Redirecting carbon flux through exogenous pyruvate kinase to achieve high ethanol yields in Clostridium thermocellum

In Clostridium thermocellum, a thermophilic anaerobic bacterium able to rapidly ferment cellulose to ethanol, pyruvate kinase (EC 2.7.1.40) is absent based on both the genome sequence and enzymatic assays. Instead, a new pathway converting phosphoenolpyruvate to pyruvate via a three-step pathway involving phosphoenolpyruvate carboxykinase, NADH-linked malate dehydrogenase, and NADP-dependent malic enzyme has been found. We examined the impact of targeted modification of enzymes associated with this pathway, termed the “malate shunt”, including expression of the pyruvate kinase gene from Thermoanaerobacterium saccharolyticum, mutation of the phosphoenolpyruvate carboxykinase and deletion of malic enzyme gene. Strain YD01 with exogenous pyruvate kinase, in which phosphoenolpyruvate carboxykinase expression was diminished by modifying the start codon from ATG to GTG, exhibited 3.25-fold higher ethanol yield than the wild-type strain. A second strain, YD02 with exogenous pyruvate kinase, in which the gene for malic enzyme and part of malate dehydrogenase were deleted, had over 3-fold higher ethanol yield than the wild-type strain. ► The absence of pyruvate kinase in Clostridium thermocellum has been confirmed. ► Malate shunt converts phosphoenolpyruvate to pyruvate via a three-step pathway. ► The ethanol yield was increased over 3-fold with genetic modifications.