Characterization of Dichloroisoeverninic Acid Biosynthesis and Chemoenzymatic Synthesis of New Orthosomycins

The orthosomycins are highly modified oligosaccharide natural products with a broad spectrum and potent antimicrobial activities. These include everninomicins and avilamycins, which inhibit protein translation by binding a unique site on the bacterial ribosome. Notably, ribosomal bound structures reveal a network of interactions between the 50S subunit and dichloroisoeverninic acid (DCIE), the aromatic A1-ring conserved across orthosomycins, but the relationship of these interactions to their antimicrobial activity remains undetermined. Genetic functional analysis of three genes putatively associated with DCIE biosynthesis in the everninomicin producer Micromonospora carbonacea delineates the native biosynthetic pathway and provides previously unreported advanced biosynthetic intermediates. Subsequent in vitro biochemical analyses demonstrate the complete DCIE biosynthetic pathway and provide access to novel everninomicin analogs. In addition to the orsellinate synthase EvdD3 and a flavin-dependent halogenase EvdD2, our results identified a key acyltransferase, EvdD1, responsible for transferring orsellinate from the acyl carrier protein domain of EvdD3 to a heptasaccharide orthosomycin biosynthetic intermediate. We have also shown that EvdD1 is able to transfer unnatural aryl groups via their N-acyl cysteamine thioesters to the everninomicin scaffold and used this as a biocatalyst to generate a panel of unnatural aryl analogs. The impact of diverse aryl functional group substitution on both ribosome inhibition and antibacterial activities demonstrates the importance of the DCIE moiety in the pharmacology of orthosomycins, notably revealing an uncoupling between ribosomal engagement and antibiotic activity. Control of A1-ring functionality in this class of molecules provides a potential handle to explore and address pharmacological roles of the DCIE ring in this potent and unique class of antibiotics.