Expression of the Isochrysis C18-Δ9 Polyunsaturated Fatty Acid Specific Elongase Component Alters Arabidopsis Glycerolipid Profiles

Abstract

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...

Elongation of polyunsaturated fatty acids in trypanosomatids

“…Yeasts expressing the predicted L. major D5 PUFA elongase (ELO5) and supplemented with the n6 substrate AA showed an additional peak when compared with the fatty acid profile of the control harboring the empty vector. This peak was identified as the fatty acid methyl ester (FAME) of 22:4D 7,10,13,16 , and this was confirmed by unequivocal determination of the double bond positions through analysis of the mass spectrum of the dimethyloxazoline (DMOX) derivative (Fig. 3, insert).…”

“…From the resultant phylogenetic tree, it is clear that a majority of putative trypanosomatid elongases group together in a branch exclusive to these parasites ( Fig. 1, sequences [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. Their sequences are related to those of known elongases of saturated and monounsaturated fatty acids, such as human ELOVL3 and ELOVL6 and the fungal elongases ( Fig.…”

“…cruzi XP_809644; 8, L. major CAJ03035; 9, L. major CAJ03028; 10, L. major CAJ03016; 11, L. major CAJ03023; 12, Try. cruzi XP_818062; 13 The other ELO-like elongases could be functionally equivalent to Sac. cerevisiae ELO1, ELO2 and ELO3, enzymes involved in the elongation of saturated and monounsaturated fatty acids [20].…”

“…The PUFA biosynthetic pathways found in lower eukaryotes usually involve the desaturation of oleate (18:1D 9 ), by an oleate or D12 desaturase, to linoleate (18:2D 9,12 4,7,10,13,16,19 (docosahexaenoic acid), respectively, by a D4 desaturase [10]. An alternative pathway has been described in Euglena gracilis.…”

“…This allowed us to describe the biosynthetic pathways present in these parasitic flagellates. L. major is able to synthesize 22:5D 4,7,10,13,16 and docosahexaenoic acid by the action of D12, x3, D6, D5 and D4 desaturases. Both Trypanosoma species contain functional D12 and D4 desaturases but lack x3, D6 and D5 desaturases.…”