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 beta (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 beta (1-2) glucosylation. The enzyme's ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze beta (1-2) glucosylation in two distinct orientations as well as beta (1-6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable beta (1-2) glucosylation, eliminate beta (1-6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides. Steviol glycosides from the plant Stevia rebaudiana are already used as lowcalorie sweeteners, but the most abundant naturally occurring compounds have a bitter aftertaste. Here, the authors characterize and engineer rice glycosyltransferase OsUGT91C1 to facilitate the large-scale production of naturally rare but palatable glycosides Reb D and Reb M

Automated summary

This study characterizes a rice enzyme OsUGT91C1 that efficiently catalyzes beta (1-2) glucosylation, and engineers it to enhance the production of naturally rare but palatable steviol glycosides Reb D and Reb M. Through structural insights, the enzyme's flexibility in binding steviol glycoside substrates allows for targeted modifications, making it a promising catalyst for industrial-scale production.