Genome Directed Mining Of Meroditerpenoids From Endophytic Fungi Of Paris Polyphylla Var.unnanensis

Endophytic fungi coexist with host plants for a long time and have evolved a unique metabolic mechanism.It is an important way to find active molecules with medicinal value from these metabolites.The endophytic fungi of medicinal plants,come from traditional medicinal plants,are more likely to produce bioactive metabolites.Among them,meroditerpenoids are widely concerned due to their anticancer and antitumor activities.In this paper,polyketide-diterpenoids(PK-DTs)hybrid molecule was used as the target,the gene clusters suitable for our study were selected from 34 endophytic fungi isolated from Paris polyphylla var.yunnanensis,which was collected from Liang Wang mountain of Yunnan Province.After genome sequencing and bioinformatics analysis,and compared with the known biosynthesis pathway of meroditerpenoids,we found that the 19 th gene cluster of one endophytic fungus,Aspergillus versicolor 0312,may be related to the biosynthesis of meroditerpenoids.So,we used heterologous expression strategy to study the natural products of the gene cluster: chevalon E(1)and its oxidized derivatives were identified in the genome of endophytic fungus 0312 from Paris polyphylla var.yunnanensis,and the biosynthetic pathway was successfully reconstructed in the heterologous expression host Aspergillus oryzae NSAR1.We have found two cytochrome P450 monooxygenase,cle2 and cle4,which can transform chevalon E(1)into seven new analogues(2-8),including 7 and 8 that possess a unique five-membered lactone ring,which has never been found in the structures of fungal meroterpenoids,Then,we speculated the metabolic pathway of natural products synthesized about the gene cluster through substrates feeding and other strategies.In addition,a gene cluster similar to chevalon E(1)was found in another endophytic fungus,Aspergillus felis 0260,which also separated from Paris polyphylla var.yunnanensis.It was found that sre3,a terpene cyclase gene encoded by this gene cluster,could accept the same substrate with chevalone E,but produced different cyclization products sartorypyrone D(11),which proved the effectiveness of this strategy for mining of meroditerpenoids.Finally,some compounds(1,2,5-8)in this study synergistically enhance the cytotoxicity of DOX in breast cancer cells.