A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum.

Abstract

ABSTRACTEngineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacterium Clostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model, iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering of C. acetobutylicum to develop a commercial process for the production of n-butanol.