Edwards-Jones, BrynAw, RochelleBarton, Geraint RTredwell, GregoryBundy, Jacob GLeak, David J2018-11-292018-11-291932-6203http://hdl.handle.net/1885/153439Results 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.application/pdfKeywords: methanol; trypsinogen; adaptation; AOX1 gene; Article; controlled study; fed batch culture; fungal gene; fungal strain; fungus culture; fungus growth; growth rate; Komagataella pastoris; metabolomics; microarray analysis; nonhuman; promoter region; protei201510.1371/journal.pone.01196372018-11-29