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

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.