49 −1.13 −1.17 −1.00 1.92 2.52 1.36 Cthe_2975 RNA polymerase sigma-I factor 1.24 1.47 −7.26 −2.59 −2.09 −1.94 1.15 1.45 4.32 1.96 Cthe_0403 RNA polymerase sigma-I factor −1.65

−1.92 −5.17 −3.64 1.89 1.76 −1.66 −1.08 −1.12 1.83 Bold values indicate significantly different expression levels as determined by ANOVA. For the PM vs. WT in 0% and 10% v/v Populus hydrolysate a positive/negative BGB324 concentration value represents a higher/lower level of expression in the PM compared to the WT. For the standard medium (0%) versus Populus hydrolysate media (10 or 17.5%) a positive/negative value represents a higher/lower expression in the hydrolysate media compared to standard medium. Values are indicated for samples collected during the mid-log (ML) and late-log (LL) growth phases. Categories of gene with increased Trichostatin A supplier expression in the PM The PM increases the gene expression in only two categories compared to the WT in standard and Populus hydrolysate media: energy production and conversion, and amino acid transport and metabolism (Figure 1). In addition to these, the PM also increases the expression of inorganic ion metabolism and transport genes compared to the WT in 10% v/v Populus hydrolysate medium. The increased expression in the energy production and conversion genes may allow for the increased growth phenotype observed in the PM strain. Increases in

glycolysis would lead to increases in reducing power (in the form of NADH) being available for downstream electron transport and ethanol production. The increase in ethanol production and increase in electron flux may generate sufficient NAD+ to ensure increased PLEKHB2 cellular metabolism [8]. The assemblage of genes encoding proteins involved in pyruvate metabolism and end-product synthesis dictate, in part, how carbon and electrons

flux is distributed between the catabolic, anabolic, and energy producing pathways of the cell [25]. C. thermocellum catabolizes glucose via the Embden-Meyerhof pathway using the “malate shunt” (Figure 2) [26–28]. Compared to the WT, the PM had a higher expression of 23 and 44 genes belonging to the energy production and conversion category in standard and Populus hydrolysate media, respectively. The PM upregulated 8 genes specific to the central metabolism and mixed-acid fermentation compared to the WT in standard medium (Figure 2 and Table 2). In 10% v/v Populus hydrolysate medium, the PM upregulated 10 genes along the central metabolism and mixed acid fermentation pathways compared to the WT. The PM has a mutation in the non-coding region upstream of the Cthe_0422-Cthe_0423 operon which encodes the rex (redox) repressor and the adhE alcohol dehydrogenase. This mutation may cause the observed increase in ethanol production [17,18]. A study of the effect of cellulose fermentation found that the central metabolism genes are typically upregulated during cellulose fermentation compared to cellobiose fermentation that the cells were grown on in this study [12,25].