CN106085937B - A kind of bacillus subtilis recombinant bacterial strain and the preparation method and application thereof - Google Patents
A kind of bacillus subtilis recombinant bacterial strain and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of bacillus subtilis recombinant bacterial strains and the preparation method and application thereof, belong to field of biotechnology.Recombinant bacterial strain provided by the present invention is that ten protease genes in bacillus subtilis are knocked out or inactivated to obtain, this ten protease genes are as follows: nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA, spollAC and srfAC.The present invention carries out the knockout of aforementioned 10 protease genes by methods of homologous recombination, and obtained bacillus subtilis recombinant bacterial strain growth is fast and exogenous protein expression amount is high.Therefore, which can be used as the host of foreign protein high efficient expression.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of bacillus subtilis recombinant bacterial strain and preparation method thereof with
Using.
Background technique
Bacillus subtilis (Bacillus subtilis) belongs to bacillus, is gram-positive bacteria, without pathogenic
Property, it is acknowledged as safe bacterial strain (GRAS).B.subtilis only has cell monolayer outer membrane, and the speed of growth is fast, nutritional need is simple
It is single, it is easy to survive, colonizes and breed；With clearly legacy background, good protein secretion ability and a series of efficient letters
Number peptide, is used for the production of food fermentation and important enzyme preparation for a long time；It is that foreign protein is expressed and secreted in prokaryotic expression system
Ideal host.
For B.subtilis since itself extracellular proteinase is more, the influence for the factors such as the expression plasmid of building is unstable makes it
Using receiving a degree of limitation.Transformation for bacillus subtilis expression system focuses primarily upon and knocks out withered grass bud
Spore bacillus extracellular protease gene.At present by gene Knockout, obtained DB104, DB403, WB600, WB700 and
The protease deficient strains such as WB800.WB800 is that professor Wong tests team constructs type strain B.subtilis168 eight
A Deficient In Extracellular Proteases strain has knocked out neutral proteinase (npr), neutral proteinase B (nprB), alkali protease (apr), metal egg
White enzyme (mpr), three kinds of serine proteases (bpf, epr and vpr) and protein in cell wall enzyme (wprA) [WuSC, YeungJC,
WongSL,etal.Functionalproduction and characterization of a fibrin-specific
single-chain antibody fragment from Bacillus subtilis:effects of molecular
chaperones and a wall-bound Protease on antibody fragment production[J]
.Appl.Environ.Microbiol, many genes 2002,68:3261-269.], is made to realize stable expression.But WB800
Grow slower, remaining protease also can effectively degrade the foreign protein of high sensitivity, and host carries Multiple Classes of Antibiotics base
Cause.
Therefore, it is necessary to further transformation is continued to bacillus subtilis, it is fast to obtain the speed of growth, and foreign protein
The high bacterial strain of expression quantity.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of bacillus subtilis recombination
Bacterial strain.
Another object of the present invention is to provide the preparation methods of the bacillus subtilis recombinant bacterial strain.
A further object of the present invention is to provide the applications of the bacillus subtilis recombinant bacterial strain.
The purpose of the invention is achieved by the following technical solution: a kind of bacillus subtilis recombinant bacterial strain, is by withered grass bud
Ten protease genes in spore bacillus knock out or inactivation obtains, this ten protease genes are as follows: neutral protein enzyme coding gene
(nprE), neutral proteinase B encoding gene (nprB), basic protein enzyme coding gene (aprE), metalloproteinases (mpr), three
Kind serine stretch protein enzyme coding gene (bpf, epr and vpr), protein in cell wall enzyme coding gene (wprA), sporelating factor F are compiled
Code gene (spollAC) and surfactin synthetase-coding gene (srfAC).
The bacillus subtilis is bacillus subtilis (Bacillus subtilis) ATCC6051.
The nucleotide sequence of the nprE is as shown in Gene ID:935981；The nucleotide sequence of the nprB is such as
Shown in Gene ID:936384；The nucleotide sequence of the aprE is as shown in Gene ID:939313；The nucleosides of the mpr
Acid sequence is as shown in Gene ID:938430；The nucleotide sequence of the bpf is as shown in Gene ID:939695；Described
The nucleotide sequence of epr is as shown in Gene ID:937332；The nucleotide sequence of the vpr such as Gene ID:937291 institute
Show；The nucleotide sequence of the wprA is as shown in Gene ID:936350；The nucleotide sequence of the spollAC such as Gene
Shown in ID:938729；The nucleotide sequence of the srfAC is as shown in Gene ID:938308.
The inactivation is that gene correctly cannot be transcribed or be translated.
The preparation method of the bacillus subtilis recombinant bacterial strain is by hindering ten protease genes to turn
Record, the correct technology transcribed or translated obtain.
The technology includes gene Knockout, frameshift mutation technology and GEM 132 perturbation technique.
The gene Knockout is preferably methods of homologous recombination.
The methods of homologous recombination preferably comprises following steps:
(1) the upstream homology arm and downstream homology arm of ten protease genes are constructed respectively, then by upstream homology arm under
Homology arm fusion is swum, upstream and downstream homology arm is obtained；Ten protease-baseds are because of neutral proteinase encoding gene (nprE), neutral egg
White enzyme B encoding gene (nprB), basic protein enzyme coding gene (aprE), metalloproteinases (mpr), three kinds of serine proteases
Encoding gene (bpf, epr and vpr), protein in cell wall enzyme coding gene (wprA), sporelating factor F encoding gene
(spollAC) and surfactin synthetase-coding gene (srfAC)；
(2) the upstream and downstream homology arm of ten protease genes is cloned into respectively in Thermo-sensitive plasmid pKS2, obtains ten
The knockout carrier of protease gene: pKS2-nprE, pKS2-nprB, pKS2-aprE, pKS2-mpr, pKS2-bpf, pKS2-
epr,pKS2-vpr,pKS2-wprA,pKS2-spollAC,pKS2-srfAC；
(3) any of the knockout carrier of ten protease genes is transferred in bacillus subtilis starting strain, is passed through
Screening, obtains recombinant bacterium 1；It is transferred to recombinant bacterium 1 with the knockout carrier of another protease gene again, screens, obtains recombinant bacterium 2；
The knockout carrier for the protease gene that one is not transferred to is transferred in recombinant bacterium obtained in the previous step respectively, obtains recombinant bacterium 3, again
Group bacterium 4, recombinant bacterium 5, recombinant bacterium 6, recombinant bacterium 7, recombinant bacterium 9, finally obtain the bacillus subtilis for knocking out ten protease genes
Bacterium.
The upstream homology arm of the nprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.12 and SEQ ID NO.13 obtains.
The downstream homology arm of the nprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.14 and SEQ ID NO.15 obtains.
The upstream homology arm of the nprB is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.16 and SEQ ID NO.17 obtains.
The downstream homology arm of the nprB is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.18 and SEQ ID NO.19 obtains.
The upstream homology arm of the aprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.20 and SEQ ID NO.21 obtains.
The downstream homology arm of the aprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.22 and SEQ ID NO.23 obtains.
The upstream homology arm of the mpr is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.24 and SEQ ID NO.25 obtains.
The downstream homology arm of the mpr is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.26 and SEQ ID NO.27 obtains.
The upstream homology arm of the bpf is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.28 and SEQ ID NO.29 obtains.
The downstream homology arm of the bpf is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.30 and SEQ ID NO.31 obtains.
The upstream homology arm of the epr is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.32 and SEQ ID NO.33 obtains.
The downstream homology arm of the epr is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.34 and SEQ ID NO.35 obtains.
The upstream homology arm of the vpr is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.36 and SEQ ID NO.37 obtains.
The downstream homology arm of the vpr is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.38 and SEQ ID NO.39 obtains.
The upstream homology arm of the wprA is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.40 and SEQ ID NO.41 obtains.
The downstream homology arm of the wprA is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.42 and SEQ ID NO.43 obtains.
The upstream homology arm of the spollAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.44 and SEQ ID NO.45 obtains.
The downstream homology arm of the spollAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.46 and SEQ ID NO.47 obtains.
The upstream homology arm of the srfAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.48 and SEQ ID NO.49 obtains.
The downstream homology arm of the srfAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.50 and SEQ ID NO.51 obtains.
The step of described fusion, is preferably as follows: same to expand upstream using upstream homology arm and downstream homology arm as template
The upstream primer of source arm and the downstream primer of amplification downstream homology arm are primer pair amplifies, obtain upstream and downstream homology arm.
The sequence of the upstream and downstream homology arm of the nprE is as shown in SEQ ID NO.1.
The sequence of the upstream and downstream homology arm of the nprB is as shown in SEQ ID NO.2.
The sequence of the upstream and downstream homology arm of the aprE is as shown in SEQ ID NO.3.
The sequence of the upstream and downstream homology arm of the mpr is as shown in SEQ ID NO.4.
The sequence of the upstream and downstream homology arm of the bpf is as shown in SEQ ID NO.5.
The sequence of the upstream and downstream homology arm of the epr is as shown in SEQ ID NO.6.
The sequence of the upstream and downstream homology arm of the vpr is as shown in SEQ ID NO.7.
The sequence of the upstream and downstream homology arm of the wprA is as shown in SEQ ID NO.8.
The sequence of the upstream and downstream homology arm of the spollAC is as shown in SEQ ID NO.9.
The sequence of the upstream and downstream homology arm of the srfAC is as shown in SEQ ID NO.10.
The upstream and downstream homology arm by ten protease genes be cloned into respectively be in Thermo-sensitive plasmid pKS2 will be described
The upstream and downstream homology arms of ten protease genes be cloned into the EcoRV restriction enzyme site in Thermo-sensitive plasmid pKS2.
The bacillus subtilis starting strain is preferably bacillus subtilis ATCC6051.
The recombinant bacterium 1 is preferably the bacillus subtilis for lacking nprE gene.
The recombinant bacterium 2 is preferably the bacillus subtilis for lacking nprE, nprB gene.
The recombinant bacterium 3 is preferably the bacillus subtilis for lacking nprE, nprB, aprE gene.
The recombinant bacterium 4 is preferably the bacillus subtilis for lacking nprE, nprB, aprE, mpr gene.
The recombinant bacterium 5 is preferably the bacillus subtilis for lacking nprE, nprB, aprE, mpr, bpf gene.
The recombinant bacterium 6 is preferably the bacillus subtilis for lacking nprE, nprB, aprE, mpr, bpf, epr gene.
The recombinant bacterium 7 is preferably the bacillus subtilis for lacking nprE, nprB, aprE, mpr, bpf, epr, vpr gene
Bacterium.
The recombinant bacterium 8 is preferably the withered grass for lacking nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA gene
Bacillus.
The recombinant bacterium 9 preferably lacks nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA, spollAC base
The bacillus subtilis of cause.
The screening is to cultivate by 37 DEG C of heating cultures and 30 DEG C of coolings, then by PCR and sequence verification, sieved
Choosing；Preferably include following steps:
It is coated on 1. the knockout carrier of protease gene is transferred to bacillus subtilis and obtains bacterium solution containing 5 μ g/mL erythromycin
(erm) the LB plate of resistance is cultivated, the positive colony tentatively confirmed in 30 DEG C of inversions；
2. the positive colony that picking tentatively confirms is inoculated in the LB liquid medium containing 5 μ g/mL Erythromycinresistants, in
30 DEG C, 200rpm culture 12h, the verifying of upgrading grain have been transferred in host, the positive colony reaffirmed；
3. the bacterium solution for the positive colony for taking 100 μ L to reaffirm is inoculated in 37 DEG C of cultures in 10mL LB liquid medium
12h dilutes 1000 times, takes 100 μ L bacterium solutions to be coated on the LB plate containing 5 μ g/mL Erythromycinresistants, 37 DEG C of culture 12h；
4. 3. picking step contains the transformant on the LB plate of 5 μ g/mL Erythromycinresistants, it is inoculated in the training of 10mL LB liquid
It supports in base, 30 DEG C, 200rpm cultivates 12h；Culture solution dilution 105Times, take 100 μ L bacterium solutions to be coated on LB solid plate, 37 DEG C
Cultivate 12h；
5. the picking step single colonie that 4. LB solid plate is grown at the same switching on LB plate and contain 5 μ g/mL erythromycin
On the LB plate of resistance, Phenotypic Expression is to grow on LB nonreactive plate, and not long in LB resistant panel is putative clone
Son, PCR are further verified, and obtain recombinant bacterium.
Application of the bacillus subtilis recombinant bacterial strain in exogenous protein expression.
The foreign protein is preferably transglutaminase (microbial transglutaminase, MTG).
The present invention has the following advantages and effects with respect to the prior art: in the transformation to bacillus subtilis, by
In the expression for changing bacillus subtilis oneself protein, although reducing the degradation degree to foreign protein, often
Influence whether the growth and breeding speed of bacillus subtilis.Although present invention inactivation ten exocytosis albumen of bacillus subtilis
Enzyme gene, but obtain the recombinant bacterial strain that growth is fast and exogenous protein expression amount is high.To which bacterial strain provided by the invention can be used as
The host of foreign protein high efficient expression.
Detailed description of the invention
Fig. 1 is gene knockout plasmid construction schematic diagram.
Fig. 2 is that the PCR of bacillus subtilis Bacillus subtilis ATCC6051 gene knockout mutant strain identifies knot
Fruit figure；Wherein, swimming lane 1-10 is B.subtilis ATCC6051, swimming lane 11 is recombinant bacterium 9, and swimming lane 12 is recombinant bacterium 1, swimming lane
13 be recombinant bacterium 3, and swimming lane 14 is recombinant bacterium 10, and swimming lane 15 is recombinant bacterium 2, and swimming lane 16 is recombinant bacterium 6, and swimming lane 17 is recombinant bacterium 4,
Swimming lane 18 is recombinant bacterium 7, and swimming lane 19 is recombinant bacterium 8, and swimming lane 20 is recombinant bacterium 5, and swimming lane M is DNA marker.
Fig. 3 be bacterium germination B.subtilis ATCC6051, recombinant bacterium Bacillus subtilis ATCC6051 △ 10,
Bacillus subtilis ATCC6051 (pBEp43-proMTG) and Bacillus subtilis ATCC6051 Δ 10
(pBEp43-proMTG) growth curve chart.
Fig. 4 is the secreting, expressing result that SDS-PAGE protein electrophoresis analyzes transglutaminase in recombinant bacterium fermented liquid supernatant
Figure；Wherein, swimming lane 1 is Bacillus subtilis ATCC6051, and swimming lane 2 is recombinant bacterium Bacillus subtilis
ATCC6051 △ 10, swimming lane 3 are the Bacillus subtilis ATCC6051 with pBEp43-proMTG plasmid, and swimming lane 4 is
Bacillus subtilis ATCC6051 △ 10 with pBEp43-proMTG plasmid.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The building of 1 bacillus subtilis Bacillus subtilis ten gene knockout carriers of ATCC6051 of embodiment
Genomic DNA with Bacillus subtilis ATCC6051 (being purchased from NBRC, article No. is NBRC 13719) is
Template is expanded with primer pair F-X-L, R-X-L and F-X-R, the R-X-R (X is gene to be knocked out, similarly hereinafter) in table 1 respectively,
Obtain amplified production X-L (gene to be knocked out upstream homology arm) and X-R (gene to be knocked out downstream homology arm).
Using X-L segment and X-R segment as template, using F-X-L and R-X-R as primer, fusion DNA vaccine is carried out, is obtained corresponding
Ten fusion products (L+R segment).Ten fusion products are cloned into respectively in pMD18-T carrier, by sequencing, this ten fusions
Segment is to have nucleotide shown in sequence 1 in sequence table (DNA molecular of nprE encoding gene homology arm), 2 institute of sequence respectively
Nucleotide (aprE encoding gene homology arm shown in the nucleotide (DNA molecular of nprB encoding gene homology arm) that shows, sequence 3
DNA molecular), nucleotide shown in nucleotide (DNA molecular of mpr encoding gene homology arm), sequence 5 shown in sequence 4
Nucleotide shown in (DNA molecular of bpf encoding gene homology arm), sequence 6 (DNA molecular of epr encoding gene homology arm), sequence
(wprA encoding gene is same for nucleotide shown in nucleotide shown in column 7 (DNA molecular of vpr encoding gene homology arm), sequence 8
The DNA molecular of source arm), shown in nucleotide (DNA molecular of spollAC encoding gene homology arm) and sequence 10 shown in sequence 9
Nucleotide (DNA molecular of srfAC encoding gene homology arm).
By above-mentioned fusion product be inserted into respectively temperature-sensitive plasmid pKS2 (plasmid in patent CN201210052578.2 it is public
It opens, is that three BamHI restriction enzyme sites in PKS1 are eliminated and are obtained, the nucleotides sequence of the carrier is classified as the sequence in sequence table
11) EcoRV restriction enzyme site obtains recombinant plasmid pKS2-X (L+R) (see Fig. 1), i.e., the knockout carrier of ten genes: pKS2-
nprE、pKS2-nprB、pKS2-aprE、pKS2-mpr、pKS2-bpf、pKS2-epr、pKS2-vpr、pKS2-wprA、pKS2-
spollAC、pKS2-srfAC。
It is as follows that 1 gene knockout carrier of table constructs relevant primer sequence:
PCR reaction system is as follows:
PCR program is 98 DEG C of 5min；35 circulations: 98 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 40s；72℃10min.
Embodiment 2
(1) building of recombinant bacterium 1 (Bacillus subtilis ATCC6051 Δ nprE)
1. recombinant plasmid pKS2-nprE is first transferred to e. coli tg1 (being purchased from precious bioengineering (Dalian) Co., Ltd),
Plasmid is extracted, other recombinant plasmids also carry out same operation.Then plasmid pKS2-nprE is transferred to by electrotransformation method
In Bacillus subtilis ATCC6051 (specific method records Natalia P, Zakataeva with reference to non-patent literature,
Oksana V et al.A simple method to introduce marker-free genetic modification
into chromosome of naturally nontransformable Bacillus amyloliquefaciens
Strains [J] .Appl Microbiol Biotechnol.2010,85:1201-1209.), it is cultivated in 30 DEG C of inversions, with 5 μ
The LB flat screen of g/mL erythromycin (erm) resistance selects positive clone molecule.
2. the positive clone molecule in picking resistant panel, being inoculated in 50mL triangular flask liquid amount is 10mL LB Liquid Culture
In base (5 μ g/mL erm), 12h is cultivated in 30 DEG C, 200rpm, the verifying of upgrading grain has been transferred in host, obtains conversion bacterial strain
Bacillus subtilis ATCC6051(pKS2-nprE)。
3. taking bacterium solution of the 100 μ L after plasmid identification, it is inoculated in 37 DEG C of trainings in 10mL LB liquid medium (non-resistant)
Support 12h, dilution 10-3, take 100 μ L bacterium solutions to be coated in LB resistant panel (5 μ g/mL erm), 37 DEG C of culture 12h.
4. the transformant in picking resistant panel is inoculated in 10mL LB liquid medium (non-resistant), 30 DEG C,
200rpm cultivates 12h；Culture solution dilution 10-5100 μ L bacterium solutions are taken to be coated on LB (non-resistant) solid plate, 37 DEG C of culture 12h.
5. the single colonie that is grown on nonreactive plate (step is 4.) while switching is on LB nonreactive plate and in LB resistant panel
(5 μ g/mL erm), Phenotypic Expression are to grow on LB nonreactive plate, and not long in LB resistant panel is putative clone,
It is further verified with primers F-nprE-L (SEQ ID NO.12) and R-nprE-R (SEQ ID NO.15) by PCR, PCR identification
As a result see Fig. 2；It is sequenced, obtains recombinant bacterium 1 (Bacillus subtilis ATCC6051 Δ nprE).
(2) building of recombinant bacterium 2:Bacillus subtilis ATCC6051 Δ 2 (nprE, nprB)
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 1 of above-mentioned steps, is used
PKS2-nprB plasmid obtains recombinant bacterium 2.With primers F-nprB-L (SEQ ID NO.16) and R-nprB-R (SEQ ID
NO.19 it) is further verified by PCR, PCR qualification result is shown in Fig. 2.
(3) building of recombinant bacterium 3:Bacillus subtilis ATCC6051 Δ 3 (nprE, nprB, aprE)
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 2 of above-mentioned steps, is used
PKS2-aprE plasmid obtains recombinant bacterium 3.With primers F-aprE-L (SEQ ID NO:20) and R-aprE-R (SEQ ID NO:
23) it is further verified by PCR, PCR qualification result is shown in Fig. 2.
(4) building of recombinant bacterium 4:Bacillus subtilis ATCC6051 Δ 4 (nprE, nprB, aprE, mpr)
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 3 of above-mentioned steps, is used
PKS2-mpr plasmid obtains recombinant bacterium 4.It is logical with primers F-mpr-L (SEQ ID NO.24) and R-mpr-R (SEQ ID NO.27)
It crosses PCR further to verify, PCR qualification result is shown in Fig. 2.
(5) structure of recombinant bacterium 5:Bacillus subtilis ATCC6051 Δ 5 (nprE, nprB, aprE, mpr, bpf)
It builds
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 4 of above-mentioned steps, is used
PKS2-bpf plasmid obtains recombinant bacterium 5.It is logical with primers F-bpf-L (SEQ ID NO.28) and R-bpf-R (SEQ ID NO.31)
It crosses PCR further to verify, PCR qualification result is shown in Fig. 2
(6) recombinant bacterium 6:Bacillus subtilis ATCC6051 Δ 6 (nprE, nprB, aprE, mpr, bpf, epr)
Building
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 5 of above-mentioned steps, is used
PKS2-epr plasmid obtains recombinant bacterium 6.It is logical with primers F-epr-L (SEQ ID NO.32) and R-epr-R (SEQ ID NO.35)
It crosses PCR further to verify, PCR qualification result is shown in Fig. 2
(7) recombinant bacterium 7:Bacillus subtilis ATCC6051 Δ 7 (nprE, nprB, aprE, mpr, bpf, epr,
Vpr building)
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 6 of above-mentioned steps, is used
PKS2-vpr plasmid obtains recombinant bacterium 7.It is logical with primers F-vpr-L (SEQ ID NO.36) and R-vpr-R (SEQ ID NO.39)
It crosses PCR further to verify, PCR qualification result is shown in Fig. 2
(8) recombinant bacterium 8:Bacillus subtilis ATCC6051 Δ 8 (nprE, nprB, aprE, mpr, bpf, epr,
Vpr, wprA) building
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 7 of above-mentioned steps, is used
PKS2-wprA plasmid obtains recombinant bacterium 8.With primers F-wprA-L (SEQ ID NO.40) and R-wprA-R (SEQ ID
NO.43 it) is further verified by PCR, PCR qualification result is shown in Fig. 2
(9) recombinant bacterium 9:Bacillus subtilis ATCC6051 Δ 9 (nprE, nprB, aprE, mpr, bpf, epr,
Vpr, wprA, spollAC) building
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 8 of above-mentioned steps, is used
PKS2-spollAC plasmid obtains recombinant bacterium 9.With primers F-spollAC-L (SEQ ID NO.44) and R-spollAC-R (SEQ
ID NO.47) it is further verified by PCR, PCR qualification result is shown in Fig. 2
(10)Bacillus subtilis ATCC6051Δ10(nprE、nprB、aprE、mpr、bpf、epr、vpr、
WprA, spollAC, srfAC) building
Operating method basic synchronization is rapid (1), and difference is: starting strain is the resulting recombinant bacterium 9 of above-mentioned steps, is used
PKS2-aprE plasmid, with obtaining mesh bacterial strain Bacillus subtilis ATCC6051 Δ 10 (nprE, nprB, aprE, mpr,
bpf,epr,vpr,wprA,spollAC,srfAC).With primers F-srfAC-L (SEQ ID NO.48) and R-srfAC-R (SEQ
ID NO.51) it is further verified by PCR, PCR qualification result is shown in Fig. 2.
3 transglutaminase of embodiment Bacillus subtilis ATCC6051 Δ 10 (nprE, nprB, aprE,
Mpr, bpf, epr, vpr, wprA, spollAC, srfAC) expression
With plasmid pBEp43-proMTG (by CN201210052578.2, the bacillus subtilis of a plant weight group and its production
The method of transglutaminase constructs to obtain) it is expression plasmid, electricity consumption method for transformation is transferred to Bacillus subtilis
ATCC6051 and Bacillus subtilis ATCC6051 Δ 10 obtains two conversion bacterial strain Bacillus subtilis
ATCC6051 (pBEp43-proMTG) and Bacillus subtilis ATCC6051 Δ 10 (pBEp43-proMTG).
Picking Bacillus subtilis ATCC6051 (pBEp43-proMTG) and Bacillus subtilis
The single colonie of ATCC6051 Δ 10 (pBEp43-proMTG) is inoculated in 10mL LB culture medium (20 μ g/mL of kanamycins) respectively
In 37 DEG C, 200rpm activates 12h, and the seed liquor of activation is inoculated in 50mL LB culture medium (20 μ g/mL of kanamycins), is connect
Kind amount is 1% (volume ratio), in 37 DEG C, 200rpm fermentation 48h；Simultaneously with starting strain Bacillus subtilis
ATCC6051 and Bacillus subtilis ATCC6051 Δ 10 is as control (the not culture medium of resistance, other culture items
Part is consistent).Point six point in time sampling, 3h, 6h, 12h, for 24 hours, 36h and 48h measure OD600Value, recombinant bacterium Bacillus
The speed of growth of subtilis ATCC6051 Δ 10 is higher than bacterium germination Bacillus subtilis ATCC6051, and
The speed of growth of Bacillus subtilis ATCC6051 Δ 10 (pBEp43-proMTG) is also to compare Bacillus
Fastly, and 48h reaches peak to subtilis ATCC6051 (pBEp43-proMTG) (see Fig. 3).
Above-mentioned 48h fermented sample centrifuging and taking supernatant is taken, supernatant runs SDS-PAGE electrophoresis.As a result as shown in figure 4,
Bacillussubtilis ATCC6051 (pBEp43-proMTG) and Bacillus subtilis ATCC6051 Δ 10
(pBEp43-proMTG) supernatant, which is shown in 44-46KDa, has band and MTG albumen in the same size；And starting strain
Bacillus subtilis ATCC6051 and Bacillus subtilis ATCC6051 Δ 10 is in this magnitude range without item
Band, and 10 (pBEp43-proMTG) band of Bacillus subtilis ATCC6051 Δ is obviously richer than Bacillus
subtilis ATCC6051(pBEp43-proMTG).Experimental result illustrates MTG proproteinase in Bacillus subtilis
The expression quantity of ATCC6051 Δ 10 is higher than Bacillus subtilis ATCC6051 (see Fig. 4).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (9)
1. a kind of bacillus subtilis recombinant bacterial strain, it is characterised in that: be by ten eggs in bacillus subtilis starting strain
White enzyme gene knocks out or inactivation obtains, this ten protease genes are as follows: nprE, nprB, aprE, mpr, bpf, epr, vpr,
WprA, spollAC and srfAC；
The bacillus subtilis starting strain be bacillus subtilis (Bacillus subtilis) ATCC6051.
2. the preparation method of bacillus subtilis recombinant bacterial strain described in claim 1, it is characterised in that: be by described in obstruction
Ten protease genes transcription or translation technology obtain.
3. the preparation method of bacillus subtilis recombinant bacterial strain according to claim 2, it is characterised in that: the technology
Including gene Knockout, frameshift mutation technology and GEM 132 perturbation technique.
4. the preparation method of bacillus subtilis recombinant bacterial strain according to claim 3, it is characterised in that: the gene
Knockout technology is methods of homologous recombination.
5. the preparation method of bacillus subtilis recombinant bacterial strain according to claim 4, it is characterised in that: described is homologous
Recombination method includes the following steps:
(1) the upstream homology arm and downstream homology arm of ten protease genes are constructed respectively, then upstream homology arm and downstream is same
Source arm fusion, obtains upstream and downstream homology arm；Ten protease-baseds because nprE, nprB, aprE, mpr, bpf, epr, vpr,
WprA, spollAC and srfAC；
(2) the upstream and downstream homology arm of ten protease genes is cloned into respectively in Thermo-sensitive plasmid pKS2, obtains ten albumen
The knockout carrier of enzyme gene: pKS2-nprE, pKS2- nprB, pKS2- aprE, pKS2- mpr, pKS2-bpf, pKS2-epr,
pKS2-vpr,pKS2-wprA,pKS2-spollAC,pKS2- srfAC；
(3) any of the knockout carrier of ten protease genes is transferred in bacillus subtilis starting strain, through screening,
Obtain recombinant bacterium 1；It is transferred to recombinant bacterium 1 with the knockout carrier of another protease gene again, screens, obtains recombinant bacterium 2；Exist respectively
The knockout carrier for the protease gene that one is not transferred to is transferred in recombinant bacterium obtained in the previous step, obtain recombinant bacterium 3, recombinant bacterium 4,
Recombinant bacterium 5, recombinant bacterium 6, recombinant bacterium 7, recombinant bacterium 9 finally obtain the bacillus subtilis for knocking out ten protease genes.
6. the preparation method of bacillus subtilis recombinant bacterial strain according to claim 5, it is characterised in that:
The upstream homology arm of the nprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.12
It is obtained with primer amplification shown in SEQ ID NO.13；
The downstream homology arm of the nprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.14
It is obtained with primer amplification shown in SEQ ID NO.15；
The upstream homology arm of the nprB is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.16
It is obtained with primer amplification shown in SEQ ID NO.17；
The downstream homology arm of the nprB is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.18
It is obtained with primer amplification shown in SEQ ID NO.19；
The upstream homology arm of the aprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.20
It is obtained with primer amplification shown in SEQ ID NO.21；
The downstream homology arm of the aprE is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.22
It is obtained with primer amplification shown in SEQ ID NO.23；
The upstream homology arm of the mpr be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.24 and
Primer amplification shown in SEQ ID NO.25 obtains；
The downstream homology arm of the mpr be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.26 and
Primer amplification shown in SEQ ID NO.27 obtains；
The upstream homology arm of the bpf be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.28 and
Primer amplification shown in SEQ ID NO.29 obtains；
The downstream homology arm of the bpf be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.30 and
Primer amplification shown in SEQ ID NO.31 obtains；
The upstream homology arm of the epr be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.32 and
Primer amplification shown in SEQ ID NO.33 obtains；
The downstream homology arm of the epr be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.34 and
Primer amplification shown in SEQ ID NO.35 obtains；
The upstream homology arm of the vpr be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.36 and
Primer amplification shown in SEQ ID NO.37 obtains；
The downstream homology arm of the vpr be using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.38 and
Primer amplification shown in SEQ ID NO.39 obtains；
The upstream homology arm of the wprA is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.40
It is obtained with primer amplification shown in SEQ ID NO.41；
The downstream homology arm of the wprA is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.42
It is obtained with primer amplification shown in SEQ ID NO.43；
The upstream homology arm of the spollAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.44 and SEQ ID NO.45 obtains；
The downstream homology arm of the spollAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID
Primer amplification shown in NO.46 and SEQ ID NO.47 obtains；
The upstream homology arm of the srfAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.48
It is obtained with primer amplification shown in SEQ ID NO.49；
The downstream homology arm of the srfAC is using bacillus subtilis starting strain DNA as template, with such as SEQ ID NO.50
It is obtained with primer amplification shown in SEQ ID NO.51.
7. the preparation method of bacillus subtilis recombinant bacterial strain according to claim 5, it is characterised in that: the fusion
The step of it is as follows: using upstream homology arm and downstream homology arm as template, under the upstream primer and amplification to expand upstream homology arm
The downstream primer for swimming homology arm is primer pair amplifies, obtains upstream and downstream homology arm；
The sequence of the upstream and downstream homology arm of the nprE is as shown in SEQ ID NO.1；
The sequence of the upstream and downstream homology arm of the nprB is as shown in SEQ ID NO.2；
The sequence of the upstream and downstream homology arm of the aprE is as shown in SEQ ID NO.3；
The sequence of the upstream and downstream homology arm of the mpr is as shown in SEQ ID NO.4；
The sequence of the upstream and downstream homology arm of the bpf is as shown in SEQ ID NO.5；
The sequence of the upstream and downstream homology arm of the epr is as shown in SEQ ID NO.6；
The sequence of the upstream and downstream homology arm of the vpr is as shown in SEQ ID NO.7；
The sequence of the upstream and downstream homology arm of the wprA is as shown in SEQ ID NO.8；
The sequence of the upstream and downstream homology arm of the spollAC is as shown in SEQ ID NO.9；
The sequence of the upstream and downstream homology arm of the srfAC is as shown in SEQ ID NO.10.
8. the preparation method of bacillus subtilis recombinant bacterial strain according to claim 5, it is characterised in that:
The bacillus subtilis starting strain is bacillus subtilis ATCC6051；
The recombinant bacterium 1 is the bacillus subtilis for lacking nprE gene；
The recombinant bacterium 2 is the bacillus subtilis for lacking nprE, nprB gene；
The recombinant bacterium 3 is the bacillus subtilis for lacking nprE, nprB, aprE gene；
The recombinant bacterium 4 is the bacillus subtilis for lacking nprE, nprB, aprE, mpr gene；
The recombinant bacterium 5 is the bacillus subtilis for lacking nprE, nprB, aprE, mpr, bpf gene；
The recombinant bacterium 6 is the bacillus subtilis for lacking nprE, nprB, aprE, mpr, bpf, epr gene；
The recombinant bacterium 7 is the bacillus subtilis for lacking nprE, nprB, aprE, mpr, bpf, epr, vpr gene；
The recombinant bacterium 8 is the bacillus subtilis for lacking nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA gene；
The recombinant bacterium 9 is the withered grass for lacking nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA, spollAC gene
Bacillus.
9. application of the bacillus subtilis recombinant bacterial strain described in claim 1 in exogenous protein expression.
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| CN108102997A (en) * | 2017-10-25 | 2018-06-01 | 江南大学 | A kind of bacillus subtilis for Pullulanase high efficient expression and High Density Cultivation |
| CN108570477B (en) * | 2018-04-18 | 2020-06-30 | 横琴仲泰生物医药有限公司 | Alkaline protease gene and construction method of recombinant bacillus subtilis strain thereof |
| CN108913706B (en) * | 2018-07-10 | 2021-04-09 | 郑州轻工业学院 | Bacillus subtilis glycerol kinase mutant gene glpK and application thereof |
| CN109554321B (en) * | 2018-12-03 | 2021-12-31 | 清华大学 | Genetically engineered bacterium for high-yield lipopeptide and application thereof |
| CN111349594A (en) * | 2018-12-21 | 2020-06-30 | 南京百斯杰生物工程有限公司 | Strain capable of producing glutamine transaminase |
| CN111349639A (en) * | 2020-01-17 | 2020-06-30 | 西宝生物科技（上海）股份有限公司 | High-efficiency biosynthesis vitamin K for improving bacillus natto2(MK-7) method |
| CN112592881B (en) * | 2021-02-25 | 2021-06-11 | 中国科学院天津工业生物技术研究所 | Engineering bacillus subtilis for high-efficiency exogenous protein expression and high-density culture |
| CN113957028B (en) * | 2021-10-25 | 2023-08-11 | 江南大学 | Extracellular protease inactivated bacillus subtilis, and construction method and application thereof |
| CN115181714B (en) * | 2022-05-31 | 2023-08-11 | 南京农业大学 | Plant immunity induced antibacterial and construction method and application thereof |
| CN116396915B (en) * | 2022-11-18 | 2023-10-31 | 西北农林科技大学 | Bacillus subtilis without specific resistance gene and application thereof |
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| WO2008048407A2 (en) * | 2006-10-13 | 2008-04-24 | Genencor International, Inc. | Expression of streptomyces subtilisin inhibitor (ssi) proteins in bacillus and streptomyces sp. |
| CN103451224A (en) * | 2013-08-26 | 2013-12-18 | 天津大学 | Traceless modification method of bacillus subtilis genome |
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| WO2008048407A2 (en) * | 2006-10-13 | 2008-04-24 | Genencor International, Inc. | Expression of streptomyces subtilisin inhibitor (ssi) proteins in bacillus and streptomyces sp. |
| CN103451224A (en) * | 2013-08-26 | 2013-12-18 | 天津大学 | Traceless modification method of bacillus subtilis genome |
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