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.

Document Type : Research Paper


1 Ph.D. Student, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

3 Assistant Professor, School of Animal and Veterinary Science, The University of Adelaide, Roseworthy, South Australia, Australia.

4 Assistant Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran


Phytases are classified as 3-phytases (E.C., 5-phytases (E.C. and 6-phytases (E.C. based on the position of the first phosphate residue removed from the myo-inositol ring of phytate. Yeasts are particularly suited to expression of foreign proteins for numerous features mean while some of them shows probiotic properties. Pichia pastoris, has been developed for the production of various recombinant proteins and growth into high cell densities in an inexpensive medium. Accordingly, Pichia pastoris is an appropriate secretory system for obtaining larger quantities of correct products, in compared to other host cells. 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. we tested the new vector, aimed to clone and coexpress the phytase genes isolated from E. coli and aspergillus niger and transformed into Pichia pastoris. Evaluations were made for the biochemical properties of the active expressed phytase.
The nucleotide sequence of phyA and appA2, 2a peptide and alpha factor were obtained from NCBI database. A coexpression system for the extracellular production of phytase of Aspergillus Niger (3-phytase) and phytase of E. coli (6-phytase) will be established in Pichia pastoris yeast. Plasmid that used in this study was pPIC9k. The genes for each enzyme are fused in-frame with the “a-factor” secretion signal and linked by the 2A-peptide-encoding sequence. After receiving the synthetic plasmid with fragments, plasmid that contained phyA and appA2 was cloned in DH5@ and after that digested by SacI and electrotransfered into Pichia pastoris. Positive cells are resuspended at BMMY containing methanol as an inducer. To follow the induction, methanol was injected into the culture every 24 hours in order to reach a definitive concentration of 0.5 percent. The recombinant protein was analyzed using sds-page.
The results showed that the recombinant phytase gene was transferred to Pichia pastoris and the results of PCR confirmed that. The molecular weight of the produced recombinant phytases was estimated to be around 45 and 80 kDa for appA and phyA, respectively. The recombinant protein has phytase activity equal to 160.97 U/ml
The designed phytase genes containing phyA and appA were successfully cloned in DH5a and the recombinant plasmid was transferred to Pichia pastoris for high expression. Recombinant yeast will be used for further experiments.


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