Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars

Multienzyme cascades enable the sequential synthesis of complex chemicals by combining multiple catalytic processes in one pot, offering considerable time and cost savings compared to a series of separate batch reactions. However, challenges related to coordination and regulatory interplay among multiple enzymes reduce the catalytic efficiency of such cascades. Herein, we genetically programmed a scaffold framework that selectively and orthogonally recruits enzymes as designed. The system was then used to generate multienzyme complexes of D-allulose 3-epimerase (DAE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) for rare sugar production. This scaffolded multienzymatic assembly achieves a 10.4-fold enhancement in the catalytic performance compared to its unassembled counterparts, obtaining allitol yield of more than 95%. Molecular dynamics simulations revealed that shorter distances between neighboring enzymes in scaffold-mounted complexes facilitated the transfer of reaction intermediates. A dual-module catalytic system incorporating (1) scaffold-bound complexes of DAE, RDH, and FDH and (2) scaffold-bound complexes of alcohol dehydrogenase and NADH oxidase expressed intracellularly in E. coli was used to synthesize D-allulose from D-fructose. This system synthesized 90.6% D-allulose from 300 g L⁻¹ D-fructose, with a space-time yield of 13.6 g L⁻¹ h⁻¹. Our work demonstrates the programmability and versatility of scaffold-based strategies for the advancement of multienzyme cascades.