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

GWP020Corynebacterium glutamicum engineered as a designerbug for the production of pyruvateW. Stefan*, B. Blombach, B.J. EikmannsInstitute of Microbiology and Biotechnology, University of Ulm, Ulm,GermanyCorynebacterium glutamicum is a non-pathogenic, Gram-positive organismthat grows on a variety of substrates and is used for the production of aminoacids, e.g. L-glutamate, L-lysine and L-valine, as well as organic acids, e.g.lactic and succinic acid. The aim of the present work was to engineer C.glutamicum to produce pyruvate. The resulting strain is supposed to be usedas a platform for production strains of amino acids and other organic acidsderived from pyruvate, e.g. dicarboxylic acids of the citric acid cycle. Thesemight be used as precursors for a variety of bulk chemicals andcommercially important polymers, which are these days produced primarilyfrom petrochemicals via chemical synthetic processes.In our study we modified C. glutamicum for the production of pyruvate anddecreased formation of byproducts (e.g. amino acids). By stepwiseinactivation of the pyruvate dehydrogenase complex, the pyruvate:quinoneoxidoreductase, the L-lactate dehydrogenase and attenuation of theacetohydroxyacid synthase [AHAS] by deleting the C-terminal domain of itsregulatory subunit, efficient pyruvate production was achieved. The deletionof the genes encoding alanine aminotransferase and pyruvate:valineaminotransferase led to a strong reduction of the side product L-alanine andtogether with the attenuation of the AHAS to decreased L-valine formation(below 5 mM). Above all, we observed efficient pyruvate formation up tonearly 200 mM in shake flask experiments, with a yield of ~0.7 g pyruvateper g of glucose. The most critical step for pyruvate formation is theattenuation of the AHAS. In fed-batch fermentations with the newlyconstructed C. glutamicum strain, final pyruvate concentrations of more than500 mM have been observed. Thus, the strain represents an efficientpyruvate producer and an ideal platform for pyruvate-derived metabolites.GWP021Extension of the substrate utilization range of Ralstoniaeutropha strain H16 for mannose and glucose bymetabolic engineeringS. Sichwart*, S. Hetzler, D. Bröker, A. SteinbüchelInstitute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, GermanyThe Gram-negative facultative chemolithoautotrophic bacterium Ralstoniaeutropha strain H16 is known for its narrow carbohydrate utilization rangewhich limits its use for biotechnological production of PHAs and possiblyother products from renewable resources. To broaden its substrate utilizationrange, which is for carbohydrates and related compounds limited to fructose,N-acetylglucosamine and gluconate, strain H16 was engineered to usemannose and glucose as sole carbon sources for growth. The genes for afacilitated diffusion protein (glf) from Zymomonas mobilis and for aglucokinase (glk), mannofructokinase (mak) and phosphomannose isomerase(pmi) from Escherichia coli were alone or in combination constitutivelyexpressed in R. eutropha strain H16 under control of the neokanamycin- orlac-promoter, respectively, using an episomal broad host range vector.Recombinant strains harboring pBBR1MCS-3::glf::mak::pmi orpBBR1MCS-3::glf::pmi grew on mannose, whereaspBBR1MCS-3::glf::mak and pBBR1MCS-3::glf did not confer the ability toutilize mannose as carbon source to R. eutropha. The recombinant strainharboring pBBR1MCS-3::glf::pmi exhibited slower growth on mannosethan the recombinant strain harboring pBBR1MCS-3::glf::mak::pmi. Thesedata indicated that phosphomannose isomerase is required to convertmannose-6-phosphate into fructose-6-phosphate for subsequent catabolismvia the Entner-Doudoroff pathway. In addition, all plasmids conferred to R.eutropha also the ability to grow in presence of glucose. Best growth wasobserved with a recombinant R. eutropha strain harboring plasmidpBBR1MCS-2::P nk::glk::glf. In addition, expression of the respectiveenzymes was demonstrated at the transcriptional and protein level and bymeasuring the activities of mannofructokinase (0.622 U mg -1 ± 0.063 U),phosphomannose isomerase (0.251 U mg -1 ± 0.017 U), and glucokinase(0.518 U mg -1 ± 0.040 U). Cells of recombinant strains of R. eutrophasynthesized poly(3-hydroxybutyrate) to about 65% - 67% (wt/wt) of cell drymass in presence of 1% (wt/vol) glucose or mannose as sole carbon sources.GWP022Conversion of 3-sulfinopropinonyl-CoA, a structuralanalogue of succinyl-CoA, to propionyl-CoA in Advenellamimigardefordensis strain DPN7 TM. Schürmann*, A. Deters, J.H. Wübbeler, A. SteinbüchelInstitute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, GermanyDegradation of 3,3-dithiodipropionate (DTDP), a sulfur containing precursorsubstrate for polythioester production, was investigated in Advenellamimigardefordensis strain DPN7 T . This bacterium was isolated due to itsability to utilize DTDP as a sole source of carbon and energy [1]. DTDP isinitially cleaved by a disulfide-reductase (LpdA) into two molecules of 3-mercaptopropionic acid [2]. In the next step a thiol dioxygenase (Mdo)catalyzes the oxidation to 3-sulfinopropionate (3-SP), which is thereafteractivated to the corresponding 3-SP-CoA thioester by a succinyl-CoAsynthetase (SCS) [3, 4]. A Tn5::mob-induced mutant, defective in growth onDTDP and 3-SP, was genotypically characterized. The transposon insertionwas mapped in an open reading frame with highest homologies to an acyl-CoA dehydrogenase (CaiA) from Verminephrobacter eiseniae strain EF01-2(63 % identical amino acids). A defined ΔcaiA mutant verified the observedeffects in the Tn5::mob induced mutant. For enzymatic studies CaiA washeterologously expressed in E. coli using pET23a::caiA. The purifiedenzyme catalyzed the conversion of 3-SP-CoA to propionyl-CoA. It istherefore a novel reaction for the abstraction of sulfur. FAD, as a putativecofactor of CaiA, has been isolated from the purified protein and its identitywas confirmed via HPLC-ESI-MS.[1] Wübbeler, J.H. et al (2006): Int J Syst Evol Microbiol. 56: 1305-10.[2] Wübbeler, J.H. et al (2010): Appl Environ Microbiol. 76:7023-8.[3] Bruland, N. et al (2009): J Biol Chem. 284:660-72.[4] Schürmann, M. et al. Submitted.GWP023Enhancing the Biodesulfurization of Dibenzothiophenewith Rhodococcus erythropolis IGTS8 using SyntheticSurfactantsW. El Moslimany*, B. Al-Nasser, R. HamzahBiotechnology, Arabian Gulf University, Manamah, BahrainBackground: Combustion of fossil fuels releases hazardous emissions likeSO 2 into the environment. This is due to the presence of high amounts oforganosulfur compounds such as dibenzothiophene (DBT) and its alkylatedderivatives. Strict environmental regulations imply that the amount of sulfurin transportation fuels be drastically reduced. The petroleum industry relieson hydrodesulfurization to remove sulfur from petroleum -derived fuels.This technique is costly, not completely efficient, and even environmentallypolluting. Biodesulfurization with dedicated microorganisms has beenproposed as an environmentally friendly and cost effective alternative orcomplement. However, large scale application of microbial desulfurizationis limited by the low biocatalytic efficacy.Aim of the work: In this work, Rhodococcus erythropolis IGTS8 wasadopted to study the influence of some synthetic surfactants on thebiodesulfurization activity using DBT as a model substrate.Methods: All experiments were conducted in mineral salts mediumcontaining glucose and dibenzothiophene as a sole sulfur source in thepresence or absence of surfactants. Cell-free culture supernatants wereanalyzed by HPLC to monitor the consumption of DPT and the formation ofthe dead end product 2-hydroxybiphenyl.Results: Among the tested surfactants, SDS was chosen to complete thestudies because it did not inhibit the growth of the IGTS8 strain. The IGTS8strain grew on glucose in the presence of SDS as a sole sulfur source after alag period of 3 days. However, The IGTS8 strain was not able to utilize SDSas a sole carbon source even after 2 weeks of incubation. The data alsoshowed that the biodesulfurization enzymes of the 4S pathway are functionalin the presence of different concentrations of SDS. Cultures growing onDBT in the presence of 1000 ppm of SDS transformed DBT faster thancontrol cultures lacking SDS.Conclusion: The surfactant SDS improved the biodesulfurization activity ofthe IGTS8 strain. The potential role of SDS in the biodesulfurization processappears to be promoting the solubility of the hydrophobic substrate, DBT, inwater. This would improve the mass transfer and, consequently, lead toenhanced biodesulfurization rates.[1] Feng, J. et al (2006): The surfactant tween 80 enhances biodesulfurization. Appl. Environ.Microbiol. 72:7390-7393.spektrum | Tagungsband 2011