Comparative genomic analysis of poly-γ-glutamic acid producing Bacillus licheniformis strains isolated from dairy products

Poly-γ-glutamic acid (γ-PGA) is a valuable biopolymer with diverse industrial applications, produced naturally by several Bacillus species. The dairy environment is an under-explored niche for identifying efficient, food-grade γ-PGA producers. In this study, four legacy dairy-derived Bacillus licheniformis strains: DPC3803, DPC6338, DPC6339, and DPC6340, producing high γ-PGA titres were examined using whole genome sequencing (WGS) and comparative genomic analysis to evaluate their suitability for future industrial applications. The genomes ranged from 4.19 to 4.29 Mb with an average GC content of 45.8-46.2%. Pangenome analysis of the four strains, together with 51 publicly available B. licheniformis genomes, identified 12,415 gene clusters, of which 18.9% and 81.1% were core and accessory genes respectively. Average nucleotide identity (ANI) analysis demonstrated >99% sequence identity among all 55B. licheniformis genomes, despite their isolation from diverse environments, indicating strong genomic conservation within the species. Experimental validation confirmed γ-PGA production by all four strains, with maximum titres (g/L) of 43.27 ± 1.49, 59.54 ± 4.33, 27.93 ± 1.87, and 47.74 ± 0.19 for DPC3803, DPC6338, DPC6339, and DPC6340, respectively. Genomic screening revealed multiple γ-PGA metabolism and CAZyme-encoding genes, as well as unique secondary metabolite clusters with potential antimicrobial activity. Although no plasmids or virulence factors were detected, twenty-one prophages were identified, sharing no significant homology with known cultivated phages, and a single β-lactamase gene suggested intrinsic resistance to β-lactams. These findings highlight the genomic and functional potential of these dairy-derived B. licheniformis as efficient, food-grade candidates for industrial γ-PGA production.