Interestingly, despite the presence xylanases, sequence homology-based annotation has not revealed the presence of xylose reductase, xylitol dehydrogenase, xylose isomerase,
or xylulokinase required for xylose selleckchem utilization. This suggests that, in the absence of cellulose, BI 2536 cost C. thermocellum may be predisposed to expressing xylanases, which typically degrade hemicellulosomal xylans, exposing buried cellulose fibres. With the exception of a 2-fold increase in cellulosomal glycosidases Cthe_0821, Cthe_2761, and Cthe_0745, and a 1.6-fold decrease in XynD (Cthe_0625), no other statistically significant changes were observed in detected cellulosomal cellulases during transition from exponential to stationary phase. While this contradicted selleck high variability in transcription of cellulosomal glycosidases of cellulose-grown cells , lack of variability in our experiment may have been attributed to differences in growth substrate used. In fact, Dror et al. found negligible changes in transcription of celB, celG, celD, and celF between exponential and stationary phase cellobiose-grown cultures . Alternatively, our processing method, which included several wash steps prior to lysing the cells,
may have imposed bias and variability by potentially washing off weakly bound cellulosomal glycosidases. In addition to cellulosomal glycosidases, 35 non-cellulosomal CAZymes that do not have a dockerin domain are encoded in the genome. Of the 19 non-cellulosomal CAZymes detected in exponential phase
cell-free extracts using 2D-HPLC-MS/MS, half Cyclin-dependent kinase 3 had RAI ratios in the top 90% (RAI > 0.1) of total peptides detected. Not surprisingly, the most abundant CAZyme cellobiose phosphorylase Cthe_0275 (glycosyltransferase family 36), which is involved in intracellular phosphorylytic cleavage of cellobiose, fell within the top 25% of detected proteins. Cellobiose phosphorylase Cthe_2989 was also found in high amounts (RAI = 0.23), whereas glycosyltransferase Cthe_1221, a putative cyclic β-1,2 glucan synthetase, was detected in the bottom 10% of all proteins detected (Figure 2a). CelI, an endo-1,4-β-glucanase (Cthe_0040) was not detected, consistent with growth on cellobiose. Other highly abundant non-cellulosomal CAZymes include amidohydrolase (Cthe_1777), glucoamylase (Cthe_1787), xylanase A precursor (Cthe_1911), α-N-arabinofuranosidase (Cthe_2548), CelC (Cthe_2807), and several less characterized glycosidases (Cthe_3163, Cthe_1911, Cthe_2989). While Raman et al.