Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSMicroscopy unit, Institut Pasteur Hellenique, Athens, Greece; 3) Department of Microbiology, Imperial College, London, United Kingdom.In the model filamentous fungus Aspergillus nidulans, PilA and PilB, two homologues of the Saccharomyces cerevisiae eisosome proteins Pil1/Lsp1, andSurG, a strict orthologue of Sur7, are assembled and form tightly packed structures in conidiospores. A. nidulans differs from the Saccharomycotina in thatit has the ability to reproduce through two different types of spores, conidiospores and ascospores, cells which have a radically different morphology andare formed through completely different developmental pathways. Ascospores are formed only after the completion of meiosis inside asci, conidiosporesarise from mitotic budding of specialized cells (phialides). We thus investigated eisosome composition and distribution in ascospores. Our results showthat core eisosome proteins PilA, PilB and SurG are not expressed in hülle cells or early ascospores, but are expressed in mature ascospores. PilA formsstatic punctate structures at the plasma membrane as does PilB (with higher concentration at the areas where the two halves of ascospores are joinedtogether), while SurG was localized both at the membrane and perinuclearly. In germlings originating from ascospores the punctate structures wereshown to be composed only of PilA. In germinated ascospores PilA foci did not colocalise with the punctate structures of AbpA, a marker for sites ofclathrin-mediated endocytosis. In the presence of myriocin -a specific inhibitor of sphingolipid biosynthesis- PilA-GFP foci of ascospore germlings were lessnumerous and their distribution was significantly altered. In this study we also investigated one of the two A. nidulans orthologues of Nce102, a proteinthat determines the structure and function of membrane microdomains in S. cerevisiae. In quiescent conidia localization of the closest orthologue,AnNce102 is detected in PilA plasma membrane associated foci as well as in 3-5 round-shaped intracellular structures. In early hyphae, a cytoplasmicfraction of Nce102 is additionally detected in highly dynamic structures that resemble Golgi equivalents. Deletion of core eisosomal genes causesmislocalization of Nce102 from the plasma membrane to these cytoplasmic structures. Ongoing experiments are investigating AnNce102 localization inresponse to sphingolipid biosynthesis and the nature of the intracellular compartments where it is located.694. Fungal-bacterial interactions: Bacillus subtilis forms biofilm on Aspergillus niger hyphae. Isabelle Benoit 1,2 , Marielle H. van den Esker 3 , MiaomiaoZhou 1 , Oscars P. Kuipers 3 , Ronald P. de Vries 1,2 , Ákos T. Kovács 4 . 1) Fungal Physiology, CBS-KNAW, Utrecht, Utrecht, Netherlands; 2) Microbiology & KluyverCentre for Genomics of Industrial Fermentation, Utrecht University, Utrecht, The Netherlands; 3) Molecular Genetics Group, Groningen BiomolecularSciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands; 4) Terrestrial Biofilms, Institute of Microbiology, FriedrichSchiller University, Jena, Germany.Pure cultures of the filamentous fungi Aspergillus niger and Aspergillus oryzae and the Gram-positive bacterium Bacillus subtilis are widely used inindustry for the production of pharmaceuticals, food ingredients and enzymes. However both by design and by accident, industrial fermentation can alsoinvolve mixed populations of micro-organisms. Moreover, in natural biotopes, these organisms live in mixed communities and have complex interactionsranging from competition to symbiosis. B. subtilis, in specific conditions, is capable of forming beneficial biofilms on surfaces and interfaces from plantroots to metal surfaces. In this study, co-cultivations of A. niger and A. oryzae together with B. subtilis were performed. A. oryzae inhibits B. subtilis growthwhile a bacterial coating was observed on A. niger hyphae. Microscopic and transcriptomic approaches were combined to study this fungal-bacterialinteraction example.695. Co-cultivations of fungi: microscopic analysis and influence on protein production. Isabelle Benoit 1,2 , Arman Vinck 2 , Jerre van Veluw 2 , ThijsGruntjes 1,2 , Han A.B. Wösten 2 , Ronald P. de Vries 1,2 . 1) Fungal Physiology, CBS-KNAW, Utrecht, Utrecht, Netherlands; 2) Microbiology & Kluyver Centre forGenomics of Industrial Fermentation, Utrecht University, Utrecht, The Netherlands.During their natural life cycle most fungi encounter other microorganisms and live in mixed communities with complex interactions, such as symbiosis orcompetition. Industrial fermentations, on purpose or by accident, can also result in mixed cultures. Fungal co-cultivations have been previously describedfor the production of specific enzymes, however, little is known about the interactions between two species that are grown together. Aspergillus niger andAspergillus oryzae are two of the most important industrial fungi worldwide and both have a long history of strain improvement to optimize enzyme andmetabolite production. We have co-cultivated the wild type strains of these two Aspergilli with each other as well as the XlnR knock out strains. XlnR is atranscription factor inducible by the presence of xylose and responsible for the regulation of a variety of genes encoding plant polysaccharide degradingenzymes. The morphology and mechanism of the interaction of these cultures on wheat bran is addressed using microscopy and proteomics.696. Improving heterologous protein production in Aspergillus vadensis . Ourdia Bouzid 1,2 , Ronald P. de Vries 1,2 . 1) Microbiology & Kluyver Centre forGenomics of Industrial Fermentation, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 2) CBS-KNAW Fungal Biodiversity Centre,Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.Aspergillus vadensis is a good candidate for heterologous protein production, because it produces very low levels of extracellular proteases and does notacidify the medium. To improve protein production in A. vadensis two strategies were tested: (i) identification of new promoters for high gene expression,and (ii) overexpression of the xylanolytic regulator, XlnR. Six new A. niger constitutive promoters were selected and compared to the gpdA promoter usingan arabinofuranosidase (abf) encoding gene from Fusarium oxysporum as a reporter. Several of the new promoters resulted in higher Abf activity thangpdA. For the second strategy, A. vadensis was transformed with xlnR, xlnD (encoding beta-xylosidase) and faeA (encoding feruloyl esterase) alone, andwith combinations of xlnD and xlnR, and faeA and xlnR. Southern blot profiles confirmed the presence of multiple copies of the genes in the transformants.XlnD and FaeA activities were measured and were compared to the control strain. This demonstrated that increased copy numbers of faeA and xlnD had amuch larger effect on the corresponding activities than increased copy numbers of xlnR. These data demonstrate that the new promoters in combinationwith high copy integration of the target genes can result in higher protein production by A. vadensis. Highlights from this study will be presented.697. Production and characterization of esterases from Chaetomium thermophilum and their applicability in biomass conversion. Xiaoxue Tong, PeterBusk, Morten Grell, Lene Lange. Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering. AalborgUniversity Copenhagen, Denmark.Xylan is the dominating hemicellulose constituent of plants and the most abundant renewable polysaccharide in nature after cellulose. Xylan and itshydrolysis products are potential resources for nutraceuticals, cosmetics, foods, bioalcohol, and industrial fine chemical production. Feruloyl esterase andacetyl xylan esterase are required for complete enzymatic hydrolysis of xylan due to its highly heterogeneous nature. The aim of this study was to produceand characterize esterases from the thermophilic fungus Chaetomium thermophilum. The esterase genes were identified by a novel bioinformatics toolPPR (Peptide pattern recognition, Busk & Lange, 2011). A Feruloyl esterase gene (CtFaeA) and a xylan esterase gene (CtAxeA) were successfully expressedin the yeast Pichia pastoris. They were purified to homogeneity from the culture supernatants. The effect of temperature and pH on the activity andstability of the esterases, as well as their substrate specificities, were studied. Both CtFaeA and CtAxeA displayed broad thermal stability and pH stability.Moreover, both esterases were active on hydrolysis of wheat arabinoxylan. These results show that Chaetomium thermophilum has a high capacity fordegradation of xylan in addition to its already described cellulolytic potential. Furthermore, the robust esterases from Chaetomium thermophilum have292