Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Clostridium acetobutylicum is a promising biocatalyst for the production of n-butanol at high yield from renewable resources. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it was previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remained unknown until this study, were necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purified, identified and characterized the proteins responsible for both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase activities and demonstrated the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrated the yield of butanol formation was limited by the level of expression of CAC_0764, the ferredoxin-NADP+ reductase encoding gene. The integration of these enzymes into metabolic engineering strategies introduces new opportunities for developing a homobutanologenic C. acetobutylicum strain.

Enforcing ATP hydrolysis enhanced anaerobic glycolysis and promoted solvent production in Clostridium acetobutylicum

Background The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. Results Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. Conclusions In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.