Higher induction temperatures and the native secretion signal peptide promote rye prolamin 75k γ-secalin production in Komagataella phaffii.

Background: Gluten proteins from wheat, rye, and barley play a substantial role in human nutrition. At the same time, they can trigger several different immune reactions. This, together with their influence on the quality of grain products and their emerging role as biomaterials, makes them an interesting target for further study. The proteins' propensity for aggregation challenges heterologous eukaryotic production systems. The yeast Komagataella phaffii has demonstrated excellent qualities as a production host for heterologous proteins and was therefore investigated as a platform strain.

Results: A gene coding for the rye (Secale cereale) prolamin 75k γ-secalin was cloned and inserted into K. phaffii; protein expression was verified via mass spectrometry and immunoblotting and quantified via ELISA. Different parameters were investigated regarding their effect on target protein production and endoplasmic reticulum (ER) homeostasis, including the induction temperature and co- and post-translational import into the ER. At 28°C, the cells produced 1.69-fold more 75k γ-secalin than at 20°C. The introduction of the MATα-pro-region, in conjunction with either the MATα-pre- or OST1-pre-signal, led to significantly lower 75k γ-secalin accumulation, 0.20- and 0.18-fold, respectively. No mutant showed significant changes in the unfolded protein response compared to a non-producing strain.

Conclusions: K. phaffii is a suitable host for prolamin production. The absence of a significant unfolded protein response during 75k γ-secalin expression indicates little challenge of ER-homeostasis by the aggregation-prone protein. It underscores K. phaffii's imminent role in protein production. The significantly decreased protein yield through the common protein secretion leader component MATα-pro demonstrates the need for further investigation into the role of secretion signals in optimizing K. phaffii as a production platform for repetitive, aggregation-prone proteins.