Ketol-acid reductoisomerase using nadh

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.

What is claimed is: 
     
         1 . A mutant ketol-acid reductoisomerase enzyme comprising the amino acid sequence as set forth in SEQ ID NO: 29. 
     
     
         2 . A nucleic acid molecule encoding the mutant ketol-acid reductoisomerase enzyme of  claim 1 . 
     
     
         3 . A nucleic acid molecule encoding a mutant ketol-acid reductoisomerase enzyme having the amino acid sequence as set forth in SEQ ID NO:19. 
     
     
         4 . A mutant ketol-acid reductoisomerase enzyme as set for in SEQ ID NO:19 
     
     
         5 . A recombinant cell comprising the mutant ketol-acid reductoisomerase enzyme of  claim 1 . 
     
     
         6 . A mutant ketol-acid reductoisomerase enzyme as set forth in SEQ ID NO:17 comprising at least one mutation at a residue selected from the group consisting of 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, 165, and 170. 
     
     
         7 . A mutant ketol-acid reductoisomerase enzyme according to  claim 6  wherein:
 a) the residue at position 47 has an amino acid substation selected from the group consisting of A, C, D, F, G, I, L, N, P, H, T, E and Y; 
 b) the residue at position 50 has an amino acid substitution selected from the group consisting of A, C, D, E, F, G, M, N, V, W and I; 
 c) the residue at position 52 has an amino acid substitution selected from the group consisting of A, C, D, G, H, N, Y, and S; 
 d) the residue at position 53 has an amino acid substitution selected from the group consisting of A, H, I, W, Y, C, and R; 
 e) the residue at position 156 has an amino acid substitution of V; 
 f) the residue at position 165 has an amino acid substitution of M; 
 g) the residue at position 61 has an amino acid substitution of F; 
 h) the residue at position 170 has an amino acid substitution of A; 
 i) the residue at position 24 has an amino acid substitution of F; 
 j) the residue at position 33 has an amino acid substitution of L; 
 k) the residue at position 80 has an amino acid substitution of I; and 
 l) the residue at position 115 has an amino acid substitution of L. 
 
     
     
         8 . A nucleic acid molecule encoding the mutant ketol-acid reductoisomerase enzyme of  claim 6 . 
     
     
         9 . A method for the evolution of an NADPH binding ketol-acid reductoisomerase enzyme to an NADH using form comprising:
 a) providing a ketol-acid reductoisomerase enzyme which uses NADPH having a specific native amino acid sequence;   b) identifying the cofactor switching residues in the enzyme of (a) based on the amino acid sequence of the  Pseudomonas fluorescens  ketol-acid reductoisomerase enzyme as set for the in SEQ ID NO:17 wherein the cofactor switching residues are at positions selected from the group consisting of: 24, 33, 47, 50, 52, 53, 61, 80, 115, 156,165, and 170; and   c) creating mutations in at least one of the cofactor switching residues of (b) to create a mutant enzyme wherein said mutant enzyme binds NADH.   
     
     
         10 . The method of  claim 9  wherein:
 a) the residue at position 47 has an amino acid substitution selected from the group consisting of A, C, D, F, G, I, L, N, P, H, T, E and Y; 
 b) the residue at position 50 has an amino acid substitution selected from the group consisting of A, C, D, E, F, G, M, N, V, W and I; 
 c) the residue at position 52 has an amino acid substitution selected from the group consisting of A, C, D, G, H, N, Y, and S; 
 d) the residue at position 53 has an amino acid substitution selected from the group consisting of A, H, I, W, Y, G, and R; 
 e) the residue at position 156 has an amino acid substitution of V; 
 f) the residue at position 165 has an amino acid substitution of M; 
 g) the residue at position 61 has an amino acid substitution of F; 
 h) the residue at position 170 has an amino acid substitution of A; 
 i) the residue at position 24 has an amino acid substitution of F; 
 j) the residue at position 33 has an amino acid substitution of L; 
 k) the residue at position 80 has an amino acid substitution of I; and 
 l) the residue at position 115 has an amino acid substitution of L. 
 
     
     
         11 . The method of  claim 9  wherein the ketol-acid reductoisomerase enzyme has the amino acid sequence as set forth in SEQ ID NO: 29. 
     
     
         12 . A method for the production of isobutanol comprising:
 a) providing a recombinant microbial host cell comprising the following genetic constructs:
 i) at least one genetic construct encoding an acetolactate synthase enzyme for the conversion of pyruvate to acetolactate; 
 ii) at least one genetic construct encoding a ketol-acid reductoisomerase enzyme of either of  claims 1  or  6 ; 
 iii) at least one genetic construct encoding an acetohydroxy acid dehydratase for the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c); 
 iv) at least one genetic construct encoding a branched-chain keto acid decarboxylase, of the conversion of α-ketoisovalerate to isobutyraldehyde, (pathway step d); 
 v) at least one genetic construct encoding a branched-chain alcohol dehydrogenase for the conversion of isobutyraldehyde to isobutanol (pathway step e); and 
   b) growing the host cell of (a) under conditions where isobutanol is produced.   
     
     
         13 . A method for the evolution and identification of an NADPH binding ketol-acid reductoisomerase enzyme to an NADH using form comprising:
 a) providing a ketol-acid reductoisomerase enzyme which uses NADPH having a specific native amino acid sequence;   b) identifying the amino acid residues in the native amino acid sequence whose side chains are in close proximity to the adenosyl 2′-phosphate of NADPH as mutagenesis targets;   c) creating a library of mutant ketol-acid reductoisomerase enzymes from the class I ketol-acid reductoisomerase enzyme of step (a), having at least one mutation in at least one of the mutagenesis target sites of step (b); and   d) screening the library of mutant ketol-acid reductoisomerase enzymes of step (c) to identify NADH binding mutant of ketol-acid reductoisomerase enzyme.   
     
     
         14 . A mutant ketol-acid reductoisomerase enzyme having the amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 67, 68, 70, 75, 79, 80, 81 and 82. 
     
     
         15 . A method for evolution of an NADPH specific ketol-acid reductoisomerase enzyme to an NADH using form comprising:
 a) providing a mutant enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 67, 68, 69, 70, and 84;   b) constructing a site-saturation library targeting amino acid positions 47, 50, 52 and 53 of the mutant enzyme of (a); and   c) screening the site-saturation library of (b) to identify mutants which accept NADH instead of NADPH as cofactor.   
     
     
         16 . A method for evolution of an NADPH specific ketol-acid reductoisomerase enzyme to an NADH using form comprising:
 a) providing a DNA fragment encoding a mutant enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98 containing mutations in cofactor specificity domain;   b) producing a DNA fragment cofactor specificity domain of (a);   c) providing a DNA fragment encoding a mutant enzyme having mutations in cofactor binding affinity domain selected from the group consisting of SEQ ID NOs: 28, 67, 68, 69, 70, 84 and 86;   d) incorporating mutations of step (b) into mutants of step (c); and   e) screening mutants of step (d) for mutant enzymes having a ratio of NADH/NADPH utilization is greater than one.   
     
     
         16 . The method of  claim 15  wherein the K M  for NADH is less than 15 μM. 
     
     
         17 . A mutant ketol-acid reductoisomerase enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOS: 75, 76, 77 and 78. 
     
     
         18 . A mutant ketol-acid reductoisomerase enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOS: 79, 80, 81, 82, and 83.