Identification and characterization of Δ;12, Δ;6, and Δ5 desaturases from the green microalga Parietochloris incisa

The freshwater microalga Parietochloris incisa accumulates, under nitrogen starvation, large amounts of triacylglycerols containing approximately 60% of the x6 very long-chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. Based on sequence homology, we isolated three cDNA sequences from P. incisa, designated PiDesD12, PiDesD6, PiDesD5. The deduced amino acid sequences of the three genes contained three conserved histidine motifs; the front-end desaturases, PiDes6 and PiDes5, contained a fused N-terminal cytochrome b5 domain. By functional characterization in the yeast Saccharomyces cerevisiae, we confirmed that PiDesD6, PiDesD5 cDNA encode membrane bound desaturases with D6, and D5 activity, respectively. Both PiDes6 and PiDes5 can indiscriminately desaturate both x6 and x3 substrates. A phylogenetic analysis showed that the three genes were homologous to the corresponding desaturases from green microalgae and lower plants that were functionally characterized. Quantitative real-time PCR revealed the concerted expression pattern of all three genes in P. incisa cells subjected to nitrogen starvation, featuring maximum expression level on day 3 of starvation, corresponding to the sharpest increase in the share of arachidonic acid.