Oleanane-type ginsenosides are highly biologically active substances in , a popular Chinese dietary plant. Lack of key enzymes for glycosylation reactions has hindered synthesis of these bioactive molecules. We mined candidate glycosyltransferases (GTs) of the ginseng database by combining key metabolites and transcriptome coexpression analyses and verified their function using enzymatic assays. The PgCSyGT1, a cellulose synthase-like GT rather than a UDP-dependent glucuronosyltransferase (UGT), was verified as the key enzyme for transferring a glucuronosyl moiety to the free C3-OH of oleanolic acid to synthesize calenduloside E. Two UGTs (PgUGT18 and PgUGT8) were first identified as, respectively, catalyzing the glycosylation reaction of the second sugar moiety of C3 and the C28 in the oleanane-type ginsenoside biosynthetic pathway. Then, we integrated these GTs in combinations into genome and realized biosynthesis of oleanane-type ginsenosides with a yield of 1.41 μg/L ginsenoside Ro in shake flasks. This report provides a basis for effective biosynthesis of diverse oleanane-type ginsenosides in microbial cell factories.
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