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

• Published in: Gene Vol. 493; no. 2; pp. 219 - 227
• Main Authors: Yu, S.Y., Li, H., Tong, M., Ouyang, L.L., Zhou, Z.G.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10.02.2012
• Subjects: Acetyltransferases - genetics; Amino Acid Sequence; Arachidonic acid; Arachidonic Acid - biosynthesis; biosynthesis; cDNA libraries; Chlorococcales; Cloning, Molecular; complementary DNA; Elongase; endoplasmic reticulum; Endoplasmic Reticulum - metabolism; expressed sequence tags; fatty acid composition; freshwater; gamma-linolenic acid; gas chromatography; gene expression regulation; Gene Transfer Techniques; genes; genetically modified organisms; mass spectrometry; Microalga; microalgae; Microalgae - genetics; nitrogen; Nitrogen - metabolism; Nitrogen starvation; Phylogeny; quantitative polymerase chain reaction; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sequence Alignment; starvation; Subcellular localization; transcription (genetics); Transformation, Genetic; yeasts; GC–MS; PUFA; Microalga; DGLA; EST; qRT-PCR; Elongase; Arachidonic acid; ER; GFP; ArA; SDA; ETA; LA; ALA; Subcellular localization; LC-PUFA; GLA; Nitrogen starvation
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2011.11.053

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 40 h 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. ► A fatty acid elongase gene has been cloned and characterized. ► Functional identification of this gene has been succeeded in yeast. ► This gene has been localized to the ER for GLA elongation. ► qRT-PCR and fatty acid contents verify its role in biosynthesis of ArA.