Ketol-acid reductoisomerase using NADH
View Patent ↗Methods for the evolution of NADPH specific ketol-add 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 host cell comprising a mutant ketol-acid reductoisomerase enzyme, 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 host cell of claim 1 , wherein the recombinant host cell is a member of a genera selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula , and Saccharomyces.
3. The recombinant host cell of claim 2 , wherein the host cell is a Saccharomyces.
4. The recombinant host cell of claim 1 , wherein the mutant ketol-acid reductoisomerase enzyme comprises a GxGxx(G/A) cofactor binding motif.
5. The recombinant host cell of claim 1 , wherein the host cell further comprises an acetolactate synthase enzyme.
6. The recombinant host cell of claim 5 , wherein the acetolactate synthase enzyme is a Bacillus subtilis acetolactate synthase enzyme.
7. A method of converting acetolactate to 2,3-dihydroxyisovalerate, the method comprising:
(a) providing a recombinant host cell comprising a mutant ketol-acid reductoisomerase enzyme, 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; and
(b) contacting the recombinant host cell with acetolactate, wherein the recombinant host cell converts acetolactate to 2,3-dihydroxyisovalerate.
8. The method of claim 7 , wherein the recombinant host cell is a member of a genera selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula , and Saccharomyces.
9. The method of claim 8 , wherein the recombinant host cell is a Saccharomyces.
10. The method of claim 7 , wherein the mutant ketol-acid reductoisomerase enzyme comprises a GxGxx(G/A) cofactor binding motif.
11. A method of producing isobutanol, the method comprising:
(a) providing a recombinant host cell comprising an isobutanol biosynthetic pathway, wherein the isobutanol biosynthetic pathway comprises a mutant ketol-acid reductoisomerase enzyme, wherein the 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; and
(b) contacting the recombinant host cell with a fermentable carbon substrate, wherein the recombinant host cell produces isobutanol.
12. The method of claim 11 , wherein the recombinant host cell is a member of a genera selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula , and Saccharomyces.
13. The method of claim 12 , wherein the recombinant host cell is a Saccharomyces.
14. The method of claim 11 , wherein the mutant ketol-acid reductoisomerase enzyme comprises a GxGxx(G/A) cofactor binding motif.
15. The method of claim 11 , wherein the recombinant host cell further comprises an acetolactate synthase enzyme.
16. The method of claim 15 , wherein the acetolactate synthase enzyme is a Bacillus subtilus acetolactate synthase enzyme.