Tandem duplication of P450 genes is involved in homoterpene biosynthesis in lima bean (Phaseolus lunatus).

Background: The irregular acyclic homoterpenes 4,8-dimethyl-1,3,7-nonatriene (DMNT) and 4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) are key volatile compounds involved in various chemical ecological interactions, particularly contributing to plant defense against phytophagous insects. However, the genomic basis and evolutionary history of homoterpene biosynthesis in legumes remain largely unexplored, limiting our understanding of their functional diversification and ecological roles.

Results: Using a yeast expression system, we identified two novel, tandemly duplicated cytochrome P450 genes, PlCYP82D47-like and PlCYP82D47, involved in DMNT and TMTT biosynthesis in lima bean (Phaseolus lunatus). PlCYP82D47-like catalyzes the conversion of (E)-nerolidol and (E,E)-geranyllinalool to DMNT and TMTT, respectively, while PlCYP82D47 specifically converts (E)-nerolidol to DMNT. Evolutionary analysis revealed syntenic tandem duplicates of these genes in lima bean, cotton, and Arabidopsis, suggesting a conserved genomic architecture for DMNT/TMTT biosynthesis among dicotyledonous plants and implying potential gene loss events in this genomic region. Computational modeling identified residues L324 and L505 as critical for substrate recognition by PlCYP82D47. Site-directed mutagenesis and in vitro enzymatic assays confirmed that substitutions at these residues significantly reduced catalytic activity.

Conclusion: Our findings provide new insights into the enzymatic and evolutionary basis of homoterpene biosynthesis in legumes and offer promising targets for future metabolic engineering applications. © 2025 Society of Chemical Industry.