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

150When Ms. mazei pWM321-p1687-uidA utilized methanol as a substrate, -glucuronidase activity was almost not detectable indicating a tightregulation of gene expression by the p1687 promoter. Induction by theaddition of trimethylamine led to a strong increase of expression of theuidA gene and -glucuronidase activity was monitored by the productionof p-nitrophenol from p-nitrophenyl--D-glucuronide.In summary, we describe the first inducible gene expression system in Ms.mazei. This will be used for the overproduction and characterization ofproteins that cannot be produced in E. coli and other simple expressionsystems. This will be of particular interest for proteins that harbourcomplex prosthetic groups that are hardly found or absent in the domainBacteria, e.g. tungsten enzymes.[1] Metcalf WW, Zhang JK, Apolinario E, Sowers KR, Wolfe RS (1997) A genetic system forArchaea of the genus Methanosarcina: liposome-mediated transformation and construction ofshuttle vectors. PNAS 94: 2626-31.[2] Krätzer C, Carini P, Hovey R, Deppenmeier U (2009) Transcriptional profiling ofmethyltransferase genes during growth of Methanosarcina mazei on trimethylamine. J Bacteriol191: 5108-15.OTP056Antibacterial investigation of Artemisia campestris L (Asteraceae)M. Salem* 1 , A. Alruba 2 , J. El-turby 21 BioTechnology Research Center, Microbiology, Tripoli, Libyan ArabJamabiriya2 Tripoli university, pharmacy Facutly, pharmacy, Tripoli, Libyan ArabJamabiriyaArtemisia campestris L. (Asteraceae) is folk Libyan medicinal, smallaromatic perennial shrub that grow in North Africa and most of Europe.The grounded of aerial parts was extracted in soxhlet apparatussuccessively, each crude extract was subjected to antibacterial evaluationagainst human pathogenic bacteria,Staph.aureus, E.coli, Salmonella spp.And Ps. aeruginosa, by using agar cup-cut diffusion assay .The resultsreported that chloroform and methanolic extracts were effectiveagainstStaph.aureus in which shown by MIC is 12.5mg .OTP057Screening for thermostable cellulases for lignocellulosicbiomass degradationC. Schröder*, V. Bockemühl, G. AntranikianTechnical University Hamburg-Harburg, Technical Microbiology,Hamburg, GermanyExisting biorefineries for ethanol production mainly use starch-biomasssuch as wheat and corn. To avoid the usage of feed- and foodstuff,lignocellulosic biomass lately attained particular interest of research.Lignocellulosic material like wheat straw is a challenging substrate due tothe compact, often crystalline structure of cellulose-, hemicellulose- andlignin-polymers. To obtain fermentable sugar-monomers from celluloseand hemicellulose by enzymatic degradation, the wheat straw has to bedecomposed, e.g. by hydrothermal processes.To discover novel thermostable cellulases for degradation of the cellulosicfraction, suitable environmental samples (T = 60-90°C, pH 5-7) wereenriched by using cellulose as sole carbon source. The DNA of thecultured microbial consortia was isolated for metagenomic libraryconstruction. Subsequently, the gene library was screened for the presenceof cellulase encoding genes by detection of endoglucanase,cellobiohydrolase and -glucosidase activity using colorimetric activityassays. Additionally, for activity-based screening, metagenomic librarieswere constructed, directly using isolated DNA from hot springs from theAzores without previous enrichment procedures. Furthermore a sequencebasedscreening approach was also applied using sequence data of ametagenome. By aligning the nucleotide sequences with known genes,potential cellulase-encoding open reading frames were identified.The activity-based screening revealed genes encoding putativeendoglucanases and -glucosidases. The sequence-based analysis resultedin the detection of one gene encoding another putative endoglucanase.Further work will be performed to express the identified genes in a suitablehost system such as E. coli and P. pastoris. The corresponding enzymeswill be tested with regard to activity towards the cellulosic fraction of thedecomposed wheat straw.OTP058Translational regulation in Haloferax volcaniiJ. Schmitt*, J. SoppaUniversität Frankfurt, Institut für molekulare Biowissenschaften/AGSoppa, Frankfurt, GermanyTranslational regulation is an important cellular mechanism for geneexpression control and is present in all three domains of life. It enables thecell to answer very rapidly to changes in environmental conditions and isthus involved in cell survival, differentiation, stress adaptation andresponse to specific stimuli.To gain a global overview of growth phase-dependent translationalregulation translatome analyses were performed with Haloferax volcaniiand Halobacterium salinarum (Lange et al., 2007). Polysome-boundmRNA was separated from free mRNA by sucrose gradient centrifugationand the two mRNA fractions were compared using DNA microarrays.Thereby it was revealed for the two species that 6% and 20%, respectively,of all genes showed growth phase-dependent differential translationalregulation (Lange et al.,2007). In H. volcanii many transcripts weretranslated with under-average efficiency in exponential as well asstationary phase, indicating that their translation might be induced inresponse to a different stimulus. Therefore, currently translatome analysesare performed after the application of various stress conditions, e.g. highand low osmolarity, high and low temperature, oxidative stress, poorcarbon sources.It was also revealed that the 5´- and 3´-UTRs are necessary and sufficientto transfer translational control from native transcripts to a reportertranscript. The 5´-UTRs are apparently necessary to down-regulateconstitutive translational initiation, while induction of translation isencoded in the 3´-UTRs (Brenneis and Soppa, 2009).However, the molecular mechanism and involved proteins are stillunknown. Therefore, the H. volcanii genome was searched for putativeRNA-binding proteins. To gain insight into their function the respectivegenes for selected proteins were deleted and a conditional overexpressionsystem was generated. Analysis of the deletion and overexpressionmutants is currently under way.C. Lange, A. Zaigler, M. Hammelmann, J. Twellmeyer, G. Raddatz, S.C. Schuster,D. Oesterhelt & J. Soppa (2007) BMC Genomics 8:415M. Brenneis, J. Soppa (2009) PLoS ONE 4(2): e4484OTP059Biocatalytical Cyclization of CitronellalG. Siedenburg* 1 , D. Jendrossek 1 , M. Breuer 2 , B. Juhl 3 , J. Pleiss 3 , M. Seitz 3 ,J. Klebensberger 3 , B. Hauer 31 University of Stuttgart, Institute for Microbiology, Stuttgart, Germany2 BASF SE, Ludwigshafen, Germany3 University of Stuttgart, Institute of Technical Biochemistry, Stuttgart, GermanyHopanoids stabilize the cytoplasm membrane of many bacteria similar tothe function of sterols in eukarotes. Key enzyme of hopanoid biosynthesisis the squalene-hopene cyclase (SHC) which catalyzes the polycyclizationreaction of squalene to the pentacyclic triterpene hopene - the precursor ofall hopanoids. The SHC-catalyzed reaction is one of the most complexbiochemical reactions and involves the formation of 5 ring structures, thealteration of 13 covalent bonds, and the formation of 9 stereo centers.Zymomonas mobilis- an important ethanol producing bacterium - harbourstwo SHC-encoding genes that were cloned and over-expressed in E. coli.Hopene-forming activity was confirmed for both SHCs. One of the SHCswas additionally able to cyclise the monoterpene citronellal to isopulegol.This finding is contrary to former results using the model SHC fromAlicyclobacillus acidocaldarius 1, 2 and several other SHCs cloned fromdifferent organisms in this study. Isopulegol is used as a flavor in differentproducts and is an important intermediate in the production of menthol.Our finding is remarkable because cyclization of mono-, sequi- andditerpenes normally requires activation of the linear precursor bydiphosphate 3, 4 . Depending on the stereo-configuration of the substratedifferent isopulegol stereoisomers were formed. Cyclization of citronellalby SHC is the first example of an enzyme-catalyzed cyclization of a notactivatedlinear monoterpene.Further work focussed on the optimization of the SHC-catalyzedcyclization of citronellal by mutagenesis of SHC active site amino acids.Several SHC muteins revealed a strong increase in isopulegol-formingactivity. Some muteins were able to catalyze an almost completeconversion of citronellal to isopulegol ( 90%) compared to only 30% forthe wild type enzyme. Interestingly, the stereo-configuration and therelative isomer composition of the product were altered in some muteins.An overview on the cyclization potential of wild type and mutant SHCsfrom different sources will be given.1. Hoshino, T.; Ohashi, S.Org.Lett.2002, 4, 2553-2556.2. Wendt, K. U.; Lenhart, A.; Schulz, G. E.J Mol Biol1999, 286, (1), 175-87.3. Bohlmann, J.; Meyer-Gauen, G.; Croteau, R.Proc Natl Acad Sci U S A1998, 95, (8), 4126-33.4. Davis, E. M.; Croteau, R.Top. Curr. Chem.2000, 209, 53-95.OTP060Development of a novel system for the functional expressionand screening of membrane proteinsA. Malach* 1 , A. Heck 1 , K.-E. Jaeger 2 , T. Drepper 11 Heinrich-Heine-Universität, Institut für molekulare Enzymtechnolgie - AGDrepper, Düsseldorf, Germany2 Heinrich-Heine-Universität, Institut für molekulare Enzymtechnolgie,Düsseldorf, GermanyThe heterologous expression of membrane proteins and enzymes usingstandard expression hosts as E. coli is often hampered by many differentfactors including low expression efficiencies, degradation of the product,product toxicity, insufficient protein folding or formation of inclusionBIOspektrum | Tagungsband 2012