Adaptive evolution of oxidosqualene cyclases has driven diversification of triterpene scaffolds 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 owing to their complex biosynthesis. Plants have evolved ecologically adaptive specialized metabolites through the amplification and functional differentiation of gene families, but our understanding of the molecular mechanisms that underlie this process remains very limited. A chromosome-level genome assembly of Melia toosendan (219.5 Mb) shows no recent whole-genome duplications and contains 14 genes predicted to encode oxidosqualene cyclases (OSCs), key enzymes in triterpenoid biosynthesis. 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 examination of the ratio of non-synonymous to synonymous substitution rates (dN/dS) suggested that this OSC lineage may have undergone neofunctionalization correlated with signals of strong positive selection. Ancestral sequence reconstruction traced the divergence of an ancestral β-amyrin synthase into two evolutionary lineages: one lineage has retained the conserved activity of tirucalla-7,24-dien-3β-ol synthase, which produces the canonical limonoid precursor, and the other has diversified into novel eupha-7,24-dien-3β-ol synthases that produce a hypothetical alternative precursor. Strikingly, four key amino acid substitutions (W258L, T413S, M730Y, and L735H) in tirucalla-7,24-dien-3β-ol synthase are sufficient to switch the product specificity of MtOSC1 from tirucalla-7,24-dien-3β-ol to eupha-7,24-dien-3β-ol. Our findings identify 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 has driven metabolic innovation across plant lineages. These findings also provide a foundation for engineering plant-derived insecticides through synthetic biology approaches.