Two putative parallel pathways for naringenin biosynthesis in <i>Epimedium wushanense</i>

Abstract

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 <i>Epimedium</i> 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 <i>Epimedium</i>. Here, we identified five genes in <i>Epimedium wushanense</i> 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, <i>in vitro</i> 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 <i>E. wushanense</i> prompted us to propose that pinocembrin is likely converted into naringenin <i>in vivo</i>, 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 <i>Epimedium</i>, paving the way for future metabolite engineering and molecular breeding of <i>E. wushanense</i> to acquire a higher titre of desired, bioactive flavonoid compounds.