Biocatalytic C3 β- O -Glycosylation of Triterpenes and Sterols to Synthesize Natural and Unnatural Saponins

Saponins are natural products that consist of triterpene or sterol cores decorated with oxidations, glycosylations, and sometimes other modifications. Many saponins are utilized as nutraceutics (e.g., glycyrrhizin) or therapeutics (e.g., QS-21 and digitoxin/digoxin). The structure–activity relationships that govern saponin bioactivity can be identified by studying structurally related saponins; however, the production of varied sets of saponins remains challenging via either chemical (semi)synthesis or native/heterologous biosynthesis. This report describes the discovery that the GT1 family enzyme GuUGT73F15 ( Glycyrrhiza uralensis ) can be used to biosynthesize many different saponins via triterpene/sterol C3 β- O -glycosylation. GuUGT73F15 utilized 22 sugar acceptors (C3 hydroxyl-containing triterpenes/sterols) and 12 sugar donors (uridine diphosphate [UDP]-sugars) as substrates to produce 130 unique monoglycosylated saponins, of which more than 100 have not been reported as natural products to the best of our knowledge. GuUGT73F15 also accepted 13 cyclohexanol- and phenol-type molecules as minimized sugar acceptors. Based on Boltz-2x predictions, the broad substrate scope of GuUGT73F15 is hypothesized to arise from its varied sugar acceptor binding poses and consistent sugar donor binding poses. Applications of broad C3 β- O -glycosylation activity were exemplified via the production of antibody–saponin conjugates as well as the in vivo microbial biosynthesis and the in vitro biosynthesis of advanced QS-21 intermediates. Together, GuUGT73F15 is a versatile biocatalytic tool that can be utilized to produce libraries of high-value saponin natural products and new-to-nature saponins.