Caratterizzazione dell'enzima glicerol-3 fosfato sn-2 aciltransferasi di Yarowia lipolytica

Synthesis, storage and degradation of neutral lipids enable cells to cope with cell metabolism when nutrients are no longer provided by the environment. As far as lipid homeostasis is concerned, imbalances yield accumulation of lipids in non-adipose tissues, a phenomenon known as lipotoxicity. Mechanisms regulating thresholds of lipid alcohols (as diacylglycerols and sterols) or fatty acids are likely to be cytoprotective. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in the synthesis of all glycerolipids [1]. Saccharomyces cerevisiae employs two GPATs, encoded by GAT1 and GAT2 genes. sn-1 GPAT doesn't exhibit substrate's preference for specific fatty acids, whilst sn-2 GPAT prefers palmitic acid. This research is focused on the characterization of glycerol 3-phosphate sn-2 acyltransferase of the oleaginous yeast Yarrowia lipolytica (Y.l.), whose gene (GAT2) has been cloned and expressed in S. cerevisiae using different episomal plasmids. The aim of the present study is to figure out whether there are at least two enzymes which accomplish this biosynthetic reaction. In Y.l. GPATs are localized mostly in the lipid particles (LP), whilst in S.c. they are located in the endoplasmatic reticulum. It has been used Y.l. due to its outstanding capacity to hoard neutral lipids to levels exceeding 50 % of cell dry weight. Studies of LPs have been performed with S.c. Conversely, information from Y.l. is limited, though it has being exploited in several biotechnological either pharmaceutical processes, such as the synthesis of β-hydroxy butyrate, L-dopa and emulsifiers, according to its efficient hydroxylation capacity as well as its rapid growth rate and environmental friendly nature . Enzimes produced by Y.l. have been studied for enantioselective transformations, such as esterases to resolve a racemic mixture of ofloxacin ester, since the (S) form is biologically active. Growth curves revealed a depressed trend for the double mutant. Neutral lipid determination via TLC and densitometric analysis unveiled a remarkable drop over the concentrations of sterols, steryl esters and triacylglicerols for the double knock-out strain. Thus, it can be surmised that Gat2 holds the major route for the downstream synthesis of neutral lipids. This suggests that null mutations of both GAT1 and GAT2 are synthetically lethal to yeast. Hence, yeast contains at least two independent systems of phosphatidic acid biosynthesis. In the phospholipid determination, alterations have been detected particularly in the synthesis of PC.