Characterization of the malic enzyme from Clostridium thermocellum reveals pyrophosphate as a possible regulator of metabolic flux during growth

Clostridium thermocellum produces ethanol plus CO₂ as end products from direct fermentation of cellulosic biomass.  Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing.  A deeper understanding of the regulatory mechanism controlling metabolic flux through competing biochemical pathways leading to H2, acetate, formate, and lactate may provide attractive targets for metabolic engineering and aid in developing reactor conditions for optimal ethanol production.  High energy metabolites, such as ATP, GTP, and pyrophosphate, can directly interact with key catabolic enzymes modulating their activities as cofactors or allosteric activators/inhibitors.  We describe the biochemical characterization of a purified NADP⁺ dependent  malic enzyme(MalE) from C. thermocellum which shows strong inhibition in the presence of relatively low concentrations of pyrophosphate.  This indicates the internal concentrations of pyrophosphate could regulate pyruvate synthesis through the malate shunt by inhibition of MalE.  The internal concentration of pyrophosphate was measured throughout growth and found to be highest in early exponential phase during growth on cellobiose.  Inorganic pyrophosphatases are believed to regulate the concentrations of pyrophosphate in actively growing cells.  C. thermocellum contains only a V-type inorganic pyrophosphatase while some clostridia contain V-type and/or soluble pyrophosphatases.  Internal concentrations of pyrophosphate were measured in other cellulolytic clostridia relative to the expression of these different types of pyrophosphatases to assess how the internal levels of pyrophosphate is influenced by the type of pyrophosphatase present in these organisms.  Understanding the fluctuations of high energy metabolites during growth will help elucidate their possible role in metabolic flux regulation.