Delta-6 Desaturase Gene Research Articles

Nontargeted metabolomic and multigene expression analyses reveal the mechanism of oil biosynthesis in sea buckthorn berry pulp rich in palmitoleic acid

Sea buckthorn berry pulp (SBP) oil is abundant in palmitoleic acid (C16:1). However, metabolic mechanisms of oil biosynthesis in SBP (non-seed tissues) are not clear. Thus, comparative nontargeted metabolomic analysis of the four developmental stages of berry pulp in two lines, Za56 and TF2-36, was performed. The results revealed that glycerol-3-phosphate (G3P) was critical for high oil accumulation in the mid-early developmental stages. In particular, the metabolism of phosphatidylcholine (PC) (16:0/16:0), PC (16:0/16:1), and PC (16:1/16:1) was also significantly altered. Sufficient supply of G3P and 16:1-CoA, coupled with upregulated expression of the glycerol-3-phosphate dehydrogenase (GPD1) and delta-9 desaturase (Δ9D) genes, were associated with high oil content enriched in C16:1 in SBP. Our results provide a scientific basis for the development of metabolic engineering strategies to increase the oil content in SBP with a high level of C16:1.

Optimizing the fatty acid profile of novel terrestrial oil blends in low fishmeal diets of rainbow trout (Oncorhynchus mykiss) yields comparable fish growth, total fillet n-3 LC-PUFA content, and health performance relative to fish oil

Identifying and effectively utilizing suitable, novel, terrestrial oil sources either alone or as blends to replace fish oil (FO) is a prerequisite for improving the sustainability of global aquaculture. Therefore, the present study evaluates several novel terrestrial oil blends (TOBs), optimized for their fatty acid profile, as alternatives to FO in low fishmeal diets fed to rainbow trout (Oncorhynchus mykiss) and describes the subsequent effects on fish growth, fatty acid composition and health. Insect oil (IO), genetically modified canola oil (CO), palm oil (PO) and linseed oil (LO) were used for the formulation of three TOBs viz., TOB-1 (30%IO+36%CO + 34%LO), TOB-2 (40%PO + 20%CO + 40%LO) and TOB-3 (50%TOB-1 + 50%TOB-2). Formulas TOB-1 and TOB-2 considered the total fatty acid profile based upon the general FO fatty acid profile, published fatty acid research for rainbow trout, and the fatty acid requirements of rainbow trout. A low fishmeal based basal diet containing 44% crude protein was formulated, and FO, TOB-1, TOB-2 and TOB-3 were incorporated in the basal diet to prepare the experimental diet groups, Control, TOB-1, TOB-2 and TOB-3, respectively. All experimental diets were fed to triplicate groups of rainbow trout juveniles (initial weight 7.9 ± 0.02 g) for 9 weeks. Growth performance (final weight, percent weight gain and specific growth rate) in TOBs fed groups were equal to the FO-based control group. Fish fed the TOB-3 diet consumed more feed followed by the control and TOB-1 diet groups. Significantly lower feed intake was observed in the TOB-2 diet group. Feed conversion ratio and protein efficiency ratio were not significantly influenced by dietary oil sources. Fish fed the control diet showed significantly higher hepatic lipid content compared to TOB groups, followed by TOB-2, TOB-3, and TOB-1, which was significantly lower in hepatic lipid content. Muscle lipid content and whole-body major nutrient compositions were not significantly influenced by the dietary oil sources. Fatty acid composition of muscle and liver reflected that of the diets. Maximum values for n3 LC-PUFAs (EPA and DHA), lauric acid (C12:0) and C18:3n-3 were observed in the FO, TOB-1 and TOB-2 groups, respectively. Except for C12:0, muscle saturated fatty acid contents were significantly lower in TOBs-based diet compared to the FO-based control diet fish. As expected, muscle C12:0 content was significantly higher in the TOB-1 group followed by the TOB-3 group. TOB-2 and control groups had significantly lower content of C12:0. The fillet total n-3 LC-PUFA was significantly higher in fish fed the control diet group followed by TOB-3 and TOB-2 groups, TOB-1 showed significantly lower content of total n-3 LC- PUFA. Hepatic delta-5 desaturase (Δ5fad), delta-6 desaturase (Δ 6fad) and fatty acid elongase-2 (Elovl-2) gene expressions were significantly influenced by dietary oil sources, where TOB-based groups showed higher Δ6fad and Elovl-2 expressions. Measured innate immunity and antioxidant markers were not affected by TOBs. Finally, we concluded that TOBs could serve as a substitute for FO in rainbow trout feed without negatively impacting performance. Moreover, fillet total n-3 LC-PUFA of TOB-fed fish satisfies the suggested daily serving for humans.

Fatty acid desaturation in red blood cell membranes of patients with type 2 diabetes is improved by zinc supplementation

Background/ObjectiveMembrane flexibility can be a determining factor in pathophysiological mechanisms of type 2 diabetes (T2D). As a cofactor of delta-5 desaturase (D5D) and delta-6 desaturase (D6D), and gene expression regulator, zinc may play a role modulating membrane flexibility by increasing membrane polyunsaturated fatty acids (PUFA) abundance. The objective of this study was to evaluate the effect of a 24-month zinc supplementation (30 mg elemental zinc) on membrane fatty acid composition in patients with T2D. Subjects/methodsSixty patients with T2D were evaluated. Thirty were randomly assigned to the zinc supplemented group and thirty to the placebo group. Fatty acid composition in red blood cell (RBC) membranes was determined by gas chromatography. Expression of gene encoding for D5D (FADS1), and D6D (FADS2) were evaluated in peripheral blood mononuclear cells by real-time polymerase chain reaction. ResultsAfter 24 months of supplementation, a greater abundance of docosapentaenoic acid (C22:5 n-3), arachidonic acid (C20:4 n-6), adrenic acid (C22:4 n-6), and total n-6 PUFA was found (p = 0.001, p = 0.007, p = 0.033, p = 0.048, respectively). The unsaturated fatty acids/saturated fatty acids ratio, and unsaturation index was increased in the zinc supplemented group at month 24 (p = 0.003 and p = 0.000, respectively). FADS1 gene was upregulated in the zinc group in relation to placebo at month 12 (p = 0.020). ConclusionsSupplementation with 30 mg/d elemental zinc during 24 months in patients with T2D had an effect on the composition of RBC membranes increasing PUFA abundance and in turn, improving membrane flexibility. This effect may be mediated by induction of D5D gene expression.

Co-expression of fat1 and fat2 in transgenic pigs promotes synthesis of polyunsaturated fatty acids.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are essential for the development and health of mammals, such as humans and livestock. n-3 PUFAs must be supplied by diet due to the absence of a key gene, namely, delta-15 desaturase (fat1), which is responsible for synthesizing n-3 PUFAs from a major type of n-6 PUFAs, linoleic acid (LA). To increase the dietary intake of n-3 PUFAs for humans, fat1-expressing transgenic (TG) livestock have been produced to provide n-3 PUFA-rich meats for humans. However, these TG livestock synthesized n-3 PUFAs from diet-derived, instead of endogenously produced, n-6 PUFAs because they still lack the delta-12 desaturase (fat2) gene for catalyzing conversion of internal oleic acid (OA) to LA. To fill the gap in the de novo n-3 PUFA biosynthesis pathway and to increase n-3 PUFA content in livestock, TG pigs co-expressing fat1-fat2 were generated in the present work. The OA content decreased in fat1-fat2 TG pigs, suggesting that OA was converted to LA by fat2 transgene-encoded delta-12 desaturase. The n-3 PUFA level was elevated, and the n-6/n-3 PUFA ratio dropped in fat1-fat2 TG pigs, revealing that fat1 transgene promoted the synthesis of n-3 PUFAs from n-6 analogs. The expression levels of fatty acid elongase-5 (ELOVL5) and fatty acid elongase-2 (ELOVL2), which are two key enzyme genes for PUFA synthesis, as well as their transcription factor peroxisome proliferator-activated receptor α, increased in fat1-fat2 TG pigs. Thus, the fat1 transgene enhanced n-3 PUFA synthesis by upregulating the expression of enzyme genes involved in the PUFA synthesis pathways. Overall, this study provided a new strategy to produce n-3 PUFA-rich meat for human consumption. The generated fat1-fat2 TG pigs can also serve as a large animal model for studying the roles of n-3 PUFAs in human development and health.

One-Carbon Metabolism and Lipid Metabolism in DOHaD.

The predisposing factors to lifestyle-associated diseases are established in the early period of life with underlying gene-environment interaction. Epigenetics is a chemical modification-based genetic mechanism that is affected by various nutritional factors. One-carbon metabolism is a metabolic system associated with methyl residue that is supplied from folic acid. Therefore, from the epigenetic point of view, proper intake of folic acid is important for pregnant women not only to prevent congenital abnormalities such as neural tube defect but also to prevent various adult disorders of the offspring. Dyslipidemia is an important risk factor of coronary heart disease, and epidemiological studies on Dutch winter famine, Jewish holocaust survivors, and Chinese famine suggested that prenatal malnutrition was associated with the dyslipidemia. Recent animal studies revealed that malnutrition in utero causes an epigenetic change in the Pparα gene, which accelerates the activity of delta-6 desaturase and delta-5 desaturase, that potentially induces dyslipidemia in adulthood. It has been known that overnutrition also increased the risk of cardiovascular diseases. Recent animal studies revealed that high-fat diet increased DNA methylation in the promoter region of delta-6 desaturase gene (Fads 2) that downregulates the gene expression in the arterial smooth muscle, which potentially contributes to cardiovascular diseases. Taken together, either insufficient or excessive nutrition alters epigenetic modification of genes that encodes enzymes associated with lipid metabolism. This altered epigenetic state persists during one's lifetime, which is potentially involved in noncommunicable diseases in adulthood.

Dietary essential α-linolenic acid and linoleic acid differentially modulate TNFα-induced NFκB activity in FADS2-deficient HEK-293 cells

The pro- or anti-inflammatory bioactivity of dietary essential linoleic acid (LA) and alpha-linolenic acid (ALA) is mainly attributed to rate-limiting delta-6 desaturase (D6D) activity. The aim of this study was to analyze mechanisms of D6D-substrates ALA, LA and D6D-product gamma-linolenic acid (GLA) under D6D-deficient conditions. Fatty acid profiles (GC-MS), D6D gene expression (real-time RT-PCR) and NFκB activity (luciferase assay) were assessed in HEK293 cells. FADS2 gene expression was approved being marginal. Incubation with ALA or LA did not increase D6D products but their elongase products C20:3n-3 and C20:2n-6. Bypassing the D6D, GLA elevated C20:3n-6 and C20:4n-6. LA significantly increased (+18% at 60 μM; p < .001), ALA reduced (−32% at 100 μM; p < .001) and GLA did not specifically change NFκB activity. Our data indicate that D6D might not be essential for the distinct effects of LA and ALA on NFκB activity.

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis.

Cloning, expression and functional analysis of a delta 6-desaturase gene from the silkworm, Bombyx mori L.

Delta 6-fatty acid desaturase is a membrane-bound enzyme, which is the rate-limiting factor in the biosynthesis of polyunsaturated fatty acids. In this study, a novel delta 6-desaturase gene was cloned from Bombyx mori (BmD6DES). Sequencing analysis revealed that BmD6DES has an open reading frame of 1357bp that encodes 448 amino acids. Heterologous expression in yeast demonstrated that BmD6DES could synthesize γ-linolenic acid (GLA, 18:3, Δ6,9,12) by utilizing the endogenous substrate linoleic acid (LA, 18:2, Δ9,12). We found that BmD6DES transcripts were distributed in almost all B. mori tissues, with high expression levels observed at the 5th instar larval, pupal, and adult moth stages. A functional analysis of BmD6DES was performed by measuring mRNA levels after temperature stress, fungal infection, and RNA interference (RNAi). The results indicated that the highest expression of BmD6DES was observed at low temperatures (0°C) and 6h to 36h after fungal infection. qPCR analysis demonstrated that BmD6DES mRNA levels in pupa after BmD6DES RNAi treatment were significantly reduced from 12h to 72h compared to those in the control group. Our findings suggest that BmD6DES not only induces the formation of the third carbon–carbon double bond in the LA carbon chain, but also leads to sensitivity to low-temperature stress and fungal infection. These results imply that BmD6DES is a key gene in the γ-linolenic acid pathway during B. mori development.

Double transgenesis of humanized fat1 and fat2 genes promotes omega-3 polyunsaturated fatty acids synthesis in a zebrafish model.

Omega-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential nutrients for human health. However, vertebrates, including humans, have lost the abilities to synthesize EPA and DHA de novo, majorly due to the genetic absence of delta-12 desaturase and omega-3 desaturase genes. Fishes, especially those naturally growing marine fish, are major dietary source of EPA and DHA. Because of the severe decline of marine fishery and the decrease in n-3 LC-PUFA content of farmed fishes, it is highly necessary to develop alternative sources of n-3 LC-PUFA. In the present study, we utilized transgenic technology to generate n-3 LC-PUFA-rich fish by using zebrafish as an animal model. Firstly, fat1 was proved to function efficiently in fish culture cells, which showed an effective conversion of n-6 PUFA to n-3 PUFA with the n-6/n-3 ratio that decreased from 7.7 to 1.1. Secondly, expression of fat1 in transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.8- and 2.4-fold, respectively. Third, co-expression of fat2, a fish codon-optimized delta-12 desaturase gene, and fat1 in fish culture cell significantly promoted n-3 PUFA synthesis with the decreased n-6/n-3 ratio from 7.7 to 0.7. Finally, co-expression of fat1 and fat2 in double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.7- and 2.8-fold, respectively. Overall, we generated two types of transgenic zebrafish rich in endogenous n-3 LC-PUFA, fat1 transgenic zebrafish and fat1/fat2 double transgenic zebrafish. Our results demonstrate that application of transgenic technology of humanized fat1 and fat2 in farmed fishes can largely improve the n-3 LC-PUFA production.

The omega smart baby project: effect of maternal DHA on infant development (269.1)

A randomized placebo controlled trial was conducted to examine the effect of DHA on gestational length and cognitive development. 115 pregnant women were supplemented with 300 mg DHA/d or placebo during pregnancy and thru the first 3 mo of breastfeeding (BF). Bayley Scales of Infant Development (BSID) were administered at 4 mo and 1y. Dietary DHA intake during pregnancy, was 81.9 +/‐ 5.8 mg/day (95% CI 72.3,91.5) and increased only slightly during BF. Some took prenatal vitamins with 0– 300 mg DHA per day resulting in total DHA intakes of 80 to 1100 mg/day. RBC and plasma phospholipid DHA at entry (expressed as % total fatty acids) were5.94 +/‐ 1.52 and 5.04 +/‐ 1.44 (mean +/‐ SD), respectively. Breastmilk (BM) % total DHA was .53 +/‐ .037 (mean +/‐ sem) at 2 mo and decreased to .35 +/‐ /039 at 4 mo. BM DHA was significantly greater in the DHA treatment ( p&lt;.02) and with the highest DHA intake compared to the lowest. BM DHA was lowest in carriers of the minor allele of the delta‐5 desaturase gene. Data were analyzed by treatment and by DHA intake. DHA supplementation resulted in a 1 wk increase in gestation with a significant (p&lt;.03) 8 d increase in gestational length comparing the highest intake of DHA( &gt; 600 mg/d) to the lowest (&lt; 300 mg/d). BSID cognitive and language scores were 10 points higher with high intake of DHA compared to the lowest (p&lt;.02). Maternal DHA impacts both pregnancy outcome and subsequent development.

Genetic variation at the FADS1-FADS2 gene locus influences delta-5 desaturase activity and LC-PUFA proportions after fish oil supplement

Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10(-19) ≤ P ≤ 4.5 × 10(-18)) and D6D (FADS2) (6.05 × 10(-8) ≤ P ≤ 4.20 × 10(-7)) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10(-16)). The significance of haplotype association with D5D activity (P = 2.19 × 10(-17)) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10(-28)) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10(-9)) and decreased D6D activity (P = 9.16 × 10(-6)) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.

Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid

BackgroundConjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains.ResultsWe have now co-expressed the delta 12-desaturase gene (FADS12, d12) from Mortierella alpina together with the codon-optimized linoleic acid isomerase (opai) gene in Y. lipolytica, expressed under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter hp4d. A multi-copy integration plasmid was used to further enhance the expression of both genes. Using glucose as the sole carbon source, the genetically-modified Y. lipolytica produced trans-10, cis-12-CLA at a level of up to 10% of total fatty acids and 0.4% of DCW. Furthermore, when the recombinant yeast was grown with soybean oil, trans-10, cis-12-CLA now accumulated at a level of up to 44% of total fatty acids, which represented 30% of DCW after 38.5 h of cultivation. In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l.ConclusionsWe have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium). Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.

Nutritional aspects of epigenetic inheritance

The impact of diet and environmental factors on genes concerned with epigenetic inheritance and the mechanism of evolution has grown significantly beyond the Modern Synthesis period. Epigenetic inheritance is the passing of phenotypic change to subsequent generations in ways that are outside the genetic code of DNA. Recently, polymorphisms of the human Delta-5 (fatty acid desaturase, FADS1) and Delta-6 (FADS2) desaturase genes have been described as being associated with the level of several long-chain n-3 and n-6 polyunsaturated fatty acids (PUFAs) in serum phospholipids. Increased consumption of refined starches and sugar increases the generation of superoxide anion in the tissues and free fatty acids (FFA) in the blood. There is an increased amount and activity of nuclear factor-κB (NF-κB), a transcriptional factor regulating the activity of at least 125 genes, most of which are pro-inflammatory. The consumption of glucose may be associated with an increase in 2 other pro-inflammatory transcription factors: activating protein-1 (AP-1), and early growth response protein-1 (Egr-1). AP-1 regulates the transcription of matrix metallo-proteinases and Egr-1 modulates the transcription of tissue factor and plasminogen activator inhibitor-1. It is possible that a complex set of factors, including nutritional factors, come into play during epigenetic inheritance.

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.

Comparison of growth, serum biochemistries and n−6 fatty acid metabolism in rats fed diets supplemented with high-gamma-linolenic acid safflower oil or borage oil for 90 days

Recently, steps have been taken to further developments toward increasing gamma-linolenic acid (GLA) concentration and lowering costs in plant seed oils using transgenic technology. Through identification and expression of a fungal delta-6 desaturase gene in the high linoleic acid safflower plant, the seeds from this genetic transformation produce oil with >40% GLA (high GLA safflower oil (HGSO)). The aim of the study was to compare the effects of feeding HGSO to a generally recognized as safe source of GLA, borage oil, in a 90day safety study in rats. Weanling male and female Sprague–Dawley rats were fed a semi-synthetic, fat free, pelleted diet (AIN93G) supplemented with a 10% (wt/wt) oil blend containing HGSO or borage oil, with equivalent GLA levels. Results demonstrated that feeding diets containing HGSO or borage oil for 90days had similar biologic effects with regard to growth characteristics, body composition, behavior, organ weight and histology, and parameters of hematology and serum biochemistries in both sexes. Metabolism of the primary n−6 fatty acids in plasma and organ phospholipids was similar, despite minor changes in females. We conclude that HGSO is biologically equivalent to borage oil and provides a safe alternative source of GLA in the diet.

Molecular cloning and expression analysis of a delta 6-fatty acid desaturase gene from Rhizopus stolonifer strain YF6 which can accumulate high levels of gamma-linolenic acid

The delta 6-desaturase gene was cloned from Rhizopus stolonifer, which could accumulate up to 49% of gamma-linolenic acid (GLA, C18:3 Δ(6,9,12)) to the total fatty acids. The cloned DNA contains a 1,380 bp open reading frame encoding a protein of 460 amino acids, which showed high similarity to those of fungal delta 6-desaturases with three conserved histidine-rich motifs and HPGG motif. Notably, this deduced sequence had a shorter C-terminus. Results demonstrated that the cDNA sequence exhibited delta 6-desaturase activity by accumulation of about 22.4 % of GLA to the total fatty acids in the recombinant Pichia pastoris strain GS115.

Fermentation Process Development of Recombinant Hansenula polymorpha for Gamma-Linolenic Acid Production

Development in the strain and the fermentation process of Hansenula polymorpha was implemented for the production of gamma-linolenic acid (GLA, C18:3 delta 6,9,12), which is an n-6 polyunsaturated fatty acid (PUFA) and has been reported to possess a number of health benefits. The mutated delta 6-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without utilization of methanol a high cell-density culture of the yeast recombinant carrying the delta 6-desaturase gene was achieved by fed-batch fermentation using glycerol-limited conditions. The delta 6-desaturated products, octadecadienoic acid (C18:2 delta6,9), GLA and stearidonic acid (C18:4 delta6,9,12,15), accumulated at high levels under the derepression condition. The GLA production was also optimized by adjusting specific growth rates. The results show that the specific growth rate affected both lipid content and fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates studied, the highest GLA concentration, which was at of 697 mg/l, was obtained in the culture with the specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor delta 6-desaturase gene was similar to that of blackcurrant oil with both containing similar proportions of n-3 and n-6 essential fatty acids.

Co-expression of heterologous desaturase genes in Yarrowia lipolytica

The hybrid promoter (hp4d) expression cassette, one of the efficient tools of Yarrowia lipolytica expression system, has been applied to produce or secrete a variety of recombinant proteins. This cassette directs a strong gene expression, because the hp4d promoter exhibits high level quasi-constitutive activity. The objective of this study is to test whether two expression cassettes inserted into a vector could function efficiently and simultaneously. Taking advantage of the well-known biosynthesis pathway of gamma-linolenic acid (GLA), we examined the performance of Y. lipolytica, transformed with two expression cassettes containing previously cloned Delta12-desaturase and Delta6-desaturase genes, by monitoring fatty acid composition of cellular lipids. Our results confirmed that each individual desaturase gene was expressed efficiently by the expression cassette. When two cassettes with respective desaturase genes, carried on the same vector, were integrated into yeast genome, a significant level of GLA was synthesized from endogenous linoleic acid (LA) and oleic acid (OA). Besides, both expression cassettes functioned effectively without influence from each other. These findings indicated that co-expression of two desaturase genes by this dual cassette vector was effective and simultaneous. Results from the present study provide an alternative approach for both the production of several proteins at the same time, and the development of single cell oil containing high-valued polyunsaturated fatty acids (PUFAs).

Expression of Acyl‐lipid Δ12‐desaturase Gene in Prokaryotic and Eukaryotic Cells and Its Effect on Cold Stress Tolerance of Potato

We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.

Isolation and Characterisation of a High-Efficiency Desaturase and Elongases from Microalgae for Transgenic LC-PUFA Production

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2omega6) and alpha-linolenic acid (ALA; 18:3omega3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Delta6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Delta6-elongase and a Delta5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39-75% conversion for omega3 substrate fatty acids), and the Delta5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5omega3) to docosapentaenoic acid (22:5omega3)) than previously reported genes. In addition, the Delta6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the omega3 substrate ALA.