Production of Archaeal Inositol Phospholipids in Engineered Saccharomyces cerevisiae

Despite phospholipid backbone divergence between Archaea (ether-isoprenoid/glycerol-1-phosphate membranes) and Bacteria/Eukarya (ester-fatty acid/glycerol-3-phosphate membranes), known as the ‘lipid divide’, inositol headgroups are conserved across all domains. Inositol lipids from thermophilic archaea form exceptionally stable liposomes but are difficult to obtain at scale. Here, we engineered eukaryotic Saccharomyces cerevisiae for heterologous biosynthesis of unsaturated archaetidylinositol, revealing unexpected promiscuity of endogenous yeast enzymes toward archaeal substrates. Metabolic engineering achieved production levels reaching 6.5% of total cellular lipids, enabling structural, physiological, and biophysical characterization. Production triggered pleiotropic lipid metabolism changes and enhanced thermotolerance, with engineered yeast retaining 44% viability after 30 minutes at 50°C, a condition baseline strains cannot survive. Purified archaeal inositol lipid formed thermostable liposomes maintaining integrity across 25-95°C. This yeast platform will enable access to versatile archaetidylinositol derivatives with tailored bioactivity and material properties for both evolutionary studies and biotechnology applications.