Genome-Guided Characterization of Weissella Species Highlights Strain-Specific Functional Traits Relevant to Food Fermentation and Probiotics

The genus Weissella comprises heterofermentative lactic acid bacteria widely distributed across fermented foods, environmental niches, and host-associated habitats, yet the genomic basis underlying their functional diversity and evolutionary plasticity remains incompletely resolved. Here, we performed a large-scale comparative genomic analysis of 347 high-quality genomes representing 16 Weissella species to characterize pan-genome architecture, phylogenomic structure, and the distribution of functional traits relevant to fermentation performance, safety, and probiotic potential. Pan-genome modeling revealed a strongly open pan-genome (P = 3419.89*N0.455), dominated by shell and cloud genes, indicating extensive genomic plasticity and ongoing gene acquisition. Average amino acid identity analysis delineated clear species boundaries while highlighting close evolutionary relationships among several taxa. Functional profiling demonstrated conserved carbohydrate-active enzyme repertoires dominated by glycoside hydrolases and glycosyltransferases, accompanied by species-specific variation in carbohydrate-binding modules. Comparative synteny analysis resolved six major architectures of exopolysaccharide biosynthesis loci, with W. cibaria enriched in regulator-rich and potentially active operons. In silico safety screening detected antimicrobial resistance genes in only three strains and no virulence-associated genes across the dataset, supporting a generally low-risk genomic profile. Probiotic-associated traits, including vitamin biosynthesis and stress resistance, were broadly conserved, whereas adhesion, {gamma}-aminobutyric acid biosynthesis, and secondary metabolite gene clusters were rare and strain-specific. Collectively, these results provide a systems-level view of genome diversification within the Weissella genus and establish a genome-guided framework for rational strain selection in food, health, and biotechnological applications. ImportanceMicrobial traits relevant to food fermentation, probiotic performance, and safety often emerge from complex interactions among core metabolism, accessory genes, and genome plasticity, yet these relationships remain poorly resolved at the genus scale. By analyzing 347 high-quality genomes spanning 16 Weissella species, this study reveals how an open pan-genome, extensive accessory gene diversity, and lineage-specific gene architectures collectively shape functional potential across this important lactic acid bacterial genus. We demonstrate that key traits, including exopolysaccharide biosynthesis, carbohydrate utilization capacity, stress resilience, and biosynthetic gene cluster distribution, are unevenly structured across species and strains, highlighting the necessity of genome-guided strain selection rather than taxonomic assumptions. The integration of phylogenomic relationships, gene content variation, and functional profiling provides a systems-level framework for linking genome evolution with applied phenotypes. These findings advance our understanding of how microbial genomic diversity underpins ecological adaptation and biotechnological performance and provide a scalable blueprint for rational selection of safe and functionally optimized strains in food and health applications.