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

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F19%3A00502942" target="_blank" >RIV/61388963:_____/19:00502942 - isvavai.cz</a>

Alternative codes found

RIV/00216208:11310/19:10393094 RIV/60461373:22330/19:43918552

Result on the web

<a href="https://www.frontiersin.org/articles/10.3389/fmicb.2019.00335/full" target="_blank" >https://www.frontiersin.org/articles/10.3389/fmicb.2019.00335/full</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3389/fmicb.2019.00335" target="_blank" >10.3389/fmicb.2019.00335</a>

Result language

angličtina

Original language name

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

Original language 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 A 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.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

10606 - Microbiology

Project

—

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2019

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Frontiers in Microbiology

ISSN

1664-302X

e-ISSN

—

Volume of the periodical

10

Issue of the periodical within the volume

Feb 27

Country of publishing house

CH - SWITZERLAND

Number of pages

18

Pages from-to

335

UT code for WoS article

000459760500001

EID of the result in the Scopus database

2-s2.0-85065901672