In this study, the EPZ015938 Cthe1053 gene displayed low expression and lactate was not detected during cellulose fermentation. Although another gene annotated as ldh (Cthe0345) was expressed at high levels, this may be related to the participation of the encoded enzyme as a malate dehydrogenase in the alternate route

for conversion of PEP to pyruvate, as discussed earlier (Figure 4). Pyruvate formate lyase (pfl) catalyzes the conversion of pyruvate to formate, along with the formation of acetyl-CoA. Sparling et al, reported formate synthesis in C. thermocellum via this pathway with detection of transcripts for pfl (Cthe0505) and the pfl activating enzyme (Cthe0506) by RT-PCR [13]. In this study, two out of four putative pfl activating enzymes (Cthe0506, Cthe0647) were expressed at relatively high levels during cellulose fermentation (Additional file 5; data not available for pfl, Cthe0505). However, formate was not detectable

in Vorinostat concentration the culture supernatant consistent with other previous reports [25, 28]. Acetyl-CoA is further catabolized to acetate with generation of ATP or to ethanol with reoxidation of NADH. C. thermocellum encodes an NADH-dependent aldehyde dehydrogenase (aldH, Cthe2238), which catalyzes the conversion of acetyl-CoA to acetaldehyde, and several iron-containing alcohol dehydrogenases (Cthe0101, Cthe0394 [adhY] and Cthe2579 [adhZ]) for alcohol synthesis from acetaldehyde; also encoded is a bi-functional acetaldehyde/alcohol dehydrogenase (Cthe0423, adhE) which catalyzes the direct conversion of acetyl-CoA to ethanol (Figure 4). AdhE has been proposed to be a key enzyme for ethanol synthesis in C. thermocellum Resminostat and transcription

of adhE, adhY and adhZ has been confirmed by RT-PCR analysis in cellobiose and cellulose cultures of C. thermocellum [11, 19]. In this study, the aldH gene showed increased expression during stationary phase while the three adh genes, Cthe0394, Che0423 and Cthe0101, were actively expressed during cellulose batch fermentation with the latter showing decreased expression in stationary phase (Figure 4, Additional file 5). Acetyl-CoA is indirectly converted to acetate via acetyl-phosphate through the action of two enzymes, encoded by the contiguous genes, phosphotransacetylase (pta, Cthe1029) and acetate kinase (ack, Cthe1028), with the generation of one ATP per acetate molecule. The reverse reaction for direct conversion of acetate to acetyl-CoA utilizes ATP and is catalyzed by acetyl-CoA synthetase (acs, Cthe0551). Previous studies have confirmed the expression of acetate kinase through RT-PCR [11] and enzyme activity measurements [25]. In this study, both pta and ack genes were expressed at low levels which further decreased in stationary phase; whereas, the acs gene was expressed at relatively higher levels over the entire course of the fermentation (Figure 4, Additional file 5).