Comparative genomic analysis of the Pediococcus genus reveals functional diversity for fermentation and probiotic applications

<i>Pediococcus</i>, a genus of lactic acid bacteria (LABs), plays key roles in food fermentations yet remains underexplored at the genomic level. This study conducted a comparative genomic analysis of 616 <i>Pediococcus</i> genomes, providing the most comprehensive evaluation of this genus to date. Genome sizes ranged from 0.56 to 2.53 Mbp with distinct GC signatures, while ANI-based clustering and phylogenomic reconstruction resolved clear species boundaries and identified a potential novel lineage within the <i>P. acidilactici</i> complex, supported by 90 % ANI and 37-38 % digital DNA-DNA hybridization (dDDH) relative to the type strains. Pan-genome analysis identified 34,011 orthologous clusters but only 32 core genes, confirming an open pan-genome and remarkable genomic flexibility. In addition, safety assessment showed a low prevalence of antimicrobial resistance genes (6.66 %) and absence of classical virulence determinants, supporting the overall safety of the genus. Functional annotation revealed a diverse arsenal of bacteriocins and secondary metabolite clusters, with <i>P. pentosaceus</i> carrying the richest repertoire, including pediocin and penocin A. Carbohydrate-active enzymes (CAZymes), dominated by glycoside hydrolases (GH) and glycosyltransferases (GT), reflected adaptation to carbohydrate-rich niches. Importantly, probiotic-associated traits were broadly distributed but species-specific. <i>P. acidilactici</i> emerged as the most versatile, harboring genes for adhesion, acid-bile tolerance, oxidative stress defense, vitamin biosynthesis, and γ-aminobutyric acid (GABA) metabolism. These findings establish <i>Pediococcus</i> as a genus with low pathogenic risk, strong probiotic potential, and broad functional diversity. This genomic framework provides valuable guidance for the rational selection of strains in food fermentations, probiotic development, and biotechnological innovation.