Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes

A method for the production of isobutanol comprising:

• (a) providing a recombinant yeast host cell comprising an isobutanol producing biosynthetic pathway comprising at least one genetic construct encoding a polypeptide having ketol-acid reductoisomerase activity, wherein the polypeptide having ketol-acid reductoisomerase activity comprises an amino acid sequence of SEQ ID NO:
• 34, SEQ ID NO:
• 35, or SEQ ID NO:
• 36 and a specific activity of greater than 1.1 μmoles/min/mg for the conversion of (S)-acetolactate to 2,3-dihydroxyisovalerate;
• (b) growing the yeast host cell of (a) under conditions where isobutanol is produced;
• wherein the ketol-acid reductoisomerase specific activity greater than 1.1 μmoles/min/mg is based on purified protein as measured by:
• A) NADPH consumption assay, run under the following conditions:
• i) pH of about 7.5,
• ii) a temperature of about 22.5° C., and
• iii) a reaction buffer comprising:
• a) 100 mM potassium-HEPES,
• b) 10 mM MgCl2,
• c) 2.25 mM (R,S)-acetolactate, and
• d) 200 μM NADPH;
• and
• B) purified protein concentration as determined by absorbance at 280 nanometers.