Processes using amino acid dehydrogenases and ketoreductase-based cofactor regenerating system
AssigneeCodexis, Inc.
InventorsFabien Louis Cabirol, Steven J. Collier, Thomas Daussmann, Naga K. Modukuru
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Abstract
The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase.
Claims16
A process for converting a compound of formula I which is a substrate for an amino acid dehydrogenase to a chiral amino acid of formula IIa:
- comprising contacting the compound of formula I with a reaction medium comprising an amino acid dehydrogenase, an ammonium ion donor selected from ammonia or an ammonium salt, a cofactor selected from NAD+/NADH or NADP+/NADPH, and a cofactor regenerating system comprising an engineered ketoreductase having at least 90% sequence identity to SEQ ID NO:
- 18, which has less than 5% of the activity of the amino acid dehydrogenase, with compound of formula I and a lower secondary alcohol, under conditions where the compound of formula I is converted to the chiral amino acid of formula IIa and the lower secondary alcohol is converted to a ketone, wherein R is a substituted or unsubstituted —(C1-C10)alkyl, —(C2-C6)alkenyl, —(C2-C6)alkynyl, —(C3-C8)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl and wherein the said ketoreductase is capable of recycling cofactor by converting isopropanol (IPA) to acetone with an activity at least 2.0-fold greater than a reference ketoreductase of SEQ ID NO:
- 2.
A process for producing (S)-2-amino-3,3-dimethylbutanoic acid, comprising:
- contacting 3,3-dimethyl-2-oxobutanoic acid with an enantioselective leucine dehydrogenase in a reaction medium comprising an ammonium ion donor, cofactor NAD+/NADH or NADP+/NADPH, and a cofactor recycling system comprising a ketoreductase and a lower secondary alcohol, under conditions where the 3,3-dimethyl-2-oxobutanoic acid is converted to (S)-2-amino-3,3-dimethylbutanoic acid,
- wherein the 3,3-dimethyl-2-oxobutanoic acid is at about 75 g/L to 125 g/L, the secondary alcohol is about 1.5 fold stoichiometric excess of the 3,3-dimethyl-2-oxobutanoic acid, the cofactor is at about 0.30 g/L to 0.70 g/L, and the leucine dehydrogenase and ketoreductase are each independently at about 0.5 to about wherein the said ketoreductase having at least 90% sequence identity to SEQ ID NO:
- 18 and capable of recycling cofactor by converting isopropanol (IPA) to acetone with an activity at least 2.0-fold greater than a reference ketoreductase of SEQ ID NO:
- 2.
A process for converting a compound mixture of formula IId which comprises a substrate for an amino acid dehydrogenase to a composition of formula I and a chiral amino acid of formula IIa:
- comprising contacting the compound mixture of formula IId with an enantioselective amino acid leucine dehydrogenase in a reaction medium comprising NAD+/NADH or NADP+/NADPH and a cofactor recycling system comprising a ketoreductase and a lower alkyl ketone, under conditions where the compound mixture of formula IId is converted to the composition of formula I and a chiral amino acid of formula IIa, and the lower alkyl ketone is converted to a lower secondary alcohol;
- wherein R is a substituted or unsubstituted —(C1-C10)alkyl, —(C2-C6)alkenyl, —(C2-C6)alkynyl, —(C3-C8)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the said ketoreductase having at least 90% sequence identity to SEQ ID NO:
- 18 and capable of recycling cofactor by converting isopropanol (IPA) to acetone with an activity at least 2.0-fold greater than a reference ketoreductase of SEQ ID NO:
- 2.
Dependencies are inferred from the claim text.