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

Biochimie. 2015 Jan:108:76-84. doi: 10.1016/j.biochi.2014.10.024. Epub 2014 Nov 5.

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.

Keywords: Biocatalysis; Cell-free; Isobutanol; Ketol-acid reductoisomerase; Meiothermus ruber; Thermophilic enzymes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biocatalysis
  • Butanols / metabolism*
  • Deinococcus / enzymology
  • Enzyme Stability
  • Ketol-Acid Reductoisomerase / chemistry*
  • Ketol-Acid Reductoisomerase / genetics
  • Ketol-Acid Reductoisomerase / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis*
  • Protein Conformation
  • Solvents / chemistry*
  • Temperature*

Substances

  • Butanols
  • Solvents
  • isobutyl alcohol
  • Ketol-Acid Reductoisomerase