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.
Highlights
Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression
As our previously reported data in Pichia pastoris indicated that co-induction of the unfolded protein response (UPR) can enhance membrane protein homogeneity and activity [8], we evaluated the combined impact of endoplasmic reticulum (ER) membrane proliferation and UPR induction on the quantity and quality of the heterologously expressed membrane proteins
The results demonstrate that the Δpah1 mutation very strongly enhanced the expression levels of the Gprotein coupled receptors (GPCRs). qPCR analysis showed that the mRNA levels for the GPCR were only slightly enhanced in the Δpah1 strain (Additional file 2: Figure S2), indicating that stronger promoter activation and/or altered mRNA metabolism in the knockout is not the main causative factor for the strongly enhanced membrane protein accumulation
Summary
Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. 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. Many eukaryotic cells can absorb longchain fatty acids (FA) and store them in cytoplasmic lipid droplets in the form of triacylglycerols (TAG) and steryl esters (SE) [2]. The uptake of fatty acids from the culture medium can directly provide the precursors necessary for phospholipid synthesis and could facilitate the biogenesis of membranes, if their incorporation into TAG and SE lipid stores could be suppressed. Informed by the massive ER/nuclear membrane proliferation seen in a Δpah strain of S. cerevisiae [4] we investigated whether we could utilize this Δpah phenotype to enhance membrane protein productivity. We chose to work with Yarrowia lipolytica because fatty acid-regulated promoters are well established for this organism [5] and we hypothesized that the ability to feed direct precursors for membrane lipids (i.e. fatty acids) would be most suitable to achieve enhanced membrane lipid biosynthesis
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