Metagenomic insights into urolithin formation from rambutan rind extract by rat faecal-derived microbiome

Ellagitannins and ellagic acid are microbially converted into urolithins, metabolites associated with antioxidant, anti-inflammatory, and mitochondrial-related activities. Although several human-derived urolithin-producing strains and their associated enzymes have recently been characterised, the diversity of microbial strategies across host systems remains poorly understood. This study investigated urolithin production in the Sprague-Dawley rat faecal-derived microbial communities supplemented with rambutan rind extract, an ellagitannin-rich agricultural by-product containing 35-40% geraniin. Rambutan rind extract supplementation was associated with reduced isobutyric acid levels at study endpoint. Ex vivo anaerobic fermentation of hydrolysed rambutan rind extract (113 µM ellagic acid equivalent) resulted in the formation of urolithin C (9.4 ± 0.6 µM) and Isourolithin A (12.5 ± 0.6 µM) by day 9. Shotgun metagenomics analysis revealed very low relative abundance of Actinobacteria (< 0.009%), despite this phylum encompassing most previously characterised urolithin-producing taxa. Canonical ellagic acid degradation genes and the MetaCyc EA degradation pathway were not detected. Comparative pathway analysis indicated overlap in general metabolic pathways with Ellagibacter isourolithinifaciens DSM 104140^T reflecting shared metabolic frameworks rather than conserved urolithin biosynthetic pathways, with highly divergent homologues (Eadh1, Eadh2, Eadh3, and Ucdh). Together, these findings demonstrate that rambutan rind extract can support urolithin formation in rat faecal-derived microbial consortia and highlight functional associations consistent with alternative or yet-uncharacterised microbial strategies for ellagitannin biotransformation. These findings support a discovery-driven framework for investigating urolithin biotransformation in non-human gut microbiomes using ellagitannin-rich agricultural substrates. KEY POINTS: • Rambutan rind extract supports urolithin formation in rat-derived gut microbiota. • Substrate concentration influences urolithin production under ex vivo conditions. • Rat gut microbiota shows homologues' divergence in urolithin-associated proteins.