Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides

Abstract

Main conclusion

Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-β-glucuronide.

Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-β-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-β-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.

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Abbreviations

OAGT:

Oleanolic acid glucuronosyltransferase

PPD:

Protopanaxadiol

PPT:

Protopanaxatriol

RPKM:

Reads per kilobase per million mapped reads

UGT:

Uridine diphosphate-dependent glycosyltransferases

UPLC–QTOF–MS:

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

References

  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    CAS  Article  Google Scholar 

  2. Augustin JM, Drok S, Shinoda T, Sanmiya K, Nielsen JK, Khakimov B, Olsen CE, Hansen EH, Kuzina V, Ekstrøm CT, Hauser T, Bak S (2012) UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance. Plant Physiol 160:1881–1895

    CAS  Article  Google Scholar 

  3. Bowles D, Isayenkova J, Lim EK, Poppenberger B (2005) Glycosyltransferases: managers of small molecules. Curr Opin Plant Biol 8:254–263

    CAS  Article  Google Scholar 

  4. Chan HH, Hwang TL, Reddy MV, Li DT, Qian K, Bastow KF, Lee KH, Wu TS (2011) Bioactive constituents from the roots of Panax japonicus var. major and development of a LC–MS/MS method for distinguishing between natural and artifactual compounds. J Nat Prod 74:796–802

    CAS  Article  Google Scholar 

  5. Chen RJ, Chung TY, Li FY, Lin NH, Tzen JT (2009) Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity. Acta Pharmacol Sin 30:61–69

    Article  Google Scholar 

  6. Chen S, Luo H, Li Y, Sun Y, Wu Q, Niu Y, Song J, Lv A, Zhu Y, Sun C, Steinmetz A, Qian Z (2011) 454 EST analysis detects genes putatively involved in ginsenoside biosynthesis in Panax ginseng. Plant Cell Rep 30:1593–1601

    CAS  Article  Google Scholar 

  7. Chen W, Kui L, Zhang GH, Zhu SS, Zhang JJ, Wang X, Yang M, Huang H, Liu Y, Wang Y, Li Y, Zeng L, Wang W, He X, Dong Y, Yang SC (2017) Whole-genome sequencing and analysis of the Chinese herbal plant Panax notoginseng. Mol Plant 10:899–902

    CAS  Article  Google Scholar 

  8. Christensen LP (2008) Ginsenosides chemistry, biosynthesis, analysis, and potential health effects. Adv Food Nutr Res 55:1–99

    Article  Google Scholar 

  9. Dai Z, Liu Y, Zhang X, Shi M, Wang B, Wang D, Huang L, Zhang X (2013) Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metab Eng 20:146–156

    CAS  Article  Google Scholar 

  10. Dai Z, Wang B, Liu Y, Shi M, Wang D, Zhang X, Liu T, Huang L, Zhang X (2014) Producing aglycons of ginsenosides in bakers’ yeast. Sci Rep 4:3698

    Article  Google Scholar 

  11. de Costa F, Barber CJS, Kim YB, Reed DW, Zhang H, Fett-Neto AG, Covello PS (2017) Molecular cloning of an ester-forming triterpenoid: UDP-glucose 28-O-glucosyltransferase involved in saponin biosynthesis from the medicinal plant Centella asiatica. Plant Sci 262:9–17

    Article  Google Scholar 

  12. Fu L, Jin J (1992) China plant red data book. Rare and endangered plants, vol 1. Science Press, Beijing

    Google Scholar 

  13. Gao ZJ (2016) Comparative transcriptome analysis of rhizome swelling molecular mechanisms in Panax japonicus var. major. Yunnan Agricultural University, Kunming

    Google Scholar 

  14. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652

    CAS  Article  Google Scholar 

  15. Han JY, Kim HJ, Kwon YS, Choi YE (2011) The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 52:2062–2073

    CAS  Article  Google Scholar 

  16. Han JY, Hwang HS, Choi SW, Kim HJ, Choi YE (2012) Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 53:1535–1545

    CAS  Article  Google Scholar 

  17. Han JY, Kim MJ, Ban YW, Hwang HS, Choi YE (2013) The involvement of β-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 54:2034–2046

    CAS  Article  Google Scholar 

  18. Haralampidis K, Trojanowska M, Osbourn AE (2002) Biosynthesis of triterpenoid saponins in plants. Adv Biochem Eng Biotechnol 75:31–49

    CAS  PubMed  Google Scholar 

  19. He SM, Song WL, Cong K, Wang X, Dong Y, Cai J, Zhang JJ, Zhang GH, Yang JL, Yang SC, Fan W (2017) Identification of candidate genes involved in isoquinoline alkaloids biosynthesis in Dactylicapnos scandens by transcriptome analysis. Sci Rep 7:9119

    Article  Google Scholar 

  20. Jung SC, Kim W, Park SC, Jeong J, Park MK, Lim S, Lee Y, Im WT, Lee JH, Choi G, Kim SC (2014) Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd. Plant Cell Physiol 55:2177–2188

    CAS  Article  Google Scholar 

  21. Kim YJ, Zhang D, Yang DC (2015) Biosynthesis and biotechnological production of ginsenosides. Biotechnol Adv 33:717–735

    CAS  Article  Google Scholar 

  22. Kurosawa Y, Takahara H, Shiraiwa M (2002) UDP-glucuronic acid: soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds. Planta 215:620–629

    CAS  Article  Google Scholar 

  23. Kushiro T, Shibuya M, Ebizuka Y (1998) β-Amyrin synthase cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur J Biochem 256:238–244

    CAS  Article  Google Scholar 

  24. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948

    CAS  Article  Google Scholar 

  25. Liang YL, Zhao SJ, Xu LX, Zhang XY (2012) Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae. Lett Appl Microbiol 55:323–329

    CAS  Article  Google Scholar 

  26. Liang C, Ding Y, Song SB, Kim JA, Cuong NM, Ma JY, Kim YH (2013) Oleanane-triterpenoids from Panax stipuleanatus inhibit NF-κB. J Ginseng Res 37:74–79

    CAS  Article  Google Scholar 

  27. Liang Z, Chen Y, Xu L, Qin M, Yi T, Chen H, Zhao Z (2015) Localization of ginsenosides in the rhizome and root of Panax ginseng by laser microdissection and liquid chromatography–quadrupole/time of flight–mass spectrometry. J Pharm Biomed Anal 105:121–133

    CAS  Article  Google Scholar 

  28. Liu MH, Yang BR, Cheung WF, Yang KY, Zhou HF, Kwok JS, Liu GC, Li XF, Zhong S, Lee SM, Tsui SK (2015) Transcriptome analysis of leaves, roots and flowers of Panax notoginseng identifies genes involved in ginsenoside and alkaloid biosynthesis. BMC Genom 16:265–276

    Article  Google Scholar 

  29. Liu X, Cheng J, Zhang GH, Ding W, Duan L, Yang J, Kui L, Cheng X, Ruan J, Fan W, Chen J, Long G, Zhao Y, Cai J, Wang W, Ma Y, Dong Y, Yang SC, Jiang HF (2018) Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches. Nat Commun 9:448–457

    Article  Google Scholar 

  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    CAS  Article  Google Scholar 

  31. Lu C, Zhao S, Wei G, Zhao H, Qu Q (2017) Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius. Plant Physiol Biochem 111:67–76

    CAS  Article  Google Scholar 

  32. Luo H, Sun C, Sun Y, Wu Q, Li Y, Song J, Niu Y, Cheng X, Xu H, Li C, Liu J, Steinmetz A, Chen S (2011) Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers. BMC Genom 12:S5

    CAS  Article  Google Scholar 

  33. Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Bélanger A, Fournel-Gigleux S, Green M, Hum DW, Iyanagi T, Lancet D, Louisot P, Magdalou J, Chowdhury JR, Ritter JK, Schachter H, Tephly TR, Tipton KF, Nebert DW (1997) The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 7:255–569

    CAS  Article  Google Scholar 

  34. Meesapyodsuk D, Balsevich J, Reed DW, Covello PS (2007) Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding β-amyrin synthase and a triterpene carboxylic acid glucosyltransferase. Plant Physiol 143:959–969

    CAS  Article  Google Scholar 

  35. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods 5:621–628

    CAS  Article  Google Scholar 

  36. Nag SA, Qin JJ, Wang W, Wang MH, Wang H, Zhang R (2012) Ginsenosides as anticancer agents: in vitro and in vivo activities, structure-activity relationships, and molecular mechanisms of action. Front Pharmacol 3:25

    Article  Google Scholar 

  37. Naoumkina MA, Modolo LV, Huhman DV, Urbanczyk-Wochniak E, Tang Y, Sumner LW, Dixon RA (2010) Genomic and coexpression analyses predict multiple genes involved in triterpene saponin biosynthesis in Medicago truncatula. Plant Cell 22:850–866

    CAS  Article  Google Scholar 

  38. Noguchi A, Horikawa M, Fukui Y, Fukuchi-Mizutani M, Iuchi-Okada A, Ishiguro M, Kiso Y, Nakayama T, Ono E (2009) Local differentiation of sugar donor specificity of flavonoid glycosyltransferase in Lamiales. Plant Cell 21:1556–1572

    CAS  Article  Google Scholar 

  39. Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, Lee Y, White J, Cheung F, Parvizi B, Tsai J, Quackenbush J (2003) TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics 19(5):651–652

    CAS  Article  Google Scholar 

  40. Qi LW, Wang CZ, Yuan CS (2011) Ginsenosides from American ginseng: chemical and pharmacological diversity. Phytochemistry 72:689–699

    CAS  Article  Google Scholar 

  41. Rai A, Yamazaki M, Takahashi H, Nakamura M, Kojoma M, Suzuki H, Saito K (2016) RNA-seq transcriptome analysis of Panax japonicus, and its comparison with other Panax species to identify potential genes involved in the saponins biosynthesis. Front Plant Sci 7:481

    PubMed  PubMed Central  Google Scholar 

  42. Sawai S, Saito K (2011) Triterpenoid biosynthesis and engineering in plants. Front Plant Sci 2:1–8

    Article  Google Scholar 

  43. Seki H, Tamura K, Muranaka T (2015) P450s and UGTs: key players in the structural diversity of triterpenoid saponins. Plant Cell Physiol 56:1463–1471

    CAS  Article  Google Scholar 

  44. Shibuya M, Nishimura K, Yasuyama N, Ebizuka Y (2010) Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max. FEBS Lett 584:2258–2264

    CAS  Article  Google Scholar 

  45. Shin HJ, Kwon HW, Cho HJ, Rhee MH, Park HJ (2016) Vasodilator-stimulated phosphoprotein-phosphorylation by ginsenoside Ro inhibits fibrinogen binding to αIIb/β3 in thrombin-induced human platelets. J Ginseng Res 40:359–365

    Article  Google Scholar 

  46. Sun C, Li Y, Wu Q, Luo H, Sun Y, Song J, Lui EM, Chen S (2010) De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX titanium platform to discover putative genes involved in ginsenoside biosynthesis. BMC Genom 11:262

    Article  Google Scholar 

  47. Tanaka O (1990) Recent studies on glycosides from plant drugs of Himalaya and south-western China: chemo-geographical correlation of Panax species. Pure Appl Chem 62:1281–1284

    CAS  Article  Google Scholar 

  48. Tansakul P, Shibuya M, Kushiro T, Ebizuka Y (2006) Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng. FEBS Lett 580:5143–5149

    CAS  Article  Google Scholar 

  49. Wang P, Wei Y, Fan Y, Liu Q, Wei W, Yang C, Zhang L, Zhao G, Yue J, Yan X, Zhou Z (2015) Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts. Metab Eng 29:97–105

    Article  Google Scholar 

  50. Wei W, Wang P, Wei Y, Liu Q, Yang C, Zhao G, Yue J, Yan X, Zhou Z (2015) Characterization of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts. Mol Plant 8:1412–1424

    CAS  Article  Google Scholar 

  51. Xu J, Chu Y, Liao B, Xiao S, Yin Q, Bai R, Su H, Dong L, Li X, Qian J, Zhang J, Zhang Y, Zhang X, Wu M, Zhang J, Li G, Zhang L, Chang Z, Zhang Y, Jia Z, Liu Z, Afreh D, Nahurira R, Zhang L, Cheng R, Zhu Y, Zhu G, Rao W, Zhou C, Qiao L, Huang Z, Cheng YC, Chen S (2017) Panax ginseng genome examination for ginsenoside biosynthesis. Gigascience 6:1–15

    PubMed  PubMed Central  Google Scholar 

  52. Yan X, Fan Y, Wei W, Wang P, Liu Q, Wei Y, Zhang L, Zhao G, Yue J, Zhou Z (2014) Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 24:770–773

    CAS  Article  Google Scholar 

  53. Yang CR, Jiang ZD, Wu MZ, Zhou J, Tanaka O (1984) Studies on saponins of rhizomes of Panax zingiberensis Wu et Feng. Acta Pharm Sin 19:232–236

    CAS  Google Scholar 

  54. Yang WZ, Ye M, Qiao X, Liu CF, Miao WJ, Bo T, Tao HY, Guo DA (2012) A strategy for efficient discovery of new natural compounds by integrating orthogonal column chromatography and liquid chromatography/mass spectrometry analysis: its application in Panax ginseng, Panax quinquefolium and Panax notoginseng to characterize 437 potential new ginsenosides. Anal Chim Acta 739:56–66

    CAS  Article  Google Scholar 

  55. Yang WZ, Hu Y, Wu WY, Ye M, Guo DA (2014) Saponins in the genus Panax L. (Araliaceae): a systematic review of their chemical diversity. Phytochemistry 106:7–24

    CAS  Article  Google Scholar 

  56. Yoshizaki K, Devkota HP, Fujino H, Yahara S (2013) Saponins composition of rhizomes, taproots, and lateral roots of Satsuma-ninjin (Panax japonicus). Chem Pharm Bull (Tokyo) 61:344–350

    CAS  Article  Google Scholar 

  57. Zhang GH, Ma CH, Zhang JJ, Chen JW, Tang QY, He MH, Xu XZ, Jiang NH, Yang SC (2015a) Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers. BMC Genom 16:159

    Article  Google Scholar 

  58. Zhang SP, Jin J, Hu BX, Wu YY, Yan Q, Zeng WY, Zheng YL, Xi-feng Zhang, Chen P (2015b) Transcriptome profiling and analysis of Panax japonicus var. major. China J Chin Mater Med 40:2084–2089

    CAS  Google Scholar 

  59. Zhang GJ, Shen Y, Meng ZG, Chen JW, Zhang GH, Yang SC (2016) Determination of 5 saponins in Panax zingiberensis by HPLC. Chin J Pharm Anal 36:500–504

    Google Scholar 

  60. Zhang D, Li W, Xia EH, Zhang QJ, Liu Y, Zhang Y, Tong Y, Zhao Y, Niu YC, Xu JH, Gao LZ (2017) The medicinal herb Panax notoginseng genome provides insights into ginsenoside biosynthesis and genome evolution. Mol Plant 10:903–907

    CAS  Article  Google Scholar 

  61. Zheng K, Li Y, Wang S, Wang X, Liao C, Hu X, Fan L, Kang Q, Zeng Y, Wu X, Wu H, Zhang J, Wang Y, He Z (2016) Inhibition of autophagosome–lysosome fusion by ginsenoside Ro via the ESR2–NCF1–ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint. Autophagy 12:1593–1613

    CAS  Article  Google Scholar 

  62. Zhu S, Zou K, Fushimi H, Cai S, Komatsu K (2004) Comparative study on triterpene saponins of ginseng drugs. Planta Med 70:666–677

    CAS  Article  Google Scholar 

  63. Zou K, Zhu S, Tohda C, Cai S, Komatsu K (2002) Dammarane-type triterpene saponins from Panax japonicus. J Nat Prod 65:346–351

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant no. U1402262), National Key R & D Plan (no. 2017YFC1702500), the Major Science and Technique Programs in Yunnan Province (Grant no. 2016ZF001), the Natural Science Foundation of Yunnan Province (Grant no. 2015FB147) and Guangxi Scientific Research and Technology Development Program (Guikezhong 14124002-1). This paper was dedicated to Mrs. Zhang, Professor Zhang’s wife, who suffered ataxia which caused serious lateral curvature. She could not stand, walk and sleep for many years. If you can help her, please contact zgh73107310@163.com.

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Correspondence to Sheng-Chao Yang.

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Tang, Q., Chen, G., Song, W. et al. Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides. Planta 249, 393–406 (2019). https://doi.org/10.1007/s00425-018-2995-6

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Keywords

  • Biosynthesis
  • Ginsenosides
  • Oleanolic acid glucuronosyltransferase (OAGT)
  • Transcriptome
  • Triterpenoid saponins