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Julia Kleetz, Georgios Vasilopoulos, Simon Czolkoss, Meriyem Aktas, Franz Narberhaus

• \(\textit {Escherichia coli}\) is the daily workhorse in molecular biology research labs and an important platform microorganism in white biotechnology. Its cytoplasmic membrane is primarily composed of the phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). As in most other bacteria, the typical eukaryotic phosphatidylcholine (PC) is not a regular component of the \(\textit {E. coli}\) membrane. PC is known to act as a substrate in various metabolic or catabolic reactions, to affect protein folding and membrane insertion, and to activate proteins that originate from eukaryotic environments. Options to manipulate the \(\textit {E. coli}\) membrane to include non-native lipids such as PC might make it an even more powerful and versatile tool for biotechnology and protein biochemistry. This article outlines different strategies how \(\textit {E. coli}\) can be engineered to produce PC and other methylated PE derivatives. Several of these approaches rely on the ectopic expression of genes from natural PC-producing organisms. These include PC synthases, lysolipid acyltransferases, and several phospholipid \(\it N\)-methyltransferases with diverse substrate and product preferences. In addition, we show that \(\textit {E. coli}\) has the capacity to produce PC by its own enzyme repertoire provided that appropriate precursors are supplied. Screening of the \(\textit {E. coli}\) Keio knockout collection revealed the lysophospholipid transporter LplT to be responsible for the uptake of lyso-PC, which is then further acylated to PC by the acyltransferase-acyl carrier protein synthetase Aas. Overall, our study shows that the membrane composition of the most routinely used model bacterium can readily be tailored on demand. \(\textbf {Key points}\) • Escherichia coli can be engineered to produce non-native methylated PE derivatives. • These lipids can be produced by foreign and endogenous proteins. • Modification of E. coli membrane offers potential for biotechnology and research.