Abstract

Komagataella phaffii(formerlyPichia 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, andPMP20—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-inducibleAOX1promoter increased the secretion of human serum albumin (HSA), withWHI3andCLPPyielding improvements of 18.3% and 17.9%, respectively. Furthermore, applying this strategy to human lactoferrin (hLF) revealed thatWHI3overexpression robustly enhanced hLF secretion by approximately 70%. Comparative analysis of different promoters (AOX1,GAP, andCAT) indicated that theAOX1promoter 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 identifyWHI3,CLPP, andPMP20as novel, effective co-expression factors for improving recombinant protein yields inK. phaffii.