Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids

The conversion of the plant-derived omega-3 (n-3) α-linolenic acid (ALA, 18:3n-3) to the long-chain eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) can be increased by ALA sufficient diets compared to ALA deficient diets. Diets containing ALA above an optimal level result in no further increase in DHA levels in animals and humans. The present study evaluates means of maximizing plasma DHA accumulation by systematically varying both linoleic acid (LA, 18:2n-6) and ALA dietary level. Weanling rats were fed one of 54 diets for three weeks. The diets varied in the percentage of energy (en%) of LA (0.07–17.1 en%) and ALA (0.02–12.1 en%) by manipulating both the fat content and the balance of vegetable oils. The peak of plasma phospholipid DHA (>8% total fatty acids) was attained as a result of feeding a narrow dietary range of 1–3 en% ALA and 1–2 en% LA but was suppressed to basal levels (～2% total fatty acids) at dietary intakes of total polyunsaturated fatty acids (PUFA) above 3 en%. We conclude it is possible to enhance the DHA status of rats fed diets containing ALA as the only source of n-3 fatty acids but only when the level of dietary PUFA is low (<3 en%).     

Substrate Specificity and Regioselectivity of Δ12 and ω3 Fatty Acid Desaturases from Saccharomyces kluyveri

Δ12 and ω3 fatty acid desaturases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs), which are important constituents of membrane glycerolipids and also precursors to signaling molecules in many organisms. In this study, we determined the substrate specificity and regioselectivity of the Δ12 and ω3 fatty acid desaturases from Saccharomyces kluyveri (Sk-FAD2 and Sk-FAD3). Based on heterologous expression in Saccharomyces cerevisiae, it was found that Sk-FAD2 converted C16–20 monounsaturated fatty acids to diunsaturated fatty acids by the introduction of a second double bond at the ν+3 position, while Sk-FAD3 recognized the ω3 position of C18 and C20. Furthermore, fatty acid analysis of major phospholipids suggested that Sk-FAD2 and Sk-FAD3 have no strong substrate specificity toward the lipid polar head group or the sn-positions of fatty acyl groups in phospholipids.     

大豆ω-3脂肪酸脱氢酶基因GmFAD3C在酿酒酵母中的表达

α亚麻酸(ALA)被称为必需脂肪酸,对人体有一系列的保健作用。ω-3脂肪酸脱氢酶(FAD)催化亚油酸(LA)生成ALA。大豆种子油中ALA含量较高,为了研究大豆ω3FAD的功能,用RTPCR方法从大豆未成熟种子中扩增出GmFAD3C的cDNA,克隆到酵母表达载体p416中,并用醋酸锂法转化酿酒酵母营养缺陷型K601,经筛选鉴定,得到阳性克隆。气相色谱分析脂肪酸成分,发现工程菌产生了新的脂肪成分ALA,含量占总脂肪酸的3.1%,LA含量与对照相比相应地下降,证明该基因编码的蛋白具有催化18碳多不饱和脂肪酸(PUFA)底物LA在Δ15位脱氢生成ALA的ω3FAD功能,首次实现大豆ω-3脂肪酸脱氢酶基因在酿酒酵母K601p416系统中的表达,建立了一种新的高效低成本的FAD酵母表达系统。     

Traits of fatty acid accumulation in dimorphic seeds of the euhalophyte Suaeda salsa in saline conditions

The mechanism on of how salinity affects seed fatty acids accumulation remains unclear in halophytes. The present results revealed that the content of total unsaturated fatty acids in black seeds was higher than in brown seeds in the euhalophyte Suaeda salsa under controlled saline conditions. Salinity (200?mM NaCl) significantly increased the total oil content, unsaturated acid/saturated acid ratio, and content of α-linolenic acid (C18:3) (ALA), especially in brown seeds. The most abundant fatty acid in dimorphic seeds is linoleic acid (C18:2) (>70%). It appears that more ALA accumulated in brown seeds compared to black seeds. The enzyme activity of omega-3 fatty acid desaturase (ω-3 FAD) in brown seeds was much higher than that in black seeds, but salinity had no significant effect on the activity of ω-3 FAD in both brown and black seeds. The relative expression of SsFAD7 was increased by salinity, and the value in brown seeds was much higher than that in black seeds. This means salinity can, salinity can improve the quantity of fatty acids in dimorphic seeds of S. salsa, and the enzyme of ω-3 FAD and SsFAD7 may involve in the accumulation of ALA in dimorphic seeds under salinity.     

Methyl-end desaturases with ∆12 and ω3 regioselectivities enable the de novo PUFA biosynthesis in the cephalopod Octopus vulgaris

The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C₂₀) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C₁₈ PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase (“ωx2”) mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase (“ωx1”) catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C₂₀ ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-¹⁴C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.     

Influence of pumpkin seed cake and extruded linseed on milk production and milk fatty acid profile in Alpine goats

The aim was to determine the effect of substituting pumpkin seed cake (PSC) or extruded linseed (ELS) for soya bean meal in goats’ diets on milk yield, milk composition and fatty acids profile of milk fat. In total, 28 dairy goats were divided into three groups. They were fed with concentrate mixtures containing soya bean meal (Control; n=9), ELS (n=10) or PSC (n=9) as main protein sources in the trial lasting 75 days. Addition of ELS or PSC did not influence milk yield and milk gross composition in contrast to fatty acid profile compared with Control. Supplementation of ELS resulted in greater branched-chain fatty acids (BCFA) and total n-3 fatty acids compared with Control and PSC (P<0.05). Total n-3 fatty acids were accompanied by increased α-linolenic acid (ALA, C18:3n-3; 0.56 g/100 g fatty acids) and EPA (C20:5n-3; 0.12 g/100 g fatty acids) proportions in milk of the ELS group. In contrast, ELS and PSC resulted in lower linoleic acid (LA, C18:2n-6; 2.10 and 2.28 g/100 g fatty acids, respectively) proportions compared with Control (2.80 g/100 g fatty acids; P<0.05). Abovementioned resulted in lower LA/ALA ratio (3.81 v. 7.44 or 6.92, respectively; P<0.05) with supplementation of ELS compared with Control or PSC. The PSC diet decreased total n-6 fatty acids compared with the Control (2.96 v. 3.54 g/100 g fatty acids, P<0.05). Oleic acid (c9-C18:1), CLA (c9,t11-18:2) and t10-,t11-C18:1 did not differ between treatments (P⩾0.08), although stearic acid (C18:0) increased in ELS diets compared with Control (12.7 v. 10.2 g/100 g fatty acids, P<0.05). Partially substituted soya bean meal with ELS in hay-based diets may increase beneficial n-3 fatty acids and BCFA accompanied by lowering LA/ALA ratio and increased C18:0. Pumpkin seed cake completely substituted soya bean meal in the diet of dairy goats without any decrease in milk production or sharp changes in fatty acid profile that may have a commercial or a human health relevancy.     

Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid

Background

Jatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission.

Results

The enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta 12 fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic Jatropha that will help reduce public concerns for environmental issues surrounding genetically modified plants.

Conclusion

In this study we produced seed-specific JcFAD2-1 RNA interference transgenic Jatropha without a selectable marker. We successfully increased the proportion of oleic acid versus linoleic in Jatropha through genetic engineering, enhancing the quality of its oil.     

Dietary linoleic acid has no effect on arachidonic acid,but increases n-6 eicosadienoic acid,and lowers dihomo-γ-linolenic and eicosapentaenoic acid in plasma of adult men

High intakes of linoleic acid (LA,18:2n-6) have raised concern due to possible increase in arachidonic acid (ARA, 20:4n-6) synthesis, and inhibition of alpha linolenic acid (ALA, 18:3n-3) desaturation to eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In healthy men, 10.5% energy compared to 3.8% energy LA with 1% energy ALA increased plasma phospholipid LA and 20:2n-6, the elongation product of LA, and decreased EPA, with no change in ARA. However, LA was inversely related to ARA at both 10.5% energy and 3.8% energy LA, (r=?0.761, r=?0.817, p<0.001, respectively). A two-fold variability in ARA among individuals was not explained by the dietary LA, ARA, ALA, or fish intake. Our results confirm LA requirements for ARA synthesis is low, <3.8% energy, and they suggest current LA intakes saturate Δ-6 desaturation and adversely affect n-3 fatty acid metabolism. Factors other than n-6 fatty acid intake are important modifiers of plasma ARA.     

Histone acetyltransferase general control non‐repressed protein 5 (GCN5) affects the fatty acid composition of Arabidopsis thaliana seeds by acetylating fatty acid desaturase3 (FAD3)

Seed oils are important natural resources used in the processing and preparation of food. Histone modifications represent key epigenetic mechanisms that regulate gene expression, plant growth and development. However, histone modification events during fatty acid (FA) biosynthesis are not well understood. Here, we demonstrate that a mutation of the histone acetyltransferase GCN5 can decrease the ratio of α‐linolenic acid (ALA) to linoleic acid (LA) in seed oil. Using RNA‐Seq and ChIP assays, we identified FAD3, LACS2, LPP3 and PLAIIIβ as the targets of GCN5. Notably, the GCN5‐dependent H3K9/14 acetylation of FAD3 determined the expression levels of FAD3 in Arabidopsis thaliana seeds, and the ratio of ALA/LA in the gcn5 mutant was rescued to the wild‐type levels through the overexpression of FAD3. The results of this study indicated that GCN5 modulated FA biosynthesis by affecting the acetylation levels of FAD3. We provide evidence that histone acetylation is involved in FA biosynthesis in Arabidopsis seeds and might contribute to the optimization of the nutritional structure of edible oils through epigenetic engineering.     

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3^cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.     

“自我剪切”2A肽介导的Δ-12和ω-3脂肪酸脱氢酶以及过氧化氢酶在转基因小鼠肌肉表达研究

哺乳动物因为缺乏Δ-12和ω-3脂肪酸脱氢酶,不能自身合成必需的多不饱和脂肪酸．目前,通过转基因技术在哺乳动物体内表达ω-3脂肪酸脱氢酶,能将长链的n-6多不饱和脂肪酸转化成n-3多不饱和脂肪酸,造成体内长链的n-6多不饱和脂肪酸含量显著减低．本研究通过自我剪切2A肽介导Δ-12和ω-3脂肪酸脱氢酶(FAT-2和FAT-1)以及人过氧化氢酶(human catalase,hCAT)在小鼠的肌肉同时表达．结果表明,转基因小鼠肌肉中长链n-3多不饱和脂肪酸含量提高2.6倍,长链n-6多不饱和脂肪酸含量没有显著变化,而n-6/n-3比例显著降低(P < 0.01)．同时蛋白质印迹检测到人过氧化氢酶hCAT在小鼠的肌肉组织中表达,且过氧化氢酶活性比野生型小鼠显著提高(P < 0.01)．     

Resistance to Magnaporthe grisea in transgenic rice with suppressed expression of genes encoding allene oxide cyclase and phytodienoic acid reductase

Linolenic acid (18:3) and its derivative jasmonic acid (JA) are important molecules in disease resistance in many dicotyledonous plants. We have previously used 18:3- and JA-deficient rice (F78Ri) to investigate the roles of fatty acids and their derivatives in resistance to the blast fungus Magnaporthe grisea [A. Yara, T. Yaeno, J.-L. Montillet, M. Hasegawa, S. Seo, K. Kusumi, K. Iba, Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid, Biochem. Biophys. Res. Commun. 370 (2008) 344-347; A. Yara, T. Yaeno, M. Hasegawa, H. Seto, J.-L. Montillet, K. Kusumi, S. Seo, K. Iba, Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of ω-3 fatty acid desaturases, Plant Cell Physiol. 48 (2007) 1263-1274]. However, because F78Ri plants are suppressed in the first step of the JA biosynthetic pathway, we could not confirm the specific contribution of JA to disease resistance. In this paper, we generated two JA-deficient rice lines (AOCRi and OPRRi) with suppressed expression of the genes encoding allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR), which catalyze late steps in the JA biosynthetic pathway. The levels of disease resistance in the AOCRi and OPRRi lines were equal to that in wild-type plants. Our data suggest that resistance to M. grisea is not dependent on JA synthesis.     

Delta-6 desaturase makes a different fatty acid profile of seed oil in Iranian borage (Echium amoenum Fisch. and Mey.)

Iranian borage (Echium amoenum) from Boraginaceae is a valuable medicinal plant native to Iran and Syria. We determined fatty acid profile and individual fatty acid contents in E. amoenum seed oil using gas chromatography. Nevertheless, the cumulative amount of ω-6 and ω-3 fatty acids in E. amoenum (78.5 %) was in great accordance with those of other species of Echium, Stearidonic acid (SDA, C18:4ω3) and gamma-linolenic acid (GLA, C18:3ω6) constituted only 9.7 % of seed oil in this plant. This observation elucidated the weak activity of delta-6 desaturase (D6DES) in E. amoenum compared with D6DESs of other species. As D6DES enzyme is responsible for converting linoleic acid (LA, C18:2ω6) and alpha-linolenic acid (ALA, C18:3ω3) to GLA and SDA, we isolated coding sequence of D6DES gene and characterized primary structure of the translated protein to probably find some evidences explaining the weak activity of D6DES enzyme in E. amoenum. Gene sequence from E. amoenum showed a high identity of 94–96 % with the other Echium species and the amino acid homology increased by 98 %. All the expected signatures including cytochrome b5 domain and three conserved histidine-rich motifs were found in the translated amino acid sequence. Protein alignment revealed that all the conserved motifs in D6DES sequence from E. amoenum are coincident with its counterparts from other Echium species. However, secondary structure of the enzyme deduced from its primary structure using computational simulation represented obvious differences with D6DES proteins of the other species.     

Variation in the Fatty‐Acid Content in Seeds of Various Black,Red, and White Currant Varieties

Currant seeds, a by‐product of juice production, are recognized as a valuable source of oil rich in polyunsaturated fatty acids. We have evaluated 28 currant varieties for their oil content and fatty‐acid composition. The oil content in the seeds ranged from 18.2–27.7%, and no statistical difference between varieties of different fruit color were recorded. Furthermore, the estimated oil yields in the field production ranged from 26.4–212.4 kg/ha. The GC and GC/MS chemical profiles of the seed oils extracted from all examined varieties were common for currants. Linoleic acid (LA) was the major component, with contents ranging from 32.7–46.9% of total fatty acids, followed by α‐linolenic acid (ALA; 2.9–32.0 %), oleic acid (OA; 9.8–19.9%), γ‐linolenic acid (GLA; 3.3–18.5%), palmitic acid (PA; 4.4–8.1%), stearidonic acid (SDA; 2.2–4.7%), and stearic acid (SA; 1.2–2.4%). Quantitative differences in the fatty‐acid profiles between varieties of different fruit color were observed. Blackcurrant varieties showed significantly higher contents of LA, GLA, and PA than red and white currant varieties, whereas significantly higher amounts of ALA and OL were detected in the red and white varieties. Cluster analysis based on the chemical oil profiles joined the blackcurrants in one group, while most of the red and white cultivars joined in a second group at the same linkage distance.