Catalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides

Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1→2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing β (1→2) glucosylation. The enzyme's ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1→2) glucosylation in two distinct orientations as well as β (1→6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1→2) glucosylation, eliminate β (1→6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides.