Abstract:[Objective] This study aimed to identify the ORM1 gene in Pichia pastoris, and to illustrate the effect of its deletion on cell growth, endoplasmic reticulum stress response, cell calcium homeostasis and reactive oxygen species accumulation. [Methods] Sequence alignment and analysis were done by the relevant bioinformatic software. The orm1Δ mutant was constructed by PCR-mediated gene disruption, and the reconstituted strain orm1Δ+ORM1 was constructed by transforming orm1Δ with the pIB1-ORM1 plasmid. The growth rates of the strains were detected using both liquid and the solid media. Gene expression related to the unfolded protein response, calcium homeostasis and antioxidant system was detected via real-time PCR. The activity of antioxidant enzymes, including catalase and superoxide dismutase, and the content of reduced glutathione were measured by relevant kits. [Results] The P. pastoris ORM1 gene sharing high homology with S. cerevisiae ORM1 and ORM2, was identified in P. pastoris genome database. Deletion of ORM1 caused growth defect, increased sensitivity to endoplasmic reticulum stress caused by tunicamycin, activation of unfolded protein response, disturbance of calcium homeostasis, reactive oxygen species accumulation, and activation of the antioxidant system. [Conclusion] Because unfolded protein response, calcium homeostasis, and production of reactive oxygen species are all correlated with ER function, P. pastoris Orm1 protein plays critical roles in cell growth and maintenance of ER functions.
