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High level expression of a recombinant acid phytase gene in Pichia pastoris
Author(s) -
Xiong A.S.,
Yao Q.H.,
Peng R.H.,
Han P.L.,
Cheng Z.M.,
Li Y.
Publication year - 2005
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2004.02476.x
Subject(s) - phytase , pichia pastoris , signal peptide , biology , biochemistry , recombinant dna , gene , microbiology and biotechnology , alcohol oxidase , saccharomyces cerevisiae , enzyme
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.