Screening And Studying Modular Structure Of PKS And NRPS Genes In Marine Sponge Hymeniacidon Perleve Associated Bacteria

This research was focused on exploring marine natural products of medical potentiality and their origin.The studies of bioactive metabolites from marine bacteria associated with marine organisms along the coast in China were carried out. Through screening of antimicrobial and cytotoxic activities,the results showed that 42 isolates had antimicrobial activity and 12 isolates had cytotoxicity.Molecular phylogenetic analysis of marine bacteria with cytotoxicity based on 16S rRNA sequences indicated that they were belong to the genera Aerococcus,Agrobacterium, Alteromona,Bacillus,Exiguobacterium,Paracoccus,Pseudoalteromons, Rheinheimera.Furthermore,marine bacteria with cytotoxicity were also screened for PKS I and NRPS genes which could be responsible for bioactive secondary metabolites biosynthesis,4 strains having KS domain or A domain were obtained, which provide strong evidence that marine bioactive bacteria can produce natural products through PKS and NRPS pathways.In order to further confirm the existence of PKS cluster in Pseudoalteromons sp. NJ632,genomic walking was used to amplify the flanking genomic DNA sequence to a KS sequence obtained from NJ632 by PCR amplification.A 10-kb continuous DNA sequence was obtained by five stepwise walking to upstream and downstream of KS fragment,respectively.The open reading frames(ORFs) were predicted and screened large multi-domain megasynthase involved in the biosynthesis of PKS.The results showed a relatively complete PKS module comprised of distinct core and auxiliary catalytic domains was obtained.Core catalytic domains include ketosynthase(KS) domain,acyltransferase(AT) domain,auxiliary domains include ketoreductase(KR), dehydratase(DH).Moreover,another auxiliary domain,cyclization(Cy),is also present in our gene walking sequence,which is generally present in NPRS module. For consideration of the existence of NRPS A domain also found in NJ632,it is suggested that there may exist a mixed or hybrid PKS/NRPS multienzyme system in the Pseudoalteromonas sp.NJ632,which could synthesize secondary metabolite with hydrid structure.In order to confirm a putative NRPS/PKS gene cluster from marine sponge associated bacterium with cytotoxic activity and elucidate the gene structural information.The genomic library of marine sponge Hymeniacidon perleve associated bacterium Pseudoalteromons.sp NJ631 is constructed using pCC1FOS fosmid.The 2880 colonies from the fosmid library were screened by hybridizing with the digoxigenin(DIG)-labeled fragment of the KS domain from PCR amplification of Pseudoalteromons sp.NJ631 genomic DNA.The results showed 14 positive clones were identified and annotated AE1,BG2,CA1,CB6,DD12,DH5,FA11,GC10,GD7, GG4,KG10,LD8,MH9,OA5,respectively.To ensure that two clones we selected to sequence located both sides of these overlapping clones,additional chromosomal walking from this locus was carried out,eventually leading to two clones,BG2 and GG4 located both sides of all of positive clones.The genome of clones BG2 and GG4 were sequenced using genome shotgun method.According to analysis of sequence,a 60-kb continuous DNA region covered by overlapping fosmids BG2 and GG4 was obtained.The ORF and BlastX analysis of this large DNA fragment indicated that there are three big ORFs,ORF14,ORF15å’ŒORF19,encoding proteins with similarities to amino acid adenylation,beta-ketoacyl synthase,and nonribosomal peptide synthase,respectively,from different organisms.The results gave us a clue there could be PKS or NRPS modules in three ORFs.Further analysis demonstrates three ORFs encoding two NPRS modules,one PKS modules and three NRPS modules.Finally,a NRPS/PKS gene cluster annotated "NJ631 NPRS-PKS" and containing seven modules was obtained.Using the specificity-conferring selection rule,the substrate specificity of four A(A₂,A₃,A₄,A₅) domains were successfully predicted,and the amino acids of the substrate specificity were Glu/Gln,Ser,Ser-D, Aeo,respectively.To raise ployclonal antibodies using KS protein produced in E.coli as antigen and test the substrate specificity of A domains from NJ631 NRPS-PKS cluster,a series of expression vector,pZPKS2,pZPA12,pZPA22,pZPA32,pZPA42,pZPA52, containing KS,A1,A2,A3,A4,A5 domains were successfully constructed using pET system(pET-28a),respectively.The expression of the target DNA was induced by the addition of 1 mM IPTG to the bacterial culture.The results showed KS,A1,A3 and A4 domains were over expressed in E.coli,successfully,which provide strong foundation for raising ployclonal antibodies using KS protein and testing the substrate specificity of A domains.This study was started with isolation of marine bacteria associated with marine organisms,continued with bioactive(antibacteria and cytotoxicity) and functional genes(PKS and NRPS) screening and bacteria identification.Then,the bacterium with bioactivity,Pseudoalteromons sp.NJ632,was further confirmed the existence of PKS cluster using genomic walking.At the same time,a NRPS/PKS gene cluster annotated "NJ631 NPRS-PKS" and containing seven modules was obtained in Pseudoalteromons sp.NJ631 using a series of methods containing the construction of genomic DNA library,screening,analysis.Finally,core domains contianing KS and A domain in NRPS/PKS gene cluster of Pseudoalteromons sp.NJ631 were tried to express in E.coli.Through these works,the integrated approach to explore the potential of marine bacteria for the production of bioactive metabolites was established and some new evidences of marine microorganisms as medical origin were provided.