Functional Analysis of H⁺-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate...

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.