Comparative analysis and natural evolution of squalene epoxidase in three Fritillaria species

Abstract

Fritillariae Bulbus are the most commonly used antitussive and edible herbs in China. Based on UPLC-QTOF-MS and UPLC-QQQ-MS, the validated MRM-based non-targeted quantitative method was applied to determinate the contents of 48 Fritillaria alkaloids (FAs) in three Fritillaria species (F. thunbergii Miq., F. unibracteata and F. ussuriensis). The RNA-Seq results showed that gene transcript levels have different expression patterns in three Fritillaria species. Based on transcriptome data, the full-length cDNA sequences of squalene epoxidase gene were cloned and characterized. Natural evolution of squalene epoxidase genes resulted in four mutations (C236R, M489L, G510A and K517R) in three Fritillaria species. Molecular docking analysis showed that the 236 residue is located inside the pocket and the binding center while other three residues are located on the surface of the protein. Functional verification indicated the mutations of SQE (C236R) could effectively increase the activity of SQE and obtain higher yield of 2,3-oxidosqualene in recombinant yeast. And the mutations of SQE (M489L and G510A), which increased the hydrophobicity of the protein surface, could also enhance the activity of SQE. This study provides major insights into the metabolites differentiation of FAs biosynthesis, and a firm foundation for the quality control and metabolic engineering of Fritillariae bulbus.

Key message

Deeper insights into the differentiation of metabolomics and transcriptomics and natural mutations of squalene epoxidase genes in three Fritillaria Bulbus.

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Abbreviations

FAs:

Fritillaria Alkaloids

Ft:

F. thunbergii Miq.

Fun:

F. unibracteata

Fus:

F. ussuriensis

UPLC-QTOF-MS:

Ultra performance liquid chromatography-quadrupole time of flight-mass spectrometry

UPLC-QQQ-MS:

Ultra performance liquid chromatography-triple quadrupole-mass spectrometry

IPP:

Isopentenyl diphosphate

DMAPP:

Dimethylallyl diphosphate

IDI:

Isopentenyl diphosphate isomerase

FPS:

Farnesyl diphosphate synthase

SQS:

Squalene synthase

SQE:

Squalene epoxidase

CAS:

Cycloartenol synthase

HMGR:

3-Hydroxy-3-methylglutaryl-CoA reductase

MK:

Mevalonate kinase

MDD:

Mevalonate pyrophosphate decarboxylase

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Acknowledgements

We acknowledge Prof. Zhang Xueli for providing the plasmid of pRS313, and the Biotechnology Research Institute at the Chinese Academy of Agricultural Sciences for its assistance in original data processing and related bioinformatics analysis. This work was supported by the National Key R&D Program of China (2019YFC1711000), National Natural Science Foundation of China (Grant Nos. 81773872, 81322051 and 81973414), Natural Science Foundation of Jiangsu Province (Grant No. BK20191319), Fundamental Research Funds for the Central Universities (2632019ZD15), "Double First-Class" University project (CPU2018GY09) and “111” project (Grant No. B16046).

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PL, GZX and YJ conceived and designed this research. XL, LNZ and JFD investigated, analyzed data and wrote the manuscript. XYZ participated in the discussion of the results. HJL contributed to the evaluation and discussion of the results and manuscript revision.

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Correspondence to Ping Li or Gui-Zhong Xin or Yan Jiang.

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Lu, X., Zhang, L., Du, J. et al. Comparative analysis and natural evolution of squalene epoxidase in three Fritillaria species. Plant Mol Biol (2020). https://doi.org/10.1007/s11103-020-01021-y

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Keywords

  • Fritillaria
  • Submetabolome
  • Transcriptomics
  • Squalene epoxidase
  • Natural evolution
  • Molecular docking