Dissecting tunicamycin biosynthesis by genome mining: cloning and heterologous expression of a minimal gene cluster

Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful activities, little is known about their biosynthesis. Here we report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. These studies unlock a comprehensive and unusual toolbox of biosynthetic machinery with which to create variants of this important natural product, allowing possible improved understanding of the mode of action and facilitating future redesign. We anticipate that these results will enable the generation of altered specific inhibitors of diverse carbohydrate-processing enzymes, including improved targeting of lipid I biosynthesis.