Systematic engineering of Komagataella phaffii for the production of hen egg ovomucoid

This study aimed to develop an eco-friendly technology to produce ovomucoid, which is not only a key contributor to egg white's unique properties but also a bioactive compound. To identify the optimal components for an ovomucoid secretion system using Komagataella phaffii, we first tested various signal peptides and promoters. Next, a major bottleneck in the secretory production of protein, endoplasmic reticulum stress, was addressed to further enhance secretion efficiency. Among various combinations, the highest concentration of ovomucoid, 23 mg/L, was obtained when a signal peptide derived from EPX1, the most abundant extracellular protein produced by K. phaffii, and the GCW14 promoter were employed along with a transcription factor involved in the unfolded protein response, HAC1, and a protein disulfide isomerase, PDI1, were co-expressed. Finally, the extracellular production of ovomucoid under high-cell density cultivation was maximized to 929 mg/L.