Li Jiao, Mu Shicheng, Yang Jiangang, Liu Cui, Zhang Yanfei, Chen Peng, Zeng Yan, Zhu Yueming, Sun Yuanxia
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, China.
National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China.
iScience. 2022 Sep 27;25(10):105222. doi: 10.1016/j.isci.2022.105222. eCollection 2022 Oct 21.
Mogrosides are widely served as natural zero-calorie sweeteners. To date, the biosynthesis of high-intensity sweetness mogrosides V from mogrol has not been achieved because of inefficient and uncontrollable multi-glycosylation process. To address this challenge, we reported three UDP-glycosyltransferases (UGTs) catalyzing the primary and branched glycosylation of mogrosides and increased the catalytic efficiency by 74-400-folds toward branched glycosylation using an activity-based sequence conservative analysis engineering strategy. The computational studies provided insights into the origin of improved catalytic activity. By virtue of UGT mutants, we provided regio- and bond-controllable multi-glycosylation routes, successfully facilitating sequential glycosylation of mogrol to three kinds of mogroside V in excellent yield of 91-99%. Meanwhile, the feasibility of the routes was confirmed in engineered yeasts. It suggested that the multi-glycosylation routes would be combined with mogrol synthetic pathway to produce mogrosides from glucose by aid of metabolic engineering and synthetic biology strategies in the future.
罗汉果甜苷被广泛用作天然零热量甜味剂。迄今为止，由于多糖基化过程效率低下且不可控，尚未实现从罗汉果醇生物合成高强度甜味的罗汉果甜苷V。为应对这一挑战，我们报道了三种催化罗汉果甜苷初级和分支糖基化的尿苷二磷酸糖基转移酶（UGT），并使用基于活性的序列保守分析工程策略将分支糖基化的催化效率提高了74至400倍。计算研究揭示了催化活性提高的根源。借助UGT突变体，我们提供了区域和键可控的多糖基化路线，成功地促进了罗汉果醇顺序糖基化为三种罗汉果甜苷V，产率高达91%至99%。同时，这些路线在工程酵母中得到了可行性验证。这表明，未来多糖基化路线将与罗汉果醇合成途径相结合，借助代谢工程和合成生物学策略从葡萄糖生产罗汉果甜苷。
