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Summary: Lipid membranes are the border between living cells and their environments. The membrane’s lipid composition defines fluidity, thickness, and protein activity and is controlled by the intricate actions of lipid gene-encoded enzymes. However, a comprehensive analysis of each protein’s contribution to the lipidome is lacking. Here, we present such a comprehensive and functional overview of lipid genes in Escherichia coli by individual overexpression or deletion of these genes. We developed a high-throughput lipidomic platform, combining growth analysis, one-step lipid extraction, rapid LC-MS, and bioinformatic analysis into one streamlined procedure. This allowed the processing of more than 300 samples per day and revealed interesting functions of known enzymes and distinct effects of individual proteins on the phospholipidome. Our data demonstrate the plasticity of the phospholipidome and unexpected relations between lipid classes and cell growth. Modeling of lipidomic responses to short-chain alcohols provides a rationale for targeted membrane engineering. : Jeucken et al. analyzed lipidomes of E. coli strains with knockout or overexpression of known lipid-related genes. They demonstrate relationships between lipid species and classes and investigate their link to cell growth. The high-throughput lipidomic method is then used to model lipidomic changes to exogenous alcohols. Keywords: lipidomics, systems biology, Escherichia coli, lipids, phospholipids, lipid genes, lipid network, high-throughput, LC-MS, bioinformatics