Plant Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India.
Plant J. 2024 Sep;119(6):2687-2705. doi: 10.1111/tpj.16942. Epub 2024 Jul 29.
Triterpenoids (C30-isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated β-amyrin-derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C-2α and C-23 hydroxylases/oxidases than C-28 oxidases. CYP716A233 and CYP716A432 catalyzed β-amyrin/erythrodiol C-28 oxidation to produce oleanolic acid. C-2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C-23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C-23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C-23 hydroxylation. Overall, P450 substrate selectivity suggested that C-28 oxidation is the first P450-catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C-2α/C-23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds.
三萜类化合物（C30-异戊二烯）是植物中具有各种生理功能的天然产物的主要类别。三萜类化合物及其衍生物具有多种生物活性，因此具有药用价值。诃子树（Terminalia arjuna）树皮中积累了高度氧化的 β-香树脂烷衍生的齐墩果烷型三萜类化合物（如，诃黎酸、诃黎宁和齐墩果酸），具有心脏保护作用。然而，其生物合成途径和酶仍然知之甚少。我们从诃子转录组中挖掘信息，并使用酿酒酵母和黄花烟草进行细胞色素 P450 单加氧酶（P450）测定，以鉴定出 6 种 P450 和 2 种 P450 还原酶，用于氧化修饰齐墩果烷型三萜类化合物。使用齐墩果烷的 P450 测定表明，C-2α 和 C-23 羟化酶/加氧酶对甾体的底物选择性高于 C-28 氧化酶。CYP716A233 和 CYP716A432 催化 β-香树脂烷/赤酮醇 C-28 氧化生成齐墩果酸。C-2α 羟化酶（CYP716C88 和 CYP716C89）将齐墩果酸和何帕烯酸转化为熊果酸和齐墩果酸。CYP716C89 还可将赤酮醇和齐墩果酸醛羟化。然而，CYP714E107a 和 CYP714E107b 催化齐墩果酸/熊果酸/诃黎酸，C-23 羟化生成何帕烯酸、齐墩果酸和诃黎宁，以及何帕烯酸 C-23 氧化生成吉普赛酸，但速率低于齐墩果酸 C-23 羟化。总体而言，P450 底物选择性表明 C-28 氧化是诃子三萜类化合物途径中第一个 P450 催化的氧化修饰。然而，此后该途径可能通过齐墩果酸的 C-2α/C-23 羟化而分支。综上所述，这些结果为 P450 的底物范围提供了新的见解，并阐明了诃子三萜类化合物的生物合成途径。此外，在酿酒酵母和黄花烟草中完整阐明和重建了齐墩果酸途径，表明诃子 P450 可用于异源生产心脏保护化合物。
