Furfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. In Escherichia coli, furfural tolerance can be increased by expressing the native fucO gene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form of fucO that confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-type fucO gene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutant fucO genes differed by less than 2-fold. The K m (furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides ؊7 to ؉37) were weak for the native gene (؊4.1 kcal mol ؊1 ) but weaker still for the fucO mutant (؊1.0 to ؊0.1 kcal mol ؊1 ). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels.T he enzyme L-1,2-propanediol oxidoreductase (encoded by fucO) is an NADH-linked, iron-dependent group III dehydrogenase. Typically, this enzyme functions only during the catabolism of deoxy sugars, where it catalyzes the reduction of lactaldehyde (1-3). FucO is a homodimer in which each subunit contains an active site (4). This enzyme has a broad substrate range (5) that includes furfural (6), a toxic side product in dilute acid hydrolysates of hemicellulose and an important inhibitor of microbial fermentations (7-9). Expression of fucO from plasmids has been used to improve furfural tolerance in Escherichia coli-based fermentations for ethanol and lactic acid (6). The reduction of furfural to the less toxic alcohol seems essential for growth and fermentation of dilute acid hydrolysates of hemicellulose (10-13).Deoxy sugars such as fucose seldom dominate in the natural environment of E. coli (14). The fuc genes encoding deoxy sugar metabolism remain silent unless induced by fucose or other related sugars in the absence of competing substrates (1, 2). Although a natural product, furfural is unlikely to be an important natural substrate for this enzyme.In this study, we have used site-specific mutagenesis and growth-based selection to identify a fucO mutation that confers a further increase in furfural tolerance.
MATERIALS AND METHODSStrains, media, and genetic manipulations. The strains, pl...