Journal of Ginseng Research

The Korean Society of Ginseng (고려인삼학회)
권호 표지
DOI QR코드

DOI QR Code

Modification of ginsenoside saponin composition via the CRISPR/Cas9-mediated knockout of protopanaxadiol 6-hydroxylase gene in Panax ginseng

  • Choi, Han Suk (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Koo, Hyo Bin (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Jeon, Sung Won (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Han, Jung Yeon (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University) ;
  • Kim, Joung Sug (Department of Biosciences and Bioinformatics, Myongji University) ;
  • Jun, Kyong Mi (Plant Molecular Genetics Institute, GreenGene Biotech Inc.) ;
  • Choi, Yong Eui (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University)
  • Received : 2020.09.04
  • Accepted : 2021.06.09
  • Published : 2022.07.01
Download PDF
⟨ Previous Next ⟩

Abstract

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

Keywords

Acknowledgement

This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01344401) of the Rural Development Administration, and by R&D Program for Forest Science Technology (Project No. 2021339A00-2123-CD02) provided by Korea Forest Service (Korea Forestry Promotion Institute), Republic of Korea.

References

  1. Hostettmann KA, Marston A. Saponins. Chemistry and pharmacology of natural products. Cambridge: United Kingdom: Cambridge University Press; 1995.
  2. Vogler BK, Pittler MH, Ernst E. The efficacy of ginseng. A systematic review of randomised clinical trials. Eur J Clin Pharmacol 1999;55:567-75. https://doi.org/10.1007/s002280050674
  3. Shibata S. Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds. J Korean Med Sci 2001;16:S28-37. https://doi.org/10.3346/jkms.2001.16.s.s28
  4. Han JY, Kwon YS, Yang DC, Jung YR, Choi YE. Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng. Plant Cell Physiol 2006;47:1653-62. https://doi.org/10.1093/pcp/pcl032
  5. Han JY, Kim HJ, Kwon YS, Choi YE. The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 2011;52:2062-73. https://doi.org/10.1093/pcp/pcr150
  6. Han JY, Hwang HS, Choi SW, Kim HJ, Choi YE. Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 2012;53:1535-45. https://doi.org/10.1093/pcp/pcs106
  7. Yan X, Fan Y, Wei W, Wang P, Liu Q, Wei Y, Zhang L, Zhao G, Yue J, Zhou Z. Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 2014;24:770-3. https://doi.org/10.1038/cr.2014.28
  8. Mao Q, Zhang PH, Wang Q, Li SL. Ginsenoside F2 induces apoptosis in humor gastric carcinoma cells through reactive oxygen species-mitochondria pathway and modulation of ASK-1/JNK signaling cascade in vitro and in vivo. Phytomedicine 2014;21(4):515-22. https://doi.org/10.1016/j.phymed.2013.10.013
  9. Ahuja A, Kim JH, Yi YS, Cho JY. Functional role of ginseng-derived compounds in cancer. J Ginseng Res 2018;42(3):248-54. https://doi.org/10.1016/j.jgr.2017.04.009
  10. Jin J, Shahi S, Kang HK, van Veen HW, Fan T-P. Metabolites of ginsenosides as novel BCRP inhibitors. Biochem Biophys Res Commun 2006;345(4):1308-14. https://doi.org/10.1016/j.bbrc.2006.04.152
  11. Shim SH, Baek K-H, Kim YS. Inhibition of human 20S proteasome by ginsenosides from Panax ginseng. Bull Korean Chem Soc 2009;30:1385-7. https://doi.org/10.5012/bkcs.2009.30.6.1385
  12. Lee JI, Ha YW, Choi TW, et al. Cellular uptake of ginsenosides in Korean white ginseng and red ginseng and their apoptotic activities in human breast cancer cells. Planta Med 2011;77(2):133-40. https://doi.org/10.1055/s-0030-1250160
  13. Chen XJ, Zhang XJ, Shui YM, Wan JB, Gao JL. Anticancer activities of protopanaxadiol- and protopanaxatriol-type ginsenosides and their metabolites. Evid-Based Complement Altern Med 2016;2016:1-19.
  14. Helms S. Cancer prevention and therapeutics: Panax ginseng. Altern Med Rev 2004;9:259-74.
  15. Wang M, Li H, Liu W, Cao H, Hu X, Gao X, Xu F, Li Z, Hua H, Li D. Dammarane-type leads panaxadiol and protopanaxadiol for drug discovery: biological activity and structural modification. Eur J Med Chem 2020;189:112087. https://doi.org/10.1016/j.ejmech.2020.112087
  16. Begemann MB, Gray BN, January E, Gordon GC, He Y, Liu H, Wu X, Brutnell TP, Mockler TC, Oufattole M. Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases. Sci Rep 2017;7:11606. https://doi.org/10.1038/s41598-017-11760-6
  17. Petolino JF, Srivastava V, Daniell H. Editing Plant Genomes: a new era of crop improvement. Plant Biotechnol J 2016;14:435-6. https://doi.org/10.1111/pbi.12542
  18. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue. Physiol Plant 1962;15:473-97. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  19. Kim JY, Adhikari PB, Ahn CH, Kim DH, Kim YC, Han JY, et al. High frequency somatic embryogenesis and plant regeneration of interspecific ginseng hybrid between Panax ginseng and Panax quinquefolius. J Ginseng Res 2019;43:38-48. https://doi.org/10.1016/j.jgr.2017.08.002
  20. Park J, Lim K, Kim JS, Bae S. Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 2017;33:286-8. https://doi.org/10.1093/bioinformatics/btw561
  21. Choi HI, Waminal NE, Park HM, Kim NH, Choi BS, Park M, Choi D, Lim YP, Kwon SJ, Park BS, et al. Major repeat components covering one-third of the ginseng (Panax ginseng C.A. Meyer) genome and evidence for allotetraploidy. Plant J 2014;77:906-16. https://doi.org/10.1111/tpj.12441
  22. Jayakodi M, Choi BS, Lee SC, Kim NH, Park JY, Jang W, et al. Ginseng Genome Database: an open-access platform for genomics of Panax ginseng. BMC Plant Biology 2018;18:62. https://doi.org/10.1186/s12870-018-1282-9
  23. Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 2014;30:1473-5. https://doi.org/10.1093/bioinformatics/btu048
  24. Choi HW, Koo DH, Bang KH, Paek KY, Seong NS, Bang JW. FISH and GISH analysis of the genomic relationships among Panax species. Genes Genom 2009;31:99-105. https://doi.org/10.1007/BF03191143
  25. Park SB, Chun JH, Ban YW, Han JY, Choi YE. Alteration of Panax ginseng saponin composition by overexpression and RNA interference of the protopanaxadiol 6-hydroxylase gene (CYP716A53v2). J Ginseng Res 2016;40:47-54. https://doi.org/10.1016/j.jgr.2015.04.010