Construction of a Self‐Assembled Multi‐Enzyme Cascade for Efficient <scp>D</scp> ‐Allulose Biosynthesis

d-Allulose is a valuable low-calorie rare sugar with diverse physiological benefits. Although phosphorylation-dephosphorylation-based multi-enzyme cascades enable efficient d-allulose biosynthesis, the free-enzyme format limits substrate channeling and promotes intermediate diffusion, resulting in byproduct accumulation and reduced cascade efficiency. In this study, peptide-mediated assembly strategies were employed to construct a dual-enzyme complex using d-allulose 6-phosphate epimerase (A6PE) and d-allulose 6-phosphate phosphatase (A6PP) as model enzymes, to mitigate reversible epimerization. Among them, the ReverseTag/ReverseCatcher system was selected due to its positive impact on enzyme activity, as evidenced by the 2.1‑fold and 27.5% increases in activity observed for RCA6PE and RTA6PP, respectively. Successful complex assembly was confirmed by dynamic light scattering and transmission electron microscopy. A five-enzyme complex (RFE) was further constructed by integrating α-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, A6PE, and A6PP to spatially organize an artificial in vitro d-allulose biosynthetic pathway. With 10 g/L maltodextrin as the substrate, the RFE system achieved a d-allulose yield of 63.1%, representing a 37.2% increase over the free-enzyme system. These findings demonstrate that ReverseTag/ReverseCatcher-mediated covalent assembly improves multi-enzyme cascade efficiency and provides a modular platform for engineering artificial in vitro biosynthetic systems.