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. 2015 Mar 18;10(3):e0119637.
doi: 10.1371/journal.pone.0119637. eCollection 2015.

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

Bryn Edwards-Jones  1 , Rochelle Aw  1 , Geraint R Barton  1 , Gregory D Tredwell  2 , Jacob G Bundy  2 , David J Leak  3
Affiliations

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

Bryn Edwards-Jones et al. PLoS One. .

Abstract

Results: 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.

Conclusion: 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 metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.

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Conflict of interest statement

Competing Interests: Funding for this research was provided by BBSRC with guidance from the "Bioprocessing Research Industry Club" (BRIC). BRIC is an umbrella organisation founded by member companies in order to _target research funding into critical aspects of (biopharmaceutical) bioprocessing. None of the BRIC member companies have any vested interest in the contents of this manuscript and it has been approved by BRIC for publication. RA received partial funding for her PhD studies from Avecia Biologics (now Fujifilm Diosynth Biotechnologies). This work did not constitute part of her PhD and Avecia/Fujifilm had no involvement with this work, except through their membership of BRIC. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Expression of RNA polymerase I, II and III core function genes, 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), TRY1-1(blue) and TRY1-3 (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the AOX1 promoter.
From top to bottom, panels represent RPA1, RPB2 and RPC2. Error bars show SEM.
Fig 2
Fig 2. Expression of genes associated with methanol oxidation and assimilation during fed-batch culture, 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), TRY1-1(blue) and TRY1-3 (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the AOX1 promoter.
From top to bottom, panels represent AOX1, FDH1, FGH1, DAS1, DAK2, TPI1 and RIB1. Error bars show SEM.
Fig 3
Fig 3. Expression of PCK1, PYC and PYK during fed-batch culture, 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), TRY1-1 (blue) and TRY1-3 (red).
Error bars show SEM.
Fig 4
Fig 4. Metabolite accumulation profiles during fed-batch culture from 0 (before) to 24 hours after the start of methanol addition to wild-type GS115 (black), TRY1-1(blue) and TRY1-3 (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the AOX1 promoter.
From top to bottom, panels represent a) formate (external), b) arabitol (external), c) trehalose (external), d) α / β-D-glucose (external), e) lactate (internal). Error bars show SEM.
Fig 5
Fig 5. Expression of HAC1 during fed-batch culture, 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), TRY1-1(blue) and TRY1-3 (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the AOX1 promoter.
Error bars show SEM.
Fig 6
Fig 6. Expression of the gene encoding D-arabitol-2-dehydrogenase (top) and “YDL124W” (bottom), 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), TRY1-1(blue) and TRY1-3 (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the AOX1 promoter.
Error bars show SEM.
Fig 7
Fig 7. Expression of starvation and stress response genes, 0 (before), 2 and 4 hours after the start of methanol addition to wild-type GS115 (black), TRY1-1(blue) and TRY1-3 (red), strains containing 1 and 3 gene copies of the human typsinogen gene, respectively, under the control of the AOX1 promoter.
From top to bottom, panels represent GCN4, TRX2, TSA1, AHP1, SOD1, SOD2. Error bars show SEM.
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