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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.

Efficient Biocatalytic Preparation of Rebaudioside KA: Highly Selective Glycosylation Coupled with UDPG Regeneration