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Front. Microbiol. | doi: 10.3389/fmicb.2019.00335

Single-cell approach to monitor the unfolded protein response during biotechnological processes with Pichia pastoris

 Hana Raschmanová1*,  Iwo Zamora2, Martina Borčinová3, Patrick Meier2, Astrid Weninger4, Dominik Mächler2, Anton Glieder4, Karel Melzoch1, Zdeněk Knejzlík5 and Karin Kovar2
  • 1Department of Biotechnology, University of Chemistry and Technology in Prague, Czechia
  • 2Department of Chemistry and Biotechnology, Zurich University of Applied Sciences, Switzerland
  • 3Department of Genetics and Microbiology, Faculty of Science, Charles University, Czechia
  • 4Institute of Molecular Biotechnology, Graz University of Technology, Austria
  • 5Institute of Organic Chemistry and Biochemistry (ASCR), Czechia

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 (EcPGA), lipase B from Candida antarctica (CaLB) and xylanase A from Thermomyces lanuginosus (TlXynA). 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 EcPGA, 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 CaLB (20%) and TlXynA (27%). The proportion of cells with up-regulated UPR in CaLB-producing (35%) and TlXynA-producing (30%) strains was lower in comparison to the EcPGA-producing strain, and a smaller proportion of CaLB and TlXynA (<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.

Keywords: unfolded protein response (UPR), stress response, Pichia pastoris, super folder green fluorescent protein (sfGFP), Flow Cytometry, single-cell, fed-batch culture, heterogeneity

Received: 31 Aug 2018; Accepted: 08 Feb 2019.

Edited by:

John P. Morrissey, University College Cork, Ireland

Reviewed by:

Pau Ferrer, Luxembourg Institute of Science and Technology, Luxembourg
Dominik Mojzita, VTT Technical Research Centre of Finland Ltd, Finland  

Copyright: © 2019 Raschmanová, Zamora, Borčinová, Meier, Weninger, Mächler, Glieder, Melzoch, Knejzlík and Kovar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Ms. Hana Raschmanová, University of Chemistry and Technology in Prague, Department of Biotechnology, Prague, Czechia,