Ketol-acid reductoisomerase using NADH
AssigneeButamax Advanced Biofuels LLC
InventorsYougen Li, Der-Ing Liao, Mark J. Nelson, Daniel P. Okeefe
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Abstract
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.
Claims16
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.
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;
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- (b) contacting the recombinant host cell with acetolactate, wherein the recombinant host cell converts acetolactate to 2,3-dihydroxyisovalerate.
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;
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- (b) contacting the recombinant host cell with a fermentable carbon substrate, wherein the recombinant host cell produces isobutanol.
Dependencies are inferred from the claim text.