Methyltransferase Contingencies In The Pathway Of Everninomicin D Antibiotics And Analogues

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation withEscherichia colito manipulateMicromonosporafor targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A(1), C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. Thein vitroactivity of A(1)- and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A(1)-ring phenol and H-ring C-4' hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis andin vitromodification of advanced biosynthetic intermediates.