Silencing of NADPH-Dependent Oxidoreductases (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia Coli

Furfural (a dehydration product of pentose sugars) is a microbial inhibitor that is formed as a side product during the acid-catalyzed hydrolysis of hemicellulose. E. coli has been previously shown to contain enzymes that catalyze the NADPH-dependent reduction of furfural although the genes were not identified. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic E. coli LY180, designated strain EMFR9. To identify candidate genes involved in resistance, mRNA expression levels in the parent and mutant were compared in the presence of a low concentration of furfural. Twelve oxidoreductases were found that varied by more than 2-fold (8 were higher in EMFR9; 4 were higher in the parent) and all were cloned. Although none of the 8 increased furfural tolerance in the parent, LY180, three of the silenced genes (yqhD, dkgA, and yqfA) decreased furfural tolerance in EMFR9 when expressed from plasmids. Both yqhD and dkgA were found to encode NADPH-dependent furfural reductase activities. Each was purified as a histidine-tagged product and characterized. Both enzymes reduced furfural to furfuryl alcohol and exhibited a low Km for NADPH (0.008 mM and 0.023 mM, respectively). No furfural reductase activity was associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the full extent observed in the mutant EMFR9. From these studies, the inhibition of biosynthesis and growth in minimal media is proposed to result partially from depletion of the NADPH pool by furfural metabolism.