The impact of ERAD on recombinant protein secretion in Pichia pastoris (syn Komagataella spp.)

The yeast Pichia pastoris (syn. Komagataella spp.) is a popular cell factory for recombinant protein production. Yeasts in general provide a good starting point for cell factory engineering. They are intrinsically robust and easy to manipulate and cultivate. However, their secretory pathway is not evolutionarily adapted to high loads of secretory protein. In particular, more complex proteins, like the antibody fragment (Fab) used in this study, overwhelm the folding and secretion capacity. This triggers cellular stress responses, which may cause excessive intracellular degradation. Previous results have shown that, in fact, about 60% of the newly synthesized Fab is intracellularly degraded. Endoplasmic reticulum-associated protein degradation (ERAD) is one possible intracellular degradation pathway for proteins aimed for secretion. We therefore targeted ERAD for cell factory engineering and investigated the impact on recombinant protein secretion in P. pastoris. Three components of the ERAD-L complex, which is involved in the degradation of luminal proteins, and a protein involved in proteasomal degradation, were successfully disrupted in Fab-secreting P. pastoris. Contrary to expectation, the effect on secretion was marginal. In the course of more detailed investigation of the impact of ERAD, we took a closer look at the intracellular variants of the recombinant protein. This enabled us to further zero in on the issue of intracellular Fab degradation and exclude an overshooting ER quality control. We propose that a major fraction of the Fab is actually degraded before entering the secretory pathway.