Characterization of a Fungal Modular Polyketide Synthase System Reveals an Unusual Strategy for Polyketide Chain Initiation

ABSTRACT Polyketides constitute a major class of natural products with remarkable structural diversity and biological activities. While bacteria predominantly produce polyketides via modular type I polyketide synthases (PKSs), eukaryotic modular PKSs remain largely unexplored. Here, we report the functional characterization of a fungal modular PKS system from Lasiodiplodia theobromae through heterologous expression in Aspergillus oryzae , leading to the production of the antimalarial macrolide strasseriolide C ( 1 ). Intriguingly, biosynthesis of 1 involves C ‐methylation of the starter malonyl unit despite the absence of a methyltransferase domain in the loading module. Furthermore, module 2 of the PKS system contains an unusual arrangement of four consecutive acyl carrier protein (ACP) domains. Although the catalytic basis of this methylation remains unresolved, our isotope‐labeling experiments, mutational analyses, and in vitro biochemical assays support a model in which the malonyl unit loaded onto the module 2 ACPs is transferred to the loading ACP, where starter‐unit methylation is proposed to occur prior to chain initiation. Our study reveals unexpected biosynthetic logic and establishes a foundation for mechanistic studies and engineering of eukaryotic modular PKSs.