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

Abstract

BackgroundPichia 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.ResultsCells 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.ConclusionsThe 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.

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

“…In eukaryotic systems, protein folding occurs in a different way; proteins are folded and processed in the endoplasmic reticulum (ER) and/or the Golgi apparatus and sometimes secreted into the extracellular environment through vesicular transport (as is in the case of recombinant expression of CH-IQ-AB in P.pastoris). In eukaryotes aberrant folding properties of the target protein and/or high level production can lead to the accumulation of unfolded or even aggregated protein in the ER, which can initiate the unfolded protein response (UPR) and ER-associated degradation [36][37][38][39][40]. This can thus possibly explain the fact that we cannot detect expression of CH-IQ-AB in P.pastoris.…”

Investigating the effect of carbon source on rabies virus glycoprotein production inPichia pastorisby a transcriptomic approach

“…Heterologous protein expression in yeasts can be affected by different factors. In P. pastoris potential limiting factors of foreign protein expression are gene dosage (Shen, Ming, Hai‐Bin, Hua, & Shu‐Qing, ), efficient transcription of the transgene using strong promoter (Gasser et al., ), protein folding in the reticulum endoplasmic (RE) (Vanz, Nimtz, & Rinas, ), and protein secretion (Pfeffer et al., ). Additionally, bioprocess parameters such as pH, temperature, growth rate, and substrate type also affect protein expression in P. pastoris (Dragosits et al., ; Files, Ogawa, Scamanb, & Baldwina, ; Xie, Zhou, Du, Gan, & Ye, ).…”

“…2012), efficient transcription of the transgene using strong promoter (Gasser et al, 2013), protein folding in the reticulum endoplasmic (RE) (Vanz, Nimtz, & Rinas, 2014), and protein secretion (Pfeffer et al, 2011). Additionally, bioprocess parameters such as pH, temperature, growth rate, and substrate type also affect protein expression in P. pastoris (Dragosits et al, 2009;Files, Ogawa, Scamanb, & Baldwina, 2001;Xie, Zhou, Du, Gan, & Ye, 2004).…”

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris

“…During the journey of a protein through different cellular compartments, namely the ER, the Golgi apparatus, and finally, vesicular transport to the extracellular environment several post-translational modifications occur (Vanz et al, 2014). However, not all recombinant proteins are efficiently secreted and ER retention during high-level production can be a problem.…”