VAAM-Jahrestagung 2012 18.–21. März in Tübingen

82FUP018FbFP as an Oxygen-Independent fluorescence reporter inSaccharomyces cerevisiae and Candida albicansI. Eichhof*, D. Tielker, J.F. ErnstHeinrich-Heine-Universität Düsseldorf, Biologie, Molekulare Mykologie,Düsseldorf, GermanyMany microbes colonize anoxic or hypoxic niches and several groups ofpathogens attain their virulence by their ability to adapt to theseconditions. Although green fluorescent protein (GFP) and its variants arevaluable tools for monitoring gene expression and protein localization,their use is limited to aerobic environments, because chromophoresynthesis of these reporters requires oxygen. Therefore we establishedflavin mononucleotide-based oxygen-independent fluorescent proteins(FbFP) as reporters for the apathogenic yeast Saccharomyces cerevisiaeand the human fungal pathogen Candida albicans by expressing thecodon-adapted gene encoding CaFbFP under the control of differentpromoters in both fungi (Eukaryot. Cell 8:913-915, 2009). Synthesis ofCaFbFP was demonstrated in S. cerevisiae and C. albicans cells byimmunoblotting and fluorescence was detected under both normoxic andhypoxic conditions in the cytoplasm of cells. To examine the use of FbFPas a reporter in other cell compartments we attempted to achieve cell walllocalization of FbFP in S. cerevisiae by generating fusions to the cell wallprotein Aga2. Fluorescence analyses and immunodetection indicated thelocalization and fluorescence of the FbFP fusion on the yeast cell surface.The ability of FbFP to fluoresce in yeast nuclei was investigated by fusionof CaFbFP to the histone H2B of S. cerevisiae and C. albicans,respectively. Fluorescence analyses of S. cerevisiae cells showed a clearFbFP-mediated fluorescence signal in the nuclei.FUP019Screening of white rot fungi from Belarus for novel dyebleaching enzymesA. Matura* 1 , M. Liebe 1 , W. Burd 2 , K.-H. van Pée 11 TU Dresden, Allgemeine Biochemie, Dresden, Germany2 University, Biology, Grodno, BelarusThe textile industry is an industrial branch with great relevance for theenvironment. During the textile dying process 30 to 40% of dyes do notbind to the cotton fibres and remain in the waste water. The costs forcleaning this waste water with different physical, chemical,electrochemical or biological methods are high. For bleaching of nonbounddyes in the waste water and bleaching of cotton, the use of enzymesfrom white rot fungi could be an environmentally friendly and also lesscost intensive alternative.We performed a screening for dye decolourisation by novel white rot fungifrom National Park Belaweschskaja Puschtscha Belarus. Because of thesimilar phenolic structure of lignin and industrial dyes, white rot fungi candegrade many of these dyes. The most effective enzymes for thisapplication are laccases and peroxidases. 17 different fungal mixedcultures were investigated for their ability to bleach 40 dyes of yellow,orange, red, blue, and black colour with different chemical structures usedin technical textile dying processes. Bleaching experiments were carriedout on agar plates and in liquid cultures. From mixed cultures with highdecolourisation rates we isolated pure fungal samples for identification.Whereas many fungi could degrade blue and black dyes, only a few ofthem could also decolourise yellow, orange, and red ones. Especially thedecolourisation of yellow dyes is a problem in many bleaching processesand often a yellow colour remains even after the successful decolourisationof blue or black dyes. Two of our new fungi aMucor hiemalis sp.silvaticusand aMortierella verticillatasp. are able to decolourise yellowdyes very effectively. The main enzymes from some of the fungi with veryhigh bleaching activity were detected and partly purified. We determinedlaccase, manganese- and ligninperoxidase activities and performed firstchromatographic purification steps for these enzymes. Thus we found newenzymes with interesting properties for the use in industrial bleachingprocesses.FUP020Alternative splicing in fungal aldo-keto reductasesK. Grützmann* 1 , K. Hoffmann 2 , M. Eckart 2 , S. Schuster 1 , K. Voigt 21 University Jena, Department of Bioinformatics, Jena, Germany2 Leibniz Institute for Natural Product Research and Infection Biology andUniversity of Jena, Jena Microbial Resource Collection, Jena, GermanyAldo-keto reductases (AKRs) are characterized by a common 3D-fold, the() 8-barrel motif and a broad substrate specificity [1]. They areNAD(P)H-dependent andrecognize broad categories of carbonylcontainingsubstrates, e.g. aldehydes, ketones, monosaccharides, andsteroids. The cofactor binding site for NAD(P)H is highly conserved.AKRs encompass a superfamily comprising approx. 120 members in 14protein families scattering through prokaryotes, plants, animals and fungi.In former studies (manuscript in prep.), we reconstructed the evolution offungal AKRs using distance, maximum parsimony, maximum likelihoodand Bayesian analyses. We are able to recognize different subgroups andparalogs and discovered different alleles. In order to understand theevolution of these different alleles we predicted the possible occurrence ofalternative splicing (AS). We applied the bioinformatics tool NetAspGene([2], http://www.cbs.dtu.dk/services/NetAspGene/), which was originallytrained for genes of the ascomycete Emericella nidulans (anamorph:Aspergillus nidulans), to AKRs from zygomycetes. Known splice sitescould be recovered. The prediction of additional splice sites with highconfidence scores ranging typically between 0.85 and 1.00 actuallysuggests a diversification through AS. The impact of AS in the evolutionof fungal AKRs are discussed.1. Barski et al. (2008) The aldo-keto reductase superfamily and its role in drug metabolism anddetoxification. Drug Metab Rev. 2008 40(4), 553-624.2. Wang et al. (2009) Analysis and prediction of gene splice sites in four Aspergillus genomes.Fungal Genet Biol. 46, 14-18.FUP021Engineering the citric/isocitric acid overproduction by theyeast Yarrowia lipolyticaV. Yovkova 1 , M. Holz 1 , A. Aurich 2 , S. Mauersberger* 1 , G. Barth 11 Technische Universität Dresden, Biology, Institute of Microbiology,Dresden, Germany2 Helmholtz Centre for Environmental Research - UFZ, Environmental andBiotechnology Centre (UBZ), Leipzig, GermanyFunctionalized carboxylic acids are highly versatile chemical species witha wide range of applications (e.g. as co-polymers, building blocks,acidulants). Therefore they are of special interest as biotechnologicallyavailable targets. The yeast Yarrowia lipolytica secretes high amounts ofvarious organic acids, like citric acid (CA) and isocitric acid (ICA) underseveral conditions of growth limitation from an excess of carbon source.Depending on the carbon source used, strains of Y. lipolytica produce amixture of CA and ICA in a characteristic ratio. On carbohydrates andglycerol, wild-type strains show a CA/ICA ratio of 90:10, and onsunflower oil and n-alkanes of 60:40. To examine, whether this CA/ICAproduct ratio can be influenced, isocitrate lyase (ICL1), aconitase (ACO1)or isocitrate dehydrogenase (IDP1) overexpressing strains wereconstructed containing multiple copies of these genes, respectively.Additionally, ICL1 disrupted strains were tested. In the ICL1overexpressing strains the part of ICA on the whole product (CA + ICA)decreased to 3-7% on all tested carbon sources [1]. In contrast, the ACO1and interestingly also the IDP1 overexpression resulted in a shift of theproduct pattern in direction of ICA [2]. On carbohydrates the ICAproportion increased from 10-12% to 14-15%, on sunflower oil even from35-45% to 65-72% of total acid produced. The loss of the isocitrate lyaseactivity in the icl1-defective strains had a comparable effect on theCA/ICA ratio like the ACO1 overexpression. On glucose and glycerol theICA proportion was 2-5% higher compared to the wild-type strain. Thus,using wild-type or engineered Y. lipolytica strains the enantiomericallypure form of isocitric acid, currently available as a speciality compound,can be produced now in large amounts and used as a building block fororganic synthesis [3].[1] Förster A, Jacobs K, Juretzek T, Mauersberger S, Barth G (2007) Appl Microbiol Biotechnol77:861-869[2] Holz M, Förster A, Mauersberger S, Barth G (2009) Appl Microbiol Biotechnol 81: 1087-1096[3] Heretzsch P, Thomas F, Aurich A, Krautscheid H, Sicker D, Giannis A (2008) Angew Chem IntEd 47: 1958-1960These studies were partially supported by the BMBF of Germany (0339822) and the SMUL of theLand Saxony, Germany (138811.61/89 and 2620000240).FUP022A molecular tool for transposon-mediated mutagenesis inAspergillus speciesE.K. Hihlal*, F. KempkenBotanisches Institut , Abt. Botanische Genetik & Molekularbiologie, Kiel,GermanyTransposons are ubiquitous genetic elements present in the genomes of allliving cells. Among the different types of transposable elements cut-andpaste transposons are particularly useful for development of transposonbasedmutagenesis systems. We previously have characterizedtransposable elements in two filamentousfungi,AspergillusnigerandPenicillium chrysogenum(ref. 1), therebyidentifiying transposonVaderas an active element inA.niger Upon selectionfor chlorate resistantA.nigercolonies, oneVadercopy was found integratedin thenirAgene. As this copy apparently contained all necessary sequenceinformation for beingtrans-activated it was used for vector developmentand fungal transformation (ref. 2).We observed a Vaderexcision frequency of about 1 in 2.2x10 5 A.nigerspores. All colonies analyzed exhibited an excision event on the DNAlevel andVader footprints were found. Employing thermal asymmetricinterlaced-PCR the reintegration sites of 21 independent excision eventswere determined. All reintegration events occurred within or very close togenes. Thus,Vader appears to be a useful tool for transposon mutagenesisinA.niger (ref. 2).BIOspektrum | Tagungsband 2012