Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS100. Inactivation of flbA results in increased secretome complexity and reduced secretion heterogeneity in colonies of Aspergillus niger. PaulineKrijgsheld 1 , Benjamin M. Nitsche 2 , Harm Post 3 , Ana M. Levin 1 , Wally H. Müller 4 , Albert J.R. Heck 3 , Arthur F.J. Ram 2 , A.F. Maarten Altelaar 3 , Han A.B.Wösten 1 . 1) Microbiology and Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University, Utrecht,The Netherlands; 2) Department ofMolecular Microbiology and Biotechnology, Institute of Biology Leiden and Kluyver Centre for Genomics of Industrial Fermentation, Leiden University,Leiden, The Netherlands; 3) Biomolecular Mass Spectrometry and Proteomics, Netherlands Proteomics Center, Bijvoet Center for Biomolecular Researchand Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; 4) Biomolecular Imaging, Utrecht University, Utrecht, TheNetherlands.Aspergilli are among the most common fungi. They colonize substrates by secreting enzymes that degrade organic polymers into small products that canbe taken up by the fungus to serve as nutrient. Hyphae at the periphery of the colony are exposed to unexplored organic material, whereas the substrateis (partly) utilized in the colony center. Aspergillus niger is known for its capacity to secrete high amounts of proteins. Interestingly, the fungus secretesproteins in the central part and at the periphery of the colony but not in the sub-peripheral zone. The sporulating zone of the colony overlaps with thenon-secreting zone, indicating that sporulation inhibits protein secretion. Indeed, strain DflbA that is affected early in the sporulation program secretedproteins throughout the colony. In contrast, the DbrlA strain that still initiates but not completes sporulation did not show an altered spatial secretion. Thesecretome of 5 concentric zones of 7-dayold xylose-grown DflbA mutant colonies of A. niger was assessed by quantitative proteomics using stable isotopedimethyl labeling. In total 171 proteins were identified in the medium of the DflbA colonies, of which 33 proteins did not have a signal sequence forsecretion. Out of the 138 secreted proteins, 101 had previously not been identified in the secretome of the 5 concentric zones of xylose-grown wild-typecolonies. Moreover, 18 proteins had never been reported to be part of the secretome of A. niger. Taken together, inactivation of flbA, but not brlA resultsin spatial changes in secretion and in a more complex secretome. The latter may be explained by the fact that strain DflbA has a thinner cell wall comparedto the wild type, enabling efficient release of proteins. These results can implemented in the industry to improve A. niger as a cell factory.This research was financed by the Kluyver Centre for Genomics of Industrial Fermentation and by the Netherlands Proteomics Centre, which are part ofthe Netherlands Genomics Initiative/ Netherlands Organisation for Scientific Research.101. Functional characterization of A. niger class III and class V chitin synthases and their role in cell wall integrity. Jean-Paul Ouedraogo 1 , Arthur Ram 1 ,Vera Meyer 2 . 1) Molecular Microbiology and Biotechnology, Institut of Biology, Leiden, Netherlands; 2) Molecular and Applied Microbiology, Institut ofBiotechnology, Berlin University of Technology, Berlin, Germany.Class III and V chitin synthases play an important role in morphogenesis and cell wall integrity in many filamentous fungi. However, their function in thefilamentous fungus, A. niger has not yet been elucidated. To address this, deletion mutants of class III and V chitin synthase-encoding genes of A.niger,chsB and csmB, and their role in cell wall integrity have been studied. Deficiency in conidiation and abnormal swollen conidiophores have been observed inchsB and csmB deletion mutants. Using cell wall inhibitor reagents, it was shown that the mutants are hypersensitive towards cell wall stress. However,there are differences between them as regards susceptibility to the antifungal protein AFP. These results suggest that ChsB and CsmB play an importantrole during asexual development and in ensuring cell wall integrity of A. niger. Interestingly, the data indicate that only chitin synthase csmB is importantto counteract AFP inhibitory effects.102. Exploiting transcriptomic signatures of Aspergillus niger to uncover key genes important for high protein traffic through its secretory pathway. MinJin Kwon 1,2 , Thomas Jørgensen 1 , Benjamin M Nitsche 1,3 , Mark Arentshorst 1 , Joohae Park 1 , Arthur F.J. Ram 1,2 , Vera Meyer 1,3 . 1) Molecular Microbiology,Institute of Biology Leiden, Leiden, Netherlands; 2) Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, TheNetherlands; 3) Institute of Biotechnology, Department Applied and Molecular Microbiology, Berlin University of Technology, Gustav-Meyer-Allee 25,13355 Berlin, Germany.The filamentous fungus Aspergillus niger is well known for its exceptional high capacity to secrete proteins. However, system-wide insights into itssecretory capacities are sparse and rational strain improvement approaches are thus limited. To gain a global view on the transcriptional basis of thesecretory pathway of A. niger, we have investigated its transcriptomic fingerprint when specifically forced to overexpress the hydrolytic enzymeglucoamylase (GLA). An A. niger wild-type strain and an GLA over-expressing strain where cultivated under maltose-limited chemostat conditions. ElevatedglaA mRNA and extracellular GLA levels in the over-expressing strain were accompanied by reinforced transcription of 772 genes and down-regulation of815 genes when compared to the wild-type situation. Using GO term enrichment analysis, four higher order categories were identified in the up-regulatedgene set: i) translocation, ii) protein glycosylation, iii) vesicle transport and iv) ion homeostasis. Among these, about 130 genes have predicted functionsfor the protein passage through the endoplasmaticum reticulum including well-known target genes of the HacA transcription factor, e.g. bipA, clxA, prpA,tigA and pdiA. To identify those genes, which are generally important for high-level secretion in A. niger, we compared the GLA transcriptome with sixother secretion stress transcriptomes of A. niger, including a constitutive active HacA transcriptome, several UPR stress transcriptomes and a carbonsourceinduced secretion transcriptome. Overall, 40 genes were commonly up-/down-regulated under these three conditions (36 genes up-regulated, 4down-regulated), thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway.103. Identification of two Golgi-localized putative UDP-galactofuranose transporters with overlapping function in Aspergillus niger. Joohae Park 1 , BorisTefsen 2 , Ellen Lagendijk 1 , Irma van Die 2 , Arthur Ram 1,3 . 1) Molecular Microbiology, Institute of Biology Leiden, Leiden, Netherlands; 2) Department ofMolecular Cell Biology and Immunology, VU University Medical Center, van den Boechorststraat 7, 1081 BT Amsterdam, The Netherlands,; 3) KluyverCentre for Genomics of Industrial Fermentation, P.O box 5057, 2600 GA Delft, The Netherlands.Galactofuranose-containing glycoconjugates are present in numerous microbes, many of which are pathogenic for humans. Metabolic aspects of themonosaccharide have proven difficult to elucidate, because galactofuranose metabolites and glycoconjugates are relatively unstable during analyses.Recent advances with genetic approaches have facilitated a better understanding of galactofuranose biosynthesis. Galactofuranose (Galf) the five-ringisomer of galactopyranose (Galp), is an essential component of the cell wall and required for a structural integrity [1-2]. Recently, it has been postulatedthat UDP-Galp, is converted to Galf by a UDP-galactopyranose mutase (UgmA) and subsequently transported into the Golgi by a putative UDP-Galftransporterfor the further biosynthesis of cell wall polymers such as galactomannan, galactoaminogalactan and cell wall glycoproteins (galactomannoproteins)[3-4]. Based on homology searches, we have identified two putative UDP-Galf-transporters in A. niger. We have studied the function of thetransporters by making deletions mutants (either single or double mutants) and by studying their localization by making GFP fusions. We conclude that thetwo putative UDP-Galf-transporters (named (UgtA and UgtB) have an overlapping function in UDP-Galf-transport and that both proteins are localized inGolgi equivalents. References: [1] Damveld, R.A. et al., 2008. Genetics 178 (2), 873-81; [2] Schmalhorst, P.S. et al. 2008, Euk. Cell 7 (8), 1268-77; [3] Engel, J.et al., 2009. J. Biol. Chem. 284; [4] Bernard, M., Latge, J. P., 2001. Med. Myc. 39, 9-17;.146