The functional roles of gut microbiota in carnivores remain poorly understood. Here, we integrated metagenomics, metabolomics, proteomics and culture-based functional assays to characterize metabolic potential of gut microbiota across 14 captive Felidae species. Comparative metagenomics analysis revealed that the Felidae gut microbiome is distinct from that of non-Felidae and reflects carnivorous dietary patterns. Genus-level core microbiota were dominated by Clostridium , Collinsella and Bacteroides , with functional enrichment in carbohydrate and amino acid metabolism. Of 219 reconstructed metagenome-assembled genomes (MAGs), 27 were identified as core MAGs containing proteases- and lipases- encoding genes, with ATP-dependent Clp proteases predominating and enriched KEGG orthologs mainly associated with amino acid metabolism. Fecal metabolomics identified 1316 metabolites shared among Felidae species, with KEGG analysis showing they were involved in amino acid and lipid metabolism and significantly enriched in protein digestion and absorption pathway. The amino acid- and lipid-related metabolites were correlated with the relative abundance of core MAGs. Culture-based assays revealed proteolytic and lipolytic activities across isolates, supported by proteomics evidence of predominant ATP-dependent proteases. In vitro fermentation with representative isolates generated fatty-acid-dominated metabolites consistent with fecal metabolomic profiles. Together, our findings demonstrate that Felidae gut microbiota play a critical role in amino acid metabolism for carnivory.
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