Ketol-acid reductoisomerase using NADH
View Patent ↗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.
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.