Identification of a Δ6 fatty acid elongase gene for arachidonic acid biosynthesis localized to the endoplasmic reticulum in the green microalga Myrmecia incisa Reisigl.

Myrmecia incisa Reisigl H4301 is a green coccoid freshwater microalga that is rich in arachidonic acid (20:4n-6, ArA) especially grown under a nitrogen starvation stress. A fatty acid elongase gene, MiFAE, was cloned based on a selected expressed sequence tag (EST) from a M. incisa cDNA library. To examine the function, the MiFAE gene was heterologously expressed in Saccharomyces cerevisiae. The fatty acid profile of the transgenic yeast was analyzed by gas chromatography-mass spectrometry (GC-MS), and the results illustrated that the enzyme encoded by MiFAE was able to elongate γ-linolenic acid (18:3n-6, GLA) and stearidonic acid (18:4n-3, SDA) to di-homo-γ-linolenic acid (20:3n-6, DGLA) and eicosatetraenoic acid (20:4n-3, ETA), respectively, suggesting that the cloned MiFAE gene seemed to encode a Δ6 fatty acid elongase. Expression of a MiFAE-GFP fusion encoded by a pYES2 vector showed that this Δ6 fatty acid elongase localized to the endoplasmic reticulum (ER) of yeast for fatty acid elongation. Quantitative real-time PCR results showed that the relative transcription level of MiFAE in M. incisa grown under a nitrogen starvation stress was increased, but it rapidly declined under conditions of nitrogen replenishment. GC-MS analysis revealed that the contents of DGLA, a direct product catalyzed by Δ6 fatty acid elongase, and ArA, the terminal product of fatty acid biosynthesis in this microalga, increased and decreased accompanying the shift from nitrogen starvation to replenishment, although there was a 40h lag time for ArA increment. The correlation between the up-regulated and down-regulated transcription of MiFAE and ArA content in response to a nitrogen starvation/replenishment shift showed that nitrogen could regulate the transcription of the MiFAE gene and that this gene is critical and responsible for the biosynthesis and accumulation of ArA in the cytoplasm of M. incisa.