The Yeast eIF2 Kinase Gcn2 Facilitates H2O2-Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production
Artikel i vetenskaplig tidskrift, 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.

translational control

ER stress

hydrogen peroxide

oxidative stress

Gcn4

H2O2

recombinant protein production

heterologous protein production

protein kinase Gcn2

Författare

Veronica Gast

Chalmers, Biologi och bioteknik, Systembiologi

Kate Campbell

Novo Nordisk Foundation Center for Biosustainability

Chalmers, Biologi och bioteknik, Systembiologi

Cecilia Picazo Campos

Universitat de Valencia

Chalmers, Biologi och bioteknik, Systembiologi

Martin Engqvist

Chalmers, Biologi och bioteknik, Systembiologi

Verena Siewers

Novo Nordisk Foundation Center for Biosustainability

Chalmers, Biologi och bioteknik, Systembiologi

Mikael Molin

Chalmers, Biologi och bioteknik, Systembiologi

Applied and Environmental Microbiology

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

Vol. 87 15 e0030121-

Ämneskategorier

Biokemi och molekylärbiologi

Mikrobiologi

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

DOI

10.1128/AEM.00301-21

PubMed

34047633

Mer information

Senast uppdaterat

2021-08-19