Substrate affinity and spatial proximity synergistically guide multi-enzyme architecture rewiring for highly...

N-acetylchitosan oligosaccharides (NACOS) show great promise in food, medicine, and agriculture. The synergistic effect of lytic polysaccharide monooxygenases (LPMOs) and chitinases can overcome kinetic barrier and boost the yield of NACOS from crystalline chitin. However, the enhancement level is highly dependent on the enzyme combination, lacking consistency and predictability. To address this challenge, this study developed a predictable multidimensional framework based on the substrate affinity of carbohydrate-binding modules (CBMs) and the spatial proximity of catalytic domains, guiding the rational design of LPMO-chitinase complex and the tunable enhancement of synergistic effect. The designed artificial fusion CBM₁₁ exhibited good catalytic activity. In the α-chitin and β-chitin systems, compared with chitinase (BcChi2) alone, the addition of LPMO (BcLPMO1_T3) increased the yield of NACOS by only 0.17-fold and 1.97-fold, while fusion CBM₁₁ (a multi-enzyme complex of BcLPMO1_T3 and BcChi2) achieved significant increases of 6.33-fold and 20.16-fold, respectively. The degradation rate of β-chitin by 3 μmol/L fusion CBM₁₁ reached over 85%. In addition, the fusion enzyme exhibited good thermostability and significant tolerance to high concentrations of H₂O₂, further demonstrating the application potential in complex reaction systems. This work presents a rational strategy for optimizing multi-enzyme synergistic systems to efficiently degrade chitin and provides a reference for the development of biocatalysts targeting other insoluble polysaccharides.