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

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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

References

  • Alseekh S, Tohge T, Wendenberg R, Scossa F, Omranian N, Li J et al (2015) Identification and mode of inheritance of quantitative trait loci for secondary metabolite abundance in tomato. Plant Cell 27:485–512

    Article  CAS  Google Scholar 

  • Benveniste P (2004) Biosynthesis and accumulation of sterols. Annu Rev Plant Biol 55:429–457

    Article  CAS  Google Scholar 

  • Brachmann CB, Davies A, Cost GJ, Caputo E, Li JC, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132

    Article  CAS  Google Scholar 

  • Burke D, Dawson D, Stearns TIM (2000) Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Cardenas PD, Sonawane PD, Pollier J, Vanden Bossche R, Dewangan V, Weithorn E et al (2016) GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nat Commun 7:10654

    Article  CAS  Google Scholar 

  • Dai ZB, Liu Y, Huang LQ, Zhang XL (2012) Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae. Biotechnol Bioeng 109:2845–2853

    Article  CAS  Google Scholar 

  • Dai ZB, Liu Y, Zhang X, Shi MY, Wang BB, Wang D et al (2013) Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metab Eng 20:146–156

    Article  CAS  Google Scholar 

  • Donald KA, Hampton RY, Fritz IB (1997) Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae. Appl Environ Microb 63:3341–3344

    Article  CAS  Google Scholar 

  • Forli S, Botta M (2007) Lennard-Jones potential and dummy atom settings to overcome the AUTODOCK limitation in treating flexible ring systems. J Chem Inf Model 47:1481–1492

    Article  CAS  Google Scholar 

  • Gietz RD, Schiestl RH, Willems AR, Woods RA (2010) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360

    Article  Google Scholar 

  • Itkin M, Heinig U, Tzfadia O, Bhide AJ, Shinde B, Cardenas PD et al (2013) Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes. Science 341:175–179

    Article  CAS  Google Scholar 

  • Jiang Y, Li P, Li HJ, Yu H (2006) New steroidal alkaloids from the bulbs of Fritillaria puqiensis. Steroids 71:843–848

    Article  CAS  Google Scholar 

  • Li HJ, Jiang Y, Li P (2007) Chemistry, bioactivity and geographical diversity of steroidal alkaloids from the Liliaceae family. Nat Prod Rep 38:735–752

    Google Scholar 

  • Lin G, Li P, Li SL, Chan SW (2001) Chromatographic analysis of Fritillaria, isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb. J Chromatogr A 935:321–338

    Article  CAS  Google Scholar 

  • Lin BQ, Ji H, Li P, Jiang Y, Fang W (2006) Selective antagonism activity of alkaloids from bulbs Fritillariae at muscarinic receptors: functional studies. Eur J Pharmacol 551:125–130

    Article  CAS  Google Scholar 

  • Pollier J, Vancaester E, Kuzhiumparambil U, Vickers CE, Vandepoele K, Goossens A et al (2019) A widespread alternative squalene epoxidase participates in eukaryote steroid biosynthesis. Nat Microbiol 4:226–233

    Article  CAS  Google Scholar 

  • Schaller H (2004) New aspects of sterol biosynthesis in growth and development of higher plants. Plant Physiol Biochem 42:465–476

    Article  CAS  Google Scholar 

  • Schäfer UA, Reed DW, Hunter DG, Yao KN, Weninger AM, Tsang EWT et al (1999) An example of intron junctional sliding in the gene families encoding squalene monooxygenase homologues in Arabidopsis thaliana and Brassica Napus. Plant Mol Biol 39:721–728

    Article  Google Scholar 

  • Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385

    Article  CAS  Google Scholar 

  • Song Y, Song Q, Liu Y, Li J, Wan JB, Wang YT et al (2017) Integrated work-flow for quantitative metabolome profiling of plants, Peucedani Radix as a case. Anal Chim Acta 953:40–47

    Article  CAS  Google Scholar 

  • Sun C, Sun YQ, Song JY, Li CJ, Li XW, Zhang XW et al (2011) Discovery of genes related to steroidal alkaloid biosynthesis in Fritillaria cirrhosa by generating and mining a dataset of expressed sequence tags (ESTs). J Med Plants Res 5:5307–5314

    CAS  Google Scholar 

  • Wang L, Yao ZP, Li P, Chen SB, So PK, Shi ZQ et al (2016) Global detection and semi-quantification of Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus by a non-targeted multiple reaction monitoring approach. J Sep Sci 39:287–295

    Article  Google Scholar 

  • Xin GZ, Hu B, Shi ZQ, Lam YC, Dong TTX, Li P et al (2014) Rapid identification of plant materials by wooden-tip electrospray ionization mass spectrometry and a strategy to differentiate the bulbs of Fritillaria. Anal Chim Acta 820:84–91

    Article  CAS  Google Scholar 

  • Zhao Q, Li R, Zhang Y, Huang KJ, Wang WG, Li J (2018) Transcriptome analysis reveals in vitro-cultured regeneration bulbs as a promising source for targeted Fritillaria cirrhosa steroidal alkaloid biosynthesis. 3 Biotech 8:191

    Article  Google Scholar 

  • Zhao FL, Bai P, Liu T, Li DS, Zhang XM, Lu WY et al (2016) Optimization of a Cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae. Biotechnol Bioeng 113(8):1787–1795

    Article  CAS  Google Scholar 

  • Zhou JL, Xin GZ, Shi ZQ, Ren MT, Qi LW, Li HJ et al (2010) Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. J Chromatogr A 1217:7109–7712

    Article  CAS  Google Scholar 

Download references

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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Author notes

  1. Xu Lu, Lin-Ning Zhang, Jin-Fa Du have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing, China

    Xu Lu, Lin-Ning Zhang, Jin-Fa Du, Xiao-Yan Zheng, Hui-Jun Li, Ping Li & Gui-Zhong Xin

  2. Nanjing Forestry University, Nanjing, 210037, China

    Yan Jiang

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  1. Xu Lu
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Contributions

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, Gui-Zhong Xin or Yan Jiang.

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Lu, X., Zhang, LN., Du, JF. et al. Comparative analysis and natural evolution of squalene epoxidase in three Fritillaria species. Plant Mol Biol 103, 705–718 (2020). https://doi.org/10.1007/s11103-020-01021-y

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