Precision fermentation of κ-casein using Pichia pastoris

The environmental burden associated with conventional cheese production has driven interest in sustainable alternatives to animal-derived dairy proteins, including recombinant animal-free caseins produced via precision fermentation. Among caseins, κ-casein is essential for the renneting step during cheese production, making its recombinant production particularly relevant. Efficient secretion of the recombinant protein is key to economic feasibility at commercial scale, as it simplifies downstream processing and thereby reduces production costs. Here, we investigated the production and secretion of bovine κ-casein in the yeast Pichia pastoris. Successful secretion was achieved, and multicopy strains gave higher titers. However, considerable protein degradation was observed, also in bioreactor cultures. A yapsin 1 knockout and a yapsin1/protease A double knockout did not fully mitigate this, indicating the involvement of additional proteolytic activities. Moreover, κ-casein was detected in the periplasmic fraction of some of the strains tested, indicating that part of the recombinant protein was not released into the culture medium. The recombinant protein displayed heterogeneous electrophoretic behavior due to extensive O-linked glycosylation. Cytoplasmic expression of κ-casein was attempted as an alternative to secretory production, but did not result in high yields or clearly improved protein integrity. These results highlight the intrinsic challenges associated with κ-casein production in P. pastoris, where limited secretion efficiency, persistent degradation, and extensive O-glycosylation constrain yield and lead to protein heterogeneity. Overcoming these barriers will be essential to enable economically viable production of κ-casein through precision fermentation using this host.