Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris,and Dictyostelium discoideum

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are bi osynthesized by a UDP-glucose: sterol glucosyltransferase (EC 2.4.1.173), W e cloned genes from three different yeasts and from Dictyostelium discoideu m, the deduced amino acid sequences of which all showed similarities with p lant sterol glucosyltransferases (Ugt80A1, Ugt80A2), These genes from Sac c haromyces cerevisiae (UGT51 = YLR189C), Pichia pas toris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coil. In vitro enzyme assays with cell-free extracts of the tr ansgenic E, coil strains showed that the genes encode UDP-glucose:sterol gl ucosyltransferases which can use different sterols such as cholesterol, sit osterol, and ergosterol as sugar accepters. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited no rmal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promo ter restored sterol glucoside synthesis in vitro, Lipid extracts of these c ells contained a novel glycolipid, This lipid was purified and identified a s ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectrosc opy. These data prove that the cloned genes encode sterol-beta-D-glucosyltr ansferases and that sterol glucoside synthesis is an inherent feature of eu karyotic microorganisms.