Distinctive scaffoldin proteins in Ruminococcoides bili FMB-CY1 and their roles in amylosome.

The gut bacterium Ruminococcoides bili FMB-CY1 employs a specialized mechanism to degrade resistant starch (RS), a pivotal carbohydrate in human gut ecosystem. This study identifies two novel scaffoldin (Sca) proteins, Rcb_Sca3 and Rcb_Sca4, which are phylogenetically distinct from conventional amylosome and cellulosome systems. In RS-rich environments, the expression of these scaffoldins was markedly upregulated, underscoring their role in RS-specific adaptation. Both proteins were found to harbor cohesin (Coh) modules and membrane-bound protein (MBP) domains. Their expression and cell membrane localization, mediated by MBPs, were validated using an eGFP fusion protein. Additionally, the binding affinity between Coh and dockerin (Doc) modules was quantified, and protein-protein interactions were corroborated through computational analysis. These results highlight the critical role of Rcb_Sca3 and Rcb_Sca4 in RS metabolism in Rc. bili FMB-CY1 and their contribution to the structural organization of the amylosome.