Lotus japonicus Triterpenoid Profile and Characterization of the CYP716A51 and LjCYP93E1 Genes Involved in Their Biosynthesis In Planta

被引:32
作者
Suzuki, Hayato [1 ]
Fukushinna, Ery Odette [1 ,2 ]
Shimizu, Yuko [1 ]
Seki, Hikaru [1 ]
Fujisawa, Yukiko [3 ]
Ishimoto, Masao [3 ]
Osakabe, Keishi [4 ]
Osakabe, Yuriko [4 ]
Muranaka, Toshiya [1 ]
机构
[1] Osaka Univ, Grad Sch Engn, Dept Biotechnol, 2-1 Yamadaoka, Suita, Osaka 5650871, Japan
[2] Univ Reg Amazon IKIAM, Km 7 Via Muyuna, Napo, Tena, Ecuador
[3] NARO, Inst Crop Sci, 2-1-2 Kannondai, Tsukuba, Ibaraki 3058518, Japan
[4] Tokushima Univ, Grad Sch Technol Ind & Social Sci, Div Biosci & Bioind, Tokushima, Japan
关键词
Betulinic acid; CRISPR/Cas9; LORE1; Oleanolic acid; Soyasapogenols; Ursolic acid; SAPONIN BIOSYNTHESIS; BETA-AMYRIN; MEDICAGO-TRUNCATULA; COMBINATORIAL BIOSYNTHESIS; OXIDOSQUALENE CYCLASES; ARABIDOPSIS-THALIANA; STRUCTURAL-ANALYSIS; SEQUENCE; GENOME; CYTOCHROME-P450;
D O I
10.1093/pcp/pcz145
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus 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 alpha-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted beta-amyrin into 24-hydroxy-beta-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 analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the 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.
引用
收藏
页码:2496 / 2509
页数:14
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