Genetic Analysis of Biosurfactant Production inUstilago maydis

Abstract

The dimorphic basidiomycete Ustilago maydis produces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated ␤-D-mannopyranosyl-D-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes, emt1 and cyp1, that are essential for the production of fungal extracellular glycolipids. emt1 is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-D-erythritol by transfer of GDP-mannose. Deletion of the gene cyp1 resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.

Nitrogen Source Affects Glycolipid Production and Lipid Accumulation in the Phytopathogen Fungus <i>Ustilago maydis</i>

“…In agreement with other reports, the preference of these two glycolipids is affected considerably by the nitrogen source [1] [37]. Additionally, in this study we showed first that UA is the main glycolipid when esterified fatty acids are accumulated in the lipid bodies, and second, that U. maydis is an important cell factory that produces not only glycolipids, but also fatty acids which may have potential application in biofuels.…”

“…The enzymes involved in the biosynthesis of the glycolipids (UA and MEL) have been described in [11] [12]. For UA a cytochrome P450 monooxygenase Cyp1 catalyzes terminal hydroxylation of palmitic acid and cyp2 is required for subterminal hydroxylation (w-1 hydroxylation) of this fatty acid [1] [12]. The expression of cyp1 is not under nitrogen regulation [22] which implies that UA production can occur without nitrogen source.…”

“…One fraction of the dried material (0.3 g) was dissolved in water. Ethyl acetate was added to a 1:1 ratio [1]. The organic phase was collected repeating the procedure to the remaining aqueous phase.…”

“…The yeast of Ustilago maydis produces extracellular glycolipids with surfactant activity that enhance the availability of hydrophobic nutrients during its interaction with the host [1] [2]. These biosurfactants are derivatives of two classes of amphiphatic glycolipids: Ustilagic acid (UA) and Mannosylerythritol lipids (MEL).…”

“…Different strains have been used for the production of glycolipids and the yield of the two glycolipids may be affected by the availability of nitrogen source [10]. The biosynthesis of UA and MEL depends on specific gene clusters [1] [11] [12]. For UA, cyp1 and cyp2 genes are required for terminal and subterminal hydroxylation of hexadecanoic acid [12].…”