Sequence diversity of gut microbial enzymes reveals strain-level associations with obesity

Background: Obesity is a complex, multifactorial condition increasingly associated with alterations in the gut microbiome. However, large-scale studies integrating taxonomic, functional, and strain-level information remain limited. Methods: We analyzed 1,543 publicly available human fecal whole-genome shotgun metagenomes from 11 independent cohorts, classifying individuals as normal-weight, overweight, or obese according to body mass index. Taxonomic and enzymatic functional profiling were integrated with multivariate analyses, correlation testing, Random Forest-based feature ranking, taxonomic back-tracing, and comparative analysis of operational protein sequence clusters. Results: Increasing body mass index (BMI) was associated with gradual changes in the relative abundance of specific microbial taxa, including enrichment of Clostridium fessum, Prevotella pectinovora, and Segatella copri. Functional profiling identified enrichment of genes involved in branched-chain amino acid catabolism, aminoacyl-tRNA-mediated biosynthesis, nucleotide metabolism, and lipid biosynthesis, together with depletion of functions related to short-chain fatty acid production, energy metabolism, and vitamin biosynthesis. Taxonomic back-tracing indicated that a limited subset of microbial taxa accounted for a substantial proportion of the reads assigned to BMI-associated enzymatic functions. Analysis of three selected enzymes further revealed differences in the richness, prevalence, and distribution of operational protein sequence clusters across BMI categories. Conclusion: Obesity-associated gut microbiome variation involves not only species-level compositional differences but also changes in microbial functional potential and strain-level enzyme sequence-cluster distribution. These findings identify candidate microbial functions and protein sequence clusters for future functional and experimental validation.