VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

GWP028Application of the soluble NAD + -reducing hydrogenase(SH) of Ralstonia eutropha H16 for solar-driven H 2 -production in cyanobacteriaK. Karstens*, B. Friedrich, O. LenzInstitute for Biology/Microbiology, Humboldt-University, Berlin, GermanyHydrogenases catalyze the reversible formation of 2e - and 2H + from H 2 [1].Hydrogen is discussed as a promising renewable fuel replacing fossil energycarriers in the future. Therefore, the H 2-production capability of theseenzymes is of significance for biotechnological applications.Theoretically cyanobacteria are ideally suited to produce H 2 from sunlightand water since they generate „high-potential” electrons duringphotosynthesis, which could be used directly by hydrogenases for hydrogenproduction.Indeed many cyanobacteria such as Synechocystis sp. PCC 6803 andSynechococcus PCC 7002, possess so called bidirectional [NiFe]-hydrogenases of the H 2:NAD(P) + -oxidoreductase type. In nature, theseenzymes evolve relatively small amounts of H 2 from accumulating reductantin form of NADH and NADPH under O 2-limiting conditions [2].In order to increase the amount of H 2 and to enable continuous production ofH 2 also in the presence of O 2, we are currently investigating the heterologoussynthesis of the soluble NAD + -reducing hydrogenase (SH) of the soilbacterium Ralstonia eutropha H16 in cyanobacteria. This bidirectional[NiFe]-hydrogenase is known to maintain H 2-conversion at highconcentrations of O 2 and has already been characterized intensively byvarious biochemical and spectroscopic methods [3,4].Conditions for heterologous production of functional R. eutropha SH inSynechocystis PCC 6803 are currently being explored. Very recently, SHproduction in the cyanobacterium Synechococcus PCC 7002 has beendemonstrated with a new heterologous expression strategy [5,6] and issubject of comprehensive characterisation and further optimisation in ourgroup.Furthermore, we aim to uncover the molecular basis for the exceptional O 2-tolerance of the R. eutropha SH. Ongoing analysis focuses on the aminoacid coordination of the Fe-S-cluster in the small e - -transferring hydrogenasesubunit of the SH. The mechanism of O 2-tolerance provides essentialknowledge to convert an O 2-sensitive cyanobacterial hydrogenase into anenzyme that produces H 2 directly from sunlight and water in the presence ofoxygen.[1] Vignais, P.M. and A. Colbeau (2004): Curr Issues Mol Biol, 6: 159-88.[2] Appel, J. et al (2000): Arch Microbiol, 173: 333-338.[3] Burgdorf, T. et al (2005): J Bacteriol, 187: 3122-32.[4] Horch, M. et al (2010): Angew Chem Int Ed Engl, 49: 8026-9.[5] Xu, Y. et al (2011): Methods Mol Biol, 684: 273-93.[6] Xu, Y. and D. Bryant (2010) personal communication.GWP029Engineered salt-induced ectoine promoter for use in H.elongata as halophilic expression systemE. Witt*, A. Grün, M. Kurz, E.A. GalinskiInstitute for Microbiology und Biotechnology, Friedrich-WestphalianWilhelms-University, Bonn, GermanyHeterologous protein expression is commonly carried out in E. coli, butoften limited by formation of inclusion bodies or unsatisfactory proteinstability. Heterologous expression systems in the presence of salt andcompatible solutes have previously been applied to demonstrate the potentialof including stabilizing/protecting solutes in the process of functionalrecombinant protein expression [1]. As E. coli´s capacity to tolerate saltstressis limited, Halomonas elongata, a moderate halophilic gammaproteobacterium of broad salt tolerance, has been proposed as an alternative[2].Halomonas elongata can grow over a salinity range of 1-20% NaCl and isable to adjust osmotic pressure by the intracellular accumulation of theprotein-protecting osmolytes ectoine and S,S-beta hydroxyectoine. Thebiosynthesis of these solutes is under the control of a salt-inducible promoterregion, promA. We present here the construction of a pBBR1-derived vectorcontaining an engineered version of the promA promoter. By modifying theribosomal binding site, we obtained a suitable vector (pWUB1) for salinitycontrolledprotein expression in H. elongata.Using GFP UV as a reporter gene, we monitored the expression patterncontrolled by the modified promoter region and also investigated theinfluence of osmotic stress and the presence of compatible solutes for finetuningof promoter regulation.[1] Barth, S. et al (2000): Compatible solute-supported periplasmic expression of functionalrecombinant proteins under stress conditions. Appl Environ Microbiol 66: 1572-1579.[2] Kurz, M. et al (2004): Heterologous protein expression in Halomonas elongata - why halophilicorganisms offer a viable alternative to E. coli. VAAM-Jahrestagung 2004; KD004.GWP030Synthesis of citrulline-rich cyanophycin by use ofPseudomonas putida ATCC 4359L. Wiefel*, A. Bröker, A. SteinbüchelInstitute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, GermanyThe provision of cyanophycin (multi-l-arginyl-poly-l-aspartic acid, CGP) asa putative precursor for biodegradable technically employed chemicalsmakes it important to synthesise CGP in recombinant organisms.Furthermore derivates of CGP, harbouring other constituents, are of specialinterests for further research. As shown previously, cyanophycin synthetaseswith wide substrate ranges are able to incorporate other amino acids thanarginine like citrulline and ornithine, but are still dependent on additionalsupplementation of these amino acids in order to achieve sufficientincorporation rates [2]. Therefore, using an organism which produces theneeded supplement by itself, was the next logical step. Pseudomonas putidastrain ATCC 4359 is such an organism because it was previously shown thatit produces large amounts of L-citrulline from L-arginine [1]. Synthesis ofCGP in this P. putida strain was achieved by expressing the cyanophycinsynthetase of Synechocystis sp. PCC 6308. Using an optimised media forcultivation, the strain was able to synthesise insoluble CGP amounting up to14.7 ± 0.7 % (w/w) and soluble CGP amounting up to 28.7 ± 0.8 % (w/w) ofthe cell dry matter, leading to an overall CGP-content of 43.5 %. Theoccurrence of soluble CGP was dependent on the temperature duringcultivation. HPLC-analysis of the soluble CGP showed that it was composedof 50.4 ± 1.3 mol % aspartic acid, 32.7 ± 2.8 mol % arginine, 8.7 ± 1.6 mol% citrulline and 8.3 ± 0.4 mol % lysine, while the insoluble CGP containedamounts of less than 1 mol % of citrulline. Using mineral salt media with1.25 or 2 % (w/v) Na-succinate, respectively, and 23.7 mM L-arginine, thisstrain synthesised amounts of 25 to 29 % of the CDM insoluble CGPshowing only a very low citrulline content of less than 1 mol %.[1] Kakimoto, T. et al (1971): Enzymatic production of l-citrulline by Pseudomonas putida. ApplMicrobiol 22:992-999.[2] Steinle, A. et al (2009): Metabolic engineering of Saccharomyces cerevisiae for production ofnovel cyanophycins with an extended range of constituent amino acids. Appl Environ Microbiol75:3437-3446.GWP031Functional genomics of the prophage CGP3 causingpopulation heterogeneity in Corynebacterium glutamicumA. Heyer*, M. Bott, J. FrunzkeInstitute of Biotechnology, Research Center Jülich, Jülich, GermanyThe Gram-positive soil bacterium Corynebacterium glutamicumATCC13032 is one of the most important organisms in WhiteBiotechnology as it is used for the industrial production of more than twomillions tons of amino acids per year. Genome sequencing of C. glutamicumrevealed the existence of three prophages (CGP1, CGP2 and CGP3) highlydiverse in size and grade of degeneration. The largest prophage CGP3 (187kbp) accounts for 6% of the genome and is inserted in a cluster of tRNAgenes.Recent studies demonstrated via a fluorescence microscopy approach thatthe CGP3 prophage exhibits spontaneous induction in a small fraction of C.glutamicum wild type cells. Upon induction CGP3 excises from the genomeand exists as a circular double-stranded DNA molecule (Frunzke et al.,2008). In several cases, phage induction was accompanied by lysis of thecells suggesting the expression of functional phage lysins. Therefore, studieswere initiated in order to understand spontaneous CGP3 induction leading topopulation heterogeneity and cell lysis in C. glutamicum cultures.A first series of experiments focused on the identification of the putativephage regulator controlling the lysogenic/lytic switch of CGP3. A potentialcandidate is the putative transcriptional regulator Cg2040 encoded by theCGP3 genome. Transcriptome comparisons of a strain overexpressingCg2040 and C. glutamicum wild type resulted in significantly reducedmRNA levels of five phage genes, which are located next to or in closevicinity of cg2040. Subsequently, binding of purified Cg2040 to putativetarget promoters was verified via electrophoretic mobility shift assay(EMSA). Furthermore, first results will be provided concerning thefunctionality of the putative phage integrase Int2 of CGP3. These effortsspektrum | Tagungsband 2011