Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf
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 re...
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ftfrontimediafig:oai:figshare.com:article/7775759 2023-05-15T13:38:04+02:00 Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf Hana Raschmanová Iwo Zamora Martina Borčinová Patrick Meier Astrid Weninger Dominik Mächler Anton Glieder Karel Melzoch Zdeněk Knejzlík Karin Kovar 2019-02-27T07:12:57Z https://doi.org/10.3389/fmicb.2019.00335.s001 https://figshare.com/articles/Data_Sheet_1_Single-Cell_Approach_to_Monitor_the_Unfolded_Protein_Response_During_Biotechnological_Processes_With_Pichia_pastoris_pdf/7775759 unknown doi:10.3389/fmicb.2019.00335.s001 https://figshare.com/articles/Data_Sheet_1_Single-Cell_Approach_to_Monitor_the_Unfolded_Protein_Response_During_Biotechnological_Processes_With_Pichia_pastoris_pdf/7775759 CC BY 4.0 CC-BY Microbiology Microbial Genetics Microbial Ecology Mycology unfolded protein response (UPR) stress response Pichia pastoris super folder green fluorescent protein (sfGFP) flow cytometry single-cell fed-batch culture heterogeneity Dataset 2019 ftfrontimediafig https://doi.org/10.3389/fmicb.2019.00335.s001 2019-02-27T23:58:13Z 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 ... Dataset Antarc* Antarctica Frontiers: Figshare |
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Open Polar |
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Frontiers: Figshare |
op_collection_id |
ftfrontimediafig |
language |
unknown |
topic |
Microbiology Microbial Genetics Microbial Ecology Mycology unfolded protein response (UPR) stress response Pichia pastoris super folder green fluorescent protein (sfGFP) flow cytometry single-cell fed-batch culture heterogeneity |
spellingShingle |
Microbiology Microbial Genetics Microbial Ecology Mycology unfolded protein response (UPR) stress response Pichia pastoris super folder green fluorescent protein (sfGFP) flow cytometry single-cell fed-batch culture heterogeneity Hana Raschmanová Iwo Zamora Martina Borčinová Patrick Meier Astrid Weninger Dominik Mächler Anton Glieder Karel Melzoch Zdeněk Knejzlík Karin Kovar Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf |
topic_facet |
Microbiology Microbial Genetics Microbial Ecology Mycology unfolded protein response (UPR) stress response Pichia pastoris super folder green fluorescent protein (sfGFP) flow cytometry single-cell fed-batch culture heterogeneity |
description |
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 ... |
format |
Dataset |
author |
Hana Raschmanová Iwo Zamora Martina Borčinová Patrick Meier Astrid Weninger Dominik Mächler Anton Glieder Karel Melzoch Zdeněk Knejzlík Karin Kovar |
author_facet |
Hana Raschmanová Iwo Zamora Martina Borčinová Patrick Meier Astrid Weninger Dominik Mächler Anton Glieder Karel Melzoch Zdeněk Knejzlík Karin Kovar |
author_sort |
Hana Raschmanová |
title |
Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf |
title_short |
Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf |
title_full |
Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf |
title_fullStr |
Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf |
title_full_unstemmed |
Data_Sheet_1_Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris.pdf |
title_sort |
data_sheet_1_single-cell approach to monitor the unfolded protein response during biotechnological processes with pichia pastoris.pdf |
publishDate |
2019 |
url |
https://doi.org/10.3389/fmicb.2019.00335.s001 https://figshare.com/articles/Data_Sheet_1_Single-Cell_Approach_to_Monitor_the_Unfolded_Protein_Response_During_Biotechnological_Processes_With_Pichia_pastoris_pdf/7775759 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
doi:10.3389/fmicb.2019.00335.s001 https://figshare.com/articles/Data_Sheet_1_Single-Cell_Approach_to_Monitor_the_Unfolded_Protein_Response_During_Biotechnological_Processes_With_Pichia_pastoris_pdf/7775759 |
op_rights |
CC BY 4.0 |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.3389/fmicb.2019.00335.s001 |
_version_ |
1766101192089272320 |