Functional characterization of a delta 6-desaturase gene from the black seabream (<Emphasis Type="Italic">Acanthopagrus schlegeli</Emphasis>)

Delta 6-fatty acid desaturase (D6DES) is used in the synthesis of polyunsaturated fatty acids (PUFAs) from microorganisms to higher animals, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA). A 1,338 bp full-length cDNA encoding D6DES was cloned from Acanthopagrus schlegeli (AsD6DES) through degenerate- and RACE-PCR methods. A recombinant vector expressing AsD6DES (pYES-AsD6DES) was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity of AsD6DES towards the production of n-6 and n-3 fatty acids. The exogenously expressed AsD6DES produced γ-linolenic acid (18:3 n-6) and stearidonic acid (18:4n-3) at 26 and 36% from exogenous linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), respectively, indicating that it is essentially a delta 6-fatty acid desaturase.     

Identification of Δ9-elongation activity from <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> by heterologous expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The Δ9-elongase isolated from Thraustochytrium aureum, which contains a high level of polyunsaturated fatty acids (PUFAs), was demonstrated to be associated with the synthesis of C20 PUFAs. The TaELO gene contains a 825 bp ORF that encodes a protein of 274 amino acids that shares a high similarity with other PUFA elongases. The expression of the TaELO gene in Pichia pastoris resulted in the elongation of linoleic acid (LA, C18:2; n-6) and α-linolenic acid (ALA, C18:3; n-3) to eicosadienoic acid (EDA, C20:2; n-6) and eicosatrienoic acid (ETrA, C20:3; n-3), respectively. The endogenous conversion rate of LA and ALA to EDA and ETrA was 32.68 and 38.57%, respectively. In addition, TaELO was also able to synthesize eicosenoic acid (C20:1; n-9) from oleic acid (OA, C18:1; n-9), even though the conversion level was low (2.81%). Furthermore, TaELO was able to carry out the 6Δ-elongation of γ-linolenic acid (GLA, C18:3; n-6) to dihomo-γ-linolenic acid (DGLA, C20:3; n-6) and Δ5-elongation of eicosapentaenoic acid (EPA, C20:5; n-3) to docosapentaenoic acid (DPA, C22:5; n-3). The conversion rate of GLA to DGLA and EPA to DPA were 93 and 28.36%, respectively. The TaELO protein was confirmed to have multifunctional activities, such as Δ9, Δ6, and Δ5-elongations as well as the elongation of monounsaturated fatty acid.     

Isolation of a novel C18-Δ9 polyunsaturated fatty acid specific elongase gene from DHA-producing <Emphasis Type="Italic">Isochrysis galbana</Emphasis> H29 and its use for the reconstitution of the alternative Δ8 pathway in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A novel gene (IgASE2) encoding a C18-Δ9 polyunsaturated fatty acids specific (C18-Δ9-PUFAs-specific) elongase was isolated and characterized from DHA-rich microalga, Isochrysis galbana H29. The IgASE2 gene was 1,653 bp in length, contained a 786 bp ORF encoding a protein of 261 amino acids that shared 87% identity with Δ9 elongase, IgASE1, and possessed a 44 bp 5′-untranslated region (5′-UTR) and a 823 bp 3′-untranslated region (3′-UTR). IgASE2, by its heterologous expression in Saccharomyces cerevisiae, elongated linoleic acid (LA, 18:2n−6) and α-linolenic (ALA, 18:3n−3) to eicosadienoic acid (EDA, 20:2n−6) and eicosatrienoic acid (ETrA, 20:3n−3), respectively. The conversions of LA to EDA and ALA to ETrA were 57.6 and 56.1%, respectively. Co-expression of this elongase with Δ8 desaturase required for the synthesis of C20-polyunsaturated fatty acids resulted in the accumulation of dihomo-γ-linolenic acid (20:3n−6) from LA and eicosatetraenoic acid (20:4n−6) from ALA. These results demonstrated that IgASE2 exhibited C18-Δ9-PUFAs-specific elongase activity and the alternative Δ8 pathway was reconstituted.     

Cloning and identification of a novel C18-Δ9 polyunsaturated fatty acid specific elongase gene from DHA-producing <Emphasis Type="Italic">Isochrysis galbana</Emphasis> H29

Isochrysis galbana, a marine prymnesiophyte microalga, is able to produce a high level of long chain polyunsaturated fatty acids such as docosahexaenoic acid (DHA, C22:6n-3). In this article, a novel gene (IgASE2) that encoded a C18-Δ9 polyunsaturase fatty acids specific (C18-Δ9-PUFAs-specific) elongase was isolated and characterized from DHA-rich microalga, I. galbana H29. A full-length cDNA of 1653 bp was cloned by rapidamplification of cDNA ends (RACE) PCR techniques. The IgASE2 contained a 786 bp ORF encoding a protein of 261 amino acids that shared 87% identity with the reported Δ9-elongase IgASE1, a 44 bp 5′ untranslated region and an 823 bp 3′ untranslated region. The function of IgASE2 was demonstrated by its heterologous expression in Saccharomyces cerevisiae. In S. cerevisiae, IgASE2 elongated linoleic acid (LA, C18:2n-6), α-linolenic (ALA, C18:3n-3) to eicosadienoic acid (EDA, C20:2n-6) and eicosatrienoic acid (ETrA, C20:3n-3). The conversion ratios of LA to EDA and ALA to ETrA were 60.47 and 58.36%, respectively. However, IgASE2 could not catalyze the elongation reactions of oleic acid (OA, C18:1n-9) and other fatty acids. These results confirmed that IgASE2 had C18-Δ9-PUFAs-specific elongase activity.     

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5 Δ^{5, 8, 11, 14, 17}). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy. Yun-Tao Li and Mao-Teng Li contributed equally to this work.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.     

Fatty acid compositional changes during the embryonic development of the swimming crab,Portunus pelagicus (Portunidae: Decapoda)

Abstract

The fatty acid composition, moisture, and total lipid of the eggs from the swimming crab, Portunus pelagicus, at three different embryonic stages (within 24 h, during the eye placode stage and the final heart beat stage), were measured. Results showed that the moisture and lipid content significantly increased and decreased (p < 0.05), respectively, as the stages progressed. The most prevalent fatty acids that were initially deposited included C16:0, C18:1n-9, and C18:0, while the most consumed fatty acids were C22:5n-6, C22:5n-3, and C20:1n-7. Among the major fatty acid groups, polyunsaturated fatty acids (PUFA) and long-chain PUFA (LC-PUFA) were consumed more than saturated fatty acids and significantly more (p < 0.05) than monounsaturated fatty acids (p < 0.05). Meanwhile, n-3 PUFA was deposited in significantly higher amounts (p < 0.05) than n-6 PUFA, but both were consumed at similar amounts at 43.4% and 41.3%, respectively. The relatively low amount of C20:5n-3 and C22:6n-3 consumption may indicate these fatty acids were conserved, while the essential fatty acids C18:3n-3 and C18:3n-6 were consumed at high amounts. These findings may have implications for broodstock nutrition in order to formulate a well-balanced diet.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in desaturase activity and the fatty acid composition of microsomal membranes from liver tissue of thermally-acclimating rainbow trout

Summary Rainbow trout (Salmo gairdneri) were acclimated to either 5 or 20°C, and then transferred to the opposite temperature, and changes in the fatty acid composition of liver microsomal membranes and the activities of the hepatic Δ9, Δ6, and Δ5 desaturases were measured at intervals of up to one month post-transfer. Inital changes (days 0–3) in fatty acid composition were: (1) an increase in the proportion of saturates and a decrease in the proportion of polyunsaturates during warm acclimation, and (2) a decrease in the proportion of saturates during cold acclimation. The activity of the Δ6 desaturase approximately doubled immediately following the changes in temperature, but alterations in Δ9 and Δ5 desaturase activities required at least 3 days to occur. The results indicate that desaturase enzymes do not play a major role in the initial adaptation of membrane fatty acid composition to changes in temperature. However, the desaturase enzymes may be involved in the later stages (3–28 days) of the acclimatory process. The proportion of monoenes was well correlated with Δ9 desaturase activity during both transfers, and appeared to be adjusted as required to offset changes in the proportion of polyunsaturates. Supported by National Science Foundation Grant PCM-8301757 to J.R.H.     

Identification of Primula “front-end” desaturases with distinct n−6 or n−3 substrate preferences

cDNA clones encoding cytochrome b₅ fusion desaturases were isolated from Primula cortusoides L. and Primula luteola Ruprecht, species previously shown to preferentially accumulate either n−6 or n−3 Δ6-desaturated fatty acids, respectively. Functional characterisation of these desaturases in yeast revealed that the recombinant Primula enzymes displayed substrate preferences, resulting in the predominant synthesis of either γ-linolenic acid (n−6) or stearidonic acid (n−3). Independent expression of the two Primula desaturases in transgenic Arabidopsis thaliana confirmed these results, with γ-linolenic acid and stearidonic acid accumulating in both leaf and seed tissues to different levels, depending on the substrate specificity of the desaturase. Targeted lipid analysis of transgenic Arabidopsis lines revealed the presence of Δ6-desaturated fatty acids in the acyl-CoA pools of leaf but not seed tissue. The implications for the transgenic synthesis of C₂₀ polyunsaturated fatty acids via the elongation of Δ6-desaturated fatty acids are discussed, as is the potential of using Primula desaturases in the synthesis of C₁₈ n−3 polyunsaturated fatty acids such as stearidonic acid.     

fat-1 transgene expression prevents cell culture-induced loss of membrane n-3 fatty acids in activated CD4+ T-cells

In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4⁺ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4⁺ T-cells from FO-fed WT mice after 3 d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4⁺ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4⁺ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3 d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4⁺ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.     

Effect of cyanocobalamin and p-toluic acid on the fatty acid composition of Schizochytrium limacinum (Thraustochytriaceae, Labyrinthulomycota)

Changes in the fatty acid composition of docosahexaenoic acid (DHA)-producing Schizochytrium limacinum SR21 were investigated. The addition of cyanocobalamin, which is an active component of vitamin B₁₂, decreased the content of odd-chain fatty acids such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). Cyanocobalamin may upregulate the cobalamin-dependent methylmalonyl-CoA mutase, which converts propionic acid to succinic acid, thereby decreasing the content of odd-chain fatty acids. The addition of p-toluic acid resulted in a decrease in docosapentaenoic acid (DPA, 22:5n-6) content and an increase in eicosapentaenoic acid (EPA, 20:5n-3) content in a dose-dependent manner. Two additional peaks of fatty acids, characterized as Δ4,7,10,14-eicosatetraenoic acid (20:4n-7) and Δ4,7,10,14-docosatetraenoic acid (22:4n-9), were detected.     

In Silico Structural Studies and Molecular Docking Analysis of Delta6-desaturase in HUFA Biosynthetic Pathway

Fish are an important source of highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid EPA (20:5 n-3) and docosahexaenoic acid DHA (22:6 n-3) and play a significant role in human nutrition. The fatty acyl delta6-desaturase (Δ6 desaturase) is a rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFA) that converts polyunsaturated fatty acids (PUFA) such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids into HUFA. In this study, fatty acyl Δ6 desaturase was identified from pangasius (Pangasianodon hypophthalmus) and further analyzed for sequenced-based characterization and 3D structural conformation. Sequenced-based analysis revealed some important secondary information such as physicochemical property. e.g., isoelectric point, extinction coefficient, aliphatic index, and grand average hydropathy, among others, and also post-translational modification sites were identified. An evolutionary-conserved stretch of amino acid residue and a functionally significant conserved structural ancestor, N-terminal cytochrome b5 and membrane FADS-like superfamily, were identified. Protein association analysis showed a high confidence score with acyl-CoA synthetase, elovl5, elovl2, and phospholipase A2. Herein, we report, for the first time, a 3D native structure of Δ6 desaturase protein by homology modeling approach; molecular docking analysis was performed with linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, which are the two key substrates in the HUFA biosynthetic pathway. This work provides insight into the structural and functional characterization of Δ6 desaturase, which is involved in HUFA biosynthesis as a rate-limiting enzyme.     

Heterologous Production of Dihomo-γ-Linolenic Acid in Saccharomyces cerevisiae

To make dihomo-γ-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Δ12 fatty acid desaturase, rat Δ6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15°C than in those grown at 20°C, and no DGLA production was observed in the cells grown at 30°C. In NSD at 15°C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15°C, the yield of DGLA reached 2.19 μg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.     

Identification and characterization of a novel enzyme related to the synthesis of PUFAs derived from <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> ATCC 34304

In Thraustochytrids, Thraustochytrium aureum ATCC 34304 was able to produce high levels of several polyunsaturated fatty acids. In the present study, a novel gene encoding protein was cloned from the DHA rich microbe, T. aureum ATCC 34304. The functional analysis of a novel gene was demonstrated by its heterologous expression in Pichia pastoris. The gene was able to synthesize C20 and C22 PUFAs, as well as, to mediate different elongations (Δ9, Δ6, and Δ5) and one Δ5 desaturation activities. The conversion rates of the Δ9 elongation (n-3) and Δ5 desaturation products were found to be higher in response to the novel enzyme than the controls (TaElo and Tad5, respectively). The other Δ9 elongation (n-6) and Δ5 elongation products were slightly lower than those of the control (TaElo). The full length of the 1,374 bp gene contained 458 amino acids that showed very limited homology with desaturases and elongases from various organisms. In addition, the rate of synthesis of PUFAs was evaluated at temperatures ranging from 10 to 30°C. The elongation products were found to decrease dramatically and the desaturation products were found to increase dramatically at 10°C. TaNE was confirmed to be a multifunctional enzyme with higher activity towards Δ6 elongations than Δ9, Δ5 elongations, and Δ5 desaturation.