Ruminiclostridium thermocellum, an anaerobic Gram-positive thermophile capable of direct lignocellulosic hydrolysis producing ethanol, H2, CO2, acetate, formate, and lactate being as major end products. The high rate of cellulose hydrolysis combined with the ability to produce ethanol makes R. thermocellum a very attractive organism for the generation of ethanol via consolidated bioprocessing. However, the branched nature of the R. thermocellum central catalysis limits the carbon and electrons available for ethanol production, therefore limiting its industrial potential when compared to current ethanol production technologies and current fossil fuel prices. Due to the branched nature of the R. thermocellum, understanding of the carbon and electron flux regulation is vital in understanding the metabolism associated with ethanol production. The understanding of flux also opens important doors in terms of genetic modifications increasing ethanol yields and cell growth rates making a more industrially competitive strain of R. thermocellum. The transcript of R. thermocellum showed that the transcription of end product synthesis genes do not necessarily correlate with the presence of the particular function in the cell. Rather, the carbon and electron flux in the cells seem to be regulated by the presence of small high-energy metabolites such as PPi since the presence of PPi directly regulates the activity of key central catabolism proteins such as the glucokinase, phosphofructokinase, and the malic enzyme, as well as being a phosphate group donor replacing the reliance ATP as an energy carrier. The glucokinase and phosphoglycerate kinase display a regulation that favors relatively low levels of cofactor, GTP or ATP, by having substrate inhibition leading to decreases in activity based on increase cofactor concentrations.
The importance of small molecule regulation is reinforced by the intracellular concentrations observed in R. thermocellum and closely related organisms. The concentration of PPi increases rapidly upon inoculation with a slow decrease reaching a minimum at the onset of stationary phase. The PPi replaces ATP as the indicator for the energetic state of the cell influencing various cellular mechanisms by direct modulation of enzyme activity or by the activation of transcriptional regulators.