Biosynthesis of Albomycin δ₂ Provides a Template for Assembling Siderophore and Aminoacyl-tRNA Synthetase Inhibitor Conjugates

“Trojan horse” antibiotic albomycins are peptidyl nucleosides consisting of a highly modified 4′-thiofuranosyl cytosine moiety and a ferrichrome siderophore that are linked by a peptide bond <i>via</i> a serine residue. While the latter component serves to sequester iron from the environment, the seryl nucleoside portion is a potent inhibitor of bacterial seryl-tRNA synthetases, resulting in broad-spectrum antimicrobial activities of albomycin δ₂. The isolation of albomycins has revealed this biological activity is optimized only following two unusual cytosine modifications, N4-carbamoylation and N3-methylation. We identified a genetic locus (named <i>abm</i>) for albomycin production in <i>Streptomyces</i> sp. ATCC 700974. Gene deletion and complementation experiments along with bioinformatic analysis suggested 18 genes are responsible for albomycin biosynthesis and resistance, allowing us to propose a potential biosynthetic pathway for installing the novel chemical features. The gene <i>abmI</i>, encoding a putative methyltransferase, was functionally assigned <i>in vitro</i> and shown to modify the N3 of a variety of cytosine-containing nucleosides and antibiotics such as blasticidin S. Furthermore, a Δ<i>abmI</i> mutant was shown to produce the descarbamoyl-desmethyl albomycin analogue, supporting that the N3-methylation occurs before the N4-carbamoylation in the biosynthesis of albomycin δ₂. The combined genetic information was utilized to identify an <i>abm</i>-related locus (named <i>ctj</i>) from the draft genome of <i>Streptomyces</i> sp. C. Cross-complementation experiments and <i>in vitro</i> studies with CtjF, the AbmI homologue, suggest the production of a similar 4′-thiofuranosyl cytosine in this organism. In total, the genetic and biochemical data provide a biosynthetic template for assembling siderophore-inhibitor conjugates and modifying the albomycin scaffold to generate new derivatives