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-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.
Claims6
A recombinant 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 having ketol-acid reductoisomerase activity.
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.
A recombinant mutant ketol-acid reductoisomerase enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOS:
- 75, 76, 77 and 78 and having ketol-acid reductoisomerase activity.
A recombinant 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 and having ketol-acid reductoisomerase activity.
Dependencies are inferred from the claim text.