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 of 45.6 ± 6.2 mg/g DCW, substantially exceeding endogenous phosphatidylinositol level measured in the same strain (4.8 ± 1.0 mg/g DCW) by >8-fold, which enabled detailed structural, physiological, and biophysical characterization of unsaturated archaetidylinositol for the first time. Production triggered pleiotropic lipid metabolism changes and enhanced thermotolerance, with engineered yeast retaining 57% viability after 40 min at 50 °C, a condition that eliminates chassis strain viability. Purified archaeal inositol lipid formed stable liposomes, which maintained integrity upon heat shock treatment at 95 °C or during 4 week storage at 4 °C, while eukaryotic lipid-derived vesicles exhibited substantial cargo leakage under identical conditions. This yeast platform will enable access to versatile archaetidylinositol derivatives with tailored bioactivity and material properties for both evolutionary studies and biotechnology applications.