© 2001 Nature Publishing Group http://biotech.nature.com
RESEARCH ARTICLE
Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control © 2001 Nature Publishing Group http://biotech.nature.com
Serguei P. Golovan1,2, M. Anthony Hayes3, John P. Phillips2*, and Cecil W. Forsberg1* 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 prolinerich 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.
duced in these transgenic mice leads to a significant reduction of fecal phosphorus levels.
Animal waste is a leading source of phosphorus pollution from agriculture1. Manure from monogastric animals such as poultry and swine is high in phosphorus, and when manure is repeatedly applied as fertilizer, phosphates can pollute surface and groundwater with severe biological consequences. Because phosphate is a limiting nutrient that restricts microbial populations in many freshwater environments2, the influx of phosphorus can lead to eutrophication. The results are cyanobacterial blooms, hypoxia and death of fish and aquatic animals3, and the production of nitrous oxide, a potent greenhouse gas4. The projected growth of the livestock industry5 is expected to accelerate such environmental problems on a global scale. It is critical that agricultural practices be modified to reduce such environmental impacts6. The high phosphorus content of manure from monogastric animals arises from the inability of these animals to hydrolyze phytate1, the major form of organic phosphate present in the typical plantbased diet7. The nutritional requirements for phosphorus needed to attain optimal growth in swine and poultry have traditionally been met through dietary supplementation with inorganic phosphate. This approach has been nutritionally successful but environmentally counterproductive. A more environmentally sound but far less common approach is the use of a microbial phytase as a feed additive. Phytase hydrolyzes phytate, and the addition of phytase to feed (250–1000 U kg-1) can fully replace phosphorus supplements at all stages of pig production8,9. However, the use of phytase as a feed additive is limited by cost, by inactivation at the high temperatures required for pelleting feed (+80°C), and by loss of activity during storage. These problems might be overcome if phytase were added to the repertoire of digestive enzymes produced endogenously by swine and poultry. Such endogenous phytase could increase the bioavailability of plant phytate and in turn lead to reduced phosphorus output from animal production. To test the feasibility of this hypothesis, we produced transgenic mice that secrete phytase in their saliva. The transgenes used in these studies contain the E. coli appA gene10,11, which was recently shown to be effective in poultry12, regulated either by the inducible proline-rich protein (PRP) R15 promoter from the rat13 or the constitutive parotid secretory protein (PSP) promoter from the mouse14. The salivary appA phytase pro-
Results Production of transgenic mice. The appA gene from E. coli was inserted downstream of the salivary-specific promoters R15-PRP and PSP, to obtain the inducible R15/APPA (Fig. 1A) and the constitutive PSP/APPA constructs (Fig. 1B). Because phytase can dephosphorylate inositol phosphates, some of which are involved in essential cellular functions15, our first model was designed with the inducible R15 promoter, in order to evaluate the possible deleterious effects of phytase expression on the animal. With the R15/APPA transgene, eight transgenic founder (G0) mice (four males and four females) were obtained, of which three did not pass the transgene to progeny and were probably mosaics. In the remaining five lines, phytase expression was induced by isoproterenol injection13. Isoproterenol injections increased the size of the salivary glands, an observation reported previously16. Phytase expression was not detected in either uninduced mice or in induced nontransgenic mice. A wide range of phytase expression was observed in the various R15/APPA transgenic lines (Table 1). We did not detect any deleterious effect of phytase expression on mice. Induced transgenic animals were fertile, producing both male and female offspring. As a second model, transgenic mice were generated using the constitutive PSP/APPA transgene (Table 1). Two transgenic founders (male and female) were produced. Despite the detection of the transgene by PCR, no phytase production was detected in saliva of the male founder or in his G1 offspring. The female founder produced a single transgenic G1 male out of 35 offspring. Phytase activity (30 U/ml) was detected in the saliva of both the G0 female founder and the G1 male offspring. The number of newborns in the G2 generation, their gender, and their viability were the same in the transgenic and nontransgenic animals, further documenting the transgene’s lack of toxicity. Expression of the phytase transgene in salivary glands. Northern blot analysis demonstrated very strong expression of phytase messenger RNA (mRNA) in parotid glands and a fivefold lower expres-
Department of Microbiology1, Department of Molecular Biology and Genetics2, Department of Pathobiology3, University of Guelph, Guelph, Ontario N1G 2W1, Canada. *Corresponding authors (
[email protected] or
[email protected]). http://biotech.nature.com
•
MAY 2001
•
VOLUME 19
•
nature biotechnology
429
© 2001 Nature Publishing Group http://biotech.nature.com
RESEARCH ARTICLE A
A
B
B D
© 2001 Nature Publishing Group http://biotech.nature.com
C Figure 1. Phytase transgene constructs. (A) Inducible R15/APPA transgene. (B) Constitutive PSP/APPA transgene. The R15-PRP and PSP promoters and the appA phytase gene are described in the text.
sion in submandibular glands following induction of R15/APPA mice (Fig. 2A). Constitutive expression in the PSP/APPA mice was also very specific for parotid and submandibular glands (Fig. 2B). No phytase mRNA transcripts were detected in other tissues of the transgenic mice or in any tissue of the nontransgenic animals (see Table 2 for tissues tested). Phytase protein was detected in parotid and submandibular glands of both R15/APPA (Fig. 2C) and PSP/APPA mice (Fig. 2D) using anti-phytase antibodies to probe western blots, but not in the other tissues of the animals. No phytase protein was detected in the nontransgenic animals. The relative molecular mass of the transgenic phytase was 55,000 (Mr 55K) compared with 45K for the E. coli enzyme, indicating post-translational modification of the enzyme. Two bands of equal intensity were detected for enzyme from parotid glands of R15/APPA, but only one in saliva, suggesting that complete modification of phytase is required for secretion. We detected 14.7 U/mg of phytase activity in parotid gland extracts and 3.5 U/mg in submandibular glands from induced R15/APPA mice (Table 2). Some increase in the level of phytase activity was detected in other tissues, but it was
