Isolation And Identification Of Lijiang-Derived Fungi Secondary Metabolites And Study On The Biosynthetic Pathway Of Myrothecisin E

Microbial germplasm resources are important basis for the chemical research of natural products,special habitats-derived filamentous fungi are important source for the discovery of lead compounds.Lijiang,Yunnan province,located in the southwest of China,possesses favorable climatic conditions along with rich species resources,so fungi originating from this place could produce natural products with novel structures and diverse activities.The mining of fungal natural products can discover more valuable compounds,which applied to the research and development of food resources;at the same time,the discovery of novel compounds provide material for the study of natural product biosynthesis oriented by structural characteristics.In this study,Lijiang-derived fungal germplasm resources were taken as the starting point and multidisciplinary methods such as microbiology,natural product chemistry and molecular biology were used to study fungal diversity,secondary metabolites and their biosynthesis,the interrelationship between "strains-compounds-genes" was initially explored.The main findings in this study were as follows:1)162 strains of fungi were isolated from 72 samples collected in Lijiang,Yunnan Province by isolation,purification and species identification.They belonged to three phyla,ten orders and 38 genera,among which Fusarium sp.,Epicoccum sp.and Trichoderma sp.were the main strains.Based on phylogenetic tree analysis 42 strains were selected and their crude extract of fermentation broth were evaluated for antimicrobial activity and chemical diversity.The results showed that Cylindrocarpon olidum MEFC350,Alfaria terrestris MEFC1004,Aspergillus terreus MEFC008 and Aspergillus austwickii MEFC1020 showed high antibacterial activity against the foodborne pathogenic bacteria Staphylococcus aureus and Escherichia coli.Alternaria tenuissima MEFC1007 displayed a rich secondary metabolite profile.Based on this,the aforementioned 5 fungal strains were selected to investigate their secondary metabolites.2)30 compounds(1-30),belonged to polyketides,meroterpenoids and indole alkaloids,were isolated and identified from five strains of fungi by chromatographic separation technology and spectroscopy.Among them,myrothecisin E(23)was a new compound and kojic acid(30)was a new natural food preservative.The cytotoxic activities of 30 compounds on five different tumor cell lines(A549,HCT 116,786-O,TE-1,and 5637)were evaluated by the cell counting kit(CCK-8)assay.The altertoxin II(6),cylindrol A4(19),cylindrol A5(20),and mellein(27)showed excellent cytotoxic activities against all five tumor cell lines,while myrothecisin E(23)exhibited weaker cytotoxicity against human bladder cancer cells 5637.3)In order to elucidate the biosynthetic pathway of the new compound myrothecisin E(23),a genetic manipulation platform was established in the producing strain A.terrestris MEFC1004.Based on the structural analysis of the myrothecisin E and the omics analysis of A.terrestris MEFC1004,two candidates for polyketide synthases(PKSs)and one Ubi A-type prenyltransferase(PTase)were screened from the genome of MEFC1004.On this basis,the core gene moc A and gene cluster moc were determined to be responsible for the biosynthesis of myrothecisin E by knockout of the core gene and analysis of the mutant metabolites.Combined with the functional annotations of other genes in moc cluster,we speculated the biosynthetic pathway of myrothecisin E,which laid a foundation for further elaborating the biosynthetic pathway of this compound.