Advertisement

Transgenic Research

, Volume 2, Issue 6, pp 336–344 | Cite as

Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

  • Jochen Berlin
  • Christiane Rügenhagen
  • Peter Dietze
  • Lothar F. Fecker
  • Oscar J. M. Goddijn
  • J. Harry C. Hoge
Papers

Abstract

Cell suspension and root cultures ofPeganum harmala were established expressing a tryptophan decarboxylase cDNA clone fromCatharanthus roseus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The tryptophan decarboxylase activity of some of the transgenic lines was greatly enhanced (25–40 pkat/mg protein) as compared to control cultures (1–5 pkat per mg protein) and remained high during the growth cycle. While the levels of tryptamine, the product of the reaction catalysed by tryptophan decarboxylase, were unchanged in the transgenic lines, their serotonin contents were enhanced up to 10-fold, reaching levels of 1.5 to 2% dry mass. Thus, tryptamine produced by the engineered reaction was apparently immediately used for enhanced serotonin biosynthesis. The yields of serotonin in transgenic lines overexpressing tryptophan decarboxylase activity were further enhanced to 3–5% dry mass by feedingl-tryptophan, while no or only minor effects were seen when control cultures were fed. These data demonstrate that the production of a plant secondary metabolite can be enhanced greatly via genetic manipulation of the level of activity of the rate-limiting enzyme. The amounts of β-carboline alkaloids, the other tryptamine-derived metabolites ofP. harmala, in contrast, were not affected by the overproduction of tryptamine. The information needed for successfully predicting manipulations that enhance production of a secondary metabolite is discussed.

Keywords

Serotonin Alkaloid Secondary Metabolite Transgenic Line Mosaic Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Berlin, J. (1988) On the formation of secondary metabolites in plant cell cultures — some general observations and some experimental approaches. In:Production of metabolites by plant cell cultures, pp. 89–98. APRIA, Paris.Google Scholar
  2. Berlin, J. and Sasse, F. (1988) β-Carboline alkaloids and indole alkylamines. In: Constabel F., Vasil I.K. (eds)Cell culture and somatic cell genetics. Vol. 5: Phytochemicals in cell cultures, pp. 357–69. Orlando, Florida: Academic Press.Google Scholar
  3. Berlin, J., Mollenschott, C., Sasse, F., Witte, L., Piehl, H.G. and Büntemeyer, H. (1987) Restoration of serotonin biosynthesis in cell suspension cultures ofPeganum harmala by selection for 4-methyltryptophan-tolerant cell lines.J. Plant Physiol. 131, 225–36.Google Scholar
  4. Berlin, J., Kuzovkina, I.N., Rügenhagen, C., Fecker, L., Commandeur, U. and Wray, V. (1992) Hairy root cultures ofPeganum harmala-II. Characterization of cell lines and effect of culture conditions on the accumulation of β-carboline alkaloids and serotonin.Z. Naturforsch. 47c, 222–230.Google Scholar
  5. Berlin, J., Rügenhagen, C., Greidziak, N., Kuzovkina, I.N., Witte, L. and Wray, V. (1993) Biosynthetic studies on serotonin and β-carboline alkaloids in hairy root cultures ofPeganum harmala.Phytochemistry 33, 593–7.Google Scholar
  6. Cahoon, E.B., Shanklin, J. and Ohlrogge J.B. (1992) Expression of a coriander desaturase results in petroselinic production in transgenic tobacco.Proc. Natl Acad. Sci. USA 89, 11184–8.Google Scholar
  7. Courtois, D., Yvernel, D., Florin, B. and Petiard, V. (1988) Conversion of tryptamine to serotonin by cell suspension cultures ofPeganum harmala.Phytochemistry 27, 3137–41.Google Scholar
  8. Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983) A plant DNA minipreparation: version II.Plant Mol. Biol. Rep. 1, 19–21.Google Scholar
  9. DeLuca, V. and Cutler, A.J. (1987) Subcellular localization of enzymes involved in indole alkaloid biosynthesis inCatharanthus roseus.Pl. Physiol. 85 1099–102.Google Scholar
  10. DeLuca, V., Marineau, C. and Brisson, N. (1989) Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: Comparison with animal dopa decarboxylases.Proc. Natl Acad. Sci. USA 86, 2582–6.Google Scholar
  11. Elkind, Y., Edwards, R., Mavandad, M., Hedrick, S.A., Ribak, O., Dixon, R.A. and Lamb, C.J. (1990) Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene.Proc. Natl Acad. Sci. USA 87, 9057–61.Google Scholar
  12. Fecker, L., Ekblom, P., Kurkinen, M. and Ekblom, M. (1990) A genomic clone encoding a novel proliferation-dependent histone H2A.1 mRNA enriched in the poly (A)+ fraction.Mol. Cell. Biol. 10, 2848–54.Google Scholar
  13. Fecker, L.F., Hillebrandt, S., Rügenhagen, C., Herminghaus, S., Landsmann, J. and Berlin, J. (1992) Metabolic effects of a bacterial lysine decarboxylase gene expressed in a hairy root culture ofNicotiana glauca.Biotechnol. Lett. 14, 1035–40.Google Scholar
  14. Galneder, E. and Zenk, M.H. (1990) Enzymology of alkaloid production in plant cell cultures. In Nijkamp, H.J.J., van der Plas, L.H.W. and van Aartrijk, J. eds,Progress in Plant Cellular and Molecular Biology, pp. 754–762, Dordrecht; Kluwer Academic Publishers.Google Scholar
  15. Gamborg, O.L., Murashige, T., Thorpe, T.A. and Vasil, I.K. (1976) Plant tissue culture media.In Vitro 12, 473–8.Google Scholar
  16. Goddijn, O.J.M. (1992) Regulation of terpenoid indole alkaloid biosynthesis inCatharanthus roseus: the tryptophan decarboxylase gene. Ph.D. Thesis, Leiden University.Google Scholar
  17. Hain, R., Bieseler, B., Kindl, H., Schröder, G. and Stöcker, R. (1990) Expression of a stilbene gene inNicotiana tabacum results in the synthesis of the phytoalexin resveratrol.Pl. Mol. Biol. 15, 325–35.Google Scholar
  18. Hamill, J.D., Robins, R.J., Parr, A.J., Evans, D.M., Furze, J.M. and Rhodes, M.J.C. (1990) Overexpressing a yeast ornithine decarboxylase gene in transgenic roots ofNicotiana rustica can lead to enhanced nicotine accumulation.Pl. Mol. Biol. 15, 27–38.Google Scholar
  19. Herminghaus, S., Schreier, P.H., McCarthy, J.E.G., Landsmann, J., Botterman, J. and Berlin, J. (1991) Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco.Pl. Mol. Biol. 17, 475–86.Google Scholar
  20. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983) A binary plant vector strategy based on separation of vir- and T-region of theAgrobacterium tumefaciens Ti-plasmid.Nature 303, 179–80.Google Scholar
  21. Johnson, D.A., Gautsch, J.W., Sportsman, J.R. and Elder, J.H. (1984) Improved technique utilizing non fat dry milk for analyses of proteins and nucleic acids transferred to nitrocellulose.Gene Analysis Techniques 3, 1–8.Google Scholar
  22. Knobloch, K.-H., Hansen, B. and Berlin, J. (1981) Medium induced formation of indole alkaloids and concomitant changes of interrelated enzyme activities in cell suspension cultures ofCatharanthus roseus.Z. Naturforsch. 36c, 40–3.Google Scholar
  23. Kyhse-Andersen, J. (1984) Electrolabelling of multiple gels. A simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose.J. Biochem. Biophys. Meth. 10, 203–9.Google Scholar
  24. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.Google Scholar
  25. Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene intoPetunia plants results in reversible co-suppression of homologous genesin trans.Pl. Cell 2, 279–89.Google Scholar
  26. Noé, W. and Berlin, J. (1985) Induction of de-novo synthesis of tryptophan decarboxylase in suspension cultures ofCatharanthus roseus.Planta 166, 500–4.Google Scholar
  27. Petit, A., David, C., Dahl, G.A., Ellis, J.G., Guyon, P., Casse-Delbart, F. and Tempe, J. (1983) Further extension of the opine concept: plasmids inAgrobacterium rhizogenes cooperate for opine degradation.Mol. Gen. Genet. 190, 204–14.Google Scholar
  28. Read, S.M. and Northcote, D.H. (1981) Minimizing of variation in the response to different proteins of Coomassie blue G dye-binding assay for protein.Anal. Biochem. 116, 53–64.Google Scholar
  29. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual Cold Spring Harbor, New York, USA: Cold Spring Harbor Laboratory Press.Google Scholar
  30. Sasse, F., Hammer, J. and Berlin, J. (1980) Fluorimetric and high-performance liquid chromatographic determination of harman alkaloids inPeganum harmala cell cultures.J. Chromatography 194, 234–8.Google Scholar
  31. Sasse, F., Witte, L. and Berlin, J. (1987) Biotransformation of tryptamine to serotonin by cell suspension cultures ofPeganum harmala.Planta Medica 53, 354–9.Google Scholar
  32. Songstad, D.V., DeLuca, V., Brisson, N., Kurz, W.G.W. and Nessler, C.L. (1990) High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase.Plant Physiol. 94, 1410–3.Google Scholar
  33. Van der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N.M. and Stuitje, A.R. (1990) Flavonoid genes inPetunia: addition of a limited number of gene copies may lead to a suppression of gene expression.Pl. Cell 2, 291–9.Google Scholar
  34. Verwoerd, T.C., Dekker, B.M.M. and Hoekema, A. (1989) A small-scale procedure for the rapid isolation of plant RNAs.Nucl. Acids Res. 17, 2362.Google Scholar
  35. Walton, N.J. and Belshaw, N.J. (1988) The effect of cadaverine on the formation of anabasine from lysine in hairy root cultures ofNicotiana hesperis.Pl. Cell Rep. 7, 115–8.Google Scholar

Copyright information

© Chapman & Hall 1993

Authors and Affiliations

  • Jochen Berlin
    • 1
    • 2
  • Christiane Rügenhagen
    • 1
    • 2
  • Peter Dietze
    • 2
  • Lothar F. Fecker
    • 2
  • Oscar J. M. Goddijn
    • 3
  • J. Harry C. Hoge
    • 3
  1. 1.Gesellschaft für Biotechnologische Forschung m.b.H.BraunschweigGermany
  2. 2.Biologische Bundesanstalt für Land- und ForstwirtschaftBraunschweigGermany
  3. 3.Institute of Molecular Plant SciencesLeiden University, Clusius LaboratoryLeidenThe Netherlands

Personalised recommendations