Programmable Scaffold-Mediated Assembly Regulation Tool for Dynamic Control of a Multienzyme Biocatalyst

Surface-displayed whole-cell biocatalysis offers significant advantages over traditional intracellular enzyme-based methods but faces challenges in complex multienzyme processes and low efficiency. We present Scaffold-Mediated Assembly Regulation Tool (SMART), a programmable platform for dynamic control of multienzyme biocatalysts on Corynebacterium glutamicum surfaces. SMART integrates orthogonal cross-linked scaffolds with the twin Catcher/Tag pairs (SpyCatcher/SpyTag and SnoopCatcher/SnoopTag), controllable molecular ratios, and high loading capacity, offering user-defined functionality for enzyme assembly. We optimized SMART through computational modeling and experimental validation and created bifunctional scaffolds and long tandem repeats for optimal enzyme loading. The versatility of SMART was demonstrated by codisplaying in a two enzyme sequential cascade with tunable stoichiometry, significantly improving the conversion of maltodextrin to trehalose. Furthermore, we integrated a Bxb1 recombinase-based state machine (RSM) genetic circuit, allowing temporal regulation of enzyme and scaffold expression within a single cell. Finally, the system’s applicability was showcased in isomaltulose production from low-cost sucrose. SMART design represents a significant advancement in surface-displayed biocatalysis, offering a programmable platform for complex multienzyme reactions with potential applications in various industrially relevant biocatalysts.