Biosynthesis of Docosahexaenoic Acid in Euglena gracilis: Biochemical and Molecular Evidence for the Involvement of a Δ4-Fatty Acyl Group Desaturase

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Abstract
Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the ω3/ω6-pathways involve the action of a Δ4-fatty acid desaturase. We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Δ4-fatty acid desaturase. For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable to convert exogenously supplied [2-14C]-docosapentaenoic acid (DPA, 22:5Δ7,10,13,16,19) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and α-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Δ4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNA library of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the Δ4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expression in yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids. The enzyme showed strict Δ4-regioselectivity and required the presence of a Δ7-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Δ4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.