The in¯uence of culture age and nitrogen concentration on the distribution of fatty acids among the dierent acyl lipid classes has been studied in continuous cultures of the microalga Phaeodactylum tricornutum. The culture age was tested in the range of 1.15±7 days, controlled by adjusting the dilution rate of fresh medium supplied. The eect of nitrogen concentration was tested from saturating conditions to starvation by modifying nitrate concentration in the fresh medium. Culture age had almost no in¯uence on the fatty acid content; 16:0, 16:3 and 20:5 increased moderately wherein the level of 16:1 decreased when the culture age decreased. Culture age had no eect on the total fatty acid content that remained around 11% of dry weight. Conversely, culture age had a greater impact on lipid classes, producing changes in amounts of triacylglycerols (TAG) which ranged between 43% and 69%, and galactolipids (GLs) that oscillated between 20% and 40%. In general, the content of polar lipids of the biomass decreased with culture age. The other factor assayed, nitrogen content, aected the fatty acid pro®le. Saturated and monounsaturated fatty acids accumulated when the nitrogen concentration was decreased. The experiments regarding the eect of nitrogen concentration on lipid species were carried out with cells of an average age of 3.5 days. A decrease of the nitrogen concentration caused the GL fraction to decrease from 21 to 12%. Conversely, both neutral lipids (NLs) and phospolipids (PLs) increased from about 73 to 79% and from 6 to 8%, respectively. In these experiments, TAG was the lipid class with the highest increase, from 69 to 75%. 7

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for the preparation of fatty acid methyl esters allows notable saving of time and reagents. The material being analysed is heated for 10 minutes with methanol, acetyl chloride and hexane.

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The synthesis of GLA (delta6,9,12-1-8:3) is carried out in a number of plant taxa by introducing a double bond at the delta6 position of its precursor, linoleic acid (delta9,12-18:2), through a reaction catalyzed by a delta6-desaturase enzyme. We have cloned genes encoding the delta6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b(5) domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other delta6-desaturases in a different cluster from that of the highly related delta8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.