Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China.
J Agric Food Chem. 2023 Aug 23;71(33):12549-12557. doi: 10.1021/acs.jafc.3c03877. Epub 2023 Aug 8.
Nucleotide sugars are essential precursors for carbohydrate synthesis but are in scarce supply. Uridine diphosphate (UDP)-glucose is a core building block in nucleotide sugar preparation, making its efficient synthesis critical. Here, a process for producing valuable UDP-glucose and functional mannose from sucrose was established and improved via a semirational sucrose synthase (SuSy) design and the accurate D-mannose isomerase (MIase) cascade. Engineered SuSy exhibited enzyme activity 2.2-fold greater than that of the WT. The structural analysis identified a latch-hinge combination as the hotspot for enhancing enzyme activity. Coupling MIase, process optimization, and reaction kinetic analysis revealed that MIase addition during the high-speed UDP-glucose synthesis phase distinctly accelerated the entire process. The simultaneous triggering of enzyme modules halved the reaction time and significantly increased the UDP-glucose yield. A maximum UDP-glucose yield of 83%, space-time yield of 70 g/L/h, and mannose yield of 32% were achieved. This novel and efficient strategy for sucrose value-added exploitation has industrial promise.
核苷酸糖是碳水化合物合成的必需前体，但供应稀缺。尿苷二磷酸（UDP）-葡萄糖是核苷酸糖制备的核心构建块，因此其高效合成至关重要。在这里，通过半理性蔗糖合酶（SuSy）设计和精确的 D-甘露糖异构酶（MIase）级联反应，建立并改进了从蔗糖生产有价值的 UDP-葡萄糖和功能性甘露糖的工艺。工程 SuSy 的酶活性比 WT 高 2.2 倍。结构分析确定了闩锁-铰链组合是提高酶活性的热点。耦合 MIase、过程优化和反应动力学分析表明，在高速 UDP-葡萄糖合成阶段添加 MIase 可明显加速整个过程。同时触发酶模块将反应时间缩短一半，并显著提高 UDP-葡萄糖的产量。UDP-葡萄糖的最大产率为 83%，时空产率为 70 g/L/h，甘露糖的产率为 32%。这种用于蔗糖增值开发的新颖而有效的策略具有工业应用前景。
