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

165In order to analyze the role of HH01 and its relation to the eukaryotic host,we established its genome sequence. The genome was determined with asize of seven Mb. The most important finding was a number ofPKS/NRPS-gene clusters that make this microbe potentially interesting forthe synthesis of novel drug molecules. In addition, it revealed the presenceof all known secretion systems (except type III) and a violaceinbiosynthesis operon. But contrary to the AHL (acyl homoserine lactone)-dependent regulation of the known Chromobacterium violaceum violaceinbiosynthesis operon [3], no evidence for an AHL-dependent mechanismwas observed.To identify structural and regulating genes linked to the violacein synthesisa Tn5 transposon mutant library of about 7,000 clones was screened forclones impaired in violacein biosynthesis. About fifty white or weaklyviolet mutant clones were obtained and subsequently analyzed by PCR andcomplementation experiments. Besides a number of mutations located instructural genes, several mutations could be linked to the regulatorypathway associated with the violacein gene expression. These mutants arecurrently being investigated in more detail to elucidate the quorum-sensingsystem of this newly discovered organism. Moreover data from thegenome annotation and complementation tests suggests that HH01 controlsthe violacein biosynthesis using a single autoinducer synthase andcorresponding receptor similar to the Vibrio cholerae CqsA/CqsS quorumsensingsystem. Thus this is first example of a violacein biosynthesispathway that is not controlled by the influence of AHL autoinducermolecules.[1] Matz et al., PLoS ONE3:e2744 (2008), [2] Becker et al.,Appl Environ Microbiol 75:6635-38(2009), [3] Hoshino, Appl Microbiol Biotechnol 91:1463-75(2011)OTP128L-lysine production by Corynebacterium glutamicum utilizingalternative renewable resourcesS. Schiefelbein*, J. Becker, N. Buschke, C. WittmannTU Braunschweig, Institute of Biochemical Engineering, Braunschweig,GermanyWith a world market of 1.5 Million tons per year, the essential amino acidL-lysine is one of the most important biotechnological products. L-lysineis mainly produced by fermentation using sugar-based feedstocksconsisting of glucose, fructose or sucrose. However the use of these sugarshas certain disadvantages. On the one hand the prices are constantly risingand on the other hand the use of sugars competes with the food industries.Therefore alternative carbon sources like lactate [1] or xylose [2] gaininterest for fermentative production. Systems metabolic engineeringprovides an excellent starting point to establish corresponding productionprocesses. Recently a superior genetically defined Corynebacteriumglutamicum strain was created with excellent production properties duringgrowth on glucose and industrially relevant feedstocks including molassesand corn steep liquor [3] .In this work we investigated the use of lactate and xylose as alternativecarbon sources. For xylose this first required metabolic engineering of thexylose assimilation pathways as previously demonstrated for theproduction of diaminopentane [2] .Lysine production from lactate also requires engineering of C. glutamicum.Consequently the L-lysine hyper producing C. glutamicum strain wasmodified to better growth on lactate. This included overexpression of thegene for quinone-dependent L-lactate dehydrogenase (LldD), by using anative strong promoter.Both producer strains were investigated for their performance to producelysine. They exhibited interesting properties and serve as a valuable proofof concept for bio based production on novel feedstocks.[1] Neuner et al. (2010): Mixed glucose and lactate uptake by Corynebacterium glutamicum throughmetabolic engineering[2] Buschke et al. (2011): Metabolic engineering of Corynebacterium glutamicum for production of 1,5-diamino-pentane from hemicellulose.[3] Becker et al. (2010): From zero to hero - Design-based systems metabolic engineering ofCorynebacterium glutamicum for L-lysine productionOTP129MenD- a biocatalyst for asymmetric C-C-ligationsS. Baier* 1 , A. Kurutsch 2 , M. Müller 2 , G. Sprenger 11 University of Stuttgart, Institute of Microbiology, Stuttgart, Germany2 Albert-Ludwig-University, Institute for Pharmaceutical Science,Freiburg, GermanyBesides their natural functions in cell metabolism, many thiaminediphosphate (ThDP) dependent enzymes can be used in vitro for C-Cligationreactions (pyruvate decarboxylase, transketolase, benzoylformatedecarboxylase and others). 1 In menaquinone biosynthesis, the ThDPdependentMenD (2-succinyl-5-enol-pyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) Synthase) performs a Stetter-like 1,4 additionreaction by ligating isochorismate and 2-ketoglutarate under non-oxidativedecarboxylation to yield SEPHCHC. 2,3 MenD (subunit size of 65 kDa)from E. coli requires ThDP and a bivalent metal ion. 4 So far, MenD is theonly described enzyme catalyzing a Stetter-like reaction which means thata nucleophilic donor substrate (2-ketoglutarate) is attached to ,unsaturatedcarbonyl acceptor (e.g. isochorismate). The reported K M andK cat values are in the M and 1 min -1 range, respectively. 3 Crystalstructures for MenD wild type and mutant proteins have been reportedfrom E. coli and B.subtilis allowing insights into amino acid residues ofthe active site which are discussed for cofactor and substrate binding. 4,5,6Besides the Stetter-like reaction, MenD also catalyzes an 1,2-addition andprovides the approach to hydroxyketones. 7 Therefore, it is a promisingbiocatalyst for C-C bond formation, but the substrate range has to beextended (e.g. 2,3-CHA) to gain access to new products. Also, in vivobiosynthesis of MenD products with recombinant E.coli cells is a goal.Work on both the clarification of structure-function-relationship and theextension of the substrate range is underway and data will be presented.[1] Pohl, M., Sprenger, G.A., & Müller, M.:Curr Op Biotech 2004,15: 335-342.[2] Jiang M, Cao Y, Guo ZF, Chen M, Chen X & Guo Z:Biochem 2007, 46. 10979- 10989[3] Emmson GT, Campell IM & Bentley R:Biochem Biophys Res Commun 1985, 131, 956- 960[4] Bhasin M, Billinsky JF, Palmer DRJ:Biochem 2003,42, 13496-13504[5] Priyadarshi A, Kim EE, Hwang KY:Biochem. Biophys. Res. Commun.2009,388, 4, 748-751[6] Dawson A,Fyfe PK& Hunter WN:J. Mol. Biol. 2008. 384, 1353-1368[7] Kurutsch A, Richter M, Brecht V,SprengerGA, Müller M:J. Mol. Catal. B, Enzym.2009, 61: 56-66.OTP130Phototrophic microbial fuel cell: Mircobial ecology forelectroactive systemsX.A. Walter*, I. Ieropoulos, J. Greenman, C. MelhuishUniversity of the West of England, Bristol Robotic Laboratory, Bristol, UnitedKingdomOne century ago (1911) Potter has shown that electricity could begenerated by anaerobic microbial respiration of organic matter: the firstmicrobial fuel cell (MFC) was born. MFC systems are composed of twocompartments: an anodic chamber and a cathodic one. The electricity isgenerated in the anodic side by the microbiologically driven transfer ofelectrons from secondary fermentation products (e.g: fatty acid, ethanol,lactate, butyrate, acetate etc) to the electrode. The anode and cathodeelectrodes are connected through a circuit, which facilitates the flow ofelectrons from the former to the latter; this results in the production ofelectrical current. The produced protons (H + ) diffuse from the anodiccompartment into the cathodic chamber, through a proton exchangemembrane (PEM) where they react with oxygen and incoming electrons,thus producing water.The project aims are to use MFC for both carbon capture and electricityproduction. To achieve our objectives, we reproduce a controlled electroncascade such as the one occurring in stratified microbial food-web (e.g.microbial mats). In such systems, carbon is fixed through photosynthesisas biomass that is further consumed by underlying anaerobic respirations.In our case, we cultivate oxygenic phototrophs for their capacity ofextracting electrons from water and utilize it to reduce inorganic carboninto biomass. In addition, the by-product of their metabolism, oxygen, willenhance cathode efficiency by an O 2 supersaturation effect. The producedbiomass will therefore serve as the electron donor for anaerobic respirationin the anodic compartment. In this system the inorganic carbon serves as atransporter to harvest light energy in the cathodic compartment and torelease it in the anodic one as electrons. The main challenge is to controlthe recycling of elements between those two compartments in order to beas close to as possible a semi-closed artificial ecosystem. Therefore, wewill obtain a carbon-neutral electroactive system. However, as we arereproducing an artificial microbial ecosystem, certain ecological conceptshave to be taken into account. Thus, we will present the ecological aspectsthat we have to control in order to produce an electroactive and carbonneutralsemi-closed system.OTP131Lake La Cruz, a Neoarchean Ecotone Ocean analogueX.A. Walter* 1 , A. Picazo-Mozo 2 , M.-R. Miracle 2 , E. Vicente 2 , A. Camacho 2 ,J. Zopfi 21 University of the West of England, Bristol Robotic Laboratory, Bristol, UnitedKingdom2 University of Valencia, Institut Cavanilles de Biodiversitat i BiologiaEvolutiva, Burjassot, SwitzerlandRecent research on the biogeochemistry of the Late Archean Ocean (2.7-2.5 Ga) showed that there was a spatial patchwork of physical-chemicalconditions. A simplified model of an Archean Ocean would thus consist oftwo distinct compartments with different dominating biogeochemicalprocesses: I) a shallow Ocean Margin compartment with oxygenicphotosynthesis in the upper water column, and euxinic conditions (anoxicand sulfidic) below the chemocline and in the sediments; II) an anoxicFe(II)-rich Open Ocean compartment with a primary productiondominated by anoxygenic photoferrotrophy and methanogenesis prevailingorganic matter degradation in bottom layers and sediments.Whilst analogues of an ocean with established sulfur cycle, correspondingto compartment I, have been described and well studied (e.g. Black Sea orLake Cadagno), only one modern analogues of the ferruginous open watercompartment has been described. Therefore, we study the microbial ironcycling, in the ferruginous water column of Lake La Cruz (Spain). WeBIOspektrum | Tagungsband 2012