Functional Analysis of H(+)-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PP(i)) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PP(i) was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H(+)-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk–malate shunt cycle, and acetate cycling in generating PP(i). Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PP(i) sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PP(i) sources reduced the growth rate by 22% (0.30 ± 0.01 h(−1)) and the biomass yield by 38% (0.18 ± 0.00 g(biomass) g(substrate)(−1)), this change was much smaller than what would be expected for stoichiometrically essential PP(i)-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PP(i)-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PP(i) altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PP(i) sources individually and combined play no significant PP(i)-supplying role, and the true source(s) of PP(i), or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H(+)-pumping membrane-bound PPase, glycogen cycling, a Ppdk–malate shunt cycle, and acetate cycling are not significant sources of PP(i) supply, this study adds functional annotation of four genes and availability of an updated PP(i) stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PP(i), or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PP(i)- and ATP-dependent glycolysis.