The Mechanism Of Enhanced Heterologous Protein Production By Low-temperature Cultivation In Pichia Pastoris

As a’cell factory’for heterologous proteins production, Pichia pastoris (P. pastoris) have lots of advantages, such as easy to genetic manipulation, cheaper and definite culture medium, high efficient secretion, post-translational modification, etc., which makes it to be a popular expression system. Low-temperature cultivation is frequently reported to improve the productivities of heterologous proteins; however, the underlying mechanisms are still unclear. Recently, the advance in misfolded protein, ER stress and autophagy indicates that protein midfolding may be the major reason for the compromised secretion yield.In this study, a P. pastoris strain expressing the recombinant human interleukin-10(rhIL-10) was used as the model in this study, which resulted in either high-yield productivity of rhIL-10with prolonged methanol-induction time at20℃or low-yield productivity with higher cell death rate at30℃. The further investigations showed that the G3-pro-rhIL10, the immature form of rhIL-10that contains the glycosylation-modified signal peptide, was accumulated and delayed for a prolonged period in ER, and accompanied with increased ROS production, Ca2+leakage, lower level of Unfolded Protein Response (UPR) and even ER-phagy when the expression strain was methanol-induced at30℃. In contrast, the level of UPR was significant higher and the immature form of G3-pro-rhIL10was quickly eliminated from the ER when the expression strain was methanol-induced at20℃.Our results clearly showed that the relative lower culture temperature effectively downregulates the level of ER stress and thus prevents yeast cell death, which can be one of the major mechanisms for the improved heterologous protein productivity in P. pastoris by low-temperature cultivation. Our research will contributes to the rational strain engineering and yeast fermentation process engineering.