Elucidation of Clostridium Acetobutylicum metabolism Using Parallel Labeling Experiments and 13c Metabolic Flux Analysis

Given their wide substrate range and high specific substrate utilization rates, solventogenic clostridia are seen as a promising class of organisms for biofuel and commodity chemical production. Clostridium acetobutylicum was historically used for industrial-scale fermentation and remains a potential candidate for butanol and butyric acid production today. However, although the biochemistry of C. acetobutylicum has been extensively reviewed, the central metabolic pathways have remained only partially resolved. Two recent reconstructions of genome-scale models have proposed different mechanisms for the biosynthesis of α-ketoglutarate, an important precursor for amino acid biosynthesis. Initial stable-isotope labeling experiments and qualitative ¹³C-isotopomer analysis supported the idea of an incomplete tricarboxylic acid (TCA) cycle and suggested a Re-stereospecificity for the citrate synthase reaction.

In this work, we have rigorously validated the metabolic network model of C. acetobutylicum to guide future efforts aimed at the metabolic engineering of this organism for biofuels/chemicals production. Using parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA), we quantitatively elucidated central carbon metabolism and amino acid metabolism. Contrary to previously proposed hypotheses, we found that while the TCA cycle runs in the oxidative direction, there is no notable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, or malate and oxaloacetate, and that the conversion of succinyl-CoA to succinate proceeds independently. Additionally, using multiple ¹³C-labeled amino acid tracers we identified a novel metabolic cycle that involves central carbon and amino acid metabolism where carbon flows from aspartate to threonine, serine, pyruvate, oxaloacetate, and back to aspartate. The physiological significance of this novel metabolic cycle in the evolution of C. acetobutylicum will be discussed.

The validated metabolic network model was used to study the metabolic stress response of C. acetobutylicum under butanol and butyric acid stresses. A major drawback to clostridia-based industrial production of these metabolites is their toxic effects on the cells, resulting in low product titers and inducing the metabolite stress response. While several studies have been carried out to understand the transcriptional and translational basis of this stress response, little is known about the impact of butanol and butyric acid stress on the metabolic pathways and fluxes of C. acetobutylicum. Here, we have used parallel labeling experiments and qualitative ¹³C-isotopomer analysis to characterize the short term and long term effects of butanol and butyric acid stress on the metabolism of C. acetobutylicum. We show that while butanol and butyric acid result in lower absolute rates of glucose uptake and cell growth rate, the relative intracellular flux distributions remain relatively unchanged under stress. As there is growing evidence that the metabolite stress response is connected to tolerance, this work contributes to a systems understanding of the stress response and its regulation, which may lead to new strategies for achieving higher tolerance in C. acetobutylicum to these and other products.