Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis.
Phaeodactylum tricornutum is an unicellular silica-less diatom in which eicosapentaenoic acid accumulates up to 30% of the total fatty acids. This marine diatom was used for cloning genes encoding fatty acid desaturases involved in eicosapentaenoic acid biosynthesis. Using a combination of PCR, mass sequencing and library screening, the coding sequences of two desaturases were identified. Both protein sequences contained a cytochrome b5 domain fused to the N-terminus and the three histidine clusters common to all front-end fatty acid desaturases. The full length clones were expressed in Saccharomyces cerevisiae and characterized as Delta5- and Delta6-fatty acid desaturases. The substrate specificity of each enzyme was determined and confirmed their involvement in eicosapentaenoic acid biosynthesis. Using both desaturases in combination with the Delta6-specific elongase from Physcomitrella patens, the biosynthetic pathways of arachidonic and eicosapentaenoic acid were reconstituted in yeast. These reconstitutions indicated that these two desaturases functioned in the omega3- and omega6-pathways, in good agreement with both routes coexisting in Phaeodactylum tricornutum. Interestingly, when the substrate selectivity of each enzyme was determined, both desaturases converted the omega3- and omega6-fatty acids with similar efficiencies, indicating that none of them was specific for either the omega3- or the omega6-pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.
Institut für Allgemeine Botanik, Universität Hamburg, Hamburg, Germany. fredDo@botanik.uni-hamburg.de
Frédéric
F
Domergue
Jens
J
Lerchl
Ulrich
U
Zähringer
Ernst
E
Heinz
D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000096:biosynthesis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D000090943:Delta-5 Fatty Acid Desaturase; D017377:Diatoms / Q000201:enzymology* / Q000235:genetics; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis*; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D015525:Fatty Acids, Omega-3 / Q000378:metabolism; D043371:Fatty Acids, Omega-6; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D000090943:Delta-5 Fatty Acid Desaturase; D015525:Fatty Acids, Omega-3; D043371:Fatty Acids, Omega-6; D005231:Fatty Acids, Unsaturated; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D015118:Eicosapentaenoic Acid; D044943:Fatty Acid Desaturases