Diacylglycerol acyltransferase type 2 cDNA from the oleaginous microalga Neochloris oleoabundans: cloning and functional characterization
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Currently, biodiesel production from microalgae is technically, but not yet economically, feasible. Increasing microalgal triacylglycerol (TAG) content via genetic engineering could be a potential approach to improve the efficiency of biodiesel production. In all organisms studied so far, diacylglycerol acyltransferase (DGAT) is the primary enzyme of TAG biosynthesis. In this study, we cloned the cDNA encoding DGAT type 2 (NeoDGAT2) from the oleaginous microalga Neochloris oleoabundans. The open reading frame of NeoDGAT2 cDNA was 978 bp encoding a protein of 325 amino acids. Although N. oleoabundans has been classified in the class Chlorophyceae, NeoDGAT2 was closely related to Treboxiophycean DGAT2 as it shared 80 % amino acid identity with CvDGAT2-2 of Chlorella variabilis, but distantly related to Chlorophycean DGAT2 as it shared only 34 % identity with CrDGAT2A (DGTT4) of Chlamydomonas reinhardtii. As suggested by hydropathy analysis, NeoDGAT2 might contain 4 to 5 transmembrane domains. We tested whether NeoDGAT2 cDNA encoded a protein with DGAT activity by functional complementation assay in yeast Saccharomyces cerevisiae mutant H1246. NeoDGAT2 was able to compensate the endogenous DGAT2 activity of mutant H1246 and restore the lipid body formation and TAG synthesis. The successful cloning of cDNA encoding a protein with DGAT activity provides a candidate for genetic manipulation in microalgae to increase TAG content for biodiesel production.
KeywordsMicroalgae Neochloris oleoabundans Biodiesel Triacylglycerol biosynthesis Diacylglycerol acyltransferase (DGAT)
We thank Prof. Sten Stymne (Scandinavian Biotechnology Research, Alnarp, Sweden) for providing the yeast strains H1246 and SCY62 and Sirirat Fa-aroonsawat for technical assistance. This work was supported by Mahidol University and The Thailand Research Fund to Wipa Chungjatupornchai. Akaraphol Watcharawipas was supported by the 60th Year Supreme Reign of His Majesty King Bhumibol Adulyadej Scholarship.
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