Plasmidic Expression ofnemAandyafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate

Hydrolysate-resistantEscherichia coli SL100 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 (nemRA operon) in SL100. This mutation increasednemA activity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increasednemA mRNA levels >200-fold. Addition of hydrolysates inducednemA expression (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 expressingnemA oryafC* 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 thenemA oryafC* plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate.