Influence of Dietary Lipids and Environmental Salinity on the n-3 Long-Chain Polyunsaturated Fatty Acids Biosynthesis Capacity of the Marine Teleost Solea senegalensis

Fish vary in their ability to biosynthesise long-chain polyunsaturated fatty acids (LC-PUFA) depending upon the complement and function of key enzymes commonly known as fatty acyl desaturases and elongases. It has been reported in Solea senegalensis the existence of a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid (DHA) from eicosapentaenoic acid (EPA), which can be modulated by the diet. The present study aims to evaluate the combined effects of the partial replacement of fish oil (FO) with vegetable oils and reduced environmental salinity in the fatty acid composition of relevant body compartments (muscle, hepatocytes and enterocytes), the enzymatic activity over α-linolenic acid (ALA) to form n-3 LC-PUFA through the incubation of isolated hepatocytes and enterocytes with [1-¹⁴C] 18:3 n-3, and the regulation of the S. senegalensis fads2 and elovl5 in the liver and intestine. The presence of radiolabelled products, including 18:4n-3, 20:4n-3 and EPA, provided compelling evidence that a complete pathway enabling the biosynthesis of EPA from ALA, establishing S. senegalensis, has at least one Fads2 with ∆6 activity. Dietary composition prevailed over salinity in regulating the expression of fads2, while salinity did so over dietary composition for elovl5. FO replacement enhanced the proportion of DHA in S. senegalensis muscle and the combination with 20 ppt salinity increased the amount of n-3 LC-PUFA in hepatocytes.     

Identification and Functional Characterization of Genes Encoding Omega-3 Polyunsaturated Fatty Acid Biosynthetic Activities from Unicellular Microalgae

In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4) from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs) in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ^6,9,12,15). These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in marine microalgae.     

Distinguishing Health Benefits of Eicosapentaenoic and Docosahexaenoic Acids

Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) are recommended for management of patients with wide-ranging chronic diseases, including coronary heart disease, rheumatoid arthritis, dementia, and depression. Increased consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is recommended by many health authorities to prevent (up to 0.5 g/day) or treat chronic disease (1.0 g/day for coronary heart disease; 1.2–4 g/day for elevated triglyceride levels). Recommendations for dietary intake of LC n-3 PUFAs are often provided for α-linolenic acid, and for the combination of EPA and DHA. However, many studies have also reported differential effects of EPA, DHA and their metabolites in the clinic and at the laboratory bench. The aim of this article is to review studies that have identified divergent responses to EPA and DHA, and to explore reasons for these differences. In particular, we review potential contributing factors such as differential membrane incorporation, modulation of gene expression, activation of signaling pathways and metabolite formation. We suggest that there may be future opportunity to refine recommendations for intake of individual LC n-3 PUFAs.     

Profiling of Polar Lipids in Marine Oleaginous Diatom Fistulifera solaris JPCC DA0580: Prediction of the Potential Mechanism for Eicosapentaenoic Acid-Incorporation into Triacylglycerol

The marine oleaginous diatom Fistulifera solaris JPCC DA0580 is a candidate for biodiesel production because of its high lipid productivity. However, the substantial eicosapentaenoic acid (EPA) content in this strain would affect the biodiesel quality. On the other hand, EPA is also known as the essential health supplement for humans. EPAs are mainly incorporated into glycerolipids in the microalgal cell instead of the presence as free fatty acids. Therefore, the understanding of the EPA biosynthesis including the incorporation of the EPA into glycerolipids especially triacylglycerol (TAG) is fundamental for regulating EPA content for different purposes. In this study, in order to identify the biosynthesis pathway for the EPA-containing TAG species, a lipidomic characterization of the EPA-enriched polar lipids was performed by using direct infusion electrospray ionization (ESI)-Q-TRAP-MS and MS/MS analyses. The determination of the fatty acid positional distribution showed that the sn-2 position of all the chloroplast lipids and part of phosphatidylcholine (PC) species was occupied by C16 fatty acids. This result suggested the critical role of the chloroplast on the lipid synthesis in F. solaris. Furthermore, the exclusive presence of C18 fatty acids in PC highly indicated the biosynthesis of EPA on PC. Finally, the PC-based acyl-editing and head group exchange processes were proposed to be essential for the incorporation of EPA into TAG and chloroplast lipids.     

Heterotrophic Production of Omega-3 Long-Chain Polyunsaturated Fatty Acids by Trophically Converted Marine Diatom Phaeodactylum tricornutum

We have created via metabolic engineering a heterotrophic strain of Phaeodactylum tricornutum that accumulates enhanced levels of the high value omega-3 long chain polyunsaturated fatty acid (LC-PUFAs) docosahexaenoic acid (DHA). This was achieved by generation of transgenic strains in which the Δ5-elongase from Ostreococcus tauri was co-expressed with a glucose transporter from the moss Physcomitrella patens. This double transformant has the capacity to grow in the dark in liquid medium supplemented with glucose and accumulate substantial levels of omega-3 LC-PUFAs. The effects of glucose concentrations on growth and LC-PUFA production of wild type and transformed strains cultivated in the light and dark were studied. The highest omega-3 LC-PUFAs accumulation was observed in cultures grown under mixotrophic conditions in the presence of 1% glucose (up to 32.2% of total fatty acids, TFA). Both DHA and EPA are detected at high levels in the neutral lipids of transgenic cells grown under phototrophic conditions, averaging 36.5% and 23.6% of TFA, respectively. This study demonstrates the potential for P. tricornutum to be developed as a viable commercial strain for both EPA and DHA production under mixo- and heterotrophic conditions.     

Evaluation of the relationship between endometriosis and omega-3 and omega-6 polyunsaturated fatty acids

Background: Endometriosis is a common chronic inflammation causing major problems including infertility. The role of omega-3 and omega-6 fatty acids as their potential anti-inflammatory effects in endometriosis needs to be further explored. The objective of this study was to compare serum phospholipid fatty acid profile in endometriosis patients with controls, and to explore the correlation of this profile with the severity of the disease. Methods: Sixty-four endometriosis patients and 74 control women, in reproductive age, participated in this study. Among the endometriosis patients, 19 cases were in stage I, 27 cases in stage II, 8 cases in stage III, and 10 cases in stage IV. Each patient underwent laparoscopy. Before surgery, 5 ml of blood was obtained. After extraction of the total lipids, serum total phospholipid fraction was isolated by thin layer chromatography. Fatty acid composition of the phospholipid fraction was determined by gas chromatography and the resulted profile was compared in endometriosis patients and controls. The profile was also compared in the endometriosis group based on the severity of disease. Results: Stearic acid was significantly lower in the endometriosis group as compared to controls (P= 0.030). No other fatty acid compositions were significantly different between patients and controls. Serum ratio of eicosapentaenoic acid (EPA) to arachidonic acid (AA) was in reasonable correlation with the severity of endometriosis (r = 0.34, P = 0.006). Conclusion: According to these findings, levels of fatty acids in serum total phospholipids seem not to be a marker for endometriosis, but the EPA to AA ratio was a relevant factor indicating severity of illness.Key Words: Endometriosis, Phospholipids, Omega-3 fatty acids     

Effect of marine polyunsaturated fatty acids on biofilm formation of Candida albicans and Candida dubliniensis

The effect of marine polyunsaturated fatty acids on biofilm formation by the human pathogens Candida albicans and Candida dubliniensis was investigated. It was found that stearidonic acid (18:4 n-3), eicosapentaenoic acid (20:5 n-3), docosapentaenoic acid (22:5 n-3) and docosahexaenoic acid (22:6 n-3) have an inhibitory effect on mitochondrial metabolism of both C. albicans and C. dubliniensis and that the production of biofilm biomass by C. dubliniensis was more susceptible to these fatty acids than C. albicans. Ultrastructural differences, which may be due to increased oxidative stress, were observed between treated and untreated cells of C. albicans and C. dubliniensis with formation of rough cell walls by both species and fibrillar structures in C. dubliniensis. These results indicate that marine polyunsaturated fatty acids may be useful in the treatment and/or prevention of biofilms formed by these pathogenic yeasts.     

Temperature Differentially Affects Gene Expression in Antarctic Thraustochytrid Oblongichytrium sp. RT2316-13

Oblongichytrium RT2316-13 synthesizes lipids rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The content of these fatty acids in the total lipids depended on growth temperature. Sequencing technology was used in this work to examine the thraustochytrid’s response to a decrease in growth temperature from 15 °C to 5 °C. Around 4% (2944) of the genes were differentially expressed (DE) and only a few of the DE genes (533 upregulated; 206 downregulated) had significant matches to those in the SwissProt database. Most of the annotated DE genes were related to cell membrane composition (fatty acids, sterols, phosphatidylinositol), the membrane enzymes linked to cell energetics, and membrane structure (cytoskeletal proteins and enzymes). In RT2316-13, the synthesis of long-chain polyunsaturated fatty acids occurred through ω3- and ω6-pathways. Enzymes of the alternative pathways (Δ8-desaturase and Δ9-elongase) were also expressed. The upregulation of the genes coding for a Δ5-desaturase and a Δ5-elongase involved in the synthesis of EPA and DHA, explained the enrichment of total lipid with these two long-chain fatty acids at the low temperature. This molecular response has the potential to be used for producing microbial lipids with a fatty acids profile similar to that of fish oils.     

Accumulation of Astaxanthin by a New Haematococcus pluvialis Strain BM1 from the White Sea Coastal Rocks (Russia)

We report on a novel arctic strain BM1 of a carotenogenic chlorophyte from a coastal habitat with harsh environmental conditions (wide variations in solar irradiance, temperature, salinity and nutrient availability) identified as Haematococcus pluvialis Flotow. Increased (25‰) salinity exerted no adverse effect on the growth of the green BM1 cells. Under stressful conditions (high light, nitrogen and phosphorus deprivation), green vegetative cells of H. pluvialis BM1 grown in BG11 medium formed non-motile palmelloid cells and, eventually, hematocysts capable of a massive accumulation of the keto-carotenoid astaxanthin with a high nutraceutical and therapeutic potential. Routinely, astaxanthin was accumulated at the level of 4% of the cell dry weight (DW), reaching, under prolonged stress, 5.5% DW. Astaxanthin was predominantly accumulated in the form of mono- and diesters of fatty acids from C16 and C18 families. The palmelloids and hematocysts were characterized by the formation of red-colored cytoplasmic lipid droplets, increasingly large in size and number. The lipid droplets tended to merge and occupied almost the entire volume of the cell at the advanced stages of stress-induced carotenogenesis. The potential application of the new strain for the production of astaxanthin is discussed in comparison with the H. pluvialis strains currently employed in microalgal biotechnology.     

Lipids and Composition of Fatty Acids of Saccharina latissima Cultivated Year-Round in Integrated Multi-Trophic Aquaculture

This study is evaluating the seasonal lipid and fatty acid composition of the brown seaweed Saccharina latissima. Biomass was sampled throughout the year (bi-monthly) at the commercial cultivation site near a fish farm in an integrated multi-trophic aquaculture (IMTA) and at a reference site in Denmark (2013–2014). Generally, there was no difference in the biomass composition between sites; however, significant seasonal changes were found. The lipid concentration varied from 0.62%–0.88% dry weight (DW) in July to 3.33%–3.35% DW in November (p < 0.05) in both sites. The fatty acid composition in January was significantly different from all the other sampling months. The dissimilarities were mainly explained by changes in the relative abundance of 20:5n-3 (13.12%–33.35%), 14:0 (11.07%–29.37%) and 18:1n-9 (10.15%–16.94%). Polyunsaturated fatty acids (PUFA’s) made up more than half of the fatty acids with a maximum in July (52.3%–54.0% fatty acid methyl esters; FAME). This including the most appreciated health beneficial PUFA’s, eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), but also arachidonic (ARA) and stearidonic acid (SDA), which are not found in land vegetables such as cabbage and lettuce. Compared to fat (salmon) and lean fish (cod) this seaweed species contains higher proportions of ARA and SDA, but lower EPA (only cod) and DHA. Conclusively, the season of harvest is important for the choice of lipid quantity and quality, but the marine vegetables provide better sources of EPA, DHA and long-chain (LC)-PUFA’s in general compared to traditional vegetables.     

Lipids of Prokaryotic Origin at the Base of Marine Food Webs

In particular niches of the marine environment, such as abyssal trenches, icy waters and hot vents, the base of the food web is composed of bacteria and archaea that have developed strategies to survive and thrive under the most extreme conditions. Some of these organisms are considered “extremophiles” and modulate the fatty acid composition of their phospholipids to maintain the adequate fluidity of the cellular membrane under cold/hot temperatures, elevated pressure, high/low salinity and pH. Bacterial cells are even able to produce polyunsaturated fatty acids, contrarily to what was considered until the 1990s, helping the regulation of the membrane fluidity triggered by temperature and pressure and providing protection from oxidative stress. In marine ecosystems, bacteria may either act as a sink of carbon, contribute to nutrient recycling to photo-autotrophs or bacterial organic matter may be transferred to other trophic links in aquatic food webs. The present work aims to provide a comprehensive review on lipid production in bacteria and archaea and to discuss how their lipids, of both heterotrophic and chemoautotrophic origin, contribute to marine food webs.     

Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum

Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.     

Advances in Technologies for Highly Active Omega-3 Fatty Acids from Krill Oil: Clinical Applications

Euphausia superba, commonly known as krill, is a small marine crustacean from the Antarctic Ocean that plays an important role in the marine ecosystem, serving as feed for most fish. It is a known source of highly bioavailable omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid). In preclinical studies, krill oil showed metabolic, anti-inflammatory, neuroprotective and chemo preventive effects, while in clinical trials it showed significant metabolic, vascular and ergogenic actions. Solvent extraction is the most conventional method to obtain krill oil. However, different solvents must be used to extract all lipids from krill because of the diversity of the polarities of the lipid compounds in the biomass. This review aims to provide an overview of the chemical composition, bioavailability and bioaccessibility of krill oil, as well as the mechanisms of action, classic and non-conventional extraction techniques, health benefits and current applications of this marine crustacean.     

Assessment of Eicosapentaenoic Acid (EPA) Production from Filamentous Microalga Tribonema aequale: From Laboratory to Pilot-Scale Study

It has long been explored to use EPA-rich unicellular microalgae as a fish oil alternative for production of the high-value omega-3 fatty acid eicosapentaenoic acid (EPA, 20:5, n-3). However, none of the efforts have ever reached commercial success. This study reported a filamentous yellow-green microalga Tribonema aequale that possesses the ability to grow rapidly and synthesize significant amounts of EPA. A series of studies were conducted in a glass column photobioreactor under laboratory culture conditions and in pilot-scale open raceway ponds outdoors. The emphasis was placed on the specific nutrient requirements and the key operational parameters in raceway ponds such as culture depth and mixing regimes. When optimized, T. aequale cells contained 2.9% of EPA (w/w) and reached a very high biomass concentration of 9.8 g L⁻¹ in the glass column photobioreactor. The cellular EPA content was increased further to 3.5% and the areal biomass and EPA productivities of 16.2 g m⁻² d⁻¹ and 542.5 mg m⁻² d⁻¹, respectively, were obtained from the outdoor pilot-scale open raceway ponds, which were the record high figures reported thus far from microalgae-based EPA production. It was also observed that T. aequale was highly resistant to microbial contamination and easy for harvesting and dewatering, which provide two additional competitive advantages of this filamentous microalga over the unicellular counterparts for potential commercial production of EPA and other derived co-products.     

Aromatic plants as a source of important phytochemicals: Vitamins, sugars and fatty acids in Cistus ladanifer, Cupressus lusitanica and Eucalyptus gunnii leaves

The phytochemical composition of three aromatic plants (Cistus ladanifer L., Cupressus lusitanica Mill. and Eucalyptus gunnii Hook. f) was evaluated, in order to valorize them as sources of nutraceuticals. Samples were analysed for ascorbic acid by spectrophotometric assay, tocopherols by high performance liquid chromatography (HPLC) coupled to a fluorescence detector, sugars by HPLC coupled to a refraction index detector (RID), and fatty acids by gas-chromatography (GC) coupled to a flame ionization detector (FID). The analysed plants contain very useful compounds such as vitamins (ascorbic acid and tocopherols), reducing sugars (glucose and fructose) and polyunsaturated fatty acids (linoleic, α-linoleic and eicosadienoic acids). Their vitamins and sugars could be useful in diseases related to oxidative stress, in dermatological applications or in cosmetics. The polyunsaturated fatty acids, including omega-3 and omega-6 families, detected in the plants constitute another important class of phytochemicals due to their generalized beneficial health effects.     

TFA and EPA productivities of Nannochloropsis salina influenced by temperature and nitrate stimuli in turbidostatic controlled experiments

The influence of different nitrate concentrations in combination with three cultivation temperatures on the total fatty acids (TFA) and eicosapentaenoic acid (EPA) content of Nannochloropsis salina was investigated. This was done by virtue of turbidostatic controlled cultures. This control mode enables the cultivation of microalgae under defined conditions and, therefore, the influence of single parameters on the fatty acid synthesis of Nannochloropsis salina can be investigated. Generally, growth rates decreased under low nitrate concentrations. This effect was reinforced when cells were exposed to lower temperatures (from 26 °C down to 17 °C). Considering the cellular TFA concentration, nitrate provoked an increase of TFA under nitrate limitation up to 70% of the biological dry mass (BDM). In contrast to this finding, the EPA content decreased under low nitrate concentrations. Nevertheless, both TFA and EPA contents increased under a low culture temperature (17 °C) compared to moderate temperatures of 21 °C and 26 °C. In terms of biotechnological production, the growth rate has to be taken into account. Therefore, for both TFA and EPA production, a temperature of 17 °C and a nitrate concentration of 1800 μmol L⁻¹ afforded the highest productivities. Temperatures of 21 °C and 26 °C in combination with 1800 μmol L⁻¹ nitrate showed slightly lower TFA and EPA productivities.     

Compare the Effect of Eicosapentaenoic Acid and Oxidized Low-Density Lipoprotein on the Expression of CD36 and Peroxisome Proliferator-Activated Receptor Gamma

Background: There is evidence that CD36 promotes foam cell formation through internalizing oxidized LDL (ox-LDL) into macrophages; therefore, it plays a key role in pathogenesis of atherosclerosis. In addition, CD36 expression seems to be mediated by nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ). The aim of the present study was to evaluate and compare the effect of PPAR-γ ligands, eicosapentaenoic acid (EPA) as an anti-atherogenic factor and ox-LDL as an atherogenic factor on CD36 expression. Mechanism of PPAR-γ action and its ligands in CD36 expression were also investigated. Methods: Raw 264.7 macrophage cell line was treated with ox-LDL (100 and 150 μg protein/LDL) and EPA (100 and 200 μM) for 24 and 48 hours in absence or presence of PPAR-γ inhibitor, T0070907. Quantitative real-time PCR and Western-blotting were used for analysis of gene and protein expression, respectively. Results: Raw 264.7 exposures to ox-LDL and EPA resulted in increased expression of CD36 mRNA and protein; however, mRNA and PPAR-γ protein were not up-regulated significantly. Pre-incubation of cells with T0070907 led to decreased expression of CD36 when treated with ox-LDL and EPA. Conclusion: It was confirmed that both EPA and ox-LDL increased CD36 expression but not PPAR-γ, and also co-treatment with PPAR-γ inhibitor decreased CD36 expression. We concluded that up-regulation of CD36 depends on PPAR-γ activation and is not related to increased expression of PPAR-γ. Induction of CD36 by EPA showed that CD36 suppression is not the means by which ω-3 fatty acids (EPA) provide protection against formation of atherosclerotic plaque. Key Words: Atherosclerosis, proliferator-activated receptor gamma (PPAR-γ), Oxidized low density lipoprotein (ox-LDL), Eicosapentaenoic acid (EPA)     

Influence of species, cutting date and cutting interval on the fatty acid composition of grasses

Two experiments were conducted to evaluate the effects of species, cutting date and cutting interval on the concentration of fatty acids in temperate grasses. The first experiment compared eight species, harvested in late autumn and summer. Levels of individual fatty acids were distinctive for some species, with low levels of C18:1 in Dactylis glomerata L. and high levels of C18:2 in Phleum pratense L. Differences in individual fatty acids could not be used to differentiate fescues and ryegrasses. However, fatty acid profiles could be used to differentiate species when material was managed similarly (i.e. at the same cut). There were large species × cut interaction effects, showing that management factors will be as important as plant breeding in manipulating fatty acid levels. Cultivars belonging to one Lolium perenne L. gene pool were identified as having significantly higher α-linolenic acid and total fatty acids in late-season (November) material.
The second experiment compared three ryegrass species over a growing season, with three or five cuts. All species had high concentrations of fatty acids and a high proportion of α-linolenic acid during vegetative growth (late April). Fatty acid levels declined markedly in all species after this date, recovering by autumn. Kunth Lolium multiflorum Lam. and Lolium  ×  boucheanum had higher levels of total fatty acids and α-linolenic acid in the early and late season when compared with perennial ryegrass. Fatty acid levels (particularly C18:2 and C18:3) declined when the regrowth interval was extended from 20 to 38 d.
These studies demonstrate opportunities to change the composition of ruminant products through breeding and management of grasses for altered levels of fatty acids.