Transgenic mice expressing AppA phytase as a model demonstrating reduced pollution in monogastric animals

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Golovan, Serguei Petrovich
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University of Guelph

A main challenge for this century is to create environmentally sustainable agriculture which will provide the growing world population with high quality food with minimal impact on the environment. Phosphorus from animal agriculture is a leading source of eutrophication in many freshwater environments. This results, in part, from the inability of monogastric animals to digest phytate, the main source of phosphorus in plants. To eliminate pollution caused by phytate I propose to introduce a phytase gene into the animal genome, which could give monogastric animals the endogenous ability to degrade phytate relieving the requirement for dietary phosphate supplements and reducing phosphorus pollution from animal agriculture. As a first step in the development of a mouse model with endogenous phytase, a highly active 'appA'-encoded acid phosphatase was isolated from 'Escherichia coli'. AppA was overexpressed, purified to homogeneity and characterized biochemically which demonstrated that AppA exhibits both acid phosphatase and phytase activities. AppA was shown to be highly active at low pH and stable in the presence of pepsin, making this enzyme an excellent candidate for generation of transgenic animals expressing phytase in saliva. Screening of a genomic library also identified a second protein with putative phytase activity encoded by the 'agp' gene and a possible transcriptional activator for the 'appA' gene. To test the feasibility of our hypothesis, transgenic mice were produced using constructs with either an inducible rat proline-rich protein (PRP) R15 promoter or a constitutive mouse parotid secretory protein (PSP) promoter. Phytase was specifically produced by salivary glands and secreted into saliva using either the inducible or constitutive transgenes. No deleterious effects of phytase expression were detected on the health, reproduction or physiology of the transgenic mice. The presence of phytase in saliva resulted in a decrease in fecal phosphorus by 10 to 11%. These results validate our original hypothesis and demonstrate that transgenic mice with salivary gland-specific transgene encoded expression can be used for efficient production of heterologous proteins.

phytase gene, gene expression, animal genome, monogastric animals, endogenous phytase, appA phytase, appA-encoded acid phosphatase, phosphorus pollution