Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Overexpression of Hyoscyamine 6${\beta}$-Hydroxylase (h6h) Gene and Enhanced Production of Tropane Alkaloids in Scopolia parviflora Hairy Root Lines

  • KANG, YOUNG-MIN (Division of Forest Science, Gyeongsang National University) ;
  • LEE, OK-SUN (Division of Applied Life Science, Gyeongsang National University) ;
  • JUNG, HEE-YOUNG (Division of Forest Science, Gyeongsang National University) ;
  • KANG, SEUNG-MI (Division of Forest Science, Gyeongsang National University) ;
  • LEE, BYUNG-HYUN (Division of Applied Life Science, Gyeongsang National University) ;
  • CHANDRAKANT KARIGAR (Division of Biochemistry, Department of Chemistry, Central College Campus, Bangalore University) ;
  • THEERTHA PRASAD (Department of Biotechnology, University of Agricultural Science) ;
  • BAHK, JUNG-DONG (Division of Applied Life Science, Gyeongsang National University) ;
  • CHOI, MYUNG-SUK (Division of Forest Science, Gyeongsang National University, Environmental Biotechnology Research Center, Gyeongsang National University)
  • Published : 2005.02.01
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Abstract

The hyoscyamine 6${\beta}$-hydroxylase (h6h) gene was introduced into the genome of Scopolia parviflora through the Agrobacterium rhizogenes binary vector system. The enzyme was expressed ally and tissue specific selectively in roots, resulting in five transgenic hairy root lines. The presence of the h6h gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction (PCR), Northern blotting, and Western blotting, respectively. In the transgenic hairy root lines which constitutively expressed the H6H enzyme, hyoscyamine and scopolamine accumulated in high concentration. Among the transgenic hairy root lines that expressed the H6H enzyme, only two were more productive. The levels of tropane alkaloids in transgenic hairy root varied greatly: The best transgenic line (#5) contained 8.12 mg of scopolamine per g dry weight, which produced the compound three times more than wild-type root. These results suggest a possibility of improving the yield of tropane alkaloids in hairy root lines by genetic and metabolic engineering.

Keywords

References

  1. Bonhomme, V., D. Laurain-Matter, and M. A. Fliniaux. 2000. Effects of the rol C gene on hairy root: Induction development and tropane alkaloid production by Atropa belladonna. J. Natural Prod. 63: 1249- 1252 https://doi.org/10.1021/np990614l
  2. Canel, C., M. I. Lopes-Cardoso, S. Whitmer, L. van-der Fits, G. Pasquali, R. van-der Heijden, J. H. C. Hoge, and R. Verpoorte. 1998. Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta 205: 414-419 https://doi.org/10.1007/s004250050338
  3. Gamberg, O. L., R. A. Miller, and K. Ojima. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 148- 151
  4. Giri, A. and M. L. Narasu. 2000. Transgenic hairy roots: Recent trends and applications. Biotechnol. Adv. 18: 1- 22 https://doi.org/10.1016/S0734-9750(99)00016-6
  5. Hashimoto, T. and Y. Yamada. 1986. Hyoscyamine $6\beta-hydroxylase$, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures. Plant Physiol. 81: 619-625 https://doi.org/10.1104/pp.81.2.619
  6. Hashimoto, T., Y. Yukimune, and Y. Yamada. 1986. Tropane alkaloid production in Hyoscyamus roots cultures. Plant Physiol. 124: 61- 75 https://doi.org/10.1016/S0176-1617(86)80178-X
  7. Hamill, J. D., A. J. Parr, M. J. C. Rhodes, R. J. Robins, and N. J. Walton. 1987. New routes to plant secondary products. Biotechnology 5: 800- 804 https://doi.org/10.1038/nbt0887-800
  8. Hilton, M. G. and M. J. C. Rhodes. 1990. Growth and hyoscyamine production of hairy root cultures of Datura stramonium in a modified stirred tank reactor. Appl. Microbiol. Biotechnol. 33: 132- 138 https://doi.org/10.1007/BF00176513
  9. Hashimoto, T., D. J. Yun, and Y. Yamada. 1993. Production of tropane alkaloids in genetically engineered root cultures. Phytochemistry 32: 713- 718 https://doi.org/10.1016/S0031-9422(00)95159-8
  10. Jouhikainen, K., L. Lindgren, T. Jokelainen, R. Hiltunen, T. H. Teeri, and K. M. Oksman-Caldentey. 1999. Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering. Planta 208: 545- 551 https://doi.org/10.1007/s004250050592
  11. Jeon, Y. H., S. P. Chang, I. Hwang, and Y. H. Kim. 2003. Involvement of growth-promoting rhizobacterium Paenibacillus polymyxa in root rot of stroded Korean ginseng. J. Microbiol. Biotechnol. 13: 881- 891
  12. Katiyar, V. and R. Goel. 2004. Improved plant growth from seed bacterization using siderophore overproducing cold resistant mutant of Pseudomonas fluorescens. J. Microbiol. Biotechnol. 14: 653- 657
  13. Kim., B. S., S. I. Jeong, S. W. Cho, J. Nikolovski, D. J. Mooney, S. H. Lee, O. Jeon, T. W. Kim, S. H. Lim, Y. S. Hong, C. Y. Choi, Y. M. Lee, S. H. Kim, and Y. H. Kim. 2003. Tissue engineering of smooth muscle under a mechanically dynamic condition. J. Microbiol. Biotechnol. 13: 881- 891
  14. Kang, Y. M., J. Y. Min, H. S. Moon, C. S. Karigar, D. T. Prasad, C. H. Lee, M. S. Choi. 2004. Rapid in vitro adventitious shoot propagation of Scopolia parviflora through rhizome cultures for enhanced production of tropane alkaloids. Plant Cell Reports 23: 128- 133 https://doi.org/10.1007/s00299-004-0820-0
  15. Selvaraj, T., C. Padrnanabhan, Y. J. Jeong, and H. Kim. 2004. Occurrence ofvesicular-arbuscular mycorrhizal (VAM) fungi and their effect on plant growth in endangered vegetations. J. Microbiol. Biotechnol. 14: 885- 890
  16. Matsuda, J., S. Okabe, T. Hashimoto, and Y. Yamada. 1998. Molecular cloning of hyoscyamine $6\beta-hydroxylase$, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger. J. Biol. Chem. 266: 9460- 9464
  17. Tsugawa, H., Y. Otsuki, and Y. Ohashi. 1996. Efficient promoter cassettes for enhanced expression of foreign genes in cotyledonous and monocotyledonous plants. Plant Cell Physiol. 37: 49- 59 https://doi.org/10.1093/oxfordjournals.pcp.a028913
  18. Rocha, P., O. Stenzel, A. Parr, N. Walton, P. Christon, B. Drager, and M. J. Leech. 2002. Functional expression of tropinone reductase I (tr I) and $hyoscyamine-6\beta-hydroxylase$ (h6h) from Hyoscyamus niger in Nicotiana tabacum. Plant Sci. 162: 905- 913 https://doi.org/10.1016/S0168-9452(02)00033-X
  19. Sambrook, J., E. E Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press, Cold Spring Harbor, New York, U.S.A.
  20. Sato, F, T. Hashimoto, A. Hachiya, K. Tamura, K. B. Choi, T. Morishige, H. Fujimoto, and Y. Yamada. 2001. Metabolic engineering of plant alkaloid biosynthesis. Proc. Natl. Acad. Sci. USA 98: 367- 372 https://doi.org/10.1073/pnas.011526398
  21. Verpoorte, R. and A. W. Alfermann. 2000. Metabolic Engineering of Plant. Secondary Metabolism. Dordrecht, Kluwer Academic Publishers, Netherlands