CN110066758A - A kind of bacillus of high yield poly-gamma-glutamic acid and its construction method and application - Google Patents
A kind of bacillus of high yield poly-gamma-glutamic acid and its construction method and application Download PDFInfo
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- 239000004220 glutamic acid Substances 0.000 title claims abstract description 57
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title claims abstract description 19
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 33
- 230000001580 bacterial effect Effects 0.000 claims abstract description 31
- 241000894006 Bacteria Species 0.000 claims abstract description 21
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 4
- 239000004098 Tetracycline Substances 0.000 claims abstract description 4
- RKLXDNHNLPUQRB-TVJUEJKUSA-N chembl564271 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]2C(C)SC[C@H](N[C@@H](CC(N)=O)C(=O)NC(=O)[C@@H](NC2=O)CSC1C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NC(=C)C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@H]1NC(=O)C(=C\C)/NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]2NC(=O)CNC(=O)[C@@H]3CCCN3C(=O)[C@@H](NC(=O)[C@H]3N[C@@H](CC(C)C)C(=O)NC(=O)C(=C)NC(=O)CC[C@H](NC(=O)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC=4C5=CC=CC=C5NC=4)CSC3)C(O)=O)C(C)SC2)C(C)C)C(C)SC1)C1=CC=CC=C1 RKLXDNHNLPUQRB-TVJUEJKUSA-N 0.000 claims abstract description 4
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- 238000000034 method Methods 0.000 claims description 17
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 238000011144 upstream manufacturing Methods 0.000 claims description 12
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- 230000029087 digestion Effects 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
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- 238000002360 preparation method Methods 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
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- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 1
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
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- 239000011574 phosphorus Substances 0.000 claims 1
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- 238000001514 detection method Methods 0.000 description 3
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- 235000013922 glutamic acid Nutrition 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
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- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
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- 206010010071 Coma Diseases 0.000 description 1
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 1
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 108700025158 Vitreoscilla hemoglobin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Abstract
The present invention provides bacillus and construction method and the application of a kind of high yield poly-gamma-glutamic acid.The bacillus is as obtained from the poly-gamma-glutamic acid synthase promoter with replacement promoter replacement bacillus licheniformis.Four plants of pgsBCAE promoter replacement bacterial strains are specifically obtained with the promoter of composing type strong promoter P43, stationary phase promoter Pylb, tetracycline inducible promoter Ptet and subtilin synzyme cluster promoter Ps rf replacement bacillus licheniformis poly-gamma-glutamic acid synthetic gene cluster pgsBCAE operon respectively.The result shows that the poly-gamma-glutamic acid yield of four plants of engineering bacterias is all remarkably higher than wild strain.The present invention uses Protocols in Molecular Biology, by preparing the engineered strain of energy high yield poly-gamma-glutamic acid with the poly-gamma-glutamic acid synthase promoter of replacement promoter replacement bacillus, a kind of new strategy is provided for large biological molecules such as bacillus high yield poly-gamma-glutamic acids.
Description
Technical field
The present invention relates to biotechnologys and fermentation arts, and in particular to a kind of bacillus of high yield poly-gamma-glutamic acid and
Its construction method and application.
Background technique
Poly-gamma-glutamic acid (γ-PGA), also known as Bacillus natto glue, poly-gamma-glutamic acid, it is a kind of by microorganism hair
Biopolymer made from ferment method is mainly polymerize by Pidolidone and (or) D-Glu monomer by γ-amido bond
It forms, molecular weight is usually 10KD-10000KD.γ-PGA has good water solubility, adsorption capacity, biodegradable, right
The characteristics such as human body and environment nonhazardous, are with a wide range of applications in various fields, in the field of medicine, can be used as drug
Sustained release agent；In terms of sewage treatment, adsorbent and flocculant can be used as；In agriculture field, water-retaining agent can be used as, fertilizer synergist increases
Imitate agent.Since γ-PGA has good economic value and broad application prospect, the production of γ-PGA is by more and more extensive
Attention.
The research of γ-PGA synthesis is concentrated mainly at present and strengthens precursor supply, the supply of oxygen, γ-PGA degradation etc.
Aspect, bear is vigorously equal to be integrated into B.licheniformis WX-02 chromosome for Vitreoscilla hemoglobin encoding gene (vgb)
In, engineering bacteria M2 energy normal expression VHb, the enhancing of thallus oxygen uptake capacity, biomass is than starting strain raising 25.5%, γ-PGA production
Amount improves 20%.Jiang etc. is overexpressed glutamate racemase gene glr in bacillus licheniformis, increases D- in γ-PGA
The content of glutamic acid, while improving the yield of γ-PGA.Tian etc. is overexpressed γ-PGA in bacillus licheniformis WX-02
Degrading enzyme gene pgdS, γ-PGA output increased 54%, and γ-PGA molecular weight becomes smaller.However to γ-PGA synzyme
It is less to express the influence research report synthesized to γ-PGA.γ-PGA is mainly by γ-PGA synzyme by glutamic acid monomer polymerization
It forms.γ-PGA synzyme is by tetra- subunit compositions of PgsB, PgsC, PgsA and PgsE, and wherein PgsBC polymerize with γ-PGA
Enzymatic activity is necessary to γ-PGA synthesis, and PgsA and PgsE may play an important role in the transport of γ-PGA, this four Asias
Base has pgsB, pgsC, pgsA and pgsE tetra- genes coding respectively, they form an operon, by the same promoter into
Row transcription.The promoter by complicated transcriptional control, by two-component regulatory factor D egS-DegU, ComP-ComA and transcription because
Sub- DegQ regulation.
Summary of the invention
In order to overcome the above-mentioned deficiency of the prior art, the present invention provides a kind of bacillus of high yield poly-gamma-glutamic acid
And its construction method and application, first passage of the present invention are closed with the poly-gamma-glutamic acid of replacement promoter replacement bacillus licheniformis
At enzyme promoters, to significantly improve the ability of bacillus licheniformis synthesis poly-gamma-glutamic acid.The present invention is bacillus
The large biological molecules such as high yield poly-gamma-glutamic acid provide a kind of new strategy.
The first purpose of this invention is to propose a kind of bacillus of high yield poly-gamma-glutamic acid, it is by with replacing
It changes obtained from the poly-gamma-glutamic acid synthase promoter of promoter replacement bacillus licheniformis and replaces bacterium.
Further, the replacement promoter is composing type strong promoter P43, stationary phase promoter Pylb, tetracycline lure
Lead any one of promoter Ptet, subtilin synzyme cluster promoter Ps rf；Poly- γ-paddy of the bacillus licheniformis
Propylhomoserin synthase promoter is pgsBCAE gene cluster promoter.
Further, the bacillus is bacillus licheniformis (Bacillus licheniformis) WX-02.
Second object of the present invention is to propose a kind of bud of high yield poly-gamma-glutamic acid as described in any one of the above embodiments
The construction method of born of the same parents bacillus, includes the following steps:
(1) the replacement carrier of poly-gamma-glutamic acid synthase promoter is obtained；
(2) the replacement carrier electricity of poly-gamma-glutamic acid synthase promoter is gone in bacillus licheniformis WX-02, is obtained
Obtain positive transformant；
(3) switching culture positive transformant, obtains single-swap zygote bacterial strain；
(4) switching culture single-swap zygote bacterial strain, and screen and obtain double crossing over bacterial strain, replacement is obtained after sequence verification
The bacillus licheniformis recombinant bacterial strain of poly-gamma-glutamic acid synthase promoter.
Further, the poly-gamma-glutamic acid synthase promoter replacement carrier the preparation method comprises the following steps:
(1) PCR amplification obtains pgsBCAE operon promoter region upstream homology arm sequence A and downstream homology arm sequence B；
(2) by pgsBCAE operon promoter region upstream homology arm sequence A, replacement promoter sequence P and pgsBCAE behaviour
It indulges sub- promoter region downstream homology arm sequence B and connects and composes A+P+B fusion segment；
(3) digestion A+P+B segment, and connected with T2 (the 2)-ori plasmid after identical endonuclease digestion；
(4) connection plasmid converts Escherichia coli, starts to poly-gamma-glutamic acid synzyme is obtained after positive transformant verifying
The replacement carrier of son.
Third object of the present invention is to propose that the bacillus of high yield poly-gamma-glutamic acid described in any of the above-described exists
Produce the application in poly-gamma-glutamic acid.
Fourth object of the present invention is a kind of method for proposing efficiently production poly-gamma-glutamic acid, with any of the above-described
The bacillus of the high yield poly-gamma-glutamic acid is starting strain, is accessed in fermentation medium after bacterial strain activation, shaking table is logical
Wind fermented and cultured 36-48h.
Further, the composition of the fermentation medium are as follows: glycerol or glucose 80g/L, 12 g/L of sodium citrate,
NaNO30-15g/L, sodium glutamate 0-60g/L, dipotassium hydrogen phosphate 0.5g/L, calcium chloride 0.5g/L, magnesium sulfate 0.5g/L, chlorination
Iron 0.004g/L, manganese sulfate 0.5g/L.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention for the first time use Protocols in Molecular Biology, by with replacement promoter replacement bacillus licheniformis it is poly-
Gamma-glutamic acid synthase promoter, to significantly improve the ability of bacillus licheniformis synthesis γ-PGA.More specifically, originally
Invention is closed respectively with composing type strong promoter P43, stationary phase promoter Pylb, tetracycline inducible promoter Ptet and subtilin
The promoter of bacillus licheniformis poly-gamma-glutamic acid synthetic gene cluster pgsBCAE operon is replaced at enzyme cluster promoter Ps rf,
Obtain four plants of pgsBCAE promoter replacement bacterial strain WX02-P43-pgsB, WX02-Pylb-pgsB, WX02-Ptet-pgsB and
WX02-Psrf-pgsB.Fermentation results show that the poly-gamma-glutamic acid yield of four plants of engineering bacterias is all remarkably higher than wild strain WX-
02, wherein WX02-P43-pgsB increase rate highest (2 times of raising).Illustrate that the genetic engineering remodeling method is improving brood cell's bar
The ability that bacterium synthesizes poly- γ glutamic acid plays a significant role.
(2) present invention uses Protocols in Molecular Biology for the first time, passes through the poly- γ-with replacement promoter replacement bacillus
Glutamate synthetase promoter prepares the replacement bacterial strain of energy high yield γ-PGA, is big point of the biology such as bacillus high yield γ-PGA
Son provides a kind of new strategy.This method can extend in the bacillus of other synthesis γ-PGA.
Detailed description of the invention
Fig. 1 is that promoter replaces carrier and promoter replaces strain construction flow diagram.
Fig. 2 is PCR amplification γ-PGA synthetase-coding gene pgsB promoter upstream and downstream homology arm and P43 in embodiment 1
Promoter, wherein swimming lane M be 5K DNA marker (from top to bottom successively are as follows: 5000bp, 3000bp, 2000bp, 1500bp,
1000bp, 750bp, 500bp, 250bp, 100bp)；Swimming lane 1 is gene pgsB promoter upstream homology arm；Swimming lane 2 is gene
PgsB promoter downstream homology arm；Swimming lane 3 is P43 promoter.
Fig. 3 is that T2 (2)-P43-pgsB plasmid construction PCR verifies glue figure in embodiment 1.Wherein swimming lane 1 is T2 (2)-
P43-pgsB plasmid construction E. coli clones PCR verifying；Swimming lane M be 5K DNA marker (from top to bottom successively are as follows:
5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Fig. 4 is that T2 (2)-Pylb-pgsB plasmid construction PCR verifies glue figure in embodiment 1.Wherein swimming lane 1 is T2 (2)-
Pylb-pgsB plasmid construction E. coli clones PCR verifying；Swimming lane M be 5K DNA marker (from top to bottom successively are as follows:
5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Fig. 5 is that T2 (2)-Ptet-pgsB plasmid construction PCR verifies glue figure in embodiment 1.Swimming lane 1:T2 (2)-Ptet-
PgsB plasmid construction E. coli clones PCR verifying；Swimming lane M:5K DNA marker (from top to bottom successively are as follows: 5000bp,
3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Fig. 6 is that T2 (2)-Psrf-pgsB plasmid construction PCR verifies glue figure in embodiment 1.Swimming lane 1:T2 (2)-Psrf-
PgsB plasmid construction E. coli clones PCR verifying；Swimming lane M:5K DNA marker (from top to bottom successively are as follows: 5000bp,
3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Fig. 7 verifies glue figure for T2 (2)-P43-pgsB plasmid electrotransformation in embodiment 2 to bacillus licheniformis PCR；Swimming lane
1,2:T2 (2)-P43-pgsB plasmid electrotransformation to bacillus licheniformis bacterium colony PCR verify；Swimming lane M:5K DNA marker (from
Top to bottm is successively are as follows: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Fig. 8 is that single exchange strains PCR is tested after T2 (2)-P43-pgsB plasmid electrotransformation to bacillus licheniformis in embodiment 2
Demonstrate,prove glue figure；Single exchange strains bacterium colony PCR is verified after swimming lane 1:T2 (2)-P43-pgsB plasmid electrotransformation to bacillus licheniformis；Swimming
Road M:5K DNA marker (from top to bottom successively are as follows: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp,
500bp, 250bp, 100bp).
Fig. 9 is that the P43 promoter of pgsBCAE operon in embodiment 2 replaces the PCR verifying glue figure of bacterial strain；Swimming lane 1:
The bacterium colony PCR of the promoter replacement bacterial strain of pgsBCAE operon verifies glue figure；Swimming lane 2: the PCR verifying of wild-type strain: swimming lane
M:5K DNA marker (from top to bottom successively are as follows: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp,
500bp, 250bp, 100bp).
Figure 10 is that the Pylb promoter of pgsBCAE operon in embodiment 2 replaces the PCR verifying glue figure of bacterial strain；Swimming lane 1:
The bacterium colony PCR of the promoter replacement bacterial strain of pgsBCAE operon verifies glue figure；Swimming lane M:5K DNA marker (from top to bottom according to
It is secondary are as follows: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Figure 11 is that the Ptet promoter of pgsBCAE operon in embodiment 2 replaces the PCR verifying glue figure of bacterial strain；Swimming lane 1:
The bacterium colony PCR of the promoter replacement bacterial strain of pgsBCAE operon verifies glue figure；Swimming lane M:5K DNA marker is (from top to bottom
Successively are as follows: 5000bp, 3000bp, 2000bp, 1500bp, 1000 bp, 750bp, 500bp, 250bp, 100bp).
Figure 12 is that the Psrf promoter of pgsBCAE operon in embodiment 2 replaces the PCR verifying glue figure of bacterial strain；Swimming lane 1,
The bacterium colony PCR of the promoter replacement bacterial strain of 2:pgsBCAE operon verifies glue figure；Swimming lane M:5K DNA marker is (from top to bottom
Successively are as follows: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Specific embodiment
It shows that example illustrates certain embodiments of the present invention, and should not be construed as limiting model of the invention
It encloses.Present disclosure can be improved from material, method and reaction condition simultaneously, all these improvement should all
It falls within spirit and scope of the invention.
Experimental method used in the following example is conventional method unless otherwise specified.
Material used in the following example, reagent etc., are commercially available unless otherwise specified.
The present invention provides a kind of brood cell by replacing poly-gamma-glutamic acid synthase promoter high yield poly-gamma-glutamic acid
The preparation method of bacillus, with pgsBCAE operon in P43, Pylb, Ptet and Psrf promoter replacement bacillus licheniformis
Promoter.In this method, the bacillus licheniformis (Bacillus licheniformis) is bacillus licheniformis
(Bacillus licheniformis) WX-02 is preserved in China typical culture collection center on April 28th, 2008,
Preservation is written as CCTCC NO:M208065.Specific preparation method includes the following steps:
(1) above and below according to γ glutamate synthetase encoding gene promoters poly- in bacillus licheniformis WX-02 genome
500bp sequence is swum, pgsB promoter upstream and downstream homology arm sequence A and B are amplified using the method for PCR；With Bacillus subtillis
168 genomic DNAs be template, the sequence of P43, Pylb, Ptet and Psrf promoter is amplified using the method for PCR, and lead to
A, B, P (P43, Pylb, Ptet and Psrf) are connected to together by the method for crossing SOE, are constituted A+P+B, are then used XbaI and BamHI
Double digestion A+P+B segment, and connected with T2 (the 2)-ori plasmid after identical endonuclease digestion, bacillus coli DH 5 alpha is converted,
Positive transformant is cloned and takes out plasmid progress double digestion and sequence verification, finally obtains replacing for the promoter of pgsBCAE operon
Change carrier T2 (2)-P43-pgsB, T2 (2)-Pylb-pgsB, T2 (2)-Ptet-pgsB, T2 (2)-Psrf-pgsB.
P43, Pylb, Ptet and Psrf promoter sequence is SEQ ID NO.1, SEQ ID NO.10, SEQ ID
Shown in NO.11 and SEQ ID NO.12.
(2) by the recombinant plasmid of above-mentioned building by electrotransformation into bacillus licheniformis WX-02, to positive transformant into
Row bacterium colony PCR verifying and pumping plasmid verifying；After positive transformant switching culture for several times, single exchange strains are obtained.Select single cross
It changes strain inoculated and carries out screening double crossing over by switching culture, PCR for several times in 37 DEG C, the culture medium without containing that penicillin of card
Bacterial strain carries out carry out sequence verification to positive strain, successfully obtains the bacillus licheniformis of replacement pgsBCAE gene cluster promoter
Recombinant bacterial strain WX02-P43-pgsB, WX02-Pylb-pgsB, WX02-Ptet-pgsB and WX02-Psrf-pgsB.
The application of the bacillus of high yield poly-gamma-glutamic acid of the invention, refer specifically to the bacillus licheniformis that will be obtained into
Row industrial fermentation is to produce γ-PGA.
In above-described application, the culture medium prescription of the fermentation, comprising: glycerol or glucose 80g/L, sodium citrate
12g/L, NaNO30-15g/L, sodium glutamate 0-60g/L, dipotassium hydrogen phosphate 0.5g/L, calcium chloride 0.5g/L, magnesium sulfate 0.5g/
L, iron chloride 0.004g/L, manganese sulfate 0.5g/L.
Embodiment 1: by taking P43 as an example, the promoter of building bacillus licheniformis pgsBCAE operon replaces carrier
1, by taking P43 as an example, the promoter of building bacillus licheniformis pgsBCAE operon replaces carrier
Step 1:
According to the upstream and downstream sequence of the pgsB promoter in bacillus licheniformis WX-02 genomic dna sequence, design
Upstream homology arm primer (A-F and A-P43-R), the downstream homology arm primer (B-P43-F and B-R) of pgsB promoter；And with ground
The genomic DNA of clothing bacillus WX-02 is template, respectively with upstream homology arm primer, the downstream homology arm of pgsB promoter
Primer carries out PCR amplification and obtains the upstream homology arm (535bp) of pgsB promoter and the downstream homology arm of pgsB promoter
(499bp).According to the sequence of P43 promoter in 168 genomic dna sequence of Bacillus subtillis (shown in SEQ ID NO.1), if
Count the primer (P43-F and P43-R) of P43 promoter；Using 168 genomic DNA of Bacillus subtillis as template, with P43-F and
P43-R is that primer progress PCR amplification obtains P43 promoter (300bp), as shown in Figure 2.
Wherein, the sequence of A-F, A-P43-R, B-F, B-R, P43-F, P43-R are as follows:
SEQ ID NO.2:A-F:GCGAGCTCTTAATACCCGGGAAACCGTAGCGA
SEQ ID NO.3:A-P43-R:
TACCACCTATCAGAATTCTCCCCTTTAAATGGGCTGTC
SEQ ID NO.4:B-P43-F:
TCTAAAGGAGGAAGGATCAATGTGGGTAATGCTATTAG
SEQ ID NO.5:B-R:CGGGATCCAATGTCCATTATAAGGAAT
SEQ ID NO.6:P43-F:
GACAGCCCATTTAAAGGGGAGAATTCTGATAGGTGGTA
SEQ ID NO.7:P43-R:
CTAATAGCATTACCCACATTGATCCTTCCTCCTTTAGA
Step 2:
The upstream homology arm of pgsB promoter, the downstream homology arm of pgsB promoter and P43 are opened by Overlap extension PCR
Promoter fragment connects together (the primer is A-F and B-R), constitutes target gene fragment (1434bp) (as shown in Figure 2).
Step 3:
Double digestion is carried out to target gene fragment and T2 (2)-ori plasmid using BamHI and XbaI restriction enzyme to obtain
To digestion genetic fragment (1434bp), wherein restriction enzyme BamHI and XbaI restriction enzyme is purchased from the full formula in Beijing
Golden Bioisystech Co., Ltd.
Obtained digestion genetic fragment and linear plasmid segment are attached through DNA ligase, obtain connection product；It is logical
The connection product is transferred to bacillus coli DH 5 alpha by superchlorination calcium conversion method, through containing that penicillin resistance of card under conditions of 37 DEG C
Culture medium screened, screening obtain transformant, to transformant choose plasmid carry out bacterium colony PCR verifying (the primer are as follows: T2-F
And T2-R).The PCR verification result of transformant are as follows: occur electrophoretic band (see Fig. 3) at 1537bp, illustrate integrating expression vector
It constructs successfully, above-mentioned transformant is positive transformant, is named as promoter replacement carrier T2 (2)-P43-pgsB；
SEQ ID NO.8:T2-F:ATGTGATAACTCGGCGTA
SEQ ID NO.9:T2-R:GCAAGCAGCAGATTACGC.
2, the building process of the promoter replacement carrier of tri- promoters of Pylb, Ptet, Psrf is opened with embodiment 1
Mover replaces carrier, and wherein Pylb, Ptet and Psrf promoter sequence are T2 (2)-Pylb- shown in SEQ ID NO.10-12
PgsB, T2 (2)-Ptet-pgsB, T2 (2)-Psrf-pgsB plasmid construction result are respectively such as Fig. 4-6.
In the embodiment 1, primer sequence used is as follows:
SEQ ID NO.13
A-Pylb-R:ATGGAGCGCGTTCGACGATGTCCCCTTTAAATGGGCTGTC
SEQ ID NO.14
B-Pylb-F:AACAAAGGGGGAGATTTGTGAATTCTGATAGGTGGTA
SEQ ID NO.15
Pylb-F:GACAGCCCATTTAAAGGGGACATCGTCGAACGCGCTCCAT
SEQ ID NO.16
Pylb-R:TACCACCTATCAGAATTCACAAATCTCCCCCTTTGTT
SEQ ID NO.17
A-Ptet-R:TGTGAAAGTGGGTCTTAATCCCCTTTAAATGGGCTGTC
SEQ ID NO.18
B-Ptet-F:AGAGGAGAAAGGATCTCCCATGTGGGTAATGCTATTAG
SEQ ID NO.19
Ptet-F:GACAGCCCATTTAAAGGGGATTAAGACCCACTTTCACA
SEQ ID NO.20
Ptet-R:CTAATAGCATTACCCACATGGGAGATCCTTTCTCCTCT
SEQ ID NO.21
A-Psrf-R:ACCAAACTGCTGCGTTCATCCCCTTTAAATGGGCTGTC
SEQ ID NO.22
B-Psrf-F:TTAGGGGAGGTATGACAATATGTGGGTAATGCTATTAG
SEQ ID NO.23
Psrf-F:GACAGCCCATTTAAAGGGGATGAACGCAGCAGTTTGGT
SEQ ID NO.24
Psrf-R:CTAATAGCATTACCCACATATTGTCATACCTCCCCTAA
The building of the promoter replacement bacterial strain of embodiment 2:pgsBCAE operon
1, by taking P43 as an example, the promoter of building pgsBCAE operon replaces bacterial strain
Step 1:
Integrating expression vector T2 (2)-P43-pgsB is transferred in bacillus licheniformis WX-02, under conditions of 37 DEG C, is contained
The culture medium for blocking that penicillin resistance is screened, and screening obtains transformant, is chosen plasmid to transformant and is carried out bacterium colony PCR verifying
(the primer are as follows: T2-F and T2-R).The PCR verification result of transformant are as follows: occur electrophoretic band at 1527bp, it was demonstrated that integration
Expression vector T2 (2)-P43-pgsB is successfully transferred in bacillus licheniformis WX-02.At this point, the transformant is positive transformant,
It is transferred to the bacillus licheniformis WX-02 of integrating expression vector T2 (2)-P43-pgsB, as a result as shown in Figure 7.
Step 2:
The positive transformant that step 1 obtains is transferred under the conditions of 45 DEG C, on the culture medium containing that penicillin resistance of card
Culture 3 times cultivates 12h every time, and carries out bacterium colony PCR as primer using T2-F and pgsB-YR and detect single exchange strains, amplifies
It is as shown in Figure 8 to prove single exchange strains result for the band of 1527bp or 2027bp length.Wherein pgsB-YF sequence is as follows:
SEQ ID NO.25pgsB-YF:GATCAGTCTGGACTCCCTT。
Step 3:
Is there is into the single exchange strains inoculated and cultured of 1527bp band in PCR that step 2 obtains detection, 37 DEG C, do not contain
Block in the culture medium of that penicillin by for several times switching culture, choose transformant carry out bacterium colony PCR verifying (primer be pgsB-YF and
P43-R).The PCR verification result of transformant are as follows: when not occurring electrophoretic band, illustrate gene reversion, which is lichens
Bacillus WX-02；Occur electrophoretic band at 944bp, illustrates that P43 promoter successfully replaces the starting of pgsBCAE operon
Son.DNA sequencing then is carried out for positive transformant further to verify, and obtains opening for the successful pgsBCAE operon of double crossing over
Mover replacement bacterial strain, i.e. bacillus licheniformis WX02-P43-pgsB, as a result as shown in Figure 9.
2, the building process of the promoter replacement bacterial strain of tri- promoters of Pylb, Ptet, Psrf is opened with embodiment 2
Mover replaces bacterial strain, WX02-Pylb-pgsB, WX02-Ptet-pgsB, WX02-Psrf-pgsB.Result is constructed respectively such as Figure 10-
Shown in 12.
Embodiment 3: production poly-gamma-glutamic acid
Bacillus licheniformis WX02-P43-pgsB, WX02-Ptet-pgsB that the present embodiment is prepared with Examples 1 and 2,
WX02-Pylb-pgsB, WX02-Psrf-pgsB are starting strain, produce γ-PGA.
1, seed fermentation
Bacillus licheniformis WX-02 and bacillus licheniformis WX02-P43-pgsB, WX02-Ptet- are activated on plate
PgsB, WX02-Pylb-pgsB, WX02-Psrf-pgsB, choose bacterium and are connected to and be seeded to the 250mL tri- containing 50mL liquid LB respectively
In the bottle of angle, 37 DEG C, 230rpm cultivates 8h.Fermentation medium (fermented and cultured is then seeded to the inoculum concentration of 1.5% (volume ratio)
Glycerol concentration is 80g/L, citric acid 12g/L, sodium glutamate 20g/L in base) in, 37 DEG C, 230rpm culture 48h.Measurement fermentation
Terminal γ-PGA yield.
2, γ-PGA measuring method
Fermentation liquid pre-treatment: fermentation liquid is diluted 30 times using deionized water, bactofugation body, then through 0.22 μm of water phase
Gel permeation chromatography detection is carried out after membrane filtration.Gel permeation chromatography testing conditions are as follows: use TSK Gel G6000PWXL
Gel permeation chromatographic column, Detection wavelength 220nm, 10 μ L of sample volume, mobile phase are the mixing of the anhydrous sodium sulfate and acetonitrile of 25mM
Liquid, volume ratio 8:1, flow velocity 0.5mL/min.γ-in fermentation liquid is calculated according to the standard curve that the standard items of γ-PGA make
The content of PGA.
Table 1 P43, Pylb, Psrf, Ptet promoter replace the γ-PGA yield and biomass comparison of bacterial strain
| Bacterial strain | Poly-gamma-glutamic acid yield (g/L) | Biomass (g/L) |
| WX-02 | 9.34 | 4.50 |
| WX02-P43-pgsB | 28.93 | 3.70 |
| WX02-Pylb-pgsB | 22.32 | 4.10 |
| WX02-Psrf-pgsB | 11.23 | 4.60 |
| WX02-Ptet-pgsB | 17.54 | 4.30 |
The result shows that P43, Pylb, Ptet and Psrf displacement pgsBCAE promoter can significantly improve poly- γ-paddy ammonia
The yield of acid, and WX02-P43-pgsB γ-PGA yield highest, improve 2 times.
Embodiment 4:
Best engineering bacteria WX02-P43-pgsB is fermented in following 18 kinds of culture mediums.Applicant has selected 18 kinds
Culture medium prescription, number 1-18 are γ-PGA fermentation medium (being shown in Table 2-3).It include dipotassium hydrogen phosphate in every kind of culture medium
0.5g/L, calcium chloride 0.5g/L, magnesium sulfate 0.5g/L, iron chloride 0.004 g/L, manganese sulfate 0.5g/L.
The condition of culture of γ-PGA fermentation medium is 37 DEG C, and 230rpm cultivates 48h, measures γ-PGA after fermentation and produces
Amount.
The culture medium prescription of 2 γ-PGA of table fermentation
The culture medium prescription of 3 γ-PGA of table fermentation
| Number | Glucose (g/L) | Sodium citrate (g/L) | Sodium nitrate (g/L) | Sodium glutamate (g/L) |
| 10 | 80 | 12 | 0 | 0 |
| 11 | 80 | 12 | 0 | 20 |
| 12 | 80 | 12 | 0 | 40 |
| 13 | 80 | 12 | 0 | 60 |
| 14 | 80 | 12 | 10 | 20 |
| 15 | 80 | 12 | 15 | 0 |
| 16 | 80 | 12 | 15 | 20 |
| 17 | 80 | 12 | 15 | 40 |
| 18 | 80 | 12 | 15 | 60 |
The yield that it is γ-PGA in 1-18 in above-mentioned culture medium number that applicant, which determines, the results are shown in Table 4.
γ-PGA the yield and biomass comparison of 4 WX02-P43-pgsB of table and control bacterium WX-02
As can be known from Table 4, under conditions of identical seed fermentation and production fermentation, using lichens brood cell's bar of the invention
Bacterium WX02-P43-pgsB can significantly improve bacterial strain synthesis γ-PGA, and increase rate most down to 16.69%, reaches as high as
577.10%.Technical solution of the present invention has important significance of scientific research and application value in bacillus high yield γ-PGA.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
<110>Wuhan Jun An Biotechnology Co., Ltd
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<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
tgtgaaagtg ggtcttaatc ccctttaaat gggctgtc 38
<210> 18
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
agaggagaaa ggatctccca tgtgggtaat gctattag 38
<210> 19
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gacagcccat ttaaagggga ttaagaccca ctttcaca 38
<210> 20
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ctaatagcat tacccacatg ggagatcctt tctcctct 38
<210> 21
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
accaaactgc tgcgttcatc ccctttaaat gggctgtc 38
<210> 22
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
ttaggggagg tatgacaata tgtgggtaat gctattag 38
<210> 23
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gacagcccat ttaaagggga tgaacgcagc agtttggt 38
<210> 24
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
ctaatagcat tacccacata ttgtcatacc tcccctaa 38
<210> 25
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gatcagtctg gactccctt 19
Claims (8)
1. a kind of bacillus of high yield poly-gamma-glutamic acid, which is characterized in that it be by with replacement promoter replacement lichens
Bacterium is replaced obtained from the poly-gamma-glutamic acid synthase promoter of bacillus.
2. a kind of bacillus of high yield poly-gamma-glutamic acid according to claim 1, which is characterized in that the replacement is opened
Mover is composing type strong promoter P43, stationary phase promoter Pylb, tetracycline inducible promoter Ptet, subtilin synzyme
Any one of cluster promoter Ps rf；The poly-gamma-glutamic acid synthase promoter of the bacillus licheniformis is pgsBCAE base
Because of cluster promoter.
3. a kind of bacillus of high yield poly-gamma-glutamic acid according to claim 1, which is characterized in that brood cell's bar
Bacterium is bacillus licheniformis (Bacillus licheniformis) WX-02.
4. a kind of construction method of the bacillus of high yield poly-gamma-glutamic acid as described in any one of claims 1-3, special
Sign is, includes the following steps:
(1) the replacement carrier of poly-gamma-glutamic acid synthase promoter is obtained；
(2) the replacement carrier electricity of poly-gamma-glutamic acid synthase promoter is gone in bacillus licheniformis WX-02, is obtained positive
Transformant；
(3) switching culture positive transformant, obtains single-swap zygote bacterial strain；
(4) switching culture single-swap zygote bacterial strain, and screen and obtain double crossing over bacterial strain, it obtains and is substituted after sequence verification
The bacillus licheniformis recombinant bacterial strain of poly-gamma-glutamic acid synthase promoter.
5. a kind of construction method of the bacillus of high yield poly-gamma-glutamic acid according to claim 4, which is characterized in that
The poly-gamma-glutamic acid synthase promoter replacement carrier the preparation method comprises the following steps:
(1) PCR amplification obtains pgsBCAE operon promoter region upstream homology arm sequence A and downstream homology arm sequence B；
(2) by pgsBCAE operon promoter region upstream homology arm sequence A, replacement promoter sequence P and pgsBCAE operon
Promoter region downstream homology arm sequence B connects and composes A+P+B fusion segment；
(3) digestion A+P+B segment, and connected with T2 (the 2)-ori plasmid after identical endonuclease digestion；
(4) connection plasmid converts Escherichia coli, to acquisition poly-gamma-glutamic acid synthase promoter after positive transformant verifying
Replace carrier.
6. the bacillus of the described in any item high yield poly-gamma-glutamic acids of claim 1-3 is producing answering in poly-gamma-glutamic acid
With.
7. a kind of method of efficiently production poly-gamma-glutamic acid, which is characterized in that with the described in any item high yields of claim 1-3
The bacillus of poly-gamma-glutamic acid is starting strain, is accessed in fermentation medium after bacterial strain activation, shaking table ventilating fermentation culture
36-48h。
8. a kind of method of efficiently production poly-gamma-glutamic acid according to claim 7, which is characterized in that the fermentation training
Support the composition of base are as follows: glycerol or glucose 80g/L, sodium citrate 12g/L, NaNO30-15g/L, sodium glutamate 0-60g/L, phosphorus
Sour hydrogen dipotassium 0.5g/L, calcium chloride 0.5g/L, magnesium sulfate 0.5g/L, iron chloride 0.004g/L, manganese sulfate 0.5g/L.
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| WO2023111304A1 (en) | 2021-12-16 | 2023-06-22 | Danmarks Tekniske Universitet | Temperature-inducible expression system for thermophilic organisms |
| CN114517193A (en) * | 2021-12-23 | 2022-05-20 | 广州远想医学生物技术有限公司 | Gamma-polyglutamic acid degrading enzyme and preparation method thereof |
| CN114517193B (en) * | 2021-12-23 | 2023-07-07 | 广州远想医学生物技术有限公司 | Gamma-polyglutamic acid degrading enzyme and preparation method thereof |
| CN114369561A (en) * | 2022-01-11 | 2022-04-19 | 江南大学 | Strain for regulating expression level of CapbCA monomer and application of strain in production of polyglutamic acid |
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| CN114874965B (en) * | 2022-06-14 | 2023-04-14 | 安徽工程大学 | Bacillus subtilis engineering bacterium and construction method and application thereof |
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