Heterologous Expression Of Disorazole Z And Construction Of Attenuated P.aeruginosa PAO1

Natural products from myxobacteria are great source for pharmaceutical intermediate with diverse structures and active bioactivities,typical examples of which are epothilones and disorazoles produced by Sorangium cellulosum.The former has been in clinical research for anticancer application,however the latter is mainly focused on preclinical studies.Disorazoles represent great potential for development of anticancer drugs due to their novel structures and potent cytotoxicitiy against several cancer cell lines including multidrug-resistant KB line at low picomolar levels through depolymerization of microtubules.Especially for the newly discovered member,disorazole Z isolated from Sorangium cellulosum So ce 427,coupled with luteinizing hormone-releasing hormone was tried for targeted cancer therapy.Due to the intriguing biological potency,along with the complex and unusual molecular architecture,massive efforts have been made to the synthesis study of disorazoles.Total chemical synthesis has been facing a huge challenge due to the presence of both heterocyclic scaffolds and labile polyene linkages.Biological synthesis is presenting advantages towards this kind of complex natural products by fermentation.However,myxobacteria especially for the genus of Sorangium are usually not easy for genetic manipulation and extraordinary slow-growing thus also not suitable for large-scale fermentation.Therefor heterologous expression in an advantageous host is a feasible way to analyze the biosynthetic pathway and regulation mechanism even to improve the yields.Together with combinatorial biosynthesis,rational synthetic pathway refactoring of these biosynthetic pathway may provide more derivates for studies on structure-activity relationships and therapeutic applications.Besides,novel recombineering system for Pseudomonas was used for genetic manipulation of PAO1 to construct attenuated strain for further application.Achievements about these studies are summarized below:1.Based on genome sequencing and mining strategies,BGC of disorazole Z was identified in native host So ce 427 and cloned into p15 vector containing conjugation and transposition cassettes via Red/ET recombineering.2.The diaorazole Z biosynthetic gene cluster was integrated into the chromosome of Myxococcus xanthus DK1622,Burkholderia DSM7029 and PG1 through conjugation/electroporation and transposition.Disorazole Z and its derivatives were successfully produced in Myxococcus xanthus DK1622.3.Two genes with unknown function within the cloned cluster was knocked out respectively to analyze its influence on the biosynthesis of disorazoles Z and its possible functional mechanism.4.Further reconstitution of the gene cluster using tetracycline inducible promoter and a strong constitutive promoter resulted in obvious increment of disorazole Z production in heterologous host.5.Based on detailed comparative studies between dis427 and dis12 gene cluster,rational design and reconstruction of dis427 even hybrid of these two gene clusters for combinatorial biosynthesis were explored hoping to produce desired compounds for structure-activity relationship studies and therapeutic applications.6.Attenuated strains of Pseudomonas aeruginosa PAO1 were constructed by genetic manipulation using novel recombineering system and also validated by animal infection experiments.In conclution,this study explored the application of Red/ET recombineering technique on discovery,direct cloning,reconstitution and heterologous expression of active natural products biosynthetic pathway from myxobacteria,and on the genome editing of pathogenic bacteria,etc.All achivements established foundations for further studies on anticancer and antiinfection research.