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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.

Edwards-Jones B, Aw R, Barton GR, Tredwell GD, Bundy JG, Leak DJ - PLoS ONE (2015)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0119637.g001: 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.

Mentions: Surprisingly, in addition to the down-regulation of expression of RNA pol I and III subunits, some RNA pol II core and specific subunits were down-regulated in all strains 2h after switching to methanol (S4 Fig. PAS_chr2-1_0125, chr4_0906, chr3_0244, chr1-4_0359), indicating a reduction in net protein synthesis. This is not typically linked to growth rate [33] but significant in the context of heterologous protein expression. However, by 4h expression of all RNA polymerase genes was starting to pick up in GS115, consistent with adaptation to a lower growth rate on methanol in GS115. Compared to their levels at 2h of induction many of the tRNA synthases were also up-regulated at 4h supporting the indication from pol II dynamics for a greater demand for net protein synthesis. However, in TRY1-3 in addition to reduction in expression of RNA polymerase I and III subunits, some core and specific RNA polymerase II subunits (Fig. 1) and tRNA synthases (S5 Fig.) remained down-regulated in comparison with GS115 after 4h. Not only does this reflect the complete cessation of growth but a significant down-regulation of the potential for de-novo protein synthesis.


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.

Edwards-Jones B, Aw R, Barton GR, Tredwell GD, Bundy JG, Leak DJ - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4364781&req=5

pone.0119637.g001: 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.
Mentions: Surprisingly, in addition to the down-regulation of expression of RNA pol I and III subunits, some RNA pol II core and specific subunits were down-regulated in all strains 2h after switching to methanol (S4 Fig. PAS_chr2-1_0125, chr4_0906, chr3_0244, chr1-4_0359), indicating a reduction in net protein synthesis. This is not typically linked to growth rate [33] but significant in the context of heterologous protein expression. However, by 4h expression of all RNA polymerase genes was starting to pick up in GS115, consistent with adaptation to a lower growth rate on methanol in GS115. Compared to their levels at 2h of induction many of the tRNA synthases were also up-regulated at 4h supporting the indication from pol II dynamics for a greater demand for net protein synthesis. However, in TRY1-3 in addition to reduction in expression of RNA polymerase I and III subunits, some core and specific RNA polymerase II subunits (Fig. 1) and tRNA synthases (S5 Fig.) remained down-regulated in comparison with GS115 after 4h. Not only does this reflect the complete cessation of growth but a significant down-regulation of the potential for de-novo protein synthesis.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus