ABSTRACT

Quercetin-3-O-glucuronide (Q3G) is a bioactive quercetin metabolite with potential applications in functional foods and nutraceuticals. However, its low natural abundance and reliance on UDP-glucuronic acid (UDP-GlcA) hinder scalable production. Here, we developed a three-enzyme cascade that couples UDP-GlcA generation from GlcA with VvGT5-catalyzed quercetin glucuronidation. Integrated sequence and expression optimization enhanced soluble VvGT5 expression, while AiCE-guided engineering generated the variant M1 (M189L/P251T/ R307P). M1 exhibited 1.63-fold higher apparent activity, improved thermostability, and a 1.42-fold increase in apparent kcat, with kcat/Km remaining nearly unchanged. Under optimized 1-L cascade conditions, M1 produced 1162 mg/L Q3G from 3 mM quercetin, corresponding to 81.0% conversion. Quercetin-3,7-Odiglucuronide was also enzymatically synthesized for the first time and structurally confirmed by MS and multidimensional NMR. Compared with quercetin, Q3G and quercetin-3,7-O-diglucuronide showed 2.9- and 3.1- fold higher residual levels after simulated intestinal digestion, respectively, while retaining appreciable ABTS and DPPH radical-scavenging activities. These findings establish an efficient in situ UDP-GlcA supply strategy for producing structurally defined quercetin glucuronides and highlight their potential as bioaccessible flavonoid derivatives for food-related applications.