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

BglPp Q7D/G189A achieved catalytic efficiency 2.3-fold that of the wild type. • Rare ginsenoside F 2 achieved 18.01  g/L in a 5-L bioreactor. • Activity enhancement and sequential degradation mechanism were investigated. 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 F 2 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 F 2 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 F 2 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. Download: Download high-res image (269KB) Download: Download full-size image