Exploring oxidosqualene cyclases and cytochrome P450s from Aralia elata for the synthesis of diverse pentacyclic triterpene sapogenins in Nicotiana benthamiana.

Pentacyclic triterpene sapogenins (PTSs) represent a ubique class of natural products for their diverse structures and bioactivities, holding potential applications across the food, cosmetic, and pharmaceutical industries. Aralia elata, a shrub that accumulates PTSs such as oleanolic and echinocystic acids, has been used in traditional folk medicine in East Asia (China, Japan, Korea, Russia). However, the limited accumulation of PTSs in A. elata, along with the complexity of their biosynthesis, restricts its use for large-scale production and poses significant challenges for industrial applications. Furthermore, the unknown biosynthetic enzymes underlying PTS production hinder their broader utilization through synthetic biology strategies. Here, we systematically mined and functionally characterized oxidosqualene cyclases (OSCs) and cytochrome P450s (CYPs) for synthesizing PTSs in A. elata. Our combined metabolic and transcriptomic analysis of A. elata, coupled with Agrobacterium tumefaciens-mediated heterologous expression in N. benthamiana, identified the α/β-amyrin and lupeol synthases, as well as the CYP716 enzymes, responsible for catalyzing the C-6, C-16, or C-28 oxidation of β-amyrin. Through combinatorial biosynthesis by strategic pairing the expression of OSCs and P450s, 19 distinct PTSs, fourteen of which were previously unidentified in A. elata, were synthesized in N. benthamiana. Our approach not only clarifies the biosynthetic pathways of PTSs in A. elata but also enhances their structural diversity and facilitates the generation of specific PTSs with potential application in various industries.