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High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris

  • Applied Genetics and Molecular Biotechnology
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Abstract

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 α-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°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.

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Acknowledgements

We are grateful to Dr. Jian Zhang, Dr. Wen-Jin Yu (University of Guelph, Canada) and Prof. Zong-Ming Cheng (University of Tennessee, USA) for their careful reading and valuable comments on the manuscript. This research was supported by the Fund of National Natural Science Foundation of China (30471258), Shanghai Natural Science Foundation (04ZR14116), Shanghai Rising-Star Program (05QMX1445), Key Project of Science and Technology Commission Foundation of Shanghai (993913002). We thank Shanghai YongYe Bio-engineering for providing some instruments and technical help.

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  1. Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, 2901 Beidi Rd, Shanghai, 201106, People’s Republic of China

    Ai-Sheng Xiong, Quan-Hong Yao, Ri-He Peng & Pei-Lai Han

  2. College of Bioscience and Biotechnology, Yangzhou University, 88 S. Daxue Road, Yangzhou, 225009, People’s Republic of China

    Jian-Min Chen

  3. Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, People’s Republic of China

    Ai-Sheng Xiong, Zhen Zhang, Fang Xu & Jin-Ge Liu

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  1. Ai-Sheng Xiong
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  3. Ri-He Peng
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  4. Zhen Zhang
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Correspondence to Quan-Hong Yao or Jian-Min Chen.

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Xiong, AS., Yao, QH., Peng, RH. et al. High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris . Appl Microbiol Biotechnol 72, 1039–1047 (2006). https://doi.org/10.1007/s00253-006-0384-8

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