Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

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 Meiothertnus 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. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.