Abstract
The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.
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Berlin, J., Rügenhagen, C., Dietze, P., Fecker, L.F., Goddijn, O.J.M. and Hoge, J.H.C. (1993) Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA fromCatharanthus roseus.Transgenic Res. 2, 336–344.
Datla, R.S.S., Bekkaoui, F., Hammerlindl, J.K., Pilate, G., Dunstan, D.I. and Crosby, W.L. (1993) Improved high-level constitutive foreign gene expression in plants using an AMVRNA4 untranslated leader sequence.Plant Science 94, 139–149.
De Almeida, E.R.P., Gossele, V., Muller, C.G., Dockx, J., Reynaerts, A., Boterman, J., Krebbers, J. and Timko, M.P. (1989) Transgenic expression of two marker genes under the control of anArabidopsis rbcS promoter: Sequences encoding the rubisco transit peptide increase expression levels.Mol. Gen. Genet. 218, 78–86.
Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983) A plant DNA minipreparation. Version II.Plant Mol. Biol. Rep. 1, 19–21.
Fecker, L.F., Beier, H. and Berlin, J. (1986) Cloning and characterization of a lysine decarboxylase gene fromHafnia alvei.Mol. Gen. Genet. 203, 177–184.
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.Biotech. Lett. 14, 1035–40.
Fecker, L.F., Rügenhagen, C. and Berlin, J. (1993) Increased production of cadaverine and anabasine in tobacco hairy root cultures expressing a bacterial lysine decarboxylase gene.Plant Mol. Biol. 23, 11–21.
Fiedler, E. and Schultz, G. (1985) Localization, purification, and characterization of shikimate oxidoreductase-dehydroquinate hydrolase from stroma of spinach chloroplasts.Plant Physiol. 79, 212–8.
Gallie, D.T. and Wilbot, V. (1992) Identification of the motifs within in the tobacco mosaic virus 5′-leader responsible for enhancing translation.Nucl. Acids Res. 20, 4631–8.
Hamill, J.D., Robins, R.J., Parr, A.J., Evans, D.M., Furze, J.M. and Rhodes, M.J.C. (1990) Overexpressing a yeast ornthine decarboxylase gene in transgenic roots ofNicotiana rustica can lead to enhanced nicotine accumulation.Plant Mol. Biol. 15, 27–38.
Hartmann, T., Schoofs, G. and Wink, M. (1980) A chloroplast-localized lysine decarboxylase ofLupinus polyphyllus.FEBS Letters 115, 35–8.
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.Plant Mol. Biol. 17, 475–86.
Hibi, N., Fujita, T., Hatano, M., Hashimoto, T. and Yamada, Y. (1992) Putrescine N-methyltransferase in cultured cells ofHyoscyamus albus. n-Butylamine as a potent inhibitor of the transferase both invitro and invivo.Plant Physiol. 100, 826–835.
Kishore, G.M. and Somerville, C.R. (1993) Genetic engineering of commercially useful biosynthetic pathways in transgenic plants.Current Opinion Biotechnology 4, 152–8.
Landsmann, L., Llewellyn, D., Dennis, E.S. and Peacock, W.J. (1988) Organ regulated expression of theParasponia andersonii hemoglobin gene in trangenic tobacco plants.Mol. Gen. Genet. 214, 203–9.
Perl, A., Shaul, O. and Galili, G. (1992) Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts.Plant Mol. Biol. 19, 815–23.
Phan, A.P.H., Ngo, T.T. and Lenhoff, H.M. (1982) Spectrophotometric assay for lysine decarboxylase.Anal. Biochem. 120, 193–7.
Redmond, J.W. and Tseng, A. (1979) A high pressure liquid chromatography determination of putrescine, cadaverine, spermidine and spermine.J. Chromatogr. 170, 479–81.
Shaul, O. and Galili, G. (1991) Increased lysine synthesis in transgenic tobacco plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts.Plant. J. 2, 203–9.
Töpfer, R., Matzeit, V., Gronenborn, B., Schell, J. and Steinbiss, H.H. (1987) A set of plant expression vectors for transcriptional and translational fusions.Nucl. Acids Res. 15, 5890.
Velten, J. and Schell, J. (1985) Selection-expression plasmid vectors for use in genetic transformation of higher plants.Nucl. Acids Res. 13, 6981–98.
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.Plant Cell Rep. 7, 115–8.
Walton, N.J., Peerless, A.C.J., Robins, R.J., Rhodes, M.J.C., Boswell, H.D. and Robins, D.J. (1994) Purification and properties and putrescine N-methyltransferase from transformed roots ofDatura stramonium L.Planta 193, 9–15.
Widholm, J.M. (1976) Selection and characterization of cultured carrot and tobacco cells resistant to lysine, methionine, and proline analogs.Can. J. Bot. 54, 1523–9.
Wong, E.Y., Hironaka, C.M. and Fischhoff, D.A. (1992)Arabidopsis thaliana small subunit leader and transit peptide enhance the expression ofBacillus thuringiensis proteins in transgenic plants.Plant Mol. Biol. 20, 81–93.
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Herminghaus, S., Tholl, D., Rügenhagen, C. et al. Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence. Transgenic Research 5, 193–201 (1996). https://doi.org/10.1007/BF01969709
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DOI: https://doi.org/10.1007/BF01969709