High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris.
Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6'-phytase, which owns high specific activity. To achieve a high expression level of 6'-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene (phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml(-1). The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50 degrees C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system.
Xiong
Ai-Sheng
AS
Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, 2901 Beidi Rd, Shanghai, 201106, PR China.
Yao
Quan-Hong
QH
Peng
Ri-He
RH
Zhang
Zhen
Z
Xu
Fang
F
Liu
Jin-Ge
JG
Han
Pei-Lai
PL
Chen
Jian-Min
JM
D010832:6-Phytase / Q000235:genetics* / Q000378:metabolism*; D001487:Basidiomycota / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005813:Genes, Synthetic; D006863:Hydrogen-Ion Concentration; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D010833:Phytic Acid; D010832:6-Phytase