TY - CONF A1 - Schumacher, Julia A1 - Brandhorst, Antonia A1 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Voigt, Oliver A1 - Gorbushina, Anna T1 - DHN melanin synthesis in the rock inhabitant Knufia petricola N2 - DHN (1,8-dihydroxynaphthalene) melanin is produced by different Ascomycetes via slightly differing biosynthetic routes. The polyketide synthases (PKS) release the heptaketide YWA1, the hexaketide AT4HN or the pentaketide T4HN. The first two products are deacetylated by ‘yellowish-green’ hydrolases to T4HN, and T4HN is further converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases (MCOs). The involved genes are tightly clustered, partially clustered or widely distributed in the genomes of DHN melanin-producing fungi. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeast) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits constitutive melanogenesis. Here, we report on the identification and functional characterization of the DHN melanogenic genes of Knufia petricola as a representative of the Chaetothyriales, the sister order of the Eurotiales. Orthologs for all melanogenic genes were identified in the genome of K. petricola A95, including one gene encoding the polyketide synthase (KpPKS1), two genes encoding ‘yellowish-green’ hydrolases (KpYGH1,2), two genes encoding THN reductases (KpTHR1,2) and one gene encoding a scytalone dehydratase (KpSDH1). Ten genes encoding MCOs were identified, all MCOs are predicted to be secreted. The genes are not clustered in the genome but are highly expressed. Gene functions are studied by generation of single, double, and multiple deletion mutants in K. petricola and by heterologous expression in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. T2 - VAAM Fachgruppentagung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - DHN melanin KW - Genetics KW - Biodegradation PY - 2022 AN - OPUS4-55676 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nitsche, Sarah A1 - Gerrits, Ruben A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - The role of the stress-activated MAP kinase SAK1 in Knufia petricola N2 - Mitogen-activated protein (MAP) kinases are highly conserved in animals, plants, and fungi and represent fundamental parts of signaling networks in eukaryotic cells. Black DHN (1,8-dihydroxynaphthalene) melanin and orange carotenoids are produced by many fungi in specific cell types/under certain conditions for protecting cells from abiotic and/or biotic stresses. Microcolonial black fungi constitutively produce DHN melanin, contain the conserved carotenogenic gene cluster, exhibit slow yeast-like growth and survive in extreme environments. Thus, the question arises to which extent pigment formation and responses mediated by the stress-activated MAP kinase module contribute to the observed extremotolerance. We address this question in the rock inhabitant Knufia petricola, the only representative of the extremotolerant black fungi that is genetically amendable. Here, the mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively (Voigt Knabe et al. 2020, Sci Rep). The gene encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. In addition, strains were generated that express a GFP-SAK1 fusion protein from the sak1 locus to follow the cytosolic/nuclear shuttling of SAK1 upon stress. Growth of the obtained single, double and triple deletion mutants was tested by dropping cell suspensions on solid media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even under non-stress conditions and are hypersensitive to different stress conditions: reduced growth is observed on media inducing, for instance, osmotic, oxidative, membrane, and pH stress, and upon incubation at 30 °C (heat stress). Melanin-free Δsak1 mutants are more sensitive than black Δsak1 mutants to some but not all stress conditions, suggesting that melanin and the SAK1 pathway have complementary roles in protecting K. petricola from stress. T2 - VAAM Fachgruppentragung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - Fungus KW - Extremotolerance KW - Signal transduction PY - 2022 AN - OPUS4-55677 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - Tools for Knufia petricola: new techniques for CRISPR/Cas9-based genome editing N2 - Black microcolonial fungi represent a group of ascomycetes with similar adaptations for existing in natural and anthropogenically created extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) in the multilayered cell walls. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a representative for developing methods for genetic manipulation, simulation of mineral weathering and study of symbiotic interactions. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and three additional resistance selection markers. The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter, while the black-white screening due to the concurrent elimination of pks1 (melanin) and phs1 (carotenoids) was used to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. In addition, two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series (Schumacher, 2012) and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for use in other fungal systems as well. T2 - 31st Fungal Genetics Conference CY - USA, CA, Pacific Grove DA - 15.03.2022 KW - Microcolonial fungi KW - Genetic engineering KW - Fluorescent proteins PY - 2022 AN - OPUS4-54586 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -