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

GWV012Autotrophic Production of Stable Isotope-labelled AminoAcidsS. Lütte* 1 , A. Pohlmann 1 , H. Heumann 2 , A. Steinbüchel 3 , B. Friedrich 11 Institute für Biology/Microbiology, Humboldt-University, Berlin, Germany2 Silantes GmbH , München, Germany3 Westphalian Wilhelms-University, Münster, GermanyStable isotope (SI)-labelled biomolecules are increasingly in demand asstandards for quantitative mass spectrometry and multidimensional NMR.The production of 13 C-labelled substances by autotrophic bacteria is apopular method since it uses the competitively priced 13 C-carbon source,13 CO 2. The β-proteobacterium Ralstonia eutropha H16 is able to growlithoautotrophically with H 2 and CO 2 as sole sources of energy and carbon,respectively [1]. The industrial production of SI-biomaterial with R.eutropha is already established [2] and R. eutropha-based SI-enriched diethas been used successfully in quantitative proteomic analyses of mousemodels [3]. Of particular interest for quantitative proteomics are SI-labelledamino acids, e.g. SI-arginine, which are required for amino acid specificlabelling of proteins in cell free assays as well as for standards inquantitative proteomics. In the current study arginine enrichment inautotrophically grown R. eutropha cells was accomplished by accumulationof the arginine-containing polymer cyanophycin [multi-L-arginyl-poly-(Lasparticacid)]. Overproduction of cyanophycin was achieved byheterologous expression of the cyanophycin synthase gene (cphA) ofSynechocystis sp. strain PCC6308 under control of the ribulose-1,5-bisphosphate carboxylase (cbb) promoter. The constructed strainaccumulates cyanophycin under lithoautotrophic growth conditions ascytoplasmic inclusions. The cyanophycin content of the cells reached up to5.5% of cellular dry weight (CDW). The plasmid-based overexpressionstrain showed a decrease of cyanophycin yield when grown withoutantibiotic, most likely due to the loss of the corresponding plasmid duringcultivation. Plasmid stability is crucial for enhanced cyanophycin synthesis[4]. To overcome plasmid curing, we integrated the cphA gene together withthe appropriate cbb promoter sequence into chromosome 2 of R. eutropha toensure stable production of cyanophycin, resulting in a strain offeringpossibilities for cost-effective production of SI-arginine, a particularlymarketable product.[1] Pohlmann, A. (2006): Nat. Biotechnol.[2] Heumann, H. (2000): PCT Int. Appl. WO0012140.[3] Frank, E. et al. (2009): PLoS One 11:e7821.[4] Voss, I. and A. Steinbüchel (2005): Metabol. Eng. 8:66-78.GWV013Systems Metabolic Engineering of Basfiasucciniciproducens for Biobased Production of SuccinicAcidJ. Hangebrauk* 1 , R. Stellmacher 1 , R. Schäfer 1 , J. Becker 1 , G. vonAbendroth 2 , H. Schröder 2 , S. Haefner 2 , C. Wittmann 11 Institute of Biochemical Engineering, University of Technology,Braunschweig, Germany2 Research Fine Chemicals & Biotechnology, BASF SE, Ludwigshafen,GermanySuccinic acid, a key building block for important bulk chemicals, iscurrently derived from petrochemical origin. With regard to the shortage andincreasing prices for fossil resources, the biotechnological production ofsuccinic acid becomes an attractive alternative to the traditional route.Hereby, the bio-based production process, using renewable resources andfixing carbon dioxide, a prominent greenhouse gas, appears to be moresustainable.Towards, an economically competitive bio-based production process forsuccinic acid we focus on the recently isolated microorganism Basfiasucciniciproducens. It belongs to the Pasteurellaceae family having theability to naturally overproduce succinic acid. It grows on a variety ofdifferent carbon sources (e.g. glucose, glycerol) [1] . Volumetric productivityfor succinic acid of up to 1.3 g L -1 h -1 and a yield of 0.6 g g -1 are reached onglucose as sole carbon source [2] . By using glycerol as sole carbon source,remarkably increased succinic acid yields of up to 1.2 g g -1 can be obtained[1] . To determine genetic and metabolic targets for the elevation of yield andproduction efficiency these studies focus on the metabolic network of B.succiniciproducens.Taking the development of a minimal medium as a starting point, severalgenetic modifications were introduced into the organism to improve theproduction efficiency. The systems biotechnological approach ofcomprehensive 13 C metabolic flux analysis program led to a detailed insightinto the metabolic network of B. succiniciproducens.[1] Scholten, E. and D. Dägele (2008): Biotechnol. Lett. 30(12), 2143-2146.[2] Stellmacher, R. (2010): CIT 82 (8), 1223-1229.GWV014Chemoenzymatic synthesis and microbial degradation ofenantiopure aromatic beta-amino acidsU. Engel*, B. Brucher, C. Syldatk, J. Rudat*Technical Biology, Karlsruhe Institute of Technology (KIT), Karlsruhe,GermanyChiral beta-amino acids are valuable building blocks for the production offine chemicals and pharmaceuticals.As their chemical synthesis is still inefficient and costly our approach is amodification of the well studied hydantoinase/carbamoylase system. Arylsubstituteddihydropyrimidines were synthesized as substrates for whole cellbiotransformation experiments with different wild type bacteria andrecombinant E. coli strains expressing hydantoinases with known activityfor aryl-substituted hydantoins. Most strains tested were able to hydrolyzethe substrates to the corresponding N-carbamoyl beta-amino acids [1]. Twoisolates showed enantioselective conversion of the model substratephenyldihydrouracil and also were able to hydrolyze p-chlorophenyldihydrouracil. The gene sequences of two novel hydantoinasesand one carbamoylase were elucidated.Furthermore we investigated the microbial degradation of beta-Phenylalanine. In all bacteria tested so far, the initial reaction is a (S)-selective transamination to the corresponding beta-keto acid by induciblePLP dependent transaminases. We established a chiral HPLC analysissuitable for the enantioseparation of several aromatic beta-amino acids tostudy the substrate spectrum of these enzymes [2].[1] Bretschneider, U. et al (2010): Chem Ing Tech 82 (1,2), 161.[2] Brucher, B. et al (2010): Chromatographia 71, 1063.GWV015Natural Product Synthesis by Squalene-Hopene Cyclases(SHCs)M. Seitz 1 , J. Klebensberger 1 , M. Breuer 2 , B. Hauer* 11 Faculty of Chemistry, Institute of Technical Biochemistry, University ofStuttgart, Stuttgart, Germany2 BASF GE, Ludwigshafen, GermanyConsidering the membrane fraction of cells, one difference between bacteriaand eukaryotes is the absence of sterols as membrane constituents. Incontrast to eukaryotes, it is considered that some eubacteria producepentacyclic triterpenes of the hopanoid class as structural and functionalequivalents of sterols [1, 2]. Hopanoids are synthesized by squalene-hopenecyclases (SHC; EC 5.4.99.17), which catalyze the cyclization of triterpenesvia cationic intermediates in one of the most complex and powerful one-stepreactions known in biochemistry. Most of our understanding about thebiochemical and molecular mechanism of this reaction has been obtained bythe characterization of a SHC from Alicyclobacillus acidocaldarius(AaSHC; GI: 1435434).In our study, we characterized a novel SHC from the gram-negative, alcoholproducing bacterium Zymomonas mobilis (ZmSHC1; GI: 56552444) andcompared its activity and substrate spectrum with another, previouslydescribed squalene-hopene cyclase (ZmSHC2; GI: 6466213) from the sameorganism [1, 3]. In order to do this, we optimized the expression system forthese enzymes in Escherichia coli and the conditions for the enzymaticreactions. Subsequently, we determined the enzymatic activity of ZmSHC1with a variety of substrates including citronellal, homofarnesol and squalene.Despite the differences in chain length (C 10-C 30) and the presence of C=Cdouble bounds or functional groups like aldehydes at the position whereprotonation needs to occur for the initiation of the reaction, conversion couldbe found for all of these substrates. Beside the conversion of squalene tohopene, the cyclization of homofarnesol to ambroxan and citronellal toisopulegol is of particular interest, as these compounds are commonly usedin the manufacturing of fragrance and flavour concentrates or could providea bio-catalytic access for the production of menthol, respectively.Furthermore, our results revealed significantly higher rates of substrateconversion of ZmSHC1 in comparison to those, previously described for thesqualene-hopene cyclase AaSHC from A. acidocaldarius [4].spektrum | Tagungsband 2011