Identification of Hydroxylation Enzymes and the Metabolic Analysis of Dihydromyricetin Synthesis in <i>Ampelopsis grossedentata</i>

Ampelopsis grossedentata leaves are highly rich in dihydromyricetin. They have been used to make tea in China for centuries. Dihydromyricetin has many potential applications in foods and medicine. This are because it has five phenolic hydroxyl groups. However, the hydroxylases involving the biosynthesis of dihydromyricetin have not been identified and characterized. In this study, a series of hydroxylases genes, including flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and cytochrome P450 reductase (CPR), were identified after RNA sequencing. The full-length CDSs of AgF3H, AgF3′H, AgF3′5′H, and AgCPR genes were amplified from the cDNA library of leaves. The aforementioned enzymes were expressed and verified in Saccharomyces cerevisiae. Through the substrate specificity assay, the functional AgF3′H, AgF3′5′H, and AgCPR in A. grossedentata were identified. The dihydromyricetin hydroxylation process in A. grossedentata was successfully identified. We found that substantial carbon flux occurred through the Naringenin (NAR)–Eriodictyol (ERI)–Dihydroquercetin (DHQ)–Dihydromyricetin (DHM) and NAR–Dihydrokaempferol (DHK)–DHQ–DHM pathways. This study provides some reference for the development and utilization of the germplasm resources and molecular breeding of A. grossedentata