TY - JOUR
T1 - 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
AU - Edwards-Jones, Bryn
AU - Aw, Rochelle
AU - Barton, Geraint R.
AU - Tredwell, Gregory D.
AU - Bundy, Jacob G.
AU - Leak, David J.
PY - 2015/3/18
Y1 - 2015/3/18
N2 - Results: We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using bothmicroarray 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 withmethanol. 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.
AB - Results: We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using bothmicroarray 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 withmethanol. 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.
UR - http://www.scopus.com/inward/record.url?scp=84925597892&partnerID=8YFLogxK
UR - http://dx.doi.org/10.1371/journal.pone.0119637
U2 - 10.1371/journal.pone.0119637
DO - 10.1371/journal.pone.0119637
M3 - Article
AN - SCOPUS:84925597892
VL - 10
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 3
M1 - e0119637
ER -