CN110093367A - A kind of bacillus subtilis oscillation mode gene expression system and its construction method and application - Google Patents
A kind of bacillus subtilis oscillation mode gene expression system and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of bacillus subtilis oscillation mode gene expression system and its construction method and applications, belong to field of biotechnology.The bacillus subtilis oscillation mode gene expression system is the knockout response regulatory factor gene comA in Bacillus subtilis genes group, is transferred to what oscillation gene expression regulation plasmid pOSC was obtained.The periodic switch process expressed by controlling gene, oscillation mode gene expression system can alleviate the supply pressure of stable growth period metabolism substrate and energy, supply gene expression and substrate and reach balance, and then improve expression yield.Using the system expression hyaluronic acid synthesis related gene, the recombined bacillus subtilis of high yield hyaluronic acid is successfully constructed.
Description
Technical field:
The present invention relates to a kind of bacillus subtilis oscillation mode gene expression system and its construction method and applications, specifically relate to
And by the building of the pitch period switch of comA/PsrfA and tetR/Ptet element regulation in B. subtilis cell
Journey belongs to field of biotechnology.
Background technique:
With molecular biology, the development of the subjects such as microbiology, a variety of protokaryons and eukaryotic expression system are developed,
To produce recombinant protein.Wherein bacillus subtilis has non-cause a disease as a kind of important Gram-positive type strain
Property, the characteristics such as genetic background is clear, can be easily separated culture, secretory protein ability is strong, be the ideal of heterologous protein expression and secretion
Host.So far, a large amount of protokaryon and eukaryotic gene clone and express in B. subtilis cell, middle part
Divide and has been applied to industrialized production.Gene recombination technology is rationally utilized, bacillus subtilis metabolic pathway is optimized, improves target egg
There is white yield important industrial application to be worth.
Efficiently controllable promoter be guarantee industrial bacillus subtilis high efficient expression foreign protein key factor it
One.Zhou Zhemin etc. constructs the self-induction gene expression system in bacillus subtilis by promoter Ps rfA exploitation, in index
Growth period promoter activity is lower, shows strong promoter activity stablizing growth period.
Hyaluronic acid (Hyaluronic acid, HA) is a kind of polymeric acidic being widely present in vertebrate body
Mucopolysaccharide, by D-Glucose aldehydic acid (D-glucuroic acid, GlcA) and N-Acetyl-D-glucosamine (N-acetyl-D-
Glucosamine, GlcNAc) it is alternately formed by connecting by β -1,3 and 1,4 glycosidic bond of β -.Due to its unique physicochemical property,
Including good moisture retention, permeability, ductility, without any immunogenicity and toxicity, hyaluronic acid is in cosmetics, food, doctor
The fields such as treatment are widely used.
Hyaluronic acid is widely present in animal tissue cell's interstitial, wherein cockscomb, umbilical cord, joint is slided with vitreum
Liquid hold-up is higher.Before the 1980s, hyaluronic acid is mainly extracted by cockscomb, and raw material sources are limited, and yield is very low.And it mentions
The hyaluronic acid taken is often mixed with the mucopolysaccharides such as foreign protein and chondroitin sulfate, causes yield low, and purification is difficult, it is difficult to be applied to big rule
Mould production.Since 1970s, China starts to produce hyaluronic acid with microbe fermentation method.Common microorganism includes epizootic disease chain
Coccus, streptococcus equi and streptococcus equisimilis etc..Demand of the hyaluronic acid fermentation to safety limits streptococcic fermentation application,
And streptococcus, to nutritional requirement height, culture medium raw material is at high cost.Therefore, a kind of safety economy is developed, nutritional requirement is low, yield
High genetic engineering bacterium has become problem in the urgent need to address.
The present invention is placed in oscillation mode gene table by the way that hyaluronic acid expression approach related gene is transferred to bacillus subtilis
Up to system target gene site, high-performance bio synthesis of the hyaluronic acid in food-grade microorganisms bacillus subtilis is realized.
Summary of the invention:
The purpose of the present invention is to provide a kind of oscillation gene expression regulation plasmid pOSC and its construction methods.
Another object of the present invention is to provide a kind of bacillus subtilis oscillation mode gene expression system and its building sides
Method.
Another object of the present invention is to provide above-mentioned bacillus subtilis oscillation mode gene expression system in recombinant protein
Application in expression.
It is saturating another object of the present invention is to provide a kind of application bacillus subtilis oscillation mode gene expression system production
The method of bright matter acid.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of oscillation gene expression regulation plasmid pOSC, oscillation gene expression regulation plasmid pOSC is in carrier pHT01
What upper parallel connection was obtained by two metabolic regulation accesses that promoter Ptet and promoter Ps rfA starts respectively.
Preferably, the metabolic regulation access of promoter Ptet starting includes promoter Ptet, response regulatory factor
Gene comA and protein degradation label gene ssrA；The metabolic regulation access of the promoter Ps rfA starting includes promoter
PsrfA, repressor gene tetR and target gene T.
The construction method of above-mentioned oscillation gene expression regulation plasmid pOSC, comprising the following steps:
(1) using 168 genomic DNA of bacillus subtilis Bacillus substilis as template, primer is utilized
PsrfAFor, PsrfARev expand srfA upstream region of gene promoter sequence；
(2) using Escherichia coli DH10B genomic DNA as template, tetR gene is expanded using primer tetRFor, tetRRev
Segment；
(3) NheI/XbaI double digestion plasmid pHT43, recovery purifying 5.4Kb carrier segments；
(4) by Gibson assembly by the promoter Ps rfA segment of purification and recovery in step (1) and step (2),
TetR genetic fragment is connected to the plasmid vector of step (3) purification and recovery, i.e. acquisition plasmid pHT43-PsrfA-tetR；TetR base
Because downstream reservation has a BamH1 restriction enzyme site, facilitate the insertion of subsequent target gene；
(5) ssrA-comA-Ptet Genetic elements are synthesized, 20bp pHT43-PsrfA-tetR carrier has respectively been merged at both ends
The same former sequence of the site EcoRV two sides；
(6) EcoRV single endonuclease digestion plasmid pHT43-PsrfA-tetR, recovery purifying 7.5Kb carrier segments；
(7) genetic fragment in step (5) is connected to by the linearisation of step (6) single endonuclease digestion by Gibson assembly
Plasmid vector, i.e., acquisition plasmid pOSC；
The primer and gene order are as follows:
PsrfAFor:5’-gccagtcactatggcgtgctgatatcgacaaaaatgtcatg-3’
PsrfARev:5’-cttttatctaatctagacatattgtcatacctcccctaatc-3’
tetRFor:5’-gattaggggaggtatgacaatatgtctagattagataaaag-3’
tetRRev:5’-ctgccccggggacgtcgactttaagacccactttcacat-3’
SsrA-comA-Ptet:
cagtcactatggcgtgctgaTGCTGCTAATGCTACATTTTGATTAAATGAATTTGTTTTTCCTGCtta ggcagctaatgctacgttttggttaaaactgttagttttgccaagtacaccgtctgatttcgcaatcaaaaccgct
tccgtccgtgaaccgacattcagcttattgaaaatcgatgtcaagctgtattcaatggaccgcttgcttaaatgaa
gggcatctgcgatttcttggtttgtaaatcccttttcaacttcttgaagaatcaggcattctctaggtgtgagcac
atcttgttctttttgagaggaaggagccggctttgttttttgctgagtcatcagctgtttaaagtaagcaaaatcg
actaaaatttctccgttgagtacgtggtatatgtattgggtgatcttttctttagattccgttttgctgatggcac
cgtgcagacccgcacgaatcgcttcctcgaaataatcctcgacctcataaccggtatacacgataattttacaatg aggattctcttgtaaaatctgtttagaaagctccatcccattgacctcgccgcctagattcagatccattaaaatg agatcatatgacgagaaatcatgctgcttgataaactgttcgctcggttcaggactgagacaatcaacagacaaat tcgaatccgtttccaaaattgtcttggtgccttccatgacagccggatggtcatcaatcactagtatctttttcat acccatctctttatccttacctattgtttgtcgctagcgtcgaatgcaagcagcagtatctctatcactgataggg atgtcaaagcAtcgacaaaaatgtcatgaaag。
A kind of bacillus subtilis oscillation mode gene expression system, the gene expression system are in Bacillus subtilis genes
Response regulatory factor gene comA is knocked out in group, is transferred to what above-mentioned oscillation gene expression regulation plasmid pOSC was obtained.
The construction method of above-mentioned bacillus subtilis oscillation mode gene expression system, comprising the following steps:
(I) building knocks out the recombinant plasmid of response regulatory factor gene comA, is transferred to bacillus subtilis, is knocked out
The bacillus subtilis of comA gene；
(II) the oscillation mode gene expression regulation plasmid pOSC constructed by comA/Psrf and tetR/Ptet element is transferred to
The bacillus subtilis for the knockout comA gene that step (I) obtains, building obtain bacillus subtilis oscillation mode gene expression system
System.
As a kind of optimal technical scheme, building knocks out the process tool of the bacillus subtilis of comA gene in step (I)
Body the following steps are included:
(1) using Bacillus subtilis genes group DNA as template, be utilized respectively primer comAupFor, comAupRev and
ComAdnFor, comAdnRev expand comA upstream region of gene and segments downstream；
The primer sequence is as follows:
ComAupFor:5 '-CAGACGCGTCGACGTCATATGatcctgcttgggattctc-3 '
ComAupRev:5 '-gtgagtaaaagggaggaaaacatgttggggggtgtagagatg-3 '
ComAdnFor:5 '-catctctacaccccccaacatgttttcctcccttttactcac-3 '
ComAdnRev:5 '-CCTCGCGTCGGGCGATATCGatccgtgccgtgaagagg-3 '
(2) BamHI digested plasmid pMAD, recovery purifying；
(3) by the plasmid of the comA gene upstream and downstream homology arm of purification and recovery in step (1) and step (2) linearization for enzyme restriction
Carrier pMAD carries out Gibson assembly, i.e. acquisition recombinant plasmid pMAD- △ comA；
(4) the recombinant plasmid pMAD- △ comA that step (3) construct is transferred to bacillus subtilis, screening positive clone obtains
To the bacillus subtilis BS168 Δ comA for knocking out response regulatory factor gene comA.
In step (II) building oscillation mode gene expression regulation plasmid pOSC process specifically includes the following steps:
(1) using 168 genomic DNA of bacillus subtilis Bacillus substilis as template, primer is utilized
PsrfAFor, PsrfARev expand srfA upstream region of gene promoter sequence；
(2) using Escherichia coli DH10B genomic DNA as template, tetR gene is expanded using primer tetRFor, tetRRev
Segment；
(3) NheI/XbaI double digestion plasmid pHT43, recovery purifying 5.4Kb carrier segments；
(4) by Gibson assembly by the promoter Ps rfA segment of purification and recovery in step (1) and step (2),
TetR genetic fragment is connected to the plasmid vector of step (3) purification and recovery, i.e. acquisition plasmid pHT43-PsrfA-tetR；TetR base
Because downstream reservation has a BamH1 restriction enzyme site, facilitate the insertion of subsequent target gene；
(5) ssrA-comA-Ptet Genetic elements are synthesized, 20bp pHT43-PsrfA-tetR carrier has respectively been merged at both ends
The same former sequence of the site EcoRV two sides；
(6) EcoRV single endonuclease digestion plasmid pHT43-PsrfA-tetR, recovery purifying 7.5Kb carrier segments；
(7) genetic fragment in step (5) is connected to by the linearisation of step (6) single endonuclease digestion by Gibson assembly
Plasmid vector, i.e., acquisition plasmid pOSC；
The primer and gene order are as follows:
PsrfAFor:5’-gccagtcactatggcgtgctgatatcgacaaaaatgtcatg-3’
PsrfARev:5’-cttttatctaatctagacatattgtcatacctcccctaatc-3’
tetRFor:5’-gattaggggaggtatgacaatatgtctagattagataaaag-3’
tetRRev:5’-ctgccccggggacgtcgactttaagacccactttcacat-3’
SsrA-comA-Ptet:
cagtcactatggcgtgctgaTGCTGCTAATGCTACATTTTGATTAAATGAATTTGTTTTTCCTGCtta ggcagctaatgctacgttttggttaaaactgttagttttgccaagtacaccgtctgatttcgcaatcaaaaccgct
tccgtccgtgaaccgacattcagcttattgaaaatcgatgtcaagctgtattcaatggaccgcttgcttaaatgaa
gggcatctgcgatttcttggtttgtaaatcccttttcaacttcttgaagaatcaggcattctctaggtgtgagcac
atcttgttctttttgagaggaaggagccggctttgttttttgctgagtcatcagctgtttaaagtaagcaaaatcg
actaaaatttctccgttgagtacgtggtatatgtattgggtgatcttttctttagattccgttttgctgatggcac
cgtgcagacccgcacgaatcgcttcctcgaaataatcctcgacctcataaccggtatacacgataattttacaatg aggattctcttgtaaaatctgtttagaaagctccatcccattgacctcgccgcctagattcagatccattaaaatg agatcatatgacgagaaatcatgctgcttgataaactgttcgctcggttcaggactgagacaatcaacagacaaat tcgaatccgtttccaaaattgtcttggtgccttccatgacagccggatggtcatcaatcactagtatctttttcat acccatctctttatccttacctattgtttgtcgctagcgtcgaatgcaagcagcagtatctctatcactgataggg atgtcaaagcAtcgacaaaaatgtcatgaaag。
The plasmid pOSC of appeal building is transferred to bacillus subtilis BS168 Δ comA, screening positive clone obtains withered grass
Bacillus oscillation mode gene expression system.
A kind of production hyaluronic acid recombined bacillus subtilis based on oscillation mode gene expression system, hyaluronic acid is expressed
Approach related gene is placed in the above-mentioned site bacillus subtilis oscillation mode gene expression system target gene T.
As a kind of optimal technical scheme, the hyaluronic acid expression approach related gene includes that encoding hyaluronan closes
At the gene hasA of enzyme, the base of gene tuaD and UTP- the Cori ester uridyltransferase of UDP-glucose dehydrogenase
Because of gtaB.It is further preferred that the hyaluronan synthase gene hasA derives from streptococcus pyogenes (Streptococcus
Pyogenes), UDP-glucose dehydrogenase gene tuaD and UDP-glucose dehydrogenase gene gtaB derives from bacillus subtilis
Bacterium 168 (Bacillus substilis 168).
Above-mentioned oscillation gene expression regulation plasmid pOSC, above-mentioned bacillus subtilis oscillation mode gene expression system exist
Application in recombinant protein expression.
Above-mentioned bacillus subtilis oscillation mode gene expression system or above-mentioned recombined bacillus subtilis are transparent in production
Application in matter acid.
A method of hyaluronic acid, including following step are produced using bacillus subtilis oscillation mode gene expression system
It is rapid:
(1) using streptococcus pyogenes genomic DNA as template, hyaluronan synthase is expanded with primer hasAFor, hasARev
Gene hasA；
(2) using Bacillus subtilis genes group DNA as template, with primer tuaDFor, tuaDRev and gtaBFor,
GtaBRev expands UDP-glucose dehydrogenase gene tuaD and UTP- Cori ester uridyltransferase gene respectively
gtaB；
The sequence of the primer is as follows:
HasAFor:5 '-gtcttaaagtcgacgtccccaaggagaaaggtacccatgcctatcttcaagaagac-
3’
HasARev:5 '-gtaccactttcctcctttacttgaagatcgttaccttc-3 '
TuaDFor:5 '-taaaggaggaaagtggtacatgaaaaaaatagctgtcattg-3 '
TuaDRev:5 '-atcgagcctagcagccggcagcttcaaccaagtaacactttttctttttataaatt g
acgcttccc-3’
GtaBFor:5 '-ctttacgtacttttttcatttaaaaggcaccttccttatgatcgagcctagcagcc g
gc-3’
GtaBRev:5 '-ctcattaggcgggctgcccctagatgaatgtccaatagcctttttgtttagatttc t
tctttgtttag-3'；
(3) the above-mentioned oscillation gene expression regulation plasmid pOSC of BamH1 digestion, recovery purifying；
(4) pass through Gibson assembly for the hyaluronan synthase gene of purification and recovery in step (1) and step (2)
HasA, UDP-glucose dehydrogenase gene tuaD and UTP- Cori ester uridyltransferase gene gtaB are connected to step
Suddenly the oscillation gene expression regulation plasmid pOSC of (3) single endonuclease digestion linearisation, i.e. acquisition plasmid pOSC-HA；
(5) above-mentioned plasmid pOSC-HA electricity is transferred to the bacillus subtilis BS168 Δ comA competence of comA gene delection
Cell, screening positive clone obtain the high yield hyaluronic acid recombined bacillus subtilis regulated and controled by oscillation mode gene expression system
BS168ΔcomA/pOSC-HA；
(6) recombined bacillus subtilis that step (5) obtains is accessed in LB liquid medium and is cultivated, obtain preculture
Engineering bacteria bacterium solution；
(7) the engineering bacteria bacterium solution access fermentation medium dosage form culture of the preculture is obtained by the volume ratio of 1%-10%
To the fermentation liquid containing hyaluronic acid.Condition of culture are as follows: 37 DEG C, 200r/min, 24-72h.
A method of hyaluronic acid is produced using above-mentioned recombination engineering, comprising the following steps:
(1) recombined bacillus subtilis is accessed in LB liquid medium and is cultivated, obtain the engineering bacteria bacterium solution of preculture.
(2) by 1%-10% volume ratio by above-mentioned preculture bacterium solution access fermentation medium, 37 DEG C, 200r/min 24-
72h obtains the fermentation liquid containing hyaluronic acid.Fermentation medium composition: glucose 20-80g/L, yeast extract 5-20g/L,
Potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 2g/L.
Promoter element of the present invention by clone's PsrfA sequence as foreign protein.Utilize comA/Psrf and tetR/
Ptet element realizes the periodic switch process of exogenous protein expression.Its basic principle is as follows:
The comA controlled by Ptet promoter the and tetR controlled by PsrfA promoter, target gene constitute oscillation
Gene expression circuit.The pheromones comX that Bacillus subtilis genes group comX and comQ expression generates.As cell density
Signaling molecule, comX are gradually expressed and are accumulated with the growth of cell.When cell grows into stable growth period, comX reaches threshold value
After concentration, in conjunction with simultaneously phosphorylation memebrane protein histidine kinase comP, then its phosphate group is transferred to it by the comP of phosphorylation
In conjunction with promoter Ps rfA, activation tetR and target gene are transcribed by effect protein comA, the comA of phosphorylation.Increase when TetR is expressed
Add, tetR repressor will inhibit the expression by the Ptet comA started can not reactivation so that the comA of phosphorylation be made to reduce
The expression of tetR and expression of target gene, tetR is suppressed, and is again expressed Bacillus subtilis genes group comX and comQ and is generated
Pheromones comX activate by the PsrfA tetR started and target gene, form the gene expression circuit of oscillation mode.Pass through this system
The supply pressure that stable growth period bacillus subtilis metabolic intermediate and energy can be alleviated, make the expression of target gene with
Substrate supply reaches balance, and then improves expressed foreign protein yield.
Beneficial effects of the present invention:
The present invention constructs oscillation mode gene expression system in B. subtilis cell, which passes through periodicity
The expression of target gene is switched, alleviates the supply pressure for stablizing growth period bacillus subtilis metabolic intermediate and energy, makes mesh
Gene expression and substrate supply reach balance, and then foreign protein yield expressed by improving.It is closed by being transferred to hyaluronic acid
At approach, building obtains the recombined bacillus subtilis of high yield hyaluronic acid, and fermentation yield reaches 21.8g/L.
Specific embodiment:
Embodiment 1: the recombinant bacillus gemma of response regulatory factor gene comA is knocked out by double crossing over homologous recombination construction
Bacillus BS168 Δ comA.
1. the building of bacillus subtilis recombinant plasmid pMAD- △ comA, comprising the following steps:
(1) using bacillus subtilis Bacillus substilis 168 (commercially available routine bacterial strain) genomic DNA as template,
It is utilized respectively primer comAupFor, comAupRev and comAdnFor, comAdnRev expands comA upstream region of gene and downstream piece
Section.
(2) BamHI digested plasmid pMAD, recovery purifying.
(3) by the plasmid of the comA gene upstream and downstream homology arm of purification and recovery in step (1) and step (2) linearization for enzyme restriction
Carrier pMAD carries out Gibson assembly, i.e. acquisition recombinant plasmid pMAD- △ comA；Gibson assembly system is
ComA upstream region of gene and each 1.2 μ l of segments downstream, 0.6 μ l, the 2x Gibson assembly Mix 3 of carrier pMAD of linearisation
μ l, reaction condition are 50 DEG C of 1h, convert Escherichia coli DH10B competent cell, ampicillin 100ug/ml screening, picking list
Colony inoculation extracts plasmid and carries out digestion verification, and positive recombinant is sequenced.
The primer sequence is as follows:
ComAupFor:5 '-CAGACGCGTCGACGTCATATGatcctgcttgggattctc-3 '
ComAupRev:5 '-gtgagtaaaagggaggaaaacatgttggggggtgtagagatg-3 '
ComAdnFor:5 '-catctctacaccccccaacatgttttcctcccttttactcac-3 '
ComAdnRev:5 '-CCTCGCGTCGGGCGATATCGatccgtgccgtgaagagg-3 '
PCR reaction system are as follows:
PCR reaction condition are as follows:
Double digestion system are as follows:
Double digestion condition is generally 37 DEG C, digestion 4h.
2. recombinant plasmid pMAD- △ comA electricity is turned bacillus subtilis bacterium competence cell, using containing erythromycin and X-gal
LB safety screen single-swap positive recombinant, choose blue colonies be inoculated in LB culture medium culture 16h, be then coated on containing X-
The LB plate screening of gal chooses white colony, obtains double crossing over positive colony.Double crossing over positive colony is inoculated with to train in LB liquid
Base culture 12h is supported, genomic DNA is extracted, is that primer carries out PCR verifying with comAupFor, comAdnRev.
The electricity of bacillus subtilis turn competent cell the preparation method comprises the following steps:
(1) 20 μ l are taken to apply sky LB plate, 37 DEG C of overnight incubations the bacillus subtilis glycerol stock that -80 DEG C of refrigerators save.It chooses
Single bacterium of making even on plate in LB liquid medium, 37 DEG C, 200r/min culture 16h obtain bacillus subtilis seed liquor.
(2) in the ratio of 1:100 (v:v) inoculation seed liquor, into 200ml culture medium, (LB of addition 0.5M D-sorbite is trained
Support base) 37 DEG C, 200r/min cultivate 3h to OD600=0.6.
(3) bacterium solution is transferred to 50ml centrifuge tube ice bath 5min, 4 DEG C, 5000g is centrifuged 10min.
(4) turn culture medium (0.5M trehalose, 0.5M D-sorbite, 0.5M mannitol, 10% glycerol) weight with the electricity of pre-cooling
Outstanding 4 DEG C of thallus, 5000g are centrifuged 10min.
(5) it repeats step (4) 3 times.
(6) turn culture medium with 2.5ml electricity and thallus is resuspended, the 1.5ml centrifuge tube of pre-cooling is dispensed by every 100 μ l of pipe, in liquid
5-10min is cooled down in nitrogen rapidly, is saved in -80 DEG C of refrigerators.
The concrete operations of electrotransformation are as follows:
Turn competence bacillus subtilis to the 100 μ l electricity for being divided in centrifuge tube and 2 μ l recombinant plasmids are added, and is transferred to pre-
Cold 2mm electricity revolving cup, ice bath 5min, adjusting electroporation apparatus voltage are 2.5kV, and electric revolving cup is packed into electroporation, presses shock button.
After electric shock, 1ml recovery medium is added to electric revolving cup immediately, and (LB culture medium adds 0.5M D-sorbite, 0.38M sweet dew
Alcohol), thallus is resuspended, and be transferred to 1.5ml centrifuge tube, 37 DEG C, 200r/min recovery 3h.
Embodiment 2: bacillus subtilis mutant BS168 Δ is converted by oscillation mode gene expression regulation plasmid pOSC
ComA constructs bacillus subtilis oscillation mode gene expression system.
1. the building of oscillation mode gene expression regulation plasmid pOSC, comprising the following steps:
(1) using 168 genomic DNA of bacillus subtilis Bacillus substilis as template, design primer
PsrfAFor, PsrfARev expand srfA upstream region of gene 0.6kb promoter sequence segment, recovery purifying.
(2) using Escherichia coli DH10B (commercially available routine bacterial strain), genomic DNA is template, design primer tetRFor,
TetRRev expands tetR genetic fragment, recovery purifying.
(3) NheI/XbaI double digestion plasmid pHT43, recovery purifying 5.4Kb carrier segments；
(4) by Gibson assembly by the promoter Ps rfA segment of purification and recovery in step (1) and step (2),
TetR genetic fragment is connected to the plasmid vector of step (3) purification and recovery, i.e. acquisition plasmid pHT43-PsrfA-tetR.Gibson
Assembly system is PsrfA segment, 0.6 μ l, 2x Gibson of tetR segment each 1.2 μ l, double digestion plasmid pHT43
3 μ l of assembly Mix, reaction condition are 50 DEG C of 1h, convert Escherichia coli DH10B competent cell, 100 μ of ampicillin
G/ml screening, the inoculation of picking single colonie extract plasmid and carry out digestion verification, and positive recombinant is sequenced.
(5) ssrA-comA-Ptet Genetic elements are synthesized, 20bp pHT43-PsrfA-tetR carrier has respectively been merged at both ends
The same former sequence of the site EcoRV two sides；
(6) EcoRV single endonuclease digestion plasmid pHT43-PsrfA-tetR, recovery purifying 7.5Kb carrier segments；
(7) genetic fragment in step (5) is connected to by the linearisation of step (6) single endonuclease digestion by Gibson assembly
Plasmid vector, i.e., acquisition plasmid pOSC；Gibson assembly system is 2.4 μ l of ssrA-comA-Ptet Genetic elements, is carried
0.6 μ l, 2x Gibson assembly Mix of body pHT43-PsrfA-tetR, 3 μ l, reaction condition are 50 DEG C of 1h, convert large intestine
Bacillus DH10B competent cell, 100 μ g/ml of ampicillin screening, the inoculation of picking single colonie are extracted plasmid progress digestion and are tested
Card, positive recombinant are sequenced.
2. above-mentioned oscillation mode gene expression regulation plasmid pOSC electricity is transferred to bacillus subtilis strain BS168 Δ comA electricity
Turn competence, 5 μ g/ml of chloramphenicol screening, the inoculation of picking single colonie proposes genomic DNA, PCR verifying.It obtains being built with oscillation mode
The recombined bacillus subtilis BS168 Δ comA/pOSC of gene expression system.
The primer and gene order are as follows:
PsrfAFor:5’-gccagtcactatggcgtgctgatatcgacaaaaatgtcatg-3’
PsrfARev:5’-cttttatctaatctagacatattgtcatacctcccctaatc-3’
tetRFor:5’-gattaggggaggtatgacaatatgtctagattagataaaag-3’
tetRRev:5’-ctgccccggggacgtcgactttaagacccactttcacat-3’
SsrA-comA-Ptet:
cagtcactatggcgtgctgaTGCTGCTAATGCTACATTTTGATTAAATGAATTTGTTTTTCCTGCtta ggcagctaatgctacgttttggttaaaactgttagttttgccaagtacaccgtctgatttcgcaatcaaaaccgct
tccgtccgtgaaccgacattcagcttattgaaaatcgatgtcaagctgtattcaatggaccgcttgcttaaatgaa
gggcatctgcgatttcttggtttgtaaatcccttttcaacttcttgaagaatcaggcattctctaggtgtgagcac
atcttgttctttttgagaggaaggagccggctttgttttttgctgagtcatcagctgtttaaagtaagcaaaatcg
actaaaatttctccgttgagtacgtggtatatgtattgggtgatcttttctttagattccgttttgctgatggcac
cgtgcagacccgcacgaatcgcttcctcgaaataatcctcgacctcataaccggtatacacgataattttacaatg aggattctcttgtaaaatctgtttagaaagctccatcccattgacctcgccgcctagattcagatccattaaaatg agatcatatgacgagaaatcatgctgcttgataaactgttcgctcggttcaggactgagacaatcaacagacaaat tcgaatccgtttccaaaattgtcttggtgccttccatgacagccggatggtcatcaatcactagtatctttttcat acccatctctttatccttacctattgtttgtcgctagcgtcgaatgcaagcagcagtatctctatcactgataggg atgtcaaagcAtcgacaaaaatgtcatgaaag。
PCR reaction step, double enzyme digestion reaction step, competent cell preparation step and electrotransformation step and 1 phase of embodiment
Together.
Embodiment 3: hyaluronic acid is expressed using bacillus subtilis oscillation mode gene expression system
1. the method for building expression hyaluronic acid oscillation mode gene regulation plasmid, specifically includes:
(1) using streptococcus pyogenes (Streptococcus pyogenes) (commercially available routine bacterial strain) genomic DNA as template,
Design primer hasAFor, hasARev expand hyaluronan synthase gene hasA, recovery purifying.
(2) using Bacillus subtilis genes group DNA as template, design primer tuaDFor, tuaDRev and gtaBFor,
GtaBRev expands UDP-glucose dehydrogenase gene tuaD and UTP- Cori ester uridyltransferase gene respectively
GtaB, recovery purifying.
(3) BamH1 digested plasmid pOSC, recovery purifying.
2. passing through Gibson assembly for the hyaluronan synthase gene of purification and recovery in step (1) and step (2)
HasA, UDP-glucose dehydrogenase gene tuaD and UTP- Cori ester uridyltransferase gene gtaB are connected to step
Suddenly the plasmid vector pOSC of (3) single endonuclease digestion linearisation, i.e. acquisition plasmid pOSC-HA.Gibson assembly system is hasA piece
Section, 3 μ l of tuaD segment and 0.3 μ l, 2x Gibson assembly Mix of gtaB segment each 0.9 μ l, carrier pOSC react item
Part is 50 DEG C of 1h, converts Escherichia coli DH10B competent cell, 100 μ g/ml of ampicillin screening, and picking single colonie is inoculated with,
Upgrading grain, the verifying of Sac1 single endonuclease digestion, positive recombinant are sequenced.
The bacillus subtilis BS168 Δ comA electricity that above-mentioned plasmid pOSC-HA electricity is transferred to comA gene delection is turned into impression
State, screening positive clone obtain the high yield hyaluronic acid recombined bacillus subtilis regulated and controled by oscillation mode gene expression system
BS168ΔcomA/pOSC-HA。
The sequence of the primer is as follows:
HasAFor:5 '-gtcttaaagtcgacgtccccaaggagaaaggtacccatgcctatcttcaagaagac-
3’
HasARev:5 '-gtaccactttcctcctttacttgaagatcgttaccttc-3 '
TuaDFor:5 '-taaaggaggaaagtggtacatgaaaaaaatagctgtcattg-3 '
TuaDRev:5 '-atcgagcctagcagccggcagcttcaaccaagtaacactttttctttttataaatt g
acgcttccc-3’
GtaBFor:5 '-ctttacgtacttttttcatttaaaaggcaccttccttatgatcgagcctagcagcc g
gc-3’
GtaBRev:5 '-ctcattaggcgggctgcccctagatgaatgtccaatagcctttttgtttagatttc t
tctttgtttag-3'；
PCR reaction step, double enzyme digestion reaction step, competent cell preparation step and electrotransformation step and 1 phase of embodiment
Together.
The recombined bacillus subtilis BS168 Δ comA/pOSC-HA built is inoculated in LB liquid medium (containing chlorine
5 μ g/ml of mycin), 37 DEG C, 200rpm culture 16h does seed liquor.Seed fermentation culture medium (glucose is inoculated with by 5% volume ratio
50g/L, yeast extract 10g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 2g/L), 37 DEG C, 200rpm
24-72h obtains the fermentation liquid containing hyaluronic acid.
The detection method of hyaluronic acid contents in fermentation liquid are as follows:
(1) it takes the above-mentioned fermentation liquid of 1ml, 1ml 0.1% (w/v) SDS solution is added, mix, be stored at room temperature 20min, 4 DEG C,
10000rpm is centrifuged 20min.Precipitating is abandoned, the dehydrated alcohol of 2ml pre-cooling, 4 DEG C of standing 1h are added.
(2) 4 DEG C, 10000rpm is centrifuged 20min, abandons supernatant, after the volatilization of remaining ethyl alcohol is clean, with deionized water weight
It is outstanding.
(3) 300 μ l HA solution examples are taken, 700 μ l acetate buffer (0.2mol/L sodium acetates, 0.15mol/L chlorine are added
Change sodium, with second acid for adjusting pH to 6.0), (2.5g/L CTAB, the sodium hydroxide for being dissolved in 0.5mol/L are molten for 2ml CTAB solution
Liquid), OD400 is surveyed after reacting 5min.
Testing result shows, high yield hyaluronic acid recombined bacillus subtilis BS168 Δ comA/pOSC-HA of the invention
Fermenting and producing hyaluronic acid fermentation yield reaches 21.8g/L.And the method for prior art production hyaluronic acid has animal tissue's extraction
It ferments with streptococcus zooepidemicus, the former contains heavy-metal residual and sensibiligen, and the latter is pathogen, risk of curing the disease.It is reported that beast
The yield that epidemic disease Streptococcal fermentation produces hyaluronic acid is generally 5-8g/L.
Sequence table
<110>Wang Yang
<120>a kind of bacillus subtilis oscillation mode gene expression system and its construction method and application
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gccagtcact atggcgtgct gatatcgaca aaaatgtcat g 41
<210> 2
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cttttatcta atctagacat attgtcatac ctcccctaat c 41
<210> 3
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gattagggga ggtatgacaa tatgtctaga ttagataaaa g 41
<210> 4
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ctgccccggg gacgtcgact ttaagaccca ctttcacat 39
<210> 5
<211> 860
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cagtcactat ggcgtgctga tgctgctaat gctacatttt gattaaatga atttgttttt 60
cctgcttagg cagctaatgc tacgttttgg ttaaaactgt tagttttgcc aagtacaccg 120
tctgatttcg caatcaaaac cgcttccgtc cgtgaaccga cattcagctt attgaaaatc 180
gatgtcaagc tgtattcaat ggaccgcttg cttaaatgaa gggcatctgc gatttcttgg 240
tttgtaaatc ccttttcaac ttcttgaaga atcaggcatt ctctaggtgt gagcacatct 300
tgttcttttt gagaggaagg agccggcttt gttttttgct gagtcatcag ctgtttaaag 360
taagcaaaat cgactaaaat ttctccgttg agtacgtggt atatgtattg ggtgatcttt 420
tctttagatt ccgttttgct gatggcaccg tgcagacccg cacgaatcgc ttcctcgaaa 480
taatcctcga cctcataacc ggtatacacg ataattttac aatgaggatt ctcttgtaaa 540
atctgtttag aaagctccat cccattgacc tcgccgccta gattcagatc cattaaaatg 600
agatcatatg acgagaaatc atgctgcttg ataaactgtt cgctcggttc aggactgaga 660
caatcaacag acaaattcga atccgtttcc aaaattgtct tggtgccttc catgacagcc 720
ggatggtcat caatcactag tatctttttc atacccatct ctttatcctt acctattgtt 780
tgtcgctagc gtcgaatgca agcagcagta tctctatcac tgatagggat gtcaaagcat 840
cgacaaaaat gtcatgaaag 860
<210> 6
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cagacgcgtc gacgtcatat gatcctgctt gggattctc 39
<210> 7
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gtgagtaaaa gggaggaaaa catgttgggg ggtgtagaga tg 42
<210> 8
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
catctctaca ccccccaaca tgttttcctc ccttttactc ac 42
<210> 9
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cctcgcgtcg ggcgatatcg atccgtgccg tgaagagg 38
<210> 10
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gtcttaaagt cgacgtcccc aaggagaaag gtacccatgc ctatcttcaa gaagac 56
<210> 11
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gtaccacttt cctcctttac ttgaagatcg ttaccttc 38
<210> 12
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
taaaggagga aagtggtaca tgaaaaaaat agctgtcatt g 41
<210> 13
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
atcgagccta gcagccggca gcttcaacca agtaacactt tttcttttta taaattgacg 60
cttccc 66
<210> 14
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ctttacgtac ttttttcatt taaaaggcac cttccttatg atcgagccta gcagccggc 59
<210> 15
<211> 68
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ctcattaggc gggctgcccc tagatgaatg tccaatagcc tttttgttta gatttcttct 60
ttgtttag 68
Claims (13)
1. a kind of oscillation gene expression regulation plasmid pOSC, which is characterized in that oscillation gene expression regulation plasmid pOSC be
Parallel connection is obtained by two metabolic regulation accesses that promoter Ptet and promoter Ps rfA starts respectively on carrier pHT01.
2. oscillation gene expression regulation plasmid pOSC according to claim 1, which is characterized in that the promoter Ptet
The metabolic regulation access of starting includes promoter Ptet, response regulatory factor gene comA and protein degradation label gene ssrA；
The metabolic regulation access of the promoter Ps rfA starting includes promoter Ps rfA, repressor gene tetR and target gene T.
3. it is of any of claims 1 or 2 oscillation gene expression regulation plasmid pOSC construction method, which is characterized in that including with
Lower step:
(1) using 168 genomic DNA of bacillus subtilis Bacillus substilis as template, using primer PsrfAFor,
PsrfARev expands srfA upstream region of gene promoter sequence；
(2) using Escherichia coli DH10B genomic DNA as template, tetR gene piece is expanded using primer tetRFor, tetRRev
Section；
(3) NheI/XbaI double digestion plasmid pHT43,5.4 Kb carrier segments of recovery purifying；
(4) pass through Gibson assembly for the promoter Ps rfA segment of purification and recovery in step (1) and step (2), tetR
Genetic fragment is connected to the plasmid vector of step (3) purification and recovery, i.e. acquisition plasmid pHT43-PsrfA-tetR；Under tetR gene
Trip is reserved with a BamH1 restriction enzyme site, facilitates the insertion of subsequent target gene；
(5) ssrA-comA-Ptet Genetic elements are synthesized, 20bp pHT43-PsrfA-tetR carrier EcoRV has respectively been merged at both ends
The same former sequence of site two sides；
(6) EcoRV single endonuclease digestion plasmid pHT43-PsrfA-tetR, 7.5 Kb carrier segments of recovery purifying；
(7) genetic fragment in step (5) is connected to the matter of step (6) single endonuclease digestion linearisation by Gibson assembly
Grain carrier, i.e. acquisition plasmid pOSC；
The primer and gene order are as follows:
PsrfAFor:5’-gccagtcactatggcgtgctgatatcgacaaaaatgtcatg-3’
PsrfARev:5’-cttttatctaatctagacatattgtcatacctcccctaatc-3’
tetRFor:5’-gattaggggaggtatgacaatatgtctagattagataaaag-3’
tetRRev:5’-ctgccccggggacgtcgactttaagacccactttcacat-3’
SsrA-comA-Ptet:
cagtcactatggcgtgctgaTGCTGCTAATGCTACATTTTGATTAAATGAATTTGTTTTTCCTGCttaggca gctaatgctacgttttggttaaaactgttagttttgccaagtacaccgtctgatttcgcaatcaaaaccgcttccg
tccgtgaaccgacattcagcttattgaaaatcgatgtcaagctgtattcaatggaccgcttgcttaaatgaagggc
atctgcgatttcttggtttgtaaatcccttttcaacttcttgaagaatcaggcattctctaggtgtgagcacatct
tgttctttttgagaggaaggagccggctttgttttttgctgagtcatcagctgtttaaagtaagcaaaatcgacta
aaatttctccgttgagtacgtggtatatgtattgggtgatcttttctttagattccgttttgctgatggcaccgtg
cagacccgcacgaatcgcttcctcgaaataatcctcgacctcataaccggtatacacgataattttacaatgagga ttctcttgtaaaatctgtttagaaagctccatcccattgacctcgccgcctagattcagatccattaaaatgagat catatgacgagaaatcatgctgcttgataaactgttcgctcggttcaggactgagacaatcaacagacaaattcga atccgtttccaaaattgtcttggtgccttccatgacagccggatggtcatcaatcactagtatctttttcataccc atctctttatccttacctattgtttgtcgctagcgtcgaatgcaagcagcagtatctctatcactgatagggatgt caaagcAtcgacaaaaatgtcatgaaag。
4. a kind of bacillus subtilis oscillation mode gene expression system, which is characterized in that the gene expression system is in withered grass bud
Response regulatory factor gene comA is knocked out in spore bacillus gene group, is transferred to oscillation gene expression regulation of any of claims 1 or 2
Plasmid pOSC is obtained.
5. the construction method of bacillus subtilis oscillation mode gene expression system as claimed in claim 4, it is characterised in that including
Following steps:
(I) building knocks out the recombinant plasmid of response regulatory factor gene comA, is transferred to bacillus subtilis, obtains knocking out comA base
The bacillus subtilis of cause；
(II) the oscillation mode gene expression regulation plasmid pOSC constructed by comA/Psrf and tetR/Ptet element is transferred to step
(I) bacillus subtilis of the knockout comA gene obtained, building obtain bacillus subtilis oscillation mode gene expression system.
6. the construction method of bacillus subtilis oscillation mode gene expression system according to claim 5, it is characterised in that
In step (I) building knock out the bacillus subtilis of comA gene process specifically includes the following steps:
(1) using Bacillus subtilis genes group DNA as template, be utilized respectively primer comAupFor, comAupRev and
ComAdnFor, comAdnRev expand comA upstream region of gene and segments downstream；
The primer sequence is as follows:
ComAupFor:5 '-CAGACGCGTCGACGTCATATGatcctgcttgggattctc-3 '
ComAupRev:5 '-gtgagtaaaagggaggaaaacatgttggggggtgtagagatg-3 '
ComAdnFor:5 '-catctctacaccccccaacatgttttcctcccttttactcac-3 '
ComAdnRev:5 '-CCTCGCGTCGGGCGATATCGatccgtgccgtgaagagg-3 '
(2) BamHI digested plasmid pMAD, recovery purifying；
(3) by the plasmid vector of the comA gene upstream and downstream homology arm of purification and recovery in step (1) and step (2) linearization for enzyme restriction
PMAD carries out Gibson assembly, i.e. acquisition recombinant plasmid pMAD- △ comA；
(4) the recombinant plasmid pMAD- △ comA that step (3) construct is transferred to bacillus subtilis, screening positive clone is struck
Except the bacillus subtilis BS168 Δ comA of response regulatory factor gene comA.
7. the construction method of bacillus subtilis oscillation mode gene expression system according to claim 5, it is characterised in that
In step (II) building oscillation mode gene expression regulation plasmid pOSC process specifically includes the following steps:
(1) using 168 genomic DNA of bacillus subtilis Bacillus substilis as template, using primer PsrfAFor,
PsrfARev expands srfA upstream region of gene promoter sequence；
(2) using Escherichia coli DH10B genomic DNA as template, tetR gene piece is expanded using primer tetRFor, tetRRev
Section；
(3) NheI/XbaI double digestion plasmid pHT43,5.4 Kb carrier segments of recovery purifying；
(4) pass through Gibson assembly for the promoter Ps rfA segment of purification and recovery in step (1) and step (2), tetR
Genetic fragment is connected to the plasmid vector of step (3) purification and recovery, i.e. acquisition plasmid pHT43-PsrfA-tetR；Under tetR gene
Trip is reserved with a BamH1 restriction enzyme site, facilitates the insertion of subsequent target gene；
(5) ssrA-comA-Ptet Genetic elements are synthesized, 20bp pHT43-PsrfA-tetR carrier EcoRV has respectively been merged at both ends
The same former sequence of site two sides；
(6) EcoRV single endonuclease digestion plasmid pHT43-PsrfA-tetR, recovery purifying 7.5Kb carrier segments；
(7) genetic fragment in step (5) is connected to the matter of step (6) single endonuclease digestion linearisation by Gibson assembly
Grain carrier, i.e. acquisition plasmid pOSC；
The primer and gene order are as follows:
PsrfAFor:5’-gccagtcactatggcgtgctgatatcgacaaaaatgtcatg-3’
PsrfARev:5’-cttttatctaatctagacatattgtcatacctcccctaatc-3’
tetRFor:5’-gattaggggaggtatgacaatatgtctagattagataaaag-3’
tetRRev:5’-ctgccccggggacgtcgactttaagacccactttcacat-3’
SsrA-comA-Ptet:
cagtcactatggcgtgctgaTGCTGCTAATGCTACATTTTGATTAAATGAATTTGTTTTTCCTGCttaggca gctaatgctacgttttggttaaaactgttagttttgccaagtacaccgtctgatttcgcaatcaaaaccgcttccg
tccgtgaaccgacattcagcttattgaaaatcgatgtcaagctgtattcaatggaccgcttgcttaaatgaagggc
atctgcgatttcttggtttgtaaatcccttttcaacttcttgaagaatcaggcattctctaggtgtgagcacatct
tgttctttttgagaggaaggagccggctttgttttttgctgagtcatcagctgtttaaagtaagcaaaatcgacta
aaatttctccgttgagtacgtggtatatgtattgggtgatcttttctttagattccgttttgctgatggcaccgtg
cagacccgcacgaatcgcttcctcgaaataatcctcgacctcataaccggtatacacgataattttacaatgagga ttctcttgtaaaatctgtttagaaagctccatcccattgacctcgccgcctagattcagatccattaaaatgagat catatgacgagaaatcatgctgcttgataaactgttcgctcggttcaggactgagacaatcaacagacaaattcga atccgtttccaaaattgtcttggtgccttccatgacagccggatggtcatcaatcactagtatctttttcataccc atctctttatccttacctattgtttgtcgctagcgtcgaatgcaagcagcagtatctctatcactgatagggatgt caaagcAtcgacaaaaatgtcatgaaag。
8. a kind of production hyaluronic acid recombined bacillus subtilis based on oscillation mode gene expression system, which is characterized in that will be saturating
Bright matter acid expression approach related gene is placed in bacillus subtilis oscillation mode gene expression system target gene as claimed in claim 4
The site T.
9. the production hyaluronic acid recombined bacillus subtilis according to claim 8 based on oscillation mode gene expression system,
It is characterized in that, the hyaluronic acid expression approach related gene includes the gene hasA of encoding hyaluronan synzyme,
The gene gtaB of gene tuaD and UTP- the Cori ester uridyltransferase of UDP-glucose dehydrogenase.
10. the production hyaluronic acid recombined bacillus subtilis according to claim 9 based on oscillation mode gene expression system,
It is characterized in that, the hyaluronan synthase gene hasA derives from streptococcus pyogenes (Streptococcus
Pyogenes), UDP-glucose dehydrogenase gene tuaD and UDP-glucose dehydrogenase gene gtaB derives from bacillus subtilis
Bacterium 168 (Bacillus substilis 168).
11. withered grass bud described in oscillation gene expression regulation plasmid pOSC as stated in claim 1 or 2, claim 4
Application of the spore bacillus oscillation mode gene expression system in recombinant protein expression.
12. any in bacillus subtilis oscillation mode gene expression system as described in claim 4 or claim 8~10
The recombined bacillus subtilis is producing the application in hyaluronic acid.
13. a kind of method using bacillus subtilis oscillation mode gene expression system production hyaluronic acid, it is characterised in that packet
Include following steps:
(1) using streptococcus pyogenes genomic DNA as template, hyaluronan synthase gene is expanded with primer hasAFor, hasARev
hasA；
(2) using Bacillus subtilis genes group DNA as template, with primer tuaDFor, tuaDRev and gtaBFor, gtaBRev divides
It Kuo Zeng not UDP-glucose dehydrogenase gene tuaD and UTP- Cori ester uridyltransferase gene gtaB；
The sequence of the primer is as follows:
HasAFor:5 '-gtcttaaagtcgacgtccccaaggagaaaggtacccatgcctatcttcaagaagac -3 '
HasARev:5 '-gtaccactttcctcctttacttgaagatcgttaccttc-3 '
TuaDFor:5 '-taaaggaggaaagtggtacatgaaaaaaatagctgtcattg-3 '
TuaDRev:5 '-atcgagcctagcagccggcagcttcaaccaagtaacactttttctttttataaatt gacgc
ttccc-3’
GtaBFor:5 '-ctttacgtacttttttcatttaaaaggcaccttccttatgatcgagcctagcagcc ggc-3 '
GtaBRev:5 '-ctcattaggcgggctgcccctagatgaatgtccaatagcctttttgtttagatttc ttctt
tgtttag-3'；
(3) BamH1 digestion oscillation gene expression regulation plasmid pOSC of any of claims 1 or 2, recovery purifying；
(4) by Gibson assembly by the hyaluronan synthase gene hasA of purification and recovery in step (1) and step (2),
UDP-glucose dehydrogenase gene tuaD and UTP- Cori ester uridyltransferase gene gtaB is connected to step (3)
The oscillation gene expression regulation plasmid pOSC of single endonuclease digestion linearisation, i.e. acquisition plasmid pOSC-HA；
(5) the bacillus subtilis BS168 Δ comA competence for above-mentioned plasmid pOSC-HA electricity being transferred to comA gene delection is thin
Born of the same parents, screening positive clone obtain the high yield hyaluronic acid recombined bacillus subtilis regulated and controled by oscillation mode gene expression system
BS168ΔcomA/pOSC-HA；
(6) recombined bacillus subtilis that step (5) obtains is accessed in LB liquid medium and is cultivated, obtain the engineering of preculture
Bacterium bacterium solution；
(7) the engineering bacteria bacterium solution access fermentation medium dosage form culture of the preculture is contained by the volume ratio of 1%-10%
There is the fermentation liquid of hyaluronic acid.
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