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

96various carotenoids instead of decaprenoxanthin on the growth behavior,sensitivity towards UV or oxidants will be assessed.MEP016Determination of influencing factors on mycotoxin productionin Alternaria alternataK. Brzonkalik*, D. Hümmer, C. Syldatk, A. NeumannKarlsruhe Institute of Technology (KIT), Institute of Process Engineeringin Life Sciences, Section II: Technical Biology, 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) and tenuazonic acid (TA) have been described ascytotoxic, genotoxic and mutagenic in vivo and in vitro. These mycotoxinswere detected in many foodstuffs even under refrigeration conditions. Tominimize the health risks of the consumers it is absolutely essential todetermine factors which influence mycotoxin production of Alternariaalternata.For the determination of influencing parameters a robust and reliableplatform process was developed 1 . The system proofed to be highlyreproducible and set the conditions for the monitoring of substrateconsumption and mycotoxin production. Additionally, variation of singleprocess parameters was possible. The influences of carbon and nitrogensource 2 , aeration rate 1 and pH value were examined. By the choice ofcarbon and nitrogen source mycotoxin concentration and composition canbe altered whereas due to the variation of aeration rate and pH value over abroad range optimum curves can be obtained. This study provides essentialdata to elucidate mycotoxin production in Alternaria alternata.1 K. Brzonkalik, T. Herrling, C. Syldatk, A. Neumann. International Journal of Food Microbiology 147(2011), p. 120-126.2 K. Brzonkalik, T. Herrling, C. Syldatk, A. Neumann, AMB Express 1:27 (2011).MEP017Production of cytotoxic tryprostatin B analogues by using theprenyltransferase FtmPT1B. Wollinsky* 1 , A. Hamacher 2 , M. Kassack 2 , S.-M. Li 11 Philipps-Universität Marburg, Institut für Pharmazeutische Biologie undBiotechnologie, Marburg, Germany2 Heinrich-Heine Universität Düseldorf, Institut für Pharmazeutische undMedizinische Chemie, Düsseldorf, GermanyThe prenyltransferase FtmPT1 from Aspergillus fumigatus is involved inthe biosynthesis of verruculogen [1] . This enzyme catalyzes the regularprenylation of cyclo-L-Trp-L-Pro (brevianamide F) of the indole nucleusat C-2 position, resulting in the formation of tryprostatin B, which wasreported to be active as a cell cycle inhibitor [2;3] . It has been shown thatFtmPT1 accepted, in addition to its natural substrate brevianamid F, sevenother tryptophan-containing cyclic dipeptides [2;4] .In this study fourteen tryptophan-containing cyclic dipeptides, includingall the four diastereomers of cyclo-Trp-Pro and cyclo-Trp-Ala, wereconverted to their C2-prenylated derivatives by using the overproducedand purified FtmPT1. The enzyme products were isolated on HPLC inpreparative scales and their structures were elucidated by NMR and MSanalyses. The cytotoxic effects of the produced compounds were testedwith several human cell lines. The prenylated products showedsignificantly higher cytotoxicity against these cell lines than the respectivenon-prenylated cyclic dipeptides. Therefore we provided additionalevidence that the prenylation is essential for the biological activity oftryprostatin analogues [5] .[1.] N. Steffan, A. Grundmann, W.-B. Yin, A. Kremer, S.-M. Li, Curr.Med.Chem.2009,16, 218-231.[2.] A. Grundmann, S.-M. Li, Microbiology 2005,151, 2199-2207.[3.] C. B. Cui, H. Kakeya, G. Okada, R. Onose, H. Osada, J.Antibiot. 1996,49, 527-533.[4.] L. Wang, W.-B. Yin, S.-M. Li, X.-Q. Liu,Chin. J.Biochem.Mol.Biol. 2009,25, 580-584.[5.] H. D. Jain, C. Zhang, S. Zhou, H. Zhou and others, Bioorg.Med.Chem. 2008,16, 4626-4651.MEP018Identification of PyrG1 as a glycosyltransferase involved in thebiosynthesis of pyrroindomycinsE.P. Patallo* 1 , K.H. van Pée 1 , A.F. Brana 2 , C.J. Moody 31 University, Biochemistry, Dresden, Germany2 University, Microbiology, Oviedo, Spain3 University, Nottingham, United KingdomStreptomyces rugosporus LL-42D005 produces pyrroindomycin A and itschlorinated derivative, pyrroindomycin B [1]. Pyrroindomycins are activeagainst Gram-positive bacteria such as methicillin-resistantStaphylococcus aureus and vancomycin-resistant Enterococci strains [2].Pyrroindomycins are related to other compounds containing a tetramic ortetronic acid moiety spiro-linked to a cyclohexene ring.Little is known about the biosynthesis of pyrroindomycins. Inpyrroindomycin B biosynthesis PyrH, a FADH 2-dependent tryptophan 5-halogenase, chlorinates tryptophan to yield 5-Cl-tryptophan the firstintermediate in the biosynthesis of a three-ring pyrroloindole structure. Nofurther information about the biosynthesis of pyrroindomycin B isavailable. We cloned around 30 kb of the pyrroindomycin biosyntheticgene cluster and we proposed the function of the ORFs we found. In orderto obtain information about the function of these putative genes,inactivation experiments were performed. A putative glycosyltransferasegene (pyrG1) was identified and a deletion mutant was constructed. Theresultant mutant strain Streptomyces rugosporus pyrG1 neither producespyrroindomycin A nor pyrroindomycin B anymore. Instead, a new maincompound with no pyrroindomycin UV-spectrum was detected. Isolation,purification and structure elucidation of the accumulated product allowedthe characterisation of this compound as the aglycon of the polyketidemoiety of pyrroindomycin A and B and provides first insight into thepyrroindomycin biosynthetic pathway.Ding et al.J. Antibiotics199447, 1250-1257Singh et al.J. Antibiotics199447, 1258-1265Zehner et al.Chem. Biol.200512, 445-52MEP019Prenylation of hydroxynaphthalenes and flavonoids by indoleprenyltransferases from fungiX. Yu* 1 , X. Xie 2 , S.-M. Li 11 Philipps-Universität Marburg, Institut für Pharmazeutische Biologie undBiotechnologie, Marburg, Germany2 Philipps-Universität Marburg, Fachbereich Chemie, Marburg, GermanyFungal indole prenyltransferases of the dimethylallyltryptophan synthase(DMATS) superfamily are involved in the biosynthesis of prenylatedindole alkaloids, and catalyze the prenylation of diverse indolederivatives.(1) These enzymes share no sequence, but structure similaritywith the prenyltransferases of the CloQ/NphB group, which acceptedhydroxynaphthalenes, 4-hydroxyphenylpyruvate, phenazine andflavonoids as substrates. We have demonstrated that some indoleprenyltransferases accepted also hydroxynaphthalenes and flavonoids assubstrates.(2,3) Nine prenylated flavonoids and twenty prenylatedhydroxynaphthalenes have been isolated, and their structures wereelucidated by MS and NMR analyses. It has been shown that, for anaccepted hydroxynaphthalene, different enzymes produced usually thesame major prenylated product, i.e. with a regular C-prenyl moiety atpara- or ortho- position to a hydroxyl group. For hydroxynaphthaleneswith low conversion rates and regioselectivity, O-prenylated anddiprenylated derivatives were also identified as enzyme products. Forflavonoids accepted by 7-DMATS, C-6 between two hydroxyl groups wasthe favorable prenylation position. The K M values and turnover numbers(k cat) of some prenyltransferases towards selected hydroxynaphthalenes,are comparable to those obtained by using indole derivatives. These resultsexpand the potential usage of prenyltransferases of the DMATSsuperfamily as catalysts for chemical synthesis, and meanwhile, increasethe structural diversity of prenylated compounds.1. Li, S.-M. (2010) Nat. Prod. Rep. 27, 57-782. Yu, X., Xie, X., and Li, S.-M. (2011) Appl. Microbiol. Biotechnol. 92, 737-7483. Yu, X. and Li, S.-M. (2011) Chembiochem 12, 2280-2283MEP020Ergot alkaloid gene cluster in the fungal family ofArthrodermataceaeC. Wallwey* 1 , C. Heddergott 2 , X. Xie 3 , A. Brakhage 2 , S.-M. Li 11 Philipps-Universität Marburg, Institut für Pharmazeutische Biologie undBiotechnologie, Marburg, Germany2 Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e.V., Jena,Germany3 Philipps-Universität Marburg, Fachbereich Chemie, Marburg, GermanyErgot alkaloids play an important role as pharmaceuticals as well as toxinsin food and feed industry.[1;2] Ergot alkaloids with a characteristictetracyclic ergoline ring can be divided into three groups: clavine-typealkaloids, ergoamides and ergopeptines.[1] Comparison of the gene clusterfor ergopeptines from Claviceps purpurea with those for clavine-typealkaloids from Aspergillus fumigatus and Penicillium commune revealedthe presence of seven orthologous/homologous genes, which werespeculated to be responsible for the formation of the ergoline system.Blasting genome sequences of different fungi with enzymes for ergotalkaloid biosynthesis, led to the identification of a putative ergot alkaloidgene cluster in fungi of the family Arthrodermataceae. The gene clusterconsists of five genes with clear sequence similarity to those assigned tothe early common steps of the ergot alkaloid biosynthesis, i.e. fromprenylation of tryptophan to formation of chanoclavine-I aldehyde, abranch point for clavine-type ergot alkaloid and ergopeptine biosynthesis.The homologous genes being responsible for the conversion ofchanoclavine-I aldehyde, i.e. fgaOx3 and fgaFS in A. fumigatus[3] or easGin C. purpurea[4], were not found in arthrodermataceous fungi, nor furthergenes in the biosynthesis of later special steps in both fungi.The function of one gene ChaDH, coding a chanoclavine-I dehydrogenase,was proven by gene cloning, expression and biochemical characterizationof the overproduced enzyme. NMR and MS analyses of the isolatedBIOspektrum | Tagungsband 2012