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. 2019 Feb 27:10:335.
doi: 10.3389/fmicb.2019.00335. eCollection 2019.

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

Hana Raschmanová  1   2 , Iwo Zamora  2 , Martina Borčinová  2   3 , Patrick Meier  2 , Astrid Weninger  4 , Dominik Mächler  2 , Anton Glieder  4 , Karel Melzoch  1 , Zdeněk Knejzlík  5 , Karin Kovar  2
Affiliations

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

Hana Raschmanová et al. Front Microbiol. .

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 (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: Pichia pastoris; fed-batch culture; flow cytometry; heterogeneity; single-cell; stress response; super folder green fluorescent protein (sfGFP); unfolded protein response (UPR).

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Figures

Figure 1
Figure 1
The upstream region of the KAR2 CDS in P. pastoris X33. The nucleotide sequence located upstream of the KAR2 CDS and downstream of the CDS of the previous gene (BUD7) in the genome of P. pastoris X33. One potential UPRE (CAGCGTG) was identified in the KAR2 upstream region, starting −84 bp upstream of the KAR2 CDS. The following variants of the KAR2 upstream region were used to control the expression of sfGFP:

  1. full length KAR2 upstream region (FL, −324 bp);

  2. truncated variant of KAR2 upstream region, still containing native UPRE sequence (−191 bp);

  3. truncated variant of KAR2 upstream region with two single mutations (84C→A, 78G→A) in the UPRE sequence (−191 bp mut.); truncated variant of KAR2 upstream region lacking the UPRE sequence (−77 bp).

Figure 2
Figure 2
The activity of different variants of the KAR2 upstream region in P. pastoris X33. The promoter activity of the different variants of the KAR2 upstream region was measured as a green fluorescence of sfGFP (channel FL1) with flow cytometry, as the gene of sfGFP was inserted downstream of the KAR2 upstream region. A variant with no promoter in front of the sfGFP gene (x) was used as a negative control. P. pastoris strains with the different variants of the KAR2 upstream region (FL, −190 bp, −190 bp mut., −77 bp) controlling sfGFP expression, and the negative control strain (x-sfGFP) were cultured overnight in shake flasks with YPD, then split into three parallels and further incubated for another 2 h. One parallel was cultured under normal conditions without experimental stress (black bars), the second parallel was cultured in the presence of 3 mM DTT (dark gray bars) and the third parallel was cultured at an increased temperature of 39°C (light gray bars). The displayed values are median FL1 values of all events belonging to a gate defined in a FSC-SSC dot plot (distinguishing P. pastoris cells from the background, data not shown). The error bar is showing a standard deviation of three measurements.
Figure 3
Figure 3
Fold-change of KAR2 expression in the P. pastoris strain producing EcPGA and the non-producing control strain as obtained by qPCR analysis. The P. pastoris strains producing EcPGA (green) and the control non-production strain (black) were cultured under the same cultivation conditions (30°C, pH 5.5), maintaining the specific growth rate of biomass with methanol at 0.016 h−1. All relative expression data are related to the expression of KAR2 in the non-producing control strain in the sample taken 3 h after induction.
Figure 4
Figure 4
The enzymatic activity of the different recombinant proteins in the centrifuged culture medium and cell extracts. The production of EcPGA in the strain with the expression cassette PKAR2(FL)-sfGFP (A) and in the control strain (B), CaLB (C), and TlXynA (D) was assessed as enzymatic activity (U L−1) both in the culture medium, i.e., in the supernatant (dark green, dark gray, dark blue, dark red for EcPGA, EcPGA, CaLB, TlXynA, respectively), as well as in the cell extracts, i.e., inside the cells (light green, light gray, light blue, light red for EcPGA, EcPGA, CaLB, TlXynA, respectively). The concentrations were recalculated to the volume of the culture broth. The activities of the extracellular (secreted) enzyme were related to the mass of total extracellular protein (U gtot.protein-1, black in A–D). The strains with the integrated PKAR2(FL)-sfGFP cassette for the monitoring of UPR and producing EcPGA (A), CaLB (C), or TlXynA (D), and the control strain with the integrated x-sfGFP cassette producing EcPGA (B) were cultured under the same cultivation conditions (30°C, pH 5.5, μmethanol 0.016 h−1).
Figure 5
Figure 5
The four sub-populations of P. pastoris cells defined with the use of PCA. The flow cytometric data (FSC, SSC, FL1, FL2, FL3) of 10,000 random cells from the bioreactor cultivations of the strains producing EcPGA and CaLB, and the non-producing strain (all at μmethanol 0.016 h−1) were analyzed with PCA. The lowest level of redundancy was observed when the data were displayed as the combination of PC2 and PC3 (describing ~39.8% of the population variability), since the flow cytometric data (black vectors) were the most de-correlated. The length of each vector is 50 AU. The combination of PC3 and PC2 was used to establish the clustering method (population centers are displayed in gray). According to the FC signals (black arrows), the identified four clusters can be described as follows: smaller and less complex viable cells with no UPR up-regulation (red); larger and more complex viable cells with no UPR up-regulation (green); viable cells with up-regulated UPR (black); and cells with impaired viability (blue).
Figure 6
Figure 6
Change of cell size and complexity, UPR up-regulation and viability during cultivations of P. pastoris strains producing different recombinant proteins. The P. pastoris strains producing EcPGA (A), CaLB (B), or TlXynA (C), as well as a control non-production strain (D) were cultured under the same cultivation conditions (30°C, pH 5.5), maintaining the specific growth rate of biomass with methanol at 0.016 h−1. Additionally, the P. pastoris strain producing CaLB was cultured at 30°C, pH 5.5 and at a specific growth rate of biomass with methanol of 0.008 h−1 (E) and 0.032 h−1 (F). The four sub-populations identified with the PCA of the flow cytometric data were observed in all the cultivation processes: smaller and less complex viable cells with no UPR up-regulation (red ovals); larger and more complex viable cells with no UPR up-regulation (green ovals); viable cells with up-regulated UPR (black ovals); and cells with an impaired viability (blue ovals).
Figure 7
Figure 7
Specific fluorescence of the culture medium and cell lysates of the P. pastoris strain producing TlXynA, and the control strains. The P. pastoris strain producing TlXynA was cultured at 30°C and pH 5.5, maintaining the specific growth rate of biomass with methanol during the production phase at 0.016 h−1. Fluorescence of the centrifuged culture medium and of the cell lysates were measured with a fluorimeter. Specific extracellular fluorescence (dark red) and specific intracellular fluorescence (light red) were calculated from the measured fluorescence (AU) and CDW (g) values. As controls, the specific extracellular fluorescence of the EcPGA-producing strain with no sfGFP production (gray), and the non-producing strain (black) are shown. The control strains were cultured under the same cultivation conditions as the TlXynA-producing strain.
Figure 8
Figure 8
CaLB/biomass yield during cultivations of P. pastoris producing recombinant CaLB under different specific growth rates with methanol. The P. pastoris strain producing CaLB was cultured at 30°C and pH 5.5, maintaining the specific growth rate of biomass with methanol during the production phase at either 0.008 h−1 (purple), 0.016 h−1 (blue), or 0.032 h−1 (brown). The secreted (dark purple/blue/brown circles) as well as the intracellular (light purple/blue/brown squares) CaLB/biomass yield were calculated.
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