Recombinant and endogenous ways to produce methylated phospholipids in Escherichia coli

Abstract

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

Analysis of Bacterial Phosphorylcholine-Related Genes Reveals an Association between Type-Specific Biosynthesis Pathways and Biomolecules Targeted for Phosphorylcholine Modification

“…The acylation-dependent PC biosynthesis pathway has been reported only for the Gram-negative bacterium E. coli ( 26 ), the plant pathogen X. campestris ( 27 ), and the Gram-positive pathogens S. pneumoniae , S. mitis , and S. oralis ( 66 ). X. campestris converts exogenous glycerophosphocholine (GPC) to lysophosphatidylcholine (lyso-PC) via two acylation reactions on GPC ( Fig.…”

“…Similarly, the recently described acylation-dependent PC biosynthesis pathway uses exogenous choline in the form of glycerophosphocholine or lysophosphatidylcholine. This pathway has been described for only three bacterial species ( 26 , 27 ), but it is well-characterized in yeast. For a more complete description of the Pcs, PmtA, and acylation-dependent PC biosynthesis pathways, see the recent review by Zhang et al ( 28 ).…”

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris

“…BLAST analyses with the two LPAAT from E. coli (PlsC and YihG) as queries suggested five PlsC candidates in X. campestris (Xc_4099, Xc_0188, Xc_0238, Xc_0517, and Xc_0505). Xc_0188 and Xc_0238 were previously shown to be involved in lyso-PC (LPC)-dependent PC biosynthesis in a yeastlike acylation pathway [18,24].…”

Associations between cord serum antibodies against phosphorylcholine and bacterial infections in neonates: a prospective cohort study in singletons and twins

“…For microorganisms, PC could be synthesized using choline from the host and serve as a resource of nutrients and an osmoprotectant [5, 6]. Moreover, PC allows bacteria to evade host immune responses and subsequently colonize in the upper respiratory tract [7, 8]. Antibodies against PC (anti-PC) were initially recognized for their anti-infective properties [9] and were later reported to be a protection marker of cardiovascular disease [10], autoimmune diseases [11], as well as fetal growth and premature birth [12].…”