[Functional characterization and product identification of sesquiterpene synthases in Alisma orientale (Sam.) Juz.]

The Chinese herbal medicine Rhizoma Alismatis is the dried tuber of Alisma orientale (Sam.) Juzep. or Alisma plantago-aquatica Linn., perennial aquatic or marsh herbaceous plants belonging to the family Alismataceae. It is a commonly used herb for inducing diuresis and eliminating dampness. It is rich in terpenoids, especially sesquiterpenoids as important active ingredients, which exhibit various pharmacological activities such as anticancer and anti-inflammatory effects. To date, studies on the sesquiterpenoid biosynthetic pathway in Alisma orientale (Sam.) Juzep. remain insufficient. This study aimed to elucidate the sesquiterpenoid biosynthetic pathway in Rhizoma Alismatis, identify and characterize key functional genes involved in sesquiterpenoid biosynthesis, and provide a theoretical basis for clarifying its pharmacodynamic material basis and the subsequent screening and application of quality markers. Therefore, employing approaches of transcriptomics and molecular biology, identified and cloned nine functional sesquiterpene synthase genes (AoTPSs) from A. orientale. Using a yeast heterologous expression system, we characterized their biochemical functions, confirming that these genes can catalyze the biosynthesis of sesquiterpenoids such as β-elemene, α-caryophyllene, (E)-nerolidol, β-caryophyllene, and α-farnesene. This study lays a foundation for in-depth elucidation of the biosynthetic mechanism and resource development of Alisma sesquiterpenoids.