A Novel Potential Signal Peptide Sequence and Overexpression of ER-Resident Chaperones Enhance Heterologous Protein Secretion in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica
2016
Roongsawang, Niran | Puseenam, Aekkachai | Kitikhun, Supattra | Sae-Tang, Kittapong | Harnpicharnchai, Piyanun | Ohashi, Takao | Fujiyama, Kazuhito | Tirasophon, Witoon | Tanapongpipat, Sutipa
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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