Abstract
ResultsWe 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.ConclusionAlthough 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.
Highlights
The methylotrophic yeast, Pichia pastoris is a commonly used host for protein expression [1, 2]
Without detailed analysis of the underlying processes it could have been mis-interpreted as secretion stress, transmitted through the unfolded protein response (UPR)
Genes to be expressed are chromosomally integrated downstream of AOX1 by either recombination at a single site, generating a mut+ strain, in which a functional copy of the AOX1 gene is retained, or transplacement, in which the AOX1 gene is disrupted. This can yield a muts or mut- strain depending on the availability of a functional AOX2 gene which encodes a second alcohol oxidase with allows slow growth on methanol [5]
Summary
The methylotrophic yeast, Pichia pastoris is a commonly used host for protein expression [1, 2]. Genes to be expressed are chromosomally integrated downstream of AOX1 by either recombination at a single site, generating a mut+ (methanol utilisation positive) strain, in which a functional copy of the AOX1 gene is retained, or transplacement, in which the AOX1 gene is disrupted. This can yield a muts (slow) or mut- strain depending on the availability of a functional AOX2 gene which encodes a second alcohol oxidase with allows slow growth on methanol [5]. Production is usually done in fed-batch in which cells are grown to high densities using glycerol as the carbon source, induced initially with a pulse of methanol and switched to a continuous methanol feed which may be maintained for up to 96h [1]
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