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

Hana Raschmanová,Karin Kovar,Iwo Zamora,Dominik Mächler,Martina Borčinová,Patrick Meier,Anton Glieder,Astrid Weninger,Karel Melzoch,Zdeněk Knejzlík

doi:10.3389/fmicb.2019.00335

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.

Highlights

The yeast Pichia pastoris (Komagataella phaffii) is an established host for the biotechnological production of wide range of heterologous proteins, most of which are secreted (Cereghino and Cregg, 2000; Ahmad et al, 2014; Meehl and Stadheim, 2014; Juturu and Wu, 2018)
A BD AccuriTM C6 flow cytometer (BD Biosciences, Franklin Lakes, USA) equipped with a 20 mW 488 nm solid state blue laser was employed for measurements of the green fluorescence of super folder green fluorescent protein (sfGFP) (FL1 530 ± 15 nm) during the flask experiments for the characterization of the KAR2 upstream region and the for the characterization of the population producing recombinant EcPGA
We constructed a reporter for monitoring unfolded protein response (UPR) up-regulation in living P. pastoris cells, which was based on the production of sfGFP upon the activation of the P. pastoris KAR2 promoter

Summary

Introduction

The yeast Pichia pastoris (Komagataella phaffii) is an established host for the biotechnological production of wide range of heterologous proteins, most of which are secreted (Cereghino and Cregg, 2000; Ahmad et al, 2014; Meehl and Stadheim, 2014; Juturu and Wu, 2018). The knowledge of this host and its use for the production of heterologous proteins is already advanced, but a more systematic and complex understanding of the P. pastoris cell factory is still needed, especially of intracellular metabolite fluxes, regulatory pathways and secretory machinery (Puxbaum et al, 2015; Zahrl et al, 2017). Though an important aspect of productivity and approach to a strain’s optimization, little attention is typically paid to intracellular product fluxes, stress responses (UPR, ERAD), strain heterogeneity in terms of growth and production/secretion, and cell physiology in microbial cultivation processes under industrially relevant cultivation conditions (Theron et al, 2018)

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