-7 route of synthesis, realize efficiently synthesizing for MK-7, do not need additional inducer, realize target product efficiently synthesize cell growth between dynamic equilibrium.The yield of finally obtained bacillus subtilis recombinant bacterium BS19, BS20 shake flask fermentation production MK-7 is up to 170mg/L, 360mg/L respectively, is 17.89,37.89 times of bacillus subtilis (Bacillus subtilis) 168.

Description

Using the method for bacillus subtilis group response regulator control system synthesis MK-7
Technical field
The present invention relates to the methods of application bacillus subtilis group response regulator control system synthesis MK-7, belong to genetic engineering Field.
Background technique
Menaquinone (MK-7) is a kind of important liposoluble vitamin, as the co-factor of gamma-glutamic acid carboxylase, In the distribution for adjusting calcium, skeleton development is promoted to reverse osteoporosis and in protection blood vessel, atherosclerosis and painstaking effort It plays an important role in pipe disease reaction.Since menaquinone has better compatibility and longer half-life period to human body, People are obtained more to pay close attention to.
Bacillus subtilis (Bacillus subtilis) is often widely used in Food enzyme and important nutrient chemistry product Production in, product by FDA certification be " generally regarded as safe " (GRAS) security level.Therefore, it transports It is the effective way for efficiently synthesizing MK-7 with metabolic engineering means building recombined bacillus subtilis.However, the synthesis way of MK-7 Diameter is sufficiently complex, and important as precursors phosphoenolpyruvate PEP is caused largely to flow to tricarboxylic acids TCA circulation, and makes branch The synthesis of acid is restricted, in addition, Isoprenoid is also the important substance of Cell wall synthesis, can be inhibited if knocking out The growth of cell；MEP approach intermediate product 1- hydroxy-2-methyl -2- cyclobutenyl 4- bisphosphate (HMBPP) and dimethallyl Base bisphosphate (DMAPP) is toxic to cell.The supply of bacillus subtilis metabolic fluxes how is adjusted, the synthesis of MK-7 is increased The problem of being a worth further investigated.
Kyushu University Hanai.T teaches seminar and uses the LuxR group from Fermi operator (Vibrio fischeri) Regulator control system is responded, carbon flow is realized in Escherichia coli from central metabolic pathway to the automatic switchover of isopropanol approach.Massachusetts Institute of Technology Prather.K teaches seminar and utilizes the Esa from P.stwartii subsp.stewartii (Pantoea stewartii) The binding site of EsaR is integrated into the promoter region of Escherichia coli phosphofructokinase (pfkA) gene by system, it is suppressed that The transcription of pfkA gene, the output increased of product inositol 5.5 times.However these researchs use heterologous group's response system Regulated and controled, cell metabolism burden, system stability is weaker, and if using group's response system of cell itself to metabolism Network carries out dynamic regulation, then will not cause added burden to the metabolism of cell.Not having also in the research of menaquinone makes The research report regulated and controled with group's response dynamics.
Summary of the invention
In order to solve above-mentioned technical problem, group's response that the present invention constructs Phr60-Rap60-Spo0A is dynamic State regulator control system, and P is transformedspoiiaAnd PabrbPromoter obtains the starting word bank different by Spo0A-P regulatory transcription activity, and By key gene HepS/T, ispH, pyk and uppS in the promoter dynamic regulation menaquinone route of synthesis of acquisition, finally The yield of obtained bacillus subtilis recombinant bacterium BS19, BS20 shake flask fermentation production MK-7 is up to 170mg/L, 360mg/ respectively L is 17.89,37.89 times of bacillus subtilis (Bacillus subtilis) 168 respectively, and recombinant bacterium BS20 is fermenting It ferments 3 days in tank, the yield of MK-7 is up to 230mg/L.
The first purpose of the invention is to provide a kind of application bacillus subtilis group response regulator control systems to synthesize MK-7 Method, the method includes following transformation has been carried out on the chromosome of bacillus subtilis:
(1) gene of histidine kinase KinA is subjected to truncated mutant and carries out constitutive expression, while knockout group The gene of histidine kinase KinB；
(2) P will be containedhagOn the Rap60 gene integration to chromosome of promoter, and Rap60 downstream of gene on chromosome Integrate the expression cassette of the signaling molecule Phr60 of two copies；
(3) P is usedabrB(cs-1)Promoter replaces gene encoding for pyruvate kinase and undecenyl pyrophosphate synthetase gene Promoter；The PabrB(cs-1)The sequence of promoter is as shown in SEQ ID NO.14.
In one embodiment, the genebank ID of histidine kinase KinA described in step (1) is 939230；Institute It states truncated mutant and refers to the region PAS-A for knocking out the signal transduction part of histidine kinase KinA gene, the histidine after knockout The sequence of kinases KinA gene kinA- Δ PAS-A is as shown in SEQ ID NO.9；The genebank of the histidine kinase KinB ID is 937167.
In one embodiment, P described in step (2)hagPromoter sequence is as shown in SEQ ID NO.16；Described turn The genebank ID for recording factor R ap60 is 1115983；The sequence of the expression cassette of the signaling molecule Phr60 such as SEQ ID Shown in NO.17.
In one embodiment, the genebank ID of gene encoding for pyruvate kinase pyk described in step (3) is 936596； The genebank ID of the undecenyl pyrophosphate synthetase gene uppS is 939640.
In one embodiment, the bacillus subtilis is bacillus subtilis (Bacillus subtilis) 168。
In one embodiment, the bacillus subtilis is Bacillus subtilis 168P43-menF P43- menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs- ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena-menA::thrC Pmena- menA::dacA is on the basis of bacillus subtilis (Bacillus subtilis) 168, with promoter P43It replaces Change the natural promoter of menF, menB gene；Use PhbsThe natural promoter of promoter replacement menE gene；With containing P43Starting The entC gene replacement dhbB gene of son；Use PhbsThe natural promoter of promoter replacement tkt gene；With containing P43Promoter On ppsA gene integration to bacillus subtilis chromosome, and knock out ptsG gene；By aroGfbrGene and promoter PhbsFusion After be integrated on chromosome；Utilize P43The natural promoter of promoter replacement aroK gene；Utilize PhbsPromoter replaces ispA base The natural promoter of cause；Utilize P43The natural promoter of promoter replacement hepS/T gene；By kdpG gene and promoter PhbsMelt It is integrated on chromosome after conjunction；Use P43Promoter replaces the natural promoter of dxr gene, dxs gene, fni gene；In withered grass bud The open reading frame of ganA, thrC, dacA integrations expression menA of spore bacillus gene group, the Strain Designation that will be finally obtained For BS17.
In one embodiment, the genebank ID of the menF is 937190；The genebank ID of the menB It is 937195；The PhbsThe sequence of promoter is as shown in SEQ ID NO.5；The genebank ID of the menE is 937132； The entC gene source is 945511 in E.coli K12, genebank ID；The genebank ID of the dhbB is 936582；The genebank ID of the tkt gene is 937377；The ppsA derives from E.coli K12, genebank ID is 946209；The genebank ID of the ptsG is 939255；The aroGfbrThe sequence of gene such as SEQ ID NO.6 institute Show；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 the dxr is 939636；The genebank ID of the dxs is 938609；The genebank ID of the fni is 938985；The menA's opens The sequence of reading frame is put as shown in SEQ ID NO.8；The genebank ID of the ganA is 936313；The thrC's Genebank ID is 936660；The genebank ID of the dacA is 940000.
In one embodiment, the method also includes using Pspoiia(cs-1,3)Promoter expresses seven polydiene propyl two Phosphate synthase gene and 4- hydroxy-3-methyl but-2-ene base diphosphonic acid reductase gene；The Pspoiia(cs-1,3)Promoter Sequence is as shown in SEQ ID NO.12.
In one embodiment, the genebank ID of the seven polydiene propyl diphosphate synthase gene HepS/T It is 938998；The genebank ID of the 4- hydroxy-3-methyl but-2-ene base diphosphonic acid reductase gene ispH is 937900.
A second object of the present invention is to provide a kind of recombinant bacteriums for producing menaquinone, which is characterized in that in withered grass Following transformation has been carried out on the chromosome of bacillus:
(1) gene of histidine kinase KinA is subjected to truncated mutant and carries out constitutive expression, while knockout group The gene of histidine kinase KinB；
(2) P will be containedhagOn the Rap60 gene integration to chromosome of promoter, and Rap60 downstream of gene on chromosome Integrate the expression cassette of the signaling molecule Phr60 of two copies；
(3) P is usedabrB(cs-1)Promoter replaces gene encoding for pyruvate kinase and undecenyl pyrophosphate synthetase gene Promoter；The PabrB(cs-1)The sequence of promoter is as shown in SEQ ID NO.14.
In one embodiment, the recombinant bacterium has also done following transformation on the basis of BS17: by histidine kinase The gene of KinA carries out truncated mutant and has carried out constitutive expression, knocks out the gene of histidine kinase KinB；P will be containedhag On the Rap60 gene integration to chromosome of promoter, and Rap60 downstream of gene integrates the signal point of two copies on chromosome The expression cassette of sub- Phr60；Finally building obtains bacterial strain Bacillus subtilis 168, P43-menF P43-menB Phbs- menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena-menA::thrC Pmena- menA::dacA Pveg-kinA-ΔPAS-AΔkinB Phag-Rap60(Pnative-Phr60)2:: hag is named as BS18。
In one embodiment, the recombinant bacterium has also done following transformation on the basis of BS18: using PabrB(cs-1) Promoter replaces pyruvate kinase (pyk, genebank ID:936596) gene and undecenyl pyrophosphate synthetase The promoter of (uppS, genebank ID:939640) gene, finally obtains bacterial strain Bacillus subtilis 168, P43- menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena-menA:: thrC Pmena-menA::dacA Pveg-kinA-ΔPAS-AΔkinB Phag-Rap60(Pnative-Phr60)2::hag PabrB(cs-1)-pyk::pyk PabrB(cs- 1)-uppS::uppS is named as BS19.
In one embodiment, the recombinant bacterium has also done following transformation on the basis of BS19: using Pspoiia(cs-1,3)Promoter express seven polydiene propyl diphosphate synthase (HepS/T, genebank ID:938998) genes and 4- hydroxy-3-methyl but-2-ene base diphosphonic acid reductase (ispH, genebank ID:937900) gene, finally obtains bacterial strain Bacillus subtilis 168,P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs-aroGfbr P43-aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena-menA::thrC Pmena-menA::dacA Pveg-kinA-ΔPAS-AΔkinB Phag- Rap60(Pnative-Phr60)2::hag PabrB(cs-1)-pyk::pyk PabrB(cs-1)-uppS::uppS Pspoiia(cs-1,3)- ispH::ispH Pspoiia(cs-1,3)- HepS/T::HepS/T is named as BS20.
Third object of the present invention is to provide a kind of methods for producing menaquinone, and the method is using described Recombinant bacterium carries out fermenting and producing.
Beneficial effects of the present invention:
(1) by phosphokinase KinA truncated mutant, and with constitutive promoter PvegExpression, enables cell training Gemma signal is generated in the case that feeding base is full of nutrition.
(2) the Spo0A system that regulation is responded by Phr60-Rap60 group is obtained using the method for the present invention, by MK-7 The dynamic regulation of route of synthesis realizes efficiently synthesizing for MK-7.
(3) do not need additional inducer, realize target product efficiently synthesize cell growth between dynamic equilibrium.
(4) in the case where adding sucrose, the yield of MK-7 significantly improves the bacterial strain obtained using the present invention.
(5) yield of finally obtained bacillus subtilis recombinant bacterium BS19, BS20 shake flask fermentation production MK-7 is respectively 170mg/L, 360mg/L are 17.89,37.89 times of bacillus subtilis (Bacillus subtilis) 168.And recombinant bacterium BS20 ferments 3 days in the fermenter, and the yield of MK-7 is up to 230mg/L.
Biomaterial
Bacillus subtilis (Bacillus subtilis) 168 of the invention is purchased from American Type Culture collection, Deposit number is ATCC No.27370.
1 strain genotype of table
2 sequence table of table
Detailed description of the invention
Fig. 1: phosphokinase KinA structural schematic diagram and truncated mutant.
Fig. 2: influence of the signaling molecule Phr60-Rap60 different proportion to Spo0A ability of regulation and control；Pspoiia- GFP: bacterial strain BS168-Pspoiia-GFP；Rap60: bacterial strain BS17-2-Rap60-GFP；Phr60-Rap60: bacterial strain BS17-2-Phr60- Rap60-GFP；2*Phr60-Rap60: bacterial strain BS17-2- (Phr60) * 2-Rap60-GFP.
The variation of Fig. 3: Spo0A binding site number and sequence variation to promoter regulation ability.
Fig. 4: influence of the cell concentration to Spo0A ability of regulation and control.
Fig. 5: Phr60-Rap60-Spo0A dynamic regulation MK-7 route of synthesis.
Fig. 6: the transcriptional level of key gene.
Fig. 7: recombinant bacterium shake flask fermentation produces MK-7.
Fig. 8: recombinant bacterium ferment tank produces MK-7.
Specific embodiment
The extraction of MK-7 and HPLC detection: 4 times of volume isopropanols and normal hexane mixture (1:2V/ are added in fermentation liquid V), after vortex oscillation extracts 30min, extracting solution is filtered out, 8000r/min is centrifuged 15min.Supernatant is collected, MK-7 dissolves at this time It in the phase, is placed in -80 DEG C of refrigerators and freezes, remove lipid material crystallization, collect filtrate, HPLC detects the content of MK-7.
HPLC detect MK-7 yield: using Agilent ZORBAX EclipseXDB-C18 splitter (5 μm, 250 × 4.6mm), for the temperature of detection at 40 DEG C, mobile phase uses methanol: methylene chloride (9:1, v/v), and flow velocity 1mL/min detects wave Long 254nm, 10 μ L of sample volume.
Embodiment 1: the building of recombinant bacterium BS17
(1) building of recombinant bacterium BS1
Pass through constitutive promoter P43The natural promoter of menF on bacillus subtilis chromosome is replaced to enhance methyl The expression of the different chorismic acid synthetase of naphthoquinones (menF, genebank ID:937190) gene.Use unmarked genetic modification plan Slightly, referring to article (Yan, X., Yu, H.-J., Hong, Q., Li, S.P., 2008.Cre/lox system and PCR-based Genome engineering in Bacillus subtilis.Appl Environ Microb.74,5556-5562), tool Body building process is as follows:
(a) clone of gene
1. using 168 genome of bacillus subtilis as template, using primer menFupAnd menF .FORup.REV amplification obtains The upstream homology arm sequence menF of menF geneup(sequence is as shown in SEQ ID NO.1).
2. the artificial synthesized lox71-zeo-lox66 box containing bleomycin gene (sequence is as shown in SEQ ID NO.2).
3. using 168 genome of bacillus subtilis as template, using primer P43And P .For43.Rev amplification obtains P43Starting Subsequence (sequence is as shown in SEQ ID NO.3).
4. expanding to obtain using primer menF.FOR and menF.REV using 168 genome of bacillus subtilis as template MenF genetic fragment (sequence is as shown in SEQ ID NO.4).
(b) acquisition of segment is merged
The four segment menF that will be obtained in step (a)up, lox71-zeo-lox66 box, P43Promoter sequence, menF base Because segment carries out Overlap extension PCR, PCR condition: 98 DEG C, 5min initial denaturation, is denaturalized 10s by then 98 DEG C, and 55 DEG C, anneal 5s, and 72 DEG C, extend 2min, 30 circulations, the correct segment of gel extraction size obtain fusion segment menF in totalup-lox71- zeo-lox66-P43-menF。
(c) homologous recombination
Fusion segment obtained in step (b) is transformed into 168 competence of wild-type strain Bacillus subtilis In cell.Since there are the upstreams of the menF on the upstream sequence of menF and 168 chromosome of bacillus subtilis in fusion segment Sequence gene is homologous, and there are the menF DNA homologs on menF gene and 168 chromosome of bacillus subtilis in fusion segment, leads to Homologous recombination is crossed, blasticidin resistance gene zeo and P in segment are merged43Promoter replaces 168 chromosome of bacillus subtilis Upper menF gene natural promoter.Specific steps are as follows:
1. the competent cell of the fusion segment electrotransformation bacillus subtilis 168 built in step (b) is merged piece Section additive amount is 100-300ng, and electrotransformation condition: voltage 2.5kV, shock by electricity time 5ms, 37 DEG C of recovery 5h coatings final concentration of 10 The LB plate of the blasticidin resistance of μ g/mL, 37 DEG C of Anaerobic culturel 48h.It is that conversion is successful that blasticidin resistance, which is positive, Bacillus subtilis.
2. selecting the single colonie grown on plate, bacterium colony PCR verifying is carried out using primer BS1YZ.FOR and BS1YZ.REV, The fragment length expanded after replacement is 1350bp.And be sequenced, sequencing is correctly lox71-zeo-lox66-P43 Fusion successfully replaces the menF gene natural promoter on 168 chromosome of bacillus subtilis.Finally by Cre/lox weight Group system knocks out blasticidin resistance gene zeo, obtains bacterial strain Bacillus subtilis 168, P after verifying is correct43- MenF is named as BS1.
3 primer sequence table of table
(2) building of recombinant bacterium BS2
On the basis of the bacterial strain BS1 that step (1) obtains, using the method similar with step (1), P is utilized43Promoter is replaced Change the day of dihydroxy naphthlene formic acid synzyme (menB, genebank ID:937195) gene on 168 chromosome of bacillus subtilis Right promoter obtains bacterial strain Bacillus subtilis 168, P after verifying is correct43-menF P43- menB is named as BS2。
(3) building of recombinant bacterium BS3
On the basis of the bacterial strain BS2 that step (2) obtains, using the method similar with step (1), P is utilizedhbsPromoter (sequence is as shown in SEQ ID NO.5) replace bacillus subtilis in O- succinylbenzoic acid-CoA ligase (menE, Genebank ID:937132) gene natural promoter, obtain bacterial strain Bacillus subtilis 168 after verifying is correct, P43-menF P43-menB Phbs- menE is named as BS3.
(4) building of recombinant bacterium BS4
On the basis of the bacterial strain BS3 that step (3) obtains, with containing P43Different point from E.coli K12 of promoter On branch acid synthase (entC, genebank ID:945511) gene replacement bacillus subtilis chromosome different chorismic acid (dhbB, Genebank ID:936582) gene.Specific building process is as follows:
(a) acquisition of segment is merged
Using 168 genome of bacillus subtilis as template, amplification obtains the upstream homology arm sequence of dhbB gene respectively dhbBup, the downstream dhbB homology arm sequence dhbBdown、P43Promoter sequence；Artificial synthesized lox71-zeo-lox66 box sequence (sequence Column are as shown in SEQ ID NO.2)；Using E.coli K12 genome as template, amplification obtains entC gene order, then by five Segment dhbBup, lox71-zeo-lox66 box, P43Promoter sequence, entC gene order, dhbBdownGenetic fragment is overlapped Extension PCR obtains fusion segment dhbBup-lox71-zeo-lox66-P43-entC-dhbBdown。
(b) homologous recombination
Fusion segment obtained in step (a) is transformed into BS3 competent cell, Cre/lox recombination system is then passed through It unites, blasticidin resistance gene zeo in knock-out bacterial strain obtains bacterial strain Bacillus subtilis 168, P43-menF P43- menB Phbs-menE P43- entC Δ dhbB, is named as BS4.
(5) building of recombinant bacterium BS5
On the basis of the bacterial strain BS4 that step (4) obtains, using the method similar with step (1), P is utilizedhbsPromoter The natural promoter of transketolase (tkt, genebank ID:937377) gene on bacillus subtilis chromosome is replaced, verifying is just Bacterial strain Bacillus subtilis 168, P is obtained after really43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs- tkt is named as BS5.
(6) building of recombinant bacterium BS6
On the basis of the bacterial strain BS5 that step (5) obtains, P will be contained43The phosphoric acid from E.coli K12 of promoter Polysaccharide on enolpyruvyl acid synthase (ppsA, genebank ID:946209) gene integration to bacillus subtilis chromosome Between deacetylase C (yjeA, genebank ID:936440) gene and yjfA (genebank ID:939830) gene, and strike Except the glucose phosphate on chromosome transports enzyme (ptsG, genebank ID:939255) gene.Specific building process are as follows:
(a) acquisition of segment is merged
Using E.coli K12 genome as template, amplification obtains ppsA gene, using 168 genome of bacillus subtilis as mould Plate, amplification obtains yjeA gene and yjfA gene, P respectively43Promoter sequence；Artificial synthesized lox71-zeo-lox66 box sequence (sequence is as shown in SEQ ID NO.2).Then by five segment yjeA, lox71-zeo-lox66 boxes, P43, ppsA, yjfA carry out Overlap extension PCR obtains fusion segment yjeA-lox71-zeo-lox66-P43-ppsA-yjfA。
Expand the upstream ptsG homology arm ptsG respectively using the genome of bacillus subtilis 168 as templateupIt is homologous with downstream Arm ptsGdown, by ptsGup、lox71-zeo-lox66、ptsGdownOverlap extension PCR is carried out, fusion segment is obtained ptsGup-lox71-zeo-lox66-ptsGdown。
(b) homologous recombination
Segment yjeA-lox71-zeo-lox66-P will be merged obtained in step (a)43- ppsA-yjfA and ptsGup- lox71-zeo-lox66-ptsGdownIt is transformed into BS5 competent cell, then by Cre/lox recombination system, knocks out bacterium Blasticidin resistance gene zeo in strain obtains bacterial strain Bacillus subtilis 168P after verifying is correct43-menF P43- menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43- ppsA Δ ptsG, is named as BS6.
(7) building of recombinant bacterium BS7
On the basis of the bacterial strain BS6 that step (6) obtains, by artificial synthesized aroGfbrGene (sequence such as SEQ ID Shown in NO.6) and promoter PhbsIt is integrated on bacillus subtilis chromosome and answers after (sequence is as shown in SEQ ID NO.5) fusion Shock protein (ytxj, genebank ID:937308) gene and 3- deoxidation-D-arabinose-sulfonate 7- in heptan phosphate synthase enzyme Between (aroA, genebank ID:937853) gene, specific building process are as follows:
(a) acquisition of segment is merged
Artificial synthesized aroGfbrGene (sequence is as shown in SEQ ID NO.6)；Using 168 genome of bacillus subtilis as mould Plate, amplification obtains ytxj gene and aroA gene, P respectivelyhbsPromoter sequence.Then by five segments ytxj, lox71-zeo- lox66、Phbs、aroGfbr, aroA carry out Overlap extension PCR, obtain fusion segment ytxj-lox71-zeo-lox66- Phbs-aroGfbr-aroA。
(b) homologous recombination
Fusion segment obtained in step (a) is transformed into BS6 competent cell, Cre/lox recombination system is then passed through It unites, blasticidin resistance gene zeo in knock-out bacterial strain obtains bacterial strain Bacillus subtilis 168P after verifying is correct43- menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr, ordered Entitled BS7.
(8) building of recombinant bacterium BS8
On the basis of the bacterial strain BS7 that step (7) obtains, using the method similar with step (1), P is utilized43Promoter is replaced The natural promoter for changing shikimate kinase on bacillus subtilis chromosome (aroK, genebank ID:938343) gene, is tested Bacterial strain Bacillus subtilis 168P is obtained after card is correct43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43- aroK is named as BS8.
(9) building of recombinant bacterium BS9
On the basis of the bacterial strain BS8 that step (8) obtains, using the method similar with step (1), P is utilizedhbsPromoter Farnesyl diphosphate synthase (ispA, genebank ID:938652) gene naturally opens on replacement bacillus subtilis chromosome Mover obtains bacterial strain Bacillus subtilis 168P after verifying is correct43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbrP43-aroK Phbs- ispA is named as BS9.
(10) building of recombinant bacterium BS10
On the basis of the bacterial strain BS9 that step (9) obtains, using the method similar with step (1), P is utilized43Promoter is replaced Change seven polydiene propyl diphosphate synthase (hepS/T, genebank ID:938998) gene on bacillus subtilis chromosome Natural promoter, obtain bacterial strain Bacillus subtilis 168P after verifying is correct43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbrP43-aroK Phbs-ispA P43- hepS/T, will It is named as BS10.
(11) building of recombinant bacterium BS11
On the basis of the bacterial strain BS10 that step (10) obtains, zymomonas mobilis (Zymomonas will be derived from Mobilis 2- dehydrogenation -3- deoxidation-phosphogluconate aldolase (kdpG, genebank ID:33073472) gene in) with Promoter PhbsUrokinase on bacillus subtilis chromosome (yclG, genebank ID:938292) gene is integrated into after fusion It is sprouted between response protein (gerkA, genebank ID:938285) gene with gemma, specific building process are as follows:
(a) acquisition of segment is merged
Using zymomonas mobilis (Zymomonas mobilis) genome as template, kdpG gene is synthesized；With withered grass bud 168 genome of spore bacillus is template, and amplification obtains yclG gene, P respectivelyhbsPromoter sequence and gerkA gene；It is artificial synthesized Lox71-zeo-lox66 box sequence (sequence is as shown in SEQ ID NO.2).Then by five segments yclG, lox71-zeo- Lox66 box, PhbsPromoter sequence, kdpG, gerkA carry out Overlap extension PCR, obtain fusion segment yclG-lox71- zeo-lox66-Phbs-kdpG-gerkA
(b) homologous recombination
Fusion segment obtained in step (a) is transformed into BS10 competent cell, Cre/lox recombination system is then passed through It unites, blasticidin resistance gene zeo in knock-out bacterial strain obtains bacterial strain Bacillus subtilis 168P after verifying is correct43- menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr:: lox72P43-aroK Phbs-ispA P43-hepS/T Phbs- kdpG is named as BS11.
(12) building of recombinant bacterium BS12
On the basis of the bacterial strain BS11 that step (11) obtains, using the method similar with step (1), P is utilized43Promoter Replace bacillus subtilis chromosome on 1-deoxy-D-xylulose -5- phosphoric acid reduction isomerase enzyme (dxr, genebank ID: 939636) natural promoter of gene obtains bacterial strain Bacillus subtilis 168P after verifying is correct43-menF P43- menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs- ispA P43-hepS/T Phbs-kdpG P43- dxr is named as BS12.
(13) building of recombinant bacterium BS13
On the basis of the bacterial strain BS12 that step (12) obtains, using the method similar with step (1), P is utilized43Promoter Replace 1-deoxyxylulose 5-phosphate synthase (dxs, genebank ID:938609) gene in bacillus subtilis 168 Natural promoter obtains bacterial strain Bacillus subtilis 168P after verifying is correct43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43- dxs is named as BS13.
(14) building of recombinant bacterium BS14
On the basis of the bacterial strain BS13 that step (13) obtains, using the method similar with step (1), P is utilized43Promoter Gene in isopentenylpyrophosphate isomerase enzyme (fni, genebank ID:938985) on replacement bacillus subtilis chromosome Natural promoter obtains bacterial strain Bacillus subtilis 168P after verifying is correct43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43- fni is named as BS14.
(15) building of recombinant bacterium BS15
On the basis of the bacterial strain BS14 that step (14) obtains, menA is inserted into the Bacillus subtilis genes site group ganA Reading frame.GanA (genebank ID:936313) expresses the circumscribed hydrolase of arabogalactan I type oligomer.Specific structure Build process are as follows:
(a) clone of gene
1. using 168 genome of bacillus subtilis as template, using primer mena-up::gana.FOR and mena-up:: Gana.rev expands to obtain the upstream homology arm sequence ganA of ganA geneup。
2. the artificial synthesized lox71-zeo-lox66 box containing bleomycin gene (sequence is as shown in SEQ ID NO.2).
3., as template, utilizing primer menA- using bacillus subtilis (Bacillus subtilis) 168 genomes The open reading frame of orf.FOR and menA-orf.REV amplification seven amylene based transferase (menA) of 1,4-dihydroxy-2-naphthsaisyuoic acid, It is 1537bp that the genetic fragment that amplification is obtained, which carries out Ago-Gel detected magnitude, and the correct segment of gel extraction size obtains Obtain the menA reading frame sequence P of nucleotide sequence such as SEQ ID NO.8mena-menA。
4. using 168 genome of bacillus subtilis as template, using primer menA-down::gana.FOR and mena- Down:gana.rev expands to obtain the downstream homology arm sequence ganA of ganA genedown。
(b) acquisition of segment is merged
The four segment ganA that will be obtained in step (a)up, lox71-zeo-lox66 box, Pmena-menA、ganAdownBase Because segment carries out Overlap extension PCR, fusion segment ganA is obtainedup-lox71-zeo-lox66-Pmena-menA- ganAdown。
(c) homologous recombination
Fusion segment obtained in step (b) is transformed into BS14 competent cell, and is coated on blasticidin resistance LB plate, select the single colonie grown on plate, carry out bacterium colony PCR verifying and be simultaneously sequenced.It is recombinated finally by Cre/lox System, blasticidin resistance gene zeo in knock-out bacterial strain, finally obtains bacterial strain Bacillus subtilis 168P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr::lox72P43- aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena- menA::ganA is ordered Entitled BS15.
4 primer sequence table of table
(16) building of recombinant bacterium BS16
On the basis of the bacterial strain BS15 that step (15) obtains, using the method similar with step (15), in withered grass gemma The reading frame of the bacillus gene site group thrC insertion menA.The synthesis of thrC (genebank ID:936660) accurate translation threonine Enzyme.Bacterial strain Bacillus subtilis 168P is obtained after verifying is correct43-menF P43-menB Phbs-menE P43-entCΔ dhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr::lox72P43-aroK Phbs-ispA P43-hepS/T Phbs- kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena- menA::thrC is named as BS16.
(17) building of recombinant bacterium BS17
On the basis of the bacterial strain BS16 that step (16) obtains, using the method similar with step (15), in withered grass gemma The reading frame of the bacillus gene site group dacA insertion menA.DacA (genebank ID:940000) expresses alanine carboxypeptidase. Bacterial strain Bacillus subtilis 168P is obtained after verifying is correct43-menF P43-menB Phbs-menE P43-entCΔ dhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr::lox72P43-aroK Phbs-ispA P43-hepS/T Phbs- kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena-menA::thrC Pmena- menA::dacA, will It is named as BS17.
Embodiment 2: the truncated mutant and utilization constitutive promoter of histidine kinase KinA is expressed
Only in the case where carbon starvation, then itself ability phosphorylation ties DNA by histidine kinase KinA, KinB Hop protein Spo0A phosphorylation is to be activated, and the Spo0A of phosphorylation is in conjunction with promoter, to raise or lower corresponding base Because of expression, the ability of controlling gene expression is played.But efficiently synthesizing for purpose product, need the lasting supply of effective carbon source. Therefore, by truncating histidine kinase KinA, and constitutive promoter P is utilizedvegTruncated phosphokinase KinA is expressed, is truncated The mutation method of PAS-A domain is as shown in Figure 1, the expression for enabling histidine phosphokinase KinA to continue.
(1) acquisition of segment is merged
Using 168 genome of bacillus subtilis as template, amplification obtains histidine kinase (kinA, genebank respectively ID:939230 upstream homology arm sequence kinA)up, (sequence is such as by histidine kinase genetic fragment kinA- Δ PAS-A after truncation Shown in SEQ ID NO.9) and PvegPromoter sequence (sequence is as shown in SEQ ID NO.10), artificial synthesized lox71-zeo- Lox66 box sequence (sequence is as shown in SEQ ID NO.2), then by four segment kinAup, lox71-zeo-lox66 box, Pveg Promoter sequence, kinA- Δ PAS-A genetic fragment carry out Overlap extension PCR, obtain fusion segment kinAup-lox71- zeo-lox66-Pveg-kinA-ΔPAS-A。
2. homologous recombination
By step 1. obtained in fusion segment be transformed into BS17 competent cell obtained in embodiment 1, and be coated with In the LB plate of blasticidin resistance, the single colonie grown on plate is selected, carry out bacterium colony PCR verifying and is sequenced.Finally By Cre/lox recombination system, blasticidin resistance gene zeo in knock-out bacterial strain obtains bacterial strain Bacillus subtilis 168,P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs- aroGfbr::lox72P43-aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena- menA::ganA Pmena-menA::thrC Pmena-menA::dacA Pveg- kinA- Δ PAS-A, is named as BS17-1.
Embodiment 3: the knockout of histidine kinase KinB
In order to reduce other influences of histidine phosphokinase to regulator control system, another main phosphokinase is knocked out KinB (kinB, genebank ID:937167), to guarantee the unicity of regulator control system.The recombinant bacterium obtained in example 2 On the basis of BS17-1, the phosphokinase gene kinB on bacillus subtilis chromosome is knocked out.
(1) acquisition of segment is merged
Using 168 genome of bacillus subtilis as template, amplification obtains kinB respectivelyup、kinBdown, it is artificial synthesized Lox71-zeo-lox66 box sequence, then by three segment kinBup, lox71-zeo-lox66 box, kinBdownOverlapping is carried out to prolong PCR is stretched, fusion segment kinB is obtainedup-lox71-zeo-lox66-kinBdown。
2. homologous recombination
By step 1. obtained in fusion segment be transformed into BS17-1 by state cell, and be coated on blasticidin resistance LB plate selects the single colonie grown on plate, carries out bacterium colony PCR verifying and is sequenced.Finally by Cre/lox recombination system It unites, blasticidin resistance gene zeo in knock-out bacterial strain finally obtains bacterial strain Bacillus subtilis 168, P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr::lox72P43- aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena- menA::thrC Pmena-menA::dacA Pveg- kinA- Δ PAS-A Δ kinB, is named as BS17-2.
The building of group's response dynamics regulator control system of embodiment 4:Phr60-Rap60-Spo0A
The phosphorylation level of Spo0A receives the regulation of histidine kinase KinA, in the bacterial strain BS17-2 that embodiment 3 obtains In, KinA has been subjected to truncated mutant and has carried out constitutive expression while having knocked out another main phosphokinase KinB, this enables Spo0A to obtain phosphorylation modification in the case where full of nutrition, to function.But withered grass gemma The response regulator control system of Phr-Rap group present in bacillus is merely capable of the phosphorylation level of control Spo0A-P, can not regulate and control The phosphorylation degree of KinA, this makes KinA just be provided with the ability of phosphorylation early period in cell growth, this is unfavorable for seven alkene first The dynamic regulation of naphthoquinones synthesizes, and settling mode is that the phosphorylation of KinA can be suppressed early period in cell growth, with cell Growth can release.It can not only inhibit Spo0A according to relevant report (DOI:10.1111/mmi.12939) Phr60-Rap60 Phosphorylation level, and be able to suppress the phosphorylation level of KinA.So introducing the response of Phr60-Rap60 group in cell System.And it is determined by experiment the proportionate relationship of the Phr60 and Rap60 of introducing, detailed process is as follows:
(1) recombinant vector pHT01-PspoiiaThe building of-GFP
In order to verify influence of the Phr60-Rap60 to Spo0A-P phosphorylation, the P regulated and controled by Spo0A-P is usedspoiiaStarting Son fusion green fluorescent protein (GFP) passes through the ability of regulation and control of the variation characterization Spo0A-P of fluorescent value.
1. with primer PspoiiaAnd P .forspoiia.rev promoter P is expandedspoiia(sequence is as shown in SEQ ID NO.11), It expands to obtain shown in green fluorescent protein GFP gene (sequence such as SEQ ID NO.15) using primer GFP.for and GFP.rev, Two segments are connected on pHT01 plasmid by Gibson multiple clips package technique, it is final to obtain recombinant vector pHT01- Pspoiia-GFP。
5 primer sequence table of table
(2) it on the basis of the recombinant bacterium BS17-2 that embodiment 3 obtains, is inserted on bacillus subtilis chromosome heterologous Transcription factor Rap60 (genebank ID:1115983).
1. merging the acquisition of segment
Using 168 genome of bacillus subtilis as template, amplification obtains the upstream homology arm sequence of Rap60 gene respectively Rap60up, Rap60 genetic fragment and PhagPromoter sequence (sequence is as shown in SEQ ID NO.16), artificial synthesized lox71- Zeo-lox66 box sequence (sequence is as shown in SEQ ID NO.2), then by four segment Rap60up、lox71-zeo-lox66 Box, PhagPromoter sequence, Rap60 genetic fragment carry out Overlap extension PCR, obtain fusion segment Rap60up-lox71- zeo-lox66-Phag-Rap60。
2. homologous recombination
By step 1. obtained in fusion segment be transformed into BS17-2 competent cell, and be coated on blasticidin resistance LB plate, select the single colonie grown on plate, carry out bacterium colony PCR verifying and be simultaneously sequenced.It is recombinated finally by Cre/lox System, blasticidin resistance gene zeo in knock-out bacterial strain, finally obtains bacterial strain Bacillus subtilis 168, P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr::lox72P43- aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::lacA Pmena- menA::thrC Pmena-menA::dacA Pveg-kinA-ΔPAS-AΔkinB Phag- Rap60 is named as BS17-2- Rap60。
(3) on the basis of the recombinant bacterium BS17-2-Rap60 that step (2) obtains, in bacillus subtilis chromosome Rap60 downstream of gene is not inserted into respectively, is inserted into the expression cassette (sequence of two copy, insertion signaling molecule Phr60 copied Column are as shown in SEQ ID NO.17), the genebank ID of Phr60 is 1115984, three plants of bacterium BS17-2-Rap60 are obtained, BS17-2-Phr60-Rap60, BS17-2- (Phr60) * 2-Rap60.
(4) obtained recombinant plasmid pHT01-P will be constructed in (1)spoiia- GFP converts wild type Bacillus respectively After three plants of bacterium obtained in subtilis 168 and step (3), four plants of bacterium are obtained, BS168-P is respectively designated asspoiia- GFP, BS17-2-Rap60-Pspoiia- GFP, BS17-2-Phr60-Rap60-Pspoiia- GFP, BS17-2- (Phr60) * 2-Rap60- Pspoiia-GFP。
(5) measurement of GFP fluorescent value
The 4 plants of bacterium constructed in step (4) are used into the culture of LB culture medium, are examined using 96 orifice plates by microplate reader within each hour Survey the variation of fluorescent value, fluorescin excitation wavelength: 395nm, launch wavelength 509nm.As a result as shown in Fig. 2, only expressing Rap60 Bacterial strain, fluorescent value is very weak to illustrate that Spo0A cannot activate promoter PspoiiaExpression, express the cell of two Phr60, fluorescence Intensity is apparently higher than the cell of one copy of expression, illustrates that the higher activation Spo0A phosphorylation degree of signaling molecule concentration is higher, more The expression of promoter can be activated.Therefore, go out bacterium germination using bacterial strain BS17-2- (Phr60) * 2-Rap60 as what is next tested Strain, is named as BS18, and genotype is Bacillus subtilis 168, P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs-ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena-menA::thrC Pmena-menA::dacA Pveg-kinA-ΔPAS-AΔkinB Phag-Rap60(Pnative-Phr60)2:: hag is used for subsequent experiment.
Relationship between the expression of embodiment 5:Spo0A-P controlling gene and cell concentration
Since the present invention is based on the relationships between cell concentration and Gene expression intensities, in order to determine cell concentration pair The influence of Spo0A-P detects the relationship between the group response Phr60-Rap60-Spo0A built and cell growth, P is used on pHT01 plasmidspoiiaAnd PabrbPromoter expresses GFP albumen, and building obtains recombinant plasmid pHT01-P respectivelyspoiia- GFP and pHT01-PabrbThen recombinant plasmid is transferred in bacterial strain BS18 obtained in embodiment 4 by-GFP, detect with thin The variation of intracellular growth fluorescent value, as a result such as Fig. 4, as can be seen from the figure when cell grows into 1.433 or so, promoter turns Regulation of the record ability by Spo0A-P.
Embodiment 6: the acquisition of mutant promoters
(1) acquisition of promoter is raised
In order to obtain the promoter different by Phr60-Rap60 ability of regulation and control, the promoter that will be raised by Spo0A-P PspoiiaFour Spo0A binding sites on (sequence is as shown in SEQ ID NO.11) are mutated respectively, are obtained 3 differences and are turned The promoter P of record abilityspoiia(cs-3)、Pspoiia(OA-3)、PSpoiia (cs-1,3), by these promoters fusion GFP gene, (sequence is such as Shown in SEQ ID NO.15), use the transcriptional capability of the variation characterization different promoters of fluorescent value.
The result shows that: the P after mutationSpoiia (cs-1,3)The transcriptional capability ratio of promoter (sequence is as shown in SEQ ID NO.12) It is original to improve about 7 times, therefore by promoter PSpoiia (cs-1,3), for enhancing the key gene table in menaquinone route of synthesis It reaches.
(2) acquisition of promoter is lowered
The promoter P that will be lowered by Spo0A-PabrbSpo0A-P bound site on (sequence is as shown in SEQ ID NO.13) Point carries out conserved sequence mutation, obtains the promoter P of 3 different transcriptional capabilitiesabrB(OA-2)、PabrB(cs-1)、PabrB(cs-2), and will These promoters merge GFP gene (sequence is as shown in SEQ ID NO.15), characterize different promoters using the variation of fluorescent value Transcriptional capability.
The result shows that: as shown in figure 3, PabrB(cs-1)The transcriptional capability of (sequence is as shown in SEQ ID NO.14) promoter is only There is original 18%, therefore by promoter PabrB(cs-1), for inhibiting the expression of menaquinone competition pathway key gene.
Embodiment 7: the building of recombinant bacterium BS19
In order to increase the synthesis of MK-7, P is used on the basis of recombinant bacterium BS18abrB(cs-1)Promoter replaces acetone in situ Acid kinase (pyk, genebank ID:936596) gene and undecenyl pyrophosphate synthetase enzyme (uppS, genebank ID:939640) promoter of gene obtains recombinant bacterial strain BS19, as the growth of cell gradually limits carbon flow to competition approach Flow.The transcriptional level of final related gene is as shown in Figure 6.Specific building process is as follows:
(1) clone of gene
1. amplification obtains the upstream homology arm sequence of pyk gene respectively using 168 genome of bacillus subtilis as template pykup, pyk genetic fragment, artificial synthesized PabrB(cs-1)Promoter sequence (sequence is as shown in SEQ ID NO.14), lox71- Zeo-lox66 box sequence (sequence is as shown in SEQ ID NO.2), then by four segment pykup, lox71-zeo-lox66 box, PabrB(cs-1)Promoter sequence, pyk genetic fragment carry out Overlap extension PCR, obtain fusion segment pykup-lox71-zeo- lox66-PabrB(cs-1)-pyk。
2. amplification obtains the upstream homology arm sequence of uppS gene respectively using 168 genome of bacillus subtilis as template uppSup, uppS genetic fragment, artificial synthesized PabrB(cs-1)Promoter sequence (sequence is as shown in SEQ ID NO.14), lox71- Zeo-lox66 box sequence (sequence is as shown in SEQ ID NO.2), then by four segment uppSup, lox71-zeo-lox66 box, PabrB(cs-1)Promoter sequence, uppS genetic fragment carry out Overlap extension PCR, obtain fusion segment uppSup-lox71- zeo-lox66-PabrB(cs-1)-uppS。
(2) homologous recombination
Fusion segment obtained in step (1) is transformed into BS18 competent cell, and is coated on blasticidin resistance LB plate, select the single colonie grown on plate, carry out bacterium colony PCR verifying and be simultaneously sequenced.It is recombinated finally by Cre/lox System, blasticidin resistance gene zeo in knock-out bacterial strain, finally obtains bacterial strain Bacillus subtilis 168, P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs- ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::lacA Pmena-menA::thrC Pmena-menA::dacA Pveg-kinA-ΔPAS-AΔkinB Phag-Rap60(Pnative-Phr60)2::hag PabrB(cs-1)- pyk::pyk PabrB(cs-1)- uppS::uppS is named as BS19.
Embodiment 8: the building of recombinant bacterium BS20
Using method similar to Example 7, embodiment 6 is used on the basis of the recombinant bacterium BS19 that embodiment 7 obtains Obtained in Pspoiia(cs-1,3)Seven polydiene propyl diphosphate synthases of promoter expression (HepS/T, genebank ID: 938998) gene and 4- hydroxy-3-methyl but-2-ene base diphosphonic acid reductase (ispH, genebank ID:937900) gene, Recombinant bacterial strain BS20 is obtained, and with the growth of cell, Carbon flux is made to flow to the route of synthesis of MK-7.Final related gene turns Record is horizontal as shown in Figure 6.
(1) acquisition of segment is merged
Using 168 genome of bacillus subtilis as template, amplification obtains the upstream homology arm sequence of HepS/T gene respectively HepS/Tup, HepS/T genetic fragment, lox71-zeo-lox66 box sequence (sequence is as shown in SEQ ID NO.2), then by four A segment HepS/Tup, lox71-zeo-lox66 box, Pspoiia(cs-1,3)Promoter sequence, HepS/T genetic fragment are overlapped Extension PCR obtains fusion segment HepS/Tup-lox71-zeo-lox66-Pspoiia(cs-1,3)-HepS/T。
Using 168 genome of bacillus subtilis as template, amplification obtains the upstream homology arm sequence of ispH gene respectively ispHup, ispH genetic fragment, lox71-zeo-lox66 box sequence (sequence is as shown in SEQ ID NO.2), then by four pieces Section ispHup, lox71-zeo-lox66 box, Pspoiia(cs-1,3)Promoter sequence, ispH genetic fragment carry out Overlap extension PCR, Obtain fusion segment ispHup-lox71-zeo-lox66-Pspoiia(cs-1,3)-ispH。
(2) homologous recombination
Fusion segment obtained in step (1) is transformed into BS19 competent cell, and is coated on blasticidin resistance LB plate, select the single colonie grown on plate, carry out bacterium colony PCR verifying and be simultaneously sequenced.It is recombinated finally by Cre/lox System, blasticidin resistance gene zeo in knock-out bacterial strain, finally obtains bacterial strain Bacillus subtilis 168, P43-menF P43-menB Phbs-menE P43-entCΔdhbB Phbs-tkt P43-ppsAΔptsG Phbs-aroGfbr P43-aroK Phbs- ispA P43-hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA::ganA Pmena-menA::thrC Pmena-menA::dacA Pveg-kinA-ΔPAS-AΔkinB Phag-Rap60(Pnative-Phr60)2::hag PabrB(cs-1)- pyk::pyk PabrB(cs-1)-uppS::uppS Pspoiia(cs-1,3)-ispH::ispH Pspoiia(cs-1,3)- HepS/T::HepS/T, It is named as BS20.
Embodiment 9: the fermentation of bacterial strain
Seed culture based formulas (mass percent): tryptone 1%, yeast extract 0.5%, sodium chloride 1%.
Medium of shaking flask fermentation formula (mass percent): soy peptone 3-8%, glucose 3-8%, sucrose 3-8%, KH2PO30.06%.
Ferment tank culture medium prescription (mass percent): soy peptone 3-8%, glucose 3-8%, sucrose 3- 8%, KH2PO30.06%.
(1) prepared by seed liquor:
Recombinant bacterium BS1~BS17, BS19, BS20 for constructing in embodiment 1,7,8 are inoculated into seed culture respectively In base, 12h is cultivated at 37 DEG C, 220rpm and obtains bacillus subtilis seed liquor.
(2) shake flask fermentation culture
Seed liquor obtained in step (1) is transferred in 250mL shaking flask with 5% inoculum concentration, liquid amount 15mL.Fermentation 40 DEG C of condition, 220rpm cultivate 6 days, fermentation results as shown in fig. 7, BS19 fermentation 6 days after yield reach 170mg/L, BS20 yield after fermentation 6 days reaches 360mg/L, and MK-7 yield is bacillus subtilis (Bacillus subtilis) 37.89 times of 168.
(3) fermented and cultured
Seed liquor obtained in step (1) is transferred to fermentation medium with 15% inoculum concentration, uses 15-L fermentor pair Recombinant bacterial strain BS20 ferments, and 40 DEG C of fermentation condition, tank presses 0.05Mpa, ventilatory capacity 10vvm, stirs 500rpm, is added by stream The mode of glucose controls the concentration of glucose in 5g/L.Fermentation results are as shown in figure 8, yield reaches 230mg/L, and after fermenting The pH meta-alkali of phase fermentation liquid.
6 recombinant bacterial strain fermenting and producing MK-7 of table
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>using the method for bacillus subtilis group response regulator control system synthesis MK-7
<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 synthesized
<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 synthesized
<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 synthesized
<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 synthesized
<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 synthesized
<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 synthesized
<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 synthesized
<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 synthesized
<400> 18
gcattcatcg tcatatcatc atagctgagc 30
<210> 19
<211> 59
<212> DNA
<213>artificial synthesized
<400> 19
tgtgaaattg ttatccgctc gagacattcc tccataatcc ttaaaatgct tttaatacc 59
<210> 20
<211> 27
<212> DNA
<213>artificial synthesized
<400> 20
tgataggtgg tatgttttcg cttgaac 27
<210> 21
<211> 28
<212> DNA
<213>artificial synthesized
<400> 21
gtgtacattc ctctcttacc tataatgg 28
<210> 22
<211> 53
<212> DNA
<213>artificial synthesized
<400> 22
tttaaggatt atggaggaat gtctcgagcg gataacaatt tcacacagga aac 53
<210> 23
<211> 30
<212> DNA
<213>artificial synthesized
<400> 23
aatcatacct accacaatat catgctcaag 30
<210> 24
<211> 22
<212> DNA
<213>artificial synthesized
<400> 24
ttccatatcc tgcggcgttt gt 22
<210> 25
<211> 25
<212> DNA
<213>artificial synthesized
<400> 25
acgaagttat tcagtcctgc tcctc 25
<210> 26
<211> 25
<212> DNA
<213>artificial synthesized
<400> 26
cttgacagcg ggtttttcat acagc 25
<210> 27
<211> 23
<212> DNA
<213>artificial synthesized
<400> 27
gctgtccatg tactagacgt ccc 23
<210> 28
<211> 19
<212> DNA
<213>artificial synthesized
<400> 28
actggcttgg acggtggtt 19
<210> 29
<211> 47
<212> DNA
<213>artificial synthesized
<400> 29
gtatgaaaaa cccgctgtca agtcctcctt gttctcttag cccttcg 47
<210> 30
<211> 39
<212> DNA
<213>artificial synthesized
<400> 30
ctgggaaaac cctggcggct gatgctccgc tcgatatgg 39
<210> 31
<211> 20
<212> DNA
<213>artificial synthesized
<400> 31
cgccaatcgc tgacctgaag 20
<210> 32
<211> 24
<212> DNA
<213>artificial synthesized
<400> 32
ctggtgtaga cggcgtaaaa acag 24
<210> 33
<211> 32
<212> DNA
<213>artificial synthesized
<400> 33
gctcattcct ccttgatatg atcggataat ga 32
<210> 34
<211> 41
<212> DNA
<213>artificial synthesized
<400> 34
tatcaaggag gaatgagcat gggtaaggga gaagaacttt t 41
<210> 35
<211> 42
<212> DNA
<213>artificial synthesized
<400> 35
ccaagcttct gcagttattt gtacagttca tccatgccat gt 42
<210> 36
<211> 36
<212> DNA
<213>artificial synthesized
<400> 36
ctgtacaaat aactgcagaa gcttggcgta atcatg 36
<210> 37
<211> 44
<212> DNA
<213>artificial synthesized
<400> 37
gccgtctaca ccagttgctc aaaaaaatct cggtcagatg ttac 44

Claims (10)

1. a kind of method using bacillus subtilis group response regulator control system synthesis MK-7, which is characterized in that in withered grass bud Following transformation has been carried out on the chromosome of spore bacillus:
(1) gene of histidine kinase KinA is subjected to truncated mutant and has carried out constitutive expression, while knocking out histidine The gene of kinases KinB；
(2) P will be containedhagOn the Rap60 gene integration to chromosome of promoter, and Rap60 downstream of gene is integrated on chromosome The expression cassette of the signaling molecule Phr60 of two copies；
(3) P is usedabrB(cs-1)The starting of promoter replacement gene encoding for pyruvate kinase and undecenyl pyrophosphate synthetase gene Son；The PabrB(cs-1)The sequence of promoter is as shown in SEQ ID NO.14.
2. the method according to claim 1, wherein histidine kinase KinA described in step (1) Genebank ID is 939230；The truncated mutant refers to the signal transduction part for knocking out histidine kinase KinA gene The region PAS-A, the sequence of the histidine kinase KinA gene kinA- Δ PAS-A after knockout is as shown in SEQ ID NO.9；It is described The genebank ID of histidine kinase KinB is 937167.
3. the method according to claim 1, wherein P described in step (2)hagPromoter sequence such as SEQ ID Shown in NO.16；The genebank ID of the transcription factor Rap60 is 1115983；The expression cassette of the signaling molecule Phr60 Sequence is as shown in SEQ ID NO.17.
4. the method according to claim 1, wherein gene encoding for pyruvate kinase pyk described in step (3) Genebank ID is 936596；The genebank ID of the undecenyl pyrophosphate synthetase gene uppS is 939640.
5. the method according to claim 1, wherein the method also includes using Pspoiia(cs-1,3)Start sublist Up to seven polydiene propyl diphosphate synthase genes and 4- hydroxy-3-methyl but-2-ene base diphosphonic acid reductase gene；It is described Pspoiia(cs-1,3)The sequence of promoter is as shown in SEQ ID NO.12.
6. a kind of recombinant bacterium for producing menaquinone, which is characterized in that carried out on the chromosome of bacillus subtilis as Lower transformation:
(1) gene of histidine kinase KinA is subjected to truncated mutant and has carried out constitutive expression, while knocking out histidine The gene of kinases KinB；
(2) P will be containedhagOn the Rap60 gene integration to chromosome of promoter, and Rap60 downstream of gene is integrated on chromosome The expression cassette of the signaling molecule Phr60 of two copies；
(3) P is usedabrB(cs-1)The starting of promoter replacement gene encoding for pyruvate kinase and undecenyl pyrophosphate synthetase gene Son；The PabrB(cs-1)The sequence of promoter is as shown in SEQ ID NO.14.
7. recombinant bacterium according to claim 6, which is characterized in that histidine kinase KinA's described in step (1) Genebank ID is 939230；The truncated mutant refers to the signal transduction part for knocking out histidine kinase KinA gene The region PAS-A, the sequence of the histidine kinase KinA gene kinA- Δ PAS-A after knockout is as shown in SEQ ID NO.9；It is described The genebank ID of histidine kinase KinB is 937167.
8. recombinant bacterium according to claim 6, which is characterized in that P described in step (2)hagPromoter sequence such as SEQ ID Shown in NO.16；The genebank ID of the transcription factor Rap60 is 1115983；The expression cassette of the signaling molecule Phr60 Sequence is as shown in SEQ ID NO.17.
9. recombinant bacterium according to claim 6, which is characterized in that gene encoding for pyruvate kinase pyk's described in step (3) Genebank ID is 936596；The genebank ID of the undecenyl pyrophosphate synthetase gene uppS is 939640.
10. recombinant bacterium according to claim 6, which is characterized in that the recombinant bacterium is done on the basis of claim 6 Following transformation: P is usedspoiia(cs-1,3)Promoter expresses seven polydiene propyl diphosphate synthase genes and 4- hydroxy-3-methyl But-2-ene base diphosphonic acid reductase gene；The Pspoiia(cs-1,3)The sequence of promoter is as shown in SEQ ID NO.12.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110229772A (en) * 2019-06-04 2019-09-13 南通励成生物工程有限公司 A kind of recombined bacillus subtilis of seven dilute menadione yield of raising and its application
CN111471634A (en) * 2020-03-14 2020-07-31 天津大学青岛海洋技术研究院 Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain
CN111549045A (en) * 2020-01-17 2020-08-18 西宝生物科技（上海）股份有限公司 Vitamin K is improved by utilizing recombinant bacillus natto2Method of producing
CN112481278A (en) * 2020-12-09 2021-03-12 江南大学 Biosensor based on AIP induction and application thereof
CN112852691A (en) * 2019-11-28 2021-05-28 中国科学院微生物研究所 Recombinant escherichia coli producing MK-7 and construction method and application thereof
WO2021175759A1 (en) * 2020-03-04 2021-09-10 Basf Se Method for the production of constitutive bacterial promoters conferring low to medium expression
CN114350699A (en) * 2021-12-02 2022-04-15 江南大学 Strain for producing D-psicose 3-epimerase and application thereof
CN116064633A (en) * 2022-11-03 2023-05-05 安徽工程大学 Construction of efficient biosynthesis of vitamin K 2 Engineering bacteria method
CN116286574A (en) * 2023-02-09 2023-06-23 中国农业大学 CRISPRa method for accurately regulating Bacillus subtilis endogenous polygene expression and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002034554A (en) * 2000-07-28 2002-02-05 Mitsukan Group Honsha:Kk Bacillus natto producing vitamin k at high concentration, method for breeding the same and fermented soybean
CN106222190A (en) * 2016-08-25 2016-12-14 江南大学 A kind of bacillus subtilis logarithmic (log) phase expression system
CN107058316A (en) * 2016-12-09 2017-08-18 江南大学 A kind of bacillus subtilis self-induction expression system and its application
KR20180076414A (en) * 2016-12-27 2018-07-06 민병규 Novel Bacillus subtilis BSM-KO2 having improved menaquinone-7 productivity and method for mass production of menaquinone-7 by using thereof
CN108715825A (en) * 2018-06-01 2018-10-30 天津大学 Gene overexpression and the bacterial strain of acquisition, application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002034554A (en) * 2000-07-28 2002-02-05 Mitsukan Group Honsha:Kk Bacillus natto producing vitamin k at high concentration, method for breeding the same and fermented soybean
CN106222190A (en) * 2016-08-25 2016-12-14 江南大学 A kind of bacillus subtilis logarithmic (log) phase expression system
CN107058316A (en) * 2016-12-09 2017-08-18 江南大学 A kind of bacillus subtilis self-induction expression system and its application
KR20180076414A (en) * 2016-12-27 2018-07-06 민병규 Novel Bacillus subtilis BSM-KO2 having improved menaquinone-7 productivity and method for mass production of menaquinone-7 by using thereof
CN108715825A (en) * 2018-06-01 2018-10-30 天津大学 Gene overexpression and the bacterial strain of acquisition, application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CUI S.等: "Engineering a Bifunctional Phr60-Rap60-Spo0A Quorum-Sensing Molecular Switch for Dynamic Fine-Tuning of Menaquinone‑7 Synthesis in Bacillus subtilis", 《ACS SYNTH. BIOL.》 *

Cited By (15)

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CN110229772A (en) * 2019-06-04 2019-09-13 南通励成生物工程有限公司 A kind of recombined bacillus subtilis of seven dilute menadione yield of raising and its application
CN112852691A (en) * 2019-11-28 2021-05-28 中国科学院微生物研究所 Recombinant escherichia coli producing MK-7 and construction method and application thereof
CN112852691B (en) * 2019-11-28 2022-09-16 中国科学院微生物研究所 Recombinant escherichia coli producing MK-7 and construction method and application thereof
CN111549045B (en) * 2020-01-17 2023-09-19 西宝生物科技（上海）股份有限公司 Vitamin K improvement by utilizing recombinant bacillus natto 2 Method for producing yield
CN111549045A (en) * 2020-01-17 2020-08-18 西宝生物科技（上海）股份有限公司 Vitamin K is improved by utilizing recombinant bacillus natto2Method of producing
WO2021175759A1 (en) * 2020-03-04 2021-09-10 Basf Se Method for the production of constitutive bacterial promoters conferring low to medium expression
CN111471634A (en) * 2020-03-14 2020-07-31 天津大学青岛海洋技术研究院 Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain
CN112481278A (en) * 2020-12-09 2021-03-12 江南大学 Biosensor based on AIP induction and application thereof
CN112481278B (en) * 2020-12-09 2023-02-21 江南大学 Biosensor based on AIP induction and application thereof
CN114350699A (en) * 2021-12-02 2022-04-15 江南大学 Strain for producing D-psicose 3-epimerase and application thereof
CN114350699B (en) * 2021-12-02 2024-03-26 江南大学 Strain for producing D-psicose 3-epimerase and application thereof
CN116064633A (en) * 2022-11-03 2023-05-05 安徽工程大学 Construction of efficient biosynthesis of vitamin K 2 Engineering bacteria method
CN116064633B (en) * 2022-11-03 2024-06-11 安徽工程大学 Construction of efficient biosynthesis of vitamin K2Engineering bacteria method
CN116286574A (en) * 2023-02-09 2023-06-23 中国农业大学 CRISPRa method for accurately regulating Bacillus subtilis endogenous polygene expression and application thereof
CN116286574B (en) * 2023-02-09 2023-12-12 中国农业大学 CRISPRa method for accurately regulating Bacillus subtilis endogenous polygene expression and application thereof

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