A new method for gene synthesis and its high-level expression.
An optimized Citrobacter braakii phytase gene, appA-c, was chemically synthesized by oligonucleotides synthesis and over-lap PCR method. The appA-c gene encoding 423 amino acids was cloned into expression vector pPIC9 and transformed into methylotropic yeast Pichia pastoris. From about 2000 transformants, 400 transformants exhibiting phytase activity were obtained. One transformant showing the strongest phytase activity was selected for detailed analyses in 5 L bioreactor. Under control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the transformant was able to secrete 3.85 mg/ml protein to the culture supernatant in about 110 h methanol induction, which comprises of 12,116 U ml(-1) phytase activity. Further characterization of the recombinant phytase was conducted. The optimal pH and temperature for this recombinant phytase was about 4.0 and 50 degrees C, respectively. Fe3+, Zn2+ and Cu2+ could significantly inhibit the recombinant phytase enzyme activity. The specific activity of this recombinant enzyme was 3147 U mg(-1). The K(m) and V(max) values for sodium phytate were determined to be 0.5 mM and 3085 U/mg, respectively. To our knowledge, this is the first report of a chemically synthesized C. braakii appA gene heterologous expression with the highest expression level and highest phytase activity achieved. The novel gene optimization and synthesis method can be applied to other related researches.
Cao
Shi-shu
SS
Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA. caoshishu@yahoo.com
Hu
Zhi-qiu
ZQ
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002954:Citrobacter / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression; D005813:Genes, Synthetic; D005818:Genetic Engineering / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D013696:Temperature
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008670:Metals; D010833:Phytic Acid; D010832:6-Phytase