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

摘要

Methods for the fermentative production of isobutanol is provided by the fermentative growth of a recombinant microorganism expressing a highly active ketol-acid reductoisomerase enzyme in addition to other enzymes required for conversion of glucose to isobutanol.

主权项

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 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., andiii) a reaction buffer comprising:
a) 100 mM potassium-HEPES,b) 10 mM MgCl2,c) 2.25 mM (R,S)-acetolactate, andd) 200 μM NADPH; andB) purified protein concentration as determined by absorbance at 280 nanometers.