Chemically defined lipid diets reveal the versatility of lipidome remodeling in genomically minimal cells

All cells are encapsulated in a lipid membrane that provides a responsive interface between life and its environment. Although simple membranes can be built from a single type of lipid, cellular membranes contain 10s to 100s of unique lipid species. Deciphering the significance of lipidome complexity is a central challenge in understanding the design principles of living membranes. While functions of individual lipids have been extensively studied, understanding how lipidomes collectively contribute to membrane function and cell phenotypes is experimentally challenging in most organisms. To address this challenge, we turned to the simple pathogenic organism Mycoplasma mycoides and its genomically derived "Minimal Cell" JCVI-syn3B, to establish a living minimal membrane model system in which lipidome complexity can be experimentally manipulated. By complexing lipids with cyclodextrins, we introduce a chemically defined approach to deliver lipid diets with different chemistries to cells, resulting in cellular lipidomes with as few as seven to nearly 30 lipids species. We explored how lipidome size and composition influences cell growth, osmotic sensitivity, and membrane adaptability to changes in growth temperature. Our findings indicate that lipidome composition dictates membrane adaptation to temperature change. Moreover, we show that lipidome diversity enhances cellular robustness to hypoosmotic shock. We further show that impaired acyl chain remodeling in the minimal cell is associated with impaired membrane temperature adaptation. Finally, we demonstrate as a proof of principle, how cells with tuneable lipidomes can be used as experimental chassis for screening membrane active antimicrobial peptides. Our study introduces an experimental resource and foundation for deciphering the role of lipidome complexity in membrane function and cellular fitness.