Cloning, Yeast Expression, and Characterization of a beta-Amyrin C-28 Oxidase (CYP716A249) Involved in Triterpenoid Biosynthesis in Polygala tenuifolia

文献类型： 外文期刊

第一作者： Zhang, Fu-Sheng

作者： Zhang, Fu-Sheng;Zhang, Xuan;Wang, Qian-Yu;Pu, Ya-Jie;Qin, Xue-Mei;Zhang, Xuan;Wang, Qian-Yu;Pu, Ya-Jie;Du, Chen-Hui;Ma, Cun-Gen;Tian, Hong-Ling;Lian, Yun-Lan;Li, Min-Sheng;Chen, Yu

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关键词： Polygala tenuifolia; C-28 oxidase; CYP monooxygenase; yeast; biosynthesis; triterpene saponin

期刊名称：BIOLOGICAL & PHARMACEUTICAL BULLETIN （ 影响因子：2.233； 五年影响因子：2.309 ）

ISSN： 0918-6158

年卷期： 2020 年 43 卷 12 期

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收录情况： SCI

摘要： Polygala tenuifolia Willd. is a traditional Chinese herbal medicine that is widely used in treating nervous system disorders. Triterpene saponins in P. tenuifolia (polygala saponins) have excellent biological activity. As a precursor for the synthesis of presenegin, oleanolic acid (OA) plays an important role in the biosynthesis of polygala saponins. However, the mechanism behind the biosynthesis of polygala saponins remains to be elucidated. In this study, we found that CYP716A249 (GenBank: ASB17946) oxidized the C-28 position of beta-amyrin to produce OA. Using quantitative real-time PCR, we observed that CYP716A249 had the highest expression in the roots of 2-year-old P. tenuifolia, which provided a basis for the selection of samples for gene cloning. To identify the function of CYP716A249, the strain R-BE-20 was constructed by expressing beta-amyrin synthase in yeast. Then, CYP716A249 was co-expressed with beta-amyrin synthase to construct the strain R-BPE-20 by using the lithium acetate method. Finally, we detected beta-amyrin and OA by ultra-HPLC-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry and GC-MS. The results of this study provide insights into the biosynthesis pathway of polygala saponins.

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