Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase

Abstract

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2±3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.

Cloning of DGAT2, a Second Mammalian Diacylglycerol Acyltransferase, and Related Family Members

“…It is somewhat surprising, however, that the two identified DGATs are members of gene families that do not share obvious homology. Interestingly, both the DGAT1 and the DGAT2 gene families have phylogenetic relatives dating back to yeast, and both enzymes have homologues that function as DGATs in plants (9,10). 2 It seems likely that a DGAT2 homologue in yeast (GenBank TM accession number CAA99466) contributes to the triglyceride synthesis activity not accounted for by lecithin:diacylglycerol acyltransferase activity, which appears to be the major triglyceride synthesis activity in this organism (17,18).…”

“…Recently, a DGAT was isolated by protein purification in the fungus M. rammaniana (26). The gene encoding this DGAT (MrDGAT2A) has no sequence homology with members of the DGAT1 family, including genes related to plant DGAT1 (9,10). Using sequences of the newly identified fungal DGAT to perform homology searches of EST data bases, we identified a family of related genes in mammalian species (Fig.…”

Compartmentation of Triacylglycerol Accumulation in Plants

“…Two classes of genes encode DGATs in plants, DGAT1 and DGAT2, and these are homologous to those found in fungi and mammals (42). Genetic studies with mutants have confirmed that DGAT1 is required for normal TAG accumulation in oilstoring tissues of Arabidopsis (43), and its overexpression in vegetative tissues or seeds can lead to enhanced TAG accumulation (44,45). However, disruption of DGAT1 gene function results in only a 20 -40% reduction in Arabidopsis seed oil, so other mechanisms must cooperate in the accumulation of TAG in plants.…”

Bioengineering Recombinant Diacylglycerol Acyltransferases

“…Animals with reduced DGAT activity are resistant to diet-induced obesity Smith et al, 2000) and lack milk production (Smith et al, 2000). Over-expression of DGAT enzymes increases TAG content in plants (Andrianov et al, 2010;Bouvier-Nave et al, 2000;Burgal et al, 2008;Durrett et al, 2010;Jako et al, 2001;Lardizabal et al, 2008;Xu et al, 2008), animals (Kamisaka et al, 2010;Liu et al, 2009;Liu et al, 2007;Roorda et al, 2005), and yeast (Kamisaka et al, 2007). DGATs have nonredundant functions in TAG biosynthesis in species such as mice (Stone et al, 2004) and tung tree (Vernicia fordii) (Shockey et al, 2006).…”