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.
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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.
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.
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.
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.
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.
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.
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.
Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983) A plant DNA minipreparation: version II.Plant Mol. Biol. Rep. 1, 19–21.
DeLuca, V. and Cutler, A.J. (1987) Subcellular localization of enzymes involved in indole alkaloid biosynthesis inCatharanthus roseus.Pl. Physiol. 85 1099–102.
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.
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.
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.
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.
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.
Gamborg, O.L., Murashige, T., Thorpe, T.A. and Vasil, I.K. (1976) Plant tissue culture media.In Vitro 12, 473–8.
Goddijn, O.J.M. (1992) Regulation of terpenoid indole alkaloid biosynthesis inCatharanthus roseus: the tryptophan decarboxylase gene. Ph.D. Thesis, Leiden University.
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.
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.
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.
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.
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.
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.
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.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.
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.
Noé, W. and Berlin, J. (1985) Induction of de-novo synthesis of tryptophan decarboxylase in suspension cultures ofCatharanthus roseus.Planta 166, 500–4.
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.
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.
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.
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.
Sasse, F., Witte, L. and Berlin, J. (1987) Biotransformation of tryptamine to serotonin by cell suspension cultures ofPeganum harmala.Planta Medica 53, 354–9.
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.
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.
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.
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.
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Berlin, J., Rügenhagen, C., Dietze, P. et al. Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus . Transgenic Research 2, 336–344 (1993). https://doi.org/10.1007/BF01976175
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DOI: https://doi.org/10.1007/BF01976175