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 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 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 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 N-methyltransferases with diverse substrate and product preferences. In addition, we show that E. coli has the capacity to produce PC by its own enzyme repertoire provided that appropriate precursors are supplied. Screening of the 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.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. Graphical abstract
One of the most common pathways for the biosynthesis of the phospholipid phosphatidylcholine (PC) in bacteria is the successive three-fold N -methylation of phosphatidylethanolamine (PE) catalyzed by phospholipid N -methyltransferases (Pmts). Pmts with different activities have been described in a number of mesophilic bacteria. In the present study, we identified and characterized the substrate and product spectrum of four Pmts from thermophilic bacteria. Three of these enzymes were purified in an active form. The Pmts from Melghirimyces thermohalophilus , Thermochromogena staphylospora and Thermobifida fusca produce monomethyl-PE (MMPE) and dimethyl-PE (DMPE). T. fusca encodes two Pmt candidates, one is mutationally inactivated and the other is responsible for the accumulation of large amounts of MMPE. The Pmt enzyme from Rubellimicrobium thermophilum catalyzes all three methylation reactions to synthesize PC. Moreover, we show that PE, previously reported to be absent in R. thermophilum , is in fact produced and serves as precursor for the methylation pathway. In an alternative route, the strain is able to produce PC by the PC synthase pathway when choline is available. The activity of all purified thermophilic Pmt enzymes was stimulated by anionic lipids suggesting membrane recruitment of these cytoplasmic proteins via electrostatic interactions. Our study provides novel insights into the functional characteristics of phospholipid N -methyltransferases in a previously unexplored set of thermophilic environmental bacteria. Importance In recent years, the presence of phosphatidylcholine (PC) in bacterial membranes has gained increasing attention, partly due to its critical role in the interaction with eukaryotic hosts. PC biosynthesis via a three-step methylation of phosphatidylethanolamine, catalyzed by phospholipid N -methyltransferases (Pmts), has been described in a range of mesophilic bacteria. Here, we expand our knowledge on bacterial PC formation by the identification, purification and characterization of Pmts from phylogenetically diverse thermophilic bacteria, and thereby provide insights into the functional characteristics of Pmt enzymes in thermophilic actinomycetes and proteobacteria.
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