Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

Abstract

BackgroundThe increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers.ResultsBased on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion.ConclusionsThe current study presents a systems biotechnology-based strategy for the engineering of the industrially important yeast P. pastoris combining the use of host specific DNA microarray technologies and physiological studies under well defined environmental conditions. Such studies allowed for the identification of novel targets related with protein trafficking and ergosterol biosynthesis for improved recombinant protein production. Nevertheless, further studies will be required to elucidate the precise mechanisms whereby membrane biogenesis and composition impact on protein secretion in P. pastoris.

Novel helper factors influencing recombinant protein production in Pichia pastoris based on proteomic analysis under simulated microgravity

“…However, co-expression of the helper gene could also compete with the target gene for transcription. Baumann et al reported that the reporter gene mRNA levels among the recombinant protein production improved strains of overexpressing helper genes were similar to the reference strain (Baumann et al 2011). In our research, the transcriptional level of PGUS was decreased when the helper gene TPX was overexpressed.…”

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion

“…This phenomenon was unexpected, because increased protein production will demand more ATP and reduction power in the form of NADPH. Although proteome data and actual metabolic activities were well in agreement in a study using recombinant P. pastoris (45), this is not generally true for S. cerevisiae (46 -47). However, a reduction of TCA cycle fluxes has been described for a human superoxide dismutase producing S. cerevisiae strain (48).…”

“…Manipulation of cellular membrane by addition of surfactants has been shown to improve protein secretion in S. pombe (11). The decrease of ergosterol levels by addition of the fungicide fluconazole has been shown to enhance protein secretion in P. pastoris (45). Addition of fluconazole to S. pombe NW10 decreased ergosterol levels by more than 30% whereas at the same time improving maltase secretion 1.3-fold.…”

Flow Cytometric Enumeration of Bacteria Using TO-PRO®-3 Iodide as a Single-Stain Viability Dye

“…This nonionic surfactant enhances TP3 binding to bacterial cells by increasing membrane fluidity and therefore influences the position of the viable population in the SSC-FL4 dotplot. 18 It must be noted that despite the advantages of FCM, the technique also has its drawbacks compared with VPC. Although the latter is cost-effective and easy to perform, skilled technicians are needed to operate the flow cytometer and interpret the often complex data.…”