Research On Heterologous Expression And Their Function Of Several Important Bacterial Polyketide Gene Clusters

An immense variety of natural compounds with good biological activities which come from bacteria,especially actinomycetes and myxobacteria,are synthesized by multi-enzyme complex known as polyketide synthases and nonribosomal peptide synthetases,respectively.Although assembled from simple building blocks,there is an extraordinary structural diversity among PKS-,NRPS-and mixed PKS/NRPS derived compounds.The efficient synthetic production of polyketides and nonribosomal peptides were highly effected by this complex structural and stereochemistry.Besides,some objective conditions such as growth rate,culture conditions,compound yield of original microbial producer and genetic manipulation tools limited by DNA size and independent of restriction sites also pose an obstacle to the traditional predominant economical route of producing secondary metabolites.Therefore,using advanced molecular biology techniques,the secondary metabolic pathways from the original producer transferred to the appropriate host strain to express active secondary metabolites heterologously become research hotspot of the biotechnology and pharmaceutical research gradually.Red/ET recombination,which was invented at the end of last century,allows DNA with homology arms?35-100 bp?recombine target DNA under the functional interaction between RecE and RecT,or between Red? and Red?.This technique represents a revolutionary DNA engineering platform that shows advantadges during large PKS-NRPS gene clusters' manipulation and has been used as the dominant strategy here.In this study,the mchs gene cluster synthesizing myxochromide in Stigmatella aurantiaca was cloned into p15A-oriV-apr vector via Red/ET recombineering technology and integrated into genomic DNA of Agrobacterium tumefaciens for heterologous expression.Native promoter?P_m?was insteaded by inducible promoter(P_tetR)to compare their individual fuction.The heterologous expression of the engineered biosynthetic gene cluster was confirmed via HPLC-MS.Subsequently,epo gene cluster spanning 56kb was" direcloned " by p 15A-oriT-BSD backbone flatted with epo homologous arms from digested Sorangium cellulosum genomice DNA.This " Direcloning"method to extract the target DNA segment from a primary DNA preparation,exactly and directly into an expression vector,would circumvent the game of chance inherent in library screening and facilitate functional investigation of sequence data.The p15A-oriT-BSD backbone constructed here showed advantages in modifying and heterologous expressing PKS/NRPS gene clusters.Red/ET recombination was applied to change original epoK for successful expression in Pseudomonas putida.According to the well-runned approach we used above,the 22kb syl gene cluster comes from Pseudomonas syringae pv.Syringae?Pss?was intergrated into Streptomyces coelicolor and Streptomyces lividans strains respectively under the control of new promoter P_snpA.The yield of heterologous compounds were identified by RT-PCR and HPLC-MS.Their anticancer activity were showed well in MTT trails against B16?4T1?Meth-A?Hela cells and mice experiments with B16 or 4T1 model.The newly-developed techniques of Red/ET recombination,termed as"Direcloning" has been improved by directly cloning chosen DNA segments into the expression vector carrying Tpase-oriT cassette.This is a great idea for speedy and flexible ways to obtain large DNA segments and heterologous express PKS/NRPS gene cluster.Three important bacterial polyketide gene clusters were directly cloned and successful heterologous expressed in this work.This research provided us with new avenues and ideas for the discovery and production of new antitumor compounds.