title: An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.
abstract: The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.
authors.affiliation: Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University, Am Botanischen Garten 5, 24118, Kiel, Germany.
authors.forename: Linda
authors.initials: L
authors.lastname: Paun
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Nitsche
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
authors.forename: Tim
authors.initials: T
authors.lastname: Homan
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
authors.forename: Arthur F
authors.initials: AF
authors.lastname: Ram
authors.affiliation: Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University, Am Botanischen Garten 5, 24118, Kiel, Germany. fkempken@bot.uni-kiel.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Kempken
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics*; D003470:Culture Media / Q000737:chemistry; D004251:DNA Transposable Elements / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006921:Hygromycin B / Q000494:pharmacology; D016296:Mutagenesis; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; D004251:DNA Transposable Elements; D004271:DNA, Fungal; D005656:Fungal Proteins; D006921:Hygromycin B; D019895:Transposases; D004318:Doxycycline
keywords: Aspergillus niger; Tet-on promoter system; Transposon-based mutagenesis; Vader