Cloning And Heterologous Expression Of Natural Product Gene Clusters From Bacillus Using Red/ET Recombineering And The Separation Of Antagonistic Bacterium From Marine Environment

Bacillus are spore-forming,Gram-positive bacterium,which produce diverse secondary metabolites.Generally their biosynthesis gene clusters are larger than 10 kb and are difficult to be cloned by PCR amplification.In this thesis,we direct cloned and heterologous expressed four different natural product gene clusters from Bacillus amyloliquefaciens FZB42,an important plant-growth-promoting rhizobacteria(PGPR)and biocontrol agent(BC),via Red/ET recombineering,and heterologous expressed these gene clusters in B.subtilis 1A751:sfp and E.coli.Thus,we established the heterologus expression system of Bacillus gene clustes laying the foundation to mine Bacillus natural products.The major results of this thesis are as follows:The direct cloning and heterologous expression of bacillomycin D using Red/ET recombineering.Bacillomycin D,which synthesed by non-ribosomal peptide synthetase(NRPS),is responsible for the main antifungal activity of B.amyloliquefaciens FZB42.Besides,it plays an important role in root colonization and the stimulation of plant resistance.By linear-linear homologous recombination(LLHR),the 39.7 kb bmy gene cluster was cloned,and transformed into B.subtilis 1A751:sfp and E.coli GB05-MtaA for heterologous expression.The HPLC-MS results showed that bmy was expressed successfully in B.subtilis 1A751:sfp,three derivates was detected(C14?C15?C16-bacillomycin D),which was consistent with its native host FZB42.The direct cloning and heterologous expression of plantazolicin using Red/ET recombineering.Plantazolicin,a ribosomal microcin from B.amyloliquefaciens FZB42,displays antibacterial activity toward closely related Bacillus(specially the B.anthrax)and inhibition of nematodes.Pzn cluster covers 9.9 kb,is essential for the production,modification,export,and self-immunity of this natural product.We cloned and modifid pzn,and then transformed it into B.subtilis 1A751:sfp and E.coli GB2005,the result showed that this gene cluster can be expressed in both hosts.The production in B.subtilis 1A751:sfp was higher than that in FZB42,which suggested heterologous expression could be an effective way to increase production.The medium optimization led to the yield of plantazolicin A increase nearly ten times.In a fellow-up experiment,we apply ccdB counter-selection to change the core peptide of pzn to generate more active plantazolicin analogues.The direct cloning and heterologous expression of difficidin and the detection of a series of intermediates.Difficidin,which synthesed by trans-AT polyketides,displays antibacterial activity towards plant pathogens.Its biosynthesis gene cluster dif covers 71.1 kb.After cloned and modified,the gene cluster was introduced into B.subtilis 1A751:sfp,HPLC-MS results showed that several biosynthetic intermediates of difficidin were producted in B.subtilis 1A751:sfp::dif.The direct cloning and heterologous expression of unknown gene cluster nrs and the analysis of its products.Nrs is an unknown gene cluster,and its product is still unclear.It was successfully cloned by LLHR and transferred into B.subtilis 1A751:sfp for heterologous expression.The HPLC-MS data showed that some compounds related to nrs were detected.Combind with the bioinformatic prediction of this cluster,the possible biosynthetic pathway and structure of putative peptide were speculated:its chemical formula was C18H19N3O2S3,molecular weight was 405.06.The enrichment and separation of antagonist bacterium from Qingdao Shilaoren bay.For the special nature of the marine environment,it is great potential to screen antimicrobial substances which are efficient,innovative and low toxic from marine.In this resreach,more than 120 strains were isolated from the enrichment culture sample which was collected from the Qingdao Shilaoren bay.We identified the taxonomic position of those strains by sequencing the 16S rRNA gene,based on sequencing results,there were four strains were potentially new species.There were 25%strains showed antimicrobial activity,mainly distributing in Bacillus and Actinomycetes.We detected the natural products of SHZI1401T via HPLC-MS,a compound was similarly to bacillomycin D was detected,with the molecular weight of 1056.56,it may be a new member of iturin family.