Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

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

References

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 23:3070–3081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 52:2050–2061

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hayashi H, Sudo H (2009) Economic importance of licorice. Plant Biotechnol 26:101–104

    Article  CAS  Google Scholar 

  • Hayashi H, Fukui H, Tabata M (1988) Examination of triterpenoids produced by callus and cell suspension cultures of Glycyrrhiza glabra. Plant Cell Rep 7:508–511

    Article  CAS  PubMed  Google Scholar 

  • Hayashi H, Fukui H, Tabata M (1993) Distribution pattern of saponins in different organs of Glycyrrhiza glabra. Planta Med 59:351–353

    Article  CAS  PubMed  Google Scholar 

  • 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 14:2016–2031

    Article  PubMed  Google Scholar 

  • 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 27:59–66

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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 11:268

    Article  Google Scholar 

  • 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 27:286–301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nelson D, Werck-Reichhart D (2011) A P450-centric view of plant evolution. Plant J 66:194–211

    Article  CAS  PubMed  Google Scholar 

  • 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 9:433–440

    Article  CAS  PubMed  Google Scholar 

  • Ramachandra Rao S, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153

    Article  CAS  Google Scholar 

  • 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 54:697–710

    Article  CAS  PubMed  Google Scholar 

  • 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 105:14204–14209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 23:4112–4123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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 26:105–107

    Article  CAS  Google Scholar 

  • Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257

    Article  CAS  PubMed  Google Scholar 

  • Yasumoto S, Fukushima EO, Seki H, Muranaka T (2016) Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes. FEBS Lett 590:533–540

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are very grateful to the Takeda Garden for Medicinal Plant Conservation, Kyoto, Japan (Takeda Pharmaceutical Company Limited) for the supply of licorice plants. We thank Dr. David R. Nelson (University of Tennessee) for the naming of CYP716A179 according to the P450 nomenclature. We also thank Mr. Tsutomu Hosouchi and Ms. Sayaka Shinpo (Kazusa DNA Research Institute) for technical support with the Illumina sequencing, Dr. Kazuto Mannen (Kazusa DNA Research Institute) and Dr. Ryosuke Sano (Nara Institute of Science and Technology) for technical advice with bioinformatics tools, Dr. Kiyoshi Ohyama (Tokyo Institute of Technology) for providing oleanolic aldehyde, ursolic aldehyde and betulinic aldehyde standards, and Dr. Ery Odette Fukushima (Osaka University) for helpful discussion on phylogenetic analysis of CYP716A subfamily enzymes. This work was supported by the “Health and Labour Sciences Research Grant” on the enhancement of “Comprehensive Medicinal Plant Database”, JSPS KAKENHI Grant Number JP26450123 and research funding from the Yamada Science Foundation to H. Seki, JSPS KAKENHI Grant Number JP15H04485 to TM, and the Doctor 21 scholarship from the Yoshida Scholarship Foundation to KT.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Keita Tamura, Hikaru Seki & Toshiya Muranaka

  2. Department of Research and Development, Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba, 292-0818, Japan

    Hideyuki Suzuki

  3. Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu, Hokkaido, 061-0293, Japan

    Mareshige Kojoma

  4. Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan

    Kazuki Saito

Authors
  1. Keita Tamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hikaru Seki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hideyuki Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mareshige Kojoma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kazuki Saito
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Toshiya Muranaka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toshiya Muranaka.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by F. Sato.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material (DOCX 2697 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tamura, K., Seki, H., Suzuki, H. et al. CYP716A179 functions as a triterpene C-28 oxidase in tissue-cultured stolons of Glycyrrhiza uralensis . Plant Cell Rep 36, 437–445 (2017). https://doi.org/10.1007/s00299-016-2092-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-016-2092-x

Keywords

Search

Navigation