13C metabolic flux analysis identifies a novel metabolic cycle in Clostridium acetobutylicum that involves central carbon and amino acid metabolism

Given their wide substrate range, solventogenic clostridia are seen as a promising class of organisms for biofuel production. Clostridium acetobutylicum was historically used for industrial-scale fermentation and remains a potential candidate for butanol 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. Initial stable-isotope labeling experiments and qualitative ¹³C-isotopomer analysis have supported the idea of an incomplete tricarboxylic acid (TCA) cycle and suggested a Re-stereospecificity for the citrate synthase reaction. Further insights into the metabolism of C. acetobutylicum may guide future efforts aimed at the metabolic engineering of this organism for biofuels production.

In this work, we have rigorously validated the metabolic network model of C. acetobutylicum. Using parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA), we quantitatively elucidated the 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 cycle in the evolution of C. acetobutylicum will be discussed.