Plant Cell Reports

, Volume 36, Issue 3, pp 437–445 | Cite as

CYP716A179 functions as a triterpene C-28 oxidase in tissue-cultured stolons of Glycyrrhiza uralensis

  • Keita Tamura
  • Hikaru Seki
  • Hideyuki Suzuki
  • Mareshige Kojoma
  • Kazuki Saito
  • Toshiya Muranaka
Original Article

Abstract

Key message

CYP716A179, a cytochrome P450 monooxygenase expressed predominantly in tissue-cultured stolons of licorice (Glycyrrhiza uralensis), functions as a triterpene C-28 oxidase in the biosynthesis of oleanolic acid and betulinic acid.

Abstract

Cytochrome P450 monooxygenases (P450s) play key roles in the structural diversification of plant triterpenoids. Among these, the CYP716A subfamily, which functions mainly as a triterpene C-28 oxidase, is common in plants. Licorice (Glycyrrhiza uralensis) produces bioactive triterpenoids, such as glycyrrhizin and soyasaponins, and relevant P450s (CYP88D6, CYP72A154, and CYP93E3) have been identified; however, no CYP716A subfamily P450 has been isolated. Here, we identify CYP716A179, which functions as a triterpene C-28 oxidase, by RNA sequencing analysis of tissue-cultured stolons of G. uralensis. Heterologous expression of CYP716A179 in engineered yeast strains confirmed the production of oleanolic acid, ursolic acid, and betulinic acid from β-amyrin, α-amyrin, and lupeol, respectively. The transcript level of CYP716A179 was about 500 times higher in tissue-cultured stolons than in intact roots. Oleanolic acid and betulinic acid were consistently detected only in tissue-cultured stolons. The discovery of CYP716A179 helps increase our understanding of the mechanisms of tissue-type-dependent triterpenoid metabolism in licorice and provides an additional target gene for pathway engineering to increase the production of glycyrrhizin in licorice tissue cultures by disrupting competing pathways.

Keywords

Glycyrrhiza uralensis Licorice P450 RNA-seq Triterpenoid 

Abbreviations

aAS

α-Amyrin synthase

bAS

β-Amyrin synthase

CAS

Cycloartenol synthase

CDS

Coding sequence

CPR

Cytochrome P450 reductase

EST

Expressed sequence tag

LUS

Lupeol synthase

OSC

Oxidosqualene cyclase

Supplementary material

299_2016_2092_MOESM1_ESM.docx (2.6 mb)
Supplementary material (DOCX 2697 kb)

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 25:3389–3402CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120CrossRefPubMedPubMedCentralGoogle Scholar
  3. Carelli M, Biazzi E, Panara F, Tava A, Scaramelli L, Porceddu A, Graham N, Odoardi M, Piano E, Arcioni S, May S, Scotti C, Calderini O (2011) Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins. Plant Cell 23:3070–3081CrossRefPubMedPubMedCentralGoogle Scholar
  4. Fukushima EO, Seki H, Ohyama K, Ono E, Umemoto N, Mizutani M, Saito K, Muranaka T (2011) CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis. Plant Cell Physiol 52:2050–2061CrossRefPubMedGoogle Scholar
  5. 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 29:644–652CrossRefPubMedPubMedCentralGoogle Scholar
  6. Hayashi H, Sudo H (2009) Economic importance of licorice. Plant Biotechnol 26:101–104CrossRefGoogle Scholar
  7. Hayashi H, Fukui H, Tabata M (1988) Examination of triterpenoids produced by callus and cell suspension cultures of Glycyrrhiza glabra. Plant Cell Rep 7:508–511CrossRefPubMedGoogle Scholar
  8. Hayashi H, Fukui H, Tabata M (1993) Distribution pattern of saponins in different organs of Glycyrrhiza glabra. Planta Med 59:351–353CrossRefPubMedGoogle Scholar
  9. Jäger S, Trojan H, Kopp T, Laszczyk MN, Scheffler A (2009) Pentacyclic triterpene distribution in various plants—rich sources for a new group of multi-potent plant extracts. Molecules 14:2016–2031CrossRefPubMedGoogle Scholar
  10. Kojoma M, Ohyama K, Seki H, Hiraoka Y, Asazu SN, Sawa S, Sekizaki H, Yoshida S, Muranaka T (2010) In vitro proliferation and triterpenoid characteristics of licorice (Glycyrrhiza uralensis Fischer, Leguminosae) stolons. Plant Biotechnol 27:59–66CrossRefGoogle Scholar
  11. 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 23:2947–2948CrossRefPubMedGoogle Scholar
  12. Li Y, Luo H-M, Sun C, Song J-Y, Sun Y-Z, Wu Q, Wang N, Yao H, Steinmetz A, Chen S-L (2010) EST analysis reveals putative genes involved in glycyrrhizin biosynthesis. BMC Genom 11:268CrossRefGoogle Scholar
  13. Moses T, Pollier J, Shen Q, Soetaert S, Reed J, Erffelinck M-L, Van Nieuwerburgh FCW, Vanden Bossche R, Osbourn A, Thevelein JM, Deforce D, Tang K, Goossens A (2015) OSC2 and CYP716A14v2 catalyze the biosynthesis of triterpenoids for the cuticle of aerial organs of Artemisia annua. Plant Cell 27:286–301CrossRefPubMedPubMedCentralGoogle Scholar
  14. Nelson D, Werck-Reichhart D (2011) A P450-centric view of plant evolution. Plant J 66:194–211CrossRefPubMedGoogle Scholar
  15. Oksman-Caldentey K-M, Inzé D (2004) Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolites. Trends Plant Sci 9:433–440CrossRefPubMedGoogle Scholar
  16. Ramachandra Rao S, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153CrossRefGoogle Scholar
  17. Ramilowski JA, Sawai S, Seki H, Mochida K, Yoshida T, Sakurai T, Muranaka T, Saito K, Daub CO (2013) Glycyrrhiza uralensis transcriptome landscape and study of phytochemicals. Plant Cell Physiol 54:697–710CrossRefPubMedGoogle Scholar
  18. Seki H, Ohyama K, Sawai S, Mizutani M, Ohnishi T, Sudo H, Akashi T, Aoki T, Saito K, Muranaka T (2008) Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proc Natl Acad Sci USA 105:14204–14209CrossRefPubMedPubMedCentralGoogle Scholar
  19. Seki H, Sawai S, Ohyama K, Mizutani M, Ohnishi T, Sudo H, Fukushima EO, Akashi T, Aoki T, Saito K, Muranaka T (2011) Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin. Plant Cell 23:4112–4123CrossRefPubMedPubMedCentralGoogle Scholar
  20. Seki H, Tamura K, Muranaka T (2015) P450s and UGTs: key players in the structural diversity of triterpenoid saponins. Plant Cell Physiol 56:1463–1471CrossRefPubMedGoogle Scholar
  21. Sudo H, Seki H, Sakurai N, Suzuki H, Shibata D, Toyoda A, Totoki Y, Sakaki Y, Iida O, Shibata T, Kojoma M, Muranaka T, Saito K (2009) Expressed sequence tags from rhizomes of Glycyrrhiza uralensis. Plant Biotechnol 26:105–107CrossRefGoogle Scholar
  22. Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257CrossRefPubMedGoogle Scholar
  23. Yasumoto S, Fukushima EO, Seki H, Muranaka T (2016) Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes. FEBS Lett 590:533–540CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Keita Tamura
    • 1
  • Hikaru Seki
    • 1
  • Hideyuki Suzuki
    • 2
  • Mareshige Kojoma
    • 3
  • Kazuki Saito
    • 4
  • Toshiya Muranaka
    • 1
  1. 1.Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuitaJapan
  2. 2.Department of Research and DevelopmentKazusa DNA Research InstituteKisarazuJapan
  3. 3.Faculty of Pharmaceutical SciencesHealth Sciences University of HokkaidoTobetsuJapan
  4. 4.Graduate School of Pharmaceutical SciencesChiba UniversityChibaJapan

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