Natural sugar substitute Rebaudioside M: Efficient and cost-effective biosynthesis via host engineering, cascade...

Rebaudioside M is a desirable zero-calorie sweetener, but its low natural abundance limits industrial application. Here, enzymatic conversion of Rebaudioside D was intensified through host engineering, cascade biocatalysis, and substrate feeding. Expression of the glycosyltransferase in a eukaryotic host increased the soluble enzyme fraction from 18% to 92% and improved specific activity by 1.46-fold compared with the bacterial system. Coupling with sucrose synthase enabled in situ regeneration of the sugar donor from inexpensive sucrose. Kinetic analysis identified substrate depletion as the dominant bottleneck in batch reactions. A fed-batch feeding strategy increased the product titer to 28.38 g L^-1 with 91.6% conversion, representing a 3.99-fold improvement over batch mode. This integrated strategy provides a practical basis for cost-efficient production of next-generation natural sweeteners.