Of the 163 genes that encode for various parts of the amino acid transport and metabolism, the PM upregulated a significant number of genes (20 and 37 genes) compared to the WT in standard and Populus hydrolysate media. Most significantly, the PM increased the expression of 10 of the 15 genes along the histidine metabolism pathway compared to the WT

in standard medium (Table 4). Cthe_2880-Cthe_2889 is a single operon and is among the most highly differentially expressed genes in the PM versus WT comparison, with an find more average 23-fold to 31-fold increase in expression in standard and Populus hydrolysate media. The PM decreases the expression of one gene in this pathway, Cthe_3028 which converts histidine to histamine (Figure 3). De novo biosynthesis of histidine during fermentation may be constrained by the high NADH/NAD+ ratio during anaerobic growth and the requirement for further

reduction of NAD+ in Go6983 solubility dmso the two terminal steps of biosynthesis [17]. Histidine may be limited by the addition of furfural [17]. The PM has two mutations involved with glutamate catabolism; a possible gain in function in argD (Cthe_1866, E55G) and a possible loss in function in proB (Cthe_1766, A149T) [17]. These two mutations seem to be a beneficial shift from proline production to glutamate and arginine production in PM [17,18,32]. The shift in amino acid production may also assist in the increased expression in the histidine pathway since glutamate is utilized in the pathway. The PM also significantly increases the expression of 6 of the 18 genes belonging to valine, leucine and isoleucine biosynthesis, which may help balance carbon and electron flow. An increase in amino acid production can also help overcome weak acid stress [17,18,33]. Table

4 Fold change in gene expression in histidine metabolism pathways Gene Product PM vs. WT 0 PM vs. WT 10 PM 0 vs. 10 PM 0 vs. 17.5 WT 0 vs. 10     ML LL ML LL ML LL ML LL ML LL Cthe_2880 ATP phosphoribosyltransferase AZD6738 chemical structure regulatory subunit 98.42 29.12 98.72 80.51 1.25 1.01 1.12 −1.06 1.25 −2.73 Cthe_2881 ATP phosphoribosyltransferase 78.48 23.79 85.06 Adenosine triphosphate 100.15 1.64 1.24 1.35 −1.01 1.52 −3.40 Cthe_2882 histidinol dehydrogenase 35.86 18.44 28.45 44.69 1.49 1.33 1.37 1.40 1.88 −1.83 Cthe_2883 histidinol-phosphate aminotransferase 38.12 19.61 23.12 40.22 1.15 1.22 1.19 1.42 1.89 −1.69 Cthe_2884 Imidazoleglycerol-phosphate dehydratase 7.45 7.71 17.31 17.09 1.23 1.25 1.18 1.27 −1.89 −1.77 Cthe_2886 Imidazole glycerol phosphate synthase subunit hisH 11.99 12.29 14.84 15.87 1.19 1.12 1.09 −1.01 −1.04 −1.16 Cthe_2887 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase 13.46 11.01 10.02 14.54 1.44 1.20 1.29 1.13 1.93 −1.10 Cthe_2888 Imidazole glycerol phosphate synthase subunit hisF 12.46 14.23 10.04 18.19 1.61 1.30 1.54 1.24 1.99 1.