Fatty Acid Desaturase Activity Research Articles

Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress

Stress acclimating plants respond to abiotic and biotic stress by remodeling membrane fluidity and by releasing alpha-linolenic (18:3) from membrane lipids. The modification of membrane fluidity is mediated by changes in unsaturated fatty acid levels, a function provided in part by the regulated activity of fatty acid desaturases. Adjustment of membrane fluidity maintains an environment suitable for the function of critical integral proteins during stress. alpha-Linolenic acid, released from membrane lipid by regulated lipase activity, is the precursor molecule for phyto-oxylipin biosynthesis. The modulation of chloroplast oleic acid (18:1) levels is central to the normal expression of defense responses to pathogens in Arabidopsis. Oleic (18:1) and linolenic (18:2) acid levels, in part, regulate development, seed colonization, and mycotoxin production by Aspergillus spp.

Effect of Temperature on Microsomal Omega‐3 Linoleate Desaturase Gene Expression and Linolenic Acid Content in Developing Soybean Seeds

The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post‐translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the transcript accumulation among the three members of the soybean [Glycine max (L.) Merr.] microsomal omega‐3 fatty acid desaturase gene family in response to altered growth temperature during seed development. Microsomal omega‐3 fatty acid desaturases catalyze the insertion of a third double bond into linoleic (18:2Δ9, 12) acid to produce linolenic (18:3Δ9, 12, 15) acid. At 35 d after flowering, transcript accumulation (normalized for soybean actin) of GmFAD3A decreased by 5‐ to 15‐fold, GmFAD3B by 2‐ to 9‐fold, and GmFAD3C by 2‐ to 3‐fold in seeds that developed in a warm (day/night [D/N] = 30/26°C) versus a normal (D/N = 26/22°C) or a cool (D/N = 22/18°C) environment. At this stage of seed development, decreased omega‐3 desaturase gene expression levels were positively associated with reductions of 39 to 50% in the linolenic acid content of seeds of three soybean varieties examined. Thus, transcriptional regulation of the microsomal omega‐3 fatty acid desaturase gene family likely accounts, at least in part, for the reduced linolenic acid levels in soybean seeds grown at elevated temperature.

Identification and characterization of a novel yeast ω3‐fatty acid desaturase acting on long‐chain n‐6 fatty acid substrates from Pichia pastoris

A cDNA sequence putatively encoding a omega(3)-fatty acid desaturase gene was isolated from methylotrophic yeast Pichia pastoris GS115. The deduced amino acid sequence of this cloned cDNA showed high identity to known fungal omega(3)-fatty acid desaturases. Functional identification of this gene heterologously in Saccharomyces cerevisiae strain INVScl indicated that the deduced amino acid sequence exhibited omega(3)-fatty acid desaturase activity. The newly identified omega(3)-fatty acid desaturase, named Pp-FAD3, is novel because it showed broad n-6 fatty acid substrate specificity by its ability to convert all the 18-carbon and 20-carbon n-6 substrates examined to the corresponding n-3 fatty acids, with an approximately equivalent high conversion rate. Pp-FAD3 is the first known yeast omega(3)-fatty acid desaturase to act on long-chain n-6 fatty acid substrates. Heterologous expression of the newly identified omega(3) desaturase in different hosts will be an alternative method to increase the flow of n-6 fatty acid intermediates into their n-3 derivatives.

Essential fatty acid deficiency induces fatty acid desaturase expression in rat epididymis, but not in testis

On the molecular level, essential fatty acid deficiency (EFAD) has been associated with induced fatty acid (FA) desaturase expression and activity in several tissues. However, there seem to be exceptions. In the present study, we examine the effects of EFAD in the male rat genital tract, combining FA analysis, gene expression studies, and morphological evaluation of epididymal spermatozoa. When feeding 21-day-old Wistar rats, a fat-free diet for 6 weeks, an increase in 18:1n-9 and 20:3n-9 and a concomitant decrease in the 18:2n-6 and 20:4n-6 species are seen in testis, as well as in liver. However, with regard to desaturase expression the rat testis seems to be unresponsive to EFAD conditions, in contrast to other organs studied. In the sexually mature testis none of the desaturases (SCD1, SCD2, D5D, or D6D) are induced in response to lowered contents of polyunsaturated FAs. This also applies to caput epididymis, while EFAD sensitivity is regained in cauda epididymis, where the desaturases are upregulated. The FA profile of epididymal spermatozoa is increasingly affected by EFAD during the transport from testis to cauda epididymis. Furthermore, a significant increase in the number of abnormal spermatozoa is observed in cauda epididymis.

Lipid Metabolism in Rats is Modified by Nitric Oxide Availability Through a Ca++‐Dependent Mechanism

We studied lipid metabolism and the antioxidant defense system in plasma and liver of rats fed diets supplemented with L(omega)-nitro-L-arginine methyl ester (L-NAME), isosorbide dinitrate (DIS), L-arginine (Arg), or the associations of these drugs. Liver hydroperoxide and thiobarbituric-acid-reactive substance (TBARS) levels were decreased by Arg and increased by L-NAME or DIS treatments. Oxidized glutathione and conjugated dienes were increased by DIS. Nitrate + nitrite levels and serum calcium ([Ca(++)]) were incremented by Arg or DIS and reduced by L-NAME. Superoxide dismutase and catalase activities decreased under Arg treatment, while L-NAME or DIS caused stimulation. Liver high-density lipoprotein (HDL) cholesterol was increased by DIS or NAME (alone or associated with Arg). Free fatty acids and neutral and polar lipids were increased by Arg, L: -NAME, and DIS. However, predominating phospholipid synthesis increased the neutral/polar ratio. Decreased levels of nitric oxide (NO) (low [Ca(++)]) was directly associated with increased fatty acid synthetase, decreased phospholipase A(2), carnitine-palmitoyl transferase, and fatty acid desaturase activities. Raised NO (high [Ca(++)]) inversely correlated with increased phospholipase-A(2) and acyl-coenzyme A (CoA) synthetase and decreased fatty acid synthetase and beta-oxidation rate. Arg or DIS produced changes that were partially reverted by association with L-NAME. Based on these observations, prolonged therapeutical approaches using drugs that modify NO availability should be carefully considered.

Construction of a High-efficient Expression Vector of Δ 12 Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

A full-length sequence coding for Δ 12 fatty acid desaturase gene from peanut ( Arachis hypogaea L.) was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently transformed into expression Escherichia. coli BL21(DE3)pLysS. The Δ 12 fatty acid desaturase was highly expressed in E. coli BL21(DE3)pLysS in the presence of isopropyl-D-thiogalactopyranoside (IPTG). The fusion protein was purified and used to form a reaction system in vitro by adding oleic acid as substrate and incubating it at 20°C for 6 h. Total fatty acids was extracted and methlesterified and then analyzed with gas chromatography. A novel peak corresponding to linoleic acid methyl ester standards was detected with the same retention time. GC-MS (gas chromatogram and gas chromatogram-mass spectrometry) analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited Δ 12 fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.

Abstract 4173: Statin Use is Associated with Higher Levels of Total and Long-Chain n-3 Fatty Acids and Lower Levels of n-6 and Saturated Fatty Acids in Patients Following Acute Myocardial Infarction

Background: Higher plasma n-3 fatty acid levels are associated with improved long-term outcomes in the primary and secondary prevention of CHD. Besides anti-arrhythmic effects, n-3 fatty acids have been shown to possess other pleiotropic effects including anti-inflammatory and antithrombotic effects in a fashion similar to statins. The effect of statin use on fatty acid synthesis and metabolism has received little attention in CHD patients. The objective of this study was to evaluate the relationship between statin use, plasma fatty acid levels and estimated desaturase activity in patients following acute myocardial infarction. Methods: Clinical and metabolic parameters including fasting plasma fatty acid levels were measured in 734 subjects, approximately eight weeks following AMI. Correlations were evaluated between statin use, fatty acid levels and desaturase activity. Results: Statin use was observed in 539 of 734 patients and was associated with higher levels of total n-3 (r=0.10, P=0.005) and long-chain n-3 fatty acids (ecosapentanoeic acid r=0.11, P=0.004; docosahexanoeic acid r=0.12, P=0.001) and lower n-6 (linoleic acid r=-0.24, P<0.001) and total saturated fatty acid levels (r=-.083, P=0.025). Multivariate models adjusted for age, sex, smoking, physical activity level and the components of the metabolic syndrome did not significantly alter the observed associations. Statin use correlated negatively with estimated Δ-9 desaturase activity (r=-0.08, P=0.025) and positively with Δ-5 activity (r=0.23, P<0.001). Conclusions: Statin use is associated with an improved plasma fatty acid profile characterized by higher levels of total and long-chain n-3 fatty acids and lower levels of saturated and certain n-6 fatty acids. We cannot exclude that dietary factors may have influenced the observed associations. Our data is consistent with basic science investigations and randomized trials in non-CHD patients demonstrating that statins do influence fatty acid synthesis and metabolism. Whether the pleiotropic effects of statins may in part be due to an improved fatty acid profile requires further study in prospective clinical trials.

Improvements in glucose tolerance and insulin sensitivity after lifestyle intervention are related to changes in serum fatty acid profile and desaturase activities: the SLIM study

The aim of this study was to investigate whether lifestyle intervention-induced changes in serum fatty acid profile of cholesteryl esters and estimated desaturase activities are related to improvements in insulin sensitivity in subjects at risk of type 2 diabetes. In the Study on Lifestyle Intervention and Impaired Glucose Tolerance Maastricht (SLIM), 97 men and women with IGT were randomised to a combined diet and exercise programme (47 intervention) or a control group (50 control subjects). At baseline and after 1 year the following assessments were made: an OGTT, an exercise test to determine maximal aerobic capacity, anthropometry, and analysis of the serum fatty acid profile of cholesteryl esters. The lifestyle programme was effective in reducing the intake of total and saturated fat, increasing physical activity, reducing obesity and improving insulin sensitivity and glucose tolerance. Regression analysis of the total population showed that an increase in the C20:4 n-6/C20:3 n-6 ratio (estimated Delta5-desaturase activity) and reductions in the C18:3 n-6/C18:2 n-6 ratio (estimated Delta6-desaturase activity) and the C16:1 n-7/C16:0 ratio (estimated Delta9-desaturase activity or stearoyl-CoA desaturase-1) were significantly associated with a decrease in homeostasis model assessment for insulin resistance. After adjustment for lifestyle changes (change in percentage body fat, aerobic capacity and saturated fat intake), these associations were partly reduced, but remained statistically significant. Lifestyle-induced changes in fatty acid profile of cholesteryl esters and desaturase activities were independently related to changes in insulin sensitivity in subjects at risk of type 2 diabetes.

Isolation of Δ12 and ω3‐fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and α‐linolenic acids in Saccharomyces cerevisiae

Two clones with homology to known fatty acid desaturase genes were isolated from the yeast Kluyveromyces lactis. The first gene, which we designate KlFAD2, consists of 411 amino acids with an overall identity of 73.0% to FAD2 from Saccharomyces kluyveri. It exhibited Delta12 fatty acid desaturase activity when expressed in S. cerevisiae under the control of ADH1 promoter and produced endogenous linoleic acid. The second clone, which we designate KlFAD3, consists of 415 amino acids with an overall identity of 79.3% to FAD3 from S. kluyveri. It exhibited omega3 fatty acid desaturase activity in S. cerevisiae when expressed under the control of ADH1 promoter in the presence of the exogenous substrate linoleic acid and produced alpha-linolenic acid. Co-expression of KlFAD2 and KlFAD3 resulted in the endogenous production of both linoleic and alpha-linolenic acids. The yield of alpha-linolenic acid reached 0.8% of total fatty acids and its production was not increased by adding exogenous oleic acid; alpha-linolenic acid reached 8.7% when exogenous linoleic acid was available.

Fatty acid composition of serum lipids predicts the development of the metabolic syndrome in men

Types of dietary fat have been related to components of the metabolic syndrome. Serum fatty acid composition mainly reflects dietary fat intake, but also endogenous fatty acid synthesis catalysed by Delta-desaturases. It is not known whether alterations of fatty acid composition or desaturase activities predict metabolic syndrome. We prospectively evaluated fatty acid composition in serum cholesteryl esters and estimated desaturase activities in 1,558 50-year-old men taking part in a population-based cohort study. The follow-up time was 20 years. Stearoyl-CoA desaturase (SCD-1), Delta6 (D6D) and Delta5 (D5D) desaturases were estimated as precursor to fatty acid ratios. High activity of estimated SCD-1 (odds ratio=1.29, p<0.05) and D6D (odds ratio=1.35, p<0.05), as well as low estimated D5D activity (odds ratio=0.71, p<0.001) predicted the development of metabolic syndrome (as defined by the National Cholesterol Education Program). The predictive value of D5D activity was independent of lifestyle factors (smoking, BMI and physical activity), whereas the risk associated with higher SCD-1 and D6D activities was mainly explained by obesity. Among those developing metabolic syndrome (119 out of 706) during follow-up, the proportions of fatty acids 14:0, 16:0, 16:1 (n-7), 18:1 (n-9), 18:3 (n-6) and 20:3 (n-6) were increased at baseline, while 18:2 (n-6) was decreased (p<0.05 for all). Serum fatty acid composition predicts the long-term development of the metabolic syndrome, and D5D activity may be particularly important in this process. Our results suggest a role of dietary fat quality in the development of metabolic syndrome, but the possibility that altered fatty acid composition, partly secondary to genetic or hormonal factors, should also be considered.

Expression of Δ12 fatty acid desaturase during the induced accumulation of the antifungal diene in avocado fruits

SUMMARY The preformed (Z,Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (AFD) is the most active antifungal compound in avocado; it affects the quiescence of Colletotrichum gloeosporioides in unripe fruit. One of the genes encoding Delta(12) fatty acid desaturase (avfad12) was hypothesized to take part in the biosynthesis of AFD, and its expression pattern and enzymatic activity were determined in relation to the content of AFD. Using avfad12-3 as a probe, high levels of expression were detected in young fruits and leaves, where the level of AFD was highest. In contrast, Northern analysis of RNA from mature leaves and fruits showed no transcripts from the avfad12 gene family and lower AFD content. The transcripts from the avfad12 gene family, the enzymatic activity of Delta(12) fatty acid desaturase, and the level of AFD in unripe-resistant fruits increased transiently when the fruits were inoculated with C. gloeosporioides or exposed to ethylene (40 microL/L), low temperature (4 degrees C) or 1 mm H(2)O(2), but ripe fruits were not affected. The effect of H(2)O(2) on the transcripts from the avfad12 gene family is of specific importance, because reactive oxygen species were produced by unripe-resistant host fruit soon after inoculation of C. gloeosporioides. In addition, the fungus itself produced H(2)O(2) in culture medium at pH 5.0, which is similar to the pH of unripe-resistant fruit, but not at pH 7.0. Treatments that enhanced Delta(12) fatty acid desaturase activity increased the concentration of the AFD precursor, linoleic acid, and its incorporation into AFD; these treatments also caused a delay in decay development. The present results demonstrate temporal relationships among the transcripts from the avfad12 gene family, the synthesis of the precursor of AFD (linoleic acid), the AFD content and quiescence of C. gloeosporioides in unripe fruits.

Incorporation and metabolism of fatty acids by desaturation and elongation in the nematode, Panagrellus redivivus

AbstractThe free-living nematode Panagrellus redivivus can be mass produced in monoxenic solid culture on Saccharomyces cerevisiae and therefore could be useful as a live food for marine fish or crustacean larvae in the rapidly expanding aquaculture industry. However, this will depend on their lipid and fatty acid composition and so this was investigated in mass produced P. redivivus grown on S. cerevisiae in three different media. Live nematodes were also incubated with [1-14C]-labelled fatty acids and the desaturation and elongation of the fatty acids were determined. The combined results from the growth trials on different media and the metabolic studies with labelled fatty acids indicated the presence of Δ9, Δ12, Δ6 and Δ5 fatty acid desaturase activities, and elongase activities active towards C18, C16 and shorter chain fatty acids. The presence of Δ15, and therefore the ability to produce n-3 polyunsaturated fatty acids, was suggested by the compositional data, but could not be conclusively established from metabolic studies.

Effects of water temperature and diets containing palm oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes of rainbow trout ( Oncorhynchus mykiss)

Food grade fisheries have reached their sustainable limits while aquaculture production has increased to meet consumer demands. However, for growth in aquaculture to continue and utilise sustainable, feeding ingredients, alternatives to fish oil (FO), the predominant lipid component of fish diets, must be developed. Therefore, there is currently considerable interest in the regulation of fatty acid metabolism in fish in order to determine strategies for the best use of plant oils in diets for commercially important cultured fish species. Plant oils are characteristically rich in C 18 polyunsaturated fatty acids (PUFA) but devoid of C 20 and C 22 highly unsaturated fatty acids (HUFA) found in FO. The fatty acyl desaturase enzyme activities involved in the biosynthesis of HUFA from PUFA are known to be under nutritional regulation and can be increased in fish fed diets rich in plant oils. However, fatty acid desaturase activity is also known to be modulated by water temperature in fish. The present study aimed to investigate the interaction between water temperature and diet in the regulation of fatty acid metabolism in rainbow trout. Trout, acclimatized to 7, 11 or 15 °C, were fed for 4 weeks on diets in which the FO was replaced in a graded manner by palm oil. At the end of the trial, fatty acyl desaturation/elongation and β-oxidation activities were determined in isolated hepatocytes and intestinal enterocytes using [1- 14C]18:3n-3 as substrate, and samples of liver were collected for analysis of lipid and fatty acid composition. The most obvious effect of temperature was that fatty acid desaturation/elongation and β-oxidation were reduced in both hepatocytes and intestinal enterocytes from fish maintained at the highest water temperature (15 °C). There were differences between the two tissues with the highest desaturation/elongation and β-oxidation activities tending to be in fish held at 11 °C in the case of hepatocytes, but 7 °C in enterocytes. Correlations between fatty acid metabolism and dietary palm oil were most clearly observed in desaturation/elongation activities in both hepatocytes and enterocytes at 11 °C. The highest β-oxidation activities were generally observed in fish fed FO alone in both hepatocytes and enterocytes with palm oil having differential effects in the two cell types.

Effect of various temperatures on phosphatidylglycerol biosynthesis in thylakoid membranes.

Lipid unsaturation, the major factor to maintain thylakoid membrane fluidity, is affected by temperature. In this work, we analysed the molecular species composition of phosphatidylglycerol (PG) in thylakoid membranes during spinach (Spinacia oleracea) and squash (Cucurbita pepo) cotyledon growth to investigate how the growth temperature affects the PG biosynthesis. Of the 10 molecular species detected, temperature affected mainly the relative content of molecular species containing linolenic acid (18:3) and those containing palmitic acid (16:0) at the sn-1 position of glycerol backbone. Lowering the temperature induced an increase in the former and a decrease in the latter. The relative content of molecular species containing 18:3 or 16:0 at the sn-1 position of the glycerol backbone were correlated with temperature. Our results indicate that the substrate selectivity of the glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts towards 16:0 or oleic acid (18:1) and the activity of fatty acid desaturases are greatly affected by temperature. In addition, changes in the relative content of PG molecular species induced by variations in growth temperature depended mainly on the substrate selectivity of GPAT.

Dietary Eritadenine and Ethanolamine Depress Fatty Acid Desaturase Activities by Increasing Liver Microsomal Phosphatidylethanolamine in Rats

The effects of eritadenine, a constituent of the Lentinus edodes mushroom, and ethanolamine, the base constituent of phosphatidylethanolamine (PE), on fatty acid desaturase activities and lipid profiles were investigated comparatively in rats. Rats were fed a control diet or a diet supplemented with either eritadenine (0.05 g/kg) or ethanolamine (8 g/kg) for 14 d. Eritadenine and ethanolamine had marked hypocholesterolemic effects. The concentration of liver microsomal PE was significantly increased and the ratio of phosphatidylcholine (PC) to PE was significantly decreased by both eritadenine and ethanolamine. These changes in phospholipid profile were also observed in the mitochondria and plasma membranes in the liver. The activities of the Delta5-, Delta6- and Delta9-desaturases in liver microsomes were significantly decreased by eritadenine and ethanolamine; there was a significant correlation between the activity of Delta5- or Delta6-desaturase and the proportion of PE in the total phospholipids or the PC/PE ratio. Reflecting decreased Delta5- and Delta6-desaturase activities, the 20:4(n-6)/18:2(n-6) ratio was significantly decreased by eritadenine and ethanolamine in PC of the liver microsomes, mitochondria and plasma membranes. Although the 20:4(n-6)/18:2(n-6) ratio of liver microsomal PE was also significantly decreased by eritadenine and ethanolamine, the fatty acid composition of phosphatidylinositol and phosphatidylserine was less affected by these compounds. Eritadenine and ethanolamine increased the proportion of 16:0-18:2 and decreased the proportion of 18:0-20:4 in liver PC. The results suggest that dietary eritadenine and ethanolamine might lead to decreases in desaturase activities and changes in fatty acid and molecular species composition of PC through an increase in liver microsomal PE.

Desaturation and elongation of Fatty acids and insulin action.

Insulin resistance is characterized by specific changes of the composition of fatty acids in the serum lipids and in the skeletal muscle membranes. Impaired insulin sensitivity is associated with high proportions of palmitic (16:0) acid and low levels of linoleic (18:2 n-6) acid in serum. In addition, there are apparent changes of the fatty acid desaturase activities, suggesting an increased activity of the Delta9 and Delta6 desaturases and a decreased activity of the Delta5 desaturase. The activity of the fatty acid desaturases is regulated by long-chain polyunsaturated fatty acids and insulin and is probably also dependent on the degree of physical activity. A high ratio between arachidonic (20:4 n-6) and dihomo-gamma linolenic (20:3 n-6) acid, as a measure of Delta5 desaturase activity, in the skeletal muscle phospholipids has been related to good insulin sensitivity. Available knowledge seems to indicate that the degree of saturation of the body lipids, and especially the proportion of palmitic acid in the lipid membranes, may be critical for insulin sensitivity. The strong relationships between the Delta5 desaturase activity, a high content of long-chain polyunsaturated fatty acids in the skeletal muscle, and insulin sensitivity may be due to parallel effects of diet and/or physical activity on the fatty acid composition and on insulin sensitivity.

Dietary trans fatty acids in early life: a review.

Trans fatty acids are unsaturated fatty acids with at least a double trans configuration, resulting in a more rigid molecule close to a saturated fatty acid. These appear in dairy fat because of ruminal activity, and in hydrogenated oils; margarines, shortenings and baked goods contain relatively high levels of trans fatty acids. These fatty acids can be incorporated into both fetal and adult tissues, although the transfer rate through the placenta continues to be a contradictory subject. In preterm infants and healthy term babies, trans isomers have been inversely correlated to infantile birth weight. However, in multigenerational studies using animals, there is no correlation between birth weight, growth, and dietary trans fatty acids. Maternal milk reflects precisely the daily dietary intake of trans fatty acids, from 2% to 5% of the total fatty acids in human milk. The level of linoleic acid in human milk is increased by a high trans diet, but long-chain polyunsaturated fatty acids remain mostly unaffected. Likewise, infant tissues incorporate trans fatty acids from maternal milk, raising the level of linoleic acid and relatively decreasing arachidonic and docosahexaenoic acids. This suggests an inhibitory effect of trans fatty acid on liver Delta-6 fatty-acid desaturase activity. As opposed to blood and liver, the brain appears to be protected from the trans fatty-acid accumulation in experimental animals, but no data have yet been reported for human newborns. Further investigations in humans are needed to definitively establish the potential physiological consequences of trans fatty-acid intake during the neonatal period.

Activity and mRNA abundance of Δ-5 and Δ-6 fatty acid desaturases in two human cell lines

We analyzed fatty acid biosynthesis in Chang and ZR-75-1 cells. Both cell lines could desaturate and further elongate substrates for Δ-5 desaturase. ZR-75-1 but not Chang cells showed Δ-6 desaturation of 18:2 n-6, 18:3 n-3, 24:4 n-6 and 24:5 n-3. In both cell lines, the mRNA abundance can be related to Δ-5 or Δ-6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Δ-6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils.

We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C(18)-specific delta(9)-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C(14) and C(16) acyl-ACPs but a relative lack of C(18) acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor delta(9)-18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.

Calcium deficiency modifies polyunsaturated fatty acid metabolism in growing rats.

Fatty acid desaturase activities were determined in liver microsomes from calcium-deficient rats and compared to calcium-sufficient ones. The calcium-deprived diet (0.5 g/kg) administered for 60 d caused a 30% inhibition in the delta5 desaturase activity and a 45-55% decrease in delta6 and delta9, respectively, facts that cannot be attributed to a reduction in food intake. In vitro addition of calcium, ethyleneglycol-bis(Beta-aminoethyl ether)N,N-tetraacetic acid, and/or cytosol fractions from control or calcium-deficient rats to microsomes from both groups of animals indicates that the reduced desaturase capacities would be the consequence of an indirect effect of calcium deprivation. The present work shows that the reduced unsaturated fatty acid biosynthesis might be the result of modifications in the physicochemical properties of microsomal membranes. Such changes could also be derived from the inhibition of phospholipase A2 activity induced by calcium deficiency.