Actinobacteria have been a rich source of novel, structurally complex natural products for many decades. Although the largest genus is Streptomyces, from which the majority of antibiotics in current and past clinical use were originally isolated, other less common genera also have the potential to produce a wealth of novel secondary metabolites. One example is the Kutzneria genus, which currently contains only five reported species. One of these species is Kutzneria albida DSM 43870T, which has 46 predicted biosynthetic gene clusters and is known to produce the macrolide antibiotic aculeximycin. Here, we report the isolation and structural characterization of two novel 30-membered glycosylated macrolides, epemicins A and B, that are structurally related to aculeximycin, from a rare Kutzneria sp. The absolute configuration for all chiral centers in the two compounds is proposed based on extensive 1D and 2D NMR studies and bioinformatics analysis of the gene cluster. Through heterologous expression and genetic inactivation, we have confirmed the link between the biosynthetic gene cluster and the new molecules. These findings show the potential of rare Actinobacteria to produce new, structurally diverse metabolites. Furthermore, the gene inactivation represents the first published report to genetically manipulate a representative of the Kutzneria genus.

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The Gram-negative bacterium Yersinia ruckeri is the causative agent for enteric red mouth disease in salmonids. The genome of Y. ruckeri YRB contains a biosynthetic gene cluster encoding the biosynthesis of catechol siderophores that are diastereomeric with the known vanchrobactin class of siderophores, (DHB D Arg L Ser) (1−3) . Ruckerbactin (1), produced by Y. ruckeri YRB, was found to be the linear tris-L-serine ester composed of L-arginine and 2,3-dihydroxybenzoic acid, (DHB L Arg L Ser) 3 . The biscatechol, (DHB L Arg L Ser) 2 (2), and monocatechol, DHB L Arg L Ser (3), compounds were also isolated and characterized. The macrolactone of ruckerbactin was not detected. The presence of L Arg in ruckerbactin makes it the diastereomer of trivanchrobactin with D Arg. The electronic circular dichroism spectra of Fe(III)− ruckerbactin and Fe(III)−trivanchrobactin reveal the opposite enantiomeric configurations at the Fe(III) sites. Fe(III)− ruckerbactin adopts the Δ configuration, and Fe(III)−trivanchrobactin adopts the Λ configuration. Y. ruckeri YRB was also found to produce the antimicrobial agent holomycin (4).