Mechanism of Long-Chain Free Fatty Acid Protonation at the Membrane-Water Interface.
ABSTRACT: Long-chain free fatty acids (FFAs) play an important role in several physiological and pathological processes such as lipid fusion, adjustments of membrane permeability and fluidity, and the regulation of enzyme and protein activities. FFA-facilitated membrane proton transport (flip-flop) and FFA-dependent proton transport by membrane proteins (e.g., mitochondrial uncoupling proteins) are governed by the difference between FFA's intrinsic pKa value and the pH in the immediate membrane vicinity. Thus far, a quantitative understanding of the process has been hampered, because the pKa value shifts upon moving the FFA from the aqueous solution into the membrane. For the same FFA, pKa values between 5 and 10.5 were reported. Here, we systematically evaluated the dependence of pKa values on chain length and number of double bonds by measuring the ?-potential of liposomes reconstituted with FFA at different pH values. The experimentally obtained intrinsic pKa values (6.25, 6.93, and 7.28 for DOPC membranes) increased with FFA chain length (C16, C18, and C20), indicating that the hydrophobic energy of transfer into the bilayer is an important pKa determinant. The observed pKa decrease in DOPC with increasing number of FFA double bonds (7.28, 6.49, 6.16, and 6.13 for C20:0, C20:1, C20:2, and C20:4, respectively) is in line with a decrease in transfer energy. Molecular dynamic simulations revealed that the ionized carboxylic group of the FFAs occupied a fixed position in the bilayer independent of chain length, underlining the importance of Born energy. We conclude that pKa is determined by the interplay between the energetic costs for 1) burying the charged moiety into the lipid bilayer and 2) transferring the hydrophobic protonated FFA into the bilayer.
Project description:An inappropriate diet, particularly excessive consumption of dietary fats and oils, may have a major negative impact on beta-cell function and cause type 2 diabetes mellitus. To investigate this issue, we examined the toxicity of free fatty acid (FFA) compositions mirroring the FFA profiles of various popular edible oils in human EndoC-?H1 beta-cells and in rat islets. For this purpose, we made compositions consisting exclusively of various FFAs in different volumetric percentages mimicking these oils and additionally mixtures of these compositions. Human EndoC-?H1 beta-cells were incubated with different oil compositions and the toxicity, lipid droplet formation, ER-stress, and H<sub>2</sub>O<sub>2</sub> production were analyzed. Compositions with prominent content of saturated as well as unsaturated long-chain FFAs showed moderate but significant toxicity both in human EndoC-?H1 beta-cells and rat islets, however, without further measurable metabolic impairments. On the other hand compositions with high content of medium-chain FFAs revealed no toxicity. A composition with 50% of the very long-chain unsaturated FFA erucic acid caused high toxicity with concomitant peroxisomal H<sub>2</sub>O<sub>2</sub> production. The toxicity of FFAs to human EndoC-?H1 beta-cells was dampened in mixtures of FFA compositions with a significant content of medium-chain FFAs, but not with a significant proportion of unsaturated FFAs.
Project description:BACKGROUND:Long-chain free fatty acids (FFAs) are a type of backbone molecule that can react with alcohol to produce biodiesels. Various microorganisms have become potent producers of FFAs. Efforts have focused on increasing metabolic flux to the synthesis of either neutral fat or fatty acyl intermediates attached to acyl carrier protein (ACP), which are the source of FFAs. Membrane lipids are also a source of FFAs. As an alternative way of producing FFAs, exogenous phospholipase may be used after heterologous production and localization in the periplasmic space. In this work, we examined whether Rhodobacter sphaeroides, which forms an intracytoplasmic membrane, can be used for long-chain FFA production using phospholipase. RESULTS:The recombinant R. sphaeroides strain Rs-A2, which heterologously produces Arabidopsis thaliana phospholipase A2 (PLA2) in the periplasm, excretes FFAs during growth. FFA productivity under photoheterotrophic conditions is higher than that observed under aerobic or semiaerobic conditions. When the biosynthetic enzymes for FA (?-ketoacyl-ACP synthase, FabH) and phosphatidate (1-acyl-sn-glycerol-3-phosphate acyltransferase, PlsC) were overproduced in Rs-A2, the FFA productivity of the resulting strain Rs-HCA2 was elevated, and the FFAs produced mainly consisted of long-chain FAs of cis-vaccenate, stearate, and palmitate in an approximately equimolar ratio. The high-cell-density culture of Rs-HCA2 with DMSO in two-phase culture with dodecane resulted in an increase of overall carbon substrate consumption, which subsequently leads to a large increase in FFA productivity of up to 2.0 g L-1 day-1. Overexpression of the genes encoding phosphate acyltransferase (PlsX) and glycerol-3-phosphate acyltransferase (PlsY), which catalyze the biosynthetic steps immediately upstream from PlsC, in Rs-HCA2 generated Rs-HXYCA2, which grew faster than Rs-HCA2 and showed an FFA productivity of 2.8 g L-1 day-1 with an FFA titer of 8.5 g L-1. CONCLUSION:We showed that long-chain FFAs can be produced from metabolically engineered R. sphaeroides heterologously producing PLA2 in the periplasm. The FFA productivity was greatly increased by high-cell-density culture in two-phase culture with dodecane. This approach provides highly competitive productivity of long-chain FFAs by R. sphaeroides compared with other bacteria. This method may be applied to FFA production by other photosynthetic bacteria with similar differentiated membrane systems.
Project description:Pu-erh tea has been extensively reported to possess lipid lowering effects but the underlying mechanisms remained unclear. Free fatty acids (FFAs) are generally correlated with the development of obesity, leading to increased risk for type 2 diabetes mellitus and cardiovascular diseases. To investigate whether Pu-erh tea treatment alters FA metabolism, we treated HFD induced obese mice with Pu-erh tea for 22 weeks and analyzed FFA profiles of experimental mice using a UPLC-QTOF-MS platform. Results showed remarkable changes in metabolic phenotypes and FFA compositions in mice treated with or without Pu-erh tea. HFD induced a marked obese phenotype in mice as revealed by significantly increased body weight, liver and adipose tissue weight, lipid levels in serum and liver, and these parameters were markedly reduced by Pu-erh tea treatment. Several FFA or FFA ratios, such as DGLA, palmitoleic acid, and OA/SA ratio, were significantly increased while the levels of SA/PA and AA/DGLA were significantly reduced in HFD-induced obese mice. Interestingly, these differential FFAs or FFA ratios were previous identified as key markers in human obese subjects, and their changes observed in the HFD group were reversed by Pu-erh tea treatment. Moreover, a panel of FFA markers including C20:3 n6/C18:3 n6 and C20:3 n6/C20:2 n6, C18:3 n6/C18:2 n6, C18:3 n3/C18:2 n6 and C24:1 n9/C22:1 n9, which were previously identified as biomarkers in predicting the remission of obesity and diabetes in human subjects who underwent metabolic surgery procedures, were reversed by Pu-erh tea intervention. Pu-erh tea significantly improved glucose homeostasis and insulin tolerance compared to the HFD group. Additionally, Pu-erh tea treatment significantly decreased FFA synthesis genes and increased the expression of genes involved in FFA uptake and ?-oxidation including FATP2, FATP5, PPAR?, CPT1?, and ACOX-1. These finding confirmed the beneficial effects of Pu-erh tea on regulating lipid and glucose metabolism, and further validated a panel of FFA markers with diagnostic and prognostic value for obesity and diabetes.
Project description:Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long-chain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.
Project description:Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family Erysipelotrichaceae in the phylum Firmicutes but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited ?-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.
Project description:The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9Å towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.
Project description:Microbial synthesis of free fatty acids (FFA) is a promising strategy for converting renewable sugars to advanced biofuels and oleochemicals. Unfortunately, FFA production negatively impacts membrane integrity and cell viability in Escherichia coli, the dominant host in which FFA production has been studied. These negative effects provide a selective pressure against FFA production that could lead to genetic instability at industrial scale. In prior work, an engineered E. coli strain harboring an expression plasmid for the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase was shown to have highly elevated levels of unsaturated fatty acids in the cell membrane. The change in membrane content was hypothesized to be one underlying cause of the negative physiological effects associated with FFA production. In this work, a connection between the regulator of unsaturated fatty acid biosynthesis in E. coli, FabR, thioesterase expression, and unsaturated membrane content was established. A strategy for restoring normal membrane saturation levels and increasing tolerance towards endogenous production of FFAs was implemented by modulating acyl-ACP pools with a second thioesterase (from Geobacillus sp. Y412MC10) that primarily targets medium chain length, unsaturated acyl-ACPs. The strategy succeeded in restoring membrane content and improving viability in FFA producing E. coli while maintaining FFA titers. However, the restored fitness did not increase FFA productivity, indicating the existence of additional metabolic or regulatory barriers.
Project description:Studies of lipids in CKD, including ESRD, have been limited to measures of conventional lipid profiles. We aimed to systematically identify 17 different lipid classes and associate the abundance thereof with alterations in acylcarnitines, a metric of ?-oxidation, across stages of CKD. From the Clinical Phenotyping Resource and Biobank Core (CPROBE) cohort of 1235 adults, we selected a panel of 214 participants: 36 with stage 1 or 2 CKD, 99 with stage 3 CKD, 61 with stage 4 CKD, and 18 with stage 5 CKD. Among participants, 110 were men (51.4%), 64 were black (29.9%), and 150 were white (70.1%), and the mean (SD) age was 60 (16) years old. We measured plasma lipids and acylcarnitines using liquid chromatography-mass spectrometry. Overall, we identified 330 different lipids across 17 different classes. Compared with earlier stages, stage 5 CKD associated with a higher abundance of saturated C16-C20 free fatty acids (FFAs) and long polyunsaturated complex lipids. Long-chain-to-intermediate-chain acylcarnitine ratio, a marker of efficiency of ?-oxidation, exhibited a graded decrease from stage 2 to 5 CKD (P<0.001). Additionally, multiple linear regression revealed that the long-chain-to-intermediate-chain acylcarnitine ratio inversely associated with polyunsaturated long complex lipid subclasses and the C16-C20 FFAs but directly associated with short complex lipids with fewer double bonds. We conclude that increased abundance of saturated C16-C20 FFAs coupled with impaired ?-oxidation of FFAs and inverse partitioning into complex lipids may be mechanisms underpinning lipid metabolism changes that typify advancing CKD.
Project description:Cyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from Synechococcus elongatus PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required.Growth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g L-1 of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was overlaid with isopropyl myristate (IM), the final cell density, cellular chlorophyll content, and the photosynthetic yield of PSII were greatly improved. The total amount of extracellular FFA was more than three times larger than that in the control culture grown without IM, with most of the secreted FFAs being recovered in the IM layer. The cellular FFA content was decreased by more than 85% by the presence of the IM layer. Thus, the two-phase culture system effectively facilitated FFA secretion out of the cell. An average FFA excretion rate of 1.5 mg L-1 h-1 was attained in the 432 h of cultivation, with a total amount of excreted FFA being 0.64 g L-1 of culture. These figures were more than three times higher than those reported previously for the cyanobacteria-based FFA production systems.Removal of FFA from the culture medium is important for improving the productivity of the FFA production system using cyanobacteria. Further increase in productivity would require an increase in both the rates of FFA production in the cell and active FFA export across the plasma membrane.
Project description:It has been previously reported that photosynthetic production of extracellular free fatty acids (FFAs) in cyanobacteria was realized by thioesterases (TesA) mediated hydrolysis of fatty acyl-ACP in cytosol and excretion of the FFA outside of the cell. However, two major issues related to the genetically modified strains need to be addressed before the scale-up commercial application becomes possible: namely, the toxicity of FFAs, and the diversity of carbon lengths of fatty acids that could mimic the fossil fuel. To address those issues, we hypothesized that generating FFAs near membrane could facilitate rapid excretion of the FFA outside of the cell and thus decrease toxicity caused by intracellular FFAs in the cytosolic expression of thioesterase. To realize this, we localized a leaderless thioesterase (AcTesA) from Acinetobacter baylyi on the cytosolic side of the inner membrane of Synechocystis sp. PCC6803 using a membrane scaffolding system. The engineered strain with AcTesA on its membrane (mAcT) produced extracellular FFAs up to 171.9 ± 13.22 mg?L-1 compared with 40.24 ± 10.94 and 1.904 ± 0.158 mg?L-1 in the cytosol-expressed AcTesA (AcT) and wild-type (WT) strains, respectively. Moreover, the mAcT strain generated around 1.5 and 1.9 times less reactive oxygen species than AcT and WT, respectively. Approximately 78% of total FFAs were secreted with an average rate of 1 mg?L-1?h-1, which was higher than 0.44 mg?L-1?h-1 reported previously. In the case of mAcT strain, 60% of total secreted FFAs was monounsaturated (C18:1) which is the preferable biodiesel component. Therefore, the engineered mAcT strain shows enhanced FFAs production with less toxicity which is highly desirable for biodiesel production.