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

Abstract

Engineering 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.

Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum

“…A recent study showed that AdeE2 contributed almost 100% for butanol production under alcoholgenic conditions whilst contributing no greater than 9% under solventogenic conditions19. This suggests that the expression of adhE2 is sensitive to culture conditions, which may be one reason why our results differ from those in a previous report where there was no change of fermentation products in adhE2 mutant25.…”

Consolidated bioprocessing for butanol production of cellulolytic Clostridia: development and optimization

“…The performance of synthetic consortia significantly depends on the benefit of the interaction between strain species (Song et al, 2014), which can be further improved via genetic engineering of strains and optimization of culture conditions. However, the empirical strategy needs to be combined with model-driven analysis (Salimi et al, 2010;Yoo et al, 2015;Zomorrodi and Segre, 2016), to rationally design and regulate the synthetic consortia towards increased efficiency and robustness (Zomorrodi and Segre, 2016).…”

Development of a genome-scale metabolic model ofClostridium thermocellumand its applications for integration of multi-omics datasets and strain design

“…18 In addition to these core and genome-scale steady-state metabolic models, a kinetic model of central metabolism, k-ctherm118, was recently developed and used to elucidate the mechanisms of nitrogen limitation and ethanol stress. 15 Due to the biotechnological relevance of the Clostridium genus, GSMs have also been developed for other species, 19 including C. acetubutylicum , 20–26 C. beijerinckii , 27 C. butyricum , 28 C. cellulolyticum , 22 and C. ljungdahlii . 29…”