Two putative parallel pathways for naringenin biosynthesis in Epimedium wushanense

Area of Science:

• Plant biochemistry
• Metabolic engineering
• Medicinal plant research

Background:

• Flavonoids possess diverse biological activities and are abundant in medicinal plants.
• The genus *Epimedium* is a valuable source of prenyl-flavonoid glycosides for the dietary supplement market.
• Biosynthetic pathways for prenyl-flavonoid glycosides in *Epimedium* remain largely uncharacterized.

Purpose of the Study:

• To identify genes involved in naringenin biosynthesis in *Epimedium wushanense*.
• To elucidate the biosynthetic pathway(s) leading to naringenin, a key flavonoid precursor.
• To lay the groundwork for metabolite engineering and molecular breeding of *E. wushanense*.

Main Methods:

• Identification of five candidate genes in *E. wushanense*.
• Enzymatic assays to establish the naringenin biosynthetic pathway from l-tyrosine.
• In vitro characterization of enzyme substrate specificity to explore alternative pathways.

Main Results:

• Five genes (EwPAL, Ew4CL1, EwCHS1, EwCHI1, EwCHIL3) were identified as responsible for naringenin biosynthesis.
• Enzymatic assays confirmed a pathway from l-tyrosine to naringenin.
• Enzyme characterization revealed a parallel pathway from l-phenylalanine to pinocembrin, suggesting pinocembrin is converted to naringenin in vivo.

Conclusions:

• Two parallel biosynthetic pathways for naringenin in *E. wushanense* are proposed.
• The study provides a foundation for understanding prenyl-flavonoid glycoside biosynthesis in *Epimedium*.
• Findings facilitate future efforts in metabolite engineering and molecular breeding for enhanced flavonoid production.