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

174PSV008Physiological effects of disrupting the acetate and acetoneformation pathways in Clostridium acetobutylicumD. Lehmann*, T. Lütke-EverslohInstitute of Biological Sciences/University of Rostock, Division ofMicrobiology, Rostock, GermanyClostridial acetone-butanol-ethanol (ABE) fermentation is a natural sourcefor microbial n-butanol production and regained much interest in academiaand industry in the past years. Due to the difficult genetic accessibility ofClostridium acetobutylicum and other solventogenic clostridia, successfulmetabolic engineering approaches are still rare. In this study, a set of fiveknock-out mutants with defects in the central fermentative metabolismwere generated using the ClosTron technology, including the constructionof targeted double knock-out mutants of C. acetobtuylicum ATCC 824.While disruption of the acetate biosynthetic pathway had no significantimpact on the metabolite distribution, mutants with defects in the acetonepathway, including both acetoacetate decarboxylase- (Adc) andacetoacetyl-CoA:acyl-CoA transferase- (CtfAB) negative mutants,exhibited high amounts of acetate in the fermentation broth. Distinctbutyrate increase and decrease patterns during the course of fermentationsprovided experimental evidence that butyrate, but not acetate, is reassimilatedvia an Adc/CtfAB-independent pathway in C. acetobutylicum.Interestingly, combining the adc and ctfA mutations with a knock-out ofthe phosphotransacetylase- (Pta) encoding gene, acetate production wasdrastically reduced, resulting in an increased flux towards butyrate. Exceptfor the Pta-negative single mutant, all mutants exhibited a significantlyreduced solvent production in pH-uncontrolled batch fermentations ascompared to the wildtype.PSV0094-Hydroxybutyryl-CoA dehydratase, a radical enzyme inmetabolic pathways of anaerobic Bacteria and ArchaeaJ. Zhang* 1,2 , P. Friedrich 3 , B.M. Martins 4 , W. Buckel 1,21 Philipps-Universität, Fachbereich Biologie, Marburg, Germany2 Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany3 University of Newcastle upon Tyne, Chemistry, Newcastle, United Kingdom4 Humboldt-Universität zu Berlin, Biologie, Berlin, Germany4-Hydroxybutyryl-CoA dehydratase (AbfD) was discovered in thefermentation of 4-aminobutyrate to ammonia, acetate and butyrate inClostridium aminobutyricum [1]. Recently, this radical enzyme has beenalso identified in two new CO 2 fixation pathways in Archaea [2]. The[4Fe-4S] cluster and FAD containing AbfD catalyzes the oxygen sensitiveand chemical difficult dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, since the unactivated -proton has to be removed (pK ~ 40). Thesuccessful production of variants of recombinant AbfD in Escherichia colidemonstrated that all highly conserved amino acids around the activecenter are essential for activity. Surprisingly, the low residual activity ofthe Y296F variant (