National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308, China.
Nat Commun. 2022 Jun 23;13(1):3582. doi: 10.1038/s41467-022-31371-8.
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.
天然的，卤酸脱卤酶超家族磷酸酶具有广泛的底物混杂性；然而，为了开发热力学驱动的糖制造途径，需要对特定的底物具有很强的特异性。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”的模型仍然是一个挑战。在此，我们报告了一种磷酸酶的结构引导工程，并成功提供了具有针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）的定制偏好的变体，同时提高了催化效率。从非优选底物到专用底物的转变达到了 12000 倍。分子动力学模拟揭示了提高活性和底物特异性的起源。此外，我们开发了四个协调的多酶系统，并在 94-96%的优异收率下完成了廉价蔗糖和淀粉向果糖和甘露糖的转化。这种创新的糖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制造提供了前景。
