<scp>UGT73FB1</scp> contributes to scaffold‐selective biosynthesis of triterpenoid glucosyl esters in saponin‐rich bark of arjuna tree

Plants make structurally diverse triterpenoids for their physiological needs, which have shown numerous therapeutic applications. Arjuna tree (Terminalia arjuna) produces bioactive oleanane (β-amyrin-derived) triterpenoids arjunic acid, arjungenin, and arjunolic acid, and the respective C28-O-glucopyranosyl esters arjunetin, arjunglucoside I, and arjunglucoside II. Arjunic acid and arjunetin are the major oleananes in bark, while arjunolic acid and arjunglucoside II are found in minor levels. Although arjungenin was detected at a considerable level, arjunglucoside I was found only at a trace level, suggesting selective biosynthesis and/or accumulation of triterpenoid glucosyl esters in bark. However, the enzyme contributing to triterpenoid C28-O-glucosylation was not characterized. We mined RNA-sequencing data and identified UDP-glucosyltransferase (UGT) transcripts that were enriched in the bark transcriptome. Further, biochemical screening of UGTs identified UGT73FB1, which catalyzed triterpenoid C28-O-glucosylation in a scaffold-selective manner. Recombinant UGT73FB1 produced in Escherichia coli or Nicotiana benthamiana formed arjunic acid and arjunolic acid C28-O-glucopyranosyl esters arjunetin and arjunglucoside II, but not arjungenin C28-O-glucopyranosyl ester (arjunglucoside I). Interestingly, UGT73FB1 showed better activity using oleananes than ursanes (α-amyrin-derived), but it did not show C28-O-glucosylation activity using various lupane triterpenoids (lupeol-derived). Overall, the spatial patterns of UGT73FB1 transcript expression and triterpenoid accumulation and scaffold-selective activity of UGT73FB1 suggested a major role of UGT73FB1 in the biosynthesis of C28-O-glucopyranosyl esters in arjuna. Moreover, UGT73FB1 co-expression with β-amyrin synthase and triterpenoid C2, C23, and C28 hydroxylases/oxidases led to complete reconstruction of the arjunglucoside II pathway in N. benthamiana, suggesting the utility of arjuna enzymes for the biosynthesis of rare triterpenoid glucopyranosyl esters in heterologous hosts.