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

[2] Steffen, W. et al. (2010): Organelle-specific expression of subunit ND5 of human complex I(NADH dehydrogenase) alters cation homeostasis in Saccharomyces cerevisiae. FEMS Yeast Res10(6): 648-659.FBP007Endocytosis and toxicity of ricin A in yeastB. Becker*, M.J. SchmittDepartment of Molecular and Cell Biology, University Saarland,Saarbrücken, GermanyThe plant toxin ricin represents one of the most powerful poisons ofbiological heritage. This heterodimeric glycoprotein belongs to the class ofA/B toxins. It consists of two polypeptide chains amongst which the B-chainrepresents the cell surface binding domain mediating toxin uptake by themammalian target cell. This domain is linked via a single disulfide bond tothe A-chain (RTA) that catalyzes the N-glycosidic cleavage of a specificadenine residue in the sarcin/ricin loop of 28S ribosomal RNA. Afterendocytotic uptake and retrograde transport, ricin enters the endoplasmaticreticulum from where it is retrotranslocated into the cytosol, most likely byusing the Sec61p translocon for ER exit. Subsequently, the B-chain ispolyubiquitinated and proteasomaly degraded. It is asumed that only alimited number of A-chain molecules are able to escape degradation finallycausing cell death by inhibiting protein synthesis.In contrast to mammalian cells, yeast is not killed by external application ofricin. Following expression of biologically active ricin A subunit variantscarrying a C-terminal His (6)-tag and different ER retention signals in E. coli,we analyzed the cytotoxic effect of the purified constructs after externalapplication against yeast spheroplasts by flow cytometry and oxygen-sensormicrotiter plate measurements. We also fused ricin A to the b-subunit of theviral yeast A/B toxin K28 in order to enable the chimeric ricinA/K28βfusion protein to be taken up by yeast via receptor-mediated endocytosis. Todo so, we constructed different fusion proteins of K28β and ricin A andexpressed them in E. coli. After successful expression and purification weused these toxin fusions to study the intracellular transport of ricin A inyeast as model organism.FBP008Process optimization of alternariol production with thefilamentous fungus Alternaria alternataK. Brzonkalik*, T. Herrling, C. Syldatk, A. NeumannTechnical Biology, Karlsruhe Institute of Technology (KIT), Karlsruhe,GermanyBlack-moulds of the genus Alternaria contaminate many foodstuffs andagricultural products. In addition to the economical damage these fungi canproduce harmful secondary metabolites, the Alternaria toxins. Some of thesemycotoxins such as alternariol (AOH), alternariolmonomethylether (AME),altenuene (ALT) and tenuazonic acid (TA) have been described as cytotoxic,genotoxic and mutagenic in vivo and in vitro. Due to the fact thatmycotoxins could be detect in many foodstuffs and these fungi growingeven at low temperatures it is necessary to produce the mycotoxins in highamounts for the elucidation of the genotoxic, cytotoxic and mutagenicpotential. For better scalability and evaluation of the parameters influencinggrowth and toxin production a fluid submerse cultivation was chosen.In a first approach alternariol production was performed in a small scalebioreactor (1.5 l) in a semi-synthetic medium with glucose as carbon sourceand a mixture of ammonium and nitrate as nitrogen source. As a result 4mg/l alternariol can be produced. Several fermentations showed thatnitrogen has an important regulatory effect since alternariol production startsafter complete nitrogen consumption. To investigate the effect of nitrogenand carbon source different substances are tested in shaking flaskexperiments to optimize the production medium and to find an economicalternative to glucose.Furthermore the influence of the aeration rate on alternariol production wasdetermined. Therefore different fermentations in the bioreactor wereperformed with decreasing aeration or decreasing oxygen concentration toobserve the effect on alternariol production.FBP009Molecular analysis of secondary metabolite biosynthesisin Alternaria alternataR. Fetzner* 1 , C. Lawrence 2 , R. Fischer 11 Institute for Microbiology, Karlsruhe Institute of Technology (KIT),Karlsruhe, Germany2 Bioinformatics Facility I, Virginia Bioinformatics Institute & Departmentof Biological Sciences, Blacksburg, USASecondary metabolites are organic compounds that are not directly involvedin normal growth, development or reproduction of organisms. In the moldAlternaria alternata a major group of different secondary metabolites aremycotoxins with heterogenous structures. Important and in food and feedfrequently found mycotoxins produced by A. alternata are the polyketidesalternariol (AOH), alternariol-monomehtylether (AME) and altenuen (ALT).AOH shows cytotoxic, foetotoxic and teratogenic effects. The polyketidebiosynthesisrequires polyketide synthases, multi-domain enzymes,separated into groups dependent on the degree of reduction of their product.One example for a non-reduced or aromatic polyketide is melanin, a pigmentfound in most organisms. It is known that genes involved in the biosynthesisof polyketides are organized in gene clusters.This work describes the identification and characterization of genes involvedin the biosynthesis of secondary metabolites. In the A. alternata genomeeleven putative polyketide synthases and regulators located within thecorresponding gene clusters have been identified. One shows high similarityto melanin biosynthesis gene clusters of other organisms. Down regulationof the regulator within this cluster using the siRNA-silencing method led toa whitish phenotype, and surprisingly had a strong impact on polar growth.Additionally the formation of conidia is strongly reduced. This suggests thatthe regulator may not only control melanin synthesis but also other cellularprocesses. The analysis of four other polyketide synthases is under way.FBP010Yeast-based protein delivery to mammalian phagocyticcells is increased by coexpression of bacterial listeriolysinB. Walch 1 , T. Breinig 2 , G. Geginat 3 , M.J. Schmitt 1 , F. Breinig* 11 Department of Molecular and Cell Biology, University Saarland,Saarbrücken, Germany2 Institute for Virolog, Saarland University Hospital, Homburg, Germany3 Faculty of Clinical Medicine Mannheim, Institute for MedicalMicrobiology and Hygiene, University of Heidelberg Mannheim, GermanyYeast-mediated protein delivery to mammalian antigen-presenting cells is apowerful approach for inducing cell-mediated immune responses. We showthat coexpression of the pore-forming protein listeriolysin O from Listeriamonocytogenes leads to improved translocation of a proteinaceous antigenand subsequent activation of specific T lymphocytes. As the resulting yeastcarrier is self-attenuated and killed after antigen delivery without exhibitingany toxic effect on antigen-presenting cells, this novel carrier systemsuggests itself as promising approach for the development of yeast-basedlive vaccines.FBP011Regio- and enantioselective hydroxylation of variousalkanes catalyzed by secreted fungal peroxygenaseS. PeterUnit of Environmental Biotechnology, International Graduate School ofZittau (IHI),Zittau, GermanySelective hydroxylation of non-activated aliphatic hydrocarbons is aparticular challenging reaction in organic synthesis. However, numerousmicroorganisms are known to be capable of oxidizing and even utilizing n-alkanes as carbon source. They use specific intracellular enzymes such asmethane monooxygenase (MMO) or certain cytochrome P450monooxygenases (P450s) to transform the inert alkanes into thecorresponding alkanols, which are further oxidized to their ketones or viaaldehydes to fatty acids.Using such enzymes in vitro for biocatalytic oxygen transfer reactions wouldoffer an interesting alternative to achieve selective hydroxylation. However,these biocatalysts are intracellular and less stable enzymes, which are hardlysuitable for application in isolated form.Here we report on a secreted peroxygenase from the agaric fungus Agrocybeaegerita, which catalyzes the H 2O 2-dependent monooxygenation of severalspektrum | Tagungsband 2011