Metabolic Engineering of Clostridium Acetobutylicum with CoA Metabolite Analysis for Enhanced Butanol Production

Clostridium acetobutylicum have been traditionally used for butanol production. However, economically feasible production is still limited by low solvent titer and productivity of traditional acetone-butanol-ethanol (ABE) fermentation. Here, we report the development of hyper solvent producing C. acetobutylicum having high volumetric productivity followed by integration of adsorptive continuous ex situ butanol recovery fermentation for high solvent titer and enhanced productivity. Metabolically engineered strain was constructed by simultaneous disruption of pta and buk genes, which encode phosphotransacetylase and butyrate kinase, and overexpression of adhE1-ctfAB operon, encoding aldehyde-alcohol dehydrogenase for high volumetric productivity. The strain produced ABE with the productivity of 1.4 g/L/h, which are 230% higher than those obtained from wild type in batch fermentation. Continuous ex situ butanol recovery fermentation was performed with the engineered strain to further improve ABE titer and productivity. The solvent productivity and desorbed ABE titer of 2.28 g/L/h and 46.3 g/L, respectively, was achieved. However, ethanol was accumulated and strain degeneration was occurred during continuous fermentation. Thus, target gene for metabolic engineering was examined with CoA metabolite analysis and the strain was further engineered by overexpressing thiolase (atoB) gene of Escherichia coli, which catalyzes condensation of two acetyl-CoA to an acetoacetyl-CoA molecule to reduce ethanol accumulation. The final recombinant strain was able to produce solvent with a high yield, productivity, and desorbed ABE titer of 0.38 g/g glucose, 2.64 g/L/h, and 55.7 g/L, respectively, with continuous ex siturecovery fermentation during 100 h. Ethanol titer was maintained less than 4 g/L in fermentor throughout the fermentation. Significant strain degeneration was not observed. The achieved ABE volumetric productivity is the highest among the reported adsorptive fermentations with Clostridia.

Keywords:

Clostridium acetobutylicum, metabolic engineering; CoA metabolite, high productivity, ex situ butanol recovery fermentation

This work was supported by the Center for Organic Wastes to Energy Business under the Environmental Technology Development Program funded by the Ministry of Environment, Korea (2013001580001).