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

expressed in E. coli. The corresponding protein was purified by metalaffinitychromatography to >90 % purity and concentrated to 12 mg ml -1 .Subsequently, the protein was subjected to an enzyme activity assaydemonstrating its functionality as RNAP. Putative promoters for this RNAPwithin the bacteriophage K1E genome were predicted computationally andsummerized in a sequence logo. In in vitro transcription experiments theK1E RNAP revealed optima of pH 8, 37°C to 40°C, with a strongdependency on Mg 2+ ions and a stimulation by spermidine. Further, evenlow salt concentrations (>30 mM NaCl) inhibited enzyme activity. Based onthese results, a system for high-yield in vitro RNA synthesis using K1ERNAP was established.Additionally, new protein production systems for Bacillus megaterium weredeveloped based on the K1E RNAP. It was shown, that a system combiningthe K1E RNAP with a SP6 RNAP promoter produced highest amounts ofthe intracellular model proteins Gfp (61.4 mg g CDW -1 ) and the extracellularTfh (2971 U l -1 ; 3.2 mg l -1 ) in vivo.Now, with the help of its just published genome sequence it is possible tocharacterize bottle necks in the protein production, especially secretion,process of B. megaterium by systems biotechnology approaches utilizingmicroarrays, proteome, metabolome and fluxome data. The bioinformaticalplatform (MEGABAC, http://www.megabac.tu-bs.de) integrates obtainedtheoretical and experimental data.GWP013Isolation and characterization of methanogenic Archaeafrom on-farm biogas plantsR. Stantscheff* 1 , K. Seyfarth 1 , S. Dröge 2 , M. Klocke 3 , H. König 11 Institute for Microbiology and Wine Research, Johannes-Gutenberg-University, Mainz, Germany2 Test and Research Institute Pirmasens, Technikum, Pirmasens, Germany3 Leibniz Institute for Agricultural Engineering Potsdam-Bornim e.V. (ATB),Bioengineering, Potsdam, GermanyElectricity and heat generation from methane-rich biogas often provides theadvantages of utilizing renewable energy sources and heat thus reducing theemission of climate-relevant greenhouse gases. Various efforts in raising theefficiency of biogas production were focused on improving the technicalaspects. The microbial biocoenosis in general as well as the specificmicrobial interactions leading to methane formation in biogas plants remainslargely a black box. Methanogenic Archaea were isolated from on-farmcontinuously stirred tank reactor (CSTR) biogas plants. In this study theobtained isolates were compared with biodiversity predictions of cultureindependentmethods and morphological and physiological characterizationswere performed.Reactor samples from five biogas plants fed with corn and cattle manurewere used as an inoculum for enrichment of methanogenic Archaea. Toachieve pure cultures, anaerobic variants of the serial dilution- or solidmedia plating- techniques were applied. Selective growth ofhydrogenotrophic, methylotrophic and acetoclastic methanogens wasachieved by application of modified DSMZ culture media. Morphologicalexaminations were accomplished by fluorescence microscopy. Culturepurity and biodiversity analysis were performed by denaturating gradient gelelectrophoresis (DGGE), as well as 16S- rDNA cloning experiments inconnection with RFLP. With Methanobacterium formicicum,Methanosarcina mazei, Methanosarcina barkeri, Methanosaeta concilii andMethanoculleus bourgensis species from four different families could beisolated. Their impact in biogas formation is discussed.GWP014Biochemical and genetic characterization of ethyleneglycol metabolism in Pseudomonas putida KT2440 andJM37B. Mückschel* 1 , O. Simon 2 , J. Klebensberger 1 , N. Graf 3 , J. Altenbuchner 3 ,J. Pfannstiel 2 , A. Huber 2 , B. Hauer 11 Institute of Technical Biochemistry,University of Stuttgart, Stuttgart,Germany2 Department of Biosensorics, University of Hohenheim, Stuttgart, Germany3 Institute of Industrial Genetics, Universität Stuttgart, Stuttgart, GermanyWe used the P. putida strains KT2440 and JM37 for the characterization ofethylene glycol (EG) metabolism with the overall goal to develop abiocatalytic route for the synthesis of glyoxylic acid (GXA), a proposedintermediate in the metabolism of EG. Being an important building block forflavors and polymers, GXA is a valuable product for many industrialprocesses. Since production of GXA is currently limited to chemicalsynthesis, a biotechnical production route is of great economical interest.In contrast to strain KT2440, we could demonstrate that P. putida JM37 wasable to use EG as well as GXA as sole source of carbon and energy. Despitethis difference, dense cell suspension experiments revealed completeconversion of 50 mM EG and GXA for both strains within 50 h. Duringconversion of EG, accumulation of 4.1 mM glycolic acid (GCA), 12.8 mMGXA, and 13.7 mM oxalate (OXA) was detected in supernatants of strainKT2440. To identify enzymes involved in the metabolism of EG in KT2440and JM37, a differential proteomic approach was used.Increased expression of tartronate semialdehyde synthase (Gcl), malatesynthase (GlcB) and isocitrate lyase (AceA) in JM37 as well as AceA instrain KT2440 was found during incubations with EG or GXA. Theseproteins represent key enzymes of known pathways involved in themetabolism of GXA. The corresponding triple mutant strain harboring anadditional deletion of the gene prpB, encoding for methyl isocitrate lyase,was constructed in strain KT2440 and characterized for GXA accumulation.This mutant strain possessed a significant reduction in its EG conversionrate compared to the wildtype strain and was found to accumulate up to 15mM GCS, 11.2 mM GXS and 8.6 mM of OXA. Further analysis uncoveredthe induction of two PQQ-dependant ethanol dehydrogenases [pp_2674,pp_2679], indicating an important role for these proteins within theoxidative metabolism of EG. A double deletion mutant of the twoisoenzymes in KT2440 resulted in a dramatic decrease in EG metabolism.The absence of GCS and GXS accumulation in this mutant further highlightsthe importance of these enzymes for EG metabolism.GWP015Production of lignin-modifying enzymes via co-cultivationof white-rot fungiS. Krügener* 1 , C. Qi-he 2 , T. Hirth 1,3 , S. Rupp 1 , S. Zibek 11 Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB,Molecular Biotechnology, Stuttgart, Germany2 Department of Food Science and Nutrition, Zhejiang University,Hangzhou, China3 Institute for Interfacial Engineering, University of Stuttgart, Stuttgart,GermanyLignin, the third most abundant polymer present in nature, is expected toplay an important role as raw material for the world’s biobased economy inthe post-oil era. White-rot fungi are efficient lignin degraders, which makesthem ideally suited for industrial applications where phenolic compoundssuch as lignin must be altered or removed. Most biodegradation processes innature take place by division of work of different microorganisms incomplex ecosystems. However, most of what known about ligninbiodegradation is from pure culture studies with basidiomycete fungi. Cocultivationapproaches for production of lignin modifying enzymes withwhite-rot fungi have been paid little attention.With this work we investigated the effects of co-culturing of white-rot fungion lignin-modifying enzyme production. In a prescreening concerningpaired growth characterisation and ligninolytic ability Bjerkandera adusta,Dichomitus squalens, Hypoxylon fragiforme, Phlebia radiate, Pleurotuseryngii and Pleurotus ostreatus were cultured in pairs on PDA agar plates orrather Remazol Brilliant Blue R dye containing agar plates. The degree ofdecolourization was clearly stimulated due to mycelia interactions.Combinations of species with good prospects were studied under submergedco-cultivation concerning the production of the three main lignin-modifyingenzymes, laccases (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.14) andmanganese peroxidases (EC 1.11.1.13). Compared with the monocultures,co-cultures of P. radiate with D. squalens and P. ostreatus with P. radiateshowed positive effects on production of lignin modifying enzymes.Current work is using secretomic approaches in connection with activityoverlay detection and MALDI-TOF-MS peptide mapping to characterizemolecular differences of lignin modifying enzyme expression between theco-cultures and the monocultures.The large versatility in biosynthetic pathways together with its broadsubstrate spectra renders P. putida a model organism for various industrialapplications, such as bioremediation or biocatalysis processes.spektrum | Tagungsband 2011