Enhanced membrane protein expression by engineering increased intracellular membrane production

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Membrane protein research is challenged by low natural abundance, necessitating recombinant gene expression.
• Existing expression systems (mammalian, insect, bacterial, yeast) lack host cell customization for enhanced membrane protein yield.
• This study focuses on tailoring yeast host cells to improve membrane protein expression.

Purpose of the Study:

• To enhance membrane protein expression in yeast by increasing intracellular membrane production.
• To investigate the effect of manipulating lipid synthesis on membrane protein accumulation.
• To assess the impact of host cell engineering on membrane protein quality and function.

Main Methods:

• Engineered the oleotrophic yeast Yarrowia lipolytica by deleting the PAH1 gene.
• Induced massive proliferation of endoplasmic reticulum (ER) membranes.
• Analyzed membrane protein accumulation, proteolytic integrity, and ligand binding activity.

Main Results:

• Deletion of PAH1 led to significant ER membrane proliferation.
• Enhanced accumulation levels and proteolytic integrity were observed for eight integral membrane protein families.
• Co-induction of the unfolded protein response (UPR) in the ∆pah1 strain improved the specific ligand binding activity of the adenosine A2AR G-protein coupled receptor.
• Demonstrated an improved quality control mechanism for membrane proteins in yeast with proliferated ER.

Conclusions:

• Inactivating the PAH1 gene redirects metabolic flux towards membrane phospholipid synthesis, enhancing eukaryotic membrane protein production.
• UPR co-induction complements increased protein quantity by improving proper folding and biological activity.
• The conserved nature of these pathways suggests potential for similar engineering in insect and mammalian cells for biotechnological applications.