Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% compared with that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 were involved in protein folding and Sec1 and Sly1 were involved in vesicle trafficking. We evaluated the possible effects of individual overexpression of these secretion helper factors on the secretion of IL2-HSA in P. pastoris. Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2-HSA secreting strain. Individual co-overexpression of Ero1, Kar2, Pdi1, Sec1 and Sly1 improved the secretion level of IL2-HSA to ~2.3-, 1.9-, 2.2-, 2.5- and 1.9-fold that in the control strain respectively in shake flasks. We evaluated the changes in mRNA and protein levels of the intracellular IL2-HSA, as well as the secretion helper factor genes in the co-overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2 and SM protein Sec1 and Sly1 could improve the secretion level of IL2-HSA fusion protein in P. pastoris, which provided new candidates for combinatorial engineering in future study. Copyright © 2016 John Wiley & Sons, Ltd.

Recently, studies performed on Saccharomyces cerevisiae and Candida albicans have confirmed the importance of fungal glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases (yapsins) for cell-wall integrity. Genome sequence annotation of Pichia pastoris also revealed seven putative GPI-anchored aspartyl protease genes. The five yapsin genes assigned as YPS1, YPS2, YPS3, YPS7 and MKC7 in P. pastoris were disrupted. Among these putative GPI-linked aspartyl proteases, disruption of PpYPS7 gene confers the Ppyps7Δ mutant cell increased resistance to cell wall perturbing reagents congo red, calcofluor white (CW) and sodium dodecyl sulfate. Quantitative analysis of cell wall components shows lower content of chitin and increased amounts of β-1,3-glucan. Further staining of the cell with CW demonstrates that disruption of PpYPS7 gene causes a reduction of the chitin content in lateral cell wall. Consistently, transmission electron micrographs show that the inner layer of mutant cell wall, mainly composed of chitin and β-1, 3-glucan, is much thicker than that in parental strain GS115. Additionally, Ppyps7Δ mutant also exhibits increased osmotic resistance compared with parental strain GS115. This could be due to the dramatically elevated intracellular glycerol level in Ppyps7Δ mutant. These results suggest that PpYPS7 is involved in cell wall integrity and response to osmotic stress.

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