VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

There are several polyketide synthase genes are involved in the productionof secondary metabolites in Alternaria alternata including Alternaiol (AOH)biosynthesis. Alternariol (AOH) is one of the main mycotoxins formed invarious foods and feeds contaminated by the mold A. Alternata. Alternariolshows cytotoxic, foetotoxic, and teratogenic effects. Since alternariol andother mycotoxins have deleterious properties to humans and animals, effortshave been directed toward the understanding of the molecular mechanismsleading to its biosynthesis. However, till date very little is known about themolecular biology of alternariol synthesis.The work here describes the identification and characterization of the genesinvolved in the alternariol biosynthetic pathway. In order to understand thebiosynthetic pathway of alternariol or other toxins in A. alternata, putativepolyketide synthase genes has been identified. The identified PKS genes arelocated in clusters. Selected genes have been disrupted/deleted to study theirinvolvement in toxin production. The characterization of the toxin profile incertain mutants strains by thin layer chromatography and/or HPLC will helpto assign functions to the different PKS proteins. Because gene deletionoccurred to be rather difficult in A. alternata, RNAi mediated silencing forfour different PKS genes has also been performed for identifying in whichsecondary metabolite biosynthesis they are involved. The metabolicprofiling of secondary metabolites is under way.FBV022Analysis of the Aspergillus fumigatus proteome revealsmetabolic changes and the activation of the pseurotin Abiosynthesis gene cluster in response to hypoxiaM. Vödisch 1,2 , K. Scherlach 3 , R. Winkler 3 , C. Hertweck 3 , H.-P. Braun 4 ,M. Roth 5 , H. Haas 6 , E.R. Werner 7 , A.A. Brakhage 1,2 , O. Kniemeyer* 1,21 Molecular and Applied Microbiology, Hans Knoll Institute (HKI), Jena,Germany2 Department of Microbiology and Molecular Biology, Friedrich-Schiller-University, Jena, Germany3 Department of Biomolecular Chemistry, Hans Knoll Institute (HKI), Jena,Germany4 Institute of Plant Genetics, Leibniz University Hannover, Hannover,Germany5 Bio Pilot Plant, Hans Knoll Institute (HKI), Jena, Germany6 Division of Molecular Biology, Innsbruck Medical University, Innsbruck,Austria7 Division of Biological Chemistry, Innsbruck Medical University,Innsbruck, AustriaAspergillus fumigatus is the most important airborne fungal pathogen andthe main causative agent of the opportunistic, often lethal infection invasiveaspergillosis. To colonize the human lungs, this saprophytic fungus has toadjust its physiology to the host’s environment including the adaptation tohypoxia, which represents an important virulence attribute. Therefore, weintended to obtain a comprehensive overview about this process on theproteome level. To ensure highly reproducible growth conditions, anoxygen-controlled chemostat cultivation was established. Two-dimensionalgel electrophoresis analysis of mycelial and mitochondrial proteins as wellas two-dimensional Blue Native/SDS-gel separation of mitochondrialmembrane proteins led to the identification of 117 proteins with an alteredabundance under hypoxic in comparison to normoxic conditions. Thisproteome analysis revealed an increased activity of the glycolytic pathway,the TCA-cycle, and especially respiration and amino acid metabolism.Consistently, hypoxia elevated the cellular contents in heme, iron, copperand zinc. Furthermore, hypoxia induced biosynthesis of the secondarymetabolite pseurotin A as demonstrated at proteomic, transcriptional andmetabolite levels. The observed and so far not reported stimulation of thebiosynthesis of a secondary metabolite by oxygen depletion may also affectthe survival of A. fumigatus in hypoxic niches of the human host. Among theproteins so far not implicated in hypoxia adaptation, an NO-detoxifyingflavohemoprotein was one of the most highly up-regulated proteins whichindicates a link between hypoxia and the generation of nitrosative stress inA. fumigatus.FBV023Fungal systems - Tools for the milligram- to gram-scalepreparation of an environmentally relevant metabolite offenoprofenM. Hoffmann* 1 , J. Zimmerling 1 , S.R. Kaschabek 1 , G. Schüürmann 2 ,M. Schlömann 11 Department of Environmental Microbiology, University of Mining andTechnology, Freiberg, Germany2 Institute of Ecological Chemistry, Helmholtz Center for EnvironmentalResearch (USZ), Leipzig, GermanySince the ubiquitous distribution of pharmaceutical residues in surfaceaquifers becomes more and more obvious, questions concerning toxic effectson human health and the ecosystem are arising. Measured traceconcentrations of individual drugs were found to be several orders ofmagnitude below the acute-effect level and toxic action towards members ofthe bioscenosis is at least very unlikely. However, effects caused by longtermexposure, by synergetic effects of complex drug mixtures, and bymetabolites of microbial transformation are poorly investigated. In contrastto the metabolism of drugs in humans, little knowledge exists ontransformation mechanisms of most pharmaceuticals by microorganisms.Usually the detection and identification of a microbial intermediate gives afirst hint on a transformation mechanism. However, such investigations areconsiderably facilitated by available references for those compounds.Bioconversion of pharmaceuticals by filamentous fungi may, in certaincases, serve as a strategy to obtain such compounds. The present work dealswith the preparation of 4′-hydroxyfenoprofen (3-(4-hydroxyphenoxy)-αmethylbenzeneaceticacid) from fenoprofen by Epicoccum nigrum DSM838and Cunninghamella elegans DSM1908. This metabolite, which is known tobe a major intermediate during fenoprofen phase-I metabolism in humans,was also found in this study to be formed during the aerobicbiotransformation of the non-steroidal anti-inflammatory drug by water andsediment from a river.Transformation studies of fenoprofen with both filamentous fungi showed acomplete (co)metabolic conversion in the concentration range of 160 μMand 500 μM under aerobic conditions. Under the conditions investigated,transformation of 0.48 mmol fenoprofen in a 3 L fermenter yielded 76.7 mgof 4′-hydroxyfenoprofen, corresponding to a 62.5 % theoretical yield. Thus,C. elegans is the first reported biological system for the fermentativeregiospecific hydroxylation of fenoprofen.FBV024Nitrogen metabolism of wood decomposingbasidiomycetes and their interaction with diazotrophs asrevealed by IRMSP. Weißhaupt* 1 , W. Pritzkow 1 , M. Noll 21 Federal Institute for Materials Research and Testing, Berlin, Germany2 Federal Institute for Risk Assessment, Berlin, GermanyIsotope ratio mass spectrometry (IRMS) is an advanced method toinvestigate carbon, nitrogen, oxygen, sulphur and hydrogen in organicsamples. In particular the N-content, its isotope signature and the C/N ratioreveal important facts of nutrient cycling, niche separation and ecologicalfood webs. In this study the characteristics of nitrogen exchange of wooddecomposing microorganisms were investigated. It was revealed that thegrowth of the white rotting fungus Trametes versicolor is enhanced after theaddition of ammonia or urea, while the brown rotting fungus Oligoporusplacenta is not accelerated. In addition, a mutualistic interaction withatmospheric N 2-assimilating (diazotrophic) bacteria was investigated.Cultivation experiments under an atmosphere of 15 N 2 and O 2 and subsequentIRMS analysis of the dry biomass of the diazotrophs Azotobactercroococcum, Beijerinckia acida and Novosphingobium nitrogenifigensrevealed that they assimilated up to 12 % of their nitrogen by fixed N 2. Theexperiments reflected nitrogen availability as a prerequisite for efficientgrowth for wood decomposing fungi and diazotrophs. Co-cultivationexperiments of both revealed that depending on the growth characteristicsand bacterial N 2 assimilation activity nitrogen is transferred from bacteriainto basidiomycetal biomass. In conclusion, a first indication of aninteraction between wood decomposing basidiomycetes and diazotrophs wasobtained which is a novel pathway of fungal nitrogen acquisition.spektrum | Tagungsband 2011