Identification of 2,3-oxidosqualene cyclase gene in Eleutherococcus senticosus and its regulatory mechanism in saponin synthesis

Abstract

Oleanane-type triterpenoid saponins are the primary medicinal components of Eleutherococcus senticosus. During saponin biosynthesis in E. senticosus, various members of the 2,3-oxidosqualene cyclase (OSC) gene family can direct 2,3-oxidosqualene into triterpene saponin and sterol synthesis pathways. However, the precise molecular mechanism underlying this phenomenon remains unclear. We initially screened for β-amyrin synthase 1 (bAS1) and cycloartenol synthase 1 (CAS1) among 10 EsOSC genes using genome-wide identification and correlation analysis. Subcellular localization, catalytic experiments, and in vivo transient overexpression demonstrated that EsbAS1 and EsCAS1 catalyze the formation of the triterpene skeleton β-amyrin and sterol precursor cycloartenol exclusively in the cytoplasm, enhancing and inhibiting the in vivo biosynthesis of oleanane-type saponins, respectively. Results from site-directed mutagenesis and molecular docking indicated that W-WY and Y-WH triplets characterized the active sites of EsbAS1 and EsCAS1, respectively. GUS (β-glucuronidase) staining and electrophoretic mobility shift assay (EMSA) experiments on the promoter region revealed that various colored light quality, DNA methylation, and five transcription factors (EsNAC047, EsNAC098, EsWRKY40, EsMYB4, and EsERF66) regulated the expression of EsbAS1 and EsCAS1. This study provides preliminary insights into the molecular mechanisms by which EsbAS1 and EsCAS1 regulate saponin synthesis in E. senticosus.

Acknowledgements

The αAM2 strain of S. cerevisiae used in this study was provided by Associate Professor Yu Yuan from North China University of Science and Technology. We would like to express our gratitude here. This work was financially supported by the National Natural Science Foundation of China (32470398), the Central Guidance for Local Science and Technology Development Fund Projects (236Z2501G), and Natural Science Foundation of Hebei Province (H2020209033).

Author contributions

Y.C. designed the research, conducted the experiments, analyzed the genome and transcriptome, analyzed the data, and wrote the original draft. J.M., M.J., X.Z., J.D., C.F., and P.L. conducted the experiments. Y.L. and Z.X. provided experimental materials, designed the research, and revised and verified the manuscript.

Data availability

All data in this study were provided in the article and its supplementary materials.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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