Integrated genome and metabolome mining unveiled structure and biosynthesis of novel lipopeptides from a deep‐sea Rhodococcus

Abstract Microbial biosurfactants have garnered significant interest from industry due to their lower toxicity, biodegradability, activity at lower concentrations and higher resistance compared to synthetic surfactants. The deep‐sea Rhodococcus sp. I2R has been identified as a producer of glycolipid biosurfactants, specifically succinoyl trehalolipids, which exhibit antiviral activity. However, genome mining of this bacterium has revealed a still unexplored repertoire of biosurfactants. The microbial genome was found to host five non‐ribosomal peptide synthetase (NRPS) gene clusters containing starter condensation domains that direct lipopeptide biosynthesis. Genomics and mass spectrometry (MS)‐based metabolomics enabled the linking of two NRPS gene clusters to the corresponding lipopeptide families, leading to the identification of 20 new cyclolipopeptides, designated as rhodoheptins, and 33 new glycolipopeptides, designated as rhodamides. An integrated in silico gene cluster and high‐resolution MS/MS data analysis allowed us to elucidate the planar structure, inference of stereochemistry and reconstruction of the biosynthesis of rhodoheptins and rhodamides. Rhodoheptins are cyclic heptapeptides where the N‐terminus is bonded to a β‐hydroxy fatty acid forming a macrolactone ring with the C‐terminal amino acid residue. Rhodamides are linear 14‐mer glycolipopeptides with a serine‐ and alanine‐rich peptide backbone, featuring a distinctive pattern of acetylation, glycosylation and succinylation. These molecules exhibited biosurfactant activity in the oil‐spreading assay and showed moderate antiproliferative effects against human A375 melanoma cells.