A biosynthetic gene cluster for a secreted cellobiose lipid with antifungal activity from Ustilago maydis

Abstract

SummaryThe phytopathogenic basidiomycetous fungus Ustilago maydis secretes large amounts of the glycolipid biosurfactant ustilagic acid (UA). UA consists of 15,16-dihydroxypalmitic or 2,15,16-trihydroxypalmitic acid, which is O-glycosidically linked to cellobiose at its terminal hydroxyl group. In addition, the cellobiose moiety is acetylated and acylated with a short-chain hydroxy fatty acid. We have identified a 58 kb spanning gene cluster that contains 12 open reading frames coding for most, if not all, enzymes needed for UA biosynthesis. Using a combination of genetic and mass spectrometric analysis we were able to assign functional roles to three of the proteins encoded by the gene cluster. This allowed us to propose a biosynthesis route for UA. The Ahd1 protein belongs to the family of non-haem diiron reductases and is required for a-hydroxylation of palmitic acid. Two P450 monooxygenases, Cyp1 and Cyp2, catalyse terminal and subterminal hydroxylation of palmitic acid. We could demonstrate that infection of tomato leaves by the plant pathogenic fungus Botrytis cinerea is prevented by co-inoculation with wild-type U. maydis sporidia. U. maydis mutants defective in UA biosynthesis were unable to inhibit B. cinerea infection indicating that UA secretion is critical for antagonistic activity.

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

“…The longest lipid moiety of ustilagic acid has been reported to hydroxilated C16:0 but for mannosylerytritol lipids C 14 , C 16 and C 18 saturated and unsaturated fatty acids were found although sucrose was the carbon source [8] [33]. 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 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.…”

“…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].…”

“…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]. Mannosylerythritol lipids (MEL) are secreted in at least four different varieties (ABCD), depending on the number of acetyl groups attached to the disaccharide [3]- [11] and a glycosyltransferase is essential for the biosynthesis of MEL; expression of this enzyme is strongly induced by limitations on nitrogen availability [11].…”

The Ustilago maydis Nit2 Homolog Regulates Nitrogen Utilization and Is Required for Efficient Induction of Filamentous Growth

“…One facet of the nitrogen starvation response of U. maydis is the production and secretion of glycolipids, mainly ustilagic acid (cellobiose lipid) and ustilipid (mannosylerythritol lipid), which act as biosurfactants (31). The biosynthetic genes are organized in gene clusters that have already been characterized (78). Recently, the transcriptional activator Rua1 has been shown to control the upregulation of the ustilagic acid gene cluster under nitrogenlimiting conditions (79).…”