It is desired to use hemicellulose as a substrate for the production of commodity products through fermentation.  However, hemicellulose hydrolysate can contain small molecules, such as acetic acid and furfural, that are inhibitory to the biocatalyst.  The goal of this project is to understand the mechanism of furfural-mediated growth inhibition and increase the furfural resistance.  Directed evolution in mineral salts medium with a growth-limiting concentration of furfural was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli. Transcriptome analysis of the parent (LY180) and evolved strain (EMFR9) identified twelve oxidoreductases that had perturbed expression in the evolved strain relative to the parent.  Cloning and phenotypic analysis revealed that overexpression of three of these oxidoreductases, yqhD, dkgA and yqfA, reverted the furfural tolerance of the evolved strain back to the parental-type sensitivity.  In vitro analysis showed that the YqhD and DkgA enzymes exhibited NADPH-dependent furfural reductase activity with NADPH K_m values comparable to biosynthetic reactions.  Corresponding deletion of yqhD or dkgA from the parent strain increased furfural resistance, confirming the negative effect of these enzymes on furfural tolerance.  Thus we conclude that growth inhibition by furfural is due in part to NADPH depletion via furfural reduction by YqhD and DkgA.