Ketol-acid reductoisomerase using NADH

• US 9,238,801 B2
• Filed: 03/15/2013
• Issued: 01/19/2016
• Est. Priority Date: 12/20/2007
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Li, Yougen, Liao, Der-Ing, Nelson, Mark J., Okeefe, Daniel P.
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US13/838,869
• Publication Number:
  US 20140057329A1
• Time in Patent Office: 1,040 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 2320/13 in a process of directed evolution, e.g. SELEX, acquiring a new function
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Y 101/01086 Ketol-acid reductoisomerase (1.1.1.86)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Methods for the evolution of NADPH specific ketol-acid reductoisomerase enzymes to acquire NADH specificity are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from Pseudomonas that have undergone co-factor switching to utilize NADH are described.

Claims

1. A recombinant mutant ketol-acid reductoisomerase enzyme having the amino acid sequence of SEQ ID NO:

29.

2. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 24 wherein the substitution is an amino acid substitution of phenylalanine.

3. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 50 wherein the substitution is an amino acid substitution of alanine.

4. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 52 wherein the substitution is an amino acid substitution of aspartic acid.

5. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 53 wherein the substitution is an amino acid substitution of alanine.

6. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 61 wherein the substitution is an amino acid substitution of phenylalanine.

7. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme further comprises a substitution at position 115 wherein the substitution is an amino acid substitution of leucine.

8. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme further comprises a substitution at position 165 wherein the substitution is an amino acid substitution of methionine.

9. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 33 wherein the substitution is an amino acid substitution of leucine.

10. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 80 wherein the substitution is an amino acid substitution of isoleucine.

11. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1, wherein the enzyme comprises a substitution at position 156 wherein the substitution is an amino acid substitution of valine.