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 Δ5‐ and Δ6‐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 Δ6‐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 ω3‐ and ω6‐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 ω3‐ and ω6‐fatty acids with similar efficiencies, indicating that none of them was specific for either the ω3‐ or the ω6‐pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.