Layered engineering of sucrose phosphorylase to control substrate tunnels for efficient industrial bioproduction of 2‑O‑α‑D‑glucopyranosyl‑L‑ascorbic acid.

The scale-up industrial production of functional glucosides via enzyme-driven biocatalysis continues to face several critical challenges, including the scarcity of robust enzymes, unclear activity regulation mechanisms, and suboptimal compatibility with industrial requirements. Here, a layered mutation strategy was employed to rationally engineer the substrate tunnel of sucrose phosphorylase (SPase), achieving the efficient industrial-scale biomanufacturing of 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G). Positive mutants, namely S147L (distal layer), Y237F (intermediary layer), V233L (intermediary layer), and A84G (proximal layer) were determined and provided the foundation for constructing multipoint mutants. Further, the combined mutant S147L/Y237F/V233L/A84G (M4-1) exhibited a broadened and shortened substrate tunnel. Strikingly, the layered regulation mechanism was revealed: flexible distal layer, small intermediary layer, and constricted proximal layer collectively formed wider and shorter substrate tunnel. In the 1000 L bioreactor, about 407.3 g/L AA-2G was produced via M4-1 catalysis, which was 273.4 g/L higher than that of wild-type (WT) SPase. This study provides a deep analysis of how the spatially layered engineering of amino acid residues in SPase regulates its enzymatic activity, offering crucial insights for subsequent research on the regulated mechanism of SPase activity.