Construction of a scaffold-mediated dual-enzyme display system in Komagataella phaffii with immobilized cells for...

Scaffold-mediated yeast surface display enhances enzyme-enzyme proximity and substrate channel efficiency through the spatial self-assembly of enzyme molecules, overcoming limitations such as enzyme dispersion and mass transfer in traditional multi-enzyme systems. Despite its potential in biocatalysis, existing scaffold systems face challenges, including large molecular size, instability, and limited flexibility, hindering industrial application. Here, an engineered tetratricopeptide repeat affinity proteins (TRAPs) as the scaffold, was firstly introduced onto the surface of K. phaffii's cell, and its feasibility of proteins self-assembly was validated both in vitro and in vivo. Furthermore, the display efficiency of the TRAP scaffold was enhanced to 92.1%, through optimization of fusion linkers and surface-engineering of the cell wall proteins. We further constructed engineered yeast strains co-displaying xylanase and β-xylosidase, which exhibited better thermal and pH stability compared to free enzymes, and explored the synergistic action of scaffold-mediated dual-enzyme display cells on xylan degradation. Moreover, we co-immobilized this scaffold-mediated dual-enzyme display cells on carriers through enhanced biofilm immobilization performance, showing excellent stability and reusability during consecutive operational batch cycles. This study provides an efficient spatial-organizing strategy for multi-enzyme biocatalysis, and offers technical support for long-lasting, stable continuous catalytic processes in green biomanufacturing.