ABSTRACTHydrolysate-resistantEscherichia coliSL100 was previously isolated from ethanologenic LY180 after sequential transfers in AM1 medium containing a dilute acid hydrolysate of sugarcane bagasse and was used as a source of resistance genes. Many genes that affect tolerance to furfural, the most abundant inhibitor, have been described previously. To identify genes associated with inhibitors other than furfural, plasmid clones were selected in an artificial hydrolysate that had been treated with a vacuum to remove furfural. Two new resistance genes were discovered from Sau3A1 libraries of SL100 genomic DNA:nemA(N-ethylmaleimide reductase) and a putative regulatory gene containing a mutation in the coding region,yafC*. The presence of these mutations in SL100 was confirmed by sequencing. A single mutation was found in the upstream regulatory region ofnemR(nemRAoperon) in SL100. This mutation increasednemAactivity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increasednemAmRNA levels >200-fold. Addition of hydrolysates inducednemAexpression (mRNA and activity), in agreement with transcriptional control. NemA activity was stable in cell extracts (9 h, 37°C), eliminating a role for proteinase in regulation. LY180 with a plasmid expressingnemAoryafC*was more resistant to a vacuum-treated sugarcane bagasse hydrolysate and to a vacuum-treated artificial hydrolysate than LY180 with an empty-vector control. Neither gene affected furfural tolerance. The vacuum-treated hydrolysates inhibited the reduction ofN-ethylmaleimide by NemA while also serving as substrates. Expression of thenemAoryafC*plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate.
Direct hydrogen production from dilute-acid pretreated sugarcane bagasse hydrolysate using the newly isolated Thermoanaerobacterium thermosaccharolyticum MJ1
