The yeast Komagataella phaffii is an emerging microbial host for the production of functional recombinant proteins. However, proteolytic degradation during fermentation often compromises product yield and stability, posing a major hurdle for industrial-scale applications. This study presents a strategy to enhance the production of recombinant humanized type I collagen (rhColI) by engineering a host strain with reduced protease activity. An initial production strain, CL1, was engineered using post-transformational vector amplification (PTVA), achieving a titer of 558.86 ± 20.05 mg/L in flask culture. Subsequent scale-up fermentation, however, revealed significant rhColI degradation. To address this, we systematically deleted 11 candidate endogenous protease genes. The knockout of a serine protease gene, KpSub2, resulted in the most pronounced improvement, elevating the rhColI titer to 1039.06 ± 34.08 mg/L, a 23.47% increase over the parent strain CL1. Furthermore, the purified KpSub2 protein, obtained from inclusion bodies in Escherichia coli, demonstrated broad proteolytic activity against various types of recombinant humanized collagens. This broad substrate specificity was consistent with the observation that KpSub2 deletion also mitigated the degradation of recombinant humanized type III collagen (rhColIII). Our findings establish KpSub2 as a key mediator of collagen degradation in K. phaffii and provide an effective engineering strategy for optimizing the production of collagen and other degradation-susceptible functional proteins in this host.
Feng et al. (Thu,) studied this question.