The Yeast eIF2 Kinase Gcn2 Facilitates H2O2-Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production
Journal article, 2021

Recombinant 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.

Gcn4

heterologous protein production

recombinant protein production

oxidative stress

translational control

H2O2

ER stress

protein kinase Gcn2

hydrogen peroxide

Author

Veronica Gast

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Kate Campbell

Novo Nordisk Foundation

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Cecilia Picazo Campos

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Universitat de Valencia

Martin Engqvist

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Verena Siewers

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Novo Nordisk Foundation

Mikael Molin

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Applied and Environmental Microbiology

0099-2240 (ISSN) 1098-5336 (eISSN)

Vol. 87 15 e0030121-16

Subject Categories

Biochemistry and Molecular Biology

Microbiology

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

DOI

10.1128/AEM.00301-21

PubMed

34047633

More information

Latest update

5/26/2023