Two putative parallel pathways for naringenin biosynthesis in Epimedium wushanense

Flavonoids that exhibit various biological activities such as antioxidant, antitumor, antiviral, antibacterial and anti-inflammatory properties are found in a wide range of medicinal plants. Among the flavonoid-producing plants identified so far, the genus Epimedium is recognised as a group of prolific prenyl-flavonoid glycoside producers with high economic value in the global dietary supplement market. To date, the biosynthetic genes for prenyl-flavonoid glycosides still remain elusive in Epimedium. Here, we identified five genes in Epimedium wushanense responsible for the biosynthesis of naringenin, the common precursor for flavonoid natural products. We successfully set up the biosynthetic pathway of naringenin using l-tyrosine as the precursor through enzymatic assays of these genes' encoding products, including phenylalanine ammonia-lyase (EwPAL), 4-coumarate-CoA ligase (Ew4CL1), chalcone synthase (EwCHS1), chalcone isomerase (EwCHI1) and CHI-like protein (EwCHIL3). Intriguingly, in vitro characterisation of the above catalytic enzymes' substrate specificity indicated a route parallel to naringenin biosynthesis, which starts from l-phenylalanine and ends in pinocembrin. The fact that there is no pinocembrin or pinocembrin-derived flavonoid accumulated in E. wushanense prompted us to propose that pinocembrin is likely converted into naringenin in vivo, constituting two parallel biosynthetic pathways for naringenin. Therefore, our study provides a basis for the full elucidation of the biosynthetic logic of prenyl-flavonoid glycoside in Epimedium, paving the way for future metabolite engineering and molecular breeding of E. wushanense to acquire a higher titre of desired, bioactive flavonoid compounds.