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One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli. By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20–30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell’s phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overex- pression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting dif- ferent codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool.

The cytoplasmic membrane of a prokaryotic cell consists of a lipid bilayer or a monolayer that shields the cellular content from the environment. In addition, the membrane contains proteins that are responsible for transport of proteins and metabolites as well as for signalling and energy transduction. Maintenance of the functionality of the membrane during changing environmental conditions relies on the cell’s potential to rapidly adjust the lipid composition of its membrane. Despite the fundamental chemical differences between bacterial ester lipids and archaeal ether lipids, both types are functional under a wide range of environmental conditions. We here provide an overview of archaeal and bacterial strategies of changing the lipid compositions of their membranes. Some molecular adjustments are unique for archaea or bacteria, whereas others are shared between the two domains. Strikingly, shared adjustments were predominantly observed near the growth boundaries of bacteria. Here, we demonstrate that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria. Electronic supplementary material The online version of this article (doi:10.1007/s00792-017-0939-x) contains supplementary material, which is available to authorized users.

Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102107 pfu.mL1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and under the scope of the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). Franklin L. Nobrega and Ana Rita Costa acknowledge FCT for grants SFRH/BD/86462/2012 and SFRH/BPD/94648/2013, respectively. Melvin F. Siliakus acknowledges funding from the Biobased Ecologically Balanced Sustainable Industrial Chemistry (BE-BASIC) foundation. Electron microscopy work was performed at the Wageningen Electron Microscopy Centre (WEMC) of Wageningen University.

In archaea, the membrane phospholipids consist of isoprenoid hydrocarbon chains that are ether-linked to a sn -glycerol-1-phosphate backbone. This unique structure is believed to be vital for the adaptation of these microorganisms to extreme environments, but it also reflects an evolutionary marker that distinguishes archaea from bacteria and eukaryotes. CDP-archaeol is the central precursor for polar head group attachment. We examined various bacterial enzymes involved in the attachment of L-serine and glycerol as polar head groups for their promiscuity in recognizing CDP-archaeol as a substrate. Using a combination of mutated bacterial and archaeal enzymes, archaetidylethanolamine and archaetidylglycerol could be produced in vitro using nine purified enzymes while starting from simple building blocks. The ether lipid pathway constituted by a set of archaeal and bacterial enzymes was introduced into E. coli , which resulted in the biosynthesis of archaetidylethanolamine and archaetidylglycerol. This is a further step in the reprogramming of E. coli for ether lipid biosynthesis.

The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). While formation and utilization of acyl-CoA takes place at the membrane, Acyl-CoA Binding Domain containing proteins (ACBD) in RBC cytosol bind acyl-CoA, limiting product feedback inhibition on ACSL and distribute Acyl-CoA to the various acyl-utilizing enzymes while protecting the cells against its potent detergent character. We have identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC, a protein with several isoforms that acts as a dimer. To relate its structure to activity, we report the expression of different modified forms in E. coli. Our data indicate that, despite the observed activity, enzyme studies of these mammalian membrane proteins in the host E. coli are strongly hampered by their aggregation into inclusion bodies. While activity can be measured, data on enzyme kinetics and specificity are questionable. Oleoyl-CoA formation from oleic acid, CoA and ATP reveled that the two transmembrane spanning segments predicted at the amino-terminus of the protein are not essential for its activity. Moreover, they are not essential for dimer formation and strong association with membranes. ACSL6 appears to be an integral membrane protein. One of the five spliced isoforms of ACSL6 reported, lacks the so-called fatty acid Gate-domain, and appears to be unable to activate long chain fatty acids. We hypothesize that this form modulates activity of the other active isoforms of ACSL6 through hetero-dimer formation. An EST clone of erythroid precursor cells identified ACBD6 as a potential AcylCoA binding protein in RBC. This modular protein contains an Acyl-CoA binding domain at the amino-terminus and two Ankyrin-repeat motifs (ANK) at the C-terminus. Both the full-length protein and the N-terminus domain were soluble when expressed and purified in E. coli. Expression of the C-terminus domain by itself rendered an insoluble protein. We report that ACBD6 binds long-chain acyl-CoA with a preference for C18:1-CoA over C20:4 and C16:0-CoA and does not bind fatty acid. Truncation of the ANK domain had no effect on the binding activity of the N-terminus domain. Together our findings implicate ACBD6 as part of the Acyl-CoA turnover mechanism in RBC, its actual role on the kinetics of ACSL and/or LPLAT activity needs to be established. The description of these proteins involved in Acyl-CoA turnover in RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.