Enviropig™

Enviropig™ > Technology


Technology

What is an Enviropig™?

Enviropig™ is a trademark used to designate a genetically modified (or genetically engineered) line of Yorkshire pigs that produces phytase in the salivary glands (parotid, submaxillary and sublingual), and secretes the enzyme in the saliva.

How does it work?

A transgene construct containing the murine (mouse) parotid secretory protein promoter gene sequence and the Escherichia coli phytase gene was introduced into the pig chromosome by pronuclear microinjection. This technique does not involve the use of either viral DNA or antibiotic resistance genes. The transgene construct was integrated in a single site in the genome, and shown to be stably transmitted to offspring in a Mendelian fashion through 8 generations. The promoter directs constitutive (continuous) production of the active phytase enzyme in secretory cells of the salivary glands including the parotid, submaxillary and sublingual glands. The phytase is secreted in the saliva and enters the mouth where it mixes with feed consumed. The phytase is most active in the acidic environment of the stomach (pH range of 2.0 to 5.5 during food consumption). There the enzyme digests the phosphorus rich phytate molecules releasing phosphate molecules that are readily absorbed from the small intestine.

The phytase is highly resistant to pepsin the major protease in the stomach, but is destroyed by trypsin and chymotrypsin in the small intestine and none is detected in the ileal contents. The phytase produced by the Enviropig™ is as active as the enzyme produced in Escherichia coli. There is only one protein produced from the transgene and that is the phytase enzyme.

Phytase Action

Figure 2. The action of phytase releasing orthophosphate from phytic acid.

How was an Enviropig™ created?

The Enviropig™ was developed by the introduction of a transgene construct composed of the promoter segment of the murine parotid secretory protein gene and the Escherichia coli phytase gene (Golovan et al 2001) into a fertilized porcine embryo by pronuclear microinjection, and this embryo along with other embryos was surgically implanted into the reproductive tract of an estrous synchronized sow. After a 114 day gestation period, the sow farrowed and piglets born were checked for the presence of the transgene and for phytase enzyme activity in the saliva. When the mature genetically modified pig was crossed with a conventional pig, approximately half of the pigs contained the phytase transgene. This showed that the transgene was stably inserted into one of the chromosomes of the pigs and was inherited in a Mendelian fashion. Through breeding, this line of pigs is in the 8th generation.

Food safety

Information on swine health and welfare, tissue composition, toxicology, allergenicity and performance characteristics have been submitted to the Food and Drug Administration in the United States.

Regulatory Hurdles on the Path to the Meat Counter

International: At the international level (CODEX ALIMENTARIUS), an organization under the auspices of the United Nations has developed guidelines for the safety assessment of transgenic food animals. These guidelines were developed by consensus among representative from each member country. Individual countries use these guidelines as a framework for development of their unique set of regulations.

United States: In January 2009 the Center for Veterinary Medicine of the Food and Drug Administration released a document entitled Regulation of Genetically Engineered Animals Containing Heritable Recombinant DNA Constructs to provide guidance for organizations pursuing regulatory approval of genetically engineered food animals.

Canada: In Canada regulatory approval for commercialization of a transgenic food animal uses as the basis the CODEX ALIMENTARIUS guidelines for human food safety, however, approval is separately required for environmental, feed and fertilizer safety.

  1. Human food safety is assessed by the Novel Foods Guidelines under the auspices of Health Canada (Guidelines for the Safety Assessment of Novel Foods)
  2. Environmental Safety as prescribed by Schedule 5 of the (Canadian Environmental Protection Act) by the New Substance Division of Environment Canada
  3. Feed safety (Feeds Act) by the Canadian Food Inspection Agency (CFIA) and
  4. Fertilizer safety (Fertilizer Act) by CFIA.

 

Financial Contributors to Enviropig™ Research

  • Ontario Pork (www.ontariopork.on.ca)
  • Ontario Ministry of Agriculture, Food and Rural Affairs (www.omafra.gov.on.ca) through a contract to the University of Guelph
  • Rural Economic Development Program of the Ontario Government
  • Advanced Food Materials Network (www.afmnet.ca) (Network Centres of Excellent funded by Federal Granting Agencies)
  • Natural Sciences and Engineering Research Council of Canada (www.nserc-crsng.gc.ca)
  • Agriculture and Agri-Food Canada (www.agr.ca)
  • University of Guelph provides the expertise and facilities
History
  • 1995 Scientists at the University of Guelph first conceived of the idea of expressing phytase in the pig.
  • 1996 A gene from Escherichia coli coding for acid phosphatase, also called phytase was identified as a good candidate to produce phytase endogenously
  • 1997-1998 Purified and characterized the phytase enzyme, identified the mouse parotid secretory protein promoter and constructed the phytase transgene.
  • 1999 Created first phytase pig called Wayne, which was soon followed by Jacques and Gordy and Cassie and later 30 other transgenic pigs with the same construct
  • 2000 Patents submitted for USA and China.
  • 2001 Published expression of the phytase in the salivary glands of mice and pigs.
  • 2006 and 2007 Patents granted in the United States (http://www.patentstorm.us/patents/7115795.html) and China.
  • 2007 Submission of food and environmental safety & efficacy data to the Food and Drug Administration for regulatory approval of the Cassie line of Enviropig™ for human food consumption, and subsequent commercialization in the United States.
  • 2009 Initiated a submission to Canadian Regulatory Agencies for approval of the Cassie line of Enviropig™ for human food consumption and subsequent commercialization in Canada.
Peer Reviewed Publications on the Enviropig™
  1. Hakimov,H.A., Walters,S., Wright,T.C., Meidinger,R.G., Verschoor,C.P., Gadish,M., Chiu,D.K., Stromvik,M.V., Forsberg,C.W., and Golovan,S.P. 2009. Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization. Proteomics 9 :4000-4016, 2009.
  2. Golovan,S.P., Hakimov,H.A., Verschoor,C.P., Walters,S., Gadish,M., Elsik,C., Schenkel,F., Chiu,D.K.Y. and Forsberg,C.W. 2008. Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders, and conventional and genetically enhanced lines. Comp.Biochem.Physiol.Part D Genomics and Proteomics Part D3: 234-242.
  3. Mao, J., Ajakaiye, A., Lan, Y., Olk, D.C., Ceballos, M., Zhang, T., Fan, M.Z and Forsberg, C.W. 2008. Chemical structures of manure from conventional and phytase transgenic pigs investigated by advanced solid-state NMR spectroscopy. J. Agric. Food Chem. In Press Jan. 2008.
  4. Murray,D., Meidinger,R.G., Golovan,S.P., Phillips,J.P., O'Halloran,I.P., Fan,M.Z., Hacker,R.R. and Forsberg,C.W. 2007. Transgene and mitochondrial DNA are indicators of efficient composting of transgenic pig carcasses. Bioresour.Technol. 98: 1795-1804.
  5. Yang,X.M., C.F. Drury, T.Q. Zhang, A. Ajakaiye, C.W. Forsberg, M.Z. Fan, and J.P. Philip. 2006. Inorganic N dynamics from soils amended with low-P manure from genetically modified pigs (Enviropig TM). Nutr. Cycle. Agroecosyst. 75:297-304.
  6. Lie, X., J. P. Blake, C. W. Forsberg, D. G. Fox, E. Grabau, Z. Mroz, A. L. Sutton, W. R. Walker, and K. Web. 2006. Biotechnology of manure nutrient management. Council for Agricultural Science and Technology 33:1-20, 2006.
  7. Forsberg, C.W., Phillips, J.P., Golovan, S.P., Fan, M.Z., Meidinger, R.G., Ajakaiye A., Hilborn D., and Hacker R.R. 2003.The Enviropig physiology, performance, and contribution to nutrient management, advances in a regulated environment: The leading edge of change in the pork industry. J.Anim.Sci. 81(E. Suppl. 2):E68-E77.
  8. Golovan, S., Wang, G., Zhang, J., and Forsberg, C.W. 2000.Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme which exhibits both phytase and acid phosphatase activities. Can.J.Microbiol 46:59-71.
  9. Golovan,S.P., Hayes,M.A., Phillips,J.P. and Forsberg,C.W. 2001a. Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control. Nat.Biotechnol. 19: 429-433.
  10. Golovan,S.P., Meidinger,R.G., Ajakaiye,A., Cottrill,M., Wiederkehr,M.Z., Barney,D., Plante,C., Pollard,J., Fan,M.Z., Hayes,M.A., Laursen,J., Hjorth,J.P., Hacker,R.R., Phillips,J.P. and Forsberg,C.W. 2001b. Pigs expressing salivary phytase produce low phosphorus manure. Nat.Biotechnol. 19: 741-745.
  11. Lim, D., Golovan, S., Forsberg, C.W. and Jia, Z. 2000. Crystal structures of Escherichia coli phytase and its complex with phytate. Nature Struct. Biol. 7: 108-113.

 

Book Chapters and Conference Proceedings on the Enviropig™
  1. Forsberg, C.W., Golovan, S.P., Ajakaiye, A., Phillips, J.P., Meidinger, R.G., Fan, M.Z., Kelly, J.M. and Hacker, R.R.. 2005. Genetic opportunities to enhance sustainability of pork production in developing countries: A model for food animals. Pp429-446 In Applications of gene-based technologies for improving animal production and health in developing countries. H.P.S. Makkar and G.J. Viljoen (eds.) Springer, Dordrecht, The Netherlands.
  2. Forsberg, C.W., Golovan, S.P., Phillips, J.P., Ajakaiye, A., Fan, M. and Hacker, R.R.. 2003. Transgenic approaches to enhance the environmental and physiological characteristics of pigs and their impact on the pork industry. Manitoba Swine Seminar edited by I. Seddon, vol. 17, p. 71-88.

 

Interesting Reviews on Animal Biotechnology
  1. Gottlieb, S. and Wheeler, M.B.. 2008. Genetically engineered animals and public health: Compelling benefits for health care, nutrition, the environment, and animal welfare, Biotechnology Industry Organization, Washington, D.C. www.bio.org, 2008. http://www.bio.org/foodag/animals/ge_animal_benefits.pdf

 

Popular Press

 

Links
  1. Ontario Pork http://www.ontariopork.on.ca/
  2. Canadian Pork Council http://www.ontariopork.on.ca/
  3. National Pork Board (USA) http://www.pork.org/
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