CN117363550A - Construction method of bacillus subtilis D high-yield strain - Google Patents
Construction method of bacillus subtilis D high-yield strain Download PDFInfo
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Abstract
The invention belongs to the fields of molecular biology and microbial fermentation, and particularly relates to a construction method of a high-yield bacterial strain of bacillus subtilis D, which is characterized in that a bacillus subtilis D synthase system is modified, a competition path is knocked out by replacing a promoter in the bacillus subtilis D synthase system, and finally, the yield of the bacillus subtilis D in a superposition engineering bacterium can reach 2.55 times that of an original bacterial strain fmbJ. The method provides a systematic method for the efficient synthesis of the bacillus subtilis D and provides a theoretical basis for the research of efficient synthesis of lipopeptides.
Description
Technical Field
The invention belongs to the fields of molecular biology and microbial fermentation, relates to 1 strain of bacillus amyloliquefaciens engineering bacteria fmbJPE with high yield of bacillus amyloliquefaciens D, and in particular relates to the strain and a construction method of the strain of bacillus amyloliquefaciens.
Background
Bacillus is one of the largest sources of natural products with biological activity, and its products have a broad range of antibiotic activity. Bacillus subtilis D is an antibacterial lipopeptide produced by Bacillus amyloliquefaciens fmbJ. The bacillus has various biological activities, in particular to the inhibition of the growth of the aspergillus flavus, so the bacillus has extremely high application value in the aspects of food preservation, biological control and the like. However, the ability of the wild-type strain to ferment and synthesize bacillus D is at a low level, which greatly limits the industrial production and application of bacillus D. Both nutrient substrates and physicochemical parameters (e.g., pH, temperature, and oxygen content, etc.) affect lipopeptide production, and thus studies have been made to increase the yield of bacilomycin D by optimizing the nutrient substrates and fermentation conditions of the medium. However, these methods increase fermentation costs while increasing the yield of bacillus D, and natural high-yielding strains are extremely difficult to screen. Therefore, a method for constructing engineering bacteria with high yield of bacillus subtilis D by genetic engineering means, thereby improving the yield of bacillus subtilis D is attracting attention.
Disclosure of Invention
Aiming at the problems in the prior art, the first object of the invention is to provide a bacillus amyloliquefaciens engineering bacterium fmbJPE with high yield of bacillus amyloliquefaciens D; the second object of the invention is to provide a construction method of the engineering bacteria. The invention provides a method for applying homologous recombination, which comprises the steps of replacing a promoter Pbmy for synthesizing bacillus amyloliquefaciens fmbJ in bacillus amyloliquefaciens fmbJ, knocking out an extracellular polysaccharide synthetic gene cluster epsA-O, and screening to obtain superposition engineering bacteria fmbJPE.
The purpose of the invention is achieved by the following means:
in the first aspect, the invention firstly protects a bacillus amyloliquefaciens engineering bacterium fmbJPE, wherein the engineering bacterium takes bacillus amyloliquefaciens fmbJ as an original strain and has the following characteristics:
(1) The promoter for synthesizing the bacillus D contains a promoter PbacA;
(2) The extracellular polysaccharide synthesis gene cluster epsA-O was knocked out.
In a specific embodiment, the bacillus amyloliquefaciens engineering strain can be obtained by firstly replacing a promoter Pbmy for synthesizing bacillus amyloliquefaciens D in bacillus amyloliquefaciens fmbJ with a promoter PbacA, then knocking out an extracellular polysaccharide synthesis gene cluster epsA-O and screening.
The application of the engineering bacteria in the preparation of bacillus D also belongs to the protection scope of the invention.
The engineering bacteria can generate high-yield bacillus subtilis D, and the yield of the bacillus subtilis D can reach 2.55 times of that of an original strain fmbJ.
The starting strain Bacillus amyloliquefaciens fmbJ used in the invention is obtained by autonomous screening by an enzyme engineering laboratory of Nanjing university, and has a strain preservation number of CGMCC No.0943, which is disclosed in a plurality of documents in the prior art.
In a second aspect, the invention also provides a construction method of the bacillus amyloliquefaciens engineering strain, which comprises the following steps:
(1) Designing primer pairs PT-UF/R, hcpd-F/R and PT-DF/R, and respectively amplifying an upstream gene fragment, a synthesized PbacA promoter fragment and a downstream gene fragment by taking genome DNA of bacillus amyloliquefaciens fmbJ as a template;
(2) Fusing the three fragments in the step (1) by adopting two-step overlap extension PCR, wherein the primers used are PT-UF/hcpd-R and PT-UF/DR respectively;
(3) Designing primer pairs delta eps-UF/UR and delta eps-DF/DR, and respectively amplifying upstream and downstream gene fragments by taking genome DNA of bacillus amyloliquefaciens fmbJ as a template;
(4) Fusing the two fragments in the step (3) by adopting overlap extension PCR, wherein the primer pair is delta eps-UF/DR;
(5) Designing primer pairs pKS2-F and pKS2-R, and PCR amplifying a linear pKS2 vector by taking pKS2 plasmid DNA as a template;
(6) Respectively connecting the fusion fragments in (2) and (4) with a linear pKS2 vector by using a recombinase to finally obtain recombinant integrated plasmids pKS2-PbacA and pKS 2-delta eps;
(7) Transferring the recombinant integrated plasmid pKS2-PbacA into a bacillus amyloliquefaciens fmbJ strain, and replacing the recombinant integrated plasmid pKS2-PbacA with a promoter PbacA through homologous recombination; and then transferring the recombinant integrated plasmid pKS 2-delta eps into a PbacA promoter replacement strain, integrating the gene fragment of the deletion epsA-O into a genome through homologous recombination, and carrying out resistance screening to obtain a recombinant clone strain with sensitive resistance, namely the superposition engineering bacterium, which is named as fmbJPE.
In a specific embodiment, the primer pair sequences of step (1) are as follows:
PT-UF: as shown in SEQ ID NO. 3; PT-UR: as shown in SEQ ID NO. 4;
hcpd-F: as shown in SEQ ID NO. 5; hcpd-R: as shown in SEQ ID NO. 6;
PT-DF: as shown in SEQ ID NO. 7; PT-DR: as shown in SEQ ID NO. 8;
in a specific embodiment, the primer pair sequences of step (3) are as follows:
Δeps-UF: as shown in SEQ ID NO. 9; Δeps-UR: as shown in SEQ ID NO. 10;
Δeps-DF: as shown in SEQ ID NO. 11; Δeps-DR: as shown in SEQ ID NO. 12;
in a specific embodiment, the primer pair sequences of step (5) are as follows:
pKS2-F: as shown in SEQ ID NO. 13; pKS2-R: as shown in SEQ ID NO. 14.
According to the invention, the superposition engineering bacteria are constructed by two methods of promoter replacement and competition reduction, and the synthesis capability of bacillus amyloliquefaciens fmbJ in the strain can be greatly improved after the transformation of a system of the original strain bacillus amyloliquefaciens fmbJ.
Engineering bacteria constructed by the construction method are also within the scope of the invention.
The recombinant integrative plasmids described above are also within the scope of the invention.
Specifically, the nucleotide sequence of the pKS2-PbacA is shown as SEQ ID NO. 1;
specifically, the nucleotide sequence of the pKS 2-deltaeps is shown as SEQ ID NO. 2.
In a third aspect, the present invention also provides a method for preparing an engineering bacterium having a high yield of bacillus D, comprising: the bacillus amyloliquefaciens fmbJ is taken as an initial strain, a promoter Pbmy for synthesizing bacillus amyloliquefaciens fmbJ is replaced by a promoter PbacA, and then an extracellular polysaccharide synthesis gene cluster epsA-O is knocked out.
In a fourth aspect, the invention also provides a preparation method of high-yield bacillus amyloliquefaciens f mbjpe, which is realized by cultivating the bacillus amyloliquefaciens engineering bacteria f mbjpe.
Advantageous effects
The invention utilizes a homologous recombination method to reform a bacillus subtilis D synthase system, and obtains superposition engineering bacteria fmbJPE by replacing a promoter in the bacillus subtilis D synthase system and knocking out a competition path, wherein the yield of the bacillus subtilis D can reach 2.55 times of that of an original strain fmbJ. The method provides a systematic method for the efficient synthesis of the bacillus subtilis D and provides a theoretical basis for the research of efficient synthesis of lipopeptides.
Drawings
Construction of the superimposed engineering strain in FIG. 1 (note: M1 and M3 are 2000bp DNA Marker,M2 and M4 are 1kb DNA ladder; FIGS. a and b are verification of positive transformants, lanes 1-5 are verification of success of replacement of Pbaca promoter replacement strain, fragment theoretical size is 641bp, it is clear from FIG. a that replacement of Pbaca promoter replacement strain is successful, lanes 6-10 are verification of transfer of demethylated plasmid pKS2Δeps into promoter replacement strain, verification of fragment theoretical size is 3305bp, it is clear from FIG. b that demethylated plasmid pKS2Δeps is successfully transferred into promoter replacement strain, FIGS. c and d are verification of superimposed engineering bacteria, lanes 11-15 are verification of reversion mutation of Pbaca promoter replacement strain, fragment theoretical size is 641bp, it is clear from FIG. c that reversion of Pbaca promoter replacement strain is successful, 16-20 are verification of success of gene cluster epsA-O, verification of fragment theoretical size is 3020 bp), and it is clear from FIG. d that gene cluster epsA-O is successful.
The yield of bacillus D in the strain fmbJPE in FIG. 2 (note: x represents P <0.0001; + represents replacement of the pro-promoter of bacillus D synthase with the promoter, -represents knockout of the gene), and as can be seen in FIG. 2, the yield of bacillus D in the strain fmbJPE is significantly higher than that in the strain fmbJ.
Detailed Description
The invention will be further illustrated by the following examples, which are not intended to be limiting, in which the strains referred to in the examples are all of the prior art, generally by means known in the art or established by the manufacturer, and readily available from published commercial sources.
Example 1
1 materials and methods
1.1 Strain and plasmid
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) fmbJ, cloning host E.coli (Escherichia coli) JM 109 and the integrating plasmid pKS2 (eliminating BamHI site) were all deposited by Nanj university of agriculture enzyme engineering laboratory; demethylated host bacteria e.coli JM110 were purchased from south kyo department biotechnology.
1.2 Medium and related solutions
LB medium: 10.0g of peptone, 5.0g of yeast extract, 10.0g of NaCl, 1000mL of distilled water and pH of 7.0-7.2.
Seed culture medium: beef extract 5.0g, yeast extract 5.0g, peptone 10.0g, naCl 5.0g, distilled water 1000mL, pH 7.0-7.2.
Fermentation medium: l-glutamic acid 5.0g, yeast extract 1.0g, KH 2 PO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，KCl 0.5g，CuSO 4 ·5H 2 O 0.15mg，FeSO 4 ·7H 2 O 1.2mg，MnSO 4 5mg, glucose 20.0g, distilled water to 1000mL, pH 7.0.
LBS medium: 10.0g of tryptone, 91.0g of sorbitol, 5.0g of yeast extract and 10.0g of NaCl, the volume is fixed to 1000mL, and the mixture is sterilized at 121 ℃ for 20min.
Electrotransformation liquid: 0.5M trehalose, 0.5M sorbitol, 0.5M mannitol, and 10% glycerol, and sterilizing at 115℃for 30min.
And (3) heavy suspension: 14% PG6000 electrotransformation solution was added.
Erythromycin (Em) mother liquor: erythromycin was prepared as a stock solution of 5mg/mL with absolute ethanol and placed at-20℃until a final concentration of 5. Mu.g/mL was reached.
Kanamycin (Kanamycin, kan) solution: 0.1g Kan is dissolved in 1mL sterile water to prepare a mother solution of 100 mg.mL-1, and the mother solution is sterilized by filtration through a 0.22 μm sterile filter membrane, and then the mother solution is packaged in a 1.5mL centrifuge tube and stored at 4 ℃.
1.3 reagents
Kanamycin and erythromycin were purchased from Shanghai Bioengineering; DNA gel recovery kit, plasmid extraction kit and recombinase were purchased from Novain Biocompany; genomic extraction kits were purchased from OMEGA Bio-Tek company.
1.4 method
1.4.1 construction of strong promoter replacement plasmid
Primers were designed based on the sequencing results of the b.amyloliquefaciens fmbJ genome, and the primer sequences are shown in table 1. For plasmid pKS2-PbacA, the upstream gene fragment, the synthesized PbacA promoter fragment and the downstream gene fragment were first amplified using primer pairs PT-UF/R, hcpd-F/R and PT-DF/R, respectively. Then, the three fragments were fused by two-step overlap extension PCR using the primers PT-UF/hcpd-R and PT-UF/DR, respectively. The fusion fragment is connected with a pKS2 plasmid linear fragment through a one-step cloning kit, and the plasmid pKS2-PbacA is obtained after demethylation. The obtained plasmid was verified by sequencing by the Kirschner Bio Inc.
1.4.2 construction of competitive pathway knockout plasmids
For pKS2Δeps, the upstream and downstream gene fragments were first amplified using primer pairs Δeps-UF/UR and Δeps-DF/DR, respectively. The two fragments were then fused using a primer pair Δeps-UF/DR. The subsequent procedure was identical to the construction method of the promoter replacement plasmid. Finally obtaining plasmid pKS 2-delta eps, and verifying the obtained plasmid by sequencing of the Kirschner biological company.
1.4.3 preparation of competent Bacillus amyloliquefaciens
The strain cultured in LBS culture medium overnight is transferred to fresh LBS culture medium, 25 times dilution is carried out, after culture is carried out for 3 hours at 37 ℃ and 180rpm (OD 600 reaches 0.5), glycine is added to enable the final concentration to reach 10mg/mL, culture is continued, when the light absorption value OD600 reaches about 0.9, thalli are placed in ice water for 30-40min, centrifugation is carried out for 5min at 8000rpm to collect thalli cells, precooled electrotransformation liquid is used for 3-4 times, finally, the thalli cells are resuspended in precooled resuspension liquid according to 1:100 (v/v), at the moment, competent cells which are the bacillus amyloliquefaciens are obtained, 100 mu L of competent cells are subpackaged for each tube and placed at-80 ℃ for standby.
1.4.4 electric transformation of Bacillus amyloliquefaciens
About 500ng of plasmid to be transformed is added into 100 mu L of competent cells, the mixture is gently mixed, the mixture is placed in an electric rotating cup (2 mm) precooled on ice, the electric shock is carried out at the voltage of 2500V after 3-5min in ice bath, 1mL of LBS culture based on the electric rotating cup is immediately added, after the mixture is fully mixed, the mixture is sucked into a 2mL centrifuge tube, the mixture is subjected to mild shaking culture at 30 ℃ for 3h, 100 mu L of bacterial liquid is remained after centrifugation and is coated on an LB plate containing kanamycin and erythromycin, and the mixture is subjected to inversion culture at 30 ℃ for 36-48h. The transformants were verified by PCR amplification and electrophoresis detection.
TABLE 1 primer sequences for construction of engineering strains
Note that: the underlined is the homologous sequence
1.4.5 homologous recombination inducing Gene replacement and knockout
Inoculating bacillus amyloliquefaciens containing integrated plasmids into LB liquid medium, shake culturing at 37 ℃ and 180rpm for 24 hours, wherein the integrated plasmids undergo first single exchange at homologous arm positions and are integrated onto host strain genome; then, the colony subjected to single exchange is inoculated into LB liquid culture at 30 ℃ and 180rpm for shaking culture for 5 generations, and the homologous arm is induced to generate second single exchange. The obtained kanamycin and erythromycin sensitive strains were verified for DNA sequences by PCR analysis and sequencing.
Determination of 1.4.6Bacillomycin D yield
The single colony of the wild fmbJ and the strain constructed as above after activation is inoculated into a seed culture medium and cultured at 37℃and 180rpm to logarithmic phase (OD 600 0.8-1.0). Then, the seed solution was inoculated into a fermentation medium at an inoculum size of 5%, and cultured for 120 hours at 33℃and 180 rpm. Centrifuging the fermentation liquor at 5000g for 20min, collecting supernatant, regulating the pH of the supernatant to 2.0,4 ℃ by using 6M HCl, standing overnight, centrifuging at 5000g for 20min, discarding the supernatant to obtain precipitate, adding methanol for dissolving, regulating the pH of the precipitate to 7.0 by using NaOH, and centrifuging at 10000g for 10min to obtain lipopeptide crude extract. The crude extract was then filtered through a 0.22mm organic filter membrane and analyzed by reverse phase high performance liquid chromatography (C18 column, 4.6 mm. Times.250 mm). Water containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B) were used as mobile phases, and the loading amount was 20. Mu.L. Finally, the yield of bacillus D was calculated by peak area.
The above-described embodiments are merely illustrative of the principles of the present invention, and the present invention is not limited thereto, since various modifications and variations may be made by those skilled in the art without departing from the spirit of the invention, and such modifications and variations fall within the scope of the invention.
Claims (10)
1. The bacillus amyloliquefaciens engineering bacterium is characterized in that the engineering bacterium takes bacillus amyloliquefaciens fmbJ as an original strain and has the following characteristics:
the promoter for synthesizing the bacillus D contains a promoter PbacA;
extracellular polysaccharide synthetic gene clusterepsA-OIs knocked out.
2. The engineering bacterium of Bacillus amyloliquefaciens according to claim 1, wherein the promoter Pbmy for synthesizing Bacillus amyloliquefaciens fmJ is replaced by the promoter PbacA, and then extracellular polysaccharide synthesis gene cluster is knocked outepsA-OAnd screening to obtain the bacillus amyloliquefaciens engineering strain.
3. A construction method of bacillus amyloliquefaciens engineering bacteria is characterized in that: the method comprises the following steps:
(1) Designing primer pairs PT-UF/R, hcpd-F/R and PT-DF/R, and respectively amplifying an upstream gene fragment, a synthesized PbacA promoter fragment and a downstream gene fragment by taking genome DNA of bacillus amyloliquefaciens fmbJ as a template;
(2) Fusing the three fragments in the step (1) by adopting two-step overlap extension PCR, wherein the primers used are PT-UF/hcpd-R and PT-UF/DR respectively;
(3) Designing primer pairs delta eps-UF/UR and delta eps-DF/DR, and respectively amplifying upstream and downstream gene fragments by taking genome DNA of bacillus amyloliquefaciens fmbJ as a template;
(4) Fusing the two fragments in the step (3) by adopting overlap extension PCR, wherein the primer pair is delta eps-UF/DR;
(5) Designing primer pairs pKS2-F and pKS2-R, and PCR amplifying a linear pKS2 vector by taking pKS2 plasmid DNA as a template;
(6) Respectively connecting the fusion fragments in the step (2) and/or the step (4) with a linear pKS2 vector by using a recombinase to finally obtain recombinant integrated plasmids pKS2-PbacA and pKS 2-deltaeps;
(7) Transferring the recombinant integrated plasmid pKS2-PbacA into a bacillus amyloliquefaciens fmbJ strain, and replacing the promoter Pbmy with the promoter PbacA through homologous recombination; then transferring the recombinant integrated plasmid pKS 2-delta eps into PbacA promoter substitution strain, and using homologous recombination to make deletionepsA-O The gene fragment of (2) is integrated into a genome, and resistance screening is carried out to obtain a recombinant clone strain with sensitive resistance, namely the bacillus amyloliquefaciens engineering strain, which is named as fmbJPE.
4. A method of construction according to claim 3, wherein the primer pair sequences of step (1) are as follows:
PT-UF: as shown in SEQ ID NO. 3; PT-UR: as shown in SEQ ID NO. 4;
hcpd-F: as shown in SEQ ID NO. 5; hcpd-R: as shown in SEQ ID NO. 6;
PT-DF: as shown in SEQ ID NO. 7; PT-DR: as shown in SEQ ID NO. 8.
5. The method of claim 3, wherein the primer pair sequences of step (3) are as follows:
Δeps-UF: as shown in SEQ ID NO. 9; Δeps-UR: as shown in SEQ ID NO. 10;
Δeps-DF: as shown in SEQ ID NO. 11; Δeps-DR: as shown in SEQ ID NO. 12.
6. A method of construction according to claim 3, wherein the primer pair sequences of step (5) are as follows:
pKS2-F: as shown in SEQ ID NO. 13; pKS2-R: as shown in SEQ ID NO. 14.
7. The recombinant integrative plasmid as defined in claim 3-6.
8. The recombinant plasmid of claim 7, wherein the recombinant plasmid is a plasmid,
the nucleotide sequence of the pKS2-PbacA is shown as SEQ ID NO. 1.
9. The recombinant plasmid of claim 7, wherein the recombinant plasmid is a plasmid,
the nucleotide sequence of the pKS 2-deltaeps is shown as SEQ ID NO. 2.
10. A preparation method of high-yield bacillus amyloliquefaciens D is characterized in that the method is realized by cultivating the bacillus amyloliquefaciens engineering bacteria in claims 1-2.
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