Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins

Abstract

Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella sp.). Overexpression of recombinant genes can overwhelm the innate secretory machinery of the P. pastoris cell, and incorrectly folded proteins may accumulate inside the ER. To restore proper protein folding, the cell naturally triggers an unfolded protein response (UPR) pathway, which upregulates the expression of genes coding for chaperones and other folding-assisting proteins (e.g., Kar2p, Pdi1, Ero1p) via the transcription activator Hac1p. Unfolded/misfolded proteins that cannot be repaired are degraded via the ER-associated degradation (ERAD) pathway, which decreases productivity. Co-expression of selected UPR genes, along with the recombinant gene of interest, is a common approach to enhance the production of properly folded, secreted proteins. Such an approach, however, is not always successful and sometimes, protein productivity decreases because of an unbalanced UPR. This review summarizes successful chaperone co-expression strategies in P. pastoris that are specifically related to overproduction of foreign proteins and the UPR. In addition, it illustrates possible negative effects on the cell’s physiology and productivity resulting from genetic engineering of the UPR pathway. We have focused on Pichia’s potential for commercial production of valuable proteins and we aim to optimize molecular designs so that production strains can be tailored to suit a specific heterologous product. Key points • Chaperones co-expressed with recombinant genes affect productivity in P. pastoris. • Enhanced UPR may impair strain physiology and promote protein degradation. • Gene copy number of the target gene and the chaperone determine the secretion rate.

Effects of Temperature and pH on Recombinant Thaumatin II Production by Pichia pastoris

“…For instance, from the study of Zhong et al [38], it was observed that lowering the cultivation temperature from 30 to 20 • C aided in the upregulation of the UPR. The upregulation can increase the yield of the protein due to faster processing of the recombinant product within the ER [38,43]. A lower temperature is expected to reduce proteolysis and unfolding of the recombinant protein.…”

β-galactosidase GALA from Bacillus circulans with high transgalactosylation activity

“…P. pastoris , an established protein expression host, is mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, the available strong and tightly regulated promoters, and the options to produce gram amounts of recombinant protein per liter of culture both intracellularly and in secretory fashion [ 29 ]. In this study, a β-glucosidase from B. circulans was expressed in P. pastoris .…”

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris

“…Overexpression of Hac1p and Yap1p did not improve IgG secretion. Hac1p is a transcription factor regulating the UPR genes and its overexpression showed improvements in the production of secreted recombinant proteins [ 8 , 37 , 38 ]. YAP1 is another transcription factor involved in oxidative stress relief [ 39 ] for which an improved recombinant protein production by P. pastoris was reported [ 26 ].…”