Functional expression of an E. coli phytase in the chloroplast of transgenic Chlamydomonas reinhardtii
So-Mi Yoon1, So-Young Kim1, Kun Feng Li1, Byung-Hak Yoon1, James G. Umen2, Senyon Choe1,3 and Mario M.-C. Kuo1
1) Synthetic Biology Laboratory, joint Center for Biosciences, Incheon 406-840, South Korea
2) Plant Molecular and Cellular Biology and 3) Structural Biology Laboratories, The Salk Institute for Biological Studies, La Jolla, CA 92037, U.S.A.
Phytase is enzyme that hydrolyzes phytic acid into inorganic orthophosphates and lowers inositol phosphatates. This enzyme can improve the nutrition value of animal diet and make positive environmental impacts. Here we created a stable transgenic Chlamdymonas reinhardtii cell line, expressing functional phytase of Escherichia coli AppA in the chloroplast. Codon optimized appA gene with C-terminal His tag was cloned into an expression vector, pATPA, between the atpA 5'UTR and rbcL 3' UTR of the native C. reinhardtii chloroplast genome. The expression and the spectinomycin-resistant p228 plasmids were co-transformed into C. rehinhardtii strain CC-125 by gold particle bombardment, and the transformants were selected under 150 µg/ml spectinomycin and screened by colony PCR amplification. The activity of the transgenic AppA has been confirmed using dry cell powder prepared by freeze-drying the cell paste. It has optimal catalytic pH and temperature of 4.5 and 60 °C, respective, which is consistent with those reported from purified AppA phytase. This study demonstrates that the C. rehinhardtii chloroplast is able to produce the microbial phytase and their phytase activity can be directly shown in a form of whole cell lysate. Our results can facilitate the development of food or animal feed supplement with a goal to reduce environmental burden of phytic acid normally excreted in excess from chicken farms. This work supported by grants from IFEZ and WCU-KOREA.
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