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.
Keywords: glycosyltransferase; rubusoside; sucrose synthase; whole-cell biocatalyst; yeast.