One-pot tailored synthesis of highly monodisperse β-1,3-glucan from sucrose driven by an in vitro self-assembled dual-enzyme system via spy chemistry

β-1,3-Glucan exhibits a broad spectrum of biological activities. However, its broader application has been constrained by current production methods that are uneconomical, inefficient, and environmentally unfriendly. Here, we developed an efficient bottom-up synthesis system for β-1,3-glucan employing thermostable sucrose phosphorylase from Bifidobacterium adolescentis and β-1,3-glucan phosphorylase from Thermosipho africanus using sucrose as a substrate and generating glucose-1-phosphate as a key intermediate. The optimized free enzyme system achieved a 93.1 % conversion of 150 mM sucrose to β-1,3-glucan in 3 h. Furthermore, a self-assembled dual-enzyme system constructed using genetically fused enzymes that self-assemble via SpyCatcher/SpyTag demonstrated a 97.4 % conversion efficiency from 500 mM sucrose in 6 h—a 4-fold enhancement over the free dual-enzyme system due to the direct channeling of 68.5 % of glucose-1-phosphate. This approach enabled the synthesis of β-1,3-glucan with a tunable average degree of polymerization (DP_n) from 31 to 13 by adjusting the concentrations of glucose primer from 1 to 150 mM. At 1 mM glucose, insoluble β-1,3-glucan (DP_n = 31) with excellent monodispersity (dispersity index = 1.01) was synthesized at a productivity of 13.2 g/L/h. Overall, this study provides an economically viable and environmentally sustainable strategy for the industrial-scale production of β-1,3-glucan from renewable sucrose, facilitating its broader application.