Identification and characterization of a type-2 diacylglycerol acyltransferase in green microalga Haematococcus pluvialis

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Nguyen, Thi Viet Trinh
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Haematococcus pluvialis is a green microalga which can accumulate high amounts of storage lipids (mainly triacylglycerol, TAG) and high-value astaxanthin under various stress conditions. In wild-type H. pluvialis, approximately 95% of the astaxanthin molecules are stored in the form of astaxanthin acyl ester in lipid bodies. Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some microalgal species. Therefore, identification and characterization of HpDGATs in H. pluvialis is essential in increasing both lipid and astaxanthin productions. Additionally, as astaxanthin accumulation coincides with TAG biosynthesis in H. pluvialis and the biosynthesis of astaxanthin esters may catalyzed by DGAT-like enzymes, the characterization of HpDGATs would also benefit the production of astaxanthin. In this project, the accumulation of storage lipid in H. pluvialis under high irritation stress was confirmed by the measurement of growth curves and lipid content. Eight candidate HpDGAT genes were identified from a transcriptome shotgun sequence database and their transcriptional expression levels were determined with qRT-PCR. The results showed that two type-2 DGATs (HpDGAT2s) had high expression levels under high irritation stress, suggesting their possible significance in TAG production. To further characterize the two HpDGAT2s, their coding sequences were expressed in Saccharomyces cerevisiae H1246, a quadruple mutant strain devoid of TAG synthesizing ability. Only HpDGAT2D could restore the TAG synthesizing ability of the yeast strain. Furthermore, in vitro enzymatic assays revealed that this recombinant HpDGAT2D had strong DGAT activity and displayed sigmoidal kinetics in response to increasing acyl-CoA concentration. Therefore, HpDGAT2D may have the potential to be used in improving oil production in H. pluvialis, other microalgae and higher plants via genetic engineering.
DGAT; Algae; Haematococcus pluvialis; Triacylglycerol biosynthesis; Sigmoidal kinetics; Saccharomyces cerevisiae