The Front-end Desaturase: Structure,Function, Evolution and Biotechnological Use

Very long chain polyunsaturated fatty acids such as arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are essential components of cell membranes, and are precursors for a group of hormone-like bioactive compounds (eicosanoids and docosanoids) involved in regulation of various physiological activities in animals and humans. The biosynthesis of these fatty acids involves an alternating process of fatty acid desaturation and elongation. The desaturation is catalyzed by a unique class of oxygenases called front-end desaturases that introduce double bonds between the pre-existing double bond and the carboxyl end of polyunsaturated fatty acids. The first gene encoding a front-end desaturase was cloned in 1993 from cyanobacteria. Since then, front-end desaturases have been identified and characterized from a wide range of eukaryotic species including algae, protozoa, fungi, plants and animals including humans. Unlike front-end desaturases from bacteria, those from eukaryotes are structurally characterized by the presence of an N-terminal cytochrome b ₅-like domain fused to the main desaturation domain. Understanding the structure, function and evolution of front-end desaturases, as well as their roles in the biosynthesis of very long chain polyunsaturated fatty acids offers the opportunity to engineer production of these fatty acids in transgenic oilseed plants for nutraceutical markets.     

Surface activity of the lipid products hydrolyzed with lipase and phospholipase A-2

We examined thein vitro surface activity, immersional wettability and adhesional wettability shown by aqueous solutions of soy lysophospholipid (SLP)/monoglyceride (MG)/fatty acid (FA), SLP/FA and SLP/MG, and found that many lipid mixtures showed significant surface activity when their MG and FA components consisted of polyunsaturated FA and/or medium chain FA. The more unsaturated the FA, the higher the surface activity. A mixture of SLP/medium chain fatty acid MG (medium chain MG)/medium chain FA showed the highest surface activity, and was comparable to an Aerosol-OT surfactant, the most effective wetting agent. SLP/polyunsaturated FA monoglyceride (polyunsaturated MG)/polyunsaturated FA, SLP/medium chain FA, and SLP/polyunsaturated FA, SLP/medium chain MG, and SLP/polyunsaturated MG also showed a high degree of activity. Wettability decreased rapidly when the amount of saturated, long chain FA moieties increased. It is recognized that the degrees of unsaturation and the chain length of FAs in the lipid mixtures have a decisive influence on surface activities. Higher ratios of MG and FA to SLP gave higher activity; and solubilizers such as bile salts were necessary to dissolve them in water.     

Biosynthesis of Long Chain Polyunsaturated Fatty Acids in the Marine Ichthyosporean Sphaeroforma arctica

Sphaeroforma arctica is a unique, recently discovered marine protist belonging to a group falling close to the yeast/animal border. S. arctica is found in cold environments, and accordingly has a fatty acid composition containing a high proportion of very long chain polyunsaturated fatty acids, including the ω3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Two elongases and five desaturases, representing the complete set of enzymes necessary for the synthesis of DHA from oleic acid, were isolated from this species and characterized in yeast. One elongase showed high conversion rates on a wide range of 18 and 20 carbon substrates, and was capable of sequential elongation reactions. The second elongase had a strong preference for the 20-carbon fatty acids EPA and arachidonic acid, with over 80 % of EPA converted to docosapentaenoic acid (DPA) in the heterologous yeast host. The isolation of a Δ8-desaturase, along with the detection of eicosadienoic acid in S. arctica cultures indicated that this species uses the alternate Δ8-pathway for the synthesis of long-chain polyunsaturated fatty acids. S. arctica also carried a Δ4-desaturase that proved to be very active in the production of DHA from DPA. Finally, a long chain acyl-CoA synthetase from S. arctica improved DHA uptake in the heterologous yeast host and led to an improvement in desaturation and elongation efficiencies.     

In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa,and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.     

Spatial–Spectral Analysis of Hyperspectral Images Reveals Early Detection of Downy Mildew on Grapevine Leaves

Downy mildew is a highly destructive disease of grapevine. Currently, monitoring for its symptoms is time-consuming and requires specialist staff. Therefore, an automated non-destructive method to detect the pathogen before the visible symptoms appear would be beneficial for early targeted treatments. The aim of this study was to detect the disease early in a controlled environment, and to monitor the disease severity evolution in time and space. We used a hyperspectral image database following the development from 0 to 9 days post inoculation (dpi) of three strains of Plasmopara viticola inoculated on grapevine leaves and developed an automatic detection tool based on a Support Vector Machine (SVM) classifier. The SVM obtained promising validation average accuracy scores of 0.96, a test accuracy score of 0.99, and it did not output false positives on the control leaves and detected downy mildew at 2 dpi, 2 days before the clear onset of visual symptoms at 4 dpi. Moreover, the disease area detected over time was higher than that when visually assessed, providing a better evaluation of disease severity. To our knowledge, this is the first study using hyperspectral imaging to automatically detect and show the spatial distribution of downy mildew on grapevine leaves early over time.     

Retroconversion of Docosapentaenoic Acid (n-6): an Alternative Pathway for Biosynthesis of Arachidonic Acid in Daphnia magna

The aim of this study was to assess metabolic pathways for arachidonic acid (20:4n-6) biosynthesis in Daphnia magna. Neonates of D. magna were maintained on [¹³C] enriched Scenedesmus obliquus and supplemented with liposomes that contained separate treatments of unlabeled docosapentaenoic acid (22:5n-6), 20:4n-6, linoleic acid (18:2n-6) or oleic acid (18:1n-9). Daphnia in the control treatment, without any supplementary fatty acids (FA) containing only trace amounts of 20:4n-6 (~0.3 % of all FA). As expected, the highest proportion of 20:4n-6 (~6.3 %) was detected in Daphnia that received liposomes supplemented with this FA. Higher availability of 18:2n-6 in the diet increased the proportion of 18:2n-6 in Daphnia, but the proportion of 20:4n-6 was not affected. Daphnia supplemented with 22:5n-6 contained ~3.5 % 20:4n-6 in the lipids and FA specific stable isotope analyses validated that the increase in the proportion of 20:4n-6 was due to retroconversion of unlabeled 22:5n-6. These results suggest that chain shortening of 22:5n-6 is a more efficient pathway to synthesize 20:4n-6 in D. magna than elongation and desaturation of 18:2n-6. These results may at least partially explain the discrepancies noticed between phytoplankton FA composition and the expected FA composition in freshwater cladocerans. Finally, retroconversion of dietary 22:5n-6 to 20:4n-6 indicates Daphnia efficiently retain long chain n-6 FA in lake food webs, which might be important for the nutritional ecology of fish.     

Impact of a Standard Rodent Chow Diet on Tissue n‐6 Fatty Acids, Δ9‐Desaturation Index,and Plasmalogen Mass in Rats Fed for One Year

Although many studies focus on senescence mechanisms, few habitually consider age as a biological parameter. Considering the effect of interactions between food and age on metabolism, here we depict the lipid framework of 12 tissues isolated from Sprague–Dawley rats fed standard rodent chow over 1 year, an age below which animals are commonly studied. The aim is to define relevant markers of lipid metabolism influenced by age in performing a fatty acid (FA) and dimethylacetal profile from total lipids. First, our results confirm impregnation of adipose and muscular tissues with medium‐chain FA derived from maternal milk during early infancy. Secondly, when animals were switched to standard croquettes, tissues were remarkably enriched in n‐6 FA and especially 18:2n‐6. This impregnation over time was coupled with a decrease of the desaturation index and correlated with lower activities of hepatic Δ5‐ and Δ6‐desaturases. In parallel, we emphasize the singular status of testis, where 22:5n‐6, 24:4n‐6, and 24:5n‐6 were exceptionally accumulated with growth. Thirdly, 18:1n‐7, usually found as a discrete FA, greatly accrued over the course of time, mostly in liver and coupled with Δ9‐desaturase expression. Fourthly, skeletal muscle was characterized by a surprising enrichment of 22:6n‐3 in adults, which tended to decline in older rats. Finally, plasmalogen‐derived dimethylacetals were specifically abundant in brain, erythrocytes, lung, and heart. Most notably, a shift in the fatty aldehyde moiety was observed, especially in brain and erythrocytes, implying that red blood cell analysis could be a good indicator of brain plasmalogens.     

Essential Polyunsaturated Fatty Acids in Blood from Patients with and without Catheter-Proven Coronary Artery Disease

Coronary artery disease (CAD) is the leading cause of death worldwide. Statins reduce morbidity and mortality of CAD. Intake of n-3 polyunsaturated fatty acid (n-3 PUFAs), particularly eicosapentaenoic acid (EPA), is associated with reduced morbidity and mortality in patients with CAD. Previous data indicate that a higher conversion of precursor fatty acids (FAs) to arachidonic acid (AA) is associated with increased CAD prevalence. Our study explored the FA composition in blood to assess n-3 PUFA levels from patients with and without CAD. We analyzed blood samples from 273 patients undergoing cardiac catheterization. Patients were stratified according to clinically relevant CAD (n = 192) and those without (n = 81). FA analysis in full blood was performed by gas chromatography. Indicating increased formation of AA from precursors, the ratio of dihomo-gamma-linolenic acid (DGLA) to AA, the delta-5 desaturase index (D5D index) was higher in CAD patients. CAD patients had significantly lower levels of omega-6 polyunsaturated FAs (n-6 PUFA) and n-3 PUFA, particularly EPA, in the blood. Thus, our study supports a role of increased EPA levels for cardioprotection.     

Diatoms and Plants Acyl-CoA:lysophosphatidylcholine Acyltransferases (LPCATs) Exhibit Diverse Substrate Specificity and Biochemical Properties

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).     

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1–3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.     

Isolation of Non-methylene Interrupted or Acetylenic Fatty Acids from Seed Oils Using Semi-preparative Supercritical Chromatography

Preparative scale supercritical fluid chromatography was used for isolating and purifying uncommon non-methylene interrupted or acetylenic polyunsaturated fatty acids ethyl esters from seed oils. Fractionation of Biota orientalis seed oil ethyl esters was performed by supercritical fluid chromatography to obtain juniperonic acid [(5Z,11Z,14Z,17Z)-eicosa-5,11,14,17-tetraenoic acid], a non-methylene interrupted polyunsaturated fatty acid. Fractionation of sandalwood seed oil ethyl esters yielded ximenynic acid [(E)-octadec-11-en-9-ynoic acid], an acetylenic polyunsaturated fatty acid. The effects of CO₂ flow rate, column stationary phase and particle size were explored to optimize ximenynic and juniperonic ethyl ester recovery and purity from ethyl ester mixtures using online UV/Vis detection. Particle size, followed by the stationary phase, were found to be the most important parameters to achieving good separation. Under optimized conditions, ximenynic and juniperonic ethyl ester purities greater than 99 and 95%, respectively, were achieved in a one step process without co-solvent. The isolation and recovery of juniperonic acid from biota seed oil free fatty acids was also attempted. Using free fatty acids as the feed material, the non-methylene interrupted polyunsaturated sciadonic acid was also able to be separated from other compounds including juniperonic acid under some conditions, and gave an increase in concentration of more than 17 times.     

Isolation and Characterization of a Docosahexaenoic Acid-Phospholipids Producing Microorganism Crypthecodinium sp. D31

Thirty-four strains of docosahexaenoic acid (DHA)-producing microorganisms were newly isolated from brackish areas in Japan. These strains showing various compositions of fatty acids. Especially, the fatty acids produced by one of the strains, named D31, had a high DHA content (over 60 % of the total fatty acids) and the simple fatty acid composition (16:0, 18:0, 18:1 and DHA without any other polyunsaturated acids). Although most oleaginous microorganisms accumulate DHA as triacylglycerol, the strain D31 accumulated DHA mainly as a polar lipid (79.4 % of total DHA), especially as phosphatidylcholine (71.4 % of polar DHA). This strain D31 was identified as a related species of Crypthecodinium cohnii on the basis of phylogenetic analysis. Crypthecodinium sp. D31 showed high DHA productivity when cultivated in a medium containing glycerol as the carbon source and a mixture of yeast extract and polypeptone as the nitrogen sources, with a salinity that was equivalent to 50 % of that of seawater and a pH in the acidic range (<pH 6.0). Crypthecodinium sp. D31 is considered as a promising producer of high-purity DHA-containing phospholipids.     

Chemical composition of Pinus sibirica nut oils

The chemical composition of oil obtained from Pinus sibirica was investigated. The nonpolar lipids were the predominant lipid fraction while the triacylglycerols were the major component of this fraction. α‐ and γ‐tocopherols were the dominant tocopherols in pine oils. Eleven fatty acids (FA) were identified in pine nut oil. The unsaturated FA comprised over 90% of the total FA. Of these, polyunsaturated FA accounted for 66% of the total FA. 18:2 and 18:3 acids were the dominant unsaturated FA, while palmitic and stearic acids were the major saturated FA. Three unusual FA, namely 10,13‐octadecadienoic, gorlic and 11,13‐eicosadienoic acid, were tentatively identified in pine nut oil.     

Comparative studies on the lipid profiles of female and male gonads of abalone (Haliotis discus hannai)

Lipid profiles of the lipids from female gonads (LFG) and male gonads (LMG) of abalones (Haliotis discus hannai) were evaluated based on the analysis of phospholipid (PL) molecular species, PL class composition, fatty acid (FA) composition, and lipid class composition. These results suggested that lipids from abalone gonads were abundant in omega-3 polyunsaturated FA (n − 3 PUFA, 14.42%–18.27% of total FAs) and PLs including phosphatidylcholine (PC, 21.26–47.85 mg g⁻¹) and phosphatidylethanolamine (PE, 9.29–24.10 mg g⁻¹). Furthermore, more than 60 molecular species of PC and PE were determined. Particularly, the molecular species containing n − 3 PUFA including eicosapentaenoic acid (EPA) constituted majority of PC and PE in LFG and LMG. By contrast, LFG contained more PC and PE species containing EPA. Considering the lipid profiles, abalone gonads are a potential source of PL-form n − 3 PUFA.     

Influence of different fats in pig feed on fatty acid composition of phospholipids and physical meat quality characteristics

Two feeding experiments (i, ii) were conducted to investigate the influence of different dietary fats on the fatty acid (FA) composition of phospholipids as well as meat quality in pigs. In each experiment 12�4 siblings of Swiss Landrace or Large White breed were allocated to one of four feeding treatments according to sex, breed, and litter and fattened from about 25 to 105 kg liveweight. Pigs were fed a control diet (barley, wheat, soybean meal) or the control diet supplemented with 7% pork fat, 4.95% olive oil or 3.17% soybean oil (i) or 5% of olein or stearin fraction of pork fat or hydrogenated fat (ii). The dietary FA composition was reflected in the FA composition of phospholipids in M. long. dorsi and triceps brachii. However, the unsaturated to saturated ratio was not affected by the dietary intake of polyunsaturated FAs and was only slightly increased by the olive oil supplementation. Trans FAs including conjugated linoleic acid were incorporated into phospholipids only to a small extent. The dietary altered fatty acid composition of phospholipids did not cause any effect on pH, cooking loss, texture, or colour of pork, but meat quality as well as the proportion of saturated FA, arachidonic acid, and n‐3 fatty acids were significantly influenced by genetic effects.