title: Directed evolution of transketolase activity on non-phosphorylated substrates.
abstract: We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form L-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.
authors.affiliation: Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, UK.
authors.forename: Edward G
authors.initials: EG
authors.lastname: Hibbert
authors.forename: Tarik
authors.initials: T
authors.lastname: Senussi
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Costelloe
authors.forename: Wenling
authors.initials: W
authors.lastname: Lei
authors.forename: Mark E B
authors.initials: ME
authors.lastname: Smith
authors.forename: John M
authors.initials: JM
authors.lastname: Ward
authors.forename: Helen C
authors.initials: HC
authors.lastname: Hailes
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalby
mesh_terms: D000596:Amino Acids; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019020:Directed Molecular Evolution; D019277:Entropy; D004926:Escherichia coli / Q000201:enzymology*; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D016415:Sequence Alignment; D013379:Substrate Specificity; D014174:Transketolase / Q000302:isolation & purification / Q000378:metabolism*