Brakhage, Axel A.

Mattern, Derek J.

Mortensen, Uffe Hasbro

Nødvig, Christina Spuur

Slotkowski, Rebecca A.

Valiante, Vito

Weber, J.

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Apr 10, 2018Functional reconstitution of the Trypacidin Gene Cluster in Aspergillus fumigatus byAdvanced Gene EditingWeber, J.; Valiante, Vito ; Nødvig, Christina Spuur; Mattern,  Derek J. ; Slotkowski, Rebecca A. ;Mortensen, Uffe Hasbro; Brakhage, Axel A.Publication date:2017Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Weber, J., Valiante, V., Nødvig, C. S., Mattern, . D. J., Slotkowski, R. A., Mortensen, U. H., & Brakhage, A. A.(2017). Functional reconstitution of the Trypacidin Gene Cluster in Aspergillus fumigatus by Advanced GeneEditing. Abstract from 29th Fungal Genetics Conference, Pacific Grove, United States.ABSTRACTS  268  624F    Mycosarcoma, a resurrected generic name for corn smut (Ustilago maydis).    D. Begerow1, A. McTaggart2, R. Shivas3  1) Geobotany, Ruhr-Universität Bochum, Bochum, DE; 2) Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; 3) Plant Pathology Herbarium, Biosecurity Queensland, Department of Agriculture and Fisheries, Queensland, Australia.     Ustilago is a polyphyletic genus of smut fungi found mainly on Poaceae. The development of a taxonomy that reflects phylogeny requires subdivision of Ustilago into smaller monophyletic genera. Several separate systematic analyses have determined that Macalpinomyces mackinlayi, M. tubiformis, Tolyposporella pachycarpa, Ustilago bouriquetii and U. maydis, occupy a unique phylogenetic position within the Ustilaginaceae. A previously introduced monotypic generic name typified by U. maydis, Mycosarcoma, is available to accommodate these species, which resolves one component of polyphyly for Ustilago s. lat. in Ustilaginaceae. An emended description of Mycosarcoma is provided to reflect the morphological synapomorphies of this monophyletic group. A specimen of Ustilago maydis that has had its genome sequenced is designated as a neotype for this species. Taxonomic stability will further be provided by a forthcoming proposal to conserve the name Uredo maydis over Lycoperdon zeae, which has priority by date, in order to preserve the well-known epithet maydis.   625W    Population structure and drivers of genetic diversity in Candida glabrata.    E. Shor1, K. Healey1, C. Jimenez-Ortigosa1, S. Lockhart2, V. Loparev2, D. Perlin1  1) Public Health Research Institute, Rutgers University, Newark, NJ; 2) Centers for Disease Control, Atlanta, GA.     The prevalence of Candida glabrata infections has been rising for several decades, and it now accounts for approximately 25% of all Candida bloodstream infections in the U.S. and can in some settings predominate as the principal bloodstream fungal pathogen. Furthermore, C. glabrata exhibits elevated intrinsic resistance to triazoles and readily acquires resistance to echinocandins. It also becoming increasingly apparent that C. glabrata has a highly genetically diverse population structure. However, how this diversity arises and whether it contributes to the virulence and drug resistance of C. glabrata is unknown. A multi-locus sequence typing (MLST) scheme has thus far identified over 80 different C. glabrata sequence types (STs); however, our preliminary analysis indicates that this number is likely much higher. We also found that different STs carry different alleles of DNA repair gene MSH2, some of which cause elevated rates of drug resistance in vitro, indicating that different STs have different propensity towards mutability and acquiring drug resistant gene variants. C. glabrata also displays a high degree of variability at the level of chromosomal architecture. For instance, it has been reported that clinical C. glabrata isolates show great diversity in terms of chromosomal number and structure, displaying a high number of chromosomal rearrangements relative to the reference laboratory strain. This observation has led to the hypothesis that emergence of new chromosomes is a virulence mechanism in C. glabrata and may underlie emergence of drug resistance. However, we find that strains of the same ST have similar chromosomal patterns, suggesting that specific chromosomal configurations may pre-exist emergence of virulence and may be a feature of commensal C. glabrata populations. To identify new determinants of virulence and/or drug resistance in C. glabrata, we are performing optical mapping and long read genome sequencing (PacBio) of several STs that are prevalent in the U.S. We are also investigating the importance of the DNA replication checkpoint in facilitating chromosomal rearrangements in C. glabrata.   626T    Comparative genomics of hybrids reveal potential host specificity genes in Microbotryum.    Britta Büker1, Michael E. Hood2, Andreas Brachmann3, Sven Rahmann4, Dominik Begerow1  1) Geobotanik, Ruhr-Universität Bochum, Bochum, DE; 2) Department of Biology, Amherst College, Amherst, MA, USA ; 3) Genetics, Faculty of Biology, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany; 4) Genominformatik, Institut für Humangenetik, Medizinische Fakultät, Universität Duisburg-Essen, Essen, Germany.     The evolution of obligate plant pathogens like smut fungi is often characterized by lineage tracking, resulting in host specificity and one-to-one relationships. Therefore, adaptation and specialization to the host plant seems to be crucial and should involve genes or regulatory pathways governing host specificity. To identify genes relevant for host specificity of Microbotryum species, we produced artificial hybrids between the two host-specific species M. lychnidis-dioicae and M. silenes-acaulis and applied strong experimental selection on different host plants to identify genes necessary for successful infections. Genome comparison of the two species revealed that most gene families are shared and the majority of genes are conserved, indicating very similar biological features of both species, including host adaptation and infection processes. Lower nucleotide identity of genes encoding for secreted proteins might indicate their importance for host specific interaction, as it is known from other plant pathogens. Moreover, we identified 211 candidate genes that occur in each hybrid and backcross genome that were posed under host-driven selection and might therefore play a crucial role in host specialization. The analysis of hybrid genomes also demonstrates the effect of genetic homogeneity on the fitness of hybrid individuals including the occurrence of species-specific mating type chromosomes. In conclusion, the combination of comparative genomics with experimental selection and hybridization is a promising way to identify potential host specificity factors. Our data suggest that only a limited set of genes is required for successful infection, but also demonstrate the strong influence of intra-genomic conflicts on the viability of hybrids.   Synthetic Biology   627F    Functional reconstitution of the Trypacidin Gene Cluster in Aspergillus fumigatus by Advanced Gene Editing.    J. Weber1,2, V. Valiante3, C.S. Nødvig4, D.J. Mattern1,2, R.A. Slotkowski1,2, U.H. Mortensen4, A.A. Brakhage1,2  1) Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI) –, Jena, Germany; 2) Institute of Microbiology, Friedrich Schiller University Jena, Germany; 3) Leibniz Research Group – Biobricks of Microbial Natural Product Syntheses, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany; 4) Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.     The human pathogenic fungus Aspergillus fumigatus is known to produce various spore-borne natural products. One of these is the polyketide trypacidin which has been shown to be involved in the interactions with alveolar macrophages as well as with the amoeba Dictyostelium discoideum. Even though recent studies could elucidate the corresponding gene cluster in A. fumigatus, it still remained elusive why several isolates do not produce trypacidin. We addressed this question employing a CRISPR/Cas9-based gene editing strategy. Thus, we could link a single nucleotide insertion in the polyketide synthase of the trypacidin biosynthetic pathway and also ABSTRACTS  269  reconstitute its production in a nonproducing strain. In addition, we developed a split-marker approach for the selection of edited strains, since the selectable marker could not be directly linked to the target site. The here established tool could be useful in next generation fungal genetics e.g. for the investigation of single nucleotide polymorphism, or amino acid substitutions.   628W    Identification of gene targets for improved heterologous enzyme production in Aspergillus niger.    M.C. Reilly1, J. Kim1, J. Lynn1, J.M. Gladden1,2, J.K. Magnuson1,3, S.E. Baker1,3  1) Joint BioEnergy Institute, Emeryville, CA; 2) Sandia National Laboratory, Livermore, CA; 3) Pacific Northwest National Laboratory, Richland, WA.     Efficient and economical deconstruction of biomass is critical for the success of lignocellulosic biorefineries. The use of ionic liquids (ILs) in the pretreatment of biomass leads to increased biomass saccharification efficiency at lower cellulose loadings. However, some ILs can inhibit the activity of commercial cellulases and must be removed from the biomass prior to the application of the lignocellulosic enzyme cocktail, a costly additional step in the conversion process. To overcome this issue, cellulolytic enzymes that can maintain function in the presence of ILs have been identified. Many of these enzymes originate from bacterial species that are difficult to cultivate in the laboratory. One possible solution are filamentous fungi, which have been widely utilized for enzyme production in industry. Here, a forward genetics approach was utilized to increase heterologous enzyme production in Aspergillus niger: a strain engineered to secrete an IL-tolerant bacterial beta-glucosidase (BG) was mutagenized and the resulting progeny screened for increased BG activity. Subsequent full-genome sequencing revealed several loci associated with the hyper-production of the heterologous enzyme. Deletion of one of these, a putative sugar transporter, results in a doubling of heterologous BG enzyme activity detected in culture supernatants. Future studies will further characterize the role of this sugar transporter in heterologous enzyme production.   629T    An in-silico reconstructed gene regulation network for Aspergillus niger for the prediction of protein functions.    Sascha Jung, Norman Paege, Paul Schäpe, Vera Meyer  Institute of Biotechnology, Department Applied and Molecular Microbiology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.     The genome of A. niger belongs to the best annotated genomes among Aspergillus species; however, only 2% of its ~14,000 genes are functionally verified and 50% of the predicted open reading frames encode for hypothetical proteins. Hence, the genetic basis for almost all cellular processes in A. niger and its physiological peculiarities is unknown. On the other hand, hundreds of post-genomic data including transcriptomic and proteomic data are available for A. niger for more than 150 different growth conditions. This holistic dataset can be scrutinized and used to predict gene functions and gene interactions. For a proof-of-concept, we have focused on the anafp gene encoding the antifungal protein AnAFP known to selectively inhibit the growth of filamentous fungi. As more than 50 AnAFP orthologs have been identified in many different genera of the Ascomycota tree of life, we wished to understand which regulatory systems control expression of the anafp gene. Our analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we could demonstrate that the anafp promoter is indeed under control of CreA and FlbA as predicted by the in silico data. A co-expression network analysis further predicted that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators (1). We currently prove these predictions by respective wet-lab experiments. First results match the expected knock out phenotype in A. niger.  (1) Paege N, Jung S, et al. A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger. PlosOne 2016; DOI:10.1371/journal.pone.0165755.s009   630F    Genome wide consequences of the deletion of the Aspergilli non-homologous end joining (NHEJ) DNA repair  mechanism.    I. Álvarez-Escribano1, C. Sasse2, J. Woo Bok3, A. Lipzen4, W. Schackwitz4, J. Marin4, K. Barry4, I. Grigoriev4, A.T. Marcos1, N.P. Keller3, G.H. Braus2, D. Canovas1  1) Department of Genetics, Faculty of Biology, University of Seville, Spain; 2) Department of Molecular Microbiology and Genetics and Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University, Göttingen, Germany; 3) Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 4) DOE Joint Genome Institute, Walnut Creek, California, USA.     The relevance of Aspergillus for human daily life is immeasurable: not only because of its industrial applications but also because of its clinical implications. Most Aspergillus laboratories world-wide use mutants in the non-homologous end joining (NHEJ) pathway (KU70 or KU80) to do genetic modifications in the strains under study due to a higher frequency of homologous integrations after transformation of DNA constructs. KU70 is involved in a DNA repair mechanism highly conserved in eukaryotes, and genetic manipulations are much faster in NHEJ mutant (?KU70) strains in many fungal species. Three Aspergillus species were selected for this study to allow for comparative genetics and genomics: A. flavus, A. fumigatus and A. nidulans. Wild-type and NHEJ mutants were grown on solid media for 60 growth passages using asexual spores (ca. 3,000 mitosis). In the case of A. nidulans ten passages with sexual cleistothecia were also independently performed. Genome sequences were obtained and analyzed. In both A. flavus and A. fumigatus, the KU70 mutants accumulated on average more mutations than the wild type strains (8.37 +/- 1.80 vs 6.12 +/- 1.76 in A. flavus, and 5.75 +/- 2.33 vs 4.12 +/- 1.76 in A. fumigatus). The higher number of non-synonymous mutations in A. fumigatus and of mutations in the intergenic regions in A. flavus in the KU70 mutants was statistically significant. None of the sexual passages of the A. nidulans KU70 strain could be completed due to the lack of cleistothecia formation, while the majority of the wild type passages reached ten passages. Collectively here we provide an assessment of the effects of the NHEJ pathway in the genomic stability in Aspergilli.   631W    CRISPR/Cas9 systems can be used to manipulate gene function in the thermally dimorphic fungus Histoplasma capsulatum.    Saori Amaike Campen, Sinem Beyhan  J Craig Venter Institute, La Jolla, CA.     The thermally dimorphic fungus, Histoplasma capsulatum is a causal agent of the severe pulmonary and systemic human disease histoplasmosis. H. capsulatum can cause disease in both immunocompromised and immunocompetent individuals and is endemic in the 

Functional reconstitution of the Trypacidin Gene Cluster in Aspergillus fumigatus by Advanced Gene Editing

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Functional reconstitution of the Trypacidin Gene Cluster in Aspergillus fumigatus by Advanced Gene Editing

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