Discovery of late intermediates in methylenomycin biosynthesis active against drug-resistant Gram-positive bacterial pathogens

The methylenomycins are highly functionalized cyclopentanone antibiotics produced byStreptomyces coelicolorA3(2). A biosynthetic pathway to the methylenomycins has been proposed based on sequence analysis of the proteins encoded by the methylenomycin biosynthetic gene cluster and incorporation of labelled precursors. However, the roles played by putative biosynthetic enzymes remain experimentally uninvestigated. Here, the biosynthetic functions of enzymes encoded bymmyD, mmyO, mmyFandmmyEwere investigated by creating in-frame deletions in each gene and investigating the effect on methylenomycin production. No methylenomycin-related metabolites were produced by themmyDmutant, consistent with the proposed role of MmyD in an early biosynthetic step. The production of methylenomycin A, but not methylenomycin C, was abolished in themmyFandmmyOmutants, consistent with the corresponding enzymes catalyzing epoxidation of methylenomycin C, as previously proposed. Expression ofmmyFandmmyOin aS. coelicolorM145 derivative engineered to expressmmr, which confers methylenomycin resistance, enabled the resulting strain to convert methylenomycin C to methylenomycin A, confirming this hypothesis. A novel metabolite (pre-methylenomycin C), which readily cyclizes to form the corresponding butanolide (pre-methylenomycin C lactone), accumulated in themmyEmutant, indicating the corresponding enzyme is involved in introducing the exomethylene group into methylenomycin C. Remarkably, both pre-methylenomycin C and its lactone precursor were one to two orders of magnitude more active against various Gram-positive bacteria, including antibiotic-resistantStaphylococcus aureusandEnterococcus faeciumisolates, than methylenomycins A and C, providing a promising starting point for the development of novel antibiotics to combat antimicrobial resistance.