Mechanistic Insights Into the Biosynthesis of Pseurotin A in <i>Aspergillus fumigatus</i>

The biosynthesis of pseurotin A, a fungal metabolite with antibiotic and immunosuppressive properties, involves a group of enzymes, including a polyketide‐nonribosomal peptide synthase (PKS‐NRPS), PsoA, and a bifunctional enzyme, PsoF. PsoA consists of a highly reducing polyketide synthase (HR‐PKS) and a single nonribosomal peptide synthase (NRPS) module. However, little is known about the molecular mechanism of the PsoA and PsoF in the biosynthesis of pseurotin A. Here, we reported the cryo‐EM structures of the PKS module of PsoA from Aspergillus fumigatus ( A. fumigatus ) in the apo state and in complex with the cofactors, revealing a conserved PKS architecture similar to that of mammalian fatty acid synthase. Interestingly, a flexible motif in the active site of the ketosynthase (KS) domain was identified to govern the production of target compounds by modulating the pocket size of the active site. We also found the direct interaction between PsoA and PsoF and mapped their contact regions. Our findings provide novel insights into the biosynthesis of pseurotin A and the molecular mechanism of PKS‐NRPS enzymes.