Characterization of Sr UGT76G4 reveals a key residue for regioselectivity and efficient Reb M synthesis

Steviol glycosides (SGs) from Stevia rebaudiana are prized as noncaloric sweeteners, with rebaudioside M (Reb M)—a next-generation SG known for its sucrose-like sweetness and lack of off-tastes—standing out for its superior sensory profile. However, Reb M’s limited natural abundance impedes its commercial production. Here, we report the identification of a glucosyltransferase, UGT76G4 that efficiently catalyzes the conversion of Reb D to Reb M with a strong preference for C19 glycosylation. Structural and functional analyses, including X-ray crystallography, molecular dynamics simulations, and mutagenesis, revealed key residues in UGT76G4 that dictate its regioselectivity, with residue 200 playing a pivotal role. Engineered UGT76G4 variants, including Q199I/G200Y and H155S/Q199I/G200Y, enhanced Reb E and Reb D conversion efficiency by 1.46-fold and 23-fold, respectively, compared to UGT76G1. The engineered variants offer a promising pathway for increasing Reb M production, advancing biotechnological strategies for steviol glycoside biosynthesis and optimizing plant metabolic engineering approaches. Our findings deepen the understanding of SG biosynthesis and provide a basis for sustainable production of high-value sweeteners.