The α-mating factor secretion signals and endogenous signal peptides for recombinant protein secretion in Komagataella phaffii

Abstract

Background The budding yeast Komagataella phaffii (Pichia pastoris) is widely employed to secrete proteins of academic and industrial interest. For secretory proteins, signal peptides are the sorting signal to direct proteins from cytosol to extracellular matrix, and their secretion efficiency directly impacts the yields of the targeted proteins in fermentation broth. Although the α-mating factor (MF) secretion signal from S. cerevisiae, the most common and widely used signal sequence for protein secretion, works in most cases, limitation exists as some proteins cannot be secreted efficiently. As the optimal choice of secretion signals is often protein specific, more secretion signals need to be developed to augment protein expression levels in K. phaffii. Results In this study, the secretion efficiency of 40 α-MF secretion signals from various yeast species and 32 endogenous signal peptides from K. phaffii were investigated using enhanced green fluorescent protein (EGFP) as the model protein. All of the evaluated α-MF secretion signals successfully directed EGFP secretion except for the secretion signals of the yeast D. hansenii CBS767 and H. opuntiae. The secretion efficiency of α-MF secretion signal from Wickerhamomyces ciferrii was higher than that from S. cerevisiae. 24 out of 32 endogenous signal peptides successfully mediated EGFP secretion. The signal peptides of chr3_1145 and FragB_0048 had similar efficiency to S. cerevisiae α-MF secretion signal for EGFP secretion and expression. Conclusions The screened α-MF secretion signals and endogenous signal peptides in this study confer an abundance of signal peptide selection for efficient secretion and expression of heterologous proteins in K. phaffii.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

“…For instance, analyses of transcriptomic and proteomic data have identified in K. phaffii a signal peptide called PAS_chr3_0030, which has successfully been utilized to secrete the NIT10 enzyme, which failed to be secreted when the α-factor was used [ 106 ]. Screening of the endogenous signal peptides in K. phaffii has revealed a series of signal sequences (Scw, Dse, Exg, Dan4, Gas1, Msb2, Fre2, and others) that are comparable or even superior to the α-MF from S. cerevisiae in the production of heterologous proteins [ 107 , 108 , 109 , 110 ]. A similar analysis of α-MF signal peptides from various yeast species has shown that the efficiency of protein secretion when the α-MF from K. lactis is employed is comparable to that from S. cerevisiae , whereas with the α-MF from Wickerhamomyces ciferrii , the efficiency is even higher [ 54 , 110 ].…”

“…Screening of the endogenous signal peptides in K. phaffii has revealed a series of signal sequences (Scw, Dse, Exg, Dan4, Gas1, Msb2, Fre2, and others) that are comparable or even superior to the α-MF from S. cerevisiae in the production of heterologous proteins [ 107 , 108 , 109 , 110 ]. A similar analysis of α-MF signal peptides from various yeast species has shown that the efficiency of protein secretion when the α-MF from K. lactis is employed is comparable to that from S. cerevisiae , whereas with the α-MF from Wickerhamomyces ciferrii , the efficiency is even higher [ 54 , 110 ]. All these sequences can potentially be used instead of the S. cerevisiae α-MF to secrete heterologous proteins in K. phaffii .…”

Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris

“…To avoid variability in signal peptide e ciency for different protein secretion, the discovery of new signal peptides is essential (Liang et al 2012;Sastry et al 2014). Systematic analysis of yeast genomes and secretomes has led to the identi cation of novel signal peptides with more e cient secretion than α-MF, such as the signal peptide derived from the P. pastoris PAS_chr3_0030 gene (PC0) and the signal peptide NCW2 in Saccharomyces cerevisiae (Shen et al 2022;Wang et al 2015;Zou et al 2022).…”

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

“…The efficiency of SP significantly affects the secretory yield of target proteins [34]. Although the α-Factor SP is effective in P. pastoris, many endogenous SPs can be mined and used as an alternative to secrete heterologous proteins [35][36][37][38]. Recent research has demonstrated that endogenous SPs from S. cerevisiae can more effectively mediate the secretion of heterologous proteins compared to the α-Factor SP [39].…”