Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS5. Characterization of fumiquinazoline biosynthesis in Aspergillus fumigatus. Fang Yun Lim 1 , Brian Ames 2 , Christopher Walsh 2 , Nancy Keller 1 . 1) Medicalmicrobiology and Immunology, University of Wisconsin-Madison, Madison, WI; 2) Biological chemistry and molecular pharmacology, Harvard MedicalSchool, Boston, MA.The fumiquinazolines (FQs) comprise a related, sequentially generated family of bioactive peptidyl alkaloids that are signature metabolites of Aspergillusfumigatus. The FQ framework is built by nonribosomal peptide synthetase (NRPS) machinery with anthranilate as a key non-proteinogenic amino acidbuilding block. Despite being prevalent across the species, its gene cluster has not been characterized. Prior bioinformatic analysis coupled withheterologous expression of the putative A. fumigatus proteins termed here FmqA -FmqD led to the identification of a four-enzymatic process that buildsincreasingly complex FQ scaffolds. Briefly, FmqA, a trimodular NRPS condenses alanine, tryptophan, and anthranilic acid to form fumiquinazoline F (FQF).The tandem action of a flavoprotein (FmqB) and a monomodular NRPS (FmqC) converts FQF to fumiquinazoline A (FQA). Finally, FmqD, a FAD-dependentoxidoreductase converts FQA to the heptacyclic fumiquinazoline C (FQC). Interestingly, FmqD contains an N-terminus signal peptide predicted forextracellular transport. This study is aimed at providing in vivo validation to the FQ biosynthetic framework and characterizing how cellular localization ofFmqD affects production of FQC in A. fumigatus. We found that the conidial metabolite, FQC, is the predominant FQ moiety in two wild type isolates and isselectively accumulated in the conidia. Targeted single gene deletions of FmqA through FmqD coupled with metabolomic profiling of the singlebiosynthetic gene mutants supported previous biochemical prediction of FQ biosynthesis. Fluorescent microscopy of mutants bearing a C-terminal FmqD-GFP fusion showed that FmqD is localized to the cell wall of the fungus and this localization is abolished when the signal peptide is removed. Future studieswill elucidate if cell wall localization of FmqD is crucial for FQC production.6. Identification of local and cross-chromosomal biosynthetic gene clusters in filamentous fungi using gene expression data. Mikael R. Andersen 1 , JakobB. Nielsen 1 , Andreas Klitgaard 1 , Lene M. Petersen 1 , Tilde J. Hansen 1 , Lene H. Blicher 1 , Charlotte H. Gottfredsen 2 , Thomas O. Larsen 1 , Kristian F. Nielsen 1 ,Uffe H. Mortensen 1 . 1) Department of Systems Biology, Technical University of Denmark, Kgs Lyngby, Denmark; 2) Department of Chemistry, TechnicalUniversity of Denmark, Kgs Lyngby, Denmark.Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compoundsdue to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodological gene deletions to identifysupporting enzymes for key synthases one cluster at a time.In earlier work we presented a method for using a gene expression compendium to accurately predict co-regulated gene clusters in general, and inparticular the members of gene clusters for secondary metabolism. A benchmarking of the method in Aspergillus nidulans by comparison to previous genedeletion studies showed the method to be accurate in 13 out of 16 known clusters and nearly accurate for the remaining three.In this work, we have expanded the algorithm to identify cross-chemistry between physically separate gene clusters (super clusters), and validate thisboth with legacy data and experimentally by prediction and verification of a new supercluster consisting of the non-ribosomal peptide synthetase (NRPS)AN1242 (on chr VIII) and the prenyltransferase AN11080 (on chromosome V) as well as identication of the shared product compound nidulanin A.We also propose further implications of the gene clustering, as our analysis shows that approximately 10 % of the genes seem to be non-randomly(p