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Title: Directed Evolution of the UDP-Glycosyltransferase UGTBL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides. Author: Zhang C, Cai Y, Zhang Z, Zheng N, Zhou H, Su Y, Du S, Hussain A, Xia X. Journal: J Agric Food Chem; 2024 Jan 24; 72(3):1640-1650. PubMed ID: 38213280. Abstract: The O-glycosylation of polyphenols for the synthesis of glycosides has garnered substantial attention in food research applications. However, the practical utility of UDP-glycosyltransferases (UGTs) is significantly hindered by their low catalytic efficiency and suboptimal regioselectivity. The concurrent optimization of the regioselectivity and activity during the glycosylation of polyphenols presents a formidable challenge. Here, we addressed the long-standing activity-regioselectivity tradeoff in glycosyltransferase UGTBL1 through systematic enzyme engineering. The optimal combination of mutants, N61S/I62M/D63W/A208R/P218W/R282W (SMWRW1W2), yielded a 6.1-fold improvement in relative activity and a 17.3-fold increase in the ratio of gastrodin to para-hydroxybenzyl alcohol-4'-O-β-glucoside (with 89.5% regioselectivity for gastrodin) compared to those of the wild-type enzyme and ultimately allowed gram-scale production of gastrodin (1,066.2 mg/L) using whole-cell biocatalysis. In addition, variant SMWRW1W2 exhibited a preference for producing phenolic glycosides from several substrates. This study lays the foundation for the engineering of additional UGTs and the practical applications of UGTs in regioselective retrofitting.[Abstract] [Full Text] [Related] [New Search]