Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called -3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.     

Effect of fatty acids of ω6 series on the biosynthesis of arachidonic acid in HTC cells

Summary The influence of the preincubation of HTC cells with fatty acids of 6 series and columbinic acid (St, 9c, 12c 18:3) on the biosynthesis of arachidonic acid was studied. The cells were incubated on a chemically defined medium with or without the addition of unlabeled linoleic, -linolenic, eicosatrienoic, arachidonic, docosatetraenoic, docosapentaenoic and columbinic acids. After 24 hr of preincubation in the presence of the aforementioned fatty acids, [1-¹⁴C]eicosa-8,11,14-trienoic acid was added to the culture medium as the only lipidic source. Twenty-four hours later the synthesis of arachidonic acid and the fatty acid composition of the cells were determined. At 20 MM concentration the 6 fatty acids studied except docosapentaenoic acid produced an increase on the biosynthesis of arachidonic acid compared to the cells incubated in the absence of unlabeled fatty acids in the medium. The fatty acids added to the culture medium were incorporated into the cells and modified their fatty acid composition. Columbinic acid, with a similar structure to linoleic acid, also produced a significant increase on the conversion of eicosatrienoic acid to arachidonic acid. These results would suggest that the effect of both, linoleic and columbinic acids, may be adscribed to their configuration and not necessarily to their transformation in higher homologs, since columbinic acid is unable to be desaturated.All authors are members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientifícas y Técnicas, Argentina.     

Identification and characterization of new Δ-17 fatty acid desaturases

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54–65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.     

Role of plasma membrane lipid composition on cellular homeostasis： learning from cell line models expressing fatty acid desaturases

Experimental evidence has suggested that plasma membrane （PM）-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids （PUFAs） effect on PM properties and analyze its influence on cholesterol （Chol） homeostasis. We have previously shown that by using a cell line overexpressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and ceil viability by different mechanisms. Now, we expanded our studies to a cell model overexpressing both A5 and A6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein AI-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the ceil functionality is preserved by regulating PM organization and Chol exportation and homeostasis.     

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed β-carotene and 13-cis-retinoic acid

The fatty acid composition of microsomal lipids and the activities of Δ9- and Δ6-desaturases in liver microsomes of rats fed diets supplemented with β-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg β-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for Δ9-desaturase and Δ6-desaturase activities. The activity of Δ9-desaturase was lower in liver microsomes of rats fed β-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal Δ6-desaturase activity was, however, higher in liver of rats fed 13-cis-retinoic acid; there was no effect of β-carotene on Δ6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding β-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.     

Role ofβ-oxidation in inhibitingLactobacillus leichmanii by fatty acids

The effect of saturated fatty acids from 6∶0 to 16∶0 and oleic acid onLactobacillus leichmanii ATCC 4797 growing in non-skim-milk media was determined. The inhibition by lauric acid was higher than that obtained with any other fatty acid. A mutant (MC12) resistant to the fatty acid inhibition with high β-oxidation activity was also studied. A positive correlation between the ability ofL. leichmanii ATCC 4797 and its derivative MC12 to degrade fatty acids and their resistance to the fatty acid inhibition is shown in this report.     

Hormonal regulation of tissue-specific ectopic expression of an Arabidopsis endoplasmic reticulum-type ω-3 fatty acid desaturase (FAD3) gene

A common feature of the membrane lipids of higher plants is a large content of polyunsaturated fatty acids, which typically consist of dienoic and trienoic fatty acids. Two types of omega-3 fatty acid desaturase. which are present in the plastids and in the endoplasmic reticulum (ER), respectively, are responsible for the conversion of dienoic to trienoic fatty acids. To establish a system for investigating the tissue-specific, and hor-mone-regulated expression of the ER-type desaturase gene (FAD3), transgenic plants of Arabidopsis thaliana (L.) Heynh. containing the firefly luciferase gene (LUC) fused to the FAD3 promoter (FAD3::LUC) were constructed. At different times during plant development, FAD3::LUC was actively expressed at two major sites, the vegetative shoot meristem and the floral organs. Transgenic plants with LUC fused to the promoter of FAD7 (FAD7::LUC) which encodes plastid-type desaturase, were also constructed. FAD3::LUC and FAD7::LUC were expressed in the same organs during reproductive growth, but not during vegetative growth. In plants exposed to both auxin and cytokinin, FAD3::LUC expression was ectopically induced in the root tissues. However, this induction by auxin and cytokinin was inhibited when abscisic acid was also present. FAD3::LUC expression could be induced in the roots by auxin and cytokinin if the hormones were applied during vegetative growth, but not if they were applied during germination or reproductive growth. Analysis of the fatty acid composition in the roots of Arabidopsis fad mutant and wild-type plants confirmed that the response of FAD3::LUC expression to various hormones reflected the response of endogenous FAD3 gene expression. These results suggest that the expression of ER-type desaturase is regulated through synergistic and antagonistic hormonal interactions, and that such hormonal regulation and the tissue specificity of the expression of this gene are further modified in accordance with the growth phase in plant development.     

Cold-induced accumulation of ω-3 polyunsaturated fatty acid in a liverwort, Marchantia polymorpha L

The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid.     

Ameliorative effects of ω-3 fatty acids on stress induced alteration in rhythms of selective biochemical parameters in rats

Twenty-four adult male Sprague-Dawley rats were randomly divided into four groups of six rats in each, Control animals without stress (GI); Rats with induced physical stress (GII); Normal Rats fed with omega fish oil as a source of omega-3 fatty acid (GIII); Stressed Rats supplemented omega-3 fatty acid diet (GIV). In GII and GIV physical stress was induced by keeping six rats in a standard cage instead of four rats and in GIV fish oil was added in the feed @ 3% to ameliorate the physical stress. Sampling was done on 15th and 29th day of the study. On 15th day of study no significant difference was found between the groups with respect to blood glucose and total protein levels, but on 29th day there was significant increase in blood glucose in GII and significant decrease in GIII. There was significant increase in total protein of GIII and decrease in GII, respectively apart from the significant increase in GIII on 29th day as compared to 15th day. Serum creatinine levels were found to be significantly lower in both the samples of GIII. The total and LDL cholesterol was significantly higher in GII but on 29th day it was found to be significantly lower in GIII. The HDL cholesterol was significantly lower in GII and significantly higher in GIII on 29th day, whereas it was vice versa with respect to triglycerides. Serum ALT, was found to be significantly lower in GIII and higher in GII on 29th day with no significant changes observed on 15th day. The liver of GII revealed central vein congestion and mononuclear cell infiltration in portal triad, whereas GIV showed only mild changes. The study proves the time-dependent beneficial effects of omega-3 fatty acids during physical stress.     

Possible involvement of fatty acid binding protein in peroxisomal β-oxidation of fatty acids

The localization of β-oxidation of fatty acids in isolated peroxisomes from rat liver was investigated. The enzyme system is soluble in the luminal compartment and carnitine does not appear to be involved in the transfer of the CoA derivatives through the peroxisomal membrane. Experiments involving proteolysis, inhibitors and competitive inhibition suggest that a fatty acid binding protein is responsible for the carrier process. This carrier protein seems to be present in increased amounts both in the supernatant and in the peroxisomes after clofibrate induction.     

Δ6-Desaturase (FADS2) deficiency unveils the role of ω3- and ω6-polyunsaturated fatty acids

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and α-linolenic acid. EFAs are converted into ω3- and ω6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of Δ6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2−/− mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood–testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2−/− mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.     

Higher de novo synthesized fatty acids and lower ω3- and ω6-long-chain polyunsaturated fatty acids in umbilical vessels of women with preeclampsia and high fish intakes

Umbilical veins (UV) and arteries (UA) of preeclamptic women in Curaçao harbor lower long-chain polyunsaturated fatty acids (LCP). The present aim was to test these findings in Mwanza (Tanzania), whose inhabitants have high LCPω3 and LCPω6 intakes from Lake Victoria fish. Women with preeclampsia (n=28) in Mwanza had lower PUFA and higher 20:0 in UV and UA, compared with normotensive/non-proteinuric controls (n=31). Their UV 22:6ω3, 22:4ω6, LCPω6, ω6, and LCPω3+ω6 were lower, while saturated FA, potentially de novo synthesized FA (Σde novo) and (Σde novo)/(LCPω3+ω6) ratio were higher. Their UA had higher 16:1ω7, ω7, 18:0, and 16:1ω7/16:0. Umbilical vessels in Mwanza had higher 22:6ω3, LCPω3, ω3, and 16:0, and lower 22:5ω6, 20:2ω6, 18:1ω9, and ω9, compared to those in Curaçao. Preeclampsia in both Mwanza and Curaçao is characterized by lower LCP and higher Σde novo. An explanation of this might be placental dysfunction, while the similarity of umbilical vessel FA-abnormalities in preeclamptic and diabetic pregnancies suggests insulin resistance as a common denominator.     

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g?1 to 8.57 mg g?1 and 10.06 mg g?1, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.