Molecular Biotechnology

, Volume 60, Issue 6, pp 412–419 | Cite as

Metabolic Engineering of Glycyrrhizin Pathway by Over-Expression of Beta-amyrin 11-Oxidase in Transgenic Roots of Glycyrrhiza glabra

  • Zahra Shirazi
  • Ali Aalami
  • Masoud Tohidfar
  • Mohammad Mehdi Sohani
Original Paper


Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121GUS-9:CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content.


Agrobacterium rhizogenes Beta-amyrin 24-hydroxylase Composite plant Hairy root Triterpene saponin 


  1. 1.
    Hanrahan, C. (2001). Gale encyclopedia of alternative medicine: licorice. Thomson Gale: Farmington Hills.Google Scholar
  2. 2.
    Hayashi, H. (2009). Molecular biology of secondary metabolism: Case study for Glycyrrhiza plants. In A. Kirakosyan & P. B. Kaufman (Eds.), Recent advances in plant biotechnology (pp. 89–103). New York: Springer.Google Scholar
  3. 3.
    Chan, H. T., Chan, C., & Ho, J. W. (2003). Inhibition of glycyrrhizic acid on aflatoxin B1-induced cytotoxicity in hepatoma cells. Toxicology, 188, 211–217.CrossRefGoogle Scholar
  4. 4.
    Jeong, H. G., You, H. J., Park, S. J., Moon, A. R., Chung, Y. C., Kang, S. K., et al. (2002). Hepatoprotective effects of 18β-glycyrrhetinic acid on carbon tetrachloride-induced liver injury: Inhibition of cytochrome P450 2E1 expression. Pharmacological Research, 46, 221–227.CrossRefGoogle Scholar
  5. 5.
    Park, H. Y., Park, S. H., Yoon, H. K., Han, M. J., & Kim, D. H. (2004). Anti-allergic activity of 18β-glycyrrhetinic acid-3-O-β-d-glucuronide. Archives of Pharmacalogy Research, 27, 57–60.CrossRefGoogle Scholar
  6. 6.
    Fiore, C., Eisenhut, M., Krausse, R., Ragazzi, E., Pellati, D., Armanini, D., et al. (2008). Antiviral effects of Glycyrrhiza species. Physiotherapy Research, 22, 141–148.Google Scholar
  7. 7.
    Salvi, M., Fiore, C., Armanini, D., & Toninello, A. (2003). Glycyrrhetinic acid-induced permeability transition in rat liver mitochondria. Biochemistry and Pharmacology, 66, 2375–2379.CrossRefGoogle Scholar
  8. 8.
    Yoon, G., Do Jung, Y., & Cheon, S. H. (2005). Cytotoxic allyl retrochalcone from the roots of Glycyrrhiza inflata. Chemical & Pharmaceutical Bulletin, 53, 694–695.CrossRefGoogle Scholar
  9. 9.
    De Clercq, E. (2000). Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection. Medicinal Research Reviews, 20, 323–349.CrossRefGoogle Scholar
  10. 10.
    Cinatl, J., Morgenstern, B., Bauer, G., Chandra, P., Rabenau, H., & Doerr, H. (2003). Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. The Lancet, 361, 2045–2046.CrossRefGoogle Scholar
  11. 11.
    Lu, H. Y., Liu, J. M., Zhang, H. C., Yin, T., & Gao, S. L. (2008). Ri-mediated transformation of Glycyrrhiza uralensis with a squalene synthase gene (GuSQS1) for production of glycyrrhizin. Plant Molecular Biology Reporter, 26, 1–11.CrossRefGoogle Scholar
  12. 12.
    Haralampidis, K., Bryan, G., Qi, X., Papadopoulou, K., Bakht, S., Melton, R., et al. (2001). A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots. Proceedings of the National Academy of Sciences, 98, 13431–13436.CrossRefGoogle Scholar
  13. 13.
    Hayashi, H., Huang, P., Kirakosyan, A., Inoue, K., Hiraoka, N., Ikeshiro, Y., et al. (2001). Cloning and characterization of a cDNA encoding β-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice. Biological and Pharmaceutical Bulletin, 24, 912–916.CrossRefGoogle Scholar
  14. 14.
    Hayashi, H., Hiraoka, N., Ikeshiro, Y., Kushiro, T., Morita, M., Shibuya, M., et al. (2000). Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase. Biological and Pharmaceutical Bulletin, 23, 231–234.CrossRefGoogle Scholar
  15. 15.
    Seki, H., Ohyama, K., Sawai, S., Mizutani, M., Ohnishi, T., Sudo, H., et al. (2008). Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proceedings of the National Academy of Sciences, 105, 14204–14209.CrossRefGoogle Scholar
  16. 16.
    Hayashi, H., & Sudo, H. (2009). Economic importance of licorice. Plant Biotechnology, 26, 101–104.CrossRefGoogle Scholar
  17. 17.
    Georgiev, M. I., Ludwig-Muller, J., & Bley, T. (2010). Hairy root culture: Copying nature in new bioprocesses. In R. Arora (Ed.), Medicinal plant biotechnology (pp. 156–175). Oxon: CAB International.CrossRefGoogle Scholar
  18. 18.
    Ono, N. N., & Tian, L. (2011). The multiplicity of hairy root cultures: Prolific possibilities. Plant Science, 180, 439–446.CrossRefGoogle Scholar
  19. 19.
    Sevon, N., & Oksman-Caldentey, K. M. (2002). Agrobacterium rhizogenes-mediated transformation: Root cultures as a source of alkaloids. Planta Medica, 68, 859–868.CrossRefGoogle Scholar
  20. 20.
    Rahnama, H., Razi, Z., Dadgar, M. N., & Hasanloo, T. (2013). Enhanced production of flavonolignans in hairy root cultures of Silybum marianum by over-expression of chalcone synthase gene. Journal of Plant Biochemistry and Biotechnology, 22, 138–143.CrossRefGoogle Scholar
  21. 21.
    Sharafi, A., Sohi, H. H., Mousavi, A., Azadi, P., Khalifani, B. H., & Razavi, K. (2013). Metabolic engineering of morphinan alkaloids by over-expression of codeinone reductase in transgenic hairy roots of Papaver bracteatum, the Iranian poppy. Biotechnology Letters, 35, 445–453.CrossRefGoogle Scholar
  22. 22.
    Li, Y. L., Yang, Y., Fu, C. H., & Yu, L. J. (2012). Production of glycyrrhizin in cell suspension of Glycyrrhiza inflata Batalin cultured in bioreactor. Biotechnology and Biotechnological Equipment, 26, 3231–3235.CrossRefGoogle Scholar
  23. 23.
    Shirazi, Z., Piri, K., Asl, A. M., & Hasanloo, T. (2012). Glycyrrhizin and isoliquiritigenin production by hairy root culture of Glycyrrhiza glabra. Journal of Medicinal Plants Research, 6, 4640–4646.CrossRefGoogle Scholar
  24. 24.
    Mousa, N. A., Siaguru, P., Wiryowidagdo, S., & Wagih, M. (2007). Establishment of regenerative callus and cell suspension system of licorice (Glycyrrhiza glabra) for the production of the sweetener glycyrrhizinin vitro. Sugar Tech, 9, 72–82.CrossRefGoogle Scholar
  25. 25.
    Shirazi, Z., Aalami, A., Tohidfar, M., & Sohani, M. M. (2016). Isolation, cloning and bioinformatics analysis of beta-amyrin 11-oxidase coding sequence from licorice. Plant Omics, 9, 165.CrossRefGoogle Scholar
  26. 26.
    Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.CrossRefGoogle Scholar
  27. 27.
    Collier, R., Fuchs, B., Walter, N., Kevin, W., & Taylor, C. G. (2005). Ex vitro composite plants: An inexpensive. Rapid method for root biology. The Plant Journal, 43, 449–457.CrossRefGoogle Scholar
  28. 28.
    Zhang, H. C., Liu, J. M., Chen, H. M., Gao, C. C., Lu, H. Y., Zhou, H., et al. (2011). Up-regulation of licochalcone A biosynthesis and secretion by Tween 80 in hairy root cultures of Glycyrrhiza uralensis Fisch. Molecular Biotechnology, 47, 50–56.CrossRefGoogle Scholar
  29. 29.
    Cai, D., Kleine, M., Kifle, S., Harloff, H. J., Sandal, N. N., Marcker, K. A., et al. (1997). Positional cloning of a gene for nematode resistance in sugar beet. Science, 275, 832–834.CrossRefGoogle Scholar
  30. 30.
    Vojin, T., Snezana, M., Aleksandar, C., Marija, P., Milana, T., & Dragana, A. (2014). Production of hairy root cultures of lettuce (Lactuca sativa). Open Life Sciences, 9, 1196–1205.CrossRefGoogle Scholar
  31. 31.
    Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29, e45.CrossRefGoogle Scholar
  32. 32.
    Hayashi, H., Fukui, H., & Tabata, M. (1988). Examination of triterpenoids produced by callus and cell suspension cultures of Glycyrrhiza glabra. Plant Cell Reports, 7, 508–511.CrossRefGoogle Scholar
  33. 33.
    Sabbioni, C., Ferranti, A., Bugamelli, F., Forti, G. C., & Raggi, M. A. (2006). Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products. Phytochemical Analysis, 17, 25–31.CrossRefGoogle Scholar
  34. 34.
    Fridborg, G., Pedersen, M., Landstrom, L. E., & Eriksson, T. (1978). The effect of activated charcoal on tissue cultures: Adsorption of metabolites inhibiting morphogenesis. Physiologia Plantarum, 43, 104–106.CrossRefGoogle Scholar
  35. 35.
    Christey, M. C., & Braun, R. H. (2004). Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes-mediated transformation (pp. 47–60). Methods and Protocols: Transgenic Plants.Google Scholar
  36. 36.
    Shi, H. P., & Kintzios, S. (2003). Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots. Plant Cell Reports, 21, 1103–1143.CrossRefGoogle Scholar
  37. 37.
    Alpizar, E., Decham, E., Espeout, S., Royer, M., Lecouls, A. C., & Nicole, M. (2006). Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots. Plant Cell Reports, 25, 959–967.CrossRefGoogle Scholar
  38. 38.
    Dhakulkar, S., Ganapathi, T., Bhargava, S., & Bapat, V. (2005). Induction of hairy roots in Gmelina arborea Roxb. and production of verbascoside in hairy roots. Plant Science, 169, 812–818.CrossRefGoogle Scholar
  39. 39.
    Jose, B., Pillai, D. B., & Satheeshkumar, K. (2016). In vitro cultivation of hairy roots of Plumbago rosea L. in a customized reaction kettle for the production of plumbagin––An anticancer compound. Industrial Crops and Products, 87, 89–95.CrossRefGoogle Scholar
  40. 40.
    Ilina, E. L., Logachov, A. A., Laplaze, L., Demchenko, N. P., Pawlowski, K., & Demchenko, K. N. (2012). Composite Cucurbita pepo plants with transgenic roots as a tool to study root development. Annals of Botany, 110, 479–489.CrossRefGoogle Scholar
  41. 41.
    Hu, Z. B., & Du, M. (2006). Hairy root and its application in plant genetic engineering. Journal of Integrative Plant Biology, 48, 121–127.CrossRefGoogle Scholar
  42. 42.
    Chattopadhyay, T., Roy, S., Mitra, A., & Maiti, M. K. (2011). Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation. Plant Cell Reports, 30, 485–493.CrossRefGoogle Scholar
  43. 43.
    Kim, S. R., Sim, J. S., Ajjappala, H., Kim, Y. H., & Hahn, B. S. (2012). Expression and large-scale production of the biochemically active human tissue-plasminogen activator in hairy roots of Oriental melon (Cucumis melo). Journal of Bioscience and Bioengineering, 113, 106–111.CrossRefGoogle Scholar
  44. 44.
    Wilson, K. J., Hughes, S., & Jefferson, R. (1992). The Escherichia coli gus operon: Induction and expression of the gus operon in E. coli and the occurrence and use of GUS in other bacteria. In S. R. Gallagher (Ed.), GUS protocols: Using the GUS gene as a reporter of gene expression (pp. 125–145). San Diego: Harcourt Brace Jovanovich.Google Scholar
  45. 45.
    Dehghan, E., Reed, D. W., Covello, P. S., Hasanpour, Z., Palazon, J., Oksman-Caldentey, K. M., et al. (2017). Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: Ploidy as a promising parameter in the metabolic engineering of tropane alkaloids. Plant Cell Reports, 36, 1615–1626.CrossRefGoogle Scholar
  46. 46.
    Chandra, S. (2012). Natural plant genetic engineer Agrobacterium rhizogenes: Role of T-DNA in plant secondary metabolism. Biotechnology Letters, 34, 407–415.CrossRefGoogle Scholar
  47. 47.
    Sato, F., Hashimoto, T., Hachiya, A., Tamura, K. I., Choi, K. B., & Morishige, T. (2001). Metabolic engineering of plant alkaloid biosynthesis. Proceedings of the National Academy of Sciences, 98, 367–372.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Zahra Shirazi
    • 1
  • Ali Aalami
    • 1
  • Masoud Tohidfar
    • 2
  • Mohammad Mehdi Sohani
    • 1
  1. 1.Department of Biotechnology, Faculty of Agricultural SciencesUniversity of GuilanRashtIran
  2. 2.Department of Plant Biotechnology, Faculty of Life Science and BiotechnologyShahid Beheshti University, G.C.TehranIran

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