Recombinant Yeast Platform for Production and Verification of Diverse Quercetin Heteroconjugates

Flavonoids, such as quercetin, are extensively metabolized, producing glucuronide, sulfate, and heteroconjugate derivatives that influence their bioavailability and biological effects. In this study, we investigated quercetin heteroconjugation using recombinant yeast systems, engineered to express uridine diphosphate-glucuronosyltransferases and sulfotransferases. This strategy was inspired by the observation of diverse heteroconjugate profiles produced by hepatic and intestinal S9 fractions from both rats and humans, highlighting the metabolic complexity of quercetin biotransformation. The yeast-based <i>in vitro</i> platform successfully facilitated the biosynthesis of structurally distinct heteroconjugates, notably Q7G/4'S, Q3G/4'S, Q7G/3'S, and Q3G/3'S, under controlled conditions. The regioselective nature of heteroconjugation was rigorously validated through targeted enzymatic deconjugation using β-glucuronidase and sulfatase, followed by detailed chromatographic analysis, to ensure precise structural confirmation. This integrated approach underscores the robustness of coupling enzymatic assays with high-performance liquid chromatography profiling to verify complex conjugate structures. Collectively, these findings demonstrate the effectiveness of a yeast-based quercetin heteroconjugate biotransformation approach.