Pyrophosphate-free glycolysis in Clostridium thermocellum increases both thermodynamic driving force and ethanol titers.

Background: Clostridium thermocellum is a promising candidate for production of cellulosic biofuels, however, its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild-type organism, this reaction uses pyrophosphate (PP) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor.

Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism.

Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. C and H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to C-tracers.