Enhanced membrane protein expression by engineering increased intracellular membrane production

Abstract

BackgroundMembrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved.ResultsWe engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER.ConclusionsWe conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeastYarrowia lipolyticaand its role in lipid biosynthesis

“…pah1 Δ) causes a 90% decrease in TAG levels of stationary phase S . cerevisiae cells (Han et al ., ), while similar results have been obtained with the putative Yl PAH1 deletion mutant (Guerfal et al ., ). Also, the S .…”

“…pah1Δ ; Han et al ., ) and in a Y . lipolytica mutant where the putative Yl PAH1 was deleted (Guerfal et al ., ). Also, the lack of Sc PAH1 has severe consequences on cell physiology, as indicated by the fact that pah1Δ cells present serious growth defects (Han et al ., ; Park et al ., ).…”

The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose

“…Previous work in Y. lipolytica showed that the lack of PAH1 decreases the TAG levels 5‐fold but increases the phospholipid levels 2‐fold in cells grown in oleic acid media (Guerfal et al, ). Also, the Y. lipolytica pah1 Δ mutant presented the typical phenotype of the ER expansion due to overproduction of phospholipids and a great decrease in the number of lipid droplets (Guerfal et al, ). These findings are in line with the phenotypes reported for the well‐studied S. cerevisiae pah1 Δ mutant grown in glucose (Fakas et al, ; Han et al, ; Han, Siniossoglou, & Carman, ).…”

Recombinant protein subunit vaccine synthesis in microbes: a role for yeast?

“…As stated in a recent review,[5] identifying or engineering yeast strains with improved yield characteristics may either be targeted towards one particular pathway or may take a more global approach [98]. Examples of the targeted approach include ‘humanizing’ the yeast glycosylation[99] and sterol[100] pathways and modifying membrane phospholipid synthesis to proliferate intracellular membranes [101]. Studies taking a more global approach in both S. cerevisiae [102,103] and P. pastoris [91,104] have identified the importance of the unfolded protein response (the cellular stress response activated by the accumulation of unfolded or misfolded protein) and reduced translational activity in high‐yielding cultures.…”