Structural Characterization and Multiomics Analysis Reveal Extensive Diversity and Global Distribution of Kurstakin Lipopeptides

<i>Bacillus</i> species, particularly those investigated as biocontrol agents, are known to produce a cocktail of bioactive lipopeptides that act synergistically to shape the ecological function of these beneficial microbes. However, while certain families of lipopeptides are well-characterized, others remain elusive. Herein, we describe the characterization of the kurstakins, a family of lipopeptides associated with promising biocontrol properties but that lack adequate characterization. Metabolomic analyses of a semipurified <i>Bacillus cereus</i> EM195W extract fraction revealed the presence of approximately 50 cyclic- and linear-peptide analogs. Deeper analyses revealed that the chemical diversity stems from the diverse lipid tails, including linear, <i>iso</i>-, and <i>anteiso</i>-lipid tails ranging from C₈-C₁₈, along with several hydroxylated lipid tails. Isolation and complete structural analysis of two new analogs represented the first kurstakin analogs characterized by NMR and provided the first experimental analyses for deducing their absolute configuration. Finally, analysis of publicly available genomic and MS data provided insights into the true chemical diversity and distribution of the kurstakins. These results expand our understanding of this family of compounds, opening the door for determining their ecological functions and the role they play in the broader activity of biocontrol agents.