Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes
View Patent ↗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.
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., and
iii) a reaction buffer comprising:
a) 100 mM potassium-HEPES,
b) 10 mM MgCl 2 ,
c) 2.25 mM (R,S)-acetolactate, and
d) 200 μM NADPH; and
B) purified protein concentration as determined by absorbance at 280 nanometers.
2. The method according to claim 1 wherein the host cell is a member of a genus selected from the group consisting of Pichia, Candida, Hansenula , and Saccharomyces.
3. The method according to claim 2 wherein the cell is Saccharomyces cerevisiae.
4. The method according to claim 1 , wherein the polypeptide having ketol-acid reductoisomerase activity comprises the amino acid sequence of SEQ ID NO:36.
5. The method according to claim 1 , wherein the polypeptide having ketol-acid reductoisomerase activity comprises the amino acid sequence of SEQ ID NO:35.