Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta.

BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. RESULT: In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). CONCLUSIONS: This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

PfbZIP85 Transcription Factor Mediates ω-3 Fatty Acid-Enriched Oil Biosynthesis by Down-Regulating PfLPAT1B Gene Expression in Plant Tissues.

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the biggest TF families identified so far in the plant kingdom, functioning in diverse biological processes including plant growth and development, signal transduction, and stress responses. For Perilla frutescens, a novel oilseed crop abundant in polyunsaturated fatty acids (PUFAs) (especially α-linolenic acid, ALA), the identification and biological functions of bZIP members remain limited. In this study, 101 PfbZIPs were identified in the perilla genome and classified into eleven distinct groups (Groups A, B, C, D, E, F, G, H, I, S, and UC) based on their phylogenetic relationships and gene structures. These PfbZIP genes were distributed unevenly across 18 chromosomes, with 83 pairs of them being segmental duplication genes. Moreover, 78 and 148 pairs of orthologous bZIP genes were detected between perilla and Arabidopsis or sesame, respectively. PfbZIP members belonging to the same subgroup exhibited highly conserved gene structures and functional domains, although significant differences were detected between groups. RNA-seq and RT-qPCR analysis revealed differential expressions of 101 PfbZIP genes during perilla seed development, with several PfbZIPs exhibiting significant correlations with the key oil-related genes. Y1H and GUS activity assays evidenced that PfbZIP85 downregulated the expression of the PfLPAT1B gene by physical interaction with the promoter. PfLPAT1B encodes a lysophosphatidate acyltransferase (LPAT), one of the key enzymes for triacylglycerol (TAG) assembly. Heterogeneous expression of PfbZIP85 significantly reduced the levels of TAG and UFAs (mainly C18:1 and C18:2) but enhanced C18:3 accumulation in both seeds and non-seed tissues in the transgenic tobacco lines. Furthermore, these transgenic tobacco plants showed no significantly adverse phenotype for other agronomic traits such as plant growth, thousand seed weight, and seed germination rate. Collectively, these findings offer valuable perspectives for understanding the functions of PfbZIPs in perilla, particularly in lipid metabolism, showing PfbZIP85 as a suitable target in plant genetic improvement for high-value vegetable oil production.

Crypthecodinium cohnii Growth and Omega Fatty Acid Production in Mediums Supplemented with Extract from Recycled Biomass.

Crypthecodinium cohnii is a marine heterotrophic dinoflagellate that can accumulate high amounts of omega-3 polyunsaturated fatty acids (PUFAs), and thus has the potential to replace conventional PUFAs production with eco-friendlier technology. So far, C. cohnii cultivation has been mainly carried out with the use of yeast extract (YE) as a nitrogen source. In the present study, alternative carbon and nitrogen sources were studied: the extraction ethanol (EE), remaining after lipid extraction, as a carbon source, and dinoflagellate extract (DE) from recycled algae biomass C. cohnii as a source of carbon, nitrogen, and vitamins. In mediums with glucose and DE, the highest specific biomass growth rate reached a maximum of 1.012 h-1, while the biomass yield from substrate reached 0.601 g·g-1. EE as the carbon source, in comparison to pure ethanol, showed good results in terms of stimulating the biomass growth rate (an 18.5% increase in specific biomass growth rate was observed). DE supplement to the EE-based mediums promoted both the biomass growth (the specific growth rate reached 0.701 h-1) and yield from the substrate (0.234 g·g-1). The FTIR spectroscopy data showed that mediums supplemented with EE or DE promoted the accumulation of PUFAs/docosahexaenoic acid (DHA), when compared to mediums containing glucose and commercial YE.

Impact of low-intensity pulsed ultrasound on the growth of Schizochytrium sp. for omega-3 production.

Schizochytrium sp. is a microalga that is known for its high content of oils or lipids. It has a high percentage of polyunsaturated fatty acids in the accumulated oil, especially docosahexaenoic acid (DHA). DHA is an important additive for the human diet. Large-scale production of Schizochytrium sp. can serve as an alternative source of DHA for humans as well as for fish feed, decreasing the burden on aqua systems. Therefore, research on improving the productivity of Schizochytrium attracts a lot of attention. We studied the potential of using low-intensity pulsed ultrasound (LIPUS) in the growth cycle of Schizochytrium sp. in shake flasks. Different intensities and treatment durations were tested. A positive effect of LIPUS on biomass accumulation was observed in the Schizochytrium sp. culture. Specifically, LIPUS stimulation at the ultrasound intensity of 400 mW/cm2 with 20 min per treatment 10 times a day with equal intervals of 2.4 h between the treatments was found to enhance the growth of Schizochytrium biomass most effectively (by up to 20%). Due to the nature of cell division in Schizochytrium sp. which occurs via zoospore formation, LIPUS stimulation was inefficient if applied continuously during all 5 days of the growth cycle. Using microscopy, we studied the interval between zoospore formation in the culture and selected the optimal LIPUS application days (Days 0-1 and Days 4-5 of the 5-day growth cycle). Microscopic images have also shown that LIPUS stimulation enhances zoospore formation in Schizochytrium sp., leading to more active cell division in the culture. This study shows that LIPUS can serve as an additional tool for cost-efficiency improvement in the large-scale production of Schizochytrium as a sustainable and environmentally friendly source of omega-3 (DHA).

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.

Lowering the culture medium temperature improves the omega-3 fatty acid production in marine microalga Isochrysis sp. CASA CC 101.

Marine microalga Isochrysis sp. contains omega-3 fatty acids like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Environmental factors play a major role in PUFA biosynthesis. Hence, the study focused to optimize factors such as temperature, pH, and photoperiod by response surface methodology (RSM). RSM results showed that the model is significant (p ≤ 0.05) with a high correlation coefficient (R2 = 0.908). The optimum conditions showed that maximum biomass (327 mg/L) at the temperature of 30 °C, pH of 7.5 and 16:8 (Light: Dark cycle), whereas the higher amount of DHA (13.3%) and EPA (9.0%) was observed in the conditions of 18 °C, pH of 7.5 and 16:8 (Light: Dark cycle). The biomass content was directly proportional to the temperature whereas DHA content was inversely proportional. It was revealed that the mRNA expression of EPA and DHA specific desaturases (5Des & 4Des) were significantly elevated in low temperature (20 °C) conditions. The results were highly correlated with the fatty acid profile of Isochrysis sp. grown under low temperature (20 °C) conditions which enhanced the EPA and DHA levels. This study suggests that the temperature is the most influencing factor which can be exploited in the industrial application of DHA and EPA production from Isochrysis sp.

Effect of Nitrogen Sources on Omega-3 Polyunsaturated Fatty Acid Biosynthesis and Gene Expression in Thraustochytriidae sp.

The molecular mechanism that contributes to nitrogen source dependent omega-3 polyunsaturated fatty acid (n-3 PUFA) synthesis in marine oleaginous protists Thraustochytriidae sp., was explored in this study. The fatty acid (FA) synthesis was significantly influenced by the supplement of various levels of sodium nitrate (SN) (1-50 mM) or urea (1-50 mM). Compared with SN (50 mM) cultivation, cells from urea (50 mM) cultivation accumulated 1.16-fold more n-3 PUFAs (49.49% docosahexaenoic acid (DHA) (w/w, of total FAs) and 5.28% docosapentaenoic acid (DPA) (w/w, of total FAs)). Strikingly higher quantities of short chain FAs (<18 carbons) (52.22-fold of that in urea cultivation) were produced from SN cultivation. Ten candidate reference genes (RGs) were screened by using four statistical methods (geNorm, NormFinder, Bestkeeper and RefFinder). MFT (Mitochondrial folate transporter) and NUC (Nucleolin) were determined as the stable RGs to normalize the RT-qPCR (real-time quantitative polymerase chain reaction) data of essential genes related to n-3 PUFAs-synthesis. Our results elucidated that the gene transcripts of delta(3,5)-delta(2,4)-dienoyl-CoA isomerase, enoyl-CoA hydratase, fatty acid elongase 3, long-chain fatty acid acyl-CoA ligase, and acetyl-CoA carboxylase were up-regulated under urea cultivation, contributing to the extension and unsaturated bond formation. These findings indicated that regulation of the specific genes through nitrogen source could greatly stimulate n-3 PUFA production in Thraustochytriidae sp.

Novel Vibrio spp. Strains Producing Omega-3 Fatty Acids Isolated from Coastal Seawater.

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA) (20:5n-3) and docosahexaenoic acid (DHA) (22:6n-3), are considered essential for human health. Microorganisms are the primary producers of omega-3 fatty acids in marine ecosystems, representing a sustainable source of these lipids, as an alternative to the fish industry. Some marine bacteria can produce LC-PUFAs de novo via the Polyunsaturated Fatty Acid (Pfa) synthase/ Polyketide Synthase (PKS) pathway, which does not require desaturation and elongation of saturated fatty acids. Cultivation-independent surveys have revealed that the diversity of microorganisms harboring a molecular marker of the pfa gene cluster (i.e., pfaA-KS domain) is high and their potential distribution in marine systems is widespread, from surface seawater to sediments. However, the isolation of PUFA producers from marine waters has been typically restricted to deep or cold environments. Here, we report a phenotypic and genotypic screening for the identification of omega-3 fatty acid producers in free-living bacterial strains isolated from 5, 500, and 1000 m deep coastal seawater from the Bay of Biscay (Spain). We further measured EPA production in pelagic Vibrio sp. strains collected at the three different depths. Vibrio sp. EPA-producers and non-producers were simultaneously isolated from the same water samples and shared a high percentage of identity in their 16S rRNA genes, supporting the view that the pfa gene cluster can be horizontally transferred. Within a cluster of EPA-producers, we found intraspecific variation in the levels of EPA synthesis for isolates harboring different genetic variants of the pfaA-KS domain. The maximum production of EPA was found in a Vibrio sp. strain isolated from a 1000 m depth (average 4.29% ± 1.07 of total fatty acids at 10 °C, without any optimization of culturing conditions).

Prospective enzymes for omega-3 PUFA biosynthesis found in endoparasitic classes within the phylum Platyhelminthes.

The free-living infectious stages of macroparasites, specifically, the cercariae of trematodes (flatworms), are likely to be significant (albeit underappreciated) vectors of nutritionally important polyunsaturated fatty acids (PUFA) to consumers within aquatic food webs, and other macroparasites could serve similar roles. In the context of de novo omega-3 (n-3) PUFA biosynthesis, it was thought that most animals lack the fatty acid (FA) desaturase enzymes that convert stearic acid (18:0) into ɑ-linolenic acid (ALA; 18:3n-3), the main FA precursor for n-3 long-chain PUFA. Recently, novel sequences of these enzymes were recovered from 80 species from six invertebrate phyla, with experimental confirmation of gene function in five phyla. Given this wide distribution, and the unusual attributes of flatworm genomes, we conducted an additional search for genes for de novo n-3 PUFA in the phylum Platyhelminthes. Searches with experimentally confirmed sequences from Rotifera recovered nine relevant FA desaturase sequences from eight species in four genera in the two exclusively endoparasite classes (Trematoda and Cestoda). These results could indicate adaptations of these particular parasite species, or may reflect the uneven taxonomic coverage of sequence databases. Although additional genomic data and, particularly, experimental study of gene functionality are important future validation steps, our results indicate endoparasitic platyhelminths may have enzymes for de novo n-3 PUFA biosynthesis, thereby contributing to global PUFA production, but also representing a potential target for clinical antihelmintic applications.

Identification and Complete Stereochemical Assignments of the New Resolvin Conjugates in Tissue Regeneration in Human Tissues that Stimulate Proresolving Phagocyte Functions and Tissue Regeneration.

Resolvin conjugates in tissue regeneration (RCTRs) are new chemical signals that accelerate resolution of inflammation, infection, and tissue regeneration. Herein, using liquid chromatography-tandem mass spectrometry-based metabololipidomics, we identified RCTRs in human spleen, lymph node, bone marrow, and brain. In human spleen incubated with Staphylococcus aureus, endogenous RCTRs were increased along with conversion of deuterium-labeled docosahexaenoic acid, conferring pathway activation. Physical and biological properties of endogenous RCTRs were matched with those prepared by total organic synthesis. The complete stereochemical assignment of bioactive RCTR1 is 8R-glutathionyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, RCTR2 is 8R-cysteinylglycinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and RCTR3 is 8R-cysteinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid. These stereochemically defined RCTRs stimulated human macrophage phagocytosis, efferocytosis, and planaria tissue generation. Proteome profiling demonstrated that RCTRs regulated both proinflammatory and anti-inflammatory cytokines with human macrophages. In microfluidic chambers, the three RCTRs limited human polymorphonuclear cell migration. In hind-limb ischemia-reperfusion-initiated organ injury, both RCTR2 and RCTR3 reduced polymorphonuclear cell infiltration into lungs. In infectious peritonitis, RCTR1 shortened the resolution intervals. Each RCTR (1 nmol/L) accelerated planaria tissue regeneration by approximately 0.5 days, with direct comparison to both maresin and protectin CTRs. Together, these results identify a new bioactive RCTR (ie, RCTR3) in human tissues and establish the complete stereochemistry and rank-order potencies of three RCTRs in vivo. Moreover, RCTR1, RCTR2, and RCTR3 each exert potent anti-inflammatory and proresolving actions with human leukocytes.

Endogenous biosynthesis of n-3 long-chain PUFA in Atlantic salmon.

A more efficient utilisation of marine-derived sources of dietary n-3 long-chain PUFA (n-3 LC PUFA) in cultured Atlantic salmon (Salmo salar L.) could be achieved by nutritional strategies that maximise endogenous n-3 LC PUFA synthesis. The objective of the present study was to quantify the extent of n-3 LC PUFA biosynthesis and the resultant effect on fillet nutritional quality in large fish. Four diets were manufactured, providing altered levels of dietary n-3 substrate, namely, 18 : 3n-3, and end products, namely, 20 : 5n-3 and 22 : 6n-3. After 283 d of feeding, fish grew in excess of 3000 g and no differences in growth performance or biometrical parameters were recorded. An analysis of fatty acid composition and in vivo metabolism revealed that endogenous production of n-3 LC PUFA in fish fed a diet containing no added fish oil resulted in fillet levels of n-3 LC PUFA comparable with fish fed a diet with added fish oil. However, this result was not consistent among all treatments. Another major finding of this study was the presence of abundant dietary n-3 substrate, with the addition of dietary n-3 end product (i.e. fish oil) served to increase final fillet levels of n-3 LC PUFA. Specifically, preferential ß-oxidation of dietary C18 n-3 PUFA resulted in conservation of n-3 LC PUFA from catabolism. Ultimately, this study highlights the potential for endogenous synthesis of n-3 LC PUFA to, partially, support a substantial reduction in the amount of dietary fish oil in diets for Atlantic salmon reared in seawater.

Intravenous Lipid Emulsions to Deliver Bioactive Omega-3 Fatty Acids for Improved Patient Outcomes.

Lipids used in intravenous nutrition support (i.e., parenteral nutrition) provide energy, building blocks, and essential fatty acids. These lipids are included as emulsions since they need to be soluble in an aqueous environment. Fish oil is a source of bioactive omega-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid). Lipid emulsions, including fish oil, have been used for parenteral nutrition for adult patients post-surgery (mainly gastrointestinal). This has been associated with alterations in biomarkers of inflammation and immune defense, and in some studies, a reduction in length of intensive care unit and hospital stay. These benefits, along with a reduction in infections, are emphasized through recent meta-analyses. Perioperative administration of fish oil may be superior to postoperative administration, but this requires further exploration. Parenteral fish oil has been used in critically ill adult patients. Here, the influence on inflammatory processes, immune function, and clinical endpoints is less clear. However, some studies found reduced inflammation, improved gas exchange, and shorter length of hospital stay in critically ill patients if they received fish oil. Meta-analyses do not present a consistent picture but are limited by the small number and size of studies. More and better trials are needed in patient groups in which parenteral nutrition is used and where fish oil, as a source of bioactive omega-3 fatty acids, may offer benefits.

Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n-6 to 18:3n-6, 18:2n-6 to 20:2n-6, and 18:3n-3 to 20:3n-3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

Hexadecenoic Fatty Acid Positional Isomers and De Novo PUFA Synthesis in Colon Cancer Cells.

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 µM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.

Complete assessment of whole-body n-3 and n-6 PUFA synthesis-secretion kinetics and DHA turnover in a rodent model.

Previous assessments of the PUFA biosynthesis pathway have focused on DHA and arachidonic acid synthesis. Here, we determined whole-body synthesis-secretion kinetics for all downstream products of PUFA metabolism, including direct measurements of DHA and n-6 docosapentaenoic acid (DPAn-6, 22:5n-6) turnover, and compared n-6 and n-3 homolog kinetics. We infused labeled α-linolenic acid (ALA, 18:3n-3), linoleic acid (LNA, 18:2n-6), DHA, and DPAn-6 as 2H5-ALA, 13C18-LNA, 13C22-DHA, and 13C22-DPAn-6. Eight 11-week-old Long Evans rats fed a 10% fat diet were infused with the labeled PUFAs over 3 h, and plasma enrichment of labeled products was measured every 30 min. The DHA synthesis-secretion rate (94 ± 34 nmol/day) did not differ from other PUFA products (range, 21.8 ± 4.3 nmol/day to 408 ± 116 nmol/day). Synthesis-secretion rates of n-6 and n-3 PUFA homologs were similar, except 22:4n-6 and DPAn-6 had lower synthesis rates. However, daily turnover from newly synthesized DHA (0.067 ± 0.023%) was 56-fold to 556-fold slower than all other PUFA turnover and was 130-fold slower than that determined directly from the total plasma unesterified DHA pool. In conclusion, n-6 and n-3 PUFA synthesis-secretion kinetics suggest that differences in turnover, not in synthesis-secretion rates, primarily determine PUFA plasma levels.

An omega-3 polyunsaturated fatty acid derivative, 18-HEPE, protects against CXCR4-associated melanoma metastasis.

Melanoma has a high propensity to metastasize and exhibits a poor response to classical therapies. Dysregulation of the chemokine receptor gene CXCR4 is associated with melanoma progression, and although n-3 polyunsaturated fatty acids (PUFAs) are known to be beneficial for melanoma prevention, the underlying mechanism of this effect is unclear. Here, we used the n-3 fatty acid desaturase (Fat-1) transgenic mouse model of endogenous n-3 PUFA synthesis to investigate the influence of elevated n-3 PUFA levels in a mouse model of metastatic melanoma. We found that relative to wild-type (WT) mice, Fat-1 mice exhibited fewer pulmonary metastatic colonies and improved inflammatory indices, including reduced serum tumor necrosis factor alpha (TNF-α) levels and pulmonary myeloperoxidase activity. Differential PUFA metabolites in serum were considered a key factor to alter cancer cell travelling to lung, and we found that n-6 PUFAs such as arachidonic acid induced CXCR4 protein expression although n-3 PUFAs such as eicosapentaenoic acid (EPA) decreased CXCR4 levels. In addition, serum levels of the bioactive EPA metabolite, 18-HEPE, were elevated in Fat-1 mice relative to WT mice, and 18-HEPE suppressed CXCR4 expression in B16-F0 cells. Moreover, relative to controls, numbers of pulmonary metastatic colonies were reduced in WT mice receiving intravenous injections either of 18-HEPE or 18-HEPE-pretreated melanoma cells. Our results indicate that 18-HEPE is a potential anticancer metabolite that mediates, at least in part, the preventive effect of n-3 PUFA on melanoma metastasis.

Genome Wide Analysis of Fatty Acid Desaturation and Its Response to Temperature.

Plants modify the polyunsaturated fatty acid content of their membrane and storage lipids in order to adapt to changes in temperature. In developing seeds, this response is largely controlled by the activities of the microsomal ω-6 and ω-3 fatty acid desaturases, FAD2 and FAD3. Although temperature regulation of desaturation has been studied at the molecular and biochemical levels, the genetic control of this trait is poorly understood. Here, we have characterized the response of Arabidopsis (Arabidopsis thaliana) seed lipids to variation in ambient temperature and found that heat inhibits both ω-6 and ω-3 desaturation in phosphatidylcholine, leading to a proportional change in triacylglycerol composition. Analysis of the 19 parental accessions of the multiparent advanced generation intercross (MAGIC) population showed that significant natural variation exists in the temperature responsiveness of ω-6 desaturation. A combination of quantitative trait locus (QTL) analysis and genome-wide association studies (GWAS) using the MAGIC population suggests that ω-6 desaturation is largely controlled by cis-acting sequence variants in the FAD2 5' untranslated region intron that determine the expression level of the gene. However, the temperature responsiveness of ω-6 desaturation is controlled by a separate QTL on chromosome 2. The identity of this locus is unknown, but genome-wide association studies identified potentially causal sequence variants within ∼40 genes in an ∼450-kb region of the QTL.

Defatted Microalgae-Mediated Enrichment of n-3 Polyunsaturated Fatty Acids in Chicken Muscle Is Not Affected by Dietary Selenium, Vitamin E, or Corn Oil.

Background: We previously showed enrichments of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in broiler chicks fed defatted microalgae. Objectives: The aims of this study were to determine 1) if the enrichments affected meat texture and were enhanced by manipulating dietary corn oil, selenium, and vitamin E concentrations and 2) how the enrichments corroborated with hepatic gene expression involved in biosynthesis and oxidation of EPA and DHA. Methods: Day-old hatching Cornish Giant cockerels (n = 216) were divided into 6 groups (6 cages/group and 6 chicks/cage). Chicks were fed 1 of the 6 diets: a control diet containing 4% corn oil, 25 IU vitamin E/kg, and 0.2 mg Se/kg (4CO), 4CO + 10% microalgae (defatted Nannochloropsis oceanica; 4CO+ MA), 4CO+ MA - 2% corn oil (2CO+MA), 2CO+MA + 75 IU vitamin E/kg (2CO+MA+E), 2CO+MA + 0.3 mg Se/kg (2CO+MA+Se), and 2CO+MA+E + 0.3 mg Se/kg (2CO+MA+E+Se). After 6 wk, fatty acid profiles, DHA and EPA biosynthesis and oxidation, gene expression, lipid peroxidation, antioxidant status, and meat texture were measured in liver, muscles, or both. Results: Compared with the control diet, defatted microalgae (4CO+MA) enriched (P < 0.05) DHA and EPA by ≤116 and 24 mg/100 g tissue in the liver and muscles, respectively, and downregulated (41-76%, P < 0.01) hepatic mRNA abundance of 4 cytochrome P450 (CYP) enzymes (CYP2C23b, CYP2D6, CYP3A5, CYP4V2). Supplemental microalgae decreased (50-82%, P < 0.05) lipid peroxidation and improved (16-28%, P < 0.05) antioxidant status in the liver, muscles, or both. However, the microalgae-mediated enrichments in the muscles were not elevated by altering dietary corn oil, vitamin E, or selenium and did not affect meat texture. Conclusion: The microalgae-mediated enrichments of DHA and EPA in the chicken muscles were associated with decreased hepatic gene expression of their oxidation, but were not further enhanced by altering dietary corn oil, vitamin E, or selenium.

Growth medium sterilization using decomposition of peracetic acid for more cost-efficient production of omega-3 fatty acids by Aurantiochytrium.

Aurantiochytrium can produce significant amounts of omega-3 fatty acids, specifically docosahexaenoic acid and docosapentaenoic acid. Use of a glucose-based medium for heterotrophic growth is needed to achieve a high growth rate and production of abundant lipids. However, heat sterilization for reliable cultivation is not appropriate to heat-sensitive materials and causes a conversion of glucose via browning (Maillard) reactions. Thus, the present study investigated the use of a direct degradation of Peracetic acid (PAA) for omega-3 production by Aurantiochytrium. Polymer-based bioreactor and glucose-containing media were chemically co-sterilized by 0.04% PAA and neutralized through a reaction with ferric ion (III) in HEPES buffer. Mono-cultivation was achieved without the need for washing steps and filtration, thereby avoiding the heat-induced degradation and dehydration of glucose. Use of chemically sterilized and neutralized medium, rather than heat-sterilized medium, led to a twofold faster growth rate and greater productivity of omega-3 fatty acids.

Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b.

Thraustochytrids are marine single-cell protists that produce large amounts of PUFAs, such as DHA. They accumulate PUFAs in lipid droplets (LDs), mainly as constituent(s) of triacylglycerol (TG). We identified a novel protein in the LD fraction of Aurantiochytrium limacinum F26-b using 2D-difference gel electrophoresis. The protein clustered with orthologs of thraustochytrids; however, the cluster was evolutionally different from known PAT family proteins or plant LD protein; thus, we named it thraustochytrid-specific LD protein 1 (TLDP1). TLDP1 surrounded LDs when expressed as a GFP-tagged form. Disruption of the tldp1 gene decreased the content of TG and number of LDs per cell; however, irregular and unusually large LDs were generated in tldp1-deficient mutants. Although the level of TG synthesis was unchanged by the disruption of tldp1, the level of TG degradation was higher in tldp1-deficient mutants than in the WT. These phenotypic abnormalities in tldp1-deficient mutants were restored by the expression of tldp1 These results indicate that TLDP1 is a thraustochytrid-specific LD protein and regulates the TG accumulation and LD morphology in A. limacinum F26-b.