Covalent Warhead Assembly in Fostriecin Biosynthesis: Malonylation-Lactonisation by a Bifunctional Thioesterase and Enzymatic Demalonylation

α,β-Unsaturated δ-lactones (AUDLs) are key pharmacophores found in various reduced polyketides exhibiting potent biological activity. Fostriecin has attracted interest as an anticancer agent, but its structural characteristics have limited its development and motivated investigations into biosynthesis-based production strategies. In this study, we reconstitute the enzymatic steps responsible for AUDL formation in fostriecin biosynthesis. We demonstrate that the terminal polyketide synthase (PKS) module FosMod8 produces a 3-O-malonylthioester intermediate using the unusual bifunctional thioesterase FosTE, which catalyses O-malonylation and subsequent lactonisation. Structural modelling revealed two arginines mediating malonyl-CoA binding and transesterification analogously to PKS-acyltransferases. Backbone phosphorylation by the kinase FosH primes the compound for site-specific demalonylation by FosM. This highly regulated pathway ensures optimised formation of bioactive phosphorylated AUDL metabolites and represents a blueprint for others, like cytostatin or phoslactomycin. Understanding this unique enzymatic logic will support new synthetic strategies to AUDL polyketides using chemoenzymatic synthesis or engineered biosynthesis.