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

91MEV007Purification and Characterisation of the Flavin-DependentMonodechloroaminopyrrolnitrin 3-Halogenase fromPyrrolnitrin BiosynthesisA. Adam*, K.-H. van PéeTU Dresden, Institut für Biochemie, Dresden, GermanyPyrrolnitrin is an antifungal compound first isolated from Pseudomonaspyrrocinia. The gene cluster and the corresponding enzymes responsiblefor pyrrolnitrin biosynthesis were identified in Pseudomonas fluorescens(BL915) and other pyrrolnitrin producing bacteria. The third enzyme,monodechloroaminopyrrolnitrin (MCAP) 3-halogenase (PrnC), catalysesthe regioselective chlorination of MCAP in the 3-position of the pyrrolering. PrnC is a flavin-dependent halogenase and its reaction mechanism issuggested to be very similar to that of other flavin-dependent halogenases.However, the amino acid sequence shows hardly any similarity to theamino acid sequence of tryptophan halogenases for some of which threedimensionalstructures are known.Additionally, PrnC is, besides the tryptophan halogenases, the only knownflavin-dependent halogenase that catalyses the halogenation of a freesubstrate. Other known flavin-dependent halogenases catalysing thechlorination of a pyrrole moiety act on a substrate bound to a peptidylcarrier protein making elucidation of the 3-D structure of these enzymes,especially in the presence of substrate, very difficult. Thus, the 3-Dstructure of PrnC is of high importance to understand how substratespecificity and regioselectivity are regulated in flavin-dependenthalogenases.So far, purification of PrnC in its active form has not been achievedsatisfactorily which is partially due to the incompatibility of the tags usedand the high tendency of PrnC to form aggregates with itself and otherproteins. The lack of purified PrnC so far prevented further detailedanalysis of the enzyme. Here we report a novel purification strategyleading to purified, active PrnC. Using the GST-fusion protein strategy it ispossible to obtain homgeneous PrnC from recombinant Escherichia colicells. The purity level of the eluted fusion protein highly depends on thegrowth temperature of the E. coli strain used for expression. MALDI-TOF-MS analysis revealed that the chaperonin GroEL and other proteins are copurifiedby glutathione affinity chromatography when the growthtemperature is not reduced to 20 °C. Dilution of the crude extract as wellas the addition of Tween-20 has a high impact on the affinity of the fusionprotein to the glutathione column. The addition of detergent does notinhibit halogenating activity, neither that of the fusion protein nor that ofthe purified PrnC after thrombin digestion.Furthermore we can now report on first characterisation results giving firsthints towards the reaction mechanism of PrnC.van Pée, K. H. and Ligon, J. M., Nat. Prod. Rep., 2000, 17, 157.Hammer, P. E., Hill, D. S., Lam, S. T., van Pée, K. H., Ligon, J. M., Appl. Environ. Microbiol., 1997, 63,2147.Dorrestein, P. C., Yeh, E., Garneau-Tsodikova, S., Kelleher, N. L., Walsh, C. T., PNAS, 2005, 39, 13843.MEV008EasG and FgaFS are key enzymes in the differentiation ofergot alkaloid biosynthesis in Claviceps purpurea andAspergillus fumigatusM. Matuschek* 1 , C. Wallwey 1 , X. Xie 2 , S.-M. Li 11 Philipps-Universität Marburg, Institut für Pharmazeutische Biologie undBiotechnologie, Marburg, Germany2 Philipps-Universität Marburg, Fachbereich Chemie, Marburg, GermanyErgot alkaloids are secondary metabolites belonging to indole derivativesand are produced by a wide range of fungi with Claviceps purpurea as themost important producer for medical use. They show a broad spectrum ofbiological activities and their toxic effects were reported back to themiddle ages. The early steps of ergot alkaloid biosynthesis are shared byC. purpurea and Aspergillus fumigatus, whereas later steps differ in thetwo fungi.[1] Chanoclavine-I aldehyde[2] was proposed as branch pointfor the biosynthesis in both fungi,[3] which is converted in A. fumigatus tothe clavine-type alkaloid festuclavine by the festuclavine synthase FgaFSin the presence of the old yellow enzyme FgaOx3.[1] In C. purpureachanoclavine-I aldehyde is converted to agroclavine by EasG in thepresence of reduced glutathione without a requirement of EasA.[4] Theenzymes were purified by affinity chromatography after overproduction inE. coli and characterized biochemically. The in vitro results for theformation of festuclavine catalysed by FgaOx3 and FgaFS proved tworeduction steps. In contrast, agroclavine differs from festuclavine by adouble bond between C8 and C9. Therefore only one reduction, butadditionally an isomerisation step is necessary. We have shown that EasGwas responsible for the reduction step and a non-enzymatic adduct withreduced glutathione for the isomerisation. The structures of festuclavineand agroclavine were unequivocally elucidated by NMR and MS analyses.In summary, EasG and FgaFS are the key enzymes controlling the branchpoint of ergot alkaloid biosynthesis in C. purpurea and A. fumigatus.[1.] C. Wallwey, M. Matuschek, X.-L. Xie, S.-M. Li, Org. Biomol. Chem. 2010, 8, 3500-3508.[2.] C. Wallwey, M. Matuschek, S.-M. Li, Arch. Microbiol. 2010, 192, 127-134.[3.] C. M. Coyle, J. Z. Cheng, S. E. O'Connor, D. G. Panaccione, Appl. Environ. Microbiol. 2010, 76, 3898-3903.[4.] M. Matuschek, C. Wallwey, X. Xie, S. M. Li, Org. Biomol. Chem. 2011, 9, 4328-4335.MEV009Biosynthesis of Cephlaosporin C Through Improved strains ofAspergillus and Acremonium speciesZ.-E. Bilal*, A. YousafInstitute of Agricultural sciences, University of the Punjab, Lahore,Pakistan., Institute of Agricultural sciences, University of the Punjab,Lahore, Pakistan., Lahore, PakistanAntibiotics are secondary metabolites produced by microorganisms,extremely important to the health of our society. Cephalosporins are broadspectrumantibiotics which are very similar in structure and action topenicillins but more resistant to -lactamases. Optimization of media isuseful to increase the production of antibiotics. Induction of mutation iscommonly employed to increase the yield of secondary metabolites likeantibiotics. Chemical mutation is preferred method because of the ease inhandling and avoiding the hazardous effects of radiations.Monitoring the concentrations of antibiotics and their precursors isrequired for their optimized production. Due to higher concentrations ofproteins and other liquid phases in fermented broth aseptic sampling is adifficult task. Spectrophotometeric analysis can be used for the estimationof the antibiotic produced by microbes. Bioassay analysis can be done toconfirm the antibiotic activity of the antibiotic produced. HPLC is used todifferentiate the specific antibiotic from other secondary metabolites ofmicrobes from the fermented broth.The aim of this research work was to optimize media conditions andimprovement of fungal strains through chemical mutation for enhancedCephalosporin C (CPC) production by Aspergillus and Acremonium species.For media optimization different concentrations of media contents wereanalyzed for increased production. Best results were shown byfermentation media supplemented with sucrose 30mg/ml. While DLmethionineshows optimum yield at 3mg/ml; and fermentation mediasupplemented with ammonium sulphate 7.5mg/ml as nitrogen sources gavemaximum yield.For mutation induction fungal strains were treated with 400g/ml of Ethylmethane sulfonate (EMS) for 30-80 minutes. It was observed that time ofchemical treatment was inversely proportional to the survival of fungalstrains; minimum survival rate was obtained at treatment of 1 hour.The mutants were then further analyzed for CPC production withoptimized media conditions in similar way as done earlier before inductionof mutation. Results obtained showed that very small increase in CPCproduction, in few fungal strains but not in all. This might be due toinsufficient mutation for target genes (i.e., involved in CPC production) inremaining fungal species.The spectrophotometeric and HPLC analysis of fermented broths wereperformed to analyze CPC yields. Similarities were observed in the resultsof both analyses. On spectrophotometeric and HPLC analysis, beforemutation maximum yield of CPC (2.583 and 0. 254mg/ml respectively)was obtained with Acremonium kiliense FCBP # 162, respectively andafter mutation maximum production of CPC (2.346, 0.24mg/mlrespectively) was achieved with Acremonium furcatum FCBP # 409.The bioassay analysis of the fungal strains and survivors (mutants) wereperformed to confirm antibacterial activity of CPC. The increasedantibacterial activity was observed for some strains after mutation, forothers it was decreased and it remained constant for remaining strains.MEV010Systems biology of the marine antibiotic producer PhaeobactergallaeciensisA. Klingner*, A. Bartsch, J. Becker, C. WittmannInstitut für Bioverfahrenstechnik, TU Braunschweig, Braunschweig, GermanyMarine bacteria gain more interest since it is assumed that among thesebacteria is a great potential of secondary metabolites which may be ofindustrial or medical interest. The Roseobacter clade is one of the mostprevalent marine microorganisms, which are highly distributed in theoceans [1]. Many new species were found in the last few years and a richrepertoire of metabolic pathways has been identified. However, there islittle information about the in vivo use in the marine environment [2, 3].Phaeobacter gallaeciensis has the ability to produce a new interestingantibiotic, the tropoditiethic acid (TDA). Furthermore, other secondarymetabolites, so called “Roseobacticides”, were found, which inhibit thegrowth of diverse marine algae and bacteria [4, 5]. This makes thebacterium interesting for studies in systems biology, to developoptimization strategies and enhance the secondary metabolite production.In this work P. gallaeciensisis investigated by systems wide metabolic fluxanalysis using 13 C-labelling studies and computational flux modelling withthe software OpenFlux [6]. This provides a first insight into the in vivo useof its pathways. The first set of experiments focussed on the impact ofdifferent carbon sources. Together with transcriptome profiling this willBIOspektrum | Tagungsband 2012