Genome-mining-guided discovery of coumarubrin: A novel aminocoumarin-substituted rubromycin antibiotic

Rubromycins are bacterial aromatic polyketides containing a hallmark spiroketal pharmacophore produced by type II polyketide synthases and accessory enzymes. They generally display cytotoxic and antimicrobial properties, frequently disrupting cellular processes and proteins associated with nucleic acids, such as DNA helicase or telomerase. Among the known rubromycin congeners, hyaluromycin stands out due to a 2-amino-3-hydroxycyclopent-2-enone (C5N) substitution that is presumably installed by an amide bond synthetase (ABS). Here, we used bioinformatic analysis to identify uncharacterized biosynthetic gene clusters and potential rubromycin producer strains encoding putative ABSs but lacking the enzymes responsible for C5N formation, suggesting potentially novel substituents. One of these strains, Lentzea tibetensis, was successfully cultivated and confirmed to produce a previously undescribed aminocoumarin-substituted rubromycin polyketide, named coumarubrin, as verified by high-resolution mass spectrometry (HRMS) and comprehensive nuclear magnetic resonance (NMR) spectroscopy. Electronic circular dichroism spectroscopy indicates an absolute configuration identical to that of previously characterized rubromycins, while the first bioactivity assays demonstrated potent inhibitory activity against Gram-positive bacteria. One-Sentence Summary: This study reports the discovery of a novel member of the rubromycins, antibiotic and cytotoxic aromatic polyketides produced by Actinobacteria, which is fused to a distinct aminocoumarin moiety.