Abstract
ABSTRACTRecombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, microcultivation, and continuous measurements in batch culture, we observed H2O2 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 H2O2, we find that Gcn2-dependent phosphorylation of eIF2α increases 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 H2O2 levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2.IMPORTANCE Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great 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 that 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 (H2O2) 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 H2O2 levels in the cytosol. In 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.
Highlights
Recombinant protein production is a known source of oxidative stress
We found the roGFP2-Prx1 sensor Ox/Red ratio to increase upon H2O2 addition and decrease upon DTT addition, whereas both roGFP2-PfAOP sensors responded mainly to DTT addition (Fig. S1)
We continued our experiments only with the roGFP2-Prx1 sensor, considering that this sensor demonstrated a high sensitivity to endogenous H2O2 levels, while its signal still increased upon addition of exogenous H2O2 (Fig. S1)
Summary
Recombinant protein production is a known source of oxidative stress. knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Gcn deficiency reduced intracellular H2O2 levels and the Hac splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2. We document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H2O2) in the cytosol of yeast cells upon the production of recombinant proteins. In engineered high-producing strains in particular, this burden is speculated to increase concomitantly with production levels, leading to ER stress [11, 12] To counter this and the accumulation of unfolded proteins within this organelle, two mechanisms can be activated or upregulated, the unfolded protein response (UPR) and ER-associated degradation (ERAD). Hac1p subsequently moves to the nucleus and activates the expression of UPRassociated genes [13]
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