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Identification and functional characterization of squalene epoxidases and oxidosqualene cyclases from Tripterygium wilfordii

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Abstract

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We cloned two squalene epoxidases and five oxidosqualene cyclases, and identified their function using CRISPR/Cas9 tool and yeast heterologous expression.

Abstract

Triterpenes are the main active ingredients of Tripterygium wilfordii Hook.f., a traditional Chinese medicinal plant with many encouraging preclinical applications. However, the biosynthetic pathways of triterpenes in this plant are poorly understood. Here, we report on the isolation and identification of two squalene epoxidases (SQE6 and SQE7) and five oxidosqualene cyclases (OSC4-8) from T. wilfordii. Yeast complementation assays showed that TwSQE6 and TwSQE7 can functionally complement an erg1 yeast mutant that was constructed using the CRISPR/Cas9 system. The putative OSC genes were functionally characterized by heterologous expression in yeast. GC/MS analysis of the fermentation products of the transgenic yeast showed that both TwOSC4 and TwOSC6 are cycloartenol synthases, while TwOSC8 is a β-amyrin synthase. The discovery of these genes expands our knowledge of key enzymes in triterpenoid biosynthesis, and provides additional target genes for increasing the production of triterpenes in T. wilfordii tissue cultures by disrupting competing pathways, or in chassis cells by reconstituting the triterpenoid biosynthetic pathway.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 81773830 and no. 81973418), Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ201710025022), the Support Project for High-level Teachers in Beijing Municipal Universities in the Period of the 13th Five-year Plan (CIT&TCD20170324) to W.G., the Key Project at central government level: the ability establishment of sustainable use for valuable Chinese medicine resources (2060302-1806-03).

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Authors and Affiliations

  1. School of Traditional Chinese Medicine, Capital Medical University, Beijing, 100069, People’s Republic of China

    Yuan Liu, Jiawei Zhou, Tianyuan Hu, Yun Lu, Linhui Gao, Lichan Tu, Jie Gao & Wei Gao

  2. School of Pharmaceutical Sciences, Capital Medical University, Beijing, 100069, People’s Republic of China

    Jiawei Zhou, Tianyuan Hu, Lichan Tu & Wei Gao

  3. National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, People’s Republic of China

    Linhui Gao & Luqi Huang

  4. Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, 10069, People’s Republic of China

    Wei Gao

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  1. Yuan Liu
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  9. Wei Gao
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Contributions

YL(Liu) and WG, LH designed this study; YL, JZ, TH, YL(Lu) carried out the experiments and performed results analysis; LG, LT, JG interpreted the data; YL(Liu) and JZ wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Wei Gao.

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Communicated by Chun-Hai Dong.

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Liu, Y., Zhou, J., Hu, T. et al. Identification and functional characterization of squalene epoxidases and oxidosqualene cyclases from Tripterygium wilfordii. Plant Cell Rep 39, 409–418 (2020). https://doi.org/10.1007/s00299-019-02499-7

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  • DOI: https://doi.org/10.1007/s00299-019-02499-7

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