Engineering membrane asymmetry to increase medium-chain fatty acid tolerance in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an attractive chassis for the production of medium-chain fatty acids, but the toxic effect of these compounds often prevents further improvements in titer, yield, and productivity. To address this issue, Lem3 and Sfk1 were identified from adaptive laboratory evolution mutant strains as membrane asymmetry regulators. Co-overexpression of Lem3 and Sfk1 [Lem3(M)-Sfk1(H) strain] through promoter engineering remodeled the membrane phospholipid distribution, leading to an increased accumulation of phosphatidylethanolamine in the inner leaflet of the plasma membrane. As a result, membrane potential and integrity were increased by 131.5% and 29.2%, respectively; meanwhile, the final OD₆₀₀ in the presence of hexanoic acid, octanoic acid, and decanoic acid was improved by 79.6%, 73.4%, and 57.7%, respectively. In summary, this study shows that membrane asymmetry engineering offers an efficient strategy to enhance medium-chain fatty acids tolerance in S. cerevisiae, thus generating a robust industrial strain for producing high-value biofuels.     

龙眼叶片膜脂脂肪酸组分与龙眼耐寒性的关系

龙眼叶片膜脂不饱和脂肪酸含量和脂肪酸不饱和度与龙眼不同品种的耐寒性呈正相关;在年周期中,不饱和脂肪酸含量的变化与龙眼耐寒性的变化呈现平行关系;龙眼叶片膜脂脂肪酸组分含量反映着龙眼品种间耐寒性遗传上的差异,可作为耐寒性的鉴定指标。     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

Changes in membrane fluidity and fatty acid composition of Pseudomonas putida CN-T19 in response to toluene

A bacterial isolate, Pseudomonas putida CN-T19, could grow in a two-phase medium with toluene up to 50% (v/v). Changes in fatty acid composition and membrane fluidity of the isolate were investigated to understand how this microorganism responds toluene. The changes in the ratios of unsaturated to saturated fatty acids were insignificant between cells grown with and without toluene. The changes in the ratio of cis- to trans-fatty acids of C16:1 and C18:1 was, however, significantly lower in cells grown with toluene than cells grown without toluene, giving approximately 1.3 and 9.7, respectively. Toluene had a fluidizing effect on the membrane of cells grown without toluene, resulting in decrease in membrane polarization ratio. Less fluidizing effect of toluene on the membrane of cells grown with toluene was observed, giving 11% of polarization percentage, which was significantly lower than 53% in cells grown without toluene. These results suggest that cis/trans isomeration of C16:1 and C18:1 makes cell membranes more rigid to respond toluene, and is an adaptive strategy allowing P. putida CN-T19 to grow in the presence of organic solvent.     

Temperature- and growth-phase-dependent changes in membrane fatty acid compositions of Brevibacterium ammoniagenes

Fatty acids newly synthesized by Brevibacterium ammoniagenes grown at different temperatures were analyzed. The assay temperature, not the growth temperature, was found to be the major factor affecting the unsaturated/saturated ratio of newly synthesized fatty acids in logarithmic-phase cells. However, in the stationary-phase cells the growth temperature also affected the product profile significantly; cells grown at 7 degrees C produced relatively more oleate and stearate and less palmitate and hexadecenoate when shifted up to 37 degrees C than did cells grown and assayed at 37 degrees C. The unsaturated/saturated ratio as well as average chain length of fatty acids also varied along with the progress of isothermal growth phase. These changes in fatty acid product profiles observed in vivo could be mimicked in vitro assays of the fatty acid synthetase by changing malonyl-CoA concentrations. Our results suggest that the malonyl-CoA concentration is a factor which, in addition to temperature, determines growth-phase-dependent and growth-temperature-dependent changes in the unsaturated/saturated ratios of fatty acids.     

Survey of Extreme Solvent Tolerance in Gram-Positive Cocci: Membrane Fatty Acid Changes in Staphylococcus haemolyticus Grown in Toluene

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.     

Ethanol-induced changes in the fatty acid composition of Lactobacillus hilgardii, its effects on plasma membrane fluidity and relationship with ethanol tolerance

The effect of environmental ethanol concentration on the fatty acid composition of strains of Lactobacillus hilgardii, differing in their tolerance to ethanol, was determined. A marked increase in the proportion of lactobacillic acid (a cyclopropane fatty acid) and a decrease in oleic and vaccenic acids with increasing ethanol concentration was observed. The amount of lactobacillic acid determined at standard conditions (25°C, 0% ethanol) was found to be proportional to the ethanol tolerance of the strains studied. The effect of this alcohol on plasma membrane fluidity was studied by differential scanning calorimetry. The adaptive response to growth in the presence of high concentrations of ethanol produced membranes which, within the limits of ethanol tolerance, maintained the fluidity and integrity in an environment which tends to increase membrane rigidity. When pre-adapted cells are analysed in the absence of environmental ethanol there is a measurabie increase in fluidity. It is proposed that this phenomenon may be correlated with the increase in the proportion of lactobacillic acid. The existence of a relationship between membrane fluidity and ethanol tolerance is discussed.     

Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.     

Docosahexaenoic acid: membrane properties of a unique fatty acid

Docosahexaenoic acid (DHA) with 22-carbons and 6 double bonds is the extreme example of an omega-3 polyunsaturated fatty acid (PUFA). DHA has strong medical implications since its dietary presence has been positively linked to the prevention of numerous human afflictions including cancer and heart disease. The PUFA, moreover, is essential to neurological function. It is remarkable that one simple molecule has been reported to affect so many seemingly unrelated biological processes. Although details of a molecular mode of action remain elusive, DHA must be acting at a fundamental level common to many tissues that is related to the high degree of conformational flexibility that the multiple double bonds have been identified to confer. One likely target for DHA action is at the cell membrane where the fatty acid is known to readily incorporate into membrane phospholipids. Once esterified into phospholipids DHA has been demonstrated to significantly alter many basic properties of membranes including acyl chain order and "fluidity", phase behavior, elastic compressibility, permeability, fusion, flip-flop and protein activity. It is concluded that DHA's interaction with other membrane lipids, particularly cholesterol, may play a prominent role in modulating the local structure and function of cell membranes.     

Selective changes in fatty acid composition of phosphatidylserine in rat erythrocyte membrane induced by nitrate

The relationship between nitrate which is formed from inhaled nitrogen dioxide, a common air pollutant, and changes in fatty acid metabolism of phosphatidylserine in rat erythrocytes has been examined. When erythrocytes were incubated at 37°C for 60 min with fatty acid, the incorporation rate of [1-¹⁴C]arachidonic acid and [9,10-³H]palmitic acid into phosphatidylserine was 15% (80 pmol/h per μmol lipid phosphorus) and 20% (12 pmol/h per μmol lipid phosphorus) of those into phosphatidylethanolamine, respectively. By the addition of 1.0 mM sodium nitrate or 0.5 μM ionophore A23187 to the incubation mixture, the rate of incorporation of both arachidonic acid and palmitic acid into phosphatidylethanolamine was stimulated 1.45-fold. On the other hand, the incorporation of palmitic acid into phosphatidylserine was little affected, while that of arachidonic acid was stimulated 1.35-fold. An increase in arachidonic acid of phosphatidylserine was also found by the addition of nitrate or ionophore A23187. This increase was dependent on the concentration of extracellular calcium and observed by the addition of other chaotropic anions in the order SCN >CIO₄^? > NO₃^?. It seems likely, therefore, that nitrate causes changes in erythrocyte membranes to facilitate calcium uptake. Increasing the concentration of intracellular calcium may cause stimulation of acyl-CoA:lysophospholipid acyltransferase and/or endogenous phospholipase A₂.     

Temperature-dependent membrane fatty acid and cell physiology changes in coccoid forms of Campylobacter jejuni.

The effect of temperature and the availability of nutrients on the transition of spiral Campylobacter jejuni cells to coccoid forms was investigated. Ageing of spiral C. jejuni cells in either nutrient-poor or nutrient-rich environments resulted in the formation of nonculturable coccoid cells at 4, 12, and 25 degrees C after different periods, with the cells incubated at 4 degrees C in nutrient-deficient media remaining culturable the longest. To study the phenomenon, ATP levels, protein profiles, and fatty acid compositions were monitored under conditions where the transition from spiral to coccoid cells occurred. During storage, the levels of intracellular ATP were highest in cells incubated at low temperatures (4 and 12 degrees C) and remained constant after a small initial decrease. During the transformation from spiral to coccoid forms, no alteration in protein profiles could be detected; indeed, inhibition of protein synthesis by chloramphenicol did not influence the transition. Furthermore, DNA damage by gamma irradiation had no effect on the process. Membrane fatty acid composition of cocci formed at low temperatures was found to be almost identical to that of spiral cells, whereas that of cocci formed at 25 degrees C was clearly different. Combining these results, it is concluded that the formation of cocci is not an active process. However, distinctions between cocci formed at different temperatures were observed. Cocci formed at 4 degrees C show characteristics comparable to those of spirals, and these cocci may well play a role in the contamination cycle of C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.     

Alpha-tocopherol and gamma-tocopherol attenuate ethanol-induced changes in membrane fatty acid composition in embryonic chick brains

BACKGROUND: This project investigated whether or not EtOH-induced reductions in the levels of long-chain polyunsaturated membrane fatty acids could be attenuated by exogenous exposure to either alpha-tocopherol, gamma-tocopherol, or diallyl sulfide (DAS). METHODS: At 0 days of development, fertile chicken eggs were injected with a single dose of either saline supplemented with various concentrations of EtOH, alpha- or gamma-tocopherol and EtOH, or DAS and EtOH. At 18 days of development, brains were isolated and subjected to membrane analyses. RESULTS: When exposed to EtOH, concentrations ranging from 0-60.50 microm/Kg egg, dose-dependent decreases in the levels of brain 18:0, 18:1 (n-9), 18:2 (n-6), 18:3 (n-3), and 20:4 (n-6) were observed. These ethanol-induced changes in membrane fatty acid composition correlated with ethanol-induced reductions in brain mass, brain protein levels, acetylcholine esterase (AChE) activities and correlated with increased lipid hydroperoxide levels. Exposure to either 2.5 microm alpha-tocopherol/Kg egg and 6.050 mm EtOH/Kg egg, or 2.5 microm alpha-tocopherol/ Kg egg and 6.050 mm EtOH/Kg egg attenuated EtOH-induced changes in membrane fatty acid composition, brain mass, brain protein levels, AChE activities, and lipid hydroperoxide levels. Embryonic exposure to the cytochrome p450-2E1 inhibitor, diallyl sulfide (DAS), also attenuated EtOH-induced decreases in long-chain, unsaturated membrane fatty acids. However, embryonic exposure to DAS promoted abnormally low brain mass. CONCLUSION: EtOH-induced reductions in the levels of brain long-chain polyunsaturated fatty acid are caused by lipid peroxidation.     

Effect of membrane fatty acid changes on the radiation sensitivity of human lymphoid cells

To test the influence of changes in membrane fatty acid composition on the radiation response of mammalian cells, human LDV cells were cultured in medium containing delipidated serum supplemented with either oleic or linoleic acid. Analysis of lipid extracts of the cells by gas liquid chromatography showed that, after 3 or more days growth in oleic or linoleic acid supplemented media, there were substantial overall increases in the proportion of oleate and linoleate, respectively, in the cellular lipid. Smaller absolute changes were measured in nuclear phospholipid, which was found to be low in unsaturated phospholipid compared to the rest of the cell. Fluorescence polarization measurements using diphenylhexatriene indicated an increased membrane fluidity in cells grown in the presence of excess linoleic acid and to a lesser extent for oleic acid. The clonogenic capacity of the cells after irradiation in air or nitrogen was not altered by any of these membrane compositional changes. The lack of effect on radiation sensitivity, in contrast to that reported for bacteria (E. coli K1060), is consistent with the fact that little or no change was brought about in the nuclear membrane composition, since evidence from partial cell irradiation experiments indicates that the cell nucleus is the sensitive target for cell killing by ionizing radiation. The ability of the cell to maintain a low level of unsaturated phospholipids in its nuclear membrane may be an important general defence mechanism against free radical damage to chromatin mediated by lipid peroxidation.     

Disentangling the photochemical salinity tolerance in Aster tripolium L.: connecting biophysical traits with changes in fatty acid composition

• A profound analysis of A. tripolium photochemical traits under salinity exposure is lacking in the literature, with very few references focusing on its fatty acid profile role in photophysiology.
• To address this, the deep photochemical processes were evaluated by Pulse Amplitude Modulated (PAM) Fluorometry coupled with a discrimination of its leaf fatty acid profile.
• Plants exposed to 125–250 mm NaCl showed higher photochemical light harvesting efficiencies and lower energy dissipation rates. under higher NaCl exposure, there is evident damage of the oxygen evolving complexes (OECs). On the other hand, Reaction Centre (RC) closure net rate and density increased, improving the energy fluxes entering the PS II, in spite of the high amounts of energy dissipated and the loss of PS II antennae connectivity. Energy dissipation was mainly achieved through the auroxanthin pathway. Total fatty acid content displayed a similar trend, being also higher under 125–250 mm NaCl with high levels of omega‐3 and omega‐6 fatty acids. The increase in oleic acid and palmitic acid allows the maintenance of the good functioning of the PS II. Also relevant was the high concentration of chloroplastic C16:1t in the individuals subjected to 125–250 mm NaCl, related with a higher electron transport activity and with the organization of the Light Harvesting Complexes (LHC) and thus reducing the activation of energy dissipation mechanisms.
• All these new insights shed some light not only on the photophysiology of this potential cash‐crop, but also highlight its important saline agriculture applications of this species as forage and potential source of essential fatty acids.

     

The role of two branched‐chain amino acid transporters in Staphylococcus aureus growth,membrane fatty acid composition and virulence

The branched‐chain amino acids (BCAAs) are vital to both growth and virulence of the human pathogen Staphylococcus aureus. In addition to supporting protein synthesis, the BCAAs serve as precursors for branched‐chain fatty acids (BCFAs), which are predominant membrane fatty acids, and, in association with the global regulatory protein CodY, the BCAAs are key co‐regulators of virulence factors. Despite these critical functions, S. aureus represses Leu and Val synthesis, instead preferring to acquire them from the extracellular milieu. We previously identified BrnQ1 as a BCAA transporter, yet a brnQ1 mutant remained capable of BCAA acquisition. Here, we describe BcaP as an additional BCAA transporter, and determine that it plays a secondary role to BrnQ1 during S. aureus growth in a chemically defined medium. Furthermore, membrane fatty acid composition analysis revealed that BrnQ1, and not BcaP, is required for transporting Leu and Val to be used for iso‐BCFA synthesis. Despite a predominant role for BrnQ1 in vitro, both BrnQ1 and BcaP are required for S. aureus fitness in vivo in a hematogenous spread infection model and a nasal colonisation model. These data demonstrate the importance of BrnQ1 and BcaP for growth, environmental adaptation and virulence of S. aureus.     

Non-reducing trisaccharide fatty acid monoesters: novel detergents in membrane biochemistry

Three families of non-reducing trisaccharide fatty acid monoesters bearing C?? to C?? acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to μM. The new compounds are capable of dissolving phospholipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed β-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.     

Expression of a gene for plastid ω-3 fatty acid desaturase and changes in lipid and fatty acid compositions in light- and dark- grown wheat leaves

The leaves of monocotyledonous plants create a developmental sequence of cells and plastids from the base to the apical portion. We investigated fatty-acid and lipid compositions in successive leaf sections of light- and dark-grown wheat (Triticum aestivum L. cv. Chihoku) seedlings. The most notable change in the fatty acid composition was the increase of linolenic acid (18:3) with maturation of leaf cells, which occurred both in light- and dark-grown leaf tissues. In light-grown leaves, the increase of 18:3 with maturation was mainly attributed to the increase of monogalactosyldiacylglycerol (MGD) and also to the increase of the 18:3 level of MGD. In dark-grown leaves, the increase of 18:3 in the leaf apex was caused by the increase of the levels of MGD and digalactosyldiacylglycerol (DGD) and also by the increase of the 18:3 levels of within these two lipids. Since MGD and DGD are mainly found in plastid membranes, these findings indicate that both the synthesis of galactolipids and the formation of 18:3 these lipids take place during plastid development. The plastid ω-3 fatty acid desaturase is responsible for the formation of 18:3 in plastid membrane lipids. To investigate the regulation of desaturation, we isolated a gene for wheat plastid ω-3 fatty acid desaturase (TaFAD7). The mRNA level of TaFAD7 in light-grown leaves was much higher than that in dark-grown leaves. During the greening of etiolated leaves the level of TaFAD7 mRNA increased significantly, accompanied by an increase of the 18:3 level of total fatty acids. On the other hand, the levels of TaFAD7 mRNA were almost the same in all the leaf sections of both light- and dark-grown leaf tissues. These results suggest that the effect of the expression of the TaFAD7 gene on the increase of the 18:3 level is different between the leaf development under continuous light- or dark-conditions and the light-induced greening process of etiolated leaves. The increase of 18:3 content of MGD (or MGD and DGD) with maturation is apparently regulated not solely by the level of TaFAD7 mRNA.