Optimizing Pichia pastoris Protein Secretion: Role of N-Linked Glycosylation on the α-Mating Factor Secretion Signal Leader

The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.