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

99comprises several structurally conserved velvet-like proteins that havedistinct developmental roles, illustrating the functional plasticity of theseregulators. We performed extensive phenotypic characterizations of singleand double knockout mutants using the codon-optimized FLP/FRTrecombination system [5]. Data from penicillin bioassays andquantification of conidiospores of these knockout mutants clearly showthat all velvet-like proteins are involved in secondary metabolism andother distinct developmental processes. By detailed fluorescencemicroscopy and protein-protein interaction studies using bimolecularfluorescence complementation, tandem-affinity purification and yeast twohybrid,we want to extend the analysis of the velvet-like complex in P.chrysogenum. Our results widen the current picture of regulatory networkscontrolling both fungal secondary metabolism and morphogenesis, whichis significant for the genetic manipulation of fungal metabolism as part ofindustrial strain improvement programs.[1] Bayram et al. (2008) Science 13:1504-1506[2] Calvo AM (2008) Fungal Genet Biol 45: 1053-1061[3] Hoff B, Kamerewerd J, Sigl C, Mitterbauer R, Zadra I, Kürnsteiner H, Kück U (2010) Eukaryot Cell:9:1236-50[4] Hoff B, Kamerewerd J, Sigl C, Zadra I, Kück U (2010) Appl Microbiol Biotechnol 85: 1081-1094[5] Kopke K, Hoff B, Kück U (2010) Appl Environ Microbiol 76:4664-4674MEP030Phenguignardic acid and guignardic acid, phytotoxicsecondary metabolites from the grape black rot fungusGuignardia bidwelliiI. Buckel* 1 , D. Molitor 2 , J. Liermann 3 , B. Berkelmann-Löhnertz 4 , T. Opatz 3 ,E. Thines 11 Institute of Biotechnology and Drug Research (IBWF), Plant protection,Kaiserslautern, Germany2 Centre de Recherche Public – Gabriel Lippmann, Department Environmentand Agro-Biotechnologies, Belvaux, Luxembourg3 Johannes-Gutenberg-University, Institute of Organic Chemistry, Mainz,Germany4 Geisenheim Research Center, Department of Phytomedicine, Geisenheim,GermanyThe causal agent of black rot on grapes is the phytopathogenicfungusGuignardia bidwellii. Black rot is one of the most devastatingdiseases on grapes and since 2002 a serve outbreak of the disease wasevident in some German winegrowing regions. The infection was observedin abandoned vineyards primarily, but subsequently an expansion tocultivated vineyards was found. The disease can result in significant croplosses ranging from 5 to 80 % of the total yield.The infection cycle ofGuignardia bidwelliiis characterized by two phases,a symptomless initial phase followed by a necrotrophic phase. Thus thefungus is classified as a hemibiotrophic pathogen. Phytopathogenic fungioften produce phytotoxins for a successful colonisation of the plant. Suchlow-molecular compounds are frequently involved in disease symptomformation.Bioactivity guided isolation led to the identification of phenguignardicacid, a new secondary metabolite from submerged cultures of the grapeblack rot fungus as phytotoxic agent. The compound is structurally relatedto guignardic acid, a dioxolanone moiety containing metabolite isolatedpreviously fromGuignardiaspecies. However, in contrast to guignardicacid, which is presumably synthesised via deamination products of valineand phenylalanine, the biochemical precursors for the biosynthesis of thenew phytotoxin appears to be exclusively phenylalanine.Both compounds were characterised in biological assays by using vine leafsegments or intact plants. During fermentation optimisation sevenstructurally related secondary metabolites were detected and isolated. Fourof the seven secondary metabolites were found to be phytotoxic on vineleaf segments.MEP031The genetic potential of Streptomyces collinus Tü 365 tosynthesize secondary metabolitesS. Rohrer* 1 , D. Iftime 1 , C. Rückert 2 , J. Kalinowski 2 , W. Wohlleben 3 , T. Weber 11 Interfakultäres Institut für Mikrobiologie und Infektionsmedizin / UniversitätTübingen, Mikrobiologie/Biotechnologie - Secondary Metabolite Genomics,Tübingen, Germany2 CeBiTec / Universität Bielefeld, Bielefeld, Germany3 Interfakultäres Institut für Mikrobiologie und Infektionsmedizin / UniversitätTübingen, Mikrobiologie/Biotechnologie, Tübingen, GermanyStreptomycetes are common producers of secondary metabolites likeantibiotics. The strain Streptomyces collinus Tü 365 is known to producethe antibiotic Kirromycin (Wolf and Zähner, 1972; Weber et al., 2008). Bybioinformatic analysis using the antiSMASH software (a secondarymetabolite prediction tool, Medema et al., 2011) 26 additional secondarymetabolite gene clusters were identified in the genome of this strain, buttheir function and their biosynthesis products remain to be elucidated. Thegenome harbors five clusters for NRPSs, five for terpenes, four for PKSsand four for PKS-NRPS-hybrids. Moreover, there are clusters for threesiderophores, a bacteriocin, an ectoin, a melanin and a lantibiotic present.Most of the clusters are not expressed or expressed at very low levelsunder standard laboratory conditions, but gene expression can be inducedunder certain conditions.Here we show transcriptional analyses of some of these gene clusters.First, the strain was cultivated in different growth media to analyze thelevel of expression of the key genes from selected biosynthetic geneclusters by reverse transcriptase PCR (RT-PCR). RNA samples were takenat different time points from the various liquid cultures. The obtained geneexpression data will facilitate the identification of the desired compounds.In a parallel approach cluster 1, a lantibiotic-like gene cluster, was investigatedin heterologous expression studies. Cluster 1 consists of a two-gene transporter,a putative lantibiotic prepeptide and a rare putative class IV lantibiotics cyclase.The prepeptide was expressed in E. coli and the prepeptide in combination withthe cyclase was expressed in S. lividans Tk 23.1. Wolf and Zähner, 1972. Metabolic products of microorganisms. 99: Kirromycin. Arch.Microbiol. 83,2:147-154.2. Weber et al., 2008. Molecular analysis of the kirromycin biosynthetic gene cluster revealed beta-alanine asprecursor of the pyridone moiety. Chem Biol. 15, 2: 175-88.3. Medema et al., 2011. antiSMASH: rapid identification, annotation and analysis of secondary metabolitebiosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 39: 339-346.MEP032Identification of Gene Clusters for Biosynthesis ofBromotyrosine in Metagenomes of the Marine SpongesIanthella basta and Aplysina cavernicolaK. Kunze*, K.-H. van PeeTU Dresden, Institut für Biochemie, Dresden, GermanyMarine sponges (Verongida) are able to produce a set of bioactivemolecules. Among those compounds are bromtyrosines and bromotyrosinederivatives. Bromtyrosines (Bts) are known to have pharmacologicalrelevance. In marine sponges, Bts are typically located within thesponging/chitin based skeleton. They are supposed to protect the chitinskeleton from degradation, through chitinase inhibition. Bts from thespecies Ianthella basta and Aplysina cavernicola have already beendetected, but were not further investigated so far. From other biosyntheticpathways, for example the biosynthetic gene cluster of the peptideantibiotic balhimycin, it is known, that halogenation of tyrosine residues iscatalysed by flavin-dependent halogenases. It should thus be possible todetect the Bt-biosynthesic gene cluster of I.basta and A. cavernicola byusing the degenerated PCR primer pair TyrhalA_for/rev which is specificfor flavin-dependent tyrosine halogenases. Sponges are known to beassociated to a large amount with bacterial symbionts. Therefore, it seemsquite likely that the bromotyrosine producer is rather a bacterial or fungalsymbiont than the sponge itself. To define the origin of the detected genes,two different methods for the extraction of metagenomic DNA (eDNA; e =environmental) are used. With the first method, the whole eDNA ofsponges is isolated, whereas the second method uses an additionalsymbiont-enrichment-step prior to eDNA extraction. After detection of thehalogenase gene, it should be possible to identify the whole gene cluster byusing a DNA library (in form of a fosmid library). Finally, the flavindependenthalogenases will be characterised with respect to itshalogenating activity and substrate specificity.Miao SC, Andersen RJ, Allen TM. Cytotoxic metabolites from the sponge Ianthella basta collectedin Papua New Guinea. J Nat Prod. 1990 Nov-Dec;53(6):1441-6.Thoms C, Wolff M, Padmakumar K, Ebel R, Proksch P. Chemical defense of Mediterraneansponges Aplysina cavernicola and Aplysina aerophoba. Z Naturforsch C. 2004 Jan-Feb;59(1-2):113-22.Pelzer S, Süssmuth R, Heckmann D, Recktenwald J, Huber P, Jung G, Wohlleben W. Identificationand analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptidebiosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob Agents Chemother. 1999Jul;43(7):1565-73.Webster NS, Taylor MW. Marine sponges and their microbial symbionts: love and otherrelationships. Environ Microbiol. 2011 Mar 28. doi: 10.1111/j.1462-2920.2011.02460.x. [Epubahead of print] PubMed PMID: 21443739.MEP033Purification and cloning of the O-Methyltransferase ofAlternaria alternataF. Oswald*, K. Brzonkalik, C. Syldatk, A. NeumannKIT, Technische Biologie, Karlsruhe, GermanyBlack-moulds of the genus Alternaria contaminate many foodstuffs andagricultural products. In addition to the economical damage these fungican produce harmful secondary metabolites, the Alternaria toxins. Some ofthese mycotoxins such as alternariol (AOH), alternariolmonomethylether(AME), altenuene (ALT) are polyketides and AOH is produced via thepolyketide pathway. AOH is than methylated by the alternariol-omethyltransferase,transferring a methyl group from SAM to AOH to yieldAME. The enzyme was partially purified and characterized, but thesequence is still unknown (1, 2).The Genome of Alternaria alternata was sequenced by the group of ChrisLawrence (3). In the genome 11 putative genes coding for polyketidesynthases were identified by Blast-analyses (4). Next to some of thesepolyketide synthetase genes for methyltransferases were also found. As thegenes for secondary metabolite production are usually clustered (5), it is likely,BIOspektrum | Tagungsband 2012