The composition of the membrane lipid matrix determines the physical properties of a biological membrane including membrane surface charge, membrane fluidity and membrane intrinsic curvature. For proper membrane function it is essential that these parameters are maintained in the appropriate range. Yeast is the eukaryote of choice for investigating the regulatory mechanisms governing membrane lipid composition because the organism is extremely tolerant to manipulation of membrane lipid biosynthesis. Phosphatidylcholine (PC) is a highly abundant membrane lipid in most eukaryotes, and is generally considered essential. With the goal of elucidating the function(s) of PC we have been studying the yeast mutant cho2opi3 that lacks the methyltransferases for converting phosphatidylethanolamine (PE) into PC and relies on supplementation with choline for PC synthesis by the CDP-choline route. Recently, we isolated cho2opi3 suppressor (cho2opi3S) clones that lost the auxotrophy for choline. These clones exhibit decent growth on fermentable carbon source in the complete absence of choline or choline substitutes. Lipidome analysis demonstrated the absence of PC and revealed that the suppressors exhibit strongly increased synthesis of fatty acids and triglycerides. This is accompanied by a shortening of the average acyl chain length and increased acyl chain desaturation. The changes in fatty acid profile are consistent with the maintenance of membrane physical properties since they reduce the non-bilayer propensity of PE, which has replaced PC as most abundant membrane lipid. At the ultrastructural level, EM revealed massive lipid droplet accumulation and aberrant mitochondrial and vacuolar structure in cho2opi3S cells. After return to choline-supplemented medium the suppression of choline auxotrophy is gradually lost, indicating that it is not caused by a mutation. Whole genome sequencing confirmed the lack of common SNPs between suppressors, but instead revealed that the suppressors acquired aneuploidy. Genome-wide transcript profiling is underway to obtain additional clues as to the molecular mechanism of the adaptation that renders PC biosynthesis redundant. Based on the results obtained so far, the hypothesis is put forward that the aneuploidy confers increased synthesis of fatty acids, which is required to shorten the average acyl chain length and thus sustain yeast growth in the absence of PC.