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

98MEP025Regulation of pristinamycin biosynthesis in S. PristinaespiralisJ. Guezguez*, Y. Mast, E. SchinkoIMIT, Microbiology/Biotechnology, Tübingen, GermanyThe streptogramin antibiotic pristinamycin, produced by Streptomycespristinaespiralis, is a mixture of two types of chemically unrelatedcompounds: pristinamycin PI and PII, which are produced in a ratio of30:70. Pristinamycin PI is a cyclic hexadepsipeptide, belonging to the B-group of streptogramins, while pristinamycin PII has the structure of apolyunsaturated macrolactone of the A-group of streptogramins. Bothcompounds alone inhibit the protein biosynthesis by binding to thepeptidyl transferase domain of the 50S subunit of the ribosome and arebacteriostatic. The A-group prevents the binding of the aminoacyl-tRNAto the 50S subunit of the ribosome. In contrast, the B-group facilitates therelease of the peptidyl-tRNA from the ribosome. Together they show astrong synergistic bactericidal activity, which can reach 100 times of theseparate components. The pristinamycin biosynthetic gene cluster ischaracterized. It covers a region of about 210 kb where genes for PI andPII biosynthesis are interspersed. Moreover, the pristinamycin codingregion is interrupted by a cryptic secondary metabolite gene cluster whichprobably encodes for an actinorhodin-like compound. Seven regulatorygenes were identified within the 210 kb region:spbR, papR1, papR2,papR3, papR4, papR5 and papR6. SpbR (S.pristinaespiralisbutyrolactoneresponsivetranscriptional repressor) is a specific receptor protein for -butyrolactones and the global regulator of pristinamycin biosynthesis.papR1, papR2 and papR4 encode proteins that are homologous to SARPswhich are pathway-specific transcriptional activator proteins, whereaspapR3 and papR5 code both for proteins that belong to the family of TetRrepressors. papR6 encodes a protein belonging to the class of responseregulators. On the basis of RT-PCR, bandshift and mutant analysis, apreliminary model of the regulation mechanism of pristinamycinbiosynthesis was established.Mast YJ, Wohlleben W, Schinko E.Identification and functional characterization of phenylglycinebiosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis.J Biotechnol.2010 Dec 10Mast Y, Weber T, Gölz M, Ort-Winklbauer R, Gondran A, Wohlleben W, Schinko E.Characterization of the'pristinamycin supercluster' of Streptomyces pristinaespiralis.Microb Biotechnol. 2011 Oct 15MEP026Activation of a silent phenazine biosynthetic gene cluster fromStreptomyces reveals a novel phenazine conjugateO. Saleh 1 , T. Bonitz* 1 , A. Kulik 2 , N. Burkard 3 , A. Mühlenweg 4 , A. Vente 4 ,S. Polnick 1 , M. Lämmerhofer 1 , B. Gust 1 , H.-P. Fiedler 2 , L. Heide 11 University of Tübingen, Pharmaceutical Institute, Tübingen, Germany2 University of Tübingen, Faculty of Biology, Tübingen, Germany3 University of Tübingen, Institute for Organic Chemistry, Tübingen, Germany4 MerLion Pharmaceuticals GmbH, Berlin, GermanyThe activation of silent biosynthetic gene clusters is a principal challengefor genome mining strategies in drug discovery. In the present study, aphenazine biosynthetic gene cluster was discovered in the Gram-positivebacterium Streptomyces tendae Tü1028. This gene cluster remained silentunder a multitude of cultivation conditions, both in the genuine producerstrain and in a heterologous expression strain. However, introduction of aconstitutive promoter upstream of the phenazine biosynthesis genes led tothe production of phenazine-1-carboxylic acid (PCA) and of a newderivative thereof, i.e. a conjugate of PCA and L-glutamine. The linkage ofPCA to L-glutamine by amide bond formation was catalyzed by enzymesof the heterologous expression host Streptomyces coelicolor M512 andmay represent a detoxification mechanism. The gene cluster also containedgenes for all enzymes of the mevalonate pathway and for an aromaticprenyltransferase, thereby resembling gene clusters for prenylatedphenazines. However, purification and biochemical investigation of theprenyltransferase proved that it does not prenylate phenazines buthydroxynaphthalene substrates, showing very similar properties as NphBof naphterpin biosynthesis (Kuzuyma et al., Nature 2005; 435: 983-7).MEP027Genetical analysis of the biosynthesis and zinc-regulation of[S,S]-EDDS, a biodegradable EDTA alternative produced byAmycolatopsis japonicumM. SpohnInterfakultäres Institut für Mikrobiologie und Infektionsmedizin,Mikrobiologie/Biotechnologie, Tübingen, GermanyEDDS (Ethylene-diamine-disuccinic acid) produced by Amycolatopsisjaponicum is a suitable biodegradable alternative for the syntheticchelating agent EDTA, which has become the highest concentrated wastecompound in surface waters.EDDS is isomeric with EDTA and has similar properties. But in contrast toEDTA it contains two asymmetric carbon atoms, resulting in the existenceof three optical isomers, [S,S]-EDDS, [R,R]-EDDS and [R,S]-EDDS. A.japonicum produces the biodegradable S,S-configuration of EDDS.The biosynthesis of EDDS in A. japonicum is strictly zinc regulated. Azinc concentration of 5 M represses the production of EDDS at any timeof the fermentation [CEBULLA, 1995].In a hypothetical EDDS biosynthesis pathway oxalacetate and theaproteinogenic aminoacid diaminopropionic acid (DAP) are covalentlybonded to form an intermediate which is subsequently processed in severalsteps to finally form [S,S]-EDDS [CEBULLA, 1995]. DAP is also used asa building block in other secondary metabolites with elucidatedbiosynthesis pathway like zwittermicin A and staphyloferrin B [ZHAO, 2008;CHEUNG, 2009]. Genetic screening in A. japonicum using the sequenceencoding the DAP-synthesizing enzymes resulted in the identification of a generegion encoding putative EDDS-biosynthesis-enzymes.To confirm their involvement in the EDDS biosynthesis we compared theirtranscription patterns of A. japonicum cultures grown in zinc-containing(none EDDS production) and zinc-free (EDDS production) media. Theputative DAP-biosynthesis genes are only expressed under EDDSproduction conditions and are strictly repressed only by zinc and no otherdivalent metal ion.By directed mutagenesis and heterologous expression we want to evidencethe responsibility of these zinc-repressed genes for the EDDS production.CEBULLA, I. (1995). Gewinnung komplexbildender Substanzen mittels Amycolatopsis orientalis.Dissertation, Universität Tübingen.CHEUNG, J; BEASLEY, F; LIU, S; LAJOIE, G AND HENRICHS, D (2009). Molecular charakterizationof staphyloferrin B biosynthesis in Staphylococcus aureus. Molecular Microbiology 74(3); 594-608.ZHAO, C; SONG, C; LUO, Y; YU, Z AND SUN, M. (2008). L-2,3-Diaminopropionate: One of thebuilding blocks for the biosynthesis of Zwittermicin A in Bacillus thuringensis susp. kurstaki strain YBT-1520. FEBS Letters 582; 3125-3131.MEP028Analysis of the biosynthesis of astins from Aster tataricus andcyclochlorotine from Penicillium islandicumL. Flor*, K.-H. van PéeTU Dresden, Biochemistry, Dresden, GermanyAstins are cyclic pentapeptides isolated from roots of the plant Astertataricus.The root extract shows potent anti-tumour activity in mouse tests(1). However, the amounts of astins that can be isolated from plants arevery low and chemical synthesis is accompanied by negative impacts onthe environment. Therefore, the project ‚Multi enzyme systems involved inastin biosynthesis and their use in heterologous astin production(MESIAB)‘ aims at enhancing the production of astins using moleculargenetic tools. So far, astins A-J are known. Cyclochlorotine, a secondarymetabolite with high similarity to astins, has been isolated from the fungusPenicillium islandicum. Cyclochlorotine is a hepatotoxic compoundcausing necrosis, vacuolation of liver cells and development of blood lakes(2). Because of the high similarity of the peptides (3), similar enzymesshould be involved in the biosynthetic pathways of astins andcyclochlorotine. Both metabolites contain a dichlorinated pyrrolecarboxylic acid derivative which is most likely derived from proline. It isassumed that chlorination occurs on the level of a peptide carrier proteintethered pyrrol carboxylic acid moiety by a flavin-dependent halogenase.The anticarcinogenic activity of astins relies on the cyclic peptide and onthe chlorinated proline residue (4,5). So far, neither a flavin-dependenthalogenase nor nonribosomal peptide synthethases have been described inplants. Via HPLC-MS from extracts of dry roots of Aster tataricus alltypes of astins could be detected, as well as cyclochlorotine from culturemedia of P. islandicum. For genetic analysis we are in the process ofsequencing the genome of P. islandicum and constructing cDNA-librariesfor A. tataricus and P. islandicum.(1) Morita et al. (1995) Tetrahedron, 51, 4, 1121-1132(2) Ghoh et al. (1978) App. Environ. Microb., 35, 6, 1074-1078(3) Schumacher et al. (1999) Tet. Letters, 40, 455-458(4) Saviano et al., 2004, Biopolymers, 76, 6, 477-84(5) Cozzolino et al. (2005) Carcinogenesis, 26, 733-739MEP029Secondary metabolism and morphogenesis in the penicillinproducer Penicillium chrysogenum is regulated by the velvet-likecomplexS. Bloemendal*, B. Hoff, K. Kopke, A. Katschorowski, S. Milbredt,J. Kamerewerd, U. KückRuhr-Universität Bochum, Christian Doppler Labor für "Biotechnologieder Pilze", Bochum, GermanyThe recent discovery of a velvet complex containing several globalregulators of secondary metabolism in the model fungus Aspergillusnidulans [1,2] raises the question whether similar type complexes directfungal development and secondary metabolism in genera other thanAspergillus. The filamentous fungus Penicillium chrysogenum is the mainindustrial producer of the pharmaceutically relevant beta-lactam antibioticpenicillin. All three biosynthesis genes are found in a single cluster and theexpression of these genes is known to be controlled by a complex networkof global regulators.Here we provide a functional analysis of a velvet-like complex in a P.chrysogenum producer strain that underwent several rounds of UVmutagenesis during a strain improvement program [3,4]. This complexBIOspektrum | Tagungsband 2012