We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

Isochrysis galbana, a marine prymnesiophyte micro-

Dietary omega3-polyunsaturated fatty acids are thought to influence the risk of Alzheimer's disease (AD), and supplemental docosahexaenoic acid (DHA; 22:6n-3) has been reported to reduce neurodegeneration in mouse models of AD. We have analysed the fatty acid composition of frontal, temporal and parietal neocortex in 58 normal and 114 AD brains. Significant reductions were found for stearic acid (18:0) in frontal and temporal cortex and arachidonic acid (20:4n-6) in temporal cortex in AD, and increases in oleic acid in frontal and temporal cortex (18:1n-9) and palmitic acid (16:0) in parietal cortex. DHA level varied more in AD than controls but the mean values were not significantly different. Fatty acid composition was not related to APOE genotype, age, gender or post-mortem delay. Further research is needed to distinguish between alterations that are secondary to AD and those that contribute to the disease process.

A cDNA isolated from the prymnesiophyte micro-alga Isochrysis galbana, designated IgASE1, encodes a fatty acid elongating component that is specific for linoleic acid (C18:2n-6) and a-linolenic acid (C18:3n-3). Constitutive expression of IgASE1 in Arabidopsis resulted in the accumulation of eicosadienoic acid (EDA; C20:2n-6) and eicosatrienoic acid (ETrA; C20:3n-3) in all tissues examined, with no visible effects on plant morphology. Positional analysis of the various lipid classes indicated that these novel fatty acids were largely excluded from the sn-2 position of chloroplast galactolipids and seed triacylglycerol, whereas they were enriched in the same position in phosphatidylcholine. EDA and ETrA are precursors of arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3), and docosahexaenoic acid (C22:6n-3) synthesized via the so-called v6 D8 desaturase and v3 D8 desaturase biosynthetic pathways, respectively. The synthesis of significant quantities of EDA and ETrA in a higher plant is therefore a key step in the production of very long chain polyunsaturated fatty acid in oil-seed species. The results are further discussed in terms of prokaryotic and eukaryotic pathways of lipid synthesis in plants.The very long chain polyunsaturated fatty acids (VLCPUFAs), arachidonic acid (AA; C20:4n-6), docosahexaenoic acid (DHA; C22:6n-3), and eicosapentaenoic acid (EPA; C20:5n-3) are considered to have profound effects on cell function and development. The reduction in nonfatal and fatal cardiovascular events that is associated with the consumption of EPA and DHA may be due to the stabilization of atherosclerotic plaques (Thies et al., 2003). AA and DHA are considered to be important in pre-and postnatal development (Crawford, 2000; for review, see Lauritzen et al., 2001). Such VLCPUFAs are also precursors of the physiologically active prostaglandins, thromboxanes, and leukotrienes. No higher plants contain these fatty acids and hence it is considered desirable to genetically engineer the capacity to synthesize them in oilseed species and so provide an important source for the nutraceutical/pharmaceutical industries.Isochrysis galbana is a marine microalga that is rich in DHA and EPA (Lopez et al., 1994). Recently we isolated and characterized a cDNA from I. galbana, which encodes a novel fatty acid elongase component designated IgASE1 (Qi et al., 2002(Qi et al., , 2003. Transgenic yeastexpressing IgASE1 converted linoleic acid (LA; C18:2n-6) and a-linolenic acid (ALA; C18:3n-3) to eicosadienoic acid (EDA; C20:2n-6) and eicosatrienoic acid (ETrA; C20:3n-3), respectively (Qi et al., 2002). The substrate specificity of IgASE1 is consistent with it catalyzing the condensation reaction of fatty acid elongation (Qi et al., 2003). We have further suggested (Qi et al., 2002(Qi et al., , 2003 that the IgASE1 elongating activity is the first committed step in VLCPUFA synthesis and hence AA, EPA, and DHA formation via the so-called v3 D8 and v6 D8 pathways, respectively ( Fig. 1; Nichols and Appleby, 1969).Here we report the...

The biosynthesis of C 18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained ∆9, ∆12 and ∆6 desaturase activities. The ∆9 desaturase exhibited characteristics similar to those of the animal and yeast ∆9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate. Cytochrome b 5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH:cytochrome b 5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time-course studies with radiolabelled oleoyl-CoA showed that the oleate [18:1(9)] was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho. Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the ∆12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho. Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn-1 and sn-2 positions served as substrate for the ∆12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine: acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. ∆6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant. Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate [18:2(9,12)] and γ-linolenate [18:3(6,9,12)] on this phospholipid is similar to that reported for oil seed membranes.Keywords : Mucor ; oil biosynthesis; polyunsaturated fatty acid ; desaturase substrate; phosphatidylcholine.The biosynthesis of polyunsaturated fatty acids in plants [1Ϫ that contain high levels of 18:3(6,9,12) [12Ϫ14] and have been 3] and animals [4,5] and their assembly to produce storage fats produced on an industrial scale [15]. We have studied previously and oils has been studied extensively. By comparison, and with the biosynthesis of 18:3(6,9,12) in higher plants and characterthe exception of oleaginous yeast [6,7], few detailed biochemi-ised the substrates and stereospecificity of the desaturases incal studies on lipid synthesis in fungi have been undertaken. Of volved in its formation [16Ϫ18]. In borage seed microsomes, particular commercial interest is the production of 'speciality the ∆12 desaturase utilised oleate esterified to positi...

IgASE1, a C18-v v9-polyunsaturated fatty acid-specific fatty acid elongase component from Isochrysis galbana, contains a variant histidine box (his-box) with glutamine replacing the ¢rst histidine of the conserved histidine-rich motif present in all other known equivalent proteins. The importance of glutamine and other variant amino acid residues in the his-box of IgASE1 was determined by site-directed mutagenesis. Results showed that all the variation in amino acid sequence between this motif in IgASE1 and the consensus sequences of other elongase components was required for optimum enzyme activity. The substrate speci¢city was shown to be una¡ected by these changes suggesting that components of the his-box are not directly responsible for substrate speci¢city.

No abstract

TAG-synthesizing capacity was greatest in the microsomal membrane fraction, which accumulated high levels of phosphatidic acid in the presence of glycerol 3-phosphate and acyl-CoA substrates a t pH 7-0. Further metabolism of phosphatidic acid to diacylglycerol and TAG was achieved by increasing the pH to 80. Lysophosphatidic acid : acyl-CoA acyltransferase (LPAAT) activity was particularly high and may have accounted for the rapid accumulation of phosphatidic acid in the membranes. The glycerol-3-phosphate : acyl-CoA acyltransferase (GPAAT) and LPAAT were non-specific for a range of saturated and unsaturated species of acyl-CoA although the GPAAT showed a marked selectivity for palmitoyl-CoA and the LPAAT for oleoyl-and linoleoyl-CoA. y-Linolenic acid was detected a t all three positions of sn-TAG and was particularly enriched a t the sn-3 position. The preparation of active in vitro systems (microsomal membranes) capable of the complete biosynthetic pathway for TAG assembly may be valuable in understanding the assembly of oils in future transgenic applications.