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

software consists of 661 amino acids on 15 exons. By ESI-MS-analysis ofthe purified enzyme 9 peptides could be found covering 39% of the protein.POX1 is the only gene product expressed under the conditions tested. Aproposed POX2 gene and protein (POX2) shows some significantdifferences to POX1 and to other published c-DNA- and protein sequences.POX2 expression could not be shown and its physiological role is stillunknown.FBP039Protoplast analysis of the fungus Ashbya gossypii revealeda correlation between differentiation and vitamin overproductionS. Nieland*, K.-P. StahmannBiology, Chemistry and Process Technology, University of Applied SciencesLausitz, Senftenberg, GermanyAshbya gossypii, a filamentous hemiascomycete, is known to produce100mg vitamin B 2 (riboflavin) per g biomass. Regulation of thisoverproduction is evident since less than 1% is produced at constant dilutionrate in chemostatic culture [1]. The molecular mechanism controling thatdifference in productivity is a regulation of the genes RIB1, RIB2 and RIB3,encoding the enzymes of the biosynthesis pathway. Evidence for inductionunder nutritional stress was given by increasing RT-PCR signals comparedwith constitutively expressed TEF or ACT1. Additionally, RIB3, encodingthe first enzyme, showed increased initiation of transcription. This wasshown by fusion of its promoter with lacZ. On enzyme level, an increase ofspecific activity of dihydroxy butanone phosphate synthase, encoded byRIB3, was determined in the transition from growht to the production phase[2].The measured changes were only partly consistant with the cellmorphologies observed by microscopy. As soon as growth rate declined asignificant part of the cells formed spores, a second part accumulatedriboflavin, resulting in a green fluorescence, and a third part appearedhyaline. In the mycelium a quantification of cell types was impossible.Therefore a conversion to protoplasts by digestion of the cell walls wasperformed. Up to 10 9 protoplast were liberated per millilitre. This wasdeteced by FACS analysis. Furthermore flow cytometry distinguished 50%needle-shaped spores from spherical protoplasts. Up to 80% of the latteremitted green light when excited at 488 nm indicating riboflavinaccumulation.By FACS sorting more than 10 6 riboflavin accumulating protoplasts wereseparated from hyaline protoplasts. In a typical crude extract more than 90mU beta-galactosidase activity was determined per mg protein. In contrastless than 5 mU were detectable in hyaline protoplast showing no riboflavinaccumulation. This difference allows the conclusion that riboflavin overproductionis limited to a part of the cells. To achieve full over-productionpotential differentiation not into four but into a single cell type whichoverproduces and stores riboflavin might become a promissing approach.[1] Stahmann, K.-P. (2010): Production of vitamin B2 and a polyunsaturated fatty acid by fungi pp231-246 In: Industrial Applications Martin Hofrichter (ed) Vol. X der Serie The Mycota K. Esser (ed)Springer, Heidelberg.[2] Schlösser, T. et al (2007): Growth stress triggers riboflavin overproduction in Ashbya gossypii.Applied Microbiology and Biotechnology 76(3): 569-578.FBP040Genetic Characterisation of MCF A95, a Micro-colonialFungus that Colonises Bare RocksS. Noack* 1 , W.J. Broughton 1 , C. Nai 1,2 , S. Lucienn 1 , R. Banasiak 1 ,A.A. Gorbushina 1,21 Federal Institute for Materials Research and Testing, Materials andEnvironment (IV), Berlin, Germany2 Institute of Geological Sciences, Division Geochemistry, Hydrogeologie,Mineralogy, Free University, Berlin, GermanyMelanised microcolonial fungi (MCF) colonize bare rock surfaces in desertsand other arid areas and are unequaled among eukaryotic organisms in theirability to withstand extreme heat, dessication and UV radiation. Theseorganisms are crucial in the establishment of subaerial rock biofilms and, assuch, set the stage for a variety of interactions important for mineral/materialstability and rock weathering. MCF are a taxonomically diverse group ofascomycetes that possess simplified stress-protective morphologies. Partlyas a result of their peculiar compact colonial structure and protective cellwalls, MCF are able to survive a broad spectrum of physical stressesincluding temperature, salt, UV-irradiation and desiccation. These survivalspecialists exist because of multiple secondary metabolic productssupporting their stress tolerance - melanins, carotenoids, mycosporines andcompatible solutes. A meristematic black yeast species, Sarcinomycespetricola (A95), was isolated from the sun exposed marble monument inAthens (Greece). As many rock-inhabiting fungi, A95 is positioned in theearly diverging lineages of Chaetothyriales, which were shown to beancestral to opportunistic pathogens and lichens. A95 is a relatively fastgrowingstrain which was identified as a suitable model organism for geneticanalysis. For its stress tolerance A95 relies on a broad spectrum of stressprotection mechanisms typical for MCF. The whole genome sequence ofSarcinomyces petricola (454 and Illumina methods) is currently underway.Different methods have been tested to establish a transformation protocol forA95. A commonly used method using the binary Ti vector system ofAgrobacterium tumefaciens was employed (De Groot et al., 1998). Thestress- tolerant morphology of the black yeast, especially the thickness oftheir cell wall and melanization makes the DNA transfer from A.tumefaciens to A95 a complicated task. Several methods to circumvent thisproblem were tested. By Microprojectile Bombardment small gold particleswere coated with DNA and directly transferred into the nucleus. Otherapproaches are the transformation of protoplasts or increasing the receptivityof A95 to A. tumefaciens transformation by mechanical or chemicalweakening of the cell wall.[1] De Groot, M.J.A. et al (1998): Agrobacterium tumefaciens - mediated transformation offilamentous fungi. Nature Biotechnology 16: 839-842.FBP041Regioselective hydroxylation of diverse flavonoids by anaromatic peroxygenaseK. Barkova* 1 , M. Kinne 1 , M. Hofrichter 1 , R. Ullrich 1 , A. Fuchs 2 , L. Hennig 31 Unit of Environmental Biotechnology, International Graduate School (IHI)Zittau, Zittau, Germany2 Department of Chemistry, University of Applied Science, Zittau, Germany3 Department of Chemistry, University of Leipzig, Leipzig, GermanySelective transfer of oxygen functionalities to non- or little activated carbonatoms (e.g. aromatic rings) is a challenging problem for chemical synthesis.Biotransformations based on the activity of oxidoreductases would offer anelegant alternative. Here we report that fungal peroxygenase from Agrocybeaegerita (AaeAPO) can selectively hydroxylate a variety of flavonoids(plant ingredients with various biological functions, e.g. as strongantioxidants).The results showed that the hydroxylation reactions proceed rapidly andregioselectively yielding C6-hydroxylated reaction products of diverseflavonoids such as flavone, flavanone, apigenin, luteolin as well as daidzein.Studies using 18 O-enriched hydrogen peroxide (H 2 18 O 2) as co-substraterevealed that the oxygen incorporated into the reaction product in factderived form the peroxide, which points to a true peroxygenase mechanism.Thus, mass spectral analysis of the metabolite formed during the AaeAPOcatalyzedhydroxylation of daidzein in the presence of H 2 18 O 2 in place ofH 2O 2 showed a shift of the principal [M+H] + ion from m/z 271 to m/z 273 incase of 6-hydroxydaidzein (demethyltexasin).Interestingly, flavonoid glycosides, especially multiple glycosilatedcompounds such as rutin, are not subject of peroxygenase attack, veryprobably due to stearic hindrance.Our results raise the possibility that fungal peroxygenases may be useful forversatile, cost-effective, and scalable syntheses of hydroxylated flavonoids.FBP042Studies of wood degradation by wood-decay fungi with anew experimental setupF. Hahn* 1 , T. Arnstadt 1 , R. Ullrich 1 , C. Liers 1 , M. Hofrichter 1Unit of Environmental Biotechnology, International Graduate School (IHI)Zittau, Zittau, GermanyWood is a hard, fibrous tissue found in many plants. It is a natural compositeof cellulose fibers embedded in a matrix of hemicelluloses and lignin, whichis consisting mainly of carbon-carbon linked and ether linked phenylpropanebuilding blocks. Lignin is a natural barrier against microbial attack and ismodified only by radicalic mechanisms catalysed by peroxidases(manganese peroxidase, lignin peroxidase), phenoloxidase (laccase) orhydroxylic radicals (produced by the Fenton’s reaction). Wood-decay fungiare known as the most efficient wood degraders. A flexibel experimentalapproach was set up to investigate the spatiotemporal degradation ofspektrum | Tagungsband 2011