Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio–product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.     

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.     

Effects of perfluorocarboxylic acids on the activities of acyl-CoA elongations in vivo and in vitro

Effects of perfluorocarboxylic acids (PFCAs) on proportions of oleic acid and cis-vaccenic acid through acyl-CoA chain elongation systems have been studied in the liver of rats. Administration of PFCAs caused a significant increase in palmitoyl-CoA chain elongation activity while these chemicals did not affect palmitoleoyl-CoA chain elongation activity in vivo.Condensation for both palmitoyl-CoA and palmitoleoyl-CoA were inhibited by PFCAs in vitro at the concentrations, which were physiologically found in the liver of rats treated with the PFCAs. Δ⁹ Desaturase, which catalyzes both stearoyl-CoA desaturation and palmitoyl-CoA desaturation, was induced by the treatments of rats with the PFCAs. The administration of the PFCAs to rats caused a marked increase in proportion of oleic acid, while that of cis-vaccenic acid was not affected at all. These results strongly suggest that the induced palmitoyl-CoA chain elongation by PFCAs, which exist in the liver, effectively produces oleic acid in concert with the induced stearoyl-CoA desaturase, but the inhibitory effects of PFCAs on either palmitoyl-CoA chain elongation or palmitoleoyl-CoA chain elongation are not crucial for the formation of the elongated fatty acids in vivo.     

Metabolic transformation of labelled exogenous fatty acids by fungal cultures of the family Entomophthoraceae

Entomophthora coronata 1932 and E. conica 1716 are quite different in their fatty acid composition and the unsaturation degree of synthesized lipids. The cultures were used as models to study metabolic transformations of exogenous 14C-labeled acetic, palmitic, stearic and oleic acids as well as to compare the activities of the synthetase and desaturase enzyme complexes. The cultures were capable of transforming exogenous acetic and fatty acids into polyunsaturated arachidonic acid. E. coronata 1932 whose lipids mainly contain fatty acids with a short chain could metabolize unsaturated oleic acid to yield polyene fatty acids. However, this culture metabolized exogenous acids at a far lower rate as compared with E. conica 1716. The high content of saturated fatty acids with a short chain in the lipids might be due to the specific action of the synthetase complex and to the low activity of the desaturation enzymes. It has been demonstrated for the first time that exogenous oleic acid is converted at a high rate by the cells into arachidonic acid, a precursor of prostaglandin compounds.     

Good Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane Homeostasis in Yeast

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.     

Effects of the catalytic hydrogenation of microsomal lipids upon four enzyme activities involved in oleic acid desaturation

Catalytical hydrogenation of the unsaturated fatty acyl residues of microsomal lipids was realized for different times. Progress of the reaction was followed by calculating the progressive loss of double-bonds in 100 initial acyl residues (percentage of hydrogenation). The maximum loss observed was 45% after 60 min.The drop in polyunsaturated faty acid content was coupled with an increase in the amount of stearic acid and oleic acid.The order parameter of microsomal lipids, measured by ESR, increased parallely to the reduction of double bonds. Maximum hydrogenation of microsomal lipids strongly (200–250%) stimulated microsomal NADH-ferricyanide reductase activity. NADH-cytochrome c reductase, lysophosphatidylcholine-acyl-transferase and oleoyl-phosphatidylcholine desaturase were inhibited (40%, 100% and 100% respectively). These modifications of enzyme activities are discussed in conjunction with the changes observed in membrane fluidity, following hydrogenation of microsomal lipids     

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ?¹²-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ?¹⁵-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ?⁹-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.     

Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ⁹Z-C_16:1) and oleic acid (Δ⁹Z-C_18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C_16:0) and stearic acid (C_18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ⁹ and Δ¹¹) and substrate chain-length preferences (i.e., C_16:0 and C_18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ¹¹Z-C_18:1), whereas neither Δ¹¹Z-C_16:1 nor palmitoleic acid (Δ⁹Z-C_16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Lipid-dependent subcellular relocalization of the acyl chain desaturase in yeast

The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C. and Yaffe, M.P. (1991). J. Cell Biol. 115, 1249-1257). We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.     

Engineering Escherichia coli membrane phospholipid head distribution improves tolerance and production of biorenewables

Economically competitive microbial production of biorenewable fuels and chemicals is often impeded by toxicity of the product to the microbe. Membrane damage is often identified as a major mechanism of this toxicity. Prior efforts to strengthen the microbial membrane by changing the phospholipid distribution have largely focused on the fatty acid tails. Herein, a novel strategy of phospholipid head engineering is demonstrated in Escherichia coli. Specifically, increasing the expression of phosphatidylserine synthase (+pssA) was found to significantly increase both the tolerance and production of octanoic acid, a representative membrane-damaging solvent. Tolerance of other industrially-relevant inhibitors, such as furfural, acetate, toluene, ethanol and low pH was also increased. In addition to the increase in the relative abundance of the phosphoethanolamine (PE) head group in the +pssA strain, there were also changes in the fatty acid tail composition, resulting in an increase in average length, percent unsaturation and decreased abundance of cyclic rings. This +pssA strain had significant changes in: membrane integrity, surface potential, electrochemical potential and hydrophobicity; sensitivity to intracellular acidification; and distribution of the phospholipid tails, including an increase in average length and percent unsaturation and decreased abundance of cyclic rings. Molecular dynamics simulations demonstrated that the +PE membrane had increased resistance to penetration of ethanol into the hydrophobic core and also the membrane thickness. Further hybrid models in which only the head group distribution or fatty acid tail distribution was altered showed that the increase in PE content is responsible for the increase in bilayer thickness, but the increased hydrophobic core thickness is due to altered distribution of both the head groups and fatty acid tails. This work demonstrates the importance of consideration of the membrane head groups, as well as a modeling approach, in membrane engineering efforts.     

Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity

The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for Δ12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the Δ12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the Δ12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.     

Temperature pre-adaptation of ectothermal organisms of different organization: Role of the lipid fatty acid composition

Composition of fatty acids (FA) was studied in bacteria Vibrio anguillarum, total and lysosomal lipids of cestodes Eubothrium crassum and Diphyllobothrium dendriticum, and roe of the roach Rutilus rutilus, carp Cyprinus carpio, and salmon Salmo salar. Bacteria collected from different geographic zones but cultivated under the similar conditions have been found to maintain the FA spectra peculiarities substantially corresponding to temperature conditions of their habitat region. Unsaturation of total lipids in D. dendriticum, determined from the ratio between saturated, monoenoic, and polyenoic acids was higher than that of lysosome membrane lipids. This phenomenon might be due to pre-adaptation of the lipid FA composition of eggs produced in huge amounts by a parasite to low temperatures of environment, in which embryos are to be developed. Quantitative ratios of lipid FA in the roach, carp, and salmon roes correspond to the spawning thermal tolerance diapason specific of some particular fish species and to some extent do not depend on real temperature conditions existing at the moment of development.     

Effect of chlorophenols on the membrane lipids of bacterial cells

Chlorophenols, widespread soil and water contaminants and often degradation products of some pesticides, are a potential stress factor for survival of environmental bacteria. The effect of pentachlorophenol (PCP) and 2,4-chlorophenol (2,4-CP) on the growth, amount of lipid, and fatty acid composition in the membrane lipids was examined in a strain of the bacterium Kocuria varians, able to degrade chlorophenols. The index of fatty acid unsaturation in two main membrane lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased in the presence of chlorophenols. Transformation of stearic acid into oleic acid was significantly increased by PCP addition only in PE, but conversion of oleic acid into linoleic acid was blocked by PCP and 2,4-CP in both PC and PE. This observation may indicate that while Δ⁹ desaturase was sensitive mainly to 2,4-CP, activity of Δ¹² desaturase was inhibited by both PCP and 2,4-CP.     

Characteristics of a lipolytic and fatty acid-requiring Butyrivibrio sp. isolated from the ovine rumen.

A naturally occurring fatty acid-requiring Butyrivibrio sp. (strain S2), isolated from the ovine rumen, deacylates plant galactolipids, phospholipids and sulpholipids to obtain sufficient fatty acid for growth. Growth in vitro was promoted by adding to the growth medium a single straight-chain saturated fatty acid (C13 to C18) or vaccenic acid. Palmitoleic and oleic acids also supported growth but gave lengthy lag phases probably due to their toxicity. Linolenic and linoleic acids supported good growth but they were completely hydrogenated to trans-11-octadecenoic acid which was incorporated into the bacterial complex lipids. No chain elongation, chain shortening or desaturation of the added fatty acids occurred and all were substantially incorporated into bacterial lipids of the plasmalogen type, partially as a new type of hydrophobic grouping derived from two molecules of fatty acid. The absence of fatty acid unsaturation poses the question of the maintenance of membrane fluidity within this bacterium.     

Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Delta(9)Z-C(16:1)) and oleic acid (Delta(9)Z-C(18:1)), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C(16:0)) and stearic acid (C(18:0)), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Delta(9) and Delta(11)) and substrate chain-length preferences (i.e., C(16:0) and C(18:0)); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Delta(11)Z-C(18:1)), whereas neither Delta(11)Z-C(16:1) nor palmitoleic acid (Delta(9)Z-C(16:1)) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Enhancement of low-temperature tolerance in transgenic tomato plants overexpressing Lefad7 through regulation of trienoic fatty acids

We studied how tomato (Lycopersicon esculentum Mill.) chloroplast omega-3 fatty acid desaturase gene (Lefad7) overexpression enhanced low-temperature (LT) tolerance in transgenic tomato plants. In these plants, the content of linolenic acid (18:3) markedly increased and, correspondingly, the content of linoleic acid (18:2) decreased. Similar changes were found after 6 h under LT (4°C) treatment. Under LT stress, wild type (WT) tomato plants showed a much greater increase in relative electrolyte leakage and malondialdehyde (MDA) contents compared with transgenic plants. Transgenic plants exhibited higher activities of antioxidative enzymes and a lower content of reactive oxygen species (ROS). Transgenic plants maintained a relatively higher level of the net photosynthetic rate (P _N) and chlorophyll (Chl) content than WT plants under LT stress. Taken together, we suggested that overexpression of Lefad7 enhanced LT tolerance by changing the composition of membrane lipids in tomato plants, with the increased content of trienoic fatty acids and reduced content of dienoic fatty acids that led to series of physiological alterations.     

Rearing system and oleic acid supplementation effect on carcass and lipid characteristics of two muscles from an obese pig breed

Quality of pork depends on genotype, rearing and pre- and post-slaughter conditions. However, no information is available on rearing system changes and oleic acid supplementation on carcass characteristics and fatty acid (FA) profile of pork from the Alentejano (AL) pig, an obese breed. This study evaluates the effects of feeding low (LO) or high oleic acid diets (HO) to AL pigs reared in individual pens (IND) or outdoor (OUT) with access to pasture. Carcass composition was obtained and longissimus dorsi and semimembranosus samples were collected to analyse chemical composition and neutral and polar intramuscular lipids FA profile by gas chromatography. Statistical analysis was performed by a two-way ANOVA for rearing system and diet effects. OUT-reared pigs presented leaner carcasses than IND-reared ones. Both muscles presented lower intramuscular lipid content in OUT-reared pigs. Treatments affected the FA profile of muscles. Overall, OUT-reared pigs presented lower n-6/n-3 FA ratios, whereas pigs fed the HO diet exhibited lower saturated fatty acids (SFA), higher monounsaturated fatty acids (MUFA) levels and lower thrombogenic indexes on neutral intramuscular lipids than LO-fed pigs. On the polar fraction, OUT-reared pigs presented lower SAT and n-6/n-3 FA ratio, and higher polyunsaturated fatty acids (PUFA) levels on both muscles. Pigs fed the HO diet exhibited higher MUFA and lower PUFA levels on both muscles, and lower SAT levels on semimembranosus. This study shows rearing system and oleic acid supplementation have complementary effects and influence carcass composition and the nutritional quality of meat.     

The role of fatty acid binding protein on the metabolism of fatty acids in isolated rat hepatocytes

In isolated rat hepatocytes flavaspidic acid, a competitor with free fatty acids for the fatty-acid-binding-protein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of CO₂ and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acid-activating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake.