Identification Of The Genes Of Antimicrobial Lipopeptide Synthetases Producted By Bacillus Sp.and The Gene Deletion Research

Antibacterial lipopeptide is low molecular weight metabolites by non-ribosomal synthesis pathway,it mainly produced by Bacillus subtilis.Based on molecular cloning technology,humans have gradually understood and mastered the laws of lipopeptides biosynthesis.The synthesis pathways of most of lipopeptides are regulated by the gene clusters of non-ribosomal peptide synthases(NRPS).First of all,we identified the encoding genes of lipopeptide synthases in this study.About 11 strains,such as Bacillus subtilis.Bacillus amyloliquefaciens and Paenibacillus polymyxa,were both taken as research objects to be detected the encoding genes by PCR ampification technique.Meanwhile the High Performance Liquid Chromatography(HPLC)ensured that the PCR provided correct results.So we speculated that they might have the potential ability to synthesis the specific lipopeptides.Based on the above means,we established the rapid identification method of antimicrobial lipopeptides and screening method of antimicrobial lipopeptide-producing strains.On the other hand,the modular structure and unconventional assembling ways of NRPS determine the diversity of the final product's structures and bioactivities.Based on combinatorial biosynthesis technology,we choose a plipastatin-producing strain,Bacillus subtilis PB2,as the research object for the molecule modification.The study amied on the substtrate-specific ppsC-A2 domain?the fully functional ppsC-Glu module and the independent active ppsC subunit from plipastatin synthetase were deleted respectively.We explored the biosynthesis of the new lipopeptides by constructing the NRPS hybrid system.Meanwhile we explored the relationship between the simulating gene knockouts and module skipping.Then,because the ppsC-A2 domain is located between the ppsC-C2 domain and the ppsC-PCP2 domain,we designed the experiment to induce the same of module skipping by deleting the ppsC-C2 domain and the ppsC-VCP2 domain.Other work aimed to go a step further:to research the interaction and connection between the different domains and modules.At the same time,We also explored the relationship between the module skipping and the NRPS biosynthesis.This thesis mainly drew conclusions as follows:1.The study established the rapid identification method of antimicrobial lipopeptides and screening method of antimicrobial lipopeptide-producing strains based on PCR amplification and HPLC detection.This study verified that the Bacillus subtilis,Bacillus amyloliquefaciens and Paenibacillus polymyxa could synthesise many kinds of antimicrobial lipopeptides.For example,they could synthesise surfactin?fengycin?iturin?bacillomycin D?subtilosin A or polymyxin.They have a lot of encoding genes required for antimicrobial lipopeptides biosynthesis,such as sfp?srfAA?srfAD?fenA?fenB?fenD?ituD?ituA?ituB?albF?albA?pmxB?PmxC?pmxE?bmyA?bmyB?bmyC.However,because of low yield or impeded synthetic process,some kinds of lipopeptides were not positive for HPLC detection.But the specific reasons need to be researched.Different kinds of strains could produce different or the same antibacterial lipopeptides.At the same time,different strains could also produce several different or the same antimicrobial lipopeptides.Therefore the above method could potentially also be used to find different species or genera containing homologous genes.2.Base on combinatorial biosynthesis technology,the study built three recombinant strains by deleting the ppsC-A2 domain>the ppsC-Glu module and the ppsC subunit respectively.Gene fragments up and down the deleted gene were cloned,through SOE-PCR,the two gene fragments was ligated together to form fusion fragment.Then recombinant gene deletion plasmid was constructed using fusion fragment to linear pKS2.The recombinant plasmid was inserted to Bacillus subtilis PB2 by homologous recombination.Finally,the three recombinant strains were analyzed by HPLC.The recombinant strain without ppsC-A2 domain could produce the lipopeptide with different structure through the module skipped assembling ways of NRPS.The new lipopeptide was determinated as plipastatin analogue without Ala by HR-ESI-MS analysis and MS/MS analysis.Unfortunately,The two recombinant strains without the ppsC-Glu module and the ppsC subunit just produced little of plipastatin through the impeded synthetic process.They couldn't produce the new lipopeptides through the module skipped assembling ways of NRPS.3.The study built two recombinant strains by deleting the ppsC-C2 domain and ppsC-PCP2 domain by homologous recombination.As for the recombinant strain without the ppsC-PCP2 domain,the result of HPLC were similar to the ppsC-A2 domain deleted strain.It also produced the same new lipopeptides by the same way.But the recombinant strain without the ppsC-C2 domain produced nothing.The result showed that the unconventional assembling ways of NRPS,module skipping,was related to A domain and PCP domain.