Multi‐Enzyme Cascade Reaction of Crude Enzyme Strategy for the Economical and Efficient Bioconversion of Rebaudioside A to Rebaudioside M

ABSTRACT Rebaudioside M (Reb M) is a natural sweetener with high sweetness, low caloric content, and an excellent sensory profile. However, it's extremely low natural abundance in Stevia rebaudiana leaves renders conventional extraction processes costly, inefficient, and unsustainable, limiting its industrial utilization. In this study, we developed a one‐pot, stepwise enzyme addition cascade strategy for the efficient biosynthesis of Reb M. In the first step, a glycosyltransferase mutant YojK M0 from Bacillus subtilis 168 catalyzed the conversion of rebaudioside A (Reb A) to rebaudioside D (Reb D). In the second step, a glycosyltransferase mutant UGT76G1 M3 from Stevia rebaudiana converted Reb D to Reb M. Simultaneously, sucrose synthase from Glycine max enabled continuous in situ regeneration of UDP‐glucose from sucrose and UDP throughout both reaction steps. Following systematic optimization of reaction conditions, a scaled‐up 3 L bioconversion using crude enzymes obtained from high‐cell‐density fermentation in Escherichia coli successfully converted 30 mmol Reb A into 24.1 mmol Reb M, achieving an overall yield of 80.34%. This work demonstrates a highly efficient, scalable, and cost‐effective enzymatic biomanufacturing platform for Reb M, providing a promising route for sustainable industrial production of next‐generation natural sweeteners.