Ketol-acid reductoisomerase using NADH
View Patent ↗Methods for the evolution of NADPH binding ketol-acid reductoisomerase enzymes to acquire NADH binding functionality are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from Pseudomonas that have undergone co-factor switching to bind NADH are described.
1. A recombinant mutant ketol-acid reductoisomerase enzyme of SEQ ID NO: 17, wherein said enzyme has a ketol-acid reductoisomerase HMM search profile E value of <10 −3 using the hmmsearch program and comprises at least one mutation within the phosphate binding loop of said enzyme wherein said at least one mutation comprises a substitution at the residue corresponding to T52 of SEQ ID NO:17 wherein the substitution at residue T52 is a substitution to a polar, negatively charged amino acid.
2. The recombinant mutant ketol-acid reductoisomerase enzyme according to claim 1 wherein the enzyme comprises one or more of the following substitutions:
a) a substitution at position 47 wherein the substitution is an amino acid substitution selected from the group consisting of A, C, D, F, G, I, L, N, P, and Y;
b) a substitution at position 50 wherein the substitution is an amino acid substitution selected from the group consisting of A, C, D, E, F, G, M, N, V, and W;
c) the substitution at position 52 is D; or
d) a substitution at position 53 wherein the substitution is an amino acid substitution selected from the group consisting of A, H, I, and W.
3. The recombinant mutant ketol-acid reductoisomerase enzyme of claim 1 further comprising at least one mutation at a residue selected from the group consisting of 24, 33, 61, 80, 115, 156, 165, and 170.
4. The recombinant mutant ketol-acid reductoisomerase enzyme according to claim 3 , wherein the enzyme further comprises one or more of the following substitutions:
a) a substitution at position 47 wherein the substitution is an amino acid substitution selected from the group consisting of A, C, D, F, G, I, L, N, P, and Y;
b) a substitution at position 50 wherein the substitution is an amino acid substitution selected from the group consisting of A, C, D, E, F, G, M, N, V, and W;
d) a substitution at position 53 wherein the substitution is an amino acid substitution selected from the group consisting of A, H, I, and W;
e) a substitution at position 156 wherein the substitution is an amino acid substitution of V;
f) a substitution at position 165 wherein the substitution is an amino acid substitution of M;
g) a substitution at position 61 wherein the substitution is an amino acid substitution of F;
h) a substitution at position 170 wherein the substitution is an amino acid substitution of A;
i) a substitution at position 24 wherein the substitution is an amino acid substitution of F;
j) a substitution at position 33 wherein the substitution is an amino acid substitution of L;
k) a substitution at position 80 wherein the substitution is an amino acid substitution of I; and
l) a substitution at position 115 wherein the substitution is an amino acid substitution of L.