Oxidosqualene cyclase and CYP716 enzymes contribute to triterpene structural diversity in the medicinal tree banaba

First author: Sandeep; Affiliations: Council of Scientific and Industrial Research‐Central Institute of Medicinal and Aromatic Plants (科学与工业研究理事会-中央药用及芳香植物研究所): Lucknow, India

Corresponding author: Sumit Ghosh

Pentacyclic triterpenes (PCTs; 五环三萜类化合物) represent a major class of bioactive metabolites in banaba (Lagerstroemia speciosa) leaves; however, biosynthetic enzymes and their involvement in the temporal accumulation of PCTs remain to be studied. We use an integrated approach involving transcriptomics, metabolomics and gene function analysis to identify oxidosqualene cyclases (OSCs) and cytochrome P450 monooxygenases (P450s) that catalyzed sequential cyclization and oxidative reactions towards PCT scaffold diversification. Four monofunctional OSCs (LsOSC1,3–5) converted the triterpene precursor 2,3‐oxidosqualene to either lupeol (羽扇豆醇), β‐amyrin (β-香树脂醇) or cycloartenol (环阿乔醇), and a multifunctional LsOSC2 formed α‐amyrin as a major product along with β‐amyrin. Two CYP716 family P450s (CYP716A265, CYP716A266) catalyzed C‐28 oxidation of α‐amyrin, β‐amyrin and lupeol to form ursolic acid (熊果酸), oleanolic acid (齐墩果酸) and betulinic acid (桦木酸), respectively. However, CYP716C55 catalyzed C‐2α hydroxylation of ursolic acid and oleanolic acid to produce corosolic acid (科罗索酸) and maslinic acid (马斯里酸), respectively. Besides, combined transcript and metabolite analysis suggested major roles for the LsOSC2, CYP716A265 and CYP716C55 in determining leaf ursane and oleanane profiles. Combinatorial expression of OSCs and CYP716s in Saccharomyces cerevisiae and Nicotiana benthamiana led to PCT pathway reconstruction, signifying the utility of banaba enzymes for bioactive PCT production in alternate plant/microbial hosts that are more easily tractable than the tree species.

五环三萜类化合物代表了大花紫薇叶片中一类主要的具有生物活性的代谢物；然而，参与这类物质生物合成的酶及其在五环三萜类化合物随着时间在叶片中积累的作用还不清楚。作者通过整合转录组、代谢组及基因功能研究鉴定到了氧化角鲨烯环化酶OSC和细胞色素P450单加氧酶P450催化连续环化和氧化反应。LsOSC1、LsOSC3、LsOSC4和LsOSC5等四个单功能性OSC酶将三萜前体2,3‐氧鲨烯要么催化成羽扇豆醇，或是β-香树脂醇，亦或是环阿乔醇，而单功能性酶LsOSC2则主要催化形成α-香树脂醇和β-香树脂醇。两个CYP716家族的P450酶CYP716A265和CYP716A266将α-香树脂醇、β-香树脂醇和羽扇豆醇的第28号碳氧化，分别形成熊果酸、齐墩果酸和桦木酸。然而，CYP716C55催化熊果酸和齐墩果酸的C‐2α羟基化，分别产生科罗索酸和马斯里酸。除此之外，结合转录本和代谢物的分析显示LsOSC2、CYP716A265和CYP716C55主要参与叶片中的乌索烷和齐墩果烷谱。在酿酒酵母和烟草中表达OSCs和CYP716s能够重建PCT通路，说明了可以在其他易于处理的植物或微生物寄主中利用大花紫薇这些酶合成具有生物活性的PCT用于研究。

doi: https://doi.org/10.1111/nph.15606

Journal: New Phytologist

First Published: 24 November, 2018