Bottromycins represent a promising class of antibiotics binding to the therapeutically unexploited A-site of the bacterial ribosome. By inhibiting translation they are active against clinically important pathogens, such as vancomycin-resistant Enterococci. Structurally, bottromycins are heavily modified peptides exhibiting various unusual biosynthetic features. To set the stage for compound modification and yield optimization, we identified the biosynthetic gene cluster, used synthetic biotechnology approaches to establish and improve heterologous production, and generated analogs by pathway genetic engineering. We unambiguously identified three radical SAM methyltransferase-encoding genes required for various methylations at unactivated carbons yielding tert-butyl valine, methyl-proline, and β-methyl-phenylalanine residues, plus a gene involved in aspartate methyl-ester formation. Evidence for the formation of the exo-thiazole unit and for a macrocyclodehydration mechanism leading to amidine ring formation is provided.
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