Isolation and Characterization of the Diatom Phaeodactylum Δ5-Elongase Gene for Transgenic LC-PUFA Production in Pichia pastoris

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host.     

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.     

Polyunsaturated Fatty Acids of Marine Macroalgae: Potential for Nutritional and Pharmaceutical Applications

As mammals are unable to synthesize essential polyunsaturated fatty acids (PUFA), these compounds need to be taken in through diet. Nowadays, obtaining essential PUFA in diet is becoming increasingly difficult; therefore this work investigated the suitability of using macroalgae as novel dietary sources of PUFA. Hence, 17 macroalgal species from three different phyla (Chlorophyta, Phaeophyta and Rhodophyta) were analyzed and their fatty acid methyl esters (FAME) profile was assessed. Each phylum presented a characteristic fatty acid signature as evidenced by clustering of PUFA profiles of algae belonging to the same phylum in a Principal Components Analysis. The major PUFA detected in all phyla were C₁₈ and C₂₀, namely linoleic, arachidonic and eicosapentaenoic acids. The obtained data showed that rhodophytes and phaeophytes have higher concentrations of PUFA, particularly from the n-3 series, thereby being a better source of these compounds. Moreover, rhodophytes and phaeophytes presented “healthier” ∑n-6/∑n-3 and PUFA/saturated fatty acid ratios than chlorophytes. Ulva was an exception within the Chlorophyta, as it presented high concentrations of n-3 PUFA, α-linolenic acid in particular. In conclusion, macroalgae can be considered as a potential source for large-scale production of essential PUFA with wide applications in the nutraceutical and pharmacological industries.     

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.     

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.     

Alternative Sources of n-3 Long-Chain Polyunsaturated Fatty Acids in Marine Microalgae

The main source of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in human nutrition is currently seafood, especially oily fish. Nonetheless, due to cultural or individual preferences, convenience, geographic location, or awareness of risks associated to fatty fish consumption, the intake of fatty fish is far from supplying the recommended dietary levels. The end result observed in most western countries is not only a low supply of n-3 LC-PUFA, but also an unbalance towards the intake of n-6 fatty acids, resulting mostly from the consumption of vegetable oils. Awareness of the benefits of LC-PUFA in human health has led to the use of fish oils as food supplements. However, there is a need to explore alternatives sources of LC-PUFA, especially those of microbial origin. Microalgae species with potential to accumulate lipids in high amounts and to present elevated levels of n-3 LC-PUFA are known in marine phytoplankton. This review focuses on sources of n-3 LC-PUFA, namely eicosapentaenoic and docosahexaenoic acids, in marine microalgae, as alternatives to fish oils. Based on current literature, examples of marketed products and potentially new species for commercial exploitation are presented.     

Antibacterial Activity of Long-Chain Polyunsaturated Fatty Acids against Propionibacterium acnes and Staphylococcus aureus

New compounds are needed to treat acne and superficial infections caused by Propionibacterium acnes and Staphylococcus aureus due to the reduced effectiveness of agents used at present. Long-chain polyunsaturated fatty acids (LC-PUFAs) are attracting attention as potential new topical treatments for Gram-positive infections due to their antimicrobial potency and anti-inflammatory properties. This present study aimed to investigate the antimicrobial effects of six LC-PUFAs against P. acnes and S. aureus to evaluate their potential to treat infections caused by these pathogens. Minimum inhibitory concentrations were determined against P. acnes and S. aureus, and the LC-PUFAs were found to inhibit bacterial growth at 32–1024 mg/L. Generally, P. acnes was more susceptible to the growth inhibitory actions of LC-PUFAs, but these compounds were bactericidal only for S. aureus. This is the first report of antibacterial activity attributed to 15-hydroxyeicosapentaenoic acid (15-OHEPA) and 15-hydroxyeicosatrienoic acid (HETrE), while the anti-P. acnes effects of the six LC-PUFAs used herein are novel observations. During exposure to the LC-PUFAs, S. aureus cells were killed within 15–30 min. Checkerboard assays demonstrated that the LC-PUFAs did not antagonise the antimicrobial potency of clinical agents used presently against P. acnes and S. aureus. However, importantly, synergistic interactions against S. aureus were detected for combinations of benzoyl peroxide with 15-OHEPA, dihomo-γ-linolenic acid (DGLA) and HETrE; and neomycin with 15-OHEPA, DGLA, eicosapentaenoic acid, γ-linolenic acid and HETrE. In conclusion, LC-PUFAs warrant further evaluation as possible new agents to treat skin infections caused by P. acnes and S. aureus, especially in synergistic combinations with antimicrobial agents already used clinically.     

Biosynthesis of Polyunsaturated Fatty Acids in Marine Invertebrates: Recent Advances in Molecular Mechanisms

Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs.     

Omega-3 Fatty Acid Intervention Suppresses Lipopolysaccharide-Induced Inflammation and Weight Loss in Mice

Bacterial endotoxin lipopolysaccharide (LPS)-induced sepsis is a critical medical condition, characterized by a severe systemic inflammation and rapid loss of muscle mass. Preventive and therapeutic strategies for this complex disease are still lacking. Here, we evaluated the effect of omega-3 (n-3) polyunsaturated fatty acid (PUFA) intervention on LPS-challenged mice with respect to inflammation, body weight and the expression of Toll-like receptor 4 (TLR4) pathway components. LPS administration induced a dramatic loss of body weight within two days. Treatment with n-3 PUFA not only stopped loss of body weight but also gradually reversed it back to baseline levels within one week. Accordingly, the animals treated with n-3 PUFA exhibited markedly lower levels of inflammatory cytokines or markers in plasma and tissues, as well as down-regulation of TLR4 pathway components compared to animals without n-3 PUFA treatment or those treated with omega-6 PUFA. Our data demonstrate that n-3 PUFA intervention can suppress LPS-induced inflammation and weight loss via, at least in part, down-regulation of pro-inflammatory targets of the TLR4 signaling pathway, and highlight the therapeutic potential of n-3 PUFA in the management of sepsis.     

Extraction of Fatty Acids and Phenolics from Mastocarpus stellatus Using Pressurized Green Solvents

Polyunsaturated fatty acids are well known for their protective properties in relation to different skin diseases. Although seaweeds possess a low lipid fraction, they could act as an alternative renewable source of polyunsaturated fatty acids whenever other valuable seaweed components are also valorized. In this study, a biorefinery process using Mastocarpus stellatus as a model seaweed was proposed. The process started with the supercritical carbon dioxide extraction of the lipid and phenolic fractions. The influence of pressure during extraction with pure supercritical CO₂ was studied while operating at a selected temperature and solvent flow rate. Kinetic data obtained during the ethanol-modified supercritical CO₂ extraction were fitted to the spline model. Sequential processing was proposed with (i) pure CO₂ to obtain a product with 30% PUFA content and ω-3:ω-6 ratio 1:1, (ii) ethanol-modified CO₂ to extract phenolics, and (iii) microwave-assisted subcritical water extraction operating under previously optimized conditions for the extraction of phenolics, carrageenan and protein fractions. The composition of the supercritical extracts showed potential for use in both dietary and topical applications in skin care products. The remaining solids are suitable for the extraction of other valuable fractions.     

Marine Macroalgae as Sources of Polyunsaturated Fatty Acids

Algae from cold water (Canada) and warm water (China) were analysed for the total lipid content, and for their fatty acid (FA) composition and content. The major findings are that fatty acids (FA) from Canadian algae are generally richer in polyunsaturated FA (PUFA), with a higher n–3/n–6 FA ratio, and a higher degree of total unsaturation. The C 18:4 FA (stearidonic acid, morotic acid as synonym) was detected in greater amounts in cold water samples. The high levels of total PUFA, and especially of n–3 FA in Canadian algae, suggests possible utilization for nutritional purposes.     

Fatty Acid Production and Direct Acyl Transfer through Polar Lipids Control TAG Biosynthesis during Nitrogen Deprivation in the Halotolerant Alga Dunaliella tertiolecta

The aims of this work were to evaluate the contribution of the free fatty acid (FA) pool to triacylglyceride (TAG) biosynthesis and to try to characterize the mechanism by which FA are assimilated into TAG in the green alga Dunaliella tertiolecta. A time-resolved lipidomic analysis showed that nitrogen (N) deprivation induces a redistribution of total lipidome, particularly of free FA and major polar lipid (PL), in parallel to enhanced accumulation of polyunsaturated TAG. The steady-state concentration of the FA pool, measured by prolonged ¹⁴C-bicarbonate pre-labeling, showed that N deprivation induced a 50% decrease in total FA level within the first 24 h and up to 85% after 96 h. The abundance of oleic acid increased from 50 to 70% of total free FA while polyunsaturated FA (PUFA) disappeared under N deprivation. The FA flux, measured by the rate of incorporation of ¹⁴C-palmitic acid (PlA), suggests partial suppression of phosphatidylcholine (PC) acyl editing and an enhanced turnover of the FA pool and of total digalactosyl-diacylglycerol (DGDG) during N deprivation. Taken together, these results imply that FA biosynthesis is a major rate-controlling stage in TAG biosynthesis in D. tertiolecta and that acyl transfer through PL such as PC and DGDG is the major FA assimilation pathway into TAG in that alga and possibly in other green microalgae. Increasing the availability of FA could lead to enhanced TAG biosynthesis and to improved production of high-value products from microalgae.     

Ocean Warming and CO2-Induced Acidification Impact the Lipid Content of a Marine Predatory Gastropod

Ocean warming and acidification are current global environmental challenges impacting aquatic organisms. A shift in conditions outside the optimal environmental range for marine species is likely to generate stress that could impact metabolic activity, with consequences for the biosynthesis of marine lipids. The aim of this study was to investigate differences in the lipid content of Dicathais orbita exposed to current and predicted future climate change scenarios. The whelks were exposed to a combination of temperature and CO₂-induced acidification treatments in controlled flowthrough seawater mesocosms for 35 days. Under current conditions, D. orbita foot tissue has an average of 6 mg lipid/g tissue, but at predicted future ocean temperatures, the total lipid content dropped significantly, to almost half. The fatty acid composition is dominated by polyunsaturated fatty acids (PUFA 52%) with an n-3:6 fatty acid ratio of almost 2, which remains unchanged under future ocean conditions. However, we detected an interactive effect of temperature and pCO₂ on the % PUFAs and n-3 and n-6 fatty acids were significantly reduced by elevated water temperature, while both the saturated and monounsaturated fatty acids were significantly reduced under increased pCO₂ acidifying conditions. The present study indicates the potential for relatively small predicted changes in ocean conditions to reduce lipid reserves and alter the fatty acid composition of a predatory marine mollusc. This has potential implications for the growth and survivorship of whelks under future conditions, but only minimal implications for human consumption of D. orbita as nutritional seafood are predicted.     

The Effect of Low-Dose Marine n-3 Fatty Acids on Plasma Levels of sCD36 in Overweight Subjects: A Randomized,Double-Blind,Placebo-Controlled Trial

CD36 is a scavenger receptor involved in lipid uptake and inflammation. Recently, non-cell-bound CD36 (sCD36) was identified in plasma and suggested to be a marker of lipid accumulation in the vessel wall. Marine n-3 polyunsaturated fatty acids (PUFA) may have cardioprotective effects. This study evaluated the effect of marine n-3 PUFA on sCD36 levels in overweight subjects. Fifty overweight subjects were randomized to 1.1 g of n-3 PUFA or 2 g of olive oil daily for six weeks. Neutrophils were isolated at baseline and after six weeks of treatment while an adipose tissue biopsy was obtained at baseline. The content of n-3 PUFA in adipose tissue and neutrophils was analyzed by gas chromatography, while plasma levels of sCD36 were determined using an enzyme-linked immunosorbent assay (ELISA). After six weeks of supplement plasma sCD36 did not differ between supplements (P = 0.18). There was no significant correlation between plasma sCD36 levels and n-3 PUFA in neutrophils at baseline (r = −0.02, P = 0.88), after six weeks supplement (r = −0.03, P = 0.85) or in adipose tissue (r = 0.14, P = 0.34). This study therefore does not provide evidence for a cardioprotective effect of n-3 PUFA acting through a CD36-dependent mechanism.     

Fatty acid composition of eleven algae from coastal zones of Qatar

The fatty acid composition of eleven species of algae belonging to Rhodophyceae, collected from the coastal zones of Qatar, are reported. The major fatty acids identified were palmitic (16:0), myristic (14:0), oleic (18:1), eicosodienoic (20:2), linoleic (18:2), stearic (18:0) and hexadecaenoic acid (16:1).The fatty acids were characterized by the relatively high abundance of polyunsaturated acids, while the C₁₈ unsaturated acids were appreciably more abundant than the C₂₀ unsaturated acids.     

Characterization and Cytotoxicity Studies of the Rare 21:4 n-7 Acid and Other Polyunsaturated Fatty Acids from the Marine Opisthobranch Scaphander lignarius,Isolated Using Bioassay Guided Fractionation

The marine opisthobranch Scaphander lignarius has been analyzed in the systematic search for novel bioactive compounds in Arctic marine organisms using bioassay guided fractionation. A number of highly cytotoxic fractions were shown to contain mainly polyunsaturated fatty acids (PUFAs). Selected PUFAs were isolated and identified using both liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). It was shown that the opisthobranch contained unusual PUFAs such as several ω3 fatty acids and the ω7 heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) not isolated before. The organism was shown to be a very rich source of PUFAs and the activity of the isolated compounds against a range of human cancer cell lines (melanoma, colon carcinoma and breast carcinoma) is further reported. The ω7 PUFA was significantly more cytotoxic in comparison with reference ω6 arachidonic and ω3 eicosapentaenoic acid. A noteworthy non-selective cytotoxicity against normal lung fibroblasts was also established. The paper contains isolation protocols in addition to cytotoxicity data of the isolated compounds. The potential of marine mollusks as a source for rare PUFAs is also discussed.