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.

  • Article
  • Published:

Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control

Nature Biotechnology volume 19, pages 429–433 (2001)Cite this article

Abstract

We have developed transgenic mouse models to determine whether endogenous expression of phytase transgenes in the digestive tract of monogastric animals can increase the bioavailability of dietary phytate, a major but indigestible form of dietary phosphorus. We constructed phytase transgenes composed of the appA phytase gene from Escherichia coli regulated for expression in salivary glands by the rat R15 proline-rich protein promoter or by the mouse parotid secretory protein promoter. Transgenic phytase is highly expressed in the parotid salivary glands and secreted in saliva as an enzymatically active 55 kDa glycosylated protein. Expression of salivary phytase reduces fecal phosphorus by 11%. These results suggest that the introduction of salivary phytase transgenes into monogastric farm animals offers a promising biological approach to relieving the requirement for dietary phosphate supplements and to reducing phosphorus pollution from animal agriculture.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Phytase transgene constructs.
Figure 2: Analysis of phytase transgene expression in various tissues of transgenic mice.
Figure 3: Immunohistochemical staining of phytase in parotid glands of mice.
Figure 4: SDS–PAGE, silver staining.

Similar content being viewed by others

References

  1. Jongbloed, A.W. & Lenis, N.P. Environmental concerns of animal manure. J. Anim. Sci. 76, 2641–2648 (1998).

    Article  CAS  Google Scholar 

  2. Hudson, J.J., Taylor, W.D. & Schindler, D.W. Phosphate concentrations in lakes. Nature 406, 54–56 (2000).

    Article  CAS  Google Scholar 

  3. Mallin, M.A. Impacts of industrial animal production on rivers and estuaries. Am. Scientist 88, 26–37 (2000).

    Article  Google Scholar 

  4. Naqvi, S.W. et al. Increased marine production of N2O due to intensifying anoxia on the Indian continental shelf. Nature 408, 346–349 (2000).

    Article  CAS  Google Scholar 

  5. Delgado, C.L., Rosegrant, M.W., Steinfeld, H., Ehui, S. & Courbois, C. The coming livestock revolution. Choices 14, 40–44 (1999).

    Google Scholar 

  6. Tilman, D. Global environmental impacts of agricultural expansion: the need for sustainable and efficient practices. Proc. Natl. Acad. Sci. USA 96, 5995–6000 (1999).

    Article  CAS  Google Scholar 

  7. Reddy, N.R., Sathe, S.K. & Salunkhe, D.K. Phytates in legumes and cereals. Adv. Food Res. 28, 1–92 (1982).

    Article  CAS  Google Scholar 

  8. Murry, A.C., Lewis, R.D. & Amos, H.E. The effect of microbial phytase in a pearl millet–soybean meal diet on apparent digestibility and retention of nutrients, serum mineral concentration, and bone mineral density of nursery pigs. J. Anim. Sci. 75, 1284–1291 (1997).

    Article  CAS  Google Scholar 

  9. O'Quinn, P.R., Knabe, D.A. & Gregg, E.J. Efficacy of Natuphos in sorghum-based diets of finishing swine. J. Anim. Sci. 75, 1299–1307 (1997).

    Article  CAS  Google Scholar 

  10. Golovan, S., Wang, G., Zhang, J. & Forsberg, C.W. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can. J. Microbiol. 46, 59–71 (2000).

    Article  CAS  Google Scholar 

  11. Lim, D., Golovan, S., Forsberg, C.W., & Jia, Z. Crystal structures of Escherichia coli phytase and its complex with phytate. Nat. Struct. Biol. 7, 108–113 (2000).

    Article  CAS  Google Scholar 

  12. Leeson, S., Namkung, H., Cottrill, M. & Forsberg, C.W. Efficacy of new bacterial phytase in poultry diets. Can. J. Anim. Sci. 80, 527–528 (2000).

    Article  Google Scholar 

  13. Tu, Z.J. et al. Isoproterenol/tannin-dependent R15 expression in transgenic mice is mediated by an upstream parotid control region. Gene Expr. 3, 289–305 (1993).

    CAS  PubMed  Google Scholar 

  14. Laursen, J. & Hjorth, J.P. A cassette for high-level expression in the mouse salivary glands. Gene 198, 367–372 (1997).

    Article  CAS  Google Scholar 

  15. Chi, T.H. & Crabtree, G. R. Perspectives: signal transduction. Inositol phosphates in the nucleus. Science 287, 1937–1939 (2000).

    Article  CAS  Google Scholar 

  16. Brown-Grant, K. Enlargement of salivary gland in mice treated with isopropylnoradrenaline. Nature 191, 1076–1078 (1961).

    Article  CAS  Google Scholar 

  17. Zhang, J.X., Krell, P.J., Phillips, J.P. & Forsberg, C.W. Expression of a bacterial endo (1-4)-β-glucanase gene in mammalian cells and post translational modification of the gene product. Biochim. Biophys. Acta 1357, 215–224 (1997).

    Article  CAS  Google Scholar 

  18. Plaut, K. et al. Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice. Nat. Biotechnol. 19, 66–70 (2001).

    Article  Google Scholar 

  19. Murray, J.D. Genetic modification of animals in the next century. Theriogenology 51, 149–159 (1999).

    Article  CAS  Google Scholar 

  20. Ward, K.A. Transgene-mediated modifications to animal biochemistry. Trends Biotechnol. 18, 99–102 (2000).

    Article  CAS  Google Scholar 

  21. Ebino, K.Y., Yoshinaga, K., Saito, T.R. & Takahashi, K.W. A simple method for prevention of coprophagy in the mouse. Lab. Anim. 22, 1–4 (1988).

    Article  CAS  Google Scholar 

  22. Kemme, P.A., Radcliffe, J.S., Jongbloed, A.W. & Mroz, Z. Factors affecting phosphorus and calcium digestibility in diets for growing-finishing pigs. J. Anim. Sci. 75, 2139–2146 (1997).

    Article  CAS  Google Scholar 

  23. Scheiffele, P. & Füllekrug, J. Glycosylation and protein transport. In Molecular trafficking. (ed. Bernstein, P.) 27–36 (Portland Press, London, UK; 2000).

    Google Scholar 

  24. Corring, T. Endogenous secretions in the pig. In Current concepts of digestion and absorption in pigs. (eds. Low, A. G. & Partridge, I. G.) 136–150 (National Institute for Research in Dairying, Reading, UK; 1980).

    Google Scholar 

  25. Mikkelsen, T. R. et al. Tissue-specific expression in the salivary glands of transgenic mice. Nucleic Acids Res. 20, 2249–2255 (1992).

    Article  CAS  Google Scholar 

  26. Smith, L.C., Bordignon, V., Babkine, M., Fecteau, G. & Keefer, C. Benefits and problems with cloning animals. Can. Vet. J. 41, 919–924 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Westhusin, M. & Piedrahita, J. Three little pigs worth the huff and puff? Nat. Biotechnol. 18, 1144–1145 (2000).

    Article  CAS  Google Scholar 

  28. Kozak, M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947–950 (1987).

    Article  CAS  Google Scholar 

  29. Hogan, B., Costantini, F. & Lacy, E. Manipulating the mouse embryo. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1986).

    Google Scholar 

  30. Zhang, J.X., Meidinger, R., Forsberg, C.W., Krell, P.J. & Phillips, J.P. Expression and processing of a bacterial endoglucanase in transgenic mice. Arch. Biochem. Biophys. 367, 317–321 (1999).

    Article  CAS  Google Scholar 

  31. Church, G.M. & Gilbert, W. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991–1995 (1984).

    Article  CAS  Google Scholar 

  32. Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354 (1979).

    Article  CAS  Google Scholar 

  33. Rushmore, T.H. et al. Purification and characterization of P-52 (glutathione S-transferase-P or 7-7) from normal liver and putative preneoplastic liver nodules. Cancer Res. 48, 2805–2812 (1988).

    CAS  PubMed  Google Scholar 

  34. Canadian Council on Animal Care. Guide to the care and use of experimental animals, Vol. 1. (Canadian Council on Animal Care, Ottawa, ON, Canada; 1980).

  35. Hu, Y., Nakagawa, Y., Purushotham, K.R. & Humphreys-Beher, M.G. Functional changes in salivary glands of autoimmune disease-prone NOD mice. Am. J. Physiol. 263, E607–E614 (1992).

    Article  CAS  Google Scholar 

  36. Harlow, E. & Lane, D. Antibodies: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1988).

    Google Scholar 

  37. Jia, Z., Golovan, S., Ye, Q. & Forsberg, C.W. Purification, crystallization and preliminary X-ray analysis of the Escherichia coli phytase. Acta Crystallogr. D 54 (Pt 4), 647–649 (1998).

    Article  CAS  Google Scholar 

  38. Engelen, A.J., van der Heeft, F.C., Randsdorp, P.H. & Smit, E.L. Simple and rapid determination of phytase activity. J. AOAC Int. 77, 760–764 (1994).

    CAS  PubMed  Google Scholar 

  39. Nesterenko, M.V., Tilley, M. & Upton, S.J. A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels. J. Biochem. Biophys. Methods 28, 239–242 (1994).

    Article  CAS  Google Scholar 

  40. Association of Official Analytical Chemists (AOAC). Official methods of analysis of the Association of Official Analytical Chemists. (Association of Official Analytical Chemists, Washington, DC; 1984).

Download references

Acknowledgements

The -10R15/ CAT plasmid was kindly provided by Dr. D.K. Ann (University of Southern California, USA) and Lama 2 vector by Dr. J. Laursen and Dr. J.P. Hjorth (University of Aarhus, Denmark). We thank Roy Meidinger, Tania Archbold, Dr. Ming Fan, and staff of the Central Animal Facility for expert technical assistance. This research was supported by funds from Ontario Pork and the Natural Science and Engineering Research Council of Canada awarded to C.W.F. and J.P.P.

Author information

Authors and Affiliations

  1. Department of Microbiology, University of Guelph, Guelph, N1G 2W1, Ontario, Canada

    Serguei P. Golovan & Cecil W. Forsberg

  2. Department of Molecular Biology and Genetics, University of Guelph, Guelph, N1G 2W1, Ontario, Canada

    Serguei P. Golovan & John P. Phillips

  3. Department of Pathobiology, University of Guelph, Guelph, N1G 2W1, Ontario, Canada

    M. Anthony Hayes

Authors
  1. Serguei P. Golovan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Anthony Hayes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John P. Phillips
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cecil W. Forsberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to John P. Phillips or Cecil W. Forsberg.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Golovan, S., Hayes, M., Phillips, J. et al. Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control. Nat Biotechnol 19, 429–433 (2001). https://doi.org/10.1038/88091

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/88091

This article is cited by

Search

Advanced 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