In this study, we provide evidence to demonstrate that high-temperature cultivation of an rhIL-10 expression strain leads to prolonged accumulation of immature G3-pro-rhIL10 in ER, thereby inducing higher levels of ER stress and subsequent cell death. levels and thus greater yeast cell death rates and lower production of rhIL-10. == Electronic supplementary material == The online version of this article (doi:10.1186/s12934-014-0163-7) contains supplementary material, which is available to authorized users. Keywords:High-temperature cultivation, Misfolded protein, ER stress, Unfolded protein response, ER-phagy,Pichia pastoris == Background == The methylotrophic yeastPichia pastoris(P. pastoris) is a well-established eukaryotic host for the production of heterologous proteins [1,2]; however, the production yield widely varies PF-06700841 P-Tosylate among particular proteins. For maximal production of a target protein, increasing target protein synthesis by increasing the gene copy number is a commonly employed strategy, but it is only effective up to a certain gene copy number limit [3] above which further increases can reduce production [4,5]. In contrast, decreasing the synthesis rate of the target protein by lowering the cultivation temperature from 30C to 20C is also commonly employed, and it is frequently reported to improve production [68]. The effectiveness of these discrepant strategies suggests that heterologous protein production is limited by the capacity of intracellular processes inP. pastoris. Understanding the potential mechanisms of this bottleneck will provide information that is useful for rationally engineering strategies to optimize yeast strains and the fermentation process. Newly synthesized secretory proteins that fail to adopt their correct conformations may be retained in the endoplasmic reticulum (ER). The ER houses a specialized folding environment that includes chaperones and foldase complexes as well as high-fidelity quality control mechanisms to ensure correct protein folding [9,10]. ER CLU homeostasis is defined as the unique equilibrium between the cellular demand for protein synthesis and the folding capacity of the ER, which promotes protein transport and maturation [11]. The retention of immature (also called misfolded) proteins in the ER can induce ER stress and disrupt ER homeostasis. The unfolded protein response (UPR) pathway is activated to mitigate ER stress; the UPR transcriptionally upregulates a group of genes required to increase the ER folding capacity by providing more chaperones and foldases and by increasing the ER surface area and luminal space [12,13]. Thus, the UPR pathway is thought to be adaptive, re-establishing ER homeostasis and normalizing ER function in a changing environment [14]. However, unlike metazoans, the UPR pathway of budding yeast does not include the translational repression sensor PERK, which temporarily inhibits general translation by phosphorylating eIF2 and thus leading to reduced protein entry into the ER lumen [13,15]. Accordingly, the absence of PERK in budding yeast makes it more sensitive to the overexpression of heterologous proteins. The ER-associated protein degradation pathway (ERAD) and autophagy are two important degradation mechanisms for eliminating potentially toxic misfolded proteins. The ERAD PF-06700841 P-Tosylate pathway clears folding-defective proteins by facilitating their movement PF-06700841 P-Tosylate into the cytosol and their subsequent proteolytic degradation by the 26S proteasome [9,16]. ERAD thereby prevents the accumulation of toxic proteins and reactive oxygen species (ROS), reducing cell death [17]. Notably, the ERAD pathway can be saturated by high substrate concentrations, as during CPY* overexpression [18,19]. The alternative degradation pathway, autophagy, engulfs and delivers misfolded proteins to the vacuole for degradation [2022]. In contrast to non-specific autophagy, ER-phagy, which selectively sequesters protein aggregates containing ER fragments and returns the ER to its normal size once folding stress subsides, is suggested to provide a survival advantage in response to ER stress [20,23,24]. Although autophagy is generally considered to be a mechanism for cell survival, recent studies also indicate its involvement in cell death, as a number of genes required for autophagy will also be involved in apoptosis [25,26]. To more.