State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, 211198, China.
Sci Rep. 2020 Apr 10;10(1):6230. doi: 10.1038/s41598-020-63379-9.
Rebaudioside KA is a diterpene natural sweetener isolated in a trace amount from the leaves of Stevia rebaudiana. Selective glycosylation of rubusoside, a natural product abundantly presented in various plants, is a feasible approach for the biosynthesis of rebaudioside KA. In this study, bacterial glycosyltransferase OleD was identified to selectively transfer glucose from UDPG to 2'-hydroxyl group with a β-1,2 linkage at 19-COO-β-D-glucosyl moiety of rubusoside for the biosynthesis of rebaudioside KA. To eliminate the use of UDPG and improve the productivity, a UDPG regeneration system was constructed as an engineered Escherichia coli strain to couple with the glycosyltransferase. Finally, rubusoside at 22.5 g/L (35.0 mM) was completely converted to rebaudioside KA by the whole cells without exogenous addition of UDPG. This study provides an efficient and scalable method for highly selective biosynthesis of rebaudioside KA.
瑞鲍迪苷 KA 是一种从甜叶菊叶片中微量分离出的二萜天然甜味剂。选择地对在各种植物中大量存在的毛蕊花糖苷进行糖基化，是瑞鲍迪苷 KA 生物合成的可行方法。在这项研究中，鉴定出细菌糖基转移酶 OleD 可选择性地将葡萄糖从 UDPG 转移到毛蕊花糖苷 19-COO-β-D-葡萄糖基部分的 2'-羟基上，形成 β-1,2 键，从而合成瑞鲍迪苷 KA。为了消除 UDPG 的使用并提高生产力，构建了 UDPG 再生系统作为与糖基转移酶偶联的工程大肠杆菌菌株。最后，在没有外源添加 UDPG 的情况下，整细胞将 22.5 g/L（35.0 mM）的毛蕊花糖苷完全转化为瑞鲍迪苷 KA。本研究为瑞鲍迪苷 KA 的高效和可扩展的选择性生物合成提供了一种方法。
