Developing the Escherichia coli Platform for Efficient Glycosylation of Steviol Glycosides through Metabolic Engineering

Abstract Rare steviol glycosides (SGs), characterized by superior sensory profiles and promising bioactivities, are high-intensity sweeteners derived from Stevia rebaudiana. Their intrinsically low abundance limits extraction yields, while in vitro bioconversion is limited by the availability of UDP-glucose (UDPG). Here, an Escherichia coli chassis was reprogrammed to achieve an intracellular UDPG content of 11.72 μmol/g CDW, 2.19-fold that of BL21(DE3), by enhancing pyrimidine nucleotide synthesis, blocking competing pathways, redirecting carbon flux, and optimizing the expression of key genes. Coupled with a UGT−SuSy system, the platform enabled in vitro conversion of Reb A into rare derivativesReb L2, Reb D, Reb D2, Reb M, Reb M2, and Reb M8without exogenous UDPG, with 1.6- to 129-fold higher conversion efficiencies than the wild type. Cultured in a 5 L bioreactor, harvested cells yielded 108.57 g/L Reb L2 and 111.99 g/L Reb D via in vitro whole-cell biotransformation. This platform provides a route for rare SG production and a versatile foundation for the glycodiversification of natural products.