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Highly Regioselective and Efficient Biosynthesis of Polydatin by an Engineered UGTBL1-AtSuSy Cascade Reaction.

Polydatin, resveratrol-3-O-β-glucoside, possesses various biological activities. However, the regioselective glucosylation of resveratrol by UDP-glycosyltransferases (UGTs) constitutes a persistent problem. In this study, semi-rational design and iterative combinatorial mutagenesis were carried out to screen the mutants of UGTBL1 and the high specificity with the glycosylation of the 3-OH group of resveratrol was explored. The triple mutant I62G/M112D/K143G exhibited near-perfect control of polydatin synthesis (regioselectivity ∼ 99%), and the ratio of polydatin to resveratrol-4'-O-β-glucoside was finally enhanced by 786-fold. Molecular docking revealed that the mutant could form three H-bonds between 3-, 5-, and 4'-OH groups of resveratrol and the residues around the active center, resulting in the oriented-binding of resveratrol. Furthermore, UGTBL1 mutant coupling sucrose synthase AtSuSy can synthesize polydatin at an unprecedented high titer of 10.33 g/L, together with efficient UDPG regeneration (RCmax = 54). This study provides an efficient approach for the regioselective biosynthesis of polydatin.