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

102Knoll, C., du Toit, M., Schnell, S., Rauhut, D., Irmler, S., 2011. Cloning and characterisation of acystathionine /g-lyase from two Oenococcus oeni oenological strains. Applied Microbiology andBiotechnology, 89, 1051-1060Pripis-Nicolau, L., Revel, G., Bertrand, A. and Lonvaud-Funel, A. (2004) Methionine catabolism andproduction of volatile sulphur compounds by Oenococcus oeni. J Appl Microbiol 96 (5): 1176-1184MEP042Strategies for the recombinant production of the cyclicdepsipeptide valinomycin in Escherichia coliJ. Jaitzig* 1 , J. Li 1 , R. Süssmuth 2 , P. Neubauer 11 Technische Universität Berlin, Institut für Biotechnologie, Berlin, Germany2 Technische Universität Berlin, Institut für Chemie, Berlin, GermanyThe natural pool of biologically active nonribosomal peptides (NRPs) frombacteria and fungi is vast but still largely untapped. Reasons are thestructural complexity of NRPs that impedes chemical synthesis and thepoor cultivability of the majority of source organisms. Since nonribosomalpeptide synthetases (NRPSs) assemble NRPs from simple building blocks,the heterologous expression of NRPSs in a robust and easy to manipulateexpression host like Escherichia coli is a desirable strategy to makepharmaceutically relevant NRPs more accessible (1). However, their largesize and complexity make recombinant expression of soluble and activeNRPSs in E. coli a bottleneck.Valinomycin is a bioactive cyclodepsipeptide formed by the two NRPSs,Vlm1 (370 kDa) and Vlm2 (284 kDa) in Streptomyces tsusimaensis (2). Inorder to establish a recombinant production system for valinomycin in E.coli and further characterize the valinomycin biosynthesis, the two vlmgenes were isolated from the genomic DNA of S. tsusimaensis andintroduced into various expression vectors via parallel recombinationalcloning. A rational expression screening in 24- and 96-well plates wasperformed to test the expression constructs and relevant cultivationparameters in parallel. Correct folding and activity of the enzymes wereassayed in vitro after purification. To provide the necessaryposttranslational phosphopantetheinylation of valinomycin synthetase thesfp gene from Bacillus subtilis was genomically integrated into the targetE. coli expression strain.We could show that with a high-throughput screening and optimizationapproach even the large, initially poorly expressed, heterodimericvalinomycin synthetase could be expressed soluble in E. coli. In vitroactivity studies of the four adenylation domains gave information onsubstrate specificities and experimentally confirmed the postulated modeof action of the valinomycin biosynthetic assembly line (3). Finally,valinomycin formation was achieved by co-expressing Vlm1 and Vlm2 inan engineered E. coli strain with genomically integrated B. subtilis sfp.This paves the way to tailor the enzymatic assembly line in order toproduce nonnatural valinomycin derivatives.1. H. Zhang, B. A. Boghigian, J. Armando, B. A. Pfeifer, Nat Prod Rep 28, 125 (2011).2. Y. Q. Cheng, ChemBioChem 7, 471 (2006).3. N. A. Magarvey, M. Ehling-Schulz, C. T. Walsh, J Am Chem Soc 128, 10698 (2006).MEP043Genomic mining for novel FADH2-dependent halogenases inmarine sponge-associated microbial consortiaK. Bayer*, M. Scheuermayer, U. HentschelUniversity of Wuerzburg, Botany 2, Wuerzburg, GermanyMany marine sponges (Porifera) are known to contain large amounts ofphylogenetically diverse microorganisms. Sponges are also known fortheir large arsenal of natural products many of which are halogenated. Inthis study, 36 different FADH2-dependent halogenase gene fragmentswere amplified from various Caribbean and Mediterranean sponges usingnewly designed degenerate PCR primers. Four unique halogenase-positivefosmid clones, all containing the highly conserved amino acid motif“GxGxxG”, were identified in the microbial metagenome of Aplysinaaerophoba. Sequence analysis of one halogenase-bearing fosmid revealednotably two ORFs with high homologies to efflux and multidrug resistanceproteins. Single cell genomic analysis allowed for a taxonomic assignmentof the halogenase genes to specific symbiotic lineages. Specifically, thehalogenase cluster S1 is predicted to be produced by a deltaproteobacterialsymbiont and halogenase cluster S2 by a poribacterial sponge symbiont.An additional halogenase gene is possibly produced by an actinobacterialsymbiont of marine sponges. The identification of three novel,phylogenetically and possibly also functionally distinct halogenase geneclusters indicates that the microbial consortia of sponges are a valuableresource for novel enzymes involved in halogenation reactions.MEP044Heterologous Expression of the Lantibiotic Lichenicidin in E.coli and Generation of New Congeners by Introducing Non-Natural Amino AcidsF. Oldach* 1 , T. Caetano 2 , A. Kuthning 1 , R. Al Toma 1 , J.M. Krawczyk 1 ,E. Mösker 1 , N. Budisa 1 , S. Mendo 2 , R.D. Süssmuth 11 Technische Universität Berlin, Institut für Chemie, Berlin, Germany2 University of Aveiro, Department of Biology and CESAM, Aveiro,PortugalLantibiotics are a family of ribosomally synthesized peptide antibiotics,produced by various bacteria. Subsequent to their synthesis lantibiotics areposttranslationally modified. Thereby the thioether-containing amino acidslanthionine (Lan) and methyllanthionine (MeLan) are formed fromSer/Cys and Thr/Cys, respectively [1]. The class I and II lantibioticsexhibit antimicrobial activity against a large number of Gram-positivebacteria, e.g. Staphylococcus aureus, including MRSA [2], while class IIIlantibiotics have no antimicrobial effects and display other remarkablebioactivities, e.g. pain-suppression in mice [3].The class II-lantibiotic Lichenicidin, produced by the Gram-positiveBacillus licheniformis is composed of the two subunits Bli and Bli thatare synthetized as an inactive prepropeptide (LicA1, LicA2). The peptideis further modified by LicM1 (for LicA1) and LicM2 (for LicA2),exported by LicT and, in the case of Bli, it is cleaved by the proteaseLicP [4]. We developed a system that enabled us to successfully expressthe biosynthetic genes of Lichenicidin in the Gram-negative hostEscherichia coli [5].In order to generate novel structural diversity, we used this powerful toolfor genetic code engineering and incorporation of noncanonical aminoacids (ncAA). The possibility to express Lichenicidin variants in E. coliprovides the opportunity for novel Lantibiotics engineering. Ultimately,this will yield novel lantibiotics with new bioactivities due to dramaticallyincreased structural diversity [6].1. (a) G. Jung Angew. Chem. Int. Ed. Engl. 1991, 30, 1051 - 1068. (b) C. Chatterjee, M. Paul, L. Xie, W. A.van der Donk Chem. Rev. 2005,105, 633 - 683.2. H.-G. Sahl, G. Bierbaum Annu. Rev. Microbiol. 1998, 52, 41 - 79.3. K. Meindl, T. Schmiederer, K. Schneider, A. Reicke, D. Butz, S. Keller, H. Gühring, L. Vértesy, J. Wink,H. Hoffmann, M. Brönstrup, G. M. Sheldrick, R. D. Süssmuth Angew. Chem. Int. Ed. 2010, 49, 1151 - 1154.4. M. Begley, P. D. Cotter, C. Hill, R. P. Ross Appl. Environ. Microbiol. 2009, 75, 5451 - 5460.5. T. Caetano, J. M. Krawczyk, E. Mösker, R. D. Süssmuth, S. Mendo Chem. Biol. 2011, 18, 90 - 100.6. F. Oldach, R. Al Toma, A. Kuthning, T. Caetano, S. Mendo, N. Budisa , R. D. Süssmuth Angew. Chem.Int. Ed. 2011, in press.MEP045Characterization of new type-III lantibioticsG.H. Völler*, J.M. Krawczyk, A. Pesic, R.D. SüssmuthTechnische Universität Berlin, Institut für Chemie , Berlin, GermanyLantibiotics are a large group of ribosomally synthesized peptidescontaining the amino acid lanthionine [1]. They are mainly synthesized byBacilli, Staphylococci, Lactococci and Actinomycetes, and are classifiedaccording to their gene cluster, their biosynthetic pathway and theirbioactivity into three major subtypes. From type-III lantibiotics producedby Actinomycetes only four peptides (SapB, SapT and LabA1/A2) havebeen structurally characterized although homologous gene clusters areabundant in other Actinomycetes [2,3,4,5].All these gene clusters share a similar architecture with all of the encodedprepropeptides containing a characteristic Ser/Ser/Cys motif, which haspreviously been suggested to act as a precursor of the lanthionine andlabionin ring, respectively [4,5]. We report on the detection, analytics andcharacterization of new type-III lantibiotics. Remarkably, according to ourpreliminary findings, the new type III-lantibiotics all contain preferably theamino acid labionin. We assume, that these findings have implications forthe structures of other type III lantibiotics, [2,3] and suggest that type-IIIlantibiotics are more abundant than anticipated previously.1. G. Jung, Angew. Chem. 1991, 30(9), 1051-11922. S. Kodani, M.E. Hudson, M.C. Durrant, M.J. Buttner, J.R. Nodwell, J. M. Willey, Proc. Natl. Acad. Sci. US A 2004, 101, 11448-11453.3. S. Kodani, M.A. Lodato, M.C Durrant, F. Picart, J.M. Willey, Mol. Microbiol. 2005, 58, 1368-13804. W. M. Müller, T. Schmiederer, P. Ensle, R. D. Süssmuth, Angew. Chem. Int. Ed. 2010, 122, 2486 -2490.5. K. Meindl, T. Schmiederer, K. Schneider, A. Reicke, D. Butz, S. Keller, H. Gühring, L. Vértesy, J. Wink,H. Hoffmann, M. Brönstrup, G. M. Sheldrick, and R. D. Süssmuth, Angew. Chem. Int. Ed. 2010, 49, 1151-1154.BIOspektrum | Tagungsband 2012