Advances in enzymatic O-glycosylation of flavonoids: Strategies for control of regioselectivity and enhancement of efficiency

Glycosylation is an effective strategy to improve flavonoid bioavailability, and precise control of regioselectivity is crucial for the efficient synthesis of high-purity products with target activity. This review outlines the relationship between the structure and bioactivity of glycosylated flavonoids. The latest progress on glycosyltransferases (GTs) and glycoside hydrolases (GHs) in flavonoid O-glycosylation and transglycosylation, respectively, is systematically reviewed. Key factors influencing enzymatic regioselectivity are discussed, including the synergistic effect of the flavonoid structural features and the enzyme active-site microenvironment. Based on the features influencing regioselectivity, this review delineates cutting-edge computer-assisted biotechnology strategies, including targeted screening and molecular engineering, with special emphasis on regioselectivity control via targeted modification of the structural elements at both active and distal sites of the enzymes. In addition, a series of widely applicable innovative strategies are proposed to enhance glycosylation efficiency, focusing on the optimization of reaction conditions, the minimization of competitive inhibition by non-flavonoid molecules, and the recycling of expensive glycosyl donors. Finally, this review explores the commercial potential and persistent challenges in industrial production of glycosylated flavonoids. The prospective role of emerging computational tools in predicting and balancing regioselectivity, catalytic efficiency, and enzyme stability is discussed, showing their great promise for the development of industrially viable biocatalysts.