The 6-phosphofructokinase reaction in Acetivibrio thermocellus is both ATP- and pyrophosphate-dependent

Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PP_i-dependent 6-phosphofructokinase (PP_i-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PP_i-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PP_i can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PP_i-generating mechanism to drive glycolysis, as PP_i-Pfk can simply use whatever PP_i is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPP_i-Pfk strain, unable to valorize PP_i, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PP_i. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/g_x/h. These findings are both relevant for fundamental insight into dual ATP/PP_i Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.