title: Improving Escherichia coli FucO for furfural tolerance by saturation mutagenesis of individual amino acid positions.
abstract: 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 Km (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.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA.
authors.forename: Huabao
authors.initials: H
authors.lastname: Zheng
authors.forename: Xuan
authors.initials: X
authors.lastname: Wang
authors.forename: Lorraine P
authors.initials: LP
authors.lastname: Yomano
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Geddes
authors.forename: Keelnatham T
authors.initials: KT
authors.lastname: Shanmugam
authors.forename: Lonnie O
authors.initials: LO
authors.lastname: Ingram
mesh_terms: D000220:Adaptation, Biological; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005285:Fermentation; D005662:Furaldehyde / Q000378:metabolism* / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055503:Protein Multimerization; D059370:RNA Folding; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D005662:Furaldehyde; D000429:Alcohol Oxidoreductases; C021494:lactaldehyde reductase; D007930:Leucine