Rebaudioside D is a promising sweetener due to its zero calorie and high sweetness. Here, a transglucosylase gene eugt11 from Oryza sativa was for the first time expressed in Pichia pastoris, and transformant XE-3 showed the highest expression levels in pH 5.5 BMMY media containing 0.75% methanol. The affinity-purified EUGT11 from XE-3 displayed the highest activity at pH 6.0–6.5 and 45 °C, compared to pH 8.5 and 35 °C for EUGT11 from Escherichia coli. One-pot synthesis with orthogonal design was employed to optimize the rebaudioside D production using XE-3, and the initial pH 7.0 of the medium appears to be a significant factor and delivers the highest conversion efficiency. A two-step temperature-control strategy was developed, and a conversion rate of 95.31% was achieved at 28/35 °C vs. 62.41% in a one-step process at 28 °C. This study provides a high-efficient whole-cell biocatalysts technology for the sweetener production.

莱鲍迪甙D由于其零卡路里和高甜度而成为有前途的甜味剂。这里，transglucosylase基因eugt11从稻是用于表示所述第一时间巴斯德毕赤酵母，和转化体XE-3显示出在pH为最高表达水平含有0.75％甲醇5.5 BMMY培养基。XE-3的亲和纯化EUGT11在pH 6.0–6.5和45°C时显示出最高活性，而大肠杆菌EUGT11的pH 8.5和35°C。采用正交设计的一锅法合成技术可优化使用XE-3的莱鲍迪甙D的生产，该介质的初始pH 7.0似乎是一个重要因素，并具有最高的转化效率。制定了两步温度控制策略，在28/35°C下实现了95.31％的转化率，而在28°C下一步实现了62.41％的转化率。这项研究为甜味剂生产提供了一种高效的全细胞生物催化剂技术。