An efficient whole-cell platform for Rebaudioside M biotransformation: cascade design, expression regulation, process engineering

Abstract

Excessive sugar consumption is fueling a global health crisis, creating demand for sustainable, zero-calorie sweeteners. Rebaudioside M (Reb-M), a high-intensity sweetener with a sucrose-like taste, is attractive. However, current supply depends on low-abundance leaf extraction and in vitro enzymatic glycosylation, limited by low titers/space-time yields, costly cofactors and enzyme purification, and inefficient atom utilization with significant byproduct streams. Here, we established for the first time an integrated E. coli whole-cell cascade co-expressing UGT76G1 variant and Glycine max sucrose synthase (GmSuSy), enabling in situ UDP-glucose regeneration from sucrose to drive Reb-D conversion into Reb-M. Process bottlenecks were overcome through expression regulation (multi-copy UGT76G1, RBS engineering of GmSuSy, and medium/induction optimization) and process engineering (permeabilization-assisted transport and reaction condition refinement). A fed-batch strategy further intensified performance, delivering 30.6 g/L Reb-M with 95.9% conversion, ranking among the highest levels reported to date. Importantly, this study also demonstrates the first conversion of the fructose byproduct into value-added D-allulose (2.97 g/L) using a D-tagatose 3-epimerase module, thereby improving atom economy and sustainability. This approach offers a generalizable strategy to address fructose byproduct accumulation in multi-enzyme cascade reactions. Together, these advances establish a a greener and more economical route to Reb-M at industrially relevant titers, highlighting both efficiency and green chemistry principles.