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

Abstract

Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-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 Delta4-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-(14)C]-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-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 Delta4-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 Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.

Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis

“…There was no difference in DHA yield, but several different fatty acids that are not intermediates of the pathway were found in the yeast expressing the fish elongase. The only detectable by-product in the strain expressing the algal elongases was 22:4 ⌬10, 13,16,19 , which resulted from the elongation of 20:4 ⌬8, 11,14,17 , underlining the advantage of using specific single-step elongases.…”

Characterization and comparison of fatty acyl Δ6 desaturase cDNAs from freshwater and marine teleost fish species

“…In contrast to the higher animals, the Δ4 pathway for the production of DHA appears to operate in some lower organisms, based on the cloning and characterisation of Δ4 desaturases from Thraustchytrium sp. (Qui et al, 2001) and the algae Euglena gracilis (Meyer et al, 2003) and Pavlova lutheri (Tonon et al, 2003).…”

Isolation and functional characterization of fatty acid Δ5‐elongase gene from the liverwort Marchantia polymorpha L

“…To clone the EgDES4 ORF (AY278558) [9] from an E. gracilis cDNA pool, PCR was carried out with primers EgDES4-01F 5 0 -GCG-GATCCGAAATGTTGGTGCTGTTTGGCA-3 0 and EgDES4-02R 5 0 -GCGGATCCTTATGACTTTTTGTCCCCGT-3 0 using AccuPrime Pfx DNA polymerase (Invitrogen). The resultant PCR product was digested by BamHI and cloned into pPIC3K (with the HIS4 gene; Invitrogen) under the control of the 5 0 AOX1 promoter.…”

“…Heterologous reconstitution of the DHA synthetic pathway in P. pastoris To reconstitute the DHA synthetic pathway, MpELO2 and the E. garacilis D4-desaturase gene EgDES4 [9] were co-expressed in P. pastoris. Cells that carry both pPICZA-MpELO2 and pPIC3K-EgDES4 integrated into the genome produced DHA in addition to DPA (Fig.…”

“…In lower eukaryotes, DHA is synthesized more simply by D5-elongation from EPA to docosapentaenoic acid (DPA, 22:5D 7,10,13,16,19 ), followed by D4-desaturation. To date, D4-desaturase genes have been identified from a marine fungus and several microalgae [8][9][10][11]. Genes encoding an enzyme with D5-elongating activity have also been isolated from marine microalgae [11,12] and several vertebrates [5].…”