Biological synthesis of isorhamnetin 3-O-glucoside using engineered glucosyltransferase

The gene for one of the glycosyltransferases from Populus deltoids, PGT-3, was cloned and was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Various flavonoids were used as potential substrates of the purified recombinant PGT-3. Flavones having two adjacent hydroxyl groups were served as substrate. The regioselectivity of PGT-3 depends on the hydroxyl groups of the substrate. Flavones having two adjacent hydroxyl groups in the B ring were glucosylated at the 4′-hydroxyl group. However, PGT-3 transferred a glucose group to the 3-hydroxyl group of isorhamnetin. Molecular modeling and docking and site-directed mutagenesis were carried out to engineer a PGT-3 having a specificity for isorhamnetin but not for quercetin. Glu82Leu turned out to display this activity. Using the Glu82Leu mutant and a quercetin 3′-O-methyltransferase, isorhamnetin 3-O-glucoside was synthesized.