Discovery And Biosynthetic Study Of Novel Molecules In Three Actinomycetes

The discovery of novel bioactive natural products is the key to expand the library of natural products and provide the clinical lead drugs.The secondary metabolites of fungi and actinomycetes are the major sources of novel bioactive compounds.With the innovation of instruments,it has been never outdated at any stage to capture renewable active products from various biomaterials by different methods.Genome sequencing and modern molecular biology tools are also used to explore the biosynthetic pathway of active molecules and elucidate the mechanism of key enzymes.The thesis focused on the secondary metabolites of three different types of actinomycetes isolated from various origins.After large scale fermentation,the final crude was obtained.Taking the consideration of the physical and chemical characteristics of the crude extract,it was then subjected to normal-and reverse-phase ODS silica gel and Sephadex LH-20.Finally,semi-preparative HPLC was used for purification.Twenty-two compounds including 10 new molecules were identified based on the spectral NMR and single-crystal X-ray diffraction data.At the same time,the biosynthesis of amycolamycins was also studied.The brief summaries were as follows:Amycolamycin B,which is a nine-membered enediyne derivative and a new rifamorp ho lines(compound 9)were isolated from the mantis-associated actinomycete Amycolatopsis sp.HCa4.Amycolamycin A showed promising cytotoxicity against MDA-MB-231 cell line with its IC50 of 7.9 μM.In the next step,we focused on the biosynthesis process of amycolamycins.After sequencing,functional annotations were performed and then the acm gene cluster(about 76 kb)was identified.After that we proposed the possible biosynthetic pathway.The characterization of AcmP1 in vitro and the heterologous expression of acm gene cluster were done to verify the proposed route.During the process,compounds 2-8 were isolated.The actinomycete NA06532 was purified from the black soil sample in the northeast of China.The two novel cyclic peptides(compounds 10-11)were discovered from its secondary metabolites.After reviewing the literature,it was found that the formation mechanisms of C-N bond have not been reported.The strain NA02950 was isolated from the sea mud sample of Hainan Island.More sea salt was added to cultivate it prominently.Finally,eleven compounds(12-22)were obtained,of which two neoant imyc in-based dimers(16 and 17)and an elaiophylin derivative(18)were new compounds.The compounds 12,14 and 15 showed weak cytotoxic activity against MCF7 cell line.In addition,this thesis contains all details about the discovery,biological activities,mechanisms and biosynthesis of enediyne natural products.