In vivo characterization of the first acyl-CoA Delta6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri.
Genomic DNA of Ostreococcus tauri, a fully sequenced marine unicellular alga from the phytoplankton, was used to amplify a gene coding for a typical front-end desaturase involved in polyunsaturated fatty acid biosynthesis. Heterologous expression in Saccharomyces cerevisiae revealed very high desaturation activity with Delta6-regioselectivity. Short-time kinetic experiments showed that the desaturase product was detected in the acyl-CoA pool 5 min after addition of the exogenous substrate to the yeast medium and long before its appearance in the total fatty acids. When this desaturase was co-expressed with the acyl-CoA Delta6-elongase from Physcomitrella patens and the lipid-linked Delta5-desaturase from Phaeodactylum tricornutum, high proportions of arachidonic or eicosapentaenoic acid were obtained, because nearly all of the Delta6-desaturated products were elongated. Furthermore, the product/educt ratios calculated in each glycerolipid for the Delta6-desaturase or for the acyl-CoA Delta6-elongase were in about the same range, whereas this ratio showed a very uneven profile in the case of the lipid-linked Delta5-desaturase. Finally, a sequence-based comparison of all the functionally characterized Delta6-desaturases showed that this enzyme was not related to any previously described sequence. Altogether, our data suggest that this desaturase from O. tauri is an acyl-CoA Delta6-desaturase, the first one cloned from a photosynthetically active organism.
Domergue
Frédéric
F
Biozentrum Klein Flottbek, Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany. fredomergue@viola.fr
Abbadi
Amine
A
Zähringer
Ulrich
U
Moreau
Hervé
H
Heinz
Ernst
E
D016718:Arachidonic Acid / Q000096:biosynthesis; D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
D016718:Arachidonic Acid; D015118:Eicosapentaenoic Acid; D044943:Fatty Acid Desaturases