A Conserved Nonribosomal Peptide Synthetase in Produces Citrulline-Functionalized Lipopeptides

The entomopathogenic bacterium exists in a mutualistic relationship with nematodes of the genus . Free-living infective juveniles of prey on insect larvae and regurgitate within the hemocoel of a host larva. subsequently produces a complex array of specialized metabolites and effector proteins that kill the insect and regulate various aspects of the trilateral symbiosis. While species are rich producers of secondary metabolites, many of their biosynthetic gene clusters remain uncharacterized. Here, we describe a nonribosomal peptide synthetase (NRPS) identified through comparative genomics analysis that is widely conserved in species. Heterologous expression of this NRPS gene from in led to the discovery of a family of lipo-tripeptides that chromatographically appear as pairs, containing either a C-terminal carboxylic acid or carboxamide. Coexpression of the NRPS with the leupeptin protease inhibitor pathway enhanced production, facilitating isolation and characterization efforts. The new lipo-tripeptides were also detected in wild-type cultures. These metabolites, termed bovienimides, share an uncommon C-terminal d-citrulline residue. The NRPS lacked a dedicated chain termination domain, resulting in product diversification and release from the assembly line through reactions with ammonia, water, or exogenous alcohols.