A Programmable Bidirectional Dynamic Switch Overcomes Reversible Isomerization Reaction for Efficient d-Allulose Biosynthesis

d-Allulose, biosynthesized through D-fructose isomerization, was limited by thermodynamic equilibrium, resulting in low yields. Herein, we redesigned a redox-driven cascade pathway for d-allulose biosynthesis in single-cell systems, utilizing post-translational protein-level reprogramming tools (protease-based programmable OFF/ON-switch toolbox) to resolve enzyme incompatibility. This OFF/ON-switch was systematically optimized by adjusting the expression levels of mf-Lon, degron, and repressors, enabling the simultaneous switching of protein abundance to desired states within 2 h based on a thermosensitive regulator. To link the redox-driven two-step reactions to synthesize d-allulose, KEase, RDH, and FDH were placed under the control of the OFF-switch, while ADH and NOX were regulated using the ON-switch. The engineered strain T3 with the constructed OFF–ON modules achieved a d-allulose titer of 190.7 g/L with a conversion rate of 95.4%, overcoming the obstacle of thermodynamic equilibrium. This study provided a practical toolbox for rewiring reverse carbon flows by controlling enzyme levels, exemplifying the versatility of programmable protein switches.