Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

Pichia pastoris is a popular yeast preferably employed for secretory protein production. Secretion is not always efficient and endoplasmic retention of proteins with aberrant folding properties, or when produced at exaggerated rates, can occur. In these cases production usually leads to an unfolded protein response (UPR) and the induction of the endoplasmic reticulum associated degradation (ERAD). P. pastoris is nowadays also an established host for secretory insulin precursor (IP) production, though little is known about the impact of IP production on the host cell physiology, in particular under industrially relevant production conditions. Here, we evaluate the cellular response to aox1 promoter-controlled, secretory IP production in controlled fed-batch processes using a proteome profiling approach. Cells were first grown in a batch procedure using a defined medium with a high glycerol concentration. After glycerol depletion IP production was initiated by methanol addition which was kept constant through continuous methanol feeding. The most prominent changes of the intracellular proteome after the onset of methanol feeding were related to the enzymes of central carbon metabolism. In particular, the enzymes of the methanol dissimilatory pathway - virtually absent in the glycerol batch phase - dominated the proteome during the methanol fed-batch phase. Unexpectedly, a strong decrease of UPR and ERAD related proteins was also observed during methanol-induced IP production. Compared to non-producing control strains grown under identical conditions the UPR down-regulation was less pronounced indicating that IP production elicits a detectable but non prominent UPR response which is repressed by the general culture condition-dependent UPR down-regulation after the shift from glycerol to methanol. The passage of IP through the secretory pathway using an optimized IP vector and growing the strain at fed-batch conditions with a high initial glycerol concentration does not impose a significant burden on the secretory machinery even under conditions leading to an extracellular accumulation of ~ 3 g L-1 IP. The glycerol batch pre-induction culture conditions are associated with a high constitutive - recombinant protein production independent - induction of the UPR and ERAD pathways probably preconditioning the cells for effective IP secretion in the methanol fed-batch phase.