Zhao Liting, Ma Zhongbao, Wang Qiong, Hu Manfeng, Zhang Jingxiang, Chen Lei, Shi Guiyang, Ding Zhongyang
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 Mar 1;71(8):3832-3841. doi: 10.1021/acs.jafc.2c08642. Epub 2023 Feb 16.
The restricted availability of UDP-glucose, an essential precursor that targets oligo/polysaccharide and glycoside synthesis, makes its practical application difficult. Sucrose synthase (Susy), which catalyzes one-step UDP-glucose synthesis, is a promising candidate. However, due to poor thermostability of Susy, mesophilic conditions are required for synthesis, which slow down the process, limit productivity, and prevent scaled and efficient UDP-glucose preparation. Here, we obtained an engineered thermostable Susy (mutant M4) from through automated prediction and greedy accumulation of beneficial mutations. The mutant improved the value at 55 °C by 27-fold, resulting in UDP-glucose synthesis at 37 g/L/h of space-time yield that met industrial biotransformation standards. Furthermore, global interaction between mutant M4 subunits was reconstructed by newly formed interfaces according to molecular dynamics simulations, with residue Trp162 playing an important role in strengthening the interface interaction. This work enabled effective, time-saving UDP-glucose production and paved the way for rational thermostability engineering of oligomeric enzymes.
UDP-葡萄糖是寡糖/多糖和糖苷合成的必需前体，但其可用性有限，这使得其实际应用变得困难。催化一步法合成UDP-葡萄糖的蔗糖合酶（Susy）是一个有前景的候选者。然而，由于Susy的热稳定性较差，合成需要嗜温条件，这会减慢合成过程、限制生产力，并阻碍规模化高效制备UDP-葡萄糖。在此，我们通过自动预测和有益突变的贪婪积累，从[具体来源未给出]获得了一种工程化的热稳定Susy（突变体M4）。该突变体在55°C时将[具体数值未给出]提高了27倍，时空产率达到37 g/L/h的UDP-葡萄糖合成，符合工业生物转化标准。此外，根据分子动力学模拟，通过新形成的界面重建了突变体M4亚基之间的全局相互作用，残基Trp162在加强界面相互作用中起重要作用。这项工作实现了高效、省时的UDP-葡萄糖生产，并为寡聚酶的合理热稳定性工程铺平了道路。
