Transgenic Research 13: 29–39, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 29 Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds Chwan-Yang Hong 1,4 , Kuo-Joan Cheng 2 , Tung-Hai Tseng 3 , Chang-Sheng Wang 3 , Li-Fei Liu 4,∗ & Su-May Yu 1,∗ 1 Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC 2 Institute of BioAgricultural Sciences, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC 3 Taiwan Agricultural Research Institute, Wu-Fong, Taichung, Taiwan, ROC 4 Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC Received 24 March 2003; revised 24 June 2003; accepted 16 July 2003 Key words: α-amylase gene promoter, feed additive, germinating seeds, phytase, rice Abstract Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a ger- mination inducible α-amylase gene (αAmy8) promoter, and extracellular phytase secretion directed by an αAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46–60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0–5.5 and 2.0–6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated trans- genic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals. Introduction Phytate (myo-inositol hexakisphosphate) is the main storage form of phosphorus in many seeds. In cere- als, legumes and Brassicae, phytate accumulates in seeds during maturation and accounts for 50–80% of total phosphorus (Austin et al., 1994). Soybean and corn meals are major components of animal feed and contain adequate phosphorus levels to meet an- ∗ Authors for correspondence E-mails: sumay@ccvax.sinica.edu.tw, lfliu@ntu.edu.tw imal growth requirements if phosphorus from phytate could be made available. However, monogastric an- imals (e.g., pigs, poultry and fish) utilize phytate extremely poorly due to deficiencies in gastrointestinal tract enzymes capable of hydrolyzing phytate, which necessitates supplementation of animal rations with expensive phosphorus to meet dietary requirements (Ravindran et al., 1995). When used as feed, phytate- phosphorus passes through the gastrointestinal tract of monogastric animals and is excreted in the manure. Consequently, a significant amount of nutritionally important phosphorus is not utilized by the animals. The excreted phosphorus contributes significantly