Discovery of Late Intermediates in Methylenomycin Biosynthesis Active against Drug-Resistant Gram-Positive Bacterial Pathogens

The methylenomycins are highly functionalized cyclopentanone antibiotics produced by <i>Streptomyces coelicolor</i> A3(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 the incorporation of labeled precursors. However, the roles played by putative biosynthetic enzymes remain experimentally uninvestigated. Here, the biosynthetic functions of enzymes encoded by <i>mmyD</i>, <i>mmyO</i>, <i>mmyF,</i> and <i>mmyE</i> were investigated by creating in-frame deletions in each gene and investigating the effect on methylenomycin production. No methylenomycin-related metabolites were produced by the <i>mmyD</i> mutant, consistent with the proposed role of MmyD in an early biosynthetic step. The production of methylenomycin A, but not methylenomycin C, was abolished in the <i>mmyF</i> and <i>mmyO</i> mutants, consistent with the corresponding enzymes catalyzing the epoxidation of methylenomycin C, as previously proposed. Expression of <i>mmyF</i> and <i>mmyO</i> in a <i>S. coelicolor</i> M145 derivative engineered to express <i>mmr</i>, which confers methylenomycin resistance, enabled the resulting strain to convert methylenomycin C to methylenomycin A, confirming this hypothesis. A novel metabolite (premethylenomycin C), which readily cyclizes to form the corresponding butanolide (premethylenomycin C lactone), accumulated in the <i>mmyE</i> mutant, indicating the corresponding enzyme is involved in introducing the exomethylene group into methylenomycin C. Remarkably, both premethylenomycin C and its lactone precursor were one to two orders of magnitude more active against various Gram-positive bacteria, including antibiotic-resistant <i>Staphylococcus aureus</i> and <i>Enterococcus faecium</i> isolates, than methylenomycins A and C, providing a promising starting point for the development of novel antibiotics to combat antimicrobial resistance.