Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes

Abstract

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.

Improving Fructose Utilization and Butanol Production by Clostridium acetobutylicum via Extracellular Redox Potential Regulation and Intracellular Metabolite Analysis

“…[5] ATP is responsible for numerous physiological metabolism associated with sugar phosphorylation, cell growth, biosynthesis of lipids, and proteins as well as stress response of C. acetobutylicum. [39] It was reported that ATP could play a major role not only increasing ABE production but also regulating the metabolic shift towards solventogenesis. [26] Grupe and Gottschalk showed that low ATP level resulted in acids formation, but high ATP level responded to ABE biosynthesis in continuous fermentation.…”

Chaotropicity: a key factor in product tolerance of biofuel-producing microorganisms

“…Overexpression of genes involved in energy generation in Clostridium acetobutylicum (i.e. pfkA-6-phosphofructokinase, and pykA-pyruvate kinase) has enhanced tolerance to butanol and ethanol, increasing yields by 29.4 and 85.5% respectively [201] Metabolically engineered yeasts must be capable of improved productivity, robustness and persistence in industrial fermentations. Brazilian studies of the microbiological dynamics of bioethanol fermentations revealed a rapid succession of yeast strains leading to the replacement of the initial 'starter' yeast culture that used as inoculum by indigenous strains that were present in the raw sugar-cane juice after several weeks [65] e .…”

Metabolic engineering of clostridia for the production of chemicals

“…In another study, the pfkA and pykA genes were overexpressed in C. acetobutylicum to increase the ATP and NADH level. The engineered strain resulted in the production of 19.0 g l −1 butanol by fed‐batch fermentation …”