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

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax Advanced Biofuels LLC (International Flavors & Fragrances Incorporated)
• Inventors: Liao, Der-Ing, Nelson, Mark J., Bramucci, Michael G.
• Primary Examiner(s): Joike, Michele K
• Application Number:
  US13/031,776
• Publication Number:
  US 20110250610A1
• Time in Patent Office: 1,848 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

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.

Claims

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., 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 μ

M 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.