Total Biosynthesis of Cotylenin Diterpene Glycosides

Cotylenins (CNs) are bioactive fungal diterpene glycosides that exhibits stabilizing activity on 14-3-3 protein-protein interactions (PPIs), which has significant therapeutic potential. Despite its importance, the biosynthetic pathway of CNs have remained unclear. Here, we report the identification of the biosynthetic gene cluster and elucidation of the biosynthetic pathway of CNs. Our investigation reveals the roles of glycosyltransferase, methyltransferase, and prenyltransferase enzymes in the assemble and modification of the glycoside moiety, as well as the multifunctional oxidation activity of the P450 enzyme CtyA. We also demonstrate the synthesis of an active unnatural CN derivative through combinatorial biosynthesis, showcasing the potential of pathway enzymes as catalytic tools to expand the structural diversity of the diterpene glycosides. Additionally, we highlight the stabilization effects of pathway intermediates on 14-3-3 PPIs, providing insights into the evolutionary optimization of bioactivity within the biosynthetic pathway. These findings pave the way for future efforts to achieve the production Cotylenin A and related compounds with enhanced bioactivity through synthetic biology or semisynthesis.