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

stationary phase, indicating that the gene cluster did not contain all relevantgenes for RoF biosynthesis. The rosA transcript was detected by reversetranscription PCR in S. davawensis cells in the stationary growth phase butnot in the exponential phase.[1] Otani, S. et al (1974): J Antibiot (Tokyo) 27, 86-87.[2] Otani, S. et al (1980): Methods Enzymol 66, 235-241.[3] Juri, N. et al (1987): J Biochem (Tokyo) 101, 705-711.[4] Matsui, K. et al (1979): J Biochem (Tokyo) 86, 167-175.GWV008Enzyme Engineering of an Enoate Reductase fromZymomonas mobilis Affecting the Enzyme Activity andEnantioselectivityS. Reich*, B.M. Nestl, B. HauerInstitute of Technical Biochemistry, University of Stuttgart, Stuttgart,GermanyRecently, the stereoselective bioreduction of activated alkenes has emergedas a valuable tool for the synthesis of various enantiopure compounds.In this light, flavin-dependent enoate reductases are interesting enzymes forthe industrial production of such chiral compounds, because they are able toreduce activated alkenes exclusively in a trans-specific fashion, which goesin hand with the creation of up to two new chiral centers [1].In this project we used a site directed mutagenesis approach and theexchange of several loops between two enoate reductases, OYE1 fromSaccharomyces carlsbergenisis and NCR from Zymomonas mobilis [2] toidentify new enzyme variants that are able to reduce various α, β -unsaturated aldehydes and ketones [3,4].Three variants possessed increased activity towards all substrates testedcompared to wild type NCR. Furthermore one variant was obtained thatshowed a significant influence on the enantioselectivity of the enzyme.[1] Stuermer, R. et al (2007): Curr. Opin. Chem. Biol., 11, 203-213.[2] Müller, A. et al (2007): Biotechnol. Bioeng., 98, 22-29.[3] Williams, R. E. et al (2002): Microbiology, 148, 1607-1614.[4] Toogood, H.S.(2010): ChemCatChem, 2, 892-914.GWV009Corynebacterium glutamicum engineered for efficientisobutanol productionB. Blombach* 1 , T. Riester 1 , S. Wieschalka 1 , C. Ziert 2 , J.-W. Youn 2 ,V.F. Wendisch 2 , B.J. Eikmanns 11 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,Germany2 Faculty of Biology & CeBiTec, Genetics of Prokaryotes, University ofBielefeld, Bielefeld, GermanyWe recently engineered Corynebacterium glutamicum for aerobicproduction of 2-ketoisovalerate by inactivation of the pyruvatedehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B,and additional overexpression of the ilvBNCD genes, encodingacetohydroxyacid synthase, acetohydroxyacid isomeroreductase, anddihydroxyacid dehydratase (1). Based on this strain, we engineered C.glutamicum for the production of isobutanol from glucose under oxygendeprivation conditions by inactivation of L-lactate and malatedehydrogenases, implementation of ketoacid decarboxylase fromLactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomycescerevisiae, and expression of the transhydrogenase genes pntAB fromEscherichia coli. The resulting strain produced isobutanol with a substratespecific yield (Y P/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, achromosomally encoded alcohol dehydrogenase rather than the plasmidencodedADH2 from S. cerevisiae was involved in isobutanol formationwith C. glutamicum and overexpression of the corresponding adhA geneinstead of the ADH2 gene increased the Y P/S to 0.77 ± 0.01 mol isobutanolper mol of glucose. Inactivation of the malic enzyme significantly reducedthe Y P/S, indicating that the metabolic cycle consisting of pyruvate and/orphosphoenolpyruvate carboxylase, malate dehydrogenase and malic enzymeis responsible for the conversion of NADH+H + to NADPH+H + . In fed-batchfermentations with an aerobic growth phase and an oxygen-depletedproduction phase, the most promising strain C. glutamicum ∆aceE ∆pqo∆ilvE ∆ldhA ∆mdh (pJC4ilvBNCD-pntAB) (pBB1kivd-adhA) producedabout 175 mM isobutanol with a volumetric productivity of 4.4 mmol l -1 h -1 ,and showed an overall Y P/S of about 0.48 mol per mol of glucose in theproduction phase.[1] Krause F.S. et al (2010): Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production. Appl. Environ. Microbiol. 76:8053-8063.GWV010Phosphotransferase system (PTS) independent glucoseutilization in Corynebacterium glutamicum by inositolpermeases and glucokinases and application forimproved L-lysine productionS. Lindner* 1 , G.M. Seibold 2 , A. Henrich 2 , R. Krämer 2 , V.F. Wendisch 11 Genetics of Prokaryotes BioVI, University of Bielefeld, Bielefeld, Germany2 Institute of Biochemistry, University of Cologne, Cologne, GermanyCorynebacterium glutamicum is used for the annual production of 1.3million tons of L-lysine from starch hydrolysates and molasses. Thepredominant carbon sources in these feedstocks, glucose, sucrose, andfructose, are substrates of the phosphoenolypyruvate dependentphosphotransferase system (PTS), which is the major path of glucose uptakeand which is essential for sucrose and fructose utilization by C. glutamicum.Some growth from glucose is retained in the absence of the PTS. Thegrowth defect of a deletion mutant lacking the general PTS component Hprin glucose medium could be overcome by suppressor mutations leading tohigh expression of inositol utilization genes or by addition of inositol to thegrowth medium if a glucokinase is overproduced simultaneously. PTSindependentglucose uptake was shown to require at least one of the inositoltransporters IolT1 or IolT2 as a mutant lacking IolT1, IolT2 and the PTScomponent Hpr could not grow with glucose as sole carbon source. Efficientglucose utilization in the absence of the PTS necessitated overexpression ofa glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 arelow affinity glucose permeases with K S-values of 2.8 mM and 1.9 mM,respectively. As glucose uptake and phosphorylation via the PTS differsfrom glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinaseby the requirement for phosphoenolpyruvate, the roles of the two pathwaysfor L-lysine production were tested. The L-lysine yield by C. glutamicumDM1729 was lower than by its PTS-deficient derivate DM1729Δhpr, which,however, showed low production rates. Combined overexpression of iolT1or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, inDM1729Δhpr enabled L-lysine production as fast as by the parent strainDM1729, but with 10 to 20 % higher L-lysine yield.GWV011Biotechnological conversion of glycerol to 2-amino-1,3-propanediol (serinol) in recombinant Escherichia coliB. Andreeßen*, A. SteinbüchelInstitute for Molecular Microbiology and Biotechnology, WestphalianWilhelms-University, Münster, GermanyThe biodiesel industry is very much interested to convert the huge surplus ofglycerol, which is obtained during transesterification of the fatty acids fromvegetable oils or fats with methanol, into higher value products. Onepromising molecule is 2-amino-1,3-propanediol better known as serinol. Ithas become an important intermediate for several chemical applications inthe last years. Amino alcohols like serinol are widely used as precursers fornon-ionic contrast agents like 1-N,3-N-bis(1,3-dihydroxypropan-2-yl)-5-[(2S)-2-hydroxypropanamido]-2,4,6-triiodobenzene-1,3-dicarboxamide(iopamidol). Iopamidol is used as contrast agent for angiography throughoutthe cardiovascular system. Serinol is also an intermediate for drugs dealingwith pain treatment, and chiral (1R,2R) phenylserinols have been used asprecursors in chloramphenicol synthesis since 1947. Until now serinol isnormally produced chemically from 2-nitro-1,3-propanediol,dihydroxyacetone and ammonia, dihydroxyacetone oxime or 5-amino-1,3-dioxane. A biological approach to synthesize serinol was designed usingamino alcohol dehydrogenases like the AMDH from Streptomyces virginiaeIFO 12827 in vitro. We constructed an artificial pathway and established forthe first time an in vivo serinol production. Therefore, we expressed thebifunctional dihydroxyacetonephosphate aminotransferase/dihydrorhizobitoxine synthase RtxA from Bradyrhizobium elkanii USD94 inrecombinant Escherichia coli strains. In flask experiments these strains wereable to accumulate serinol up to 3 g/l in the supernatant. 2-amino-1,3-propanediol was isolated by converting it into the correspondinghydrochloride. Further purification was achieved by cation exchangechromatography employing a Dowex ® fine mesh resin and elution withammonium hydroxide. With this method 60 % of the product was recovered.spektrum | Tagungsband 2011