Identification of a Δ4‐desaturase from the microalga Ostreococcus lucimarinus

Very long chain PUFA (VLCPUFA) like DHA are essential and health‐beneficial components of the human diet. Due to a shortfall in VLCPUFA supply, research is ongoing to establish VLCPUFA production in heterologous systems like for example oilseed plants. For this purpose it is crucial to identify the required enzymes from primary producers of VLCPUFA. Here, we describe a cDNA from the microalga Ostreococcus lucimarinus coding for a Δ4‐fatty acid desaturase. It exhibits a cytochrome b₅ domain fused to its amino terminus and three histidine boxes that are typically found in front‐end desaturases. Heterologous expression of the partly codon‐optimized version of the cDNA in yeast revealed that the encoded protein catalyzes the desaturation of (n‐3)‐ as well as (n‐6)‐substrates with a preference for VLCPUFA. In yeast it localized at the endoplasmic reticulum (ER) membrane and analysis of the product distribution into different lipid classes suggested that the enzyme most likely acts in a lipid‐dependent manner. Practical applications : The identified Δ4‐desaturase may be useful for the production of DHA in transgenic oleaginous organisms like annual oilseed crops.     

Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes

Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our findings suggest that two biosynthetic pathways for PUFAs exist in these organisms.     

EPA and DHA in microalgae: Health benefits,biosynthesis, and metabolic engineering advances

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are ω-3 very long-chain polyunsaturated fatty acids (VLC-PUFAs) that offer a wide range of human health benefits impacting cardiovascular, anti-inflammatory, and neurological health. It is widely known that humans inefficiently synthesize these compounds and as such rely on exogenous dietary sources, such as marine fish oils. Unfortunately, the production of marine fish oils is an unsustainable process and has suffered a dramatic fall in recent years due to overfishing and climate change, as the demand for EPA and DHA continues to rise. Therefore, there is an urgent need to develop alternative, sustainable sources for consumable EPA and DHA. Metabolic engineering of marine microalgae to improve their EPA and DHA productivity is regarded as a promising option that has received increasing commercial attention in recent years. In this mini-review, we describe several notable health benefits of EPA and DHA, summarize the natural sources and biosynthesis of VLC-PUFAS, as well as the recent advances in metabolic engineering of EPA and DHA production in representative microalgal and protist species, including Schizochytrium sp., Phaeodactylum tricornutum, and Nannochloropsis oceanica.     

Dietary Linseed Oil Reduces Growth While Differentially Impacting LC-PUFA Synthesis and Accretion into Tissues in Eurasian Perch (Perca fluviatilis)

The aim of this study was to evaluate the impact of replacing dietary fish oil (FO) with linseed oil (LO) on growth, fatty acid composition and regulation of lipid metabolism in Eurasian perch (Perca fluviatilis) juveniles. Fish (17.5 g initial body weight) were fed isoproteic and isoenergetic diets containing 116 g/kg of lipid for 10 weeks. Fish fed the LO diet displayed lower growth rates and lower levels of DHA in the liver and muscle than fish fed the FO diet, while mortality was not affected by dietary treatment. However, DHA content recorded in the liver and muscle of fish fed the LO diet remained relatively high, despite a weight gain of 134 % and a reduced dietary level of long‐chain polyunsaturated fatty acids (LC‐PUFA), suggesting endogenous LC‐PUFA biosynthesis. This was supported by the higher amounts of pathway intermediates, including 18:4n‐3, 20:3n‐3, 20:4n‐3, 18:3n‐6 and 20:3n‐6, recorded in the liver of fish fed the LO diet in comparison with those fed the FO diet. However, fads2 and elovl5 gene expression and FADS2 enzyme activity were comparable between the two groups. Similarly, the expression of genes involved in eicosanoid synthesis was not modulated by dietary LO. Thus, the present study demonstrated that in fish fed LO for 10 weeks, growth was reduced but DHA levels in tissues were largely maintained compared to fish fed FO, suggesting a physiologically relevant rate of endogenous LC‐PUFA biosynthesis capacity.     

Characteristics of the Fatty Acid Composition of a Deep-Sea Vent Gastropod, <Emphasis Type="Italic">Ifremeria nautilei</Emphasis>

Neutral and polar lipids in the soft parts of a gastropod species, Ifremeria nautilei, collected from deep-sea hydrothermal vents, were examined to assess the trophic relationships in hydrothermal vents. The vent gastropod obtains many of its lipids from symbiotic chemosynthetic microorganisms. The major polyunsaturated fatty acids (PUFA) both in the triacylglycerols and phospholipids of the gastropod consist of a limited number of n-3 and n-6 PUFA: arachidonic acid (20:4n-6), icosapentaenoic acid (20:5n-3), and docosapentaenoic acid (22:5n-3), without docosahexaenoic acid (DHA, 22:6n-3). Noticeable levels of various n-6 PUFA, such as 18:2n-6,9, 20:2n-6,9, 20:3n-6,9,12, and 20:3n-6,9,15 with significant levels of 16:1n-6 and 18:1n-6 indicate the biosynthetic characteristic of the endosymbionts. The lack of DHA in all specimens suggests a limitation of its lipid biosynthesis ability with its symbionts. This finding with regard to the lipids is unusual for a marine animal in the grazing or detrital food chain because many marine animal lipids evidently contain high levels of DHA with low levels of n-6 fatty acids. Such contradictory findings lead to some new insights into the absence of a biosynthetic pathway for DHA in I. nautilei, and provide evidence that DHA in this species is dispensable. Similar to herbivorous gastropods, the lack of DHA with significant levels of n-6 PUFA in this species also indicates its selective assimilation of specific microorganisms, such as chemosynthetic bacteria in hydrothermal vents, because significant levels of DHA were found in carnivorous mollusk lipids.     

Genomic and functional characterization of polyunsaturated fatty acid biosynthesis in Caenorhabditis elegans

The biosynthetic pathway for polyunsaturated fatty acids in the model animal Caenorhabditis elegans was examined in the context of the completed genome sequence. The genomic organization and location of seven desaturase genes and one elongase activity, all previously identified by functional characterization, were elucidated. A pathway for the biosynthesis of polyunsaturated fatty acids in C. elegans was proposed based on these genes. The role of gene duplication in enzyme evolution and proliferation is discussed.     

Effects of different culture media and oxygen upon lipids ofEscherichia coli K-12

The effects of altering the chemical composition of the culture media and the oxygen content of the environment upon the lipid metabolism ofEscherichia coli K-12 were investigated. WhenE. coli cells were grown on the same culture medium but under aerobic and anaerobic conditions, an increase in the free fatty acids of anaerobically grown cells was observed with a disproportionate increase in the unsaturated fatty acids. When glucose was the sole carbon source, both fatty alcohols and hydrocarbons were detected as component lipids of these cells, whether growth occurred under aerobic or anaerobic conditions. Based upon this observation, acetate is considered the initial precursor for fatty alcohol and hydrocarbon biosynthesis. A possible metabolic pathway involving fatty alcohols in hydrocarbon synthesis has been postulated.     

破囊壶菌发酵生产DHA的研究进展

二十二碳六烯酸（DHA）是一种非常重要的多不饱和脂肪酸,能参与人及动物体内多个生理过程。破囊壶菌具有生长迅速、细胞内DHA含量高的特点,是工业化生产DHA的潜力菌。本文主要介绍破囊壶菌DHA代谢通路、影响破囊壶菌生产DHA的因素以及破囊壶菌中试发酵的研究现状。首先,对破囊壶菌合成DHA的两个代谢途径,即脂肪酸合成酶（fatty acid synthesis pathway,FAS）途径和聚酮合酶（polyketide synthesis pathway,PKS）途径进行总结和描述;其次,对影响破囊壶菌发酵生产DHA的三个主要因素（碳氮源、溶氧和温度）进行综述;随后,阐述了破囊壶菌发酵生产DHA的中试放大工艺的研究现状;最后,提出破囊壶菌发酵生产DHA过程中存在的问题,并指出进一步分离获得优质的破囊壶菌菌株、对其代谢途径和关键酶的研究以及中试放大工艺的研究是下一步研究的重点。通过对上述一系列问题进行综述,旨在为利用破囊壶菌工业化生产DHA提供一定的参考。     

Lipid peroxidation during n−3 fatty acid and vitamin E supplementation in humans

The purpose of this study was to investigate in healthy humans the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake, alone or in combination with dL-α-tocopherol acetate (vitamin E) supplements on lipid peroxidation. Eightly men were randomly assigned in a double-blind fashion to take daily for 6 wk either menhaden oil (6.26 g, n−3 fatty acids) or olive oil supplements with either vitamin E (900 IU) or its placebo. Antioxidant vitamins, phospholipid composition, malondialdehyde (MDA), and lipid peroxides were measured in the plasma at baseline and week 6. At the same time, breath alkane output was measured. Plasma α-tocopherol concentration increased in those receiving vitamin E (P<0.0001). In those supplemented with n−3 fatty acids, EPA and DHA increased in plasma phospholipids (P<0.0001) and plasma MDA and lipid peroxides increased (P<0.001 and P<0.05, respectively). Breath alkane output did not change significantly and vitamin E intake did not prevent the increase in lipid peroxidation during menhaden oil supplementation. The results demonstrate that supplementing the diet with n−3 fatty acids resulted in an increase in lipid peroxidation, as measured by plasma MDA release and lipid peroxide products, which was not suppressed by vitamin E supplementation.     

Possible Biosynthetic Pathways for all <Emphasis Type="Italic">cis</Emphasis>-3,6,9,12,15,19,22,25,28-Hentriacontanonaene in Bacteria

A very long chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9), was detected in an eicosapentaenoic acid (EPA)-producing marine bacterium, which was isolated from the mid-latitude seashore of Hokkaido, Japan, and was tentatively identified as mesophilic Shewanella sp. strain osh08 from 16S rRNA gene sequencing. The geometry and position of the double bonds in this compound were determined physicochemically to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25, and 28. Although C31:9 was detected in all of the seven EPA- or/and docosahexaenoic acid-producing bacteria tested, an EPA-deficient mutant (strain IK-1Δ8) of one of these bacteria had no C31:9. Strain IK-1Δ8 had defects in the pfaD gene, one of the five pfa genes responsible for the biosynthesis of EPA. Although Escherichia coli DH5α does not produce EPA or DHA inherently, cells transformed with the pfa genes responsible for the biosynthesis of EPA and DHA produced EPA and DHA, respectively, but not C31:9. These results suggest that the Pfa protein complex is involved in the biosynthesis of C31:9 and that pfa genes must not be the only genes responsible for the formation of C31:9. In this report, we determined for the first time the molecular structure of the C31:9 and discuss the possible biosynthetic pathways of this compound.     

Preparation of Fatty Acid Methyl Esters by Selective Methanolysis of Polar Glycerolipids

KOH in aqueous methanol catalyzes selective methanolysis of polar glycerolipids with O-ester-linked acyl residues, while triacylglycerols and sterol esters are inert in the solution. Based on these findings, a convenient and reliable method was developed for the preparation of fatty acid methyl esters (FAMEs) from polar glycerolipids in lipid mixtures without prior isolation. Methanolysis of polar glycerolipids was completed within 2.5 min by vortexing or 20 min by shaking with 0.7 M KOH/70% (v/v) methanol in the presence of hexane at 30 °C. The yields of FAMEs obtained by the present method were greater than 95%. The method was applied successfully to gas chromatographic analysis of the fatty acid compositions of polar glycerolipids in seed oil and blood. No obvious differences were found between the fatty acid compositions determined by the present method and those determined by conventional methods, including lipid extraction with chloroform/methanol followed by isolation of polar lipids by chromatography. The fatty acid composition of polar glycerolipids, including phospholipids, can be determined readily in many crude samples.     

Effect of different cell disruption methods on lipid yield of Schizochytrium sp.

In this study, it was investigated to increase the lipid yield of the microalgae Schizochytrium sp., by applying different cell disruption methods; acid treatment with HCl, osmotic shock, enzyme applications and ultrasonic homogenizer were combined with the Bligh and Dyer or Soxhlet methods. In the Soxhlet method, the lipid and fatty acid yields decreased due to the inability of the method to break down the lipid cells sufficiently and the high temperature application. Enzyme application (hemicellulase treatment at 55°C for 2 days) prior to Bligh and Dyer method (BDE) was found more efficient in terms of lipid and DHA yield compared to other methods. BDE process increased the lipid yield to 21.72 ± 0.74% and DHA content to 19.25 ± 0.09% from lipid yield of 18.87 ± 0.4% and DHA content of 18.41 ± 0.20% by the Bligh and Dyer control (BDC). Major saturated fatty acids were 14:0, 16:0, 18:0, 24:0 and the highest saturated fatty acid was 16:0 (palmitic acid). Lipid health indices such as n-6/n-3, PUFA/SFA, atherogenicity index, thrombogenicity index and hypocholesterolemic/hypercholesterolemic ratios were almost favorable. With this study, appropriate lipid extraction methods were studied to provide an economical and environmental friendly suggestion for future studies to be used in areas such as food, feed and cosmetics grade. It was concluded that the most convenient method among the cell disruption methods was BDE owing to lipid and fatty acid yield.     

Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185

As a representative of a genus with species considered to be potential commercial producers of the nutritionally important polyunsaturated fatty acid docosahexaenoic acid (DHA), Thraustochytrium sp. ATCC 26185 was investigated to determine its potential for DHA production and lipid composition. Cells from liquid shake cultures contained 32% (w/w) lipid, 18% of which was nonsaponifiable lipid. The major saturated fatty acids (14∶0 and 16∶0) comprised up to 59% of the total fatty acids, and DHA was up to 25% after 6 d incubation. Squalene represented 63% of the nonsaponifiable lipid, and cholesterol composed 41% of the total sterols. The phospholipids expected for eucaryotic microbes were detected with phosphatidylcholine as the major phospholipid at 76% of the total. The ultrastructure of this species was similar to other Thraustochytrium species except that the cells did not have surface scales and they contained unusual membrane-like structures that appeared to be associated with oil formation.     

Specific accumulation of docosahexaenoic acid (22∶6n−3) in brain lipids during development of juvenile turbotScophthalmus maximus L.

The changes in the lipid class and the fatty acid compositions of total lipids and individual glycerophospholipids which occur in brain during development of juvenile turbot (Scophthalmus maximus L.) were investigated. Fish were sampled during a 10-week period immediately following weaning from a live feed to a pellet diet. During this period, brain dry weight increased over 6-fold. The percentages of protein, cholesterol and galactolipids increased in brain during development, presumably reflecting increased membrane maturation and myelination processes. The percentages of docosahexaenoic acid (DHA; 22∶6n−3) were low at the beginning of the study period. However, DHA specifically accumulated in juvenile turbot brain during development. The percentages of DHA increased in total lipid, total diradyl glycerophosphocholine (GPC), total diradyl glycerophosphoethanolamine (GPE), phosphatidylserine (PS) and phosphatidylinositol (PI) reaching 26.1%, 25.8%, 40.8%, 47.1% and 17.9% of the total fatty acids, respectively, by the end of the 10-week period. The percentages of other n−3 polyunsaturated fatty acids (PUFA) and n−6 PUFA generally decreased during this period, as did that of monoenes; the percentages of saturated fatty acids remained relatively constant. Non-linear regression analysis showed that the increase in DHA in total lipid, GPC, GPE and PS fitted 1st order rate kinetics (plus offset) allowing maximum values for the percentages of DHA in each lipid class to be estimated.     

Hepatic microsomal and peroxisomal docosahexaenoate biosynthesis during piglet development

The roles of peroxisomes and microsomes on the biosynthetic pathway for docosahexaenoic acid (DHA) from α-linolenic acid (ALA) were investigated. Microsomes and peroxisomes were prepared from livers of fetal and neonatal piglets by a combination of differential and gradient layer centrifugation. Microsomes, peroxisomes, and combined cell fractions were incubated with [¹³C-U]18∶3n−3. The [M] and [M+18] isotopomers of the fatty acids in the long-chain polyunsaturated fatty acid (LCPUFA) n−3 pathway were detected by gas chromatography-mass spectrometry. The quantity of each fatty acid was determined by gas chromatography, and synthesis of each fatty acid was calculated for a 30-min period. Synthesis of DHA was not detected in combined fetal liver fractions. The data suggest that DHA in the fetus is probably supplied from maternal sources through the placenta. In either singly incubated microsomal or peroxisomal preparations from neonatal livers, no DHA synthesis was detected. After combination of the microsomal and peroxisomal fractions, DHA synthesis was evident and increased rapidly between birth and 2 wk of age. This is the first demonstration of the entire biosynthetic LCPUFA n−3 pathway in subcellular organelles starting from isotopically labeled ALA to the final product, DHA, with all the intermediates present and isotopically labeled. The primary importance of the data is that it unequivocally demonstrates that peroxisomes are required for biosynthesis of DHA from ALA.     

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

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

Retinal fatty acids of piglets fed docosahexaenoic and arachidonic acids from microbial sources

Docosahexaenoic acid (DHA, 22∶6n-3) and arachidonic acid (AA, 20∶4n-6) serve important roles in perinatal visual and neural development. A neonatal pig model was used to determine if dietary supplementation with DHA and AA at slightly greater concentrations than normally found in human milk would influence fatty acid accretion in retina. One-day-old piglets were assigned to one of four diets (n=5/group): (i) STD, standard diet containing fat similar to infant formula; (ii) STD+DHA, 0.7% of fatty acids as DHA; (iii) STD+AA, 0.9% as AA; and (iv) STD+BOTH, 0.8% as DHA plus 1.0% as AA. After 25 d, fatty acids in retina phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were determined. Supplementation with DHA resulted in approximately twofold increases (P<0.05) in PC-DHA (4.88% in STD vs. 10.03% in STD+DHA and 9.47% in STD+BOTH). Similarly, AA supplementation increased PC-AA 1.3–1.4-fold (4.47% in STD vs. 6.19% in STD+AA and 5.70% in STD+BOTH). For PE, supplementation with either fatty acid or in combination resulted in no significant increases, except for a 1.2-fold increase in DHA for STD+BOTH (32.66%) vs. STD (28.38%). Thus, PC responded to dietary supplementation, with addition of DHA, AA, or BOTH, resulting in increases in respective fatty acids; PE was less responsive, with only STD+BOTH resulting in increased DHA. No significant competition between DHA and AA in incorporation into phospholipids was observed. In conclusion, consumption of a combination of DHA and AA by neonatal pigs supported accretion of DHA in retina phospholipids, while simultaneously supplying the AA necessary for membrane phospholipids and eicosanoid biosynthesis. Based on a presentation at the AOCS Annual Meeting & Expo in San Antonio, Texas, May 7–11, 1995.