Engineering of β-glucosidase for rare ginsenoside biosynthesis via distal conformational fine-tuning and sequential degradation control

The structural complexity and regiospecificity of ginsenosides pose significant challenges for precise biocatalysis, necessitating enzymes with finely tuned conformational control. Here, we report the high-level biomanufacturing of the rare ginsenoside F₂ via computational redesign of β-glucosidase BglPp from Paenibacillus phyllosphaerae. By integrating evolutionary fitness landscapes with thermodynamic stability gradients, we identified a synergistic double mutant, Q7D/G189A, with catalytic efficiency 2.3-fold that of the wild type, achieving an unprecedented F₂ titer of 18.01  g/L in a 5-L bioreactor. Mechanistic analyses revealed that distal mutations induce long-range allosteric effects, remodeling the active site and converting substrate binding from nonspecific hydrophobic interactions to a precise “molecular tweezers” mode, stabilized by an enhanced hydrogen-bond network that lowers the activation energy for glycosidic cleavage. Moreover, we uncovered a sequential degradation mechanism governing regioselectivity, which constrains substrate rotational freedom and directs an orderly conversion from ginsenoside Rb1 to F₂ by partitioning catalytic trajectories. This work establishes a robust platform for industrial-scale synthesis of rare ginsenosides and provides a generalizable framework for rational engineering of complex carbohydrate-active enzymes in synthetic biology.