Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris

PloS One
Bryn Edwards-JonesDavid J Leak

Abstract

We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient ...Continue Reading

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Citations

Feb 6, 2017·Journal of Industrial Microbiology & Biotechnology·Gregory D TredwellJacob G Bundy
Sep 22, 2017·FEMS Yeast Research·Richard J ZahrlBrigitte Gasser
May 20, 2020·Applied Microbiology and Biotechnology·Martina BorčinováKarin Kovar

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Datasets Mentioned

BETA
E-MTAB-2530

Methods Mentioned

BETA
NMR
chip
histone acetylation
protein folding
glycosylation
environmental stresses

Software Mentioned

KEGG Search & Colour
Blast
rNMR
bioconductor
R
KOBAS ( KEGG Orthology Based Annotation System )
iNMR
Bioconductor package

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