Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

Abstract

Pichia pastoris ( Komagataella sp .) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured P. pastoris strains producing three different recombinant proteins: penicillin G acylase from Escherichia coli ( Ec PGA), lipase B from Candida antarctica ( Ca LB) and xylanase A from Thermomyces lanuginosus ( Tl XynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing Ec PGA, which accumulated in the cells (50–70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing Ca LB (20%) and Tl XynA (27%). The proportion of cells with up-regulated UPR in Ca LB-producing (35%) and Tl XynA-producing (30%) strains was lower in comparison to the Ec PGA-producing strain, and a smaller proportion of Ca LB and Tl XynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant P. pastoris population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity.

Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris

“…Their expression was the highest when the clones were cultivated at intermediate D of 0.10 h −1 , whereas only moderate expression increases were found at D 0.15 h −1 . These results are in-line with other published work reporting that not only recombinant protein production, but also µ, having a significant impact on UPR induction [ 36 , 53 ]. The cited studies are also similar to the data presented here, showing unremarkable UPR levels at lower µ, whereas µ increases led to UPR upregulation.…”

Role of BGS13 in the Secretory Mechanism of Pichia pastoris

“…Sensitivity to Cr and Cfw, increased levels of DTT-extractable cell wall proteins, and enhanced secretion of some recombinant proteins are phenotypes of cells with a disrupted UPR. The UPR is an endoplasmic reticulum (ER)-to-nucleus signaling pathway initiated by ER stress, such as the buildup of unfolded proteins in the ER that can result from protein overexpression (33)(34)(35). The UPR attempts to reduce this ER stress by activating a large number of target genes, whose products function in a range of activities, such as enhanced protein folding to improve secretion efficiency and protein degradation to remove unfolded aggregate peptides.…”

Application of In-Situ and Soft-Sensors for Estimation of Recombinant P. pastoris GS115 Biomass Concentration: A Case Analysis of HBcAg (Mut+) and HBsAg (MutS) Production Processes under Varying Conditions

“…Under circumstances introduced earlier, the permittivity-based biomass estimate should, accordingly, increase by 10%. Raschmanová et al [ 31 ] extensively studied the morphological changes of several P. pastoris strains under different growth rates, synthesizing extracellular proteins. The results indicate the extended range for the apoptotic (impaired viability) cells to form 10 to 30% of the population depending on the strain used and the specific biomass growth rate.…”

“…As the permittivity measurement selectively accounts for the viable cell volume (compared to DCW, in which also nonviable cells are counted), it is obvious that under varying process conditions the correlation between permittivity and DCW may differ. This can be especially true for the methanol induction phase in methylotrophic yeast P. pastoris cultivations, in which variations in cell viability and size are present at different growth rates [ 31 ]. In this case, alternatively to the DCW measurement, generally accepted as the standard biomass measure reference, evaluation of soft-sensor performance, if calibration is made to the reference permittivity-based biomass estimates, is an interesting aspect not widely investigated elsewhere.…”