title: Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis.
abstract: Due to its enhanced energy content and hydrophobicity, isobutanol is flagged as a next generation biofuel and chemical building block. For cellular and cell-free isobutanol production, NADH dependent (over NADPH dependent) enzyme systems are desired. To improve cell-free isobutanol processes, we characterized and catalytically optimized a NADH dependent, thermo- and solvent stable ketol-acid reductoisomerase (KARI) derived from the bacterium Meiothermus ruber (Mr). The wild type Mr-KARI has the most temperature tolerant KARI specific activity reported to date. The KARI screening procedure developed in this study allows accelerated molecular optimization. Thus, a KARI variant with a 350% improved activity and enhanced NADH cofactor specificity was identified. Other KARI variants gave insights into Mr-KARI structure-function relationships.
authors.affiliation: Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address: steven.reisse@tum.de.
authors.forename: Steven
authors.initials: S
authors.lastname: Reiße
authors.affiliation: Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address: daniel.garbe@tum.de.
authors.forename: Daniel
authors.initials: D
authors.lastname: Garbe
authors.affiliation: Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address: brueck@tum.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brück
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D000440:Butanols / Q000378:metabolism*; D034301:Deinococcus / Q000201:enzymology; D004795:Enzyme Stability; D050540:Ketol-Acid Reductoisomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry*; D013696:Temperature
chemical_list: D000440:Butanols; D012997:Solvents; C040507:isobutyl alcohol; D050540:Ketol-Acid Reductoisomerase
keywords: Biocatalysis; Cell-free; Isobutanol; Ketol-acid reductoisomerase; Meiothermus ruber; Thermophilic enzymes