Study On The Biosynthesis Of Polyene Macrolides From Mangrove-derived Streptomyces Sp.OUC6819

Streptomyces sp.OUC6819was isolated from reeds rhizosphere soil in Guangdong province of China, which is able to produce a series of32membered polyene macrolides (PEMS) with significant antifungal activities. For the purpose of elucidating the biosynthesis mechanism of polyene macrolides in Streptomyces sp.OUC6819, in this thesis, the following experiments were carried out:Firstly, growth conditions for Streptomyces sp.OUC6819were studied and optimized. Streptomyces sp.OUC6819was inoculated in the different medium (MS, ISP2, ISP4, COM, gause) with salt concentration ranging from0%to10%. The results of experimental showed the optimum culture conditions are ISP2,28℃, salt concentration3.3%and pH=7.2. The antibiotic resistance experiment showed Streptomyces sp.OUC6819is sensitive to Thiostrepton, Kanamycin and Erythromycin.Secondly, the genomic library of Streptomyces sp.OUC6819was constructed. Total DNA of Streptomyces sp.OUC6819was extracted and partially digested by restriction endonuclease Sau3AI, and then DNA fragments at the size of35-45kb were recovered and ligated with SuperCosl, which was digested by XbaI, dephosphorylated and redigested by BamHI. The ligation product was packaged into phage particles with Lambda Packaging Extracts, which was then transfected E. coli Trans10to yield the genomic library of Streptomyces sp.OUC6819. The titer of the library was6.0×106cfu/mL,.meeting the requirements as a standard genomic library. The clones were stored at-80℃. Construction of the genomic library of Streptomyces sp.OUC6819laid the foundation for cloning of biosynthesis gene cluster of secondary metabolites and their heterologous expression.Thirdly, the biosynthesis gene cluster encoding polyene macrolides in Streptomyces sp.OUC6819was cloned and bioinformatically analyzed. The results demonstrated the gene clusters is98kb and contains14ORFs, including4polyketide synthase gene(opeG, opeH, opeI and opeJ),5possible regulatory genes (opeA, opeC, opeD, opeE and opeF),1resistance genes (opeK),1type Ⅱ thioesterase superfamily enzyme gene (opeM),1CoA ligase(ppeN) and2unknown function gene(opeB and opeL). By using PCR targeting method, recombinant cosmids for opeA, opeC and opeF gene inactivation were constructed and named as ZH1, ZH2and ZH3, respectively. And then ZH3were introduced into Streptomyces sp.OUC6819wild type strain via conjugations, generating opeF gene mutant. Both wild type strain and opeF mutant were fermented under the same conditions, and the fermentation products were anylzed by using HPLC and HPLC-MS analyses. The results showed that inactivation of opeF gene abolished the production of polyene macrolides in the mutant, indicating that opeF’was the positive regulatory gene.Finally, the regulation mechanism of opeF gene was studied at the transcriptional level. The RT-PCR results showed that opeF regulates the expression of opeA, opeE, opeF, opeG, opeK, opeM and opeN genes in a positive manner. This result provides theoretical basis for further genetic engineering.