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 (LAB) widely distributed in fermented foods, plant-associated environments, and host-associated niches. However, the functional diversity, safety-related genomic features, and evolutionary dynamics of the genus remain incompletely understood at the genomic level. This study performed a comprehensive comparative genomic analysis of 347 high-quality Weissella genomes representing 16 assigned species to elucidate their genomic diversity, pan-genome architecture, and functional potential relevant to food fermentation, probiotics, and biotechnology. Pan-genome analysis revealed an open pan-genome following a power-law model with an exponent of 0.304, dominated by shell and cloud genes, indicating substantial genomic plasticity and ongoing gene acquisition. In silico safety screening showed that the vast majority of genomes lacked detectable antimicrobial resistance or virulence-associated genes under the applied criteria, indicating a low detectable burden of these genetic determinants across the data set. Moreover, functional annotation revealed conserved carbohydrate-active enzyme (CAZyme) repertoires dominated by glycoside hydrolases (GHs) and glycosyltransferases (GTs), alongside species-specific variation in carbohydrate-binding modules (CBMs). Comparative synteny analysis identified six distinct architectures of exopolysaccharide (EPS) biosynthesis loci, with W. cibaria more frequently harboring regulator-rich, putatively complete EPS gene clusters. Putative probiotic-associated genetic markers were unevenly distributed across species, mainly involving stress resistance, vitamin biosynthesis, GIT tolerance, and oxidative stress resistance, whereas GABA production and antimicrobial metabolite-associated markers were not detected in the data set. Hence, this genome-resolved analysis provides a systematic framework for understanding Weissella diversity and supports a genome-guided, strain-level approach for prioritizing candidate strains for future experimental validation in food, health, and biotechnological applications. IMPORTANCE Microbial traits relevant to food fermentation, probiotic performance, and safety emerge from complex interactions among core metabolism, accessory genes, and genome plasticity; however, 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 shape functional potential across this lactic acid bacterial genus. Key traits, including EPS biosynthesis, carbohydrate utilization capacity, stress resilience, and biosynthetic gene cluster distribution, are unevenly structured across species and strains, emphasizing the need for genome-guided strain selection rather than taxonomic assumptions. Integrating phylogenomics, gene content variation, and functional profiling links genome evolution with applied phenotypes. These findings advance understanding of how genomic diversity underpins ecological adaptation and biotechnological performance and provide a blueprint for selecting safe and functionally optimized strains in food and health applications.