LAUSR

Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. ABSTRACT Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were EPX1, PAS_chr3_0030, SCW10, and UTH1, suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the PAS_chr3_0030 gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the PAS_chr3_0030 gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris. IMPORTANCE Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the PAS_chr3_0030 gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the PAS_chr3_0030 gene product may facilitate the application of P. pastoris in industrial biocatalysis.