Evaluation of recombinant trypsinogen and chymotrypsinogen production in Komagataella phaffii through co-expression of HAC1 and PDI1

BACKGROUND: Trypsinogen and chymotrypsinogen are serine proteases with potential therapeutic applications in cancer. However, due to ethical, safety and variability concerns, their clinical use has been limited by the reliance on bovine or porcine pancreatic extracts. Recombinant expression in Komagataella phaffii (syn. Pichia pastoris) offers a scalable and controlled alternative. However, further optimisation is required to increase protein titer and secretion. RESULTS: Recombinant strains were engineered to express human trypsinogen and chymotrypsinogen. The effect of medium supplementation and co-expression of HAC1 and PDI1 on protein production was evaluated. Co-expression of HAC1 increased trypsinogen production by 3.2-fold, while PDI1 significantly increased chymotrypsinogen titer by 8.5-fold compared to parental strains. Medium optimisation had minimal effect on protein expression. SDS-PAGE and Western blotting analyses confirmed successful expression and secretion of both enzymes. CONCLUSIONS: The combination of genetic modification and medium optimisation significantly improved the recombinant production of trypsinogen and chymotrypsinogen in K. phaffii. HAC1 proved to be more effective in increasing the production of trypsinogen, while PDI1 co-expression significantly increased chymotrypsinogen titers. These findings provide a promising strategy for improving the production of these proteases, supporting their potential applications in biotechnology and biomedical research.