Accumulation Of Eicosapentaenoic Acid Research Articles

Identification of genes regulated by lipids from seaweed Susabinori (Pyropia yezoensis) involved in the improvement of hepatic steatosis: Insights from RNA-Seq analysis in obese db/db mice

Hepatic steatosis is an early stage in the progression of non-alcoholic fatty liver disease (NAFLD) and can lead to the development of non-alcoholic steatohepatitis (NASH), a major cause of liver-related morbidity and mortality. Identification of dietary components that can alleviate hepatic steatosis is crucial for developing effective therapeutic strategies for NAFLD. Recently, we demonstrated the impact of lipids extracted from the marine red alga Susabinori (Pyropia yezoensis) in a murine model of type 2-diabete (db/db). We found that Susabinori lipids (SNL), abundant in eicosapentaenoic acid (EPA)-containing polar lipids, protected against obesity-induced hepatic steatosis in db/db mice. To understand the specific genes or biological pathways underlying the effects of SNL, we conducted RNA-Seq analysis of the hepatic transcriptome. By performing comparative analysis of differentially expressed genes between normal mice and db/db mice consuming a control diet, as well as SNL-fed db/db mice, we identified the 15 SNL-dependent up-regulated genes that were down-regulated in db/db mice but up-regulated by SNL feeding. Gene ontology and pathway analysis on these 15 genes demonstrated a significant association with the metabolisms of arachidonic acid (AA) and linoleic acid (LA). Furthermore, we observed alterations in the expression levels of monoacylglycerol lipase (Magl) and fatty acid-binding protein 4 (Fabp4) in the SNL-fed db/db mice, both of which are implicated in AA and LA metabolism. Additionally, the livers of SNL-fed db/db mice exhibited reduced levels of AA and LA, but a high accumulation of EPA. In conclusion, the SNL diet might affect the metabolisms of AA and LA, which contribute to the improvement of hepatic steatosis. Our findings provide insights into the molecular mechanisms underlying the beneficial effects of SNL.

Effects of soya lecithin on the growth, gonad development, transcription of genes related with fatty acid synthesis and inflammation between sexes of sea urchin Strongylocentrotus intermedius (A. Agassiz, 1864)

A 28-day feeding experiment was performed to investigate the effects of soya lecithin (SL) addition on the growth, gonad development, fatty acid composition, transcription of genes related with fatty acid synthesis and inflammation between sexes of sea urchin Strongylocentrotus intermedius (A. Agassiz, 1864). Three experimental diets were prepared with increasing levels of SL (0%, 1.6%, and 4%). Each diet was randomly assigned to eight male and eight female sea urchins individually cultured in floating cages. The results showed that SL addition significantly decreased the weight gain rate of male sea urchins but had no significant effects on that of female individuals. The gametogenesis of male S. intermedius was markedly accelerated by SL addition at a moderate level (1.6%), reflected by more spermatozoa and fewer nutritive phagocytes in the testis. By comparison, the gametogenesis of female individuals was only slightly promoted by 1.6% and 4% SL addition, with no oocytes observed in the ovary. The contents of arachidonic acid (ARA) in the digestive tract and gonads, and eicosapentaenoic acid (EPA) in the gonads of male and female S. intermedius were increased by SL addition at a moderate level, although their contents were still lower than the initial values. Similarly, the transcription of most fatty acid synthesis-related genes was significantly increased by 1.6% SL addition in both sexes of S. intermedius. The transcription of almost all inflammation-related genes increased with the increase of SL addition in both male and female S. intermedius. Notably, male S. intermedius showed higher ARA contents and transcription of most fatty acid synthesis and inflammation related genes than their female counterparts, especially in the SL1.6 and/or SL4 groups. In summary, SL addition showed no beneficial or negative effects on growth performance but markedly promoted gonad development and ARA and EPA accumulation in S. intermedius, especially at a moderate level (1.6%). Males showed faster gametogenesis than females possibly due to relatively higher ARA synthesis and cyclooxygenase-2 mediated eicosanoid production. The retarded growth and gametogenesis of male S. intermedius fed diets with the addition of 4% SL could be due to excessive inflammation. These results could be helpful for understanding the dosage-dependent and sex-related physiological effects of SL on growth and gonad development of S. intermedius.

Transcriptome Analysis Reveals an Eicosapentaenoic Acid Accumulation Mechanism in a Schizochytrium sp. Mutant.

Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. Schizochytrium is a marine eukaryote that has been widely utilized for the synthesis of PUFAs. The current low potency and performance of EPA production by fermentation of Schizochytrium spp. limits its prospect in commercial production of EPA. Since the synthesis pathway of EPA in Schizochytrium spp. is still unclear, mutagenesis combined with efficient screening methods are still desirable. In this study, a novel screening strategy was developed based on a two-step progressive mutagenesis method based on atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) after multiple stresses (sethoxydim, triclosan and 2,2'-bipyridine) compound screening. Finally, the mutant strain DBT-64 with increased lipid (1.57-fold, 31.71 g/L) and EPA (5.64-fold, 1.86 g/L) production was screened from wild-type (W) strains; the docosahexaenoic acid (DHA) content of mutant DBT-64 (M) was 11.41% lower than that of wild-type strains. Comparative transcriptomic analysis showed that the expression of genes related to the polyketide synthase, fatty acid prolongation, and triglyceride synthesis pathways was significantly upregulated in the mutant strain, while the expression of genes involved in the β-oxidation pathway and fatty acid degradation pathway was downregulated in favor of EPA biosynthesis in Schizochytrium. This study provides an effective strain improvement method to enhance EPA accumulation in Schizochytrium spp. IMPORTANCE Schizochytrium, a marine eukaryotic microorganism, has emerged as a candidate for the commercial production of PUFAs. EPA is an omega-3 PUFA with preventive and therapeutic effects against cardiovascular diseases, schizophrenia, and other disorders. Currently, the low potency and performance of EPA production by Schizochytrium spp. limits its commercialization. In this study, we performed two-step progressive mutagenesis based on ARTP and DES and screened multiple stresses (sethoxydim, triclosan, and 2,2'-bipyridine) to obtain the EPA-high-yielding Schizochytrium mutant. In addition, high expression of the polyketide synthase pathway, fatty acid elongation pathway, and triglyceride synthesis pathway in the mutants was confirmed by transcriptomic analysis. Therefore, the multistress screening platform established in this study is important for breeding EPA-producing Schizochytrium spp. and provides valuable information for regulating the proportion of EPA in microalgal lipids by means of genetic engineering.

Metabolic analysis of Schizochytrium sp. mutants with high EPA content achieved with ARTP mutagenesis screening.

Eicosapentaenoic acid (EPA) belonged to the ω-3 series of polyunsaturated fatty acids and had physiological functions lipid as regulating blood lipid and preventing cardiovascular diseases. Schizochytrium sp. was considered to be a potential industrial fermentation strain of EPA because of its fast growth, high oil content, and simple fatty acid composition. However, Schizochytrium sp. produced EPA with low production efficiency and a long synthesis path. This research aims to improve the yield of EPA in Schizochytrium sp. by ARTP mutagenesis and to reveal the mechanism of high-yield EPA through transcriptome analysis. ARTP mutagenesis screening yielded the mutant M12 that whereas the productivity of EPA increased 108% reaching 0.48g/L, the total fatty acid concentration was 13.82g/L with an increase of 13.7%. The transcriptomics revealed 2995 differentially expressed genes were identified between M12 and the wild-type strain and transcripts involved in carbohydrate, amino acid, energy, and lipid metabolism were up-regulated. Among them, the hexokinase (HK) and the phosphofructokinase genes (PFK), which can catalyze pyruvate to acetyl-CoA, were increased 2.23-fold and 1.78-fold. Glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GLDH), which can both generate NADPH, were increased by 1.67-fold and 3.11-fold. Furthermore, in the EPA synthesis module, the expression of 3-oxoacyl-[acyl-carrier protein] reductase(fabG) and carbonyl reductase 4 / 3-oxoacyl-[acyl-carrier protein] reductase beta subunit(CBR4), also up-regulated 1.11-fold and 2.67-fold. These may lead to increases in cell growth. The results provide an important reference for further research on promoting fatty acid and EPA accumulation in Schizochytrium sp.

Using field evaluation and systematic iteration to rationalize the accumulation of omega-3 long-chain polyunsaturated fatty acids in transgenic Camelina sativa.

SummaryThe Brassicaceae Camelina sativa (gold of pleasure) is now an established niche crop and being used as a transgenic host for a range of novel seed traits. Most notable of these is the accumulation of omega‐3 long‐chain polyunsaturates such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), fatty acids normally only found in marine organisms. As part of continued efforts to optimize the accumulation of these non‐native fatty acids via seed‐specific expression of algal genes, a new series of iterative constructs was built and introduced into Camelina. Seed fatty acid composition was determined, and the presence of EPA and DHA was confirmed. To provide an additional level of evaluation, full environmental release was carried out on selected events, providing a real‐world gauntlet against which to assess the performance of these novel lines. Composition of the seed oil triacylglycerol was determined by mass spectrometry, allowing for conclusions as to the contribution of different activities to the final accumulation of EPA and DHA. Since these data were derived from field‐grown material, they also represent a robust demonstration of the stability of the omega‐3 LC‐PUFA trait in Camelina. We propose that field trialling should be routinely incorporated in the plant synthetic biology ‘design–build–test–learn’ cycle.

An auxin-like supermolecule to simultaneously enhance growth and cumulative eicosapentaenoic acid production in Phaeodactylum tricornutum

Phaeodactylum tricornutum, a model alga, is well known for its ability to accumulate intracellular omega-3 eicosapentaenoic acid (EPA). However, P.tricornutum cells need to have a higher EPA content if they are to be used for industrial applications. In this study, an auxin-like supermolecule (SM) was synthesised and used for the cultivation of P. tricornutum. Results show that the addition of 1 ppm of SM significantly increased the P. tricornutum cell density and boosted the P. tricornutum biomass. The experimental group treated with 5 ppm of SM, had an EPA content of 31.7%, which was a 2.09-fold increase over the EPA content in the untreated group. Overall, our results demonstrated that SM can significantly improve the microalgal growth and EPA accumulation in P. tricornutum, providing a feasible strategy to achieve efficient and cost-effective EPA production.

Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies

The marine alga Nannochloropsis oceanica has been considered as a promising photosynthetic cell factory for synthesizing eicosapentaenoic acid (EPA), yet the accumulation of EPA in triacylglycerol (TAG) is restricted to an extreme low level. Poor channeling of EPA to TAG was observed in N. oceanica under TAG induction conditions, likely due to the weak activity of endogenous diacylglycerol acyltransferases (DGATs) on EPA-CoA. Screening over thirty algal DGATs revealed potent enzymes acting on EPA-CoA. Whilst overexpressing endogenous DGATs had no or slight effect on EPA abundance in TAG, introducing selected DGATs with strong activity on EPA-CoA, particularly the Chlamydomonas-derived CrDGTT1, which resided at the outermost membrane of the chloroplast and provided a strong pulling power to divert EPA to TAG for storage and protection, led to drastic increases in EPA abundance in TAG and TAG-derived EPA level in N. oceanica. They were further promoted by additional overexpression of an elongase gene involved in EPA biosynthesis, reaching 5.9- and 12.3-fold greater than the control strain, respectively. Our results together demonstrate the concept of applying combined pulling and pushing strategies to enrich EPA in algal TAG and provide clues for the enrichment of other desired fatty acids in TAG as well.

High silicate concentration facilitates fucoxanthin and eicosapentaenoic acid (EPA) production under heterotrophic condition in the marine diatom Nitzschia laevis

Nitzschia laevis has the potential for co-production of fucoxanthin and eicosapentaenoic acid (EPA). In this study, the physiological and morphological changes of Nitzschia laevis were investigated under heterotrophic condition with different initial silicate concentrations. High silicate addition stimulated biomass growth and accumulation of fucoxanthin and EPA. The highest yields of biomass (2.41 g/L) and fucoxanthin (32.78 mg/L) were reached with 480 mg/L silicate supplementation. The EPA yield was roughly equal (~70 mg/L) with 240, 480 and 960 mg/L silicate. High silicate concentration (especially 960 mg/L) is a simple strategy to reduce cell aggregation and promote the formation of porous cell structure, which would improve the efficiency of heat and mass transfer during fermentation. In response to high silicate level, extensive metabolic changes are observed, among which the elevated glycolysis and tricarboxylic acid (TCA) cycle might be major contributors to fucoxanthin and fatty acids accumulation by providing additional carbon precursors and energy. This study provided insights for future metabolic engineering to improve fucoxanthin and EPA production in this promising diatom.

High level accumulation of EPA and DHA in field-grown transgenic Camelina - a multi-territory evaluation of TAG accumulation and heterogeneity.

The transgene‐directed accumulation of non‐native omega‐3 long chain polyunsaturated fatty acids in the seed oil of Camelina sativa (Camelina) was evaluated in the field, in distinct geographical and regulatory locations. A construct, DHA2015.1, containing an optimal combination of biosynthetic genes, was selected for experimental field release in the UK, USA and Canada, and the accumulation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) determined. The occurrence of these fatty acids in different triacylglycerol species was monitored and found to follow a broad trend irrespective of the agricultural environment. This is a clear demonstration of the stability and robust nature of the transgenic trait for omega‐3 long chain polyunsaturated fatty acids in Camelina. Examination of non‐seed tissues for the unintended accumulation of EPA and DHA failed to identify their presence in leaf, stem, flower, anther or capsule shell material, confirming the seed‐specific accumulation of these novel fatty acids. Collectively, these data confirm the promise of GM plant‐based sources of so‐called omega‐3 fish oils as a sustainable replacement for oceanically derived oils.

Monitoring of eicosapentaenoic acid (EPA) production in the microalgae Nannochloropsis oceanica

With the increase awareness for a healthier food regime and greener environmental processes, microalgae are being looked as a solution for a sustainable production of polyunsaturated fatty acids, such as omega-3 eicosapentaenoic acid (EPA). Nannochloropsis oceanica is an oleaginous microalga, well-known for the ability of EPA accumulation, although higher lipid productivities are still required to make the process competitive. Therefore, three cultivation parameters were tested in the present work (temperature, light cycles and nitrogen supply) in order to study the EPA profile in the polar and neutral fractions of the cells. In addition, an online monitoring tool based on a fluorescence spectroscopy technique was developed with the aim of increasing process knowledge at real time. The results of this work show that nitrogen depletion induces the highest variability in EPA accumulation in the neutral fraction (triacylglycerols). However, to increase the EPA content in the polar fraction a different strategy needs to be implemented, such as decreasing the cultivation temperature or the light available per cell. Chemometric models were developed through PCA (Principal Component Analysis) and PLS (Projection to Latent Structures), using only fluorescence spectra as inputs, enabling the monitoring of EPA in both fractions separately. High explained variance was observed (above 85%) in both fractions, with R2 above 0.81 and slopes above 0.93 for both validation and training data sets. Lower values of cross-validation and prediction errors were observed (between 0.29 and 0.49% g/gDW). The results obtained show that fluorescence spectroscopy is a powerful technique for online monitoring of non-fluorophore molecules, such as EPA, in complex process like microalgae cultivation.

Fatty Acid Profile of Pacific Oyster, Crassostrea gigas, Fed Different Ratios of Dietary Seaweed and Microalgae during Broodstock Conditioning.

The fatty acid (FA) profile of oysters generally reflects the dietary FA composition. Moreover, incorporation of FA into tissues is modulated by various metabolic factors, and final composition will depend upon the dietary sources, cumulative intake, and oysters' development stage. Thus, the aim of this study was to assess the impact of dietary incorporation of seaweed (SW) Ulva rigida, in replacement of traditional microalgae diet, on the FA composition of Pacific oysters Crassostrea gigas, during broodstock conditioning. The dietary conditioning consisted of direct replacement of microalgae (33% Tisochrysis lutea, 50.25% Skeletonema costatum, and 16.75% Chaetoceros calcitrans) by SW at four different substitution levels (0%, 25%, 50%, and 100% diet). The dietary docosahexaenoic acid (DHA) (22:6n-3) and eicosapentaenoic acid (EPA) (20:5n-3) contents showed a positive correlation with the dietary microalgae level. During the trial, oysters fed with higher percentages of microalgae revealed a depletion of DHA and accumulation of EPA. The 100% SW caused a significant reduction in oxygen consumption and, consequently, in the standard metabolic rate. Based on these results, a partial substitution of up to 25% of dietary microalgae seems to be a suitable alternative, because it elicited similar results to the commercial 100% microalgae diet.

Two Co-Expression Strategies for Eicosapentaenoic Acid Production in Mortierella alpina

The omega-3 long-chain Polyunsaturated Fatty Acid (PUFA) Eicosapentaenoic Acid (EPA) has beneficial effects on human health, leading to its use in health products and foods. The oleaginous microorganism Mortierella alpina has been used in the industrial production of Arachidonic Acid (AA). The omega-3 desaturase oPpFADS17 is a key enzyme in the bioconversion of AA to EPA from Phytophthora parasitica and Glucose-6-Phosphate Dehydrogenase (G6PD2) is a critical enzyme that provide reducing power NADPH for lipid biosynthesis. In this study, we used a double expression cassette and 2A peptide strategies to co-express the these two critical enzymes. Subsequently, we investigated the effects of these strategies on total lipid and EPA accumulation. The recombination strains generated using the above-described strategies exhibited no differences in cell growth, compared with the control strain. Recombination strains generated using the double expression cassette exhibited an increase in total lipids to 43% of the cell dry weight, but did not accumulate EPA. Recombination strains generated using the 2A peptide strategy exhibited increased EPA accumulation, such that this PUFA accounted for 30% of the total lipid content. These co-expression strategies provide the improvements of multi-gene modification in PUFA accumulation in M. alpina.

Lipid Production in Nannochloropsis gaditana during Nitrogen Starvation.

The microalga Nannochloropsis gaditana is a natural producer of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). TAG accumulation is induced by nitrogen starvation. The biomass-specific photon supply rate used had an effect on EPA and TAG accumulation during nitrogen starvation as well as on the localization of EPA accumulation. Clear differences in TAG yield on light were found for different biomass-specific photon supply rates and light regimes during nitrogen starvation. De novo EPA synthesis or the translocation of EPA between lipid fractions might be limiting for EPA accumulation in TAG. Further studies are needed to fully understand EPA accumulation in TAG during nitrogen starvation. To elucidate the function of EPA in TAG nitrogen recovery, experiments are suggested. The overexpression of genes involved in de novo EPA synthesis and translocation is proposed to elucidate the exact metabolic routes involved in these processes during nitrogen starvation. This work addresses future opportunities to increase EPA accumulation.

Combined production of fucoxanthin and EPA from two diatom strains Phaeodactylum tricornutum and Cylindrotheca fusiformis cultures.

Fucoxanthin and eicosapentaenoic acid (EPA) provide significant health benefits for human population. Diatom is a potential natural livestock for the combined production of EPA and fucoxanthin. In this study, first, the effects of three important parameters including light intensity, nitrogen concentration and salinity were evaluated for the production of EPA and fucoxanthin in two diatom strains Phaeodactylum tricornutum and Cylindrotheca fusiformis. And then, two steps method based on light intensity were applied to produce EPA and fucoxanthin in large scale. Higher light intensity was first adopted for the high growth rate and lipid content of diatom, and after a period of time, light intensity was lowered to enhance the accumulation of fucoxanthin and EPA. In final, the highest EPA yields were 62.55 and 27.32mg L-1 for P. tricornutum and C. fusiformis, and the fucoxanthin yield reached 8.32 and 6.05mg L-1, respectively.

Effect of nutritional status and concentration of Nannochloropsis gaditana as enrichment diet for the marine rotifer Brachionus sp

The development of strategies to improve the quality and to optimize the costs of rotifer production for fish larval feeding, particularly those related to microalgal supply, are highly desirable. In the present work, three different rations (80,000, 130,000 or 200,000 cells rotifer−1) of Nannochloropsis gaditana were assessed as enrichment diets for the marine rotifer Brachionus sp. The microalga was cultured in semi-continuous regime with two different daily renewal rates, 10% and 40%, which provided nutrient-depleted or nutrient-sufficient conditions respectively and resulted in strong differences in biochemical composition. Protein and arachidonic acid (ARA) content were twice higher and eicosapentaenoic acid (EPA) was three times higher in N. gaditana from nutrient-sufficient cultures. After 24-h enrichment, rotifers fed N. gaditana cultured in nutrient-sufficient conditions showed higher dry weight, as well as higher contents of protein, lipids and carbohydrates. Total polyunsaturated fatty acids (PUFAs) and ARA contents increased >50%, whereas EPA content doubled in rotifers fed N. gaditana from nutrient-sufficient conditions. As for feed ration, the increase from 80,000 to 130,000 cells individual−1 enhanced the accumulation of protein, lipids, carbohydrates, total PUFAs, ARA and EPA, but a further increase to 200,000 cells individual−1 only enhanced the accumulation of lipid content. Our results show that the nutritional quality of microalgae, rather than concentration, is the most important factor influencing biomass productivity and biochemical composition of rotifer cultures. Since microalgal biomass is in general a limited resource for aquaculture, the improvement of its nutritional value through the optimization of culture conditions should be considered as an adequate and cost-efficient strategy to produce high-quality live feed.

Supplementation of docosahexaenoic acid (DHA) / Eicosapentaenoic acid (EPA) in a ratio of 1/1.3 during the last trimester of pregnancy results in EPA accumulation in cord blood

Omega-3 fatty acids (n-3 FA), specifically DHA, are associated with fetal growth and development. We aimed to determine the levels of DHA and EPA in cord serum after n-3 FA supplementation during the last trimester of pregnancy. Among 55 women, 23 were administered daily one capsule of n-3 FA supplement, involving DHA/EPA in a ratio of 1/1.3. Twenty nine women were enrolled as control group. Blood samples were collected at 22–24 weeks of gestation and at delivery. Fatty acids were analyzed with the method of GC-MS. Cord DHA level increased and EPA level decreased in both groups between the days of 22–24 and delivery. However, decrease in cord EPA level was significant in control group (p < 0.001) but not in supplement group (p > 0.05). Supplementation of DHA/EPA in a ratio of 1/1.3 during the last trimester of pregnancy caused higher cord EPA level compared to control group indicating an accumulation in umbilical cord.

Identification and quantification of triacylglycerols containing n-3 long-chain polyunsaturated fatty acids in bovine milk

The n-3 long-chain polyunsaturated fatty acids (LC-PUFA) are low-abundance components in milk fat, but have great potential in promoting human health. A comprehensive survey on triacylglycerol (TAG) molecular species in milk that contain at least one type of n-3 LC-PUFA, namely eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid, was conducted in this work using HPLC-linear trap quadrupole-Orbitrap and HPLC-triple quadrupole mass spectrometry techniques. A total of 51 TAG species that contain n-3 LC-PUFA have been identified in bovine milk and their structures assigned. The TAG species containing docosahexaenoic acid were found in much smaller number and at much lower abundance compared with the other 2 types of TAG. An HPLC-triple quadrupole mass spectrometry-based method was developed, which provides relative quantification of all these TAG species in a run of 36 min. Application of this method to the quantification of n-3 LC-PUFA-incorporated TAG in 32 individual animal milk samples allowed us to determine variation between animals, identify strong metabolic relationships between TAG species, and reveal negative effect of a grape marc supplement on the accumulation of eicosapentaenoic acid in milk.

Eicosapentaenoic acid (EPA) production by an oleaginous fungus Mortierella alpina expressing heterologous the Δ17‐desaturase gene under ordinary temperature

AbstractThe oleaginous fungus Mortierella alpina is known to accumulate eicosapentaenoic acid (EPA) only when cultivated at a low temperature (below 15°C). Here, we investigated EPA production at an ordinary temperature (28°C) by expressing the Saprolegnia diclina Δ17 desaturase gene (sdd17m) in M. alpina ST1358, an ω3‐desaturation activity‐defective mutant derived from M. alpina 1S‐4. Expression of the exogenous gene was confirmed by EPA accumulation in transformants at both 28 and 12°C. The EPA content in total lipids produced by transformants was over 20% at 28°C. Bench‐scale fermentation with a 5‐L jar fermentor showed that EPA content reached 26.4% of total fatty acids, and EPA production reached 1.8 g/L. This is the first study to report the accumulation of EPA in M. alpina at an ordinary temperature, and provide a platform technology for the industrial production of EPA using M. alpina as a promising source for EPA.Practical applications: This achievement shows the potential of M. alpina for industrial production of EPA at lower cost compared to conventional transgenic organisms such as plants and yeasts. The composition of fatty acids produced by M. alpina transformants was simple and did not contain unusual fatty acids compared with that of fish oils, so it may be acceptable for general consumers. Low‐cost, large‐scale, and high‐purity EPA supply by M. alpina will also lead to development of academic research on physiological functions of EPA and its derivatives.The oleaginous fungus Mortierella alpina accumulates eicosapentaenoic acid (EPA) only when cultivated at a low temperature (below 15°C). Here, we investigated EPA production at an ordinary temperature (28°C) by expressing the Saprolegnia diclina Δ17 desaturase gene (sdd17m) in M. alpina. The transformants with the sdd17m gene produced EPA (&gt;20% in total lipids) at 28°C.

Dietary supplement enriched in antioxidants and omega-3 protects from progressive light-induced retinal degeneration.

In the present study, we have evaluated one of the dietary supplements enriched with antioxidants and fish oil used in clinical care for patient with age-related macular degeneration. Rats were orally fed by a gastric canula daily with 0.2 ml of water or dietary supplement until they were sacrificed. After one week of treatment, animals were either sacrificed for lipid analysis in plasma and retina, or used for evaluation of rod-response recovery by electroretinography (ERG) followed by their sacrifice to measure rhodopsin content, or used for progressive light-induced retinal degeneration (PLIRD). For PLIRD, animals were transferred to bright cyclic light for one week. Retinal damage was quantified by ERG, histology and detection of apoptotic nuclei. Animals kept in dim-cyclic-light were processed in parallel. PLIRD induced a thinning of the outer nuclear layer and a reduction of the b-wave amplitude of the ERG in the water group. Retinal structure and function were preserved in supplemented animals. Supplement induced a significant increase in omega-3 fatty acids in plasma by 168% for eicosapentaenoic acid (EPA), 142% for docosapentaenoic acid (DPA) and 19% for docosahexaenoic acid (DHA) and a decrease in the omega-6 fatty acids, DPA by 28%. In the retina, supplement induced significant reduction of linolenic acid by 67% and an increase in EPA and DPA by 80% and 72%, respectively, associated with significant decrease in omega-6 DPA by 42%. Supplement did not affect rhodopsin content or rod-response recovery. The present data indicate that supplement rapidly modified the fatty acid content and induced an accumulation of EPA in the retina without affecting rhodopsin content or recovery. In addition, it protected the retina from oxidative stress induced by light. Therefore, this supplement might be beneficial to slow down progression of certain retinal degeneration.

Enhancing LC-PUFA production in Thalassiosira pseudonana by overexpressing the endogenous fatty acid elongase genes

The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10–20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.