Uncovering the Molecular Landscape of Tetracycline Family Natural Products through Bacterial Genome Mining

Tetracycline (TC) family natural products have attracted significant attention due to their diverse chemical structures and important role in drug development. As one of the most successful classes of drugs, TC antibiotics have been used clinically for over 70 years and remain crucial in treating infections. Despite their importance, systematic exploration of novel TC natural products has been limited, leaving the molecular landscape of the TC family poorly understood and hindering further development of these compounds for therapeutic applications. Here, we developed a targeted strategy to identify TC biosynthetic gene clusters (BGCs) based on specific cyclase signatures involved in assembling the TC scaffold. This led to the discovery of 82 representative BGCs with the potential to produce structurally diverse TCs. Among them, we uncovered three groups of novel natural products─misiomycins, varsomycins, and hibarimicins J-L─and identified their biosynthetic pathways. These compounds display distinctive structural features, with misiomycin A and hibarimicin L among the most highly modified TCs identified to date. Misiomycin A biosynthesis involves extensive glycosylation, while biosynthesis of varsomycin A, featuring a unique six-membered lactone ring structure, requires the coordinated action of two TC BGCs. The biosynthesis of hibarimicins J-L, derived from TC monomer dimerization, undergoes complex oxidative modifications involving seven oxygenases. Several TCs exhibited potent activity against drug-resistant Gram-positive pathogens. Our work further expands the structural diversity within the TC family and underscores the potential of these BGCs for generating new TC structures, providing valuable insights for the discovery and development of novel TC-based therapeutics.