Abstract
Cotylenins (CNs) are bioactive fungal diterpene glycosides that exhibit stabilizing activity on 14-3-3 protein-protein interactions (PPIs), which has significant therapeutic potential. Although CNs were isolated as early as 1970, their biosynthetic pathway has remained unclear, and their limited supply has hindered further research. Here, we report the identification of the biosynthetic gene cluster cty and elucidation of the biosynthetic pathway of CNs. Our investigation reveals the roles of glycosyltransferase, methyltransferase, and prenyltransferase enzymes in the assembly and modification of the saccharide moiety, as well as the multifunctional oxidation activity of the P450 enzyme CtyA. We leveraged this knowledge to achieve the total biosynthesis of not only key intermediates such as CN-C, E, F, and I, but also a novel, unnatural CN derivative using heterologous expression. This showcases the potential of pathway enzymes as catalytic tools to expand the structural diversity of diterpene glycosides. Furthermore, the stabilization effects of pathway intermediates on 14-3-3 PPIs underscore the importance of saccharide modifications in bioactivity. These findings provide a foundation for future rational synthesis of cotylenin A and other structurally diverse derivatives, broadening the scope of diterpene glycoside production.