Transgenic microalgae expressing <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks

Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization and to reduce fecal phytates and inorganic phosphate (iP) outputs. To facilitate the process of application, we engineered an Escherichia coli appA phytase gene into the chloroplast genome of the model microalga, Chlamydomonas reinhardtii, and isolated homoplasmic plastid transformants. The catalytic activity of the recombinant E. coli AppA can be directly detected in the whole-cell lysate, termed Chlasate, prepared by freeze-drying the transgenic cell paste with liquid nitrogen. The E. coli AppA in the Chlasate has a pH and temperature optima of 4.5 and 60°C, respectively, which are similar to those described in the literature. The phytase-expressed Chlasate contains 10 phytase units per gram dry matter at pH 4.5 and 37°C. Using this transgenic Chlasate at 500 U/kg of diet for young broiler chicks, the fecal phytate excretion was reduced, and the iP was increased by 43% and 41%, respectively, as compared to those of the chicks fed with only the basal diet. The effectiveness of the Chlasate to break down the dietary phytates is compatible with the commercial Natuphos fungal phytase. Our data provide the first evidence of functional expression of microbial phytase in microalgae and demonstrate the proof of concept of using transgenic microalgae as a food additive to deliver dietary enzymes with no need of protein purification.