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.