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

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 <i>in vitro</i> and <i>in vivo</i>. For the <i>in vivo</i> utilization of cellobiose in <i>Escherichia coli</i>, 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 <i>Alteromonadales bacterium</i> 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 <i>k</i>_cat/<i>K</i>_m compared to AtBgl1. Structural analysis and molecular dynamics simulations provided insights into the molecular mechanisms underlying this enhanced performance. Finally, <i>E. coli</i> was given the ability to metabolize cellobiose, and the <i>E.</i> 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 <i>in vivo</i> cellobiose metabolism in <i>E. coli</i> metabolic engineering.