Identification of Hydroxylation Enzymes and the Metabolic Analysis of Dihydromyricetin Synthesis in Ampelopsis grossedentata

Abstract

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.

Genome-wide analysis of flavonoid biosynthetic genes in Musaceae (Ensete,Musella, andMusaspecies) reveals amplification of flavonoid 3’,5’-hydroxylase

“…1), generating 3’,4’,5’-hydroxylated flavonoids (Wang et al ., 2014), which are regarded as giving quality attributes, e.g., for example to tea (Jin et al ., 2017). F3’H and F3’5’H are members of the cytochrome P450 enzyme family (Park et al ., 2021; Zhang et al ., 2022). McClean et al .…”

“…1), generating 3',4',5'-hydroxylated flavonoids (Wang et al, 2014), which are regarded as giving quality attributes, e.g., for example to tea (Jin et al, 2017). F3'H and F3'5'H are members of the cytochrome P450 enzyme family (Park et al, 2021;Zhang et al, 2022). McClean et al (2022) reported that their "phylogenetic analysis determined F3'5'H first appeared in the Streptophyta and is present in only 41% of angiosperm reference genomes" while F3'H is found universally in angiosperms, due to its critical role in abiotic stress, F3'5'H was reported as a major gene for the flower and seed coat colour regulation in plants (McClean et al, 2022), with MYB and MYC elements in the promoter region (McClean et al, 2022).…”

De Novo Synthesis of Chrysin in Saccharomyces cerevisiae

“…The Arabidopsis thaliana CPR (AtCPR), Erigeron breviscapus CPR (EbCPR), Erigeron breviscapus CPR (HtCPR), Glycine max CPR (GmCPR), S. cerevisiae CPR (ScCPR), and A. grossedentata CPR (AgCPR1) genes were previously reported. 20 Plasmids in this study were constructed by the method of seamless assembly (ClonEx-pressTM II one-step cloning kit and ClonExpress MultiS one-step cloning kit, Vazyme Biotech Co., Ltd., China) and are listed in Table S2.…”

Diabetes cardiomyopathy: targeted regulation of mitochondrial dysfunction and therapeutic potential of plant secondary metabolites

“…Dihydromyricetin is a dihydroflavonol compound widely present in plants of the ampelopsis family, with pharmacological effects including scavenging free radicals, antioxidant, and anti-fibrotic properties ( Zhang et al, 2022 ). Wu et al found that treatment with dihydromyricetin (100 mg/kg/d) for 14 weeks improved mitochondrial function in STZ-induced diabetic C57BL/6J mice, increasing ATP content in, ETC., and the activity of complex I/II/III/IV/V, restoring Δψm, reducing oxidative stress, and improving mitochondrial energy metabolism.…”

“…Ginsenoside Rg1 is a saponin glycoside isolated from Panax ginseng C. A. Mey, which has various biological activities such as anti-inflammatory, anti-oxidant, anti-platelet aggregation, anti-cancer, hypoglycemic, and neuroprotective effects ( Zhang et al, 2022 ). Qin et al showed that ginsenoside Rg1 (20 mg/kg/d) can promote mitochondrial biogenesis by increasing the expression of PGC-1α, AMPK, Nrf2 and HO-1 proteins, and reduce the expression of NF-κB and NLRP3 proteins to reduce oxidative stress after 8 weeks of treatment in STZ induced diabetes Wistar rats.…”