title: Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion.
abstract: Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeastPichia pastorisas model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression ofPpYAP1leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression ofYAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression ofYAP1restores cellular redox conditions of protein-secretingP. pastorisby reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG) in theYAP1co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant) proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.
authors.affiliation: Department of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria. ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria.
authors.forename: Marizela
authors.initials: M
authors.lastname: Delic
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria. ; School of Bioengineering, University of Applied Sciences FH Campus Wien, Vienna, Austria.
authors.forename: Alexandra B
authors.initials: AB
authors.lastname: Graf
authors.affiliation: Department of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria. ; Department of Chemistry, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
authors.forename: Gunda
authors.initials: G
authors.lastname: Koellensperger
authors.affiliation: Department of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria. ; Department of Chemistry, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Haberhauer-Troyer
authors.affiliation: Department of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria. ; Department of Chemistry, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hann
authors.affiliation: Department of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria. ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria.
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.affiliation: Department of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Vienna, Austria. ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
keywords: ER; Pichia pastoris; cellular redox regulation; cytosol; glutathione; oxidative protein folding; redox sensitive roGFP