Controlled polymerization of dextran from sucrose via SpyTag/SpyCatcher-assembled enzyme cascades

Inspired by natural metabolons, spatial organization of enzymes into complexes is a promising route to enhance cascade catalysis, yet precise control over assembly, efficiency, and product specificity remains challenging. This study presents a systematic comparison between intracellular gene fusion and modular SpyTag/SpyCatcher-drivenin vitroassembly for one-pot enzymatic conversion of sucrose into dextran. The in vitro-assembled complex exhibited significantly enhanced performance, achieving 97.7% conversion of 200 mM sucrose within 7 h with a 41.7% higher reaction rate than the free enzyme mixture, while yielding low-molecular-weight dextran with narrow dispersity. This improvement was driven by efficient substrate channeling, with an intermediate transfer efficiency of 62.2%, far exceeding the 2.6% observed in the free system. By modulating assembly stoichiometry and implementing a thermal activity switch, we further achieved controlled synthesis of high-molecular-weight dextran with uniform chain length. This work provides a programmable and versatile platform for enzyme spatial organization, offering a robust biocatalytic tool with strong potential for biomanufacturing applications that require precise regulation of polymerization-hydrolysis equilibria in renewable biopolymer production.