Identification of Fungal Sphingolipid C9-Methyltransferases by Phylogenetic Profiling.
Philipp Ternes (1), Petra Sperling (1), Sandra Albrecht (1), Stephan Franke (2), James M. Cregg (3), Dirk Warnecke (1), Ernst Heinz (1)
(1) Biozentrum Klein Flottbek und Botanischer Garten, Universität Hamburg, Ohnhorststr. 18, 22609 Hamburg, Germany; (2) Organische Mikroanalytik, Universität Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany; (3) Keck Graduate Institute, 535 Watson Drive, Claremont, CA 91711, U.S.A.
Glucosylceramides, i.e. sphingolipids with a glucose headgroup, play an important role in the interactions between plants and phytopathogenic fungi. The glucosylceramides of many fungi are distinguished from those of plants and animals by the presence of a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species investigated. A Pichia pastoris knockout strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was specially engineered to produce sphingolipids suitable as substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S -adenosylmethionine-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases.