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A Novel Potential Signal Peptide Sequence and Overexpression of ER-Resident Chaperones Enhance Heterologous Protein Secretion in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica

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Abstract

The thermotolerant methylotrophic yeast Ogataea thermomethanolica is a host for heterologous protein expression via secretion to the culture medium. Efficient secretion is a major bottleneck for heterologous protein production in this strain. To improve protein secretion, we explored whether the use of a native signal peptide sequence for directing heterologous protein secretion and overexpression of native ER-resident chaperone genes could improve heterologous protein secretion in O. thermomethanolica. We cloned and characterized genes encoding α-mating factor (Otα-MF) and ER-resident chaperones OtBiP, OtCNE1, and OtPDI. The pre and pre-pro sequences of Otα-MF were shown to promote higher secretion of heterologous endoxylanase comparing with the classical pre-pro sequence of Saccharomyces cerevisiae. However, in the case of heterologous glycosylated phytase, only the Otα-MF pre-pro sequence significantly enhanced protein secretion. The effect of chaperone overexpression on heterologous protein secretion was tested in cotransformant cells of O. thermomethanolica. Overexpression of ER-resident chaperones improved protein secretion depending on heterologous protein. Overexpression of OtBiP, OtCNE1, and OtPDI significantly increased unglycosylated endoxylanase secretion at both 30 and 37 °C while only OtBiP overexpression enhanced glycosylated phytase secretion at 30 °C. These observations suggested the possibility to improve heterologous protein secretion in O. thermomethanolica.

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Acknowledgments

We are grateful to Dr. Philip J. Shaw for critically editing the manuscript. Financial support (P-12-01067) from National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand is greatly appreciated. This work was also partially supported by Mahidol University. We thank Dr. Duangdao Wichadakul for Blastp analysis of O. thermomethanolica α-MF. S.K. is thankful for the scholarship from Thailand Graduate Institute of Science and Technology (TGIST). N.R. acknowledges the International Center for Biotechnology, Osaka University for invitation as a visiting research scholar.

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Authors and Affiliations

  1. Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand

    Niran Roongsawang, Aekkachai Puseenam, Kittapong Sae-Tang, Piyanun Harnpicharnchai & Sutipa Tanapongpipat

  2. Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand

    Supattra Kitikhun & Witoon Tirasophon

  3. International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Takao Ohashi & Kazuhito Fujiyama

  4. Microbial Cell Factory Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand

    Niran Roongsawang

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  1. Niran Roongsawang
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Roongsawang, N., Puseenam, A., Kitikhun, S. et al. A Novel Potential Signal Peptide Sequence and Overexpression of ER-Resident Chaperones Enhance Heterologous Protein Secretion in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica . Appl Biochem Biotechnol 178, 710–724 (2016). https://doi.org/10.1007/s12010-015-1904-8

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