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

文献类型： 外文期刊

作者： Xiong, AS 1 ; Yao, QH 2 ; Peng, RH 2 ; Han, PL 2 ; Cheng, ZM 2 ; Li, Y 2 ;

作者机构： 1.Shanghai Acad Agr Sci, Agrobiotechnol Res Ctr, Shanghai, Peoples R China

2.Shanghai Acad Agr Sci, Agrobiotechnol Res Ctr, Shanghai, Peoples R China; Univ Tennessee, Dept Plant Sci, Knoxville, TN USA; Univ Connecticut, Dept Plant Sci, Storrs, CT 06269 USA

关键词： codon modification;Pichia pastoris;recombinant phytase gene;successive polymerase chain reaction;synthetic signal sequence;ASPERGILLUS-NIGER PHYTASE;MYOINOSITOL HEXAKISPHOSPHATE PHOSPHOHYDROLASES;CLONING;PURIFICATION;YEAST;PHYA;GLYCOSYLATION;PHOSPHORUS;PROTEINS;IMPROVES

期刊名称：JOURNAL OF APPLIED MICROBIOLOGY （ 影响因子：3.772； 五年影响因子：3.963 ）

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收录情况： SCI

摘要： 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 alpha-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 alpha-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 60degreesC.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.

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