The response to unfolded protein is involved in osmotolerance of Pichia pastoris

Abstract

BackgroundThe effect of osmolarity on cellular physiology has been subject of investigation in many different species. High osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. Several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms. Thus, the effect of osmolarity on the cellular physiology of Pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. Transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. While our main intention was to analyze the effect of different osmolarities on P. pastoris in general, this was complemented by studying it in context with recombinant protein production.ResultsIn contrast to the model yeast Saccharomyces cerevisiae, the main osmolyte in P. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. 2D Fluorescence Difference Gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. These data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a P. pastoris strain, secreting the Fab fragment, compared with the wild type strain. No transcriptional activation of the high osmolarity glycerol (HOG) pathway was observed at steady state conditions. Furthermore, no change of the specific productivity of recombinant Fab was observed at increased osmolarity.ConclusionThese data point out that the physiological response to increased osmolarity is different to S. cerevisiae. Increased osmolarity resulted in an unfolded protein response (UPR) like response in P. pastoris and lead to pre-conditioning of the recombinant Fab producing strain of P. pastoris to growth at high osmolarity. The current data demonstrate a strong similarity of environmental stress response mechanisms and recombinant protein related stresses. Therefore, these results might be used in future strain and bioprocess engineering of this biotechnologically relevant yeast.

Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

“…Although Yap1 itself did not show up within the differentially regulated genes, the Reporter Transcription Factors algorithm 30 identified Yap1 as significant based on down-regulation of Yap1 target genes in a S. cerevisiae strain secreting α-amylase, a difficult to express recombinant protein 31. On the contrary, during our transcriptomics analyses of different recombinant protein secreting strains of P. pastoris , we did not see any significant regulation of putative Yap1 target genes involved in oxidative stress defense (3233 and Stadlmayr et al unpublished data). However, a significant downregulation of YAP1 , and several of its target genes such as AHP1 , CTA1 , GLR1 , GRX3 , GSH1 , GSH2 , SOD2 , and TRX1 (among other genes) was observed in a HAC1 overexpression P. pastoris strain (except for SOD1 which was up-regulated; data from 34) correlating with ROS accumulation in this strain with constitutive UPR induction 22.…”

Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing

“…The N-glycosylation pathway in P. pastoris has been reengineered to produce heterologous pharmaceutical proteins with human-like N-glycan structures, which may further improve the importance of P. pastoris in the biopharmaceutical industry [2-4]. P. pastoris is also used as a model organism to study the protein expression machinery, such as protein folding and secretion [5-9]. Apart from applications in protein expression, P. pastoris could also be used to study the biogenesis and degradation of the peroxisome [10].…”

Genome‐scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production

“…All known biomass components from different parts of the metabolic network as well as their corresponding contributions to the synthesis of biomass were combined together to construct the biomass reaction. Data taken from literature as well as experimental data measuring the components from cultures of P. pastoris were utilized (see Supporting information S2) [34] …”