CN110157654A - A kind of bafillus natto recombinant bacterium and its construction method and application - Google Patents

A kind of bafillus natto recombinant bacterium and its construction method and application Download PDF

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CN110157654A
CN110157654A CN201910425269.7A CN201910425269A CN110157654A CN 110157654 A CN110157654 A CN 110157654A CN 201910425269 A CN201910425269 A CN 201910425269A CN 110157654 A CN110157654 A CN 110157654A
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许正宏
史劲松
李会
张晓梅
徐行
刘珍
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Jiangnan University
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Abstract

The invention discloses a kind of bafillus natto recombinant bacterium and its construction method and applications, belong to gene engineering technology field.Present system is attempted to knock out key enzyme ubiA and aroH in by-product feedback metabolic pathway using CRISPR-CAS gene editing technology for the first time, and Enhanced expressing key gene menA, menC, menD in the genome, gamma-polyglutamic acid is obtained while improving farnoquinone (MK-7) concentration, and gamma-polyglutamic acid concentration reaches higher level, accomplishes the new approaches of the Joint Production of farnoquinone (MK-7) and gamma-polyglutamic acid.

Description

A kind of bafillus natto recombinant bacterium and its construction method and application
Technical field
The present invention relates to a kind of bafillus natto recombinant bacterium and its construction method and applications, belong to technique for gene engineering neck Domain.
Background technique
Vitamin K2(menaquinone, MK) is a kind of liposoluble vitamin, the naphthoquinones base with phylloquinone bioactivity The derivative of group, is one of indispensable important vitamin in human body.Farnoquinone is aphthoquinone series compound, yellowish Color crystal is shared 14 kinds of forms according to the length difference of C-3 isoprene side chains on its molecular structure, is indicated to refer to side with MK-n The number of isoprene unit on chain), wherein MK-7 (Agua-Mephyton 2) bioactivity is the most significant.
γ-polyglutamic acid (γ-PGA) is to be polymerized by D-Glu and (or) Pidolidone monomer through γ-amido bond A kind of multifunctional bio high molecular polymer, γ-PGA be form natto viscoloid main ingredient, have promote mineral The effect that matter absorbs.γ-PGA special molecular structure, makes it have extremely strong moisture-retaining capacity, addition γ-PGA in cosmetics or It in skin care products, can effectively increase the moisture-retaining capacity of skin, promote skin health.
MK-7 and gamma-polyglutamic acid are the metabolites of bafillus natto (Bacillus subtilis natto). Now one plant of bafillus natto (Bacillus subtilis natto) ND-1-A27, Neng Gouyong has been obtained by mutagenic and breeding Come fermenting and producing farnoquinone (MK-7).But bafillus natto (Bacillus subtilis natto) ND-1-A27 exists Fermenting and producing farnoquinone (MK-7) concentration is lower in industrial fermentation medium, and gamma-polyglutamic acid concentration is lower, is not suitable for In industrial production.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of recombinant bacterium of bafillus natto, by the genome Enhanced expressing menA, menC, menD gene, and ubiA gene and aroH gene are knocked out, reach forced for vitamins K2(MK- 7) concentration, and the purpose of Joint Production gamma-polyglutamic acid.
The first purpose of the invention is to provide a kind of bafillus natto recombinant bacterium, the recombinant bacterium has knocked out 4- hydroxyl Benzoic acid polyprenyltransferase enzymes gene (ubiA) has knocked out branch's mutase gene (aroH), while in the genome Enhanced expressing 1,4- dihydroxy-2-naphthoic acid polyprenyltransferase enzymes gene (menA), adjacent succinylbenzoic acid synthesis Enzyme gene (menC) and 2- succinyl -5- enolpyruvyl acyl -6- hydroxyl -3- cyclohexene -1- carboxylic acid synthetase gene (menD).
Further, the amino acid sequence of the 4-hydroxybenzoate polyprenyltransferase such as SEQ ID NO.1 It is shown.
Further, the amino acid sequence of branch's mutase is as shown in SEQ ID NO.2.
Further, the amino acid sequence such as SEQ of the 1,4-dihydroxy-2-naphthsaisyuoic acid polyprenyltransferase enzymes Shown in ID NO.3, the amino acid sequence of neighbour's succinylbenzoic acid synzyme is as shown in SEQ ID NO.4, the 2- amber The nucleotide sequence of acyl -5- enolpyruvyl acyl -6- hydroxyl -3- cyclohexene -1- carboxylic acid synthetase is as shown in SEQ ID NO.5.
Further, the recombinant bacterium is to be with bafillus natto Bacillus subtilis nattoND-1-A27 Host strain.During the bafillus natto Bacillus subtilis nattoND-1-A27 was preserved on March 14th, 2018 State's Type Tissue Collection, preservation address are Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2018131, Bafillus natto and its application of farnoquinone (MK-7) are produced referring specifically to mono- plant height of CN201810392873.X.
Further, the recombinant bacterium is that gene knockout or gene overexpression are carried out using pMA5 as expression vector.
A second object of the present invention is to provide the construction methods of the recombinant bacterium described in one kind, include the following steps:
(1) synthesizing ribonucleotide sequence Cas9 protein expression frame as shown in SEQ ID NO.6 is designed, nucleotide sequence is such as SgRNA scaffold expression cassette shown in SEQ ID NO.7 and nucleotide sequence the sgRNA piece as shown in SEQ ID NO.8 Section；
(2) Cas9 protein expression frame is connected on pMA5 expression vector, obtains plasmid pMA5-Cas9；
(3) sgRNA scaffold expression cassette is connected on plasmid pMA5-Cas9；
(4) bafillus natto ND-1-A27 genomic DNA is extracted, each 500bp of ubiA upstream and downstream is expanded, by gene menA The homologous repairing sequence is connected on the plasmid of step (3) by amplification collectively as homologous repairing sequence, obtains natto gemma Bacillus gene editor's carrier pMAC；
(5) bafillus natto gene editing carrier pMAC electricity is gone in ND-1-A27 competence, screening obtains ubiA It knocks out, the bafillus natto of menA Enhanced expressing, and carries out non-resistant passage, eliminate plasmid experiment, finally obtain non-resistant Bafillus natto ND-1-A27A △ ubiA；
(6) bafillus natto ND-1-A27 genomic DNA is extracted, each 500bp of aroH upstream and downstream is expanded, by gene MenC, menD are expanded collectively as homologous repairing sequence, and the homologous repairing sequence is connected on the plasmid of step (3), is obtained Obtain bafillus natto gene editing carrier pMAC-1；
(7) bafillus natto gene editing carrier pMAC-1 electricity is gone in ND-1-A27A △ ubiA competence, is screened Obtain aroH knockout and the bafillus natto ND-1-A27ACD △ ubiA △ aroH of Enhanced expressing menC and menD.
Third object of the present invention is to provide the recombinant bacteriums described in one kind in fermenting and producing vitamin K2(MK-7) and γ- Application in polyglutamic acid.
Fourth object of the present invention is to provide recombinant bacterium fermenting and producing vitamin K described in one kind2(MK-7) and γ-is poly- The recombinant bacterium single colonie is inoculated in seed culture medium by the method for glutamic acid, 35~38 DEG C, 100~150r/min culture 6 ~10h obtains seed culture fluid；Seed culture fluid is forwarded to fermentation medium, 35~38 DEG C of cultures 3 with 1~5% inoculum concentration ~6 days, fermentation liquid is obtained, separation and Extraction obtains vitamin K from fermentation liquid2(MK-7) and γ-polyglutamic acid.
Further, the seed culture medium are as follows: 25~35g/L of glucose, 35~45g/L of peptone, sodium chloride 3~ 7g/L, 3~7g/L of beef extract, 3~7g/L of yeast extract.
Further, the fermentation medium are as follows: glycerol 40-60g/L, soy peptone 25-35g/L, yeast powder 0.5- 1g/L, K2HPO40.2-0.6g/L。
The beneficial effects of the present invention are:
Present system is attempted to be metabolized the general a kind of jade feedback regulation of by-product using CRISPR-CAS gene editing technology for the first time Key enzyme ubiA and by-product amino acid feedback adjust key enzyme aroH in metabolic pathway and are knocked out in approach, while in base Because organizing upper Enhanced expressing key gene menA, menC, menD, obtained while improving farnoquinone (MK-7) concentration Gamma-polyglutamic acid, and the concentration of gamma-polyglutamic acid reaches higher level, accomplishes farnoquinone (MK-7) and gamma-polyglutamic acid Joint Production new approaches.
Specific embodiment
The present invention is further explained in the light of specific embodiments, so that those skilled in the art can be preferably Understand the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Technical solution of the present invention, such as not special standby explanation, is the ordinary skill in the art；The reagent or material, If not otherwise specified, commercial channel is derived from.
Embodiment 1:
Non-resistant bafillus natto ND-1-A27A △ ubiA preparation method:
(1) the Cas9 protein expression frame (SEQ for meeting bafillus natto ND-1-A27 password sub-feature is designed and synthesized ID No.6), sgRNA expression cassette (SEQ ID No.7), sgRNA segment (SEQ ID No.8)；
The Cas protein expression frame T-A clone of synthesis obtains plasmid T-Cas9 from pMD19-T-simple carrier.It reuses Primer amplification segment, the seamless spliced pMA5 to BamH I and Mlu I digestion after glue recycling, obtains plasmid pMAC；
(2) it with primer amplification using ubiA as the sgRNA of shot design, is expanded and is obtained with Primer star archaeal dna polymerase SgRNA scaffold expression cassette with target spot N20；
Amplified production carries out connecting the site MluI into plasmid pMA5-Cas9 after MluI digestion after purification；
(3) bafillus natto ND-1-A27 genomic DNA is extracted, Primer Star archaeal dna polymerase and correlation are used Primer expands each 500bp and menA gene of ubiA upstream and downstream as homologous reparation sequence respectively, and according to the upstream ubiA 500bp, The seamless spliced pst I site in plasmid pMA5-Cas9 of the downstream menA and ubiA 500bp sequence.Obtain the natto bud Spore bacillus ND-1-A27 genome editor's carrier pMAC；
(4) Plasmid DNA is accessed in the bafillus natto ND-1-A27 competent cell of preparation；Electricity turns competent cell In, ice bath 5min；It is added in the electric revolving cup of pre-cooling, shock by electricity primary (2.0kV, 4.5~5.0ms), can if electric shock is inefficient It carries out twice；Electric shock, which finishes, is added 700 μ L recovery medium RM (LB+0.5M sorbierite+0.38M sweet dews into electric revolving cup immediately Alcohol), the thallus pressure-vaccum of bottom of a cup is got up, then sets 37 DEG C, 220rpm culture 3-3.5h makes thallus restoration ecosystem；With 6000rpm After being centrifuged 5min, supernatant is sucked, the kalamycin resistance plate of 10 μ g/ml of band, 37 DEG C of trainings are applied after staying 100 μ L or so that thallus is resuspended The transformant grown is verified after supporting 12h；
(5) by the recombination bafillus natto ND-1-A27 positive transformant for carrying pMAC plasmid of acquisition, pMA5 is used Universal primer carries out bacterium colony PCR detection, to the transformant that PCR detection ubiA clip size is changed, recycles it and expands piece Section is simultaneously sent to Nanjing Jin Weizhi biotechnology company and is sequenced, and sequencing result and protogene are compared, and detects ubiA base Because whether knock out successfully and menA whether Enhanced expressing；
(6) will knock out successful single colonie picking into the seed culture medium of non-resistant subculture to eliminate pMAC matter Grain obtains non-resistant bafillus natto ND-1-A27A △ ubiA.
Embodiment 2:
Bafillus natto ND-1-A27ACD △ ubiA △ aroH preparation method:
(1) it with primer amplification using aroH as the sgRNA of shot design, is expanded and is obtained with Primer star archaeal dna polymerase SgRNA scaffold expression cassette with target spot N20；
Amplified production carries out connecting the site MluI into plasmid pMA5-Cas9 after MluI digestion after purification；
(2) bafillus natto ND-1-A27 genomic DNA is extracted, Primer Star archaeal dna polymerase and correlation are used Primer expands each 500bp and menC gene of aroH upstream and downstream, menD gene as homologous reparation sequence respectively, and according on aroH Swim the seamless spliced pst I site in plasmid pMA5-Cas9 of the downstream 500bp, menC, menD and aroH 500bp sequence.It obtains Bafillus natto ND-1-A27 genome editor's carrier pMAC-1；
(3) Plasmid DNA is accessed in the bafillus natto ND-1-A27A △ ubiA competent cell of preparation；Electricity turns sense By in state cell, ice bath 5min；It is added in the electric revolving cup of pre-cooling, shock by electricity primary (2.0kV, 4.5~5.0ms), if electric shock effect Rate is not high to be carried out twice；Electric shock finish immediately toward electricity revolving cup in be added 700 μ L recovery medium RM (LB+0.5M sorbierite+ 0.38M mannitol), the thallus pressure-vaccum of bottom of a cup is got up, then sets 37 DEG C, 220rpm culture 3-3.5h makes thallus restoration ecosystem； After being centrifuged 5min with 6000rpm, supernatant is sucked, the kalamycin resistance that 10 μ g/ml of band is applied after staying 100 μ L or so that thallus is resuspended is flat Plate verifies the transformant grown after 37 DEG C of culture 12h；
(4) it by the recombination bafillus natto ND-1-A27 positive transformant for carrying pMAC-1 plasmid of acquisition, uses PMA5 universal primer carries out bacterium colony PCR detection, to the transformant that PCR detection aroH clip size is changed, recycles its expansion Increase segment and send to Nanjing Jin Weizhi biotechnology company and be sequenced, sequencing result and protogene are compared, detected AroH gene whether knock out successfully and menC, menD whether Enhanced expressing success；
(5) will knock out successful single colonie picking into the seed culture medium of non-resistant subculture to eliminate pMAC-1 matter Grain obtains bafillus natto ND-1-A27ACD △ ubiA △ aroH.
Embodiment 3:
Utilize the application of Metabolically engineered obtained strains Joint Production farnoquinone (MK-7) and gamma-polyglutamic acid.Including under State step:
(1) the cellular liquid culture of bafillus natto ND-1-A27ACD △ ubiA △ aroH: picking nutrition fine jade is prepared One ring of bafillus natto ND-1-A27ACD △ ubiA △ aroH bacterial strain, is seeded in equipped with 30mL seed culture on rouge culture medium In the 250mL conical flask of base, at 37 DEG C, the revolving speed culture 8h on shaking table with 120r/min is placed in logarithmic phase, obtains and receives The cellular liquid culture of beans bacillus ND-1-A27ACD △ ubiA △ aroH bacterial strain.
(2) ingredient and proportion of fermentation medium are as follows: glycerol 50g/L, soy peptone 30g/L, yeast powder 0.6g/L, K2HPO40.3g/L；121 DEG C sterilize 20 minutes.
(3) shake flask fermentation: the cellular liquid of above-mentioned bafillus natto ND-1-A27ACD △ ubiA △ aroH bacterial strain is trained Object is supported to be seeded in the 250mL conical flask equipped with sterilized 30mL fermentation medium with the inoculum concentration of 2% (w/w), at 37 DEG C, Fermented and cultured 3-6 days under the conditions of stationary culture obtain sticky fermentation liquid.
(4) concentration mensuration: use n-hexane: isopropanol (2:1) is as extractant, by above-mentioned ND-1-A27ACD △ ubiA The fermentation liquid of △ aroH bacterial strain carries out product extraction, uses C18 column；Mobile phase is 100% methanol；40 DEG C of column temperature；Flow velocity is 1.0ml does HPLC analysis.In addition dilution fermentation liquid measures the concentration of gamma-polyglutamic acid by HPLC.
When fermenting by the 4th day, farnoquinone (MK-7) concentration is increased to 80.29mg/L from the 30.87mg/L before transformation, Gamma-polyglutamic acid concentration reaches 103.53g/L.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention Protection scope within.Protection scope of the present invention is subject to claims.
Sequence table
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Gly Leu Asp Thr Ala Glu Ser Val Gly Ile Gly Gly Ala Ile Val Arg
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His Gly Met Lys Pro Lys Thr Ile Leu Gln Leu Ala Leu Ala Ser Tyr
85 90 95
Gly Ile Ala Ile Leu Leu Gly Val Tyr Ile Cys Ala Ser Ser Ser Trp
100 105 110
Trp Leu Ala Leu Ile Gly Leu Val Gly Met Ala Ile Gly Tyr Leu Tyr
115 120 125
Thr Gly Gly Pro Leu Pro Ile Ala Tyr Thr Pro Phe Gly Glu Leu Phe
130 135 140
Ser Gly Ile Cys Met Gly Ser Val Phe Val Leu Ile Ser Phe Phe Ile
145 150 155 160
Gln Thr Asp Met Ile Asn Thr Gln Ser Ile Leu Ile Ser Ile Pro Ile
165 170 175
Ala Ile Leu Val Gly Ala Ile Asn Leu Ser Asn Asn Ile Arg Asp Ile
180 185 190
Glu Glu Asp Lys Lys Gly Gly Arg Lys Thr Leu Ala Ile Leu Met Gly
195 200 205
His Lys Gly Ala Val Thr Leu Leu Ala Ala Ser Phe Ala Val Ala Tyr
210 215 220
Ile Trp Val Val Gly Leu Val Ile Thr Gly Ala Ala Ser Pro Trp Leu
225 230 235 240
Phe Val Val Phe Leu Ser Val Pro Lys Pro Val Gln Ala Val Lys Gly
245 250 255
Phe Val Gln Lys Glu Met Pro Met Asn Met Ile Val Ala Met Lys Ser
260 265 270
Thr Ala Gln Thr Asn Thr Phe Phe Gly Phe Leu Leu Ser Ile Gly Leu
275 280 285
Leu Ile Ser Tyr Phe Arg
290
<210> 4
<211> 371
<212> PRT
<213>(artificial sequence)
<400> 4
Met Ile Glu Ile Glu Lys Ile Thr Leu Tyr His Leu Ser Met Asn Leu
1 5 10 15
Lys Lys Pro Phe Lys Asn Ser Ile Glu Thr Leu Gln Glu Arg Lys Phe
20 25 30
Leu Ile Val Glu Ala Ile Asp Thr Thr Gly Val Thr Gly Trp Gly Glu
35 40 45
Val Ser Ala Phe Ser Ser Pro Trp Tyr Thr Glu Glu Thr Ile Gly Thr
50 55 60
Cys Leu His Met Leu Lys Asp Phe Phe Ile Pro Asn Val Val Gly Arg
65 70 75 80
Glu Phe Asn His Pro Ser Glu Val Pro Asp Ser Leu Ala Arg Tyr Lys
85 90 95
Gly Asn Arg Met Ala Lys Ala Gly Leu Glu Ser Ala Val Trp Asp Ile
100 105 110
Tyr Ala Lys Lys Lys Gly Val Ser Leu Ala Glu Ala Leu Gly Gly Thr
115 120 125
Arg Asp Lys Val Pro Ala Gly Val Val Val Gly Leu Ala Pro Leu Asp
130 135 140
Asp Met Leu Lys Glu Ile Glu Ser Tyr Gln Lys Glu Gly Tyr Gln Arg
145 150 155 160
Ile Lys Ile Lys Ile Gln Pro Gly Gln Asp Val Glu Leu Val Lys Ala
165 170 175
Ile Arg Ser Arg Phe Pro Asn Ile Pro Leu Met Ala Asp Ala Asn Ser
180 185 190
Ser Tyr Glu Leu Lys Asp Ile Ser Arg Leu Lys Glu Leu Asp Asp Tyr
195 200 205
His Leu Met Met Ile Glu Gln Pro Leu Gln Ala Asp Asp Ile Val Asp
210 215 220
His Arg His Leu Gln Lys His Leu Lys Thr Ala Ile Cys Leu Asp Glu
225 230 235 240
Ser Ile Cys Ser Val Asp Asp Ala Arg Arg Ala Ile Glu Leu Gly Ser
245 250 255
Cys Lys Ile Ile Asn Ile Lys Pro Ser Arg Val Gly Gly Leu Thr Glu
260 265 270
Ala Leu Lys Ile His Asp Leu Cys Lys Glu His His Met Gln Val Trp
275 280 285
Cys Gly Gly Met Leu Glu Thr Gly Ile Ser Arg Ala Gln Asn Val Ala
290 295 300
Leu Ala Ser Leu Pro Gln Phe Thr Ile Pro Gly Asp Ile Ser Ser Ser
305 310 315 320
Ser Arg Tyr Trp Asp Glu Asp Ile Val Thr Pro Asp Ile Arg Ile Asp
325 330 335
Asn Gly Phe Ile Ser Val Ser Lys Gln Pro Gly Leu Gly Val Glu Val
340 345 350
Asn Gln Asp Ile Met Arg Lys Tyr Val Thr Lys Met Asp Val Phe Thr
355 360 365
Gln His Gly
370
<210> 5
<211> 579
<212> PRT
<213>(artificial sequence)
<400> 5
Met Thr Val Asn Pro Ile Thr His Tyr Ile Gly Ser Phe Ile Asp Glu
1 5 10 15
Phe Ala Leu Ser Gly Ile Thr Asp Ala Val Val Cys Pro Gly Ser Arg
20 25 30
Ser Thr Pro Leu Ala Val Leu Cys Ala Ala His Pro Asp Ile Ser Val
35 40 45
His Val Gln Ile Asp Glu Arg Ser Ala Gly Phe Phe Ala Leu Gly Leu
50 55 60
Ala Lys Ala Lys Gln Arg Pro Val Leu Leu Ile Cys Thr Ser Gly Thr
65 70 75 80
Ala Ala Ala Asn Phe Tyr Pro Ala Val Val Glu Ala His Tyr Ser Arg
85 90 95
Val Pro Ile Ile Val Leu Thr Ala Asp Arg Pro His Glu Leu Arg Glu
100 105 110
Val Gly Ala Pro Gln Ala Ile Asn Gln His Phe Leu Phe Gly Asn Phe
115 120 125
Val Lys Phe Phe Thr Asp Ser Ala Leu Pro Glu Glu Ser Pro Gln Met
130 135 140
Leu Arg Tyr Ile Arg Thr Leu Ala Ser Arg Ala Ala Gly Glu Ala Gln
145 150 155 160
Lys Arg Pro Met Gly Pro Val His Val Asn Val Pro Leu Arg Glu Pro
165 170 175
Leu Met Pro Asp Leu Ser Asp Glu Pro Phe Gly Arg Met Arg Thr Gly
180 185 190
Arg His Val Ser Val Lys Thr Gly Thr Gln Ser Val Asp Arg Glu Ser
195 200 205
Leu Ser Asp Val Ala Glu Met Leu Ala Glu Ala Glu Lys Gly Met Ile
210 215 220
Val Cys Gly Glu Leu His Ser Asp Val Asp Lys Glu Asn Ile Ile Thr
225 230 235 240
Leu Ser Lys Ala Leu Gln Tyr Pro Ile Leu Ala Asp Pro Leu Ser Asn
245 250 255
Leu Arg Asn Gly Val His Asp Lys Ser Thr Val Ile Asp Ala Tyr Asp
260 265 270
Ser Phe Leu Lys Asp Asp Lys Leu Lys Arg Lys Leu Arg Pro Asp Val
275 280 285
Val Ile Arg Phe Gly Pro Met Pro Val Ser Lys Pro Val Phe Leu Trp
290 295 300
Leu Lys Asp Asp Pro Ala Ile Gln Gln Ile Val Ile Asp Glu Asp Gly
305 310 315 320
Gly Trp Arg Asp Pro Thr Gln Val Ser Ala His Met Ile His Cys Asn
325 330 335
Ala Ser Val Phe Ala Glu Glu Ile Met Ala Gly Leu Thr Ala Ala Arg
340 345 350
Ser Ser Glu Trp Leu Glu Lys Trp Gln Phe Val Asn Gly Arg Phe Arg
355 360 365
Glu His Leu Gln Thr Ile Ser Ser Glu Asp Val Ser Phe Glu Gly Asn
370 375 380
Leu Tyr Arg Ile Leu Gln His Leu Val Pro Glu Asn Ser Ser Leu Phe
385 390 395 400
Val Gly Asn Ser Met Pro Ile Arg Asp Val Asp Thr Phe Phe Glu Lys
405 410 415
Gln Asp Arg Pro Phe Arg Ile Tyr Ser Asn Arg Gly Ala Asn Gly Ile
420 425 430
Asp Gly Val Val Ser Ser Ala Met Gly Val Cys Glu Gly Thr Lys Ala
435 440 445
Pro Val Thr Leu Val Ile Gly Asp Leu Ser Phe Tyr His Asp Leu Asn
450 455 460
Gly Leu Leu Ala Ala Lys Lys Leu Gly Ile Pro Leu Thr Val Ile Leu
465 470 475 480
Val Asn Asn Asp Gly Gly Gly Ile Phe Ser Phe Leu Pro Gln Ala Ser
485 490 495
Glu Lys Thr His Phe Glu Asp Leu Phe Gly Thr Pro Thr Gly Leu Asp
500 505 510
Phe Lys His Ala Ala Ala Leu Tyr Gly Gly Thr Tyr Ser Cys Pro Ala
515 520 525
Ser Trp Asp Glu Phe Lys Thr Ala Tyr Ala Pro Gln Ala Asp Lys Pro
530 535 540
Gly Leu His Leu Ile Glu Ile Lys Thr Asp Arg Gln Ser Arg Val Gln
545 550 555 560
Leu His Arg Asp Met Leu Asn Glu Ala Val Arg Glu Val Lys Lys Gln
565 570 575
Trp Glu Leu
<210> 6
<211> 4107
<212> DNA
<213>(artificial sequence)
<400> 6
atggataaaa agtattccat tggcctggac atcggcacca attctgtggg ttgggcagtc 60
atcaccgacg aatacaaggt cccatccaag aagttcaagg tgctcggtaa taccgatcgc 120
cactctatca agaaaaacct gatcggcgcc ctgctcttcg actccggcga aaccgcagaa 180
gcaacccgtc tcaagcgtac cgcacgtcgc cgctacaccc gccgtaagaa tcgcatctgc 240
tacctccagg aaatcttctc taatgagatg gcaaaggtgg atgactcctt tttccaccgc 300
ctcgaagagt ccttcctggt ggaagaggac aagaaacacg agcgccatcc tatcttcggc 360
aatattgtcg atgaagtcgc atatcatgaa aaatacccaa ccatttacca tctccgtaaa 420
aaactcgtcg attccaccga taaggcagat ctccgcctga tctatctggc actggcccac 480
atgatcaagt ttcgtggcca cttcctgatc gaaggtgacc tcaatccaga caattccgac 540
gtggacaagc tgttcatcca gctggtgcaa acctacaacc agctctttga ggaaaaccca 600
atcaacgcat ctggcgtcga cgcaaaagcc atcctgtccg cccgtctctc caagtctcgt 660
cgcctcgaaa acctcattgc ccagctccct ggcgagaaga aaaacggtct gttcggcaat 720
ctgatcgccc tgtctctggg tctgacccca aatttcaaat ccaactttga tctcgcagaa 780
gatgccaagc tgcagctctc taaggacacc tacgatgatg acctggataa cctcctcgcc 840
cagatcggcg accagtacgc cgatctcttc ctcgcagcca agaacctctc tgacgcaatt 900
ctgctgtccg acatcctgcg cgtgaacacc gaaatcacta aggcaccact ctctgcctcc 960
atgattaagc gctacgacga gcatcatcag gatctcactc tcctcaaagc cctggtccgc 1020
cagcagctcc cagagaagta caaggaaatc tttttcgacc aatccaaaaa cggctacgca 1080
ggttacatcg atggcggcgc ctctcaggaa gagttttaca aattcattaa gccaatcctc 1140
gaaaagatgg acggcaccga ggaactgctg gtgaaactca accgtgaaga tctcctgcgc 1200
aaacagcgca ccttcgacaa cggttccatt cctcaccaga tccacctggg cgaactgcac 1260
gcaatcctcc gccgtcaaga ggacttctac ccattcctga aggacaaccg tgaaaagatc 1320
gaaaagattc tcaccttccg catcccttac tacgtgggtc ctctcgcccg tggcaattcc 1380
cgcttcgcct ggatgactcg caaatctgaa gagaccatca ccccttggaa ctttgaggaa 1440
gtcgtcgata aaggcgcctc tgcccaatcc ttcatcgagc gcatgaccaa cttcgacaag 1500
aacctcccaa acgagaaagt gctgcctaag cattccctcc tgtacgagta ctttactgtc 1560
tacaatgagc tgactaaggt gaagtatgtc actgaaggca tgcgtaagcc agcctttctc 1620
tccggcgagc agaaaaaggc aatcgtcgac ctcctgttta aaaccaaccg caaagtgact 1680
gtgaaacagc tcaaggaaga ttactttaaa aaaatcgaat gcttcgattc tgtggaaatc 1740
tccggcgtgg aggatcgttt caacgcctcc ctgggtacct atcacgacct cctgaagatt 1800
atcaaggaca aagattttct ggataacgag gaaaacgagg acattctcga ggacattgtg 1860
ctgaccctga ccctcttcga ggaccgcgag atgatcgagg agcgcctgaa gacctatgcc 1920
cacctctttg acgacaaggt catgaagcaa ctcaagcgcc gccgctatac cggttggggc 1980
cgtctctccc gtaagctcat caatggtatc cgcgacaagc aatccggcaa gactatcctg 2040
gactttctga agtctgacgg cttcgccaac cgcaatttta tgcaactgat ccacgacgat 2100
tccctgacct tcaaagagga catccagaaa gcccaagtgt ccggtcaagg cgactccctg 2160
cacgaacaca tcgccaatct ggcaggttcc ccagcaatca agaagggcat cctgcagacc 2220
gtcaaggtgg tggacgaact cgtcaaagtg atgggtcgcc acaaaccaga aaacatcgtc 2280
atcgagatgg cccgtgagaa ccagaccacc cagaaaggcc agaaaaactc ccgtgagcgc 2340
atgaagcgca ttgaagaagg cattaaagag ctcggctctc agatcctgaa agagcatcct 2400
gtcgagaaca cccaactgca gaatgagaag ctgtatctgt attatctcca gaacggccgc 2460
gacatgtacg tcgaccagga actggacatc aaccgtctct ctgattacga tgtggaccat 2520
atcgtccctc agtctttcct gaaagacgac tctattgaca acaaagtcct cacccgctcc 2580
gacaagaacc gcggcaagtc cgataacgtg ccatccgagg aggtcgtgaa gaagatgaag 2640
aactactggc gccagctgct caacgccaag ctgatcactc agcgcaagtt cgataacctg 2700
accaaggccg aacgtggtgg tctctccgag ctcgacaagg caggctttat caagcgccaa 2760
ctcgtggaga ctcgccaaat cactaaacac gtcgcccaga tcctcgactc ccgcatgaat 2820
accaagtacg atgaaaatga caagctcatc cgcgaagtga aagtcattac cctgaagtcc 2880
aaactggtct ctgactttcg caaggatttc cagttctaca aggtccgcga gattaataac 2940
tatcatcatg cacacgatgc atacctcaac gcagtcgtgg gtaccgcact gatcaagaag 3000
taccctaaac tggagtccga gttcgtctat ggcgactaca aggtgtacga cgtccgcaaa 3060
atgattgcca agtccgagca ggagatcggc aaagcaactg ccaaatattt cttttactcc 3120
aacatcatga acttcttcaa gaccgaaatc accctcgcca acggtgaaat ccgcaaacgt 3180
ccactcatcg agactaatgg tgaaaccggc gagatcgtct gggacaaggg ccgtgacttc 3240
gcaaccgtcc gcaaggtcct ctccatgcca caggtcaaca tcgtgaaaaa gaccgaggtg 3300
caaaccggcg gcttctccaa ggagtctatc ctgcctaaac gcaattccga taagctcatt 3360
gcacgcaaaa aggactggga ccctaaaaaa tacggcggtt tcgactcccc aactgtcgca 3420
tattctgtgc tcgtggtcgc caaagtggaa aagggcaaat ccaaaaagct caagtccgtc 3480
aaggaactcc tgggtatcac catcatggaa cgctcctcct ttgagaagaa ccctatcgat 3540
ttcctcgaag caaaaggtta caaggaggtg aagaaagatc tgatcatcaa gctccctaaa 3600
tactccctct tcgagctcga gaacggccgc aagcgtatgc tggcctccgc aggtgaactg 3660
caaaaaggta acgagctggc actcccatcc aagtatgtca actttctcta cctggcctcc 3720
cactacgaaa agctgaaagg ttccccagaa gacaacgagc agaaacagct gttcgtggag 3780
cagcacaagc actacctgga cgaaatcatc gagcagatct ccgagttctc taaacgcgtc 3840
attctggccg atgccaacct cgataaagtg ctctccgcct acaataagca tcgtgataag 3900
ccaatccgtg agcaggcaga gaacatcatt cacctgttca ctctcaccaa cctgggtgca 3960
ccagccgcct ttaagtactt cgacaccacc atcgaccgca agcgctatac ctccactaag 4020
gaggtgctcg atgcaaccct gatccaccag tctatcaccg gcctctacga gactcgcatc 4080
gatctctccc agctgggtgg cgactaa 4107
<210> 7
<211> 118
<212> DNA
<213>(artificial sequence)
<220>
<221> misc_feature
<222> (9)..(28)
<223> n is a, c, g, or t
<400> 7
cggaattcnn nnnnnnnnnn nnnnnnnngt tttagagcta gaaatagcaa gttaaaataa 60
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggatcccg 118
<210> 8
<211> 20
<212> DNA
<213>(artificial sequence)
<400> 8
tcgcggtgaa agacatgtta 20

Claims (10)

1. a kind of bafillus natto recombinant bacterium, which is characterized in that the recombinant bacterium has knocked out the poly- isoamyl two of 4-HBA Alkenyl transferase gene ubiA, knockout branch's mutase gene aroH while Enhanced expressing 1,4-dihydroxy-2-naphthsaisyuoic acid gather different Prenyl transferase gene menA, adjacent succinylbenzoic acid synthase gene menC and 2- succinyl -5- enolpyruvyl acyl -6- Hydroxyl -3- cyclohexene -1- carboxylic acid synthetase gene menD.
2. recombinant bacterium according to claim 1, which is characterized in that the 4-hydroxybenzoate polyprenyltransferase Amino acid sequence as shown in SEQ ID NO.1；The amino acid sequence of branch's mutase is as shown in SEQ ID NO.2.
3. recombinant bacterium according to claim 1, which is characterized in that the 1,4-dihydroxy-2-naphthsaisyuoic acid polyisoprene The amino acid sequence of based transferase is as shown in SEQ ID NO.3, and the amino acid sequence of neighbour's succinylbenzoic acid synzyme is such as Shown in SEQ ID NO.4, the nucleosides of the 2- succinyl -5- enolpyruvyl acyl -6- hydroxyl -3- cyclohexene -1- carboxylic acid synthetase Acid sequence is as shown in SEQ ID NO.5.
4. recombinant bacterium according to claim 1, which is characterized in that the recombinant bacterium is with bafillus natto Bacillus Subtilis nattoND-1-A27 is host strain.
5. recombinant bacterium according to claim 1, which is characterized in that the recombinant bacterium is carried out using pMA5 as expression vector The Enhanced expressing of gene knockout or gene.
6. a kind of construction method of the described in any item recombinant bacteriums of Claims 1 to 5, which comprises the steps of:
(1) synthesizing ribonucleotide sequence Cas9 protein expression frame as shown in SEQ ID NO.6, nucleotide sequence such as SEQ ID are designed The sgRNA segment as shown in SEQ ID NO.8 of sgRNA scaffold expression cassette and nucleotide sequence shown in NO.7；
(2) Cas9 protein expression frame is connected on pMA5 expression vector, obtains plasmid pMA5-Cas9；
(3) sgRNA scaffold expression cassette is connected on plasmid pMA5-Cas9；
(4) bafillus natto ND-1-A27 genomic DNA is extracted, each 500bp of ubiA upstream and downstream is expanded, gene menA is expanded Collectively as homologous repairing sequence, the homologous repairing sequence is connected on the plasmid of step (3), obtains bafillus natto Gene editing carrier pMAC；
(5) bafillus natto gene editing carrier pMAC electricity being gone in ND-1-A27 competence, screening obtains ubiA knockout, The bafillus natto of menA Enhanced expressing, and non-resistant passage is carried out, plasmid experiment is eliminated, it is final to obtain non-resistant natto bud Spore bacillus ND-1-A27A △ ubiA；
(6) extract bafillus natto ND-1-A27 genomic DNA, expand each 500bp of aroH upstream and downstream, by gene menC, MenD is expanded collectively as homologous repairing sequence, and the homologous repairing sequence is connected on the plasmid of step (3), obtains natto Bacillus gene editor's carrier pMAC-1；
(7) bafillus natto gene editing carrier pMAC-1 electricity is gone in ND-1-A27A △ ubiA competence, screening obtains AroH is knocked out and the bafillus natto ND-1-A27ACD △ ubiA △ aroH of Enhanced expressing menC and menD.
7. the described in any item recombinant bacteriums of Claims 1 to 5 are in fermenting and producing vitamin K2(MK-7) and in γ-polyglutamic acid Using.
8. a kind of described in any item recombinant bacterium fermenting and producing vitamin Ks of Claims 1 to 52(MK-7) and γ-polyglutamic acid Method, which is characterized in that the recombinant bacterium single colonie is inoculated in seed culture medium, 35~38 DEG C, 100~150r/min training It supports 6~10h and obtains seed culture fluid；Seed culture fluid is forwarded to fermentation medium, 35~38 DEG C of trainings with 1~5% inoculum concentration It supports 3~6 days, obtains fermentation liquid, separation and Extraction obtains vitamin K from fermentation liquid2(MK-7) and γ-polyglutamic acid.
9. according to the method described in claim 8, it is characterized in that, the seed culture medium are as follows: 25~35g/L of glucose, egg White 35~45g/L of peptone, 3~7g/L of sodium chloride, 3~7g/L of beef extract, 3~7g/L of yeast extract.
10. according to the method described in claim 8, it is characterized in that, the fermentation medium are as follows: glycerol 40-60g/L, soybean Peptone 25-35g/L, yeast powder 0.5-1g/L, K2HPO40.2-0.6g/L。
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