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

作者：Roongsawang Niran^*; Puseenam Aekkachai; Kitikhun Supattra; Sae Tang Kittapong; Harnpicharnchai Piyanun; Ohashi Takao; Fujiyama Kazuhito; Tirasophon Witoon; Tanapongpipat Sutipa

来源：Applied Biochemistry and Biotechnology, 2016, 178(4): 710-724.

DOI：10.1007/s12010-015-1904-8

• Methylotrophic thermotolerant yeast
• Ogataea thermomethanolica
• Molecular chaperones
• Secretion signal sequence

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 alpha-mating factor (Ot alpha-MF) and ER-resident chaperones OtBiP, OtCNE1, and OtPDI. The pre and pre-pro sequences of Ot alpha-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 Ot alpha-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 A degrees C while only OtBiP overexpression enhanced glycosylated phytase secretion at 30 A degrees C. These observations suggested the possibility to improve heterologous protein secretion in O. thermomethanolica.

• 出版日期2016-2