title: A Novel Potential Signal Peptide Sequence and Overexpression of ER-Resident Chaperones Enhance Heterologous Protein Secretion in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica.
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.
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand. niran.roo@biotec.or.th.|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.roo@biotec.or.th.
authors.forename: Niran
authors.initials: N
authors.lastname: Roongsawang
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand.
authors.forename: Aekkachai
authors.initials: A
authors.lastname: Puseenam
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand.
authors.forename: Supattra
authors.initials: S
authors.lastname: Kitikhun
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand.
authors.forename: Kittapong
authors.initials: K
authors.lastname: Sae-Tang
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand.
authors.forename: Piyanun
authors.initials: P
authors.lastname: Harnpicharnchai
authors.affiliation: International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Ohashi
authors.affiliation: International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand.
authors.forename: Witoon
authors.initials: W
authors.lastname: Tirasophon
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand.
authors.forename: Sutipa
authors.initials: S
authors.lastname: Tanapongpipat
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors; D006358:Hot Temperature; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D004718:Saccharomycetales / Q000378:metabolism* / Q000502:physiology
chemical_list: D005656:Fungal Proteins; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D011994:Recombinant Proteins
keywords: Methylotrophic thermotolerant yeast; Molecular chaperones; Ogataea thermomethanolica; Secretion signal sequence