Design of gene structure and expression of E. coli phytase and Aspergillus Niger phytase in Pichia pastoris yeast in order to increase plant phytate degradation Bahareh Pakbaten Ph.D. Student, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. E-mail address: bahareh.pakbaten@gmail.com Hassan Kermanshahi *Corresponding author. Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. E-mail address: kermansh@um.ac.ir Farhid Hemmatzadeh Assistant Professor, School of Animal and Veterinary Science, The University of Adelaide, Roseworthy, South Australia, Australia. E-mail address: farhid.hemmatzadeh@adelaide.edu.au Reza Majidzadeh Heravi Assistant Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. E-mail address: rmajidzadeh@um.ac.ir Ali Javadmanesh Assistant Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. E-mail address: javadmanesh@um.ac.ir Abstract Objective Phytases are classified as 3-phytases (E.C.3.1.3.8), 5-phytases (E.C. 3.1.3.72) and 6-phytases (E.C. 3.1.3.26) based on the position of the first phosphate residue removed from the myo-inositol ring of phytate. P. pastoris has been of interest in the last few years as an industrial expression system. Among the advantages of this expression system, can mention strong and adjustable progress, high density of cells in culture medium and convenient genetic manipulation. The methylotrophic yeast Pichia pastoris requires a simpler environment (only one carbon source and one nitrogen source) than other yeasts to produce recombinant products at high cell density. The coexpression of digestive enzymes in a single recombinant cell system would thus be advantageous. The objective of this study is to determine whether combining fungal phytases (3- phytase) with bacterial phytase (6-phytase) was more effective than each phytase alone in degrading of plant phytate. In the paper, we tested the new vector, aimed to clone and coexpress the phytase genes (appA and phyA) isolated from E. coli and aspergillus niger, respectively, and