Cytosolic Triacylglycerol Biosynthetic Pathway in Oilseeds. Molecular Cloning and Expression of Peanut Cytosolic Diacylglycerol Acyltransferase

Abstract

Triacylglycerols (TAGs) are the most important storage form of energy for eukaryotic cells. TAG biosynthetic activity was identified in the cytosolic fraction of developing peanut (Arachis hypogaea) cotyledons. This activity was NaF insensitive and acyl-coenzyme A (CoA) dependent. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG biosynthesis that acylates diacylglycerol to TAG. Soluble DGAT was identified from immature peanuts and purified by conventional column chromatographic procedures. The enzyme has a molecular mass of 41 6 1.0 kD. Based on the partial peptide sequence, a degenerate probe was used to obtain the full-length cDNA. The isolated gene shared less than 10% identity with the previously identified DGAT1 and 2 families, but has 13% identity with the bacterial bifunctional wax ester/ DGAT. To differentiate the unrelated families, we designate the peanut gene as AhDGAT. Expression of peanut cDNA in Escherichia coli resulted in the formation of labeled TAG and wax ester from [14 C]acetate. The recombinant E. coli showed high levels of DGAT activity but no wax ester synthase activity. TAGs were localized in transformed cells with Nile blue A and oil red O staining. The recombinant and native DGAT was specific for 1,2-diacylglycerol and did not utilize hexadecanol, glycerol-3-phosphate, monoacylglycerol, lysophosphatidic acid, and lysophosphatidylcholine. Oleoyl-CoA was the preferred acyl donor as compared to palmitoyl-and stearoyl-CoAs. These data suggest that the cytosol is one of the sites for TAG biosynthesis in oilseeds. The identified pathway may present opportunities of bioengineering oil-yielding plants for increased oil production.Oils and fats are glycerol triesters of fatty acids (triacylglycerols [TAGs]) and are mainly derived from plant and animal sources, respectively. Vegetable oils are the major source of edible lipids, accounting for more than 75% of the total lipids consumed across the world (Broun et al., 1999). The global demand for plant oils has intensified our efforts to genetically modify the organism to enhance oil yield.De novo biosynthesis of TAG has been shown to occur by the sequential acylation of glycerol-3-P (Kennedy, 1961;Ohlrogge et al., 1991). The first enzyme in this pathway, glycerol-3-P acyltransferase, catalyzes the formation of lysophosphatidic acid (LPA) that can be acylated to give phosphatidic acid (PA) by LPA acyltransferase. PA is the precursor for diacylglycerol (DAG) and anionic phospholipids. PA phosphatase catalyzes the dephosphorylation of PA to form DAG, which is an immediate precursor for TAG, phosphatidylcholine, and phosphatidylethanolamine.

Characterisation of DGAT1 and DGAT2 from Jatropha curcas and their functions in storage lipid biosynthesis

“…In addition to DGAT1 and DGAT2 (which have been extensively identified in plants), the third class DGAT3 gene was identified from Arabidopsis (Hernández et al 2012) and peanut (Saha et al 2006), but little is known about the DGAT3 gene in other plants. Studies have shown that the DGAT1 and DGAT2 represent different gene families with its own functional motifs in gene structure.…”

Three Acyltransferases and Nitrogen-responsive Regulator Are Implicated in Nitrogen Starvation-induced Triacylglycerol Accumulation in Chlamydomonas

“…Likewise, type-2 DGAT in mouse is important since mice lacking this enzyme are severely depleted in both tissue and plasma TAG, which leads to early death (36). Genes representing a third class (type-3) of DGAT, which is a soluble cytosolic enzyme, have been identified in Arachis hypogaea (peanut) (37) and Arabidopsis thaliana (38).…”

Defective in Cuticular Ridges (DCR) of Arabidopsis thaliana, a Gene Associated with Surface Cutin Formation, Encodes a Soluble Diacylglycerol Acyltransferase

“…Saturation kinetics for TAG formation with varying concentrations of 1,2-dioleoylglycerol was performed. The purified recombinant At5g23940 (0.5 g) was added to 10 M oleoylCoA with varying concentrations of diacylglycerol (10,20,40,50, and 100 M). Data represent mean Ϯ S.D.…”

“…A novel bifunctional DGAT/ wax ester synthase was described in Acinetobacter calcoaceticus (19). All of these acyltransferases are membrane-bound, and we have isolated a novel DGAT from developing peanut (Arachis hypogaea) cotyledons (20) and named it DGAT3; unlike all of the above reported DGATs, DGAT3 is soluble.…”