Komagataella phaffii (formerly Pichia pastoris) is a prominent platform for recombinant protein production, yet secretion efficiency often remains a critical bottleneck. In this study, we validated three candidate genes—WHI3, CLPP, and PMP20—previously identified through genome-wide CRISPR activation screening, for their potential to enhance heterologous protein secretion. Overexpression of these factors under the control of the methanol-inducible AOX1 promoter increased the secretion of human serum albumin (HSA), with WHI3 and CLPP yielding improvements of 18.3% and 17.9%, respectively. Furthermore, applying this strategy to human lactoferrin (hLF) revealed that WHI3 overexpression robustly enhanced hLF secretion by approximately 70%. Comparative analysis of different promoters (AOX1, GAP, and CAT) indicated that the AOX1 promoter remains the most effective driver for these enhancers, suggesting a threshold-dependent regulatory mechanism. These results demonstrate the protein-dependent nature of secretion optimization and identify WHI3, CLPP, and PMP20 as novel, effective co-expression factors for improving recombinant protein yields in K. phaffii.
Tao et al. (Mon,) studied this question.