State Key Laboratory of Phytochemistry and Plant Resources in West China, and Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, P. R. China.
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China.
Chem Biodivers. 2021 Jul;18(7):e2100342. doi: 10.1002/cbdv.202100342. Epub 2021 Jun 17.
Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. is a precious traditional Chinese medicine, and steroidal saponins are its major bioactive constituents possessing extensive biological activities. Squalene synthase (SQS) catalyzes the first dedicated step converting two molecular of farnesyl diphosphate (FDP) into squalene, a key intermediate in the biosynthetic pathway of steroidal saponins. In this study, a squalene synthase gene (PpSQS1) was cloned and functionally characterized from P. polyphylla var. yunnanensis, representing the first identified SQS from the genus Paris. The open reading frame of PpSQS1 is 1239 bp, which encodes a protein of 412 amino acids showing high similarity to those of other plant SQSs. Expression of PpSQS1 in Escherichia coli resulted in production of soluble recombinant proteins. Gas chromatography-mass spectrometry analysis showed that the purified recombinant PpSQS1 protein could produce squalene using FDP as a substrate in the in vitro enzymatic assay. qRT-PCR analysis indicated that PpSQS1 was highly expressed in rhizomes, consistent with the dominant accumulation of steroidal saponins there, suggesting that PpSQS1 is likely involved in the biosynthesis of steroidal saponins in the plant. The findings lay a foundation for further investigation on the biosynthesis and regulation of steroidal saponins, and also provide an alternative gene for manipulation of steroid production using synthetic biology.
云南重楼（Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz.）是一种珍贵的传统中药，甾体皂苷是其主要的生物活性成分，具有广泛的生物活性。鲨烯合酶（SQS）催化将两个法呢基二磷酸（FDP）分子转化为鲨烯的第一个专用步骤，鲨烯是甾体皂苷生物合成途径中的关键中间体。本研究从云南重楼中克隆并功能表征了一种鲨烯合酶基因（PpSQS1），这是巴黎属中第一个鉴定的 SQS。PpSQS1 的开放阅读框为 1239bp，编码 412 个氨基酸的蛋白质，与其他植物 SQS 高度相似。PpSQS1 在大肠杆菌中的表达导致产生可溶性重组蛋白。气相色谱-质谱分析表明，纯化的重组 PpSQS1 蛋白可以在体外酶促测定中使用 FDP 作为底物产生鲨烯。qRT-PCR 分析表明，PpSQS1 在根茎中高度表达，与甾体皂苷在那里的优势积累一致，表明 PpSQS1 可能参与植物中甾体皂苷的生物合成。这些发现为进一步研究甾体皂苷的生物合成和调控奠定了基础，也为使用合成生物学操纵甾体产生提供了替代基因。
