Methods for Functional Validation of Terpenoid Metabolic Clusters in Nicotiana benthamiana and Aspergillus oryzae

Terpenoids represent one of the most structurally diverse classes of natural products, with vast potential applications in medicine, food, and agriculture. However, exploiting this potential has historically been challenging due to their low abundance in natural sources and the economic inefficiency of synthetic chemistry approaches for such complex structures. Heterologous biosynthesis offers a promising solution to these limitations. To date, multiple heterologous hosts have been developed for terpenoid production. Among these, Nicotiana benthamiana has emerged as a prominent platform for reconstituting plant-derived natural product pathways. Its compatibility with Agrobacterium-mediated transient expression, a scalable and flexible method, enables rapid integration and functional analysis of genes from diverse plant species. Conversely, Aspergillus oryzae has gained attention as a preferred host for fungal terpenoid pathways, owing to its highly efficient fungal expression system. Herein we present methodologies for functionally validating terpenoid biosynthetic clusters in N. benthamiana and A. oryzae.