Self‐Resistance Gene‐Guided Discovery of the Molecular Basis for Biosynthesis of the Fatty Acid Synthase Inhibitor Cerulenin

Cerulenin (1) is the first reported natural fatty acid synthase inhibitor and has been intensively researched for its antifungal, anticancer and anti‐obesity properties. However, the molecular basis for its biosynthesis has remained a mystery for six decades. Here, we have identified the polyketide biosynthetic gene cluster (cer) responsible for the biosynthesis of 1 from two Sarocladium species using a self‐resistance gene mining approach, which we validated via heterologous reconstitution of cer cluster in an Aspergillus nidulans host. Expression of various combinations of cer genes uncovered key pathway intermediates, electrocyclisation products derived from PKS‐encoded polyenoic acids, and a suite of 13 new analogues of 1. This enabled us to establish a biosynthetic pathway to 1 that starts with a C12 polyketide precursor with both E and Z double bonds and involves a complex series of epoxidations, double bond shifts, E/Z isomerisation and epoxide reduction. Using in vitro assays, we further validated the roles of amidotransferase CerD in amidation, and oxidase CerF and reductase CerE in the final two‐electron oxidation and enone reduction steps towards 1. These findings expand our understanding of complex tailoring modifications in highly reducing PKS pathways and pave the way for the engineered biosynthesis of cerulenin analogues.