Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories

Adaptive laboratory evolution is often used to improve the performance of microbial cell factories. Reverse engineering of evolved strains enables learning and subsequent incorporation of novel design strategies via the design-build-test-learn cycle. Here, we reverse engineer a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties. Here, evolution was determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. All three mutations were required in order to reproduce the increased growth rate in the presence of 20 mM C8 and increased cell surface hydrophobicity; the WaaG and RpoC mutations both contributed to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributed to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The increase in average lipid tail length can be attributed to the RpoC^H419P mutation, which also confers tolerance to other industrially-relevant inhibitors, such as furfural, vanillin and n-butanol. The RpoC^H419P mutation may impact binding or function of the stringent response alarmone ppGpp to RpoC site 1. Each of these mutations provides novel strategies for engineering microbial robustness, particularly at the level of the microbial cell membrane.     

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.     

Shedding off specific lipid constituents from sperm cell membrane during cryopreservation

Membrane damage is one of the main reasons for reduced motility and fertility of sperm cells during cryopreservation. Using a model system of sperm cryopreservation developed in our laboratory, we have investigated the detailed changes due to cryopreservation in the plasma membrane lipid composition of the goat epididymal sperm cells. Total lipid and its components, i.e., neutral lipids, glycolipids and phospholipids decreased significantly after cryopreservation. Among neutral lipids sterols, steryl esters and 1-O-alkyl-2,3-diacyl glycerols decreased appreciably, while among phospholipids, major loss was observed for phosphatidyl choline and phosphatidyl ethanolamine. Unsaturated fatty acids bound to the phospholipids diminished while the percentage of saturated acids increased. The cholesterol:phospholipid ratio enhanced and the amount of hydrocarbon, which was unusually high, increased further on cryopreservation. The data indicates that profound increase of the hydrophobicity of the cell membrane is one of the major mechanisms by which spermatozoa acquire potential to resist or combat stress factors like cryodamage. The results are compatible with the view that for survival against cryodamage, sperm cells modulate the structure of their outer membrane by shedding off preferentially some hydrophilic lipid constituents of the cell membrane.     

Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.     

Effect of hydrophobicity of yeast cell envelopes on the rate of methyl oleate autooxidation

The relationships between the antioxidant and antiperoxide properties and the lipid composition of yeast cell envelopes before and after interaction with a liquid culture medium was studied. Correlations between the hydrophobicity of cell envelopes, their lipid composition, and parameters of kinetic oxidation curves of model methyl oleate in the presence of lipids were found. Irrespective of the total lipid content in yeast cell envelopes, the preparations with a high antiperoxide activity of lipids exhibited a higher hydrophobicity and adsorbed lipophilic prooxidants from the medium more readily, whereas the preparations with a low antiperoxide activity were less hydrophobic and adsorbed predominantly lipophilic inhibitors. It was found that analysis of kinetic oxidation curves in toto provides the most comprehensive information on the physicochemical properties of lipids adsorbed from medium.     

In silico design of robust bolalipid membranes

The robustness of microorganisms used in industrial fermentations is essential for the efficiency and yield of the production process. A viable tool to increase the robustness is through engineering of the cell membrane and especially by incorporating lipids from species that survive under harsh conditions. Bolalipids are tetraether lipids found in Archaea bacteria, conferring stability to these bacteria by spanning across the cytoplasmic membrane. Here we report on in silico experiments to characterize and design optimal bolalipid membranes in terms of robustness. We use coarse-grained molecular dynamics simulations to study the structure, dynamics, and stability of membranes composed of model bolalipids, consisting of two dipalmitoylphosphatidylcholine (DPPC) lipids covalently linked together at either one or both tail ends. We find that bolalipid membranes differ substantially from a normal lipid membrane, with an increase in thickness and tail order, an increase in the gel-to-liquid crystalline phase transition temperature, and a decrease in diffusivity of the lipids. By changing the flexibility of the linker between the lipid tails, we furthermore show how the membrane properties can be controlled. A stiffer linker increases the ratio between spanning and looping conformations, rendering the membrane more rigid. Our study may help in designing artificial membranes, with tunable properties, able to function under extreme conditions. As an example, we show that incorporation of bolalipids makes the membrane more tolerant toward butanol.     

Interaction of Hsp90AA1 with phospholipids stabilizes membranes under stress conditions

During heat shock conditions, structural changes in cellular membranes may lead to cell death. Hsp90AA1 and other heat shock proteins involved in membranes are responsible for protecting membrane stabilization. However, the membrane binding mechanism of Hsp90AA1 remains largely uncharacterized. In this study, we showed Hsp90AA1 interacts with phospholipid membrane with high affinity. Using the depth-dependent fluorescence-quenching with brominated lipids, we found Hsp90AA1 penetrated 10.7?Å into the hydrocarbon core of the lipid bilayer. Circular dichroism spectra studies showed Hsp90AA1 lost part of its α-helical structures upon interaction with phospholipid membrane. By assessing binding properties of the three Hsp90AA1 domains, we found Hsp90AA1 interacted into the lipid bilayer mainly toward its C-terminus domain (CTD). Using scanning electron microscopy, we examined the protection on host cell membrane by overexpressing Hsp90AA1. The results indicated Hsp90AA1 or Hsp90AA1-CTD expressing E. coli cells exhibited better membrane integrity compared to the control after thermal treatment. The following liposome leakage assay suggested the protection of Hsp90AA1 might due to its stabilization of the membrane lipid. Collectively, the present study demonstrates Hsp90AA1 embeds into the lipid bilayer through its C-terminal domain and the Hsp90AA1-lipid association potentially has a significant function in keeping membranes stabilization during stress conditions.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping

Membrane lipids diffuse rapidly in the plane of the membrane but their ability to flip spontaneously across a membrane bilayer is hampered by a significant energy barrier. Thus spontaneous flip-flop of polar lipids across membranes is very slow, even though it must occur rapidly to support diverse aspects of cellular life. Here we discuss the mechanisms by which rapid flip-flop occurs, and what role lipid flipping plays in membrane homeostasis and cell growth. We focus on conceptual aspects, highlighting mechanistic insights from biochemical and in silico experiments, and the recent, ground-breaking identification of a number of lipid scramblases.     

Effect of hydrophobicity of yeast cell envelope on the rate of autooxidation of methyloleate

The relationships between the antioxidant and antiperoxide properties and the lipid composition of yeast cell envelope prior to, and after the reaction with a liquid culture medium were studied. Correlations between the hydrophobicity of envelopes, their lipid composition and the parameters of the kinetic curves of the oxidation of model methyloleate in the presence of lipids were established. It was found that, irrespective of the general content of lipids in yeast cell envelope, preparations with high antiperoxide activity of lipids had a high hydrophobicity and sorbed lipophilic prooxidants from medium, whereas preparations with low antiperoxide activity were less hydrophobic and adsorbed predominantly lipophilic inhibitors. It was found that the most comprehensive information on the physicochemical properties of lipids adsorbed from medium is provided by an analysis of kinetic curves of oxidation in toto.     

α-Synuclein Senses Lipid Packing Defects and Induces Lateral Expansion of Lipids Leading to Membrane Remodeling

There is increasing evidence for the involvement of lipid membranes in both the functional and pathological properties of α-synuclein (α-Syn). Despite many investigations to characterize the binding of α-Syn to membranes, there is still a lack of understanding of the binding mode linking the properties of lipid membranes to α-Syn insertion into these dynamic structures. Using a combination of an optical biosensing technique and in situ atomic force microscopy, we show that the binding strength of α-Syn is related to the specificity of the lipid environment (the lipid chemistry and steric properties within a bilayer structure) and to the ability of the membranes to accommodate and remodel upon the interaction of α-Syn with lipid membranes. We show that this interaction results in the insertion of α-Syn into the region of the headgroups, inducing a lateral expansion of lipid molecules that can progress to further bilayer remodeling, such as membrane thinning and expansion of lipids out of the membrane plane. We provide new insights into the affinity of α-Syn for lipid packing defects found in vesicles of high curvature and in planar membranes with cone-shaped lipids and suggest a comprehensive model of the interaction between α-Syn and lipid bilayers. The ability of α-Syn to sense lipid packing defects and to remodel membrane structure supports its proposed role in vesicle trafficking.     

Influence of Membrane Lipid Composition on a Transmembrane Bacterial Chemoreceptor

Most bacterial chemoreceptors are transmembrane proteins. Although less than 10% of a transmembrane chemoreceptor is embedded in lipid, separation from the natural membrane environment by detergent solubilization eliminates most receptor activities, presumably because receptor structure is perturbed. Reincorporation into a lipid bilayer can restore these activities and thus functionally native structure. However, the extent to which specific lipid features are important for effective restoration is unknown. Thus we investigated effects of membrane lipid composition on chemoreceptor Tar from Escherichia coli using Nanodiscs, small (∼10-nm) plugs of lipid bilayer rendered water-soluble by an annulus of “membrane scaffold protein.” Disc-enclosed bilayers can be made with different lipids or lipid combinations. Nanodiscs carrying an inserted receptor dimer have high protein-to-lipid ratios approximating native membranes and in this way mimic the natural chemoreceptor environment. To identify features important for functionally native receptor structure, we made Nanodiscs using natural and synthetic lipids, assaying extents and rates of adaptational modification. The proportion of functionally native Tar was highest in bilayers closest in composition to E. coli cytoplasmic membrane. Some other lipid compositions resulted in a significant proportion of functionally native receptor, but simply surrounding the chemoreceptor transmembrane segment with a lipid bilayer was not sufficient. Membranes effective in supporting functionally native Tar contained as the majority lipid phosphatidylethanolamine or a related zwitterionic lipid plus a rather specific proportion of anionic lipids, as well as unsaturated fatty acids. Thus the chemoreceptor is strongly influenced by its lipid environment and is tuned to its natural one.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.     

The damaging effects of short chain fatty acids on Escherichia coli membranes

Carboxylic acids are an attractive biorenewable chemical. However, like many other fermentatively produced compounds, they are inhibitory to the biocatalyst. An understanding of the mechanism of toxicity can aid in mitigating this problem. Here, we show that hexanoic and octanoic acids are completely inhibitory to Escherichia coli MG1655 in minimal medium at a concentration of 40 mM, while decanoic acid was inhibitory at 20 mM. This growth inhibition is pH-dependent and is accompanied by a significant change in the fluorescence polarization (fluidity) and integrity. This inhibition and sensitivity to membrane fluidization, but not to damage of membrane integrity, can be at least partially mitigated during short-term adaptation to octanoic acid. This short-term adaptation was accompanied by a change in membrane lipid composition and a decrease in cell surface hydrophobicity. Specifically, the saturated/unsaturated lipid ratio decreased and the average lipid length increased. A fatty acid-producing strain exhibited an increase in membrane leakage as the product titer increased, but no change in membrane fluidity. These results highlight the importance of the cell membrane as a target for future metabolic engineering efforts for enabling resistance and tolerance of desirable biorenewable compounds, such as carboxylic acids. Knowledge of these effects can help in the engineering of robust biocatalysts for biorenewable chemicals production.     

Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The membrane composition and lipid physical properties have been systematically investigated as a function of fatty acid composition for a series of Acholeplasma laidlawii B membrane preparations made homogeneous in various fatty acids by growing cells on single fatty acids and avidin, a potent fatty acid synthetic inhibitor. The membrane protein molecular weight distribution is essentially constant as a function of fatty acid composition, but the lipid/protein ratio varies over a 2-fold range when different fatty acid growth supplements are used. The membrane lipid head-group composition varies somewhat under these conditions, particularly in the ratio of the two major neutral glycolipids. Differential thermal analytical investigations of the thermotropic phase transitions of various combinations of membrane components suggest that these compositional changes are unlikely to result in qualitative changes in the nature of lipid-protein or lipid-lipid interactions, although lesser changes of a quantitative nature probably do occur. The total lipids of membranes made homogeneous in their lipid fatty acyl chain composition exhibit sharper than normal gel-to-liquid-crystalline phase transitions of which midpoint temperatures correlate very well with the phase transition temperatures of synthetic hydrated phosphatidylcholines with like acyl chains. Our results indicate that using avidin and suitable fatty acids to grow A. laidlawii B, it is possible to manipulate the position and the sharpness of the membrane lipid phase transition widely and independently without causing major modifications in other aspects of the membrane composition. This fact makes the fatty acid-homogeneous A. laidlawii B membrane a very useful biological membrane preparation in which to study lipid physical properties and their functional consequences.     

The yeast plasma membrane P4-ATPases are major transporters for lysophospholipids

The transbilayer movement of phospholipids plays an essential role in establishing and maintaining the asymmetric distribution of lipids in biological membranes. The P4-ATPase family has been implicated as the major transporters of the aminoglycerophospholipids in both surface and endomembrane systems. Historically, fluorescent lipid analogs have been used to monitor the lipid transport activity of the P4-ATPases. Recent evidence now demonstrates that lyso-phosphatidylethanolamine (lyso-PtdEtn) and lyso-phosphatidylcholine (lyso-PtdCho) are bona fide biological substrates transported by the yeast plasma membrane ATPases, Dnf1p and Dnf2p, in consort with a second protein Lem3p. Subsequent to transport, the lysophospholipids are acylated by the enzyme Ale1p to produce PtdEtn and PtdCho. The transport of the lysophospholipids occurs at rates sufficient to support all the PtdEtn and PtdCho synthesis required for rapid cell growth. The lysophospholipid transporters also utilize the anti-neoplastic and anti-parasitic ether lipid substrates related to edelfosine. The identification of biological substrates for the plasma membrane ATPases coupled with the power of yeast genetics now provides new tools to dissect the structure and function of the aminoglycerophospholipid transporters.     

Inclusion Biogenesis and Reactivation of Persistent Chlamydia trachomatis Requires Host Cell Sphingolipid Biosynthesis

Chlamydiae are obligate intracellular pathogens that must coordinate the acquisition of host cell-derived biosynthetic constituents essential for bacterial survival. Purified chlamydiae contain several lipids that are typically found in eukaryotes, implying the translocation of host cell lipids to the chlamydial vacuole. Acquisition and incorporation of sphingomyelin occurs subsequent to transport from Golgi-derived exocytic vesicles, with possible intermediate transport through endosomal multivesicular bodies. Eukaryotic host cell-derived sphingomyelin is essential for intracellular growth of Chlamydia trachomatis, but the precise role of this lipid in development has not been delineated. The present study identifies specific phenotypic effects on inclusion membrane biogenesis and stability consequent to conditions of sphingomyelin deficiency. Culturing infected cells in the presence of inhibitors of serine palmitoyltransferase, the first enzyme in the biosynthetic pathway of host cell sphingomyelin, resulted in loss of inclusion membrane integrity with subsequent disruption in normal chlamydial inclusion development. Surprisingly, this was accompanied by premature redifferentiation to and release of infectious elementary bodies. Homotypic fusion of inclusions was also disrupted under conditions of sphingolipid deficiency. In addition, host cell sphingomyelin synthesis was essential for inclusion membrane stability and expansion that is vital to reactivation of persistent chlamydial infection. The present study implicates both the Golgi apparatus and multivesicular bodies as key sources of host-derived lipids, with multivesicular bodies being essential for normal inclusion development and reactivation of persistent C. trachomatis infection.     

Randomly organized lipids and marginally stable proteins: A coupling of weak interactions to optimize membrane signaling

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6–5.8 bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.