Unsaturation effects on lipid transmembrane asymmetry.

Within cell plasma membranes, unsaturated lipids are asymmetrically distributed over the inner and outer leaflets, offering an attractive local structural feature. However, the mechanism to keep lipid transmembrane asymmetry and the closely related transmembrane movement (flip-flop) for unsaturated lipids remain poorly understood. Here, we applied sum frequency generation vibrational spectroscopy to investigate this lipid transmembrane asymmetry upon mimicking the cell membrane homeostatic processes. On the one hand, unsaturated lipids were found to hinder the flip-flop process and preserve lipid transmembrane asymmetry in model cell membranes, owing to the steric hindrance caused by their bent tails. On the other hand, local unsaturated lipids in the mixed unsaturated/saturated lipid bilayer were conducive to the formation of the local asymmetry. Therefore, lipid unsaturation can be recognized as an intrinsic key factor to form and maintain lipid transmembrane asymmetry in cell membranes.

Structural investigation of interactions between halogenated flavonoids and the lipid membrane along with their role as cytotoxic agents.

This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1-D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and FT-Raman spectroscopy, the research aims to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties suggest these halogenated flavonoids potential candidates for exploring their potential for medical use.

Maintenance of bacterial outer membrane lipid asymmetry: insight into MlaA.

The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier to protect against toxic compounds. By nature, the OM is asymmetric with the highly packed lipopolysaccharide (LPS) at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla system, in which is responsible for the retrograde transport of glycerophospholipids from the OM to the inner membrane. This system is comprised of six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids that are mis-localized at the outer leaflet of the OM. Interestingly, MlaA was initially identified - and called VacJ - based on its role in the intracellular spreading of Shigella flexneri.Many open questions remain with respect to the Mla system and the mechanism involved in the translocation of mislocated glycerophospholipids at the outer leaflet of the OM, by MlaA. After summarizing the current knowledge on MlaA, we focus on the impact of mlaA deletion on OM lipid composition and biophysical properties of the OM. How changes in OM lipid composition and biophysical properties can impact the generation of membrane vesicles and membrane permeability is discussed. Finally, we explore whether and how MlaA might be a candidate for improving the activity of antibiotics and as a vaccine candidate.Efforts dedicated to understanding the relationship between the OM lipid composition and the mechanical strength of the bacterial envelope and, in turn, how such properties act against external stress, are needed for the design of new targets or drugs for Gram-negative infections.

Sum-frequency vibrational spectroscopy, a tutorial: Applications for the study of lipid membrane structure and dynamics.

Planar supported lipid bilayers (PSLBs) are an ideal model for the study of lipid membrane structures and dynamics when using sum-frequency vibrational spectroscopy (SFVS). In this paper, we describe the construction of asymmetric PSLBs and the basic SFVS theory needed to understand and make measurements on these membranes. Several examples are presented, including the determination of phospholipid orientation and measuring phospholipid transmembrane translocation (flip-flop).

Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis.

Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.

Recent Advances in the Inhibition of Membrane Lipid Peroxidation by Food-Borne Plant Polyphenols via the Nrf2/GPx4 Pathway.

Lipid peroxidation (LP) leads to changes in the fluidity and permeability of cell membranes, affecting normal cellular function and potentially triggering apoptosis or necrosis. This process is closely correlated with the onset of many diseases. Evidence suggests that the phenolic hydroxyl groups in food-borne plant polyphenols (FPPs) make them effective antioxidants capable of preventing diseases triggered by cell membrane LP. Proper dietary intake of FPPs can attenuate cellular oxidative stress, especially damage to cell membrane phospholipids, by activating the Nrf2/GPx4 pathway. Nuclear factor E2-related factor 2 (Nrf2) is an oxidative stress antagonist. The signaling pathway regulated by Nrf2 is a defense transduction pathway of the organism against external stimuli such as reactive oxygen species and exogenous chemicals. Glutathione peroxidase 4 (GPx4), under the regulation of Nrf2, is the only enzyme that reduces cell membrane lipid peroxides with specificity, thus playing a pivotal role in regulating cellular ferroptosis and counteracting oxidative stress. This study explored the Nrf2/GPx4 pathway mechanism, antioxidant activity of FPPs, and mechanism of LP. It also highlighted the bioprotective properties of FPPs against LP and its associated mechanisms, including (i) activation of the Nrf2/GPx4 pathway, with GPx4 potentially serving as a central target protein, (ii) regulation of antioxidant enzyme activities, leading to a reduction in the production of ROS and other peroxides, and (iii) antioxidant effects on LP and downstream phospholipid structure. In conclusion, FPPs play a crucial role as natural antioxidants in preventing LP. However, further in-depth analysis of FPPs coregulation of multiple signaling pathways is required, and the combined effects of these mechanisms need further evaluation in experimental models. Human trials could provide valuable insights into new directions for research and application.

Deciphering the distinct biocontrol activities of lipopeptides fengycin and surfactin through their differential impact on lipid membranes.

Lipopeptides produced by beneficial bacilli present promising alternatives to chemical pesticides for plant biocontrol purposes. Our research explores the distinct plant biocontrol activities of lipopeptides surfactin (SRF) and fengycin (FGC) by examining their interactions with lipid membranes. Our study shows that FGC exhibits a direct antagonistic activity against Botrytis cinerea and no marked immune-eliciting activity in Arabidopsis thaliana while SRF only demonstrates an ability to stimulate plant immunity. It also reveals that SRF and FGC exhibit diverse effects on membrane integrity and lipid packing. SRF primarily influences membrane physical state without significant membrane permeabilization, while FGC permeabilizes membranes without significantly affecting lipid packing. From our results, we can suggest that the direct antagonistic activity of lipopeptides is linked to their capacity to permeabilize lipid membrane while the stimulation of plant immunity is more likely the result of their ability to alter the mechanical properties of the membrane. Our work also explores how membrane lipid composition modulates the activities of SRF and FGC. Sterols negatively impact both lipopeptides' activities while sphingolipids mitigate the effects on membrane lipid packing but enhance membrane leakage. In conclusion, our findings emphasize the importance of considering both membrane lipid packing and leakage mechanisms in predicting the biological effects of lipopeptides. It also sheds light on the intricate interplay between the membrane composition and the effectiveness of the lipopeptides, providing insights for targeted biocontrol agent design.

Exploring the connections between ER-based lipid metabolism and plasma membrane nanodomain signaling.

Recent advancements in our understanding of cell membrane dynamics have shed light on the importance of plasma membrane (PM) nanodomains in plant cell signaling. Nevertheless, many aspects of membrane nanodomains, including their regulatory mechanisms and biological functions, remain enigmatic. To address this knowledge gap, our review article proposes a novel perspective wherein signaling pathways target endoplasmic reticulum (ER)-based lipid metabolism to exert control over the formation and function of membrane nanodomains. Subsequently, these nanodomains reciprocate by influencing the localization and activity of signaling molecules at the PM. We place a specific emphasis on ER-based enzymatic reactions, given the ER's central role in membrane lipid biosynthesis and its capacity to directly impact PM lipid composition, particularly with regard to saturation levels - an essential determinant of nanodomain properties. The interplay among cell signaling, glycerolipid metabolism, and PM nanodomain may create feedforward/feedback loops that fine-tune cellular responses to developmental and environmental cues.

The GPAT4/6/8 clade functions in Arabidopsis root suberization nonredundantly with the GPAT5/7 clade required for suberin lamellae.

Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant-specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4/6/8 clade in Arabidopsis thaliana, which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5/7 clade is mainly required for abscisic acid-regulated suberization. In addition, the GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. Site-directed mutagenesis revealed that the intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, contributes to suberin formation. GPAT5/7 lack an active phosphatase domain and the amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8. Thus, GPATs that lack an active phosphatase domain synthetize lysophosphatidic acids that might play a role in the formation of the lamellated structure of suberin. GPATs with active and nonactive phosphatase domains appear to have nonredundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin.

Pixelated High-Q Metasurfaces for in Situ Biospectroscopy and Artificial Intelligence-Enabled Classification of Lipid Membrane Photoswitching Dynamics.

Nanophotonic devices excel at confining light into intense hot spots of electromagnetic near fields, creating exceptional opportunities for light-matter coupling and surface-enhanced sensing. Recently, all-dielectric metasurfaces with ultrasharp resonances enabled by photonic bound states in the continuum (BICs) have unlocked additional functionalities for surface-enhanced biospectroscopy by precisely targeting and reading out the molecular absorption signatures of diverse molecular systems. However, BIC-driven molecular spectroscopy has so far focused on end point measurements in dry conditions, neglecting the crucial interaction dynamics of biological systems. Here, we combine the advantages of pixelated all-dielectric metasurfaces with deep learning-enabled feature extraction and prediction to realize an integrated optofluidic platform for time-resolved in situ biospectroscopy. Our approach harnesses high-Q metasurfaces specifically designed for operation in a lossy aqueous environment together with advanced spectral sampling techniques to temporally resolve the dynamic behavior of photoswitchable lipid membranes. Enabled by a software convolutional neural network, we further demonstrate the real-time classification of the characteristic cis and trans membrane conformations with 98% accuracy. Our synergistic sensing platform incorporating metasurfaces, optofluidics, and deep learning reveals exciting possibilities for studying multimolecular biological systems, ranging from the behavior of transmembrane proteins to the dynamic processes associated with cellular communication.

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis.

BACKGROUND: Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. METHODS: Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori-AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. RESULTS: CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC-MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1 h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16 h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. CONCLUSIONS: The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy.

Influence of pH and lipid membrane on the liquid-liquid phase separation of wheat γ-gliadin in aqueous conditions.

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid-liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

Evaluation of Lipids for the Study of Photosynthetic Membranes.

The biological role of lipids goes far beyond the formation of a structural membrane bilayer platform for membrane proteins and controlling fluxes across the membranes. For example, in photosynthetic thylakoid membranes, lipids occupy well-defined binding niches within protein complexes and determine the structural organization of membrane proteins and their function by controlling generic physicochemical membrane properties. In this chapter, two-dimensional thin-layer chromatography (2D TLC) and gas chromatography (GC) techniques are presented for quantitative analysis of lipid classes and fatty acids in thylakoid membranes. In addition, lipid extraction methods from isolated thylakoid membranes and leaves are described together with a procedure for the derivatization of fatty acids to fatty acid methyl esters (FAME) that is required for GC analysis.

Interaction of L-phenylalanine with carbonyl groups in mixed lipid membranes.

The interaction of L-Phe with the membrane components, i.e., lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl-sn-glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di-O-hexadecyl-sn-glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC.

Bioconcentration potential of ionic liquids: New data on membrane partitioning and its comparison with predictions obtained by COSMOmic.

Ionic liquids (ILs) have recently gained significant attention in both the scientific community and industry, but there is a limited understanding of the potential risks they might pose to the environment and human health, including their potential to accumulate in organisms. While membrane and storage lipids have been considered as primary sorption phases driving bioaccumulation, in this study we used an in vitro tool known as solid-supported lipid membranes (SSLMs) to investigate the affinity of ILs to membrane lipid - phosphatidylcholine and compare the results with an existing in silico model. Our findings indicate that ILs may have a strong affinity for the lipids that form cell membranes, with the key factor being the length of the cation's side chain. For quaternary ammonium cations, increase in membrane affinity (logMA) was observed from 3.45 ± 0.06 at 10 carbon atoms in chain to 4.79 ± 0.06 at 14 carbon atoms. We also found that the anion can significantly affect the membrane partitioning of the cation, even though the anions themselves tend to have weaker interactions with phospholipids than the cations of ILs. For 1-methyl-3-octylimidazolium cation the presence of tricyanomethanide anion caused increase in logMA to 4.23 ± 0.06. Although some of our data proved to be consistent with predictions made by the COSMOmic model, there are also significant discrepancies. These results suggest that further research is needed to improve our understanding of the mechanisms and structure-activity relationships involved in ILs bioconcentration and to develop more accurate predictive models.

Nanoparticle induced fusion of lipid membranes.

Membrane fusion is crucial for infection of enveloped viruses, cellular transport, and drug delivery via liposomes. Nanoparticles can serve as fusogenic agents facilitating such membrane fusion for direct transmembrane transport. However, the underlying mechanisms of nanoparticle-induced fusion and the ideal properties of such nanoparticles remain largely unknown. Here, we used molecular dynamics simulations to investigate the efficacy of spheroidal nanoparticles with different size, prolateness, and ligand interaction strengths to enhance fusion between vesicles. By systematically varying nanoparticle properties, we identified how each parameter affects the fusion process and determined the optimal parameter range that promotes fusion. These findings provide valuable insights for the design and optimization of fusogenic nanoparticles with potential biotechnological and biomedical applications.

AtMYB41 acts as a dual-function transcription factor that regulates the formation of lipids in an organ- and development-dependent manner.

The plant cuticle controls non-stomatal water loss and can serve as a barrier against biotic agents, whereas the heteropolymer suberin and its associated waxes are deposited constitutively at specific cell wall locations. While several transcription factors controlling cuticle formation have been identified, those involved in the transcriptional regulation of suberin biosynthesis remain poorly characterized. The major goal of this study was to further analyse the function of the R2R3-Myeloblastosis (MYB) transcription factor AtMYB41 in formation of the cuticle, suberin, and suberin-associated waxes throughout plant development. For functional analysis, the organ-specific expression pattern of AtMYB41 was analysed and Atmyb41ge alleles were generated using the CRISPR/Cas9 system. These were investigated for root growth and water permeability upon stress. In addition, the fatty acid, wax, cutin, and suberin monomer composition of different organs was evaluated by gas chromatography. The characterization of Atmyb41ge mutants revealed that AtMYB41 negatively regulates the production of cuticular lipids and fatty acid biosynthesis in leaves and seeds, respectively. Remarkably, biochemical analyses indicate that AtMYB41 also positively regulates the formation of cuticular waxes in stems of Arabidopsis thaliana. Overall, these results suggest that the AtMYB41 acts as a negative regulator of cuticle and fatty acid biosynthesis in leaves and seeds, respectively, but also as a positive regulator of wax production in A. thaliana stems.

Acidic electrolyzed-oxidizing water treatment mitigated the disease progression in Phomopsis longanae Chi-infected longans by modulating ROS and membrane lipid metabolism.

Postharvest harmful pathogenic infestation leads to rapid decay in longan fruit. Compared with P. longanae-infected longans, AEOW alleviated fruit disease severity and diminished the O2-. production rate and MDA content. It also increased APX, CAT, and SOD activities, delayed the decrease in the levels of GSH and AsA, as well as the reducing power and DPPH radical scavenging ability, which resulted in a decline in membrane lipid peroxidation in P. longanae-infected longans. Additionally, AEOW reduced LOX, lipase, PI-PLC, PC-PLC, and PLD activities, maintained higher levels of PC, PI, IUFA, USFAs, and U/S, while reducing levels of PA, DAG, SFAs, and CMP. These effects alleviated membrane lipid degradation and peroxidation in P. longanae-infected longans. Consequently, AEOW effectively maintained membrane integrity via improving antioxidant capacity and suppressing membrane lipid peroxidation. This comprehensive coordination of ROS and membrane lipid metabolisms improved fruit resistance and delayed disease development in longans.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.