Adaptive evolution of oxidosqualene cyclases driving triterpene scaffold diversification for limonoid biosynthesis in Melia toosendan

Limonoids are a structurally diverse class of highly oxidized triterpenoids produced by Meliaceae and Rutaceae species. They exhibit remarkable biological activities but are difficult to produce due to their complex biosynthesis. Plants evolve ecologically adaptive specialized metabolites through the amplification and functional differentiation of gene families, yet our understanding of the molecular mechanisms remains very limited. A chromosome-level genome assembly of Melia toosendan (219.5 Mb) showed no recent whole-genome duplication, and we annotated 14 key enzymes for triterpenoid biosynthesis, the oxidosqualene cyclases (OSCs). Transient expression of these OSCs in Nicotiana benthamiana produced 11 distinct triterpene skeletons, including eupha-7,24-dien-3β-ol, the product of MtOSC10. Phylogenetic analysis and dN/dS ratio assessments suggest that this OSC lineage may have undergone neofunctionalization events, correlating with signals of strong positive selection. Ancestral sequence reconstruction traced the divergence of an ancestral β-amyrin synthase into two evolutionary lineages: one lineage retains the conserved activity of tirucalla-7,24-dien-3β-ol synthase, which produces the canonical limonoid precursor, and the other diversifies into novel eupha-7,24-dien-3β-ol synthases that produce a hypothetical alternative precursor. Strikingly, four key amino acid residue substitutions (W258L, T413S, M730Y, L735H) in tirucalla-7,24-dien-3β-ol synthase were sufficient to switch MtOSC1 product specificity from tirucalla-7,24-dien-3β-ol to eupha-7,24-dien-3β-ol. Our findings uncover a potential alternative pathway for limonoid biosynthesis and reveal the molecular basis of triterpene skeleton diversification in Meliaceae. More broadly, they illustrate how neofunctionalization of OSCs under positive selection drives metabolic innovation across plant lineages. The finding also provides a foundation for synthetic biology approaches to engineer plant-derived insecticides.