Implications of Soluble Diacylglycerol Acyltransferases in Triacylglycerol Biosynthesis in Yeast and Plants

Abstract

Lipids are stored in a cell for providing energy. The main advantages of storing lipids over carbohydrates like glycogen is that, lipids yield more energy after oxidation because they represent the highly reduced form of carbon, needs less space and water for storage. Conservation of chemical energy in the form of biologically inert form is by storing molecules like triacylglycerol (TAG) and Steryl esters (SE). Triacylglycerol is the major storage form of energy in all eukaryotic cells. During the periods of nutritional excess and nutritional stress, all organisms like bacteria, yeast, animals, and plants can able to do the critical function of synthesizing the triacylglycerol. TAG is an energy store and a repository of essential and non-essential fatty acids and precursors for phospholipid biosynthesis. TAG synthesis mainly takes place in endoplasmic reticulum in mammals and in plants, it takes place in plastid and mitochondria. Triacylglycerol synthesis discovered by Kennedy starts with glycerol 3- phosphate. Glycerol 3-phosphate gets acylate to form lysophosphatic acid (LPA), which in turn acylate to form phosphatic acid (PA) and the reactions are catalyzed by glycerol 3-phosphate acyltransferase (GPAT) and LPA acyltransferase (LPAT) respectively. PA undergoes phosphorylation by PA phosphatase enzyme to give diacylglycerol (DAG). Further acylation of DAG gives rise to TAG and the reaction is catalyzed by diacylglycerol acyltransferase (DGAT). There are several DGAT classes were identified they are DGAT1, DGAT2, PDAT and bifunctional TAG/wax ester synthase. However all the enzymes involved in Kennedy TAG biosynthetic pathway as well as the enzymes of all different DGAT classes are membrane bound enzymes. Through our studies an another DGAT class that is soluble and cytosolic DGAT was first identified in peanut and also in yeast, Rhodotorula glutinis in which a soluble cytosolic complex itself has been identified. The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in R. glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of alternate TAG biosynthetic pathway in oleaginous yeasts. A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX2GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.