Biosensor-Assisted Evolution of a β-Glucosidase for Enzymatic Robustness and In Vivo Cellobiose Metabolism in Escherichia coli

β-Glucosidase is one of the essential components of the enzyme cocktail required for the degradation of lignocellulose, which catalyzes the conversion of cellobiose into glucose both in vitro and in vivo. For the in vivo utilization of cellobiose in Escherichia coli, it is crucial to express and regulate a BGL optimally with enhanced enzymatic properties. This study characterizes the enzymatic properties of a BGL named AtBgl1, derived from Alteromonadales bacterium TW-7. A cellobiose biosensor, Cbio3-R-R3, was constructed and optimized, showing a 2.27-fold increase in sensitivity compared to Cbio1. A mutant library was constructed through two rounds of error-prone mutagenesis. Using cellobiose-based screening plates and a biosensor, we identified the BGL mutant strain M5, which showed a 2.13-fold increase in kcat/Km compared to AtBgl1. Structural analysis and molecular dynamics simulations provided insights into the molecular mechanisms underlying this enhanced performance. Finally, E. coli was given the ability to metabolize cellobiose, and the E. coli BL21-M5 showed remarkable improvements in metabolic efficiency, achieving an 88.4% cellobiose utilization rate within 48 h. This study provides valuable strategies and insights for the biosensor-assisted directed evolution of BGL, enhancing its enzymatic robustness and facilitating in vivo cellobiose metabolism in E. coli metabolic engineering.