Acyl-CoA: diacylglycerol acyltransferase (DGAT) is a key enzyme responsible for triacylglycerol (TAG) synthesis in eukaryotic organisms. The present work reported DGAT genes in the green alga Myrmecia incisa, a promising candidate for arachidonic acid (ArA) production. According to the results of homology search against a transcriptome database, we cloned three cDNAs encoding putative DGAT1 and DGAT2. The 2238-bp, 1056-bp and 1068-bp of open reading frame (ORF) of these three cDNAs, designated as MiDGAT1, MiDGAT2A and MiDGAT2B, were predicted to encode proteins composed of 745, 351 and 355 amino acids. They were separated by 14, 6 and 6 introns, respectively, as revealed by comparing their corresponding cDNA and DNA sequences. Multiple sequence alignment of amino acids indicated that MiDGAT1 had a pleckstrin homology (PH) domain, whilst MiDGAT2s contained a highly conserved HPHG, a characteristic motif of DGAT2 family. To determine the function, they were expressed heterologously in a Saccharomyces cerevisiae mutant strain with impaired TAG metabolism. Results of thin-layer chromatography and BODIPY staining indicated that both MiDGAT1 and MiDGAT2s were able to restore TAG synthesis and lipid body formation. GC-MS analysis indicated that palmitic acid and stearic acid were the major components of TAGs in yeast cells, and their ratio between wild type and the transformed yeasts was not significantly different. Quantitative RT-PCR results showed that the transcript level of MiDGAT2A was regulated by nitrogen starvation, which was consistent with TAG accumulation in M. incisa.
BackgroundArachidonic acid (ArA) is important for human health because it is one of the major components of mammalian brain membrane phospholipids. The interest in ArA inspired the search for a new sustainable source, and the green microalga Myrmecia incisa Reisigl H4301 has been found a potential ArA-producer due to a high content of intracellular ArA. To gain more molecular information about metabolism pathways, including the biosynthesis of ArA in the non-model microalga, a transcriptomic analysis was performed.ResultsThe 454 pyrosequencing generated 371,740 high-quality reads, which were assembled into 51,908 unique sequences consisting of 22,749 contigs and 29,159 singletons. A total of 11,873 unique sequences were annotated through BLAST analysis, and 3,733 were assigned to Gene Ontology (GO) categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered a C4-like photosynthesis pathway in M. incisa. The biosynthesis pathways of lipid particularly those of ArA and triacylglycerol (TAG) were analyzed in detail, and TAG was proposed to be accumulated in oil bodies in the cytosol with the help of caleosin or oil globule-associated proteins. In addition, the carotenoid biosynthesis pathways are discussed.ConclusionThis transcriptomic analysis of M. incisa enabled a global understanding of mechanisms involved in photosynthesis, de novo biosynthesis of ArA, metabolism of carotenoids, and accumulation of TAG in M. incisa. These findings provided a molecular basis for the research and possibly economic exploitation of this ArA-rich microalga.
In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress.
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