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Analysis of a draft nuclear genome sequence of the diatom Thalassiosira pseudonana revealed the presence of 11 open reading frames showing significant similarity to functionally characterized fatty acid front‐end desaturases. The corresponding genes occupy discrete chromosomal locations as determined by comparison with the recently published genome sequence. Phylogenetic analysis showed that two of the T. pseudonana desaturase (Tpdes) sequences grouped with proteobacterial desaturases that lack a fused cytochrome b5 domain. Among the nine remaining gene sequences, temporal expression analysis revealed that seven were expressed in T. pseudonana cells. One of these, TpdesN, was previously characterized as encoding a Δ11‐desaturase active on palmitic acid. From the six remaining putative desaturase genes, we report here that three, TpdesI, TpdesO and TpdesK, respectively encode Δ6‐, Δ5‐ and Δ4‐desaturases involved in production of the health beneficial polyunsaturated fatty acid DHA (docosahexaenoic acid). Furthermore, we show that one of the remaining genes, TpdesB, encodes a Δ8‐sphingolipid desaturase with strong preference for dihydroxylated substrates.

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The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of healthbeneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast.Long-chain acyl-CoA synthetases (LACSs) play a critical role in the biosynthetic pathways of nearly all fatty acid (FA)-derived molecules. These enzymes esterify free FAs to CoA to form acyl-CoAs, a key activation step necessary for the utilization of FAs by most lipid metabolic enzymes (Groot et al., 1976). The enzymatic mechanism is a two-step reaction that proceeds via the formation of an acyl adenylate (acyl-AMP) intermediate (Kornberg and Pricer, 1953). Acyl-CoAs serve as important intermediates in many metabolic pathways, such as elongation and b-oxidation of FAs, enzyme activation, cell signaling, and transcriptional regulation (Watkins, 1997). Consistent with the diverse roles of acyl-CoA synthetases (ACSs) in cell metabolism, many eukaryotic organisms encode several different ACSs that specifically activate short-(C6-C8), medium-(C10-C12), long-(C14-C20), or very long-(.C22) chain FAs (Watkins, 1997). Moreover, some organisms possess multiple enzymes for each set of acyl chain lengths. In plants, LACS activity has been localized to several subcellular compartments (Schnurr et al., 2002(Schnurr et al., , 2004Shockey et al., 2002;Fulda et al., 2004), enabling acyl chains produced by de novo FA synthesis to be activated to their CoA esters and subsequently used for metabolic pathways such as those involved in the synthesis of membrane glycerolipids and storage lipids (triacylglycerols [TAGs]) in developing seeds (Ohlrogge et al., 1991). In addition, LACS enzymes play an important role in intracellular FA homeostasis (Choi and Martin, 1999) and in FA transport. This process has been studied in detail in bacteria (Black et al., 1992), yeast (Saccharomyces cerevisiae;Faergeman et al., 2001), and mammalian cells (Mashek et al., 2004). Recently, Fulda et al. (2004) demonstrated the involvement of two Arabidopsis (Arabidopsis ...