Lotus japonicus triterpenoid profile and characterization of the CYP716A51 and LjCYP93E1 genes involved in their biosynthesis in planta.

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, compared to Medicago truncatula and Glycine max, less information regarding its composition, content, and biosynthesis is available. In this study, we analyzed the triterpenoid content and composition of L. japonicus. L. japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic, and oleanolic acids) and soyasapogenols (soyasapogenol B, A, and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted β-amyrin into 24-hydroxy-β-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR (qPCR) analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.