Cryptic Methylation Enables Amino-Maleimide Formation in Ligiamycin Biosynthesis

Abstract Maleimide is a rare but powerful pharmacophore in natural products, yet its biosynthetic logic remains poorly understood. Here, we elucidate the biosynthesis of ligiamycins, bacterial decalin–amino-maleimide metabolites from Streptomyces sp. NA07271. Genetic and biochemical studies revealed an unusual dispersed PKS–NRPS pathway, in which LigO1 hydroxylates a PCP-tethered precursor, followed by LigQ-mediated Dieckmann cyclization and LigN-catalyzed Diels–Alder cyclization to construct the tetramate-containing trans-decalin scaffold. Unexpectedly, LigM catalyzes a cryptic C-2 O-methylation that is not retained in the isolated products, but instead primes the scaffold for LigO2-mediated oxidation to a highly electrophilic methoxy-maleimide species. This reactive intermediate is readily intercepted by amine nucleophiles through nonenzymatic addition–elimination, giving rise to diverse amino-maleimide congeners. These findings reveal transient methylation as a biosynthetic activation strategy, redefine the chemical end point of ligiamycin biosynthesis, and establish methoxy-maleimide as a promising reactive functional group for natural product diversification and bioconjugation.