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

92provide an insight into the regulation network in order to approach thecomplete picture of the cell.First labelling studies showed that glucose is metabolized only via theEntner-Doudoroff pathway. This is an extremely unusual flux distributionand seems to be characteristic for the Roseobacter clade. In this study theimpact of nutritional changes on the flux distribution was investigated byperforming the first systems wide metabolic flux analyses.Acknowledgements: The work is funded by the German Research Foundationwithin the subproject C4 in the SFB TRR51 “Ecology, Physiology andMolecular Biology of the Roseobacter clade: Towards a Systems BiologyUnderstanding of a Globally Important Clade of Marine Bacteria”.[1] Buchan A, González JM, Moran MA (2005), Appl Environ Microbol, 71(10): 5665-5677[2] Wagner-Döbler I, Ballhausen B, et al. (2010) ISME J, 4: 61-77[3] Fürch T, Preusse M, et al. (2009), BMC Microbiology, 9: 209[4] Seyedsayamdost, M. R., G. Carr, et al. (2011), J Am Chem Soc.[5] Martens T, Heidorn T, et al. (2006), Int J Syst Evol Microbiol, 56(6): 1293[6] Quek, L. E., C. Wittmann, et al. (2009), Microb Cell Fact 8: 25.MEV011Characterization and manipulation of the biosyntheticpathway of cyanobacterial tricyclic microviridins in E. coliA.R. Weiz* 1 , K. Ishida 2 , K. Makower 1 , N. Ziemert 3 , C. Hertweck 2 , E. Dittmann 11 Institute of Biochemistry and Biology, Microbiology, Golm, Germany2 Leibniz Institute for Natural Product Research and Infection Biology, Jena,Germany3 Scripps Institution of Oceanography, San Diego, United StatesCyanobacteria are a structurally diverse group of bacteria, making avariety of biochemically active natural products using mostly thenonribosomal machinery of large multienzyme complexes. Microviridinsare the largest known cyanobacterial oligopeptides synthesized through aunique ribosomal route (1). The unprecedented microviridin gene clusterencodes for a precursor peptide (MdnA), two novel ATP-grasp ligases(MdnB and C), a GNAT-type acetyltransferase (MdnD) and an ABCtransporter(MdnE).Microviridins comprise an unrivaled multicyclic cagelikearchitecture, carrying characteristic -ester and a secondary -amidebond. They are produced by different isolates of cyanobacteria, includingthe unicellular, bloom-forming freshwater cyanobacteriumMicrocystisaeruginosaNies843. The serine protease inhibitory activity contributes toboth ecological and pharmacological relevance of microviridins. Here wereport the construction of a stable expression platform for heterologousexpression of microviridins inE. coli. Biostatistics and mutational analysisidentified the conserved PFFARFL motif in the precursor peptide as arecognition sequence for the ATP-grasp ligases. Manipulations of the C-terminal part of the leader peptide abolished lactam ring formation ofmicroviridins.The ABC-transporter MdnE was unveiled to be crucial forcyclization and processing of microviridins, probably holding andstabilizing a putative microviridin maturation complex at the innermembrane (2). Site-directed mutagenesis in the microviridin core sequenceshowed flexibility of the microviridin biosynthetic pathway to be used forpeptide engineering. We determined residues that are important for theprotease inhibition and are currently in process to optimize the product fordifferent pharmaceutical targets. Furthermore, we developed a method toexpress cryptic microviridin precursor peptides from field and lab samples.1. Ziemert, N., K. Ishida, A. Liaimer, C. Hertweck and E. Dittmann. Angew Chem Int EdEngl47(40), 2008, p. 7756-9.2. Weiz, Annika R., K. Ishida, K. Makower, N. Ziemert, C. Hertweck and E. Dittmann. Chemistry& Biology18(11), 2011, p. 1413-1421.MEV012Evaluation of Streptomyces coelicolor as a heterologous expressionhost for natural products from marine filamentous cyanobacteriaA. Jones* 1,2 , S. Ottilie 2 , A. Eustáquio 2 , D. Edwards 3 , L. Gerwick 2 ,B. Moore 2 , W. Gerwick 21 Universität Tübingen, Pharmazeutische Biologie, Tübingen, Germany2 University of California San Diego, Scripps Institution of Oceanography,La Jolla, CA, USA, United States3 California State University , Chico, CA, USA, United StatesFilamentous marine cyanobacteria are rich sources of bioactive naturalproducts and employ highly unusual biosynthetic enzymes in theirassembly. However, the current lack of techniques for stable DNA transferinto these filamentous organisms combined with the absence ofheterologous expression strategies for non-ribosomal cyanobacterial geneclusters prohibit the creation of mutant strains or the heterologousproduction of these cyanobacterial compounds in other bacteria. In thisstudy, we evaluated the capability of a derivative of the modelactinomycete Streptomyces coelicolor A3(2) to express enzymes involvedin the biosynthesis of the protein kinase C activator lyngbyatoxin A from aHawaiian strain of Moorea producta (previously classified as Lyngbyamajuscula). Despite large differences in GC content between these twobacteria and the presence of multiple TTA/UUA leucine codons inlyngbyatoxin open reading frames, we were able to achieve expression ofLtxB and LtxC in S. coelicolor M512 and confirmed the in vitrofunctionality of S. coelicolor overexpressed LtxC. Attempts to express theentire lyngbyatoxin A gene cluster in S. coelicolor M512 were notsuccessful because of transcript termination observed for the ltxA gene,which encodes a large non-ribosomal peptide synthetase. However, theseattempts did show a detectable level of cyanobacterial promoterrecognition in Streptomyces. Successful Streptomyces expression ofbiosynthetic enzymes from marine cyanobacteria provides a new platformfor biochemical investigation of these proteins and a promising avenue forcombinatorial biosynthesis between these two bacterial phyla.MEP001Endophytic fungi, the microbial factories of associated plantsecondary metabolites: Camptothecin as an exampleS. Kusari*, M. SpitellerTU Dortmund, Institute of Environmental Research (INFU) of the Facultyof Chemistry, Dortmund, GermanyEndophytic fungi inhabit healthy tissues of plants and occasionallyproduce associated plant secondary metabolites [1-5]. We recently isolatedan endophytic fungus, Fusarium solani from the bark of Camptothecaacuminata, which is capable of producing the anticancer pro-drugcamptothecin (CPT) and two structural analogues in axenic monoculture[6]. We deciphered a cross-species biosynthetic pathway where theendophyte utilizes indigenous geraniol 10-hydroxylase, secologaninsynthase, and tryptophan decarboxylase to biosynthesize CPT precursors.However, to complete CPT biosynthesis, it requires the host strictosidinesynthase [7]. The fungal CPT biosynthetic genes destabilized ex plantaover successive subculture generations. The seventh subculture predictedproteins exhibited reduced homologies to the original enzymes provingthat such genomic instability leads to dysfunction at the amino acid level.The endophyte with an impaired CPT biosynthetic capability wasartificially inoculated into the living host plants and then recovered aftercolonization. CPT biosynthesis could still not be restored [7]. We furtherdiscovered the survival strategy of this endophyte by identifying typicalamino acid residues in the CPT-binding and catalytic domains of itstopoisomerase I [8]. Recently, it was also revealed that chrysomelidbeetles (Kanarella unicolor) feeds on the leaves of CPT-containing N.nimmoniana without any apparent adverse effect [9]. We thus envisageaddressing the following open questions: why and how do endophytesproduce plant bioactive compounds? What are the diverse interactions thatendophytes have with other coexisting endophytes, host plants, insects,and specific herbivores? Elucidating these connections can not onlyenhance the understanding of evolution of complex defense mechanisms inplants and associated organisms, but also help in the sustained productionof plant compounds using endophytes harbored within them.[1] Kusari, S. & Spiteller, M. (2011). Nat. Prod. Rep. 28, 1203-1207.[2] Kusari, S. & Spiteller, M. (2010). In Biotechnology - Its Growing Dimensions. Sonali Publications, NewDelhi, India, pp. 1-27.[3] Kusari, S., Lamshöft, M., Spiteller, M. (2009). J. Appl. Microbiol. 107, 1019-1030.[4] Kusari, S., Zühlke, S., Kosuth, J., Cellarova, E. & Spiteller, M. (2009). J. Nat. Prod. 72, 1825-1835.[5] Kusari, S., Lamshöft, M., Zühlke, S. & Spiteller, M. (2008). J. Nat. Prod. 71, 159-162.[6] Kusari, S., Zühlke, S. & Spiteller, M. (2009). J. Nat. Prod. 72, 2-7.[7] Kusari, S., Zühlke, S. & Spiteller, M. (2011). J. Nat. Prod. 74, 764-775.[8] Kusari, S., Kosuth, J., Cellarova, E. & Spiteller, M. (2011). Fungal Ecol. 4, 219-223.[9] Ramesha, B. T., Zuehlke, S., Vijaya, R., Priti, V., Ravikanth, G., Ganeshaiah, K., Spiteller, M., Shaanker,R. U. (2011). J. Chem. Ecol. 37, 533-536.MEP002Biochemical characterization of ectoine hydroxylases fromextremophilesN. Widderich*, M. Pittelkow, S. Weigand, E. BremerPhilipps-University Marburg, Biology, Marburg, GermanyEctoine and 5-hydroxyectoine are widely used by members of the Bacteriato offset the detrimental effects of high osmolarity on cellular physiology.Both compatible solutes also possess stabilizing effects formacromolecules and these properties, sometimes also referred to in theliterature as "chemical chaperones", have spurred considerablebiotechnological interest in ectoines. They have already found practicaluses in cosmetics, skin-care products, as protein- and whole cell stabilizersand medical applications are currently envisioned as well. Ectoinesynthesis is osmotically stimulated and catalyzed by the EctABC enzymes.A subset of the ectoine producers typically convert part of the newlyproduced ectoine into 5-hydoxyectoine through the enzymatic action of theEctD hydroxylase, a member of the non-heme iron (II) and 2-oxoglutaratedependentdeoxygenase super-family (1, 2). Although closely related inchemical structure, ectoine and 5-hydroxyectoine possess differentproperties, with 5-hydroxyectoine being often the more effectivestabilizing compound and the more potent cellular stress protectant (3).Ectoine hydroxylases from Virgibacillus salexigens (1) and Streptomycescoelicolor (3) have been biochemically characterized and a high-resolutioncrystal structure of the EctD protein from V. salexigens has been solved(2). This crystal structure revealed the positioning of the iron ligand withinthe active site of the EctD enzyme but it contained neither the substrateectoine nor the co-substrate 2-oxoglutarate. To advance our biochemicalunderstanding of this enzyme and to characterize EctD-type proteins forfurther crystallographic studies, we have characterized the properties ofectoine hydroxylases from microorganisms that can colonize habitats withBIOspektrum | Tagungsband 2012