Naturally, haloacid dehalogenase superfamily phosphatases have been evolved with broad substrate promiscuity; however, strong specificity to a particular substrate is required for developing thermodynamically driven routes for manufacturing sugars. How to alter the intrinsic substrate promiscuity of phosphatases and fit the “one enzyme-one substrate” model remains a challenge. Herein, we report the structure-guided engineering of a phosphatase, and successfully provide variants with tailor-made preference for three widespread phosphorylated sugars, namely, glucose 6-phosphate, fructose 6-phosphate, and mannose 6-phosphate, while simultaneously enhancement in catalytic efficiency. A 12000-fold switch from unfavorite substrate to dedicated one is generated. Molecular dynamics simulations reveal the origin of improved activity and substrate specificity. Furthermore, we develop four coordinated multienzyme systems and accomplish the conversion of inexpensive sucrose and starch to fructose and mannose in excellent yield of 94–96%. This innovative sugar-biosynthesis strategy overcomes the reaction equilibrium of isomerization and provides the promise of high-yield manufacturing of other monosaccharides and polyols.

自然地，卤代酸脱卤素酶超家族磷酸酶的进化具有广泛的底物混杂性；然而，开发热力学驱动的制糖路线需要对特定底物有很强的特异性。如何改变磷酸酶固有的底物混杂性并适应“一种酶-一种底物”模型仍然是一个挑战。在此，我们报告了一种磷酸酶的结构导向工程，并成功地提供了对三种广泛存在的磷酸化糖（即葡萄糖 6-磷酸、果糖 6-磷酸和甘露糖 6-磷酸）具有定制偏好的变体，同时增强了催化效率。产生了从不喜欢的底物到专用底物的 12000 倍转换。分子动力学模拟揭示了改善活性和底物特异性的起源。此外，我们开发了四种协调的多酶系统，并以 94-96% 的优异产率将廉价的蔗糖和淀粉转化为果糖和甘露糖。这种创新的糖生物合成策略克服了异构化的反应平衡，并为其他单糖和多元醇的高产量生产提供了希望。