Molecular characterization of the biocontrol activity of Pseudozyma flocculosa
Final Report Abstract
Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O-glycosidically linked to 3,15,16- trihydroxypalmitic acid. The sugar moiety is acylated with 2-hydroxy-octanoic acid and acetylated at two positions. In this work I was able to elucidate a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. The cluster was compared to the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl-transferase and is lacking a gene homologous to the α-hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyltransferase genes (Fat1, Fat2 and Fat3) including the additional acetyl-transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, I could show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. Further the function of the NADPH dependent hydroxylase Fhd1, the homolog of the U. maydis Uhd1, was studied. Uhd1 catalyzes β-hydroxylation of the short chain fatty acid of UA. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis ∆uhd1 mutants. Uhd1 deletion strains produced UA lacking the β-hydroxyl-group of the short chain fatty acid. Fhd1 was able to restore wild-type UA production indicating that Fhd1 is responsible for β-hydroxylation of the flocculosin short chain fatty acid. In addition a P. flocculosa homolog of the U. maydis long chain fatty acid alpha hydroxylase Ahd1 has been investigated. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis ∆ahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis ∆ahd1 phenotype nor resulted in the production of β-hydroxylated UA. This suggests that the P. flocculosa Ahd1 is not involved in flocculosin hydroxylation.
Publications
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2011. Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa. Mol Microbiol 79 (6):1483-1495
Teichmann, B., C. Labbé, F. Lefebvre, M. Bölker, U. Linne, and R. R. Bélanger
