High level expression of a recombinant acid phytase gene in Pichia pastoris

Aims:  To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. Methods and Results:  The 1347‐bp phytase gene of Aspergillus niger SK‐57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon‐modified Saccharomyces cerevisiae mating factor α ‐prepro‐leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly‐inducible alcohol oxidase gene promoter with the MF4I‐ or wild type α ‐signal sequence were used to transform Pichia pastoris . The P. pastoris strain that expressed the modified phytase gene ( phyA‐sh ) with MF4I sequence produced 6·1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml −1 . The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2·5 and pH 5·5) and an optimum temperature of 60°C. Conclusions:  The P. pastoris strain with the genetically engineered phytase gene produced 6·1 g l −1 of phytase or 865 U ml −1 phytase activity, a 14·5‐fold increase compared with the P. pastoris strain with the wild type phytase gene. Significance and Impact of the Study:  The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.