tailieunhanh - Báo cáo khoa học: 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 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. | Eur. J. Biochem. 269 4105-4113 2002 FEBS 2002 doi Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis Frederic Domergue1 Jens Lerchl2 Ulrich Zahringer3 and Ernst Heinz1 1Institut fur Allgemeine Botanik Universitat Hamburg Hamburg Germany 2BASF Plant Science GmbH BPS-A30 Ludwigshafen Germany 3Forschungszentrum Borstel Borstel Germany 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 A5- and A6-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 A6-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 x3- and ro6-pathways in good agreement with both routes coexisting in Phaeodacty-lum tricornutum. Interestingly when the substrate selectivity of each enzyme was determined both desaturases converted the x3- and ro6-fatty acids with similar efficiencies indicating that none of them was specific for either the I 3- or the ro6-pathway. To our knowledge this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms. .