While, PMF may be generated through PPi hydrolysis using a membrane RO4929097 mouse bound proton-translocating pyrophosphatase (PPase), the directionality of this PPase is unknown, and may in fact use PMF for PPi synthesis. PPi is a by-product of various endergonic biosynthetic reactions, including poly-nucleic acid synthesis from (deoxy)nucleotide triphosphates and activation of amino acids, carbohydrates, and fatty acids for protein, polysaccharide, and lipid synthesis [21].

Thus, the effective removal of PPi improves the thermodynamic feasibility of these reactions. Concentrations as low as 2 mM PPi have shown to inhibit growth of some bacteria [94]. In addition to serving as a central energy carrier, PPi serves to regulate key enzymes in carbohydrate metabolism including LDH in Ca. saccharolyticus[21], malic enzyme in C. thermocellum (Taillefer and Sparling, unpublished), ATP-dependent PFK in T. maritima[95], and PTA in C. acidiurici[96]. As mentioned above, PPi can be utilized in the glycolytic direction by (i) PPi-dependent 6-P-fructokinase, (ii) PPDK, and (iii) acetate thiokinase. Alternatively, hydrolysis of PPi via a membrane-bound PPase (Cthe_1425) can be coupled to C188-9 price PMF generation that could

be utilized for transport of nutrients, motility, and ATP synthesis. The PPi-dependent enzymes used by C. thermocellum have remarkable similarities to that of parasitic protists (ie. Trichomonas foetus, Entamoeba histolytica; [75]) and other bacteria such as Ca. saccharolyticus[97]. PPi levels in Ca. saccharolyticus have been shown to be elevated (4 ± 2 mM) during exponential phase and lower during transition

to stationary phase [97], consistent with other organisms that do not contain a cystolic PPase (C. thermoaceticum and C. pasteuranum; [98]). Conversely, PPi levels in E. coli, which possesses a cystolic PPase, were low (0.3 mM) and did not fluctuate during growth [98]. We observed a 1.9-fold increase in membrane-bound PPase expression in stationary phase cells. Conclusions A unified understanding of how gene and gene-product expression, stability, and regulation, in conjunction with intracellular metabolic Adenosine profiling and thermodynamics of product formation, are key elements for targeted metabolic engineering strategies and fermentation optimization for the economic feasibility of biofuels production via consolidated bioprocessing. Clostridium thermocellum, like many cellulolytic, fermentative, biofuel producing organisms, has multiple enzymes capable of catalyzing parallel reactions and branched product pathways. Measuring peptide spectral counts via shotgun proteomics has been shown to be a valid method for determining relative protein abundance profiles [57–60]. In turn, understanding protein expression profiles may provide genetic engineering strategies targeted at redirecting carbon and electron flux for the optimization of end-product production. Furthermore, responses of protein expression in response to physiological conditions (ie.