Identification And Functional Analysis Of Avocado Dgat1 Expressed In Yeast

The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. There is significant human nutritional demand for vegetable oil, but its use in production of renewable biomaterials and fuels has intensified the need to further increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). Both DGAT and PDAT contribute to TAG biosynthesis in an independent or overlapping manner, depending on the species. However, preferred pathway for TAG biosynthesis is not well studied in nonseed tissues such as mesocarp. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 was identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGAT1s. These data reveal that the putative DGAT1 of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For further functional analysis, the avocado DGAT1 was expressed in H1246, a TAG-deficient yeast strain and lipotoxicity rescue assays, TLC analysis, Nile Red staining were conducted. The complementation of this yeast strain confirmed enzyme activity and supported the possible role of avocado DGAT1 in TAG biosynthesis. Finally, substrate specificity of DGAT was determined by incubating microsomes with different radiolabeled substances and found that avocado DGAT1 has a preference toward oleic acid (18:1) compared to palmitic acid (16:0) while it is converting diacylglycerol (DAG) to triacylglycerol. In summary, we characterized functional DGAT1 in a basal angiosperm species, which may be metabolically engineered into crop species to produce TAG enriched in oleic acid