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

Steven Reiße, Daniel Garbe, Thomas Brück

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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.

Original languageEnglish
Pages (from-to)76-84
Number of pages9
JournalBiochimie
Volume108
DOIs
StatePublished - Jan 2015

Keywords

  • Biocatalysis
  • Cell-free
  • Isobutanol
  • Ketol-acid reductoisomerase
  • Meiothermus ruber
  • Thermophilic enzymes

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