Data Sheet 1_Comprehensive genomic and metabolomic profiling of Weissella confusa UTNCys2-2 highlights bioactive potential.docx

IntroductionThe genus Weissella comprises a diverse group of lactic acid bacteria (LAB) widely distributed across plant- and food-associated ecosystems and recognized for their functional and technological versatility. Weissella confusa UTNCys2-2, a plant-derived strain isolated from Amazonian spiral ginger (Costus sp.), that produces exopolysaccharides (EPS) with documented antioxidant activity and promising probiotic properties.
MethodsWhole-genome sequencing of UTNCys2-2 was performed to establish its taxonomic assignment, phylogenomic analysis, while genome mining was conducted to evaluate safety, metabolic potential, and biosynthetic capabilities. Carbohydrate-active enzymes (CAZymes), Kyoto Encyclopedia of Genes and Genomes (KEGG), and MetaCyc pathways were analyzed for functional insights. Moreover, the metabolite composition of the cell-free supernatant (CFS) was examined using liquid chromatography–tandem mass spectrometry (LC–MS/MS) combined with Sequential Windowed Acquisition of all Theoretical Fragment Ion Mass Spectra (SWATH-MS).
ResultsThe genome consists of a 2.32 Mb circular chromosome (44.59% GC) encoding 2,194 proteins, 76 tRNAs, and 10 rRNAs, with no plasmids. Phylogenomic analyses assigned the strain to the W. confusa clade, clustering closely with the reference strain DSM 20196. UTNCys2-2 harbors a complete Type II-A CRISPR-Cas system, intact prophages, and mobile elements, while lacking virulence determinants and transferable antimicrobial resistance genes. Functional annotation revealed 118 CAZymes supporting EPS biosynthesis, polysaccharide utilization, and carbohydrate metabolism. KEGG and MetaCyc pathways highlighted glycogen and riboflavin biosynthesis, stress tolerance, and metabolic versatility. Genome mining identified a Type III polyketide synthase (T3PKS) gene cluster with low similarity to known pathways, suggesting potential for novel secondary metabolites. Pangenome analysis showed extensive strain-specific genes linked to carbohydrate metabolism and EPS production. Metabolomic profiling of the CFS detected alkaloids, bioactive peptides, functional carbohydrates, and phenolics, supporting antimicrobial, probiotic, and host-interactive activities.
ConclusionW. confusa UTNCys2-2 represents a biosafe and metabolically versatile strain with strong genomic capacity for EPS production, potential for novel secondary metabolite biosynthesis, and diverse bioactive properties, supporting its applicability in food fermentation, probiotic development, and microbial biotechnology.