Abstract
BackgroundThe 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.ResultsFollowing 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.ConclusionsIn 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.
Highlights
The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism
We have demonstrated that the strategy of intracellular ATP depletion can be applied in anaerobic cell factories such as C. acetobutylicum, to enhance glycolysis and improve end product formation
Reduced intracellular ATP level and ATP/ADP ratio by overexpression of F1‐ATP hydrolase (ATPase) genes F1-ATPase is the water-soluble component of the F1Fo-ATP synthase and catalyzes ATP hydrolysis
Summary
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. Increasing the proportion of free-form F1 by reducing the expression of Fo or enhancing the expression of F 1 could promote ATP hydrolyzation, reduce the intracellular ATP level, and induce glycolysis. Previous studies demonstrated that in many aerobic cell factories decreasing intracellular ATP level forced the cells to maintain a higher substrate uptake rate, promoted glycolysis to regenerate NAD+, and enhanced fluxes towards end products. Increasing relative abundance of F 1 unit increased the glucose consumption in Bacillus subtilis [13] and Corynebacterium glutamicum [14] All these efforts were accomplished in aerobic or facultative aerobic microbes; whether this strategy is applicable in obligate anaerobes remains unclear
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