Genomic and functional characterization of carbohydrate-active enzymes from Ruminococcoides bili FMB-CY1 reveals...

Ruminococcoides bili FMB-CY1, a human gut bacterium, exhibits strong resistant starch (RS)-degrading ability. To elucidate its RS-degradation strategy, we performed comprehensive genomic annotation and biochemical characterization of 19 encoded carbohydrate-active enzymes (CAZymes). Genome analysis revealed glycoside hydrolases (GH13, GH31, GH77), glycosyltransferase (GT35), carbohydrate-binding modules (CBMs), and domains associated with amylosome-like multienzyme complexes, including dockerin and cohesin motifs. All 19 CAZyme genes were heterologously expressed in Escherichia coli, and their enzymatic properties were systematically characterized. Most α-amylases exhibited extracellular activity against raw RS granules, particularly those harboring CBMs. Hydrolysis profiling revealed distinct substrate preferences, leading to functional reannotation of four enzymes. Domain analyses further suggested that select CAZymes form a surface-associated complex analogous to the amylosome. Together, these enzymes suggest a putative RS-degradation system, in which extracellular α-amylases initiate starch breakdown and are followed by pullulanases, glucosidases, and transferases that complete RS degradation. The released sugars support microbial cross-feeding and potentially contribute to host energy metabolism. The study provides molecular insight into RS utilization by Rc. bili FMB-CY1 and identifies enzymatic features relevant to gut microbial ecology and functional food applications targeting RS metabolism.