CN105602876A - Surfactin and Plipastatin co-production bacillus subtilis strain and method for constructing same - Google Patents
Surfactin and Plipastatin co-production bacillus subtilis strain and method for constructing same Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology and microbial fermentation, and provides a Surfactin and Plipastatin co-production bacillus subtilis strain pB2-L, a method for constructing the same and application of the Surfactin and Plipastatin co-production bacillus subtilis strain. The method for constructing the Surfactin and Plipastatin co-production bacillus subtilis strain includes steps of amplifying target genes; amplifying fusion fragments; acquiring intermediate vectors; acquiring recombinant integration plasmids; acquiring and detecting transformants. The Surfactin and Plipastatin co-production bacillus subtilis strain, the method and the application have the advantages that a fragment of fusion genes P43-sfp-degQ are led into amyE site of B-subtilis pB2 of non-producing antibacterial substances Surfactin and Plipastatin by the aid of homologous recombination processes, so that the recombinant Surfactin and Plipastatin co-production bacillus subtilis strain B.subtilis pB2-L can be obtained, the strain has certain antibacterial activity for gram positive bacteria and filamentous fungi, and the Surfactin and Plipastatin co-production bacillus subtilis strain, the method and the application have an important significance on follow-up study.
Description
Technical field
The invention belongs to molecular biology and microorganism fermentation field, relate to a kind of coproduction Surfactin andThe bacillus subtilis strain of Plipastatin, the weight that is specifically related to this bacillus subtilis and obtains in building processConstruction method and the application thereof of group plasmid pDL-PSQ, this bacillus subtilis.
Background technology
Bacillus is distribute a class gram-positive bacterium very widely of occurring in nature, wherein bacillus subtilis andBacillus amyloliquefaciens is as the micro-ecological dominance flora of soil and plant, can produce plurality of plant diseases and prevent and treat aspect and have extensivelyThe lipopeptide antibiotic substance of general using value as: Surfactin is the beta-hydroxy fat that contains 13-14 carbon atom byThe ring-type lipopeptids of acid chain and 7 amino acid depsipeptides composition, as a kind of very strong biosurfactant, also have antibacterium,Antitumor, antiviral and prevent the biologically actives such as fibrin clot. Fengycin or Plipastatin are by 14-18 carbonFormer molecular beta-hydroxy fatty acid with become cyclic decapeptide two parts compositions; Filamentous fungi is had to good inhibition active.
Surfactin and Plipastatin are to be all that catalysis produces by the NRPS114 in bacillusSecondary metabolite, in building-up process, 4 '-Phosphopantetheinyl transferase (by sfp gene code) is mustFew functional enzyme, if disappearance can not be produced Surfactin and Plipastatin. Meanwhile, degQ gene is a kind of multiple-effectThe controlling gene of property, there are some researches show, this gene pairs Plipastatin synthetic has upper regulating and controlling effect. B.subtilisIn pB2 genome, contain complete Surfactin synzyme srfA operon and Plipastatin synzyme pps operon,But due to sfp gene and degQ gene base mutation inactivation, cause B.subtilispB2 can not produce Surfactin andPlipastatin。
Because the wild type bacillus that produces cyclic lipopeptide compound is all difficult to be converted, limit non-ribosomal peptides syntheticThe further research of enzyme system, B.subtilispB2 is as pattern bacterium bacillus subtilis, and its method for transformation is ripe, if sharpBy the method for homologous recombination, the sfp gene and the degQ gene that have functional activity are imported in B.subtilispB2 genome, makeThe ability of its resume production Surfactin and Plipastatin, can be to the structure of follow-up Plipastatin synzyme and meritThe further investigation of energy provides prerequisite, and exploitation simultaneously has the potentiality of new microbe-derived lipopeptide compound.
Summary of the invention
For problems of the prior art, the invention provides a kind of method of using homologous recombination, will there is function to liveThe sfp gene of property is incorporated into the genomic Xie Dian of bacillus subtilis (Bacillussubtilis) pB2 together with degQ geneOn powder enzyme gene amyE site, screening has obtained the recombinant bacterial strain of a strain coproduction Surfactin and PlipastatinB.subtilispB2-L。
The object of the invention obtains by following means:
1. the invention provides a kind of bacillus subtilis strain, this bacterial strain coproduction Surfactin and Plipastatin, lifePB2-L by name, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 23rd, 2015, protectTibetan number is CGMCCNo.11723, and classification is by name: Bacillus subtilis; Preservation address is: BeijingNo. 3, No. 1, North Star West Road, Chaoyang District institute.
2. the present invention also provides a kind of recombinant plasmid pDL-PSQ, and this recombinant plasmid sequence, as shown in SEQIDNO.1, is wrappedDraw together sfp gene and degQ gene.
3. the above-mentioned 2 recombinant plasmid pDL-PSQ that provide, this recombinant plasmid is taking P43 gene as promoter, and amyE is homologyArm.
4. the present invention also provides the structure of the bacillus subtilis engineering bacteria of a kind of coproduction Surfactin and PlipastatinConstruction method, the method comprises the steps:
(1) design primer pair P43-F and P43-R, sfp-F and sfp-R, degQ-F and degQ-R: with B.subtilisThe genomic DNA of fmbJ is template, pcr amplification sfp gene and degQ gene; Taking B.subtilispB2 genomic DNA as mouldPlate, pcr amplification Promoter P43 gene;
(2) design primer pair PS-F and PS-R, increase by overlapping extension PCR, obtains and merge fragment P43-sfp;
(3) carry out enzyme with restriction enzyme and cut, and by coupled reaction, degQ genetic fragment is inserted to the enzyme of plasmid pDLCut and between site, obtain plasmid pDL-degQ;
(4) carry out enzyme with restriction enzyme and cut, and will merge fragment P43-sfp insertion plasmid pDL-by coupled reactionDegQ, finally obtains recombination and integration plasmid pDL-PSQ;
(5) recombination and integration plasmid pDL-PSQ is proceeded in B.subtilispB2 bacterial strain, exchanges by homology arm site,Genes of interest P43-sfp-degQ has been incorporated in genome, and resistance screening, obtains positive transformant, is coproductionThe bacillus subtilis engineering bacteria of Surfactin and Plipastatin, called after B.subtilispB2-L.
5. above-mentioned 4 construction methods that provide, wherein, step (1), by pcr amplification, is introduced in Promoter P43 geneEcoRI restriction enzyme site is introduced BamHI restriction enzyme site in sfp gene, introduces BamHI and SacI enzyme is cut position in degQ genePoint.
6. above-mentioned 4 construction methods that provide, wherein, the described primer pair sequence of step (1) is as follows:
P43-F: as shown in SEQIDNO.2; P43-R: as shown in SEQIDNO.3;
Sfp-F: as shown in SEQIDNO.4; Sfp-R: as shown in SEQIDNO.5;
DegQ-F: as shown in SEQIDNO.6; DegQ-R: as shown in SEQIDNO.7.
7. above-mentioned 4 construction methods that provide, wherein, the described primer pair sequence of step (2) is as follows:
PS-F: as shown in SEQIDNO.8; PS-R: as shown in SEQIDNO.9.
8. the coproduction Surfactin that the method providing according to above-mentioned 4-7 any one obtains and the withered grass bud of PlipastatinSpore bacillus.
9. the application of the above-mentioned 8 bacillus subtilis engineering bacterias that provide aspect gram-positive bacterium, filamentous fungi inhibition.
10. the above-mentioned 8 bacillus subtilis engineering bacterias that provide application aspect preventing and treating at peach Bacteria erwinia.
Beneficial effect:
The present invention utilizes the method for homologous recombination, and a strain is not produced to antibacterial material Surfactin and PlipastatinB.subtilispB2, has imported one section of fusion P43-sfp-degQ in amyE site, obtained a strain coproductionThe recombinant bacterial strain B.subtilispB2-L of Surfactin and Plipastatin, this bacterial strain is to gram-positive bacterium, threadFungi has certain bacteriostatic activity.
The bacillus subtilis that the present invention adopts is classified as and can obtain at agriculture field and field of food thereof by the Ministry of Agriculture of ChinaArrive application safety bacterial strain widely. The antibacterial lipopeptid Surfactin and the Plipastatin that produce belong to microorganism natural antibacterialPeptide, high to human health and foodsafety, can not cause the pollution to environment, can be used for biological control, food processing and agricultureProduct preservation and freshness etc.
The present invention has set up a set of bacillus subtilis chemical conversion and homologous recombination method, to follow-up studyPlipastatin synthetase series structure and function basic theory are significant.
Brief description of the drawings
Fig. 1 functional gene sfp and degQ nucleotides sequence are listed in compare of analysis on ncbi database
A figure: functional gene sfp and Bacillus.amyloliquefacienssubsp.plantarumUCMB5063Compare;
B figure: functional gene degQ and Bacillus.amyloliquefacienssubsp.plantarumUCMB5063Compare.
Fig. 2 recombination and integration plasmid pDL-PSQ and homologous recombination principle schematic
Figure A: recombination and integration plasmid pDL-PSQ schematic diagram;
Figure B: homologous recombination principle schematic.
(note: M is DNAMarker in the amplification of Fig. 3 recombinant bacterial strain genes of interest P43-sfp-degQ; Swimming lane 1,2,3,4 isThe transformant DNA of picking is template, the amplification of genes of interest P43-sfp-degQ)
Fig. 4 recombinant bacterium B.subtilispB2-L produces the ESI-MS figure of Surfactin and Plipastatin
A figure: recombinant bacterium B.subtilispB2-L produces the ESI-MS figure of Surfactin;
B figure: recombinant bacterium B.subtilispB2-L produces the ESI-MS figure of Plipastatin.
Fig. 5 recombinant bacterium B.subtilispB2-L produces hemolytic activity figure and the antibacterial figure of antibacterial material
Figure A: recombinant bacterium B.subtilispB2-L produces the hemolytic activity figure of antibacterial material
Figure B: recombinant bacterium B.subtilispB2-L produces the antibacterial figure of antibacterial material
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described, the experiment side of unreceipted actual conditions in the following exampleMethod, conventionally according to the known approaches of this area, or according to the suggestion condition of manufacturer, the bacterial strain relating in embodiment all belongs toPrior art, those skilled in the art can be easily from being openly that commercial channel obtains.
Embodiment 1
1. materials and methods
1.1 bacterial strains and plasmid
Bacillus subtilis B.subtilisfmbJ (CGMCCNO.0943), Escherichia coli EscherichiacoliDH5 α cloning host (buying from Nuo Weizan biotech firm), B.subtilispB2 and bacillus subtilis integrated plasmid pDL(YuanandWong，RegulationofgroEexpressioninBacillussubtilis：theinvolvementofthesigmaA-likepromoterandtherolesoftheinvertedrepeatSequence (CIRCE), 1995) preserve by Agricultural University Of Nanjing's enzyme engineering laboratory; EscherichiacoliT-A gramGrand carrier pMD19-T (purchased from TaKaRa company). Indicator strain: peach Bacteria erwinia Rhizopusstolonifer (AS3.2336), micrococcus luteus (CMCC28001), is purchased from DSMZ of institute of microbiology of the Chinese Academy of Sciences.
1.2 culture medium
LB culture medium: peptone 10.0g, yeast extract 5.0g, NaCl10.0g, distilled water 1000mL, pH7.0～7.2;
10×Tbaase：(NH4)2SO420.0g，K2HPO4140.0g，KH2PO460.0g，Na3C6H5O7·2H2O10.0g，MgSO4·7H2O2.0g, adds water to 1000mL;
Seed culture medium: beef extract 5.0g, yeast extract 5.0g, peptone 10.0g, NaCl5.0g, distilled water1000mL，pH7.0～7.2；
Improvement Landy culture medium: glucose 40.0gL-1, Pidolidone 2.0gL-1，(NH4)2SO42.3g·L-1，K2HPO41.0g·L-1，MgSO40.5g·L-1，KCl0.5g·L-1，CuSO41.6mg·L-1，Fe2(SO4)31.2mg·L-1，MnSO40.4mg·L-1, 100mM3-N-agate coffee quinoline propane sulfonic acid (MOPS), pH7.0～7.2;
PDA culture medium: potato is cleaned peeling, is cut into 1cm3Little fourth, takes 200-300g, boils half an hour, eight layers of yarnCloth is crossed leaching juice, adds 20.0g glucose, and distilled water is settled to 1000mL, then adds 15-20g agar, and 115 DEG C, 30min is highPress steam sterilizing, pH nature;
GMI：10×Tbaase100mL，100g·L-1Glucose 50mL, 50gL-1Yeast extract 20mL, 10gL-1Acid hydrolyzed casein 20mL, 2.5gL-1Tryptophan 20mL, adds water to 1000mL;
GMII：10×Tbaase100mL，100g·L-1Glucose 50mL, 50gL-1Yeast extract 0.4mL, 10gL-1Acid hydrolyzed casein 5mL, 0.5molL-1MgCl25mL，0.05mol·L-1CaCl210mL, adds water to 1000mL;
Each component in GMI, GMII is separated sterilizing, and the used time mixes in proportion, the wherein logical filtration sterilization of amino acid, grape115 DEG C of sterilizing 30min of sugar, all the other are 121 DEG C of sterilizing 20min all.
Chloramphenicol (chloramphenicol, Cm; Sigma) storage liquid: chloramphenicol is mixed with to 10mg with absolute ethyl alcoholmL-1Storage liquid, be stored in-20 DEG C, when use, final concentration is 10 μ gmL-1。
1.3 reagent
UNIQ-10 pillar plasmid is extraction agent box in a small amount, UNIQ-10 pillar PCR product purification kit, UNIQ-10 postFormula glue reclaims kit all purchased from Shanghai Sheng Gong bioengineering Co., Ltd; Genome extracts kit, purchased from OMEGA company;Various molecular biology restriction enzymes, ligase are purchased from TaKaRa company; Chloramphenicol is purchased from Sigma company.
1.4 test method
1.4.1 the structure of bacillus subtilis integrated plasmid
The extraction of B.subtilisfmbJ genomic DNA, with reference to OMEGA genome kit description, utilizes Primer5.0 Software for Design PCR reaction special primers (table 1). Taking B.subtilispB2 genomic DNA as template, adopt primer P43-F/R amplification Promoter P43 gene, 94 DEG C of 5min of PCR reaction condition, 94 DEG C of 50s, 55 DEG C of 50s, 72 DEG C of 1min, 30 circulations; 72DEG C 10min. Taking B.subtilisfmbJ genomic DNA as template, with primer sfp-F/R and the degQ-F/R sfp base that increases respectivelyCause and degQ gene; Adopt the amplification of overlapping extension PCR method to merge fragment P43-sfp, and is connected with pMD19-T carrier and obtains weightGroup plasmid, called after pMD19-T-P43-sfp; DegQ Gene cloning to pMD19-T carrier is obtained to plasmid pMD19-degQ, sendJin Site Bioisystech Co., Ltd carries out nucleotide sequencing.
Through mensuration, taking B.subtilispB2 genomic DNA as template, the gene P43 that pcr amplification obtains, its sequenceSize is 426bp; Taking B.subtilisfmbJ genomic DNA as template, pcr amplification obtains gene sfp and degQ, and sequence is largeLittle 762bp and the 432bp of being respectively; According to gene sequencing acquired results, on ncbi database, compare, find bacterial strainThe nucleotides of sfp and degQ gene composition in fmbJ, with Bacillus.amyloliquefacienssubsp.plantarumUCMB5063 similitude reaches 99%, and comparison result is as Fig. 1. Method by overlapping extension PCR is by P43 promoter and sfp fragmentCouple together, be cloned into the pMD19-T-P43-sfp obtaining after pMD19-T carrier through order-checking, checking is correct.
Plasmid pDL and pMD19-degQ are carried out to double digestion with BamHI and SacI respectively, the linear pDL carrier after purifyingCarry out enzyme with fragment degQ and connect, connect product Transformed E .coliDH5 α competent cell and utilize the Cm resistance base carrying on itBecause filtering out resistant strain, extract plasmid and carry out PCR and double digestion qualification, obtain plasmid pDL-degQ. In like manner will merge fragmentIt is upper that P43-sfp is connected to plasmid pDL-degQ by the method for EcoRI and BamHI double digestion, obtains recombination and integration plasmidPDL-PSQ, as shown in Figure 2.
Table 1 is for the PCR primer of construction
1.4.2B.subtilis168 the competent preparation of bacterial strain
Aimed strain B.subtilispB2 is rule on LB culture medium, cultivate 2d for 37 DEG C; Picking list colony inoculation inIn 10mLGMI solution, 30 DEG C, 100rmin-1Incubated overnight; Cell culture is transferred molten in GMII by 1: 10 (volume ratio)In liquid, 37 DEG C, 200rmin-1, cultivate 3.5h; Cell culture is transferred in GMII solution by 1: 10 (volume ratio) again,30℃，100r·min-1, cultivate 90min; Take out cell culture, 5000rmin-1, centrifugal 10min collecting cell; RetainSupernatant, uses supernatant re-suspended cell by 1: 10 (volume ratio), and every pipe 100 μ L packing thalline, now do existing use.
1.4.3 the screening of the conversion of recombination and integration plasmid pDL-PSQ and integration bacterial strain
To in 100 μ L competent cells, add 10～15 μ L linear plasmid pDL-PSQ (Sca1 enzyme is cut processing), 37 DEG C,180r·min-1, 30～60min; By chemical transformation, linear plasmid pDL-PSQ is proceeded to B.subtilispB2 bacterial strainIn, there is twice double crossing in homology arm amyE site, as shown in Figure 2, genes of interest P43-sfp-degQ has been incorporated into geneIn group. Bacterial strain after transforming is coated to the LB flat board that contains chlorampenicol resistant, and 37 DEG C, incubated overnight, has obtained 5-10Individual transformant. The transformant growing on the LB flat board of picking chlorampenicol resistant, taking P43-F and degQ-R as primer, pcr amplification orderFragment P43-sfp-degQ, agarose gel electrophoresis detect, as shown in Figure 3, determined the positive transformant of bacterial strain No. 2, lifeB.subtilispB2-L by name. No. 2 strains are checked order, further confirm.
1.4.4 the production of antibacterial lipopeptid and qualification
B.subtilispB2-L inoculation, on test tube slant LB culture medium, is cultivated to 24h, activated spawn for 37 DEG C.Test tube slant bacterial classification is inoculated in the triangular flask that seed culture medium is housed, 37 DEG C, 180rpm are cultivated 20-24h again, raw to logarithmFor a long time. Seed liquor is inoculated in this good Landy fermentation medium with 5% concentration, and at 33 DEG C, 180rpm cultivates 72h, obtainsAntibacterial material zymotic fluid. Zymotic fluid centrifugal 15min under 4 DEG C, 8000g removes thalline, and supernatant regulates pH extremely with 6NHCl2.0, after acid is sunk and is spent the night, the centrifugal 5min of 12000g, collecting precipitation, and with methyl alcohol redissolution, obtain crude extract.
Crude extract utilizes electron spray ionisation, induction to collide the mass spectrum (Electrosprayionizationmass that dissociatesSpectrometry/collision-induceddissociation, ESI-MS/CID) measure its molecular weight. ESI-MS/CID adopts LCQDECAXPPLUSThermoFinnigan LC-MS (LC-MS) equipment that has been equipped with electric spray ion source(ThermoCorporation, USA). (2.6mm Φ × 250mm, 2 μ m) analyze, and in HPLC condition, flow to adopt MS postPhase constituent and ratio are: 0～30min, acetonitrile+0.1% formic acid 50～95%; 30～40min, acetonitrile+0.1% formic acid 95%;40～50min, acetonitrile+0.1% formic acid 95～50%; Flow velocity is 0.2mL/min. Electrospray ionization source operating voltage is 5kV, operation temperatureDegree is 300 DEG C, and operating pressure is 20abr.
As shown in Figure 4 A, lipopeptid compound Surfactin:m/z994.7, the m/z1008.7 of acquisition, m/z1022.8,M/z1036.8, m/z1050.7, m/z1064.7; As shown in Figure 4 B, the lipopeptid compound Plipastatin:m/z of acquisition1463.7、m/z1477.7、m/z1491.7、m/z1505.7、m/z1519.7、m/z1533.7。
1.4.5 recombinant bacterium antibacterial product bacteriostatic activity and hemolytic activity detect
Blood plate is made: behind the cooling 50 DEG C of left and right of the LB of 100mL sterilizing culture medium, add 5mL defiber sheep blood, mixEvenly, import in the culture dish of sterilizing, make blood plate. On blood plate, place the filter paper of high-temperature sterilization, respectively by 20 μThe recombinant bacterium B.subtilispB2-L of L and original strain B.subtilispB2 crude extract add in filter paper, mistake at 37 DEG CCultivate night, Taking Pictures recording.
Using peach Bacteria erwinia and micrococcus luteus as indicator bacteria, adopt agar hole diffusion method respectively, cultivate indicator bacteriaIn base, sneaking into 1.0mL bacterium amount is 106The indicator bacteria of concentration, gets respectively 100 μ L recombinant bacterium B.subtilispB2-L and original bacteriumStrain B.subtilispB2 sample adds in agar hole, 28 DEG C of cultivation 72h of mould, and 37 DEG C of cultivation 24h of bacterium, observe antibacterial effectReally.
As shown in Figure 5, original strain B.subtilispB2 is owing to can not producing Surfactin, therefore shape not on blood plateBecome haemolysis circle, peach Bacteria erwinia and micrococcus luteus are not all had to bacteriostatic activity. But recombinant bacterial strain B.subtilispB2-LFermentation crude extract on blood plate, formed obvious haemolysis circle, peach Bacteria erwinia and micrococcus luteus are had necessarilyInhibitory action.
Can know, the illustrative embodiments that above-described embodiment only adopts for inventive principle is described, but thisBrightly be not limited only to this, those skilled in the art, not departing under real situation of the present invention, can make various improvement and change, thisA little improvement and change also belong to protection scope of the present invention.
Claims (10)
1. a bacillus subtilis strain, is characterized in that: this bacterial strain coproduction Surfactin and Plipastatin, called afterPB2-L, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number on November 23rd, 2015For CGMCCNo.11723.
2. a recombinant plasmid pDL-PSQ, is characterized in that: this recombinant plasmid sequence, as shown in SEQIDNO.1, comprises sfpGene and degQ gene.
3. recombinant plasmid pDL-PSQ claimed in claim 2, is characterized in that: this recombinant plasmid is taking P43 gene as promoter,AmyE is homology arm.
4. a construction method for the bacillus subtilis of coproduction Surfactin and Plipastatin, is characterized in that, the partyMethod comprises the steps:
(1) design primer pair P43-F and P43-R, sfp-F and sfp-R, degQ-F and degQ-R; With B.subtilisfmbJ'sGenomic DNA is template, pcr amplification sfp gene and degQ gene; Taking B.subtilispB2 genomic DNA as template, PCRAmplification Promoter P43 gene;
(2) design primer pair PS-F and PS-R, increase by overlapping extension PCR, obtains and merge fragment P43-sfp;
(3) carry out enzyme with restriction enzyme and cut, and the enzyme that degQ genetic fragment is inserted to plasmid pDL by coupled reaction is cut positionBetween point, obtain plasmid pDL-degQ;
(4) carry out enzyme with restriction enzyme and cut, and will merge fragment P43-sfp insertion plasmid pDL-by coupled reactionDegQ, finally obtains recombination and integration plasmid pDL-PSQ;
(5) recombination and integration plasmid pDL-PSQ is proceeded in B.subtilispB2 bacterial strain, exchange by homology arm site, by orderGene P43-sfp-degQ be incorporated in genome, resistance screening, obtain positive transformant, be coproduction SurfactinWith the bacillus subtilis engineering bacteria of Plipastatin, called after B.subtilispB2-L.
5. construction method claimed in claim 4, is characterized in that: step (1) is by pcr amplification, in Promoter P43 geneIntroduce EcoRI restriction enzyme site, in sfp gene, introduce BamHI restriction enzyme site, in degQ gene, introduce BamHI and SacI enzymeCut site.
6. construction method claimed in claim 4, is characterized in that, the described primer pair sequence of step (1) is as follows:
P43-F: as shown in SEQIDNO.2; P43-R: as shown in SEQIDNO.3;
Sfp-F: as shown in SEQIDNO.4; Sfp-R: as shown in SEQIDNO.5;
DegQ-F: as shown in SEQIDNO.6; DegQ-R: as shown in SEQIDNO.7.
7. construction method claimed in claim 4, is characterized in that, the described primer pair sequence of step (2) is as follows:
PS-F: as shown in SEQIDNO.8; PS-R: as shown in SEQIDNO.9.
8. the coproduction Surfactin obtaining according to method described in claim 4-7 any one and the withered grass gemma of PlipastatinBacillus.
9. the application of bacillus subtilis engineering claimed in claim 8 aspect gram-positive bacterium, filamentous fungi inhibition.
10. bacillus subtilis engineering claimed in claim 8 application aspect preventing and treating at peach Bacteria erwinia.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108440655A (en) * | 2018-03-26 | 2018-08-24 | 河北科技大学 | The disease-resistant bacterial strain of anti-plant soft rot disease albumen and its encoding gene and structure |
| CN111518163A (en) * | 2020-04-08 | 2020-08-11 | 大连百奥泰科技有限公司 | Application of lipopeptide compounds in resisting novel coronavirus |
| CN113265414A (en) * | 2021-05-21 | 2021-08-17 | 天津大学 | Construction method for converting high-yield bacterial strain of fengycin by using glucose |
| CN115820705A (en) * | 2022-08-16 | 2023-03-21 | 天津大学 | Bacillus subtilis strain for high yield of fengyuan and construction method |
-
2016
- 2016-01-18 CN CN201610032668.3A patent/CN105602876A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| KENJI TSUGE等: "The Genes degQ, pps, and lpa-8 (sfp) Are Responsible for Conversion of Bacillus subtilis 168 to Plipastatin Production", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| 刘丽霞 等: "Bacillus subtilis 168 菌株产surfactin 改造", 《南京农业大学学报》 * |
| 周小虹: "Bacillus subtilis fmbj对桃软腐病菌Rhizopus stolonifer拮抗机理及应用研究", 《南京农业大学学位论文》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108440655A (en) * | 2018-03-26 | 2018-08-24 | 河北科技大学 | The disease-resistant bacterial strain of anti-plant soft rot disease albumen and its encoding gene and structure |
| CN108440655B (en) * | 2018-03-26 | 2021-11-02 | 河北科技大学 | Plant soft rot resistance protein, coding gene thereof and constructed disease-resistant strain |
| CN111518163A (en) * | 2020-04-08 | 2020-08-11 | 大连百奥泰科技有限公司 | Application of lipopeptide compounds in resisting novel coronavirus |
| CN113265414A (en) * | 2021-05-21 | 2021-08-17 | 天津大学 | Construction method for converting high-yield bacterial strain of fengycin by using glucose |
| CN113265414B (en) * | 2021-05-21 | 2022-07-08 | 天津大学 | Construction method for converting high-yield bacterial strain of fengycin by using glucose |
| CN115820705A (en) * | 2022-08-16 | 2023-03-21 | 天津大学 | Bacillus subtilis strain for high yield of fengyuan and construction method |
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