文献类型：期刊文献

英文题名：Whole-Cell Biocatalyst for Rubusoside Production in Saccharomyces cerevisiae

作者：Mao, Yaping[1,2,3];Chen, Zhuo[2,3];Ren, Yuhong[1];Sun, Yuwei[3];Wang, Yong[1,3]

机构：[1]East China Univ Sci & Technol, Shanghai 200237, Peoples R China;[2]Univ Chinese Acad Sci, Beijing 100039, Peoples R China;[3]Chinese Acad Sci, CAS Ctr Excellence Mol Plant Sci, CAS Key Lab Synthet Biol, Shanghai 200032, Peoples R China

年份：2021

卷号：69

期号：44

起止页码：13155

外文期刊名：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

收录：;EI(收录号：20214611157837);WOS:【SCI-EXPANDED(收录号:WOS:000718312300022)】；

基金：This study was funded from the National Key R&D Program of China (2018YFA0900600), the Program of Shanghai Academic Research Leader (20XD1404400), the Strategic Priority Research Program "Molecular mechanism of Plant Growth and Development" of CAS (XDB27020202), the National Natural Science Foundation of China (grant nos. 32070328, 41876084, and 22077129), the Construction of the Registry and Database of Bioparts for Synthetic Biology of the Chinese Academy of Science (no. ZSYS-016), the International Partnership Program of the Chinese Academy of Science (no. 153D31KYSB20170121), and the National Key Laboratory of Plant Molecular Genetics, CEMPS, CAS. Y.S. is supported by the Foundation of Youth Innovation Promotion Association of the Chinese Academy of Sciences.

语种：英文

外文关键词：yeast; rubusoside; whole-cell biocatalyst; glycosyltransferase; sucrose synthase

摘要：Rubusoside (Rub) is a highly sweet diterpene glycoside mainly isolated from the leaves of Rubus suavissimus (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 Saccharomyces cerevisiae. 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.