×

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

  • US 9,284,612 B2
  • Filed: 02/22/2011
  • Issued: 03/15/2016
  • Est. Priority Date: 04/18/2007
  • Status: Active Grant
First Claim
Patent Images

1. 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 μ

    moles/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 μ

    moles/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., andiii) a reaction buffer comprising;

    a) 100 mM potassium-HEPES,b) 10 mM MgCl2,c) 2.25 mM (R,S)-acetolactate, andd) 200 μ

    M NADPH; and

    B) purified protein concentration as determined by absorbance at 280 nanometers.

View all claims
  • 3 Assignments
Timeline View
Assignment View
    ×
    ×