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.

SignificanceNatural products biosynthesized by cryptic gene clusters represent a largely untapped source for drug discovery. However, mining of these products by promoter engineering is restricted by the lack of streamlined genetic tools, especially in nonmodel biosynthetic gene cluster (BGC)-rich bacteria. Here, we describe the discovery of a pair of bacteriophage recombinases and application of recombinase-assisted promoter engineering to rapidly identify and activate several cryptic biosynthetic gene clusters in two Burkholderiales strains that currently lack effective genetic tools. Construction of an efficient genome engineering platform in a natural product producer expedites mining of cryptic BGCs in their native backgrounds, and host melioration for yield or structure optimization. This strategy enables potentially scalable discovery of novel metabolites with intriguing bioactivities from many other bacteria.

The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.