The Yeast eIF2 Kinase Gcn2 Facilitates H ₂ O ₂ -Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production

Abstract

Recombinant protein production is a known source of oxidative stress. Knowledge of which ROS are involved or the specific growth phase in which stress occurs however remains lacking. Using modern, hypersensitive genetic H 2 O 2 -specific probes, micro-cultivation and continuous measurements in batch culture, we observed H 2 O 2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae , correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H 2 O 2 , we find Gcn2-dependent phosphorylation of eIF2α to increase alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains, but not in the wild-type. Gcn2-deficiency furthermore reduced intracellular H 2 O 2 levels and the Hac1 splicing ratio whilst expression of antioxidants and the ER disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding to be coupled and subject to feedback inhibition by H 2 O 2 . Importance Recombinant protein production is a multi-billion dollar industry. Optimizing the productivity of host cells is, therefore, of large interest. In several hosts oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses which could compromise the productivity of the host cell. Here we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H 2 O 2 ) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2 we observed a doubling of recombinant protein productivity in addition to reduced H 2 O 2 levels in the cytosol. By this study we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae

“…Although SIP-α(Δ55−70) and SIP-Ost1(Δ30−43) strains secreted more HRP, they still had significantly higher intracellular residues compared with the residue in the RAD14 strain (Figures 5C and 6A), activating the unfold protein response (UPR) and upregulating the expression of genes involved in protein folding and secretion. 22 The purified extracellular HRP was deglycosylated by Endo H and detected by SDS-PAGE to further confirm the increasing secretory amount of HRP during optimization (Figure S2). The titers of HRP were also calculated according to the volumetric activities in the supernatant and the specific activity of purified fermentation samples (Table S8).…”

“…To enhance the extracellular accumulation of HRP in S. cerevisiae, expression and secretory optimization of heterologous proteins have been regarded as two general but inevitable topics. , Due to the complex structure of HRP, the potential difficulty in protein folding is most likely to conflict with cell growth, , leading to the bottleneck when HRP was constitutively expressed in S. cerevisiae. , Meanwhile, HRP can cause cell stress in K.…”

Impact of Hydrogen Peroxide on Protein Synthesis in Yeast

“…However, upon stimulation, concentrations typically swiftly rise, allowing the second messengers to rapidly diffuse from their source to the ligand-specific protein sensors [89]. In this context, H 2 O 2 could be argued to perform a second messenger role in coordinating protein synthesis with, e.g., mitochondrial activity or ER-folding capacity [41,66]. Signal attenuation upon repeated stimulation (adaptation) is an inherent property of signaling systems involving adaptive changes in the equilibrium state of biological systems in response to alterations in their surrounding environment [90].…”

“…On the other hand, Gcn4 is neither activated by H 2 O 2 nor required for H 2 O 2 resistance in two other yeast strain backgrounds (S288c and CEN-PK) [65,66], suggesting that other Gcn2 targets may be more important in the ability of cells to adapt to H 2 O 2 in these strains. Interestingly, we recently showed that cells engineered to secrete increased levels of recombinant protein displayed increased levels of H 2 O 2 in the cytosol and Gcn2 activation [66]. Notably, Gcn2 deficiency restored H 2 O 2 levels, cytosolic protein synthesis and ER-folding homeostasis through an unknown mechanism, suggesting a role for H 2 O 2 and Gcn2 in balancing cytosolic protein synthesis to protein secretion in the ER.…”

“…H 2 O 2 was also shown to cause an upregulation of GCN4 expression, at least in the divergent yeast strain as well as a requirement for Gcn4 for H 2 O 2 tolerance [64]. On the other hand, Gcn4 is neither activated by H 2 O 2 nor required for H 2 O 2 resistance in two other yeast strain backgrounds (S288c and CEN-PK) [65,66], suggesting that other Gcn2 targets may be more important in the ability of cells to adapt to H 2 O 2 in these strains. Interestingly, we recently showed that cells engineered to secrete increased levels of recombinant protein displayed increased levels of H 2 O 2 in the cytosol and Gcn2 activation [66].…”