title: Improvement of thermostable aldehyde dehydrogenase by directed evolution for application in Synthetic Cascade Biomanufacturing.
abstract: The aldehyde dehydrogenase from Thermoplasma acidophilum, which was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols, was optimized by directed evolution. Improvements have been made to enhance reaction velocity and solubility. Using a random approach followed by site-directed and saturation mutagenesis, three beneficial amino acid mutations were found after screening of ca. 20,000 variants. Mutation Y399C enhanced the protein solubility after recombinant expression in Escherichia coli 6-fold. Two further mutations, F34M and S405N, enhanced enzyme activity with the cofactor NAD(+) by a factor of eight. Impacts on enzyme stability and substrate specificity were negligible. Modeling of the enzyme structure did not reveal any direct interactions between the amino acid substitutions and residues of the active site or the enzyme's substrates. Thus, a directed evolution approach allowed for the generation of improved enzyme variants which were unlikely to be found by rational or semi-rational strategies.
authors.affiliation: Chemistry of Biogenic Resources, Straubing Centre of Science, Technische Universität München, Schulgasse 16, 94315 Straubing, Germany. Electronic address: fabian.steffler@tum.de.
authors.forename: Fabian
authors.initials: F
authors.lastname: Steffler
authors.forename: Jan-Karl
authors.initials: JK
authors.lastname: Guterl
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001709:Biotechnology; D020134:Catalytic Domain / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013822:Thermoplasma / Q000201:enzymology / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D000444:Aldehyde Dehydrogenase
keywords: AlDH; Biocatalysis; Directed evolution; Enzyme engineering; Screening; Synthetic Cascade Biomanufacturing; TaAlDH; Thermostable dehydrogenase; aldehyde dehydrogenase; aldehyde dehydrogenase from Thermoplasma acidophilum