Corn stover, switchgrass, sorghum, and poplars (NM658 and P3959 (link)) were obtained from the Great Lakes Bioenergy Research Center (GLBRC) in the form of debarked, 5 mm chips, and used as received. Water (Fisher, HPLC grade), acetone (Sigma, 98%), ethanol (Fisher, HPLC grade, 200 proof), γ-valerolactone (GVL; Sigma, 95%), tetrahydrofuran (THF; Acros, 99%+ anhydrous with 200 ppm BHT inhibitor), acetonitrile (MeCN; Sigma, 98%), dimethyl sulfoxide (DMSO; Sigma, 98%), and N-methyl pyrrolidinone (NMP; Sigma, 99%) were obtained from vendors and used as received. Sulfuric acid (98 wt%), sodium hydroxide, sodium chlorite and acetic acid were obtained from Sigma and used as received. β-D-glucose, xylose, furfural, and 5-hydroxymethylfurfural (Sigma-Aldrich ACS reagent grade) were obtained from vendors, and used as calibration standards.
CelR_cbm3a was created through fusion of the Hungateiclostridium thermocellum CelR enzyme domain (GenBank accession code CAE51308.1 residues 31 – 642, i.e., GH9 and CBM3c modules only) with an additional 153-residue CBM3a from the scaffolding protein CipA, connected via the 48-residue CipA-native linker sequence. The lac operon-controlled expression plasmid was shipped to a third-party vendor, where it was expressed and purified at scale.
CelR_cbm3a was created through fusion of the Hungateiclostridium thermocellum CelR enzyme domain (GenBank accession code CAE51308.1 residues 31 – 642, i.e., GH9 and CBM3c modules only) with an additional 153-residue CBM3a from the scaffolding protein CipA, connected via the 48-residue CipA-native linker sequence. The lac operon-controlled expression plasmid was shipped to a third-party vendor, where it was expressed and purified at scale.