Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

Production of Cyclodextrins, a Novel Carbohydrate, in the Tubers of Transgenic Potato Plants

Abstract

Cyclodextrins (CDs) are cyclic oligosaccharides containing six (α), seven (β), or eight (γ) glucose molecules, respectively. The cyclodextrin glycosyltransferases (CGT), which produce CDs from starch, are found only in bacteria and are used in batch fermentors with hydrolyzed starch to produce CDs commercially. Using a CGT gene from Klebsiella, we attempted to engineer the tubers of developing potatoes to produce these novel, high–value carbohydrates. A chimeric gene, consisting of (1) the patatin promoter for tuber–specific expression, (2) the small subunit of ribulose bisphosphate carboxylase (SSU) transit peptide for plastid targeting, (3) the CGT structural gene from Klebsiella and (4) the nopaline synthase 3′ region, was introduced into potatoes. Both α and β CDs were produced in tubers of transgenic potatoes at levels corresponding to 0.001–0.01% of the starch being converted to CDs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Whistler, R.L. 1984. History and future expectation of starch use, p. 1–9. In: Starch Chemistry and Technology, second edition. Whistler, R.L., BeMiller, J.N., and Paschall, E.F. (Eds.). Academic Press, New York.

    Google Scholar 

  2. Shannon, J.C. and Garwood, D.L. 1984. Genetics and physiology of starch development, p. 25–86. In: Starch Chemistry and Technology, second edition. Ibid.

    Chapter  Google Scholar 

  3. Hovenkamp-Hermelink, J.H.M., Jacobsen, E., Ponstein, A.S., Visser, R.G.F., Vos-Scheperkeuter, G.H., Bijmolt, E.W., deVries, J.N., Witholt, B. and Feenstra, W.J. 1987. Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theor. Appl. Gen 75: 217–221.

    Article  Google Scholar 

  4. Visser, R.G.F., Somhorst, I., Kuipers, G.J., Ruys, N.J., Feenstra, W.J. and Jacobsen, E. 1991. Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Mol. Gen. Genet. 225: 289–296.

    Article  CAS  PubMed  Google Scholar 

  5. Szejtli, J. 1988. Cyclodextrin Technology. Kluwer Academic Publishers, Dordrecht, The Netherlands.

    Book  Google Scholar 

  6. Szejtli, J. 1989. Downstream processing using cyclodextrins. Trends in Biotechnology 7: 170–174.

    Article  CAS  Google Scholar 

  7. Parrish, M.A., 1987. Cyclodextrins, a review. Specialty Chemicals 7: 366–380.

    CAS  Google Scholar 

  8. Starnes, R.L. 1990. Industrial potential of Cyclodextrin glycosyl transferases. Cereal Foods World 35: 1094–1099.

    CAS  Google Scholar 

  9. Binder, F., Huber, O. and Böck, A. 1986. Cyclodextrin-glycosyltrans-ferase from Klebsiella pneumoniae M5al: cloning, nucleotide sequence and expression. Gene 47: 269–277.

    Article  CAS  PubMed  Google Scholar 

  10. Mares, D.J., Sowokinos, J.R. and Hawker, J.S. 1985. Carbohydrate metabolism in developing potato tubers, p. 279–327. In: Potato Physiology. Li, P.H. (Ed.). Academic Press, New York.

    Google Scholar 

  11. Park, W.D. 1983. Tuber proteins of potato - A new and surprising molecular system. Pl. Mol. Biol. Rept. 1: 61–66.

    Article  CAS  Google Scholar 

  12. Rocha-Sosa, M., Sonnewald, U., Frommer, W., Stratmann, M., Schell, J. and Willmitzer, L. 1989. Both developmental and metabolic signals activate the promoter of a class I patatin gene. The EMBO J. 8: 23–29.

    Article  CAS  PubMed  Google Scholar 

  13. Wenzler, H.C., Mignery, G.A., Fisher, L.M. and Park, W.D. 1989. Analysis of a chimaeric class I patatin-GUS gene in transgenic potato plants: high-level expression in tubers and sucrose-inducible expression in cultured leaf and stem explants. Plant Mol. Biol. 12: 41–50.

    Article  CAS  PubMed  Google Scholar 

  14. Jefferson, R., Goldsbrough, A. and Bevan, M. 1990. Transcriptional regulation of a patatin-1 gene in potato. Plant Mol. Bio. 14: 995–1006.

    Article  CAS  Google Scholar 

  15. Van den Broeck, G., Timko, M. P., Kausch, A. P., Cashmore, A. R., Van Montagu, M. and Herrera-Estrella, L. 1985. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bis phosphate carboxylase. Nature 313: 358–363.

    Article  CAS  PubMed  Google Scholar 

  16. Schreier, P.H., Seftor, E.A., Schell, J. and Bohnert, H.J. 1985. The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. The EMBO J. 4: 25–32.

    Article  CAS  PubMed  Google Scholar 

  17. Wasmann, C.C., Reiss, B., Bartlett, S.G. and Bohnert, H.J. 1986. The importance of the transit peptide and the transported protein for protein import into chloroplasts. Mol. Gen. Genet. 205: 446–453.

    Article  CAS  Google Scholar 

  18. Comai, L., Larson-Kelly, N., Kiser, J., Mau, C.J.D., Pokalsky, A.R., Shewmaker, C.K., McBride, K., Jones, A. and Stalker, D.M. 1988. Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyI 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide. J. Biol. Chem. 263: 15104–15109.

    CAS  PubMed  Google Scholar 

  19. Klösgen, R.B. and Weil, J.H. 1991. Subcellular location and expression level of a chimeric protein consisting of the maize waxy transit peptide and the β-glucuronidase of Escherichia coli in transgenic potato plants. Mol. Gen. Genet. 225: 297–304.

    Article  PubMed  Google Scholar 

  20. Bevan, M., Barnes, W.M. and Chilton, M.-D. 1983. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 11: 369–385.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. McBride, K.E. and Summerfelt, K. 1990. Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 14: 269–276.

    Article  CAS  PubMed  Google Scholar 

  22. Comai, L., Moran, P. and Maslyar, D. 1990. Novel and useful properties of chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol. Biol. 15: 373–381.

    Article  CAS  PubMed  Google Scholar 

  23. Mäkelä, M.J., Korpela, T.K., Puisto, J. and Laakso, S.V. 1988. Cyclodextrin assays: evaluation of sensitivity, specificity, and conversion mixture applications. J. Agric. Food Chem. 36: 83–88.

    Article  Google Scholar 

  24. Bender, H. 1982. Enzymology of the cyclodextrins, p. 77–87. In: Proc. Int. Symp. Cyclodextrins. Szejtli, J. (Ed.). Reidel, Dordrecht, The Netherlands.

    Chapter  Google Scholar 

  25. Bender, H. 1990. Studies of the mechanism of the cyclisation reaction catalysed by the wildtype and a truncated α-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain m 5 al, and the β-cyclodextrin glycosyltransferase from Bacillus circulans strain 8. Carb. Res. 206: 257–267.

    Article  CAS  Google Scholar 

  26. Hiatt, A.C. 1989. Production of antibodies in transgenic plants. Nature 342: 76–78.

    Article  CAS  PubMed  Google Scholar 

  27. Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., van den Elzen, P.J.M. and Hoekema, A. 1990. Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8: 217–221.

    CAS  Google Scholar 

  28. Goodman, R.M., Houck, C.M. and Comai, L. 1987. Molecular Farming. PCT WO 87/00865.

    Google Scholar 

  29. Pool, R. 1989. In search of the plastic potato. Science 245: 1187–1189.

    Article  CAS  PubMed  Google Scholar 

  30. Keeler, R. 1991. Don't let food go to waste-make plastic out of it. R&D Magazine 33: 52–57.

    Google Scholar 

  31. van Es, A. and Hartmans, K.J. 1981. Structure and chemical composition of the potato, p. 17–81. In: Storage of Potatoes; Post-harvest Behaviour, Store Design, Storage Practice and Handling. Rastovoski, A., and van Es., A. (Eds.). Center for Agricultural Publishing and Documentation. Wageningen, The Netherlands.

    Google Scholar 

  32. Murray, E.E., Lotzer, J. and Eberle, M. 1989. Codon usage in plants. Nuc. Acids Res. 17: 477–498.

    Article  CAS  Google Scholar 

  33. Hoekema, A., Kastelein, R.A., Vasser, M. and deBoer, H. A. 1987. Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. Mol. and Cell. Bio. 7: 2914–2924.

    Article  CAS  Google Scholar 

  34. Perlak, F.J., Deaton, R.W., Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate, J.T. and Fischoff, D.A. 1990. Insect resistant cotton plants. Bio/Technology 8: 939–943.

    CAS  Google Scholar 

  35. Perlak, F.J., Fuchs, R.L., Dean, D.A., McPherson, S.L. and Fischoff, D.A. 1991. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc. Natl. Acad. Sci. USA 88: 3324–3328.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. de Boer, D., Cremers, F., Teertstra, R., Smits, L., Hille, J., Smeekens, S. and Weisbeek, P. 1988. In vivo import of plastocyanin and a fusion protein into developmentally different plastids of transgenic plants. EMBO J. 7: 2631–2635.

    Article  CAS  PubMed  Google Scholar 

  37. Villiers, A. 1891. Sur la transformation de le fécule en dextrine par le ferment buytrique. Compt. Rend. 112: 435–437.

    Google Scholar 

  38. Bender, H. 1977. Cyclodextrin-glucanotransferase von Klebsiella pneumoniae 1. synthese, reinigung und eigenschaften des enzymes von Klebsiella pneumoniae M5 al. Arch. Microbiol. 111: 271–282.

    Article  CAS  PubMed  Google Scholar 

  39. Kimura, K., Takano, T. and Yamane, K. 1987. Molecular cloning of the β-cyclodextrin synthetase gene from an alkatophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis. Appl. Microbiol. Biotechnol. 26: 149–153.

    Article  CAS  Google Scholar 

  40. Bevan, M., Barker, R., Goldsbrough, A., Jarvis, M., Kavanagh, T. and Iturriaga, G. 1986. The structure and transcription start site of a major potato tuber protein gene. Nucleic Acids Res. 14: 4625–4638.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yanisch-Perron, C., Vieira, J. and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequence of M13mp18 and vectors. Gene 33: 103–119.

    Article  CAS  PubMed  Google Scholar 

  42. Chang, A.C.Y. and Cohen, S.N. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from p15A criptic mini-plasmid. J. Bacteriol. 134: 1141–1156.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Rose, R.E. 1988. The nucleotide sequence of pACYC184. Nucleic Acids Res. 16: 355.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoot, R.A. 1988. A binary vector based on separation of vir and T-region of the Agrobacterium tumefaciens Ti plasmids. Nature 303: 179–181.

    Article  Google Scholar 

  45. Holsters, M., DeWarle, D., Depicker, A., Messens, E., Van Montague, M. and Schell, J. 1979. Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet. 163: 181–187.

    Article  Google Scholar 

  46. Sheerman, S. and Bevan, M.W. 1988. A rapid transformation method for Solanum tuberosum using binary Agrobacterium tumefaciens vectors. Plant Cell Rep. 7: 13–16.

    Article  CAS  PubMed  Google Scholar 

  47. Radke, S.E., Andrews, B.M., Moloney, M.M., Crouch, M.L., Kridl, J.C. and Knauf, V.C. 1988. Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a re-introduced napin gene. Theor. Appl. Genet. 75: 685–694.

    Article  CAS  Google Scholar 

  48. Logemann, J., Schell, J. and Willmitzer, L. 1987. Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163: 16–20.

    Article  CAS  PubMed  Google Scholar 

  49. Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  50. Facciotti, D., O'Neal, J.K., Lee, S. and Shewmaker, C.K. 1985. Light-inducible expression of a chimeric gene in soybean tissue transformed with Agrobacterium. Bio/Technology 3: 241–246.

    CAS  Google Scholar 

  51. Shewmaker, C.K., Caton, J.R., Houck, C.M. and Gardner, R.C. 1985. Transcription of cauliflower mosaic virus integrated into plant genomes. Virology 140: 281–288.

    Article  CAS  PubMed  Google Scholar 

  52. Wiedenhof, N. 1964. Thin-layer chromatography of cyclodextrins and some other sugars using microchromatoplates. J. Chromatog. 15: 100–102.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Oakes, J., Shewmaker, C. & Stalker, D. Production of Cyclodextrins, a Novel Carbohydrate, in the Tubers of Transgenic Potato Plants. Nat Biotechnol 9, 982–986 (1991). https://doi.org/10.1038/nbt1091-982

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1091-982

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing