| dc.description.abstract | Antimicrobial resistance is a critical threat to human health that results in an estimated 100,000 deaths in the United States each year, according to the World Health Organization. This growing crisis demands the development of new classes of antimicrobials with unique mechanisms of action for bacterial inhibition. The orthosomycin class of natural products, which include everninomicin and avilamycin, are complex oligosaccharides with broad and potent activity against gram-positive pathogens, including many antibiotic resistant strains. Importantly, the orthosomycins inhibit protein translation by binding a unique site on the bacterial ribosome, which was recently detailed via crystallography and cryogenic electron microscopy. The structures reveal crucial interactions between the 50S subunit and the extended orthosomycin structure. Targeted gene deletions of four putative methyltransferases in the everninomicin gene cluster allowed us to assign the function of all ten methyltransferases and to generate twelve novel everninomicin analogs. These results also confirm the mutability of the everninomicin biosynthetic pathway for future analog generation with alterations across the oligosaccharide scaffold. Subsequent to this work, we decided to interrogate the biosynthesis of dichloroisoeverninic acid (DCIE), an aromatic moiety conserved across orthosomycins that forms discrete interactions with the 50S ribosome. Due to the difficulty in accessing analogs via synthetic methods, we sought to utilize the biosynthetic apparatus to develop designer oligosaccharide analogs to probe the structure-activity relationship at this unique ribosomal binding site. We performed targeted deletion of four genes putatively associated with DCIE biosynthesis in the everninomicin producer Micromonospora carbonacea. The genetic functional analysis confirmed vital DCIE biosynthetic steps and provided access to three novel everninomicin metabolites. Our results identified a key KAS III-like acyltransferase, EvdD1, responsible for attaching the aromatic ring to the larger oligosaccharide scaffold. We have also shown that EvdD1 is capable of transferring customized non-natural aromatic moieties to the everninomicin scaffold, with current work focused on probing the interactions of these non-natural ring systems with the ribosome via bioactivity assays. By further exploring the role of DCIE in the orthosomycins’ mechanism of action, we can propel this complex class of molecules forward as a human therapeutic for the treatment of antimicrobial resistant infections. | |
