East China University of Science and Technology, Shanghai 200237, China.
University of Chinese Academy of Sciences, Beijing 100039, China.
J Agric Food Chem. 2021 Nov 10;69(44):13155-13163. doi: 10.1021/acs.jafc.1c04873. Epub 2021 Oct 26.
Rubusoside (Rub) is a highly sweet diterpene glycoside mainly isolated from the leaves of (Rosaceae). It has been used as a low-calorie natural sweetener for decades and was recently found to be a potential drug lead. In this study, we designed a whole-cell biocatalyst to achieve the glycosylation of steviol to Rub in . The sucrose synthases were applied to construct a uridine diphosphate glucose regeneration system, which were coupled with optimal combinations of different uridine diphosphate (UDP) glycosyltransferases from multiple plant species. After optimization of reaction conditions, the residues in SrUGT74G1 probably influencing glycosylation efficiency were subjected to site-directed mutagenesis. Double mutations of S84A/E87A reduced the accumulation of intermediates, finally glucosylating 1.27 g/L steviol to 0.45 ± 0.06 g/L steviolmonoside (conversion rate = 23.3%) and 1.92 ± 0.17 g/L Rub (conversion rate = 74.9%). A high efficiency of Rub biosynthesis could be achieved without supply of additional UDPG. This work provided the first example of multi-step glycosylation reactions in whole-cell biocatalysis, which laid a foundation of scalable production of the value-added diterpene sweetener in the future.
毛蕊花糖苷（Rub）是一种高甜度的二萜糖苷，主要从（蔷薇科）的叶子中分离得到。它已被用作低热量的天然甜味剂数十年，最近被发现是一种有潜力的药物先导化合物。在本研究中，我们设计了一种全细胞生物催化剂，以实现甜菊醇向 的糖苷化。蔗糖合酶被应用于构建尿苷二磷酸葡萄糖再生系统，该系统与来自多种植物的不同尿苷二磷酸（UDP）糖基转移酶的最佳组合相偶联。在优化反应条件后，对可能影响糖苷化效率的 SrUGT74G1 中的残基进行了定点突变。S84A/E87A 的双重突变减少了中间体的积累，最终将 1.27 g/L 的甜菊醇转化为 0.45±0.06 g/L 的甜菊醇单糖苷（转化率=23.3%）和 1.92±0.17 g/L 的毛蕊花糖苷（转化率=74.9%）。在不额外提供 UDPG 的情况下，可实现毛蕊花糖苷的高效生物合成。这项工作提供了全细胞生物催化中多步糖基化反应的首例，为未来高附加值二萜甜味剂的规模化生产奠定了基础。
