Comparative Genome Mining and Metabolomics Reveal Divergent NRPS-Derived Fengycin Biosynthetic Gene Clusters in Bacillus halotolerans

Abstract Microbial multidrug resistance is a major public health concern, underscoring the urgent need for new antimicrobial natural products. In this study, strain F11, identified as Bacillus halotolerans, was selected based on its strong antimicrobial activity and taxonomic identification. Whole-genome sequencing revealed a single circular chromosome of 4.15 Mb with a GC content of 43.82%, encoding 4,122 predicted proteins. Pangenome analysis identified 17 unique genes. Genome mining predicted ten biosynthetic gene clusters (BGCs), including a complete fengycin cluster. Comparative analyses using BiG-SCAPE/CORASON and clinker revealed evolutionary divergence within the fengycin BGCs, including those identified in B. halotolerans F11 and B. halotolerans HMB20199. This divergence was further supported by NRPS substrate specificity predictions, which revealed two amino acid variations at positions 6 and 8 in the predicted fengycin decapeptide of strain B. halotolerans F11 compared to the canonical sequence. In contrast, B. halotolerans HMB20199 exhibited a mosaic fengycin–iturin hybrid organization, characterized by an extended NRPS assembly line comprising 19 modules. Furthermore, untargeted metabolomic profiling of B. halotolerans F11 detected 9,719 metabolites, of which 3,453 were successfully annotated. Integration of genomic and metabolomic datasets enabled the correlation of two compounds—bacillaene and bacillibactin—with their corresponding BGCs. However, the lack of detection of fengycin, surfactin, and subtilosin A was attributable to methodological constraints. Collectively, these findings expand our understanding of B. halotolerans strains as promising genomic reservoirs of novel NRPS-derived lipopeptides and highlight Algerian Sahara soils as a valuable source of antimicrobial natural products.