The methylotrophic yeast, Pichia pastoris (P. pastoris), known for its ability to grow to high cell densities and its strong and tightly regulated promoters, has been widely used as a robust system for the production of heterologous proteins (Ahmad et al., 2014, Karbalaei et al., 2020). In 1995, P. pastoris underwent reclassification and was placed in the newly established genus, Komagataella (Yamada et al., 1995). Subsequently, this genus was further divided into multiple species, resulting in the former P. pastoris strains being distinctively classified as two species, Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) (Kurtzman, 2009, Kurtzman, 2005, Pena et al., 2018). Both species are used for heterologous protein expression. The strains designated GS115 and X-33 have been identified as belonging to the species K. phaffii, whereas the protease-deficient strain series SMD, among which SMD1168 is the most recognized, are classified under the type species K. pastoris (Mattanovich et al., 2009). Presently, a total of six species (K. pastoris, K. phaffii, K. pseudopastoris, K. poluli, K. ulmi, and K. kurtzmanii) fall under the genus Komagataella (Naumov et al., 2013). In this study, the established name P. pastoris is employed as a synonymous reference for both K. pastoris and K, phaffii strains.

P. pastoris is a popular host organism for heterologous protein secretion expression because of its efficient secretory pathway involving the endoplasmic reticulum (ER) and Golgi apparatus, which ensures proper protein folding, processing, and modification (Bevis et al., 2002, Mogelsvang et al., 2003, Rossanese et al., 1999). This includes the formation of disulfide bonds, glycosylation, and oligomerization, resulting in the secretion of proteins into the culture medium as more native-like, soluble forms with higher physiological activity. Additionally, P. pastoris has the advantage of low endogenous protein secretion, making the purification of recombinant proteins easier (Karbalaei et al., 2020). The secretion efficiency of signal peptides is crucial for directing proteins to the extracellular matrix and affects yields of targeted proteins in the fermentation broth. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is the most commonly used signal sequence in P. pastoris for recombinant protein secretion. The α-MF secretion signal leader of S. cerevisiae consists of two regions: the pre-peptide, located at the N-terminus of the α-MF secretion signal leader, acts as a signal peptide; the pro-peptide, located at the C-terminus of the α-MF secretion signal leader, functions to facilitate protein secretion (Julius et al., 1983).

The completion of genomic sequencing of K. pastoris and K, phaffii provided valuable insights into its molecular toolbox and confirmed the presence of the α-MF genes within their genomes (De Schutter et al., 2009, Kuberl et al., 2011, Love et al., 2016, Mattanovich et al., 2009). The α-MF secretion signal leaders of K. pastoris and K. phaffii share a structural resemblance with the α-MF secretion signal leader of S. cerevisiae, with both consisting of pre-peptide and pro-peptide components. Despite the structural similarities among the α-MF secretion signal leaders from K. pastoris, K. phaffii, and S. cerevisiae, precious study had revealed that the efficacy of these signal leaders from K. pastoris and K. phaffii in directing the secretion of heterologous proteins is suboptimal (Zou et al., 2022). The factors influencing the functionality of the α-MF secretion signal leaders from K. pastoris and K. phaffii remain to be elucidated.

Improvement of heterologous protein secretion in P. pastoris is crucial for its continued dominance and competitiveness as a host for recombinant protein production. As the demands for more efficient and cost-effective production of proteins with pharmaceutical and industrial relevance grow, understanding and optimizing the secretion pathways of P. pastoris becomes increasingly vital (Besada-Lombana and Da Silva, 2019, Liu et al., 2022, Puxbaum et al., 2015, Raschmanova et al., 2021). In this study, our research aims to explore and enhance the secretion efficiency of heterologous proteins in P. pastoris. We focused on the role of N-linked glycosylation of the α-MF secretion signal leaders on the effect of heterologous protein secretion. and revealed that N-linked glycosylation of the α-MF secretion signal leaders promotes the secretion of the reporter protein by reducing the UPR in the ER through the involvement of the molecular chaperone calnexin.