Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
Inventors
AGARD, NICHOLAS J. • Alvizo, Oscar • Mayo, Melissa Ann • Minor, Stefanie Ng • Riggins, James Nicholas • Moore, Jeffrey C.
Assignees
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Publication Number
US-12312609-B2
Publication Date
2025-05-27
Expiration Date
Abstract
The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.
Core Innovation
The patent describes engineered imine reductase polypeptides derived from opine dehydrogenase (CENDH), including polypeptides having at least 90% sequence identity to SEQ ID NO:6 and two or more residue differences selected from specified X-labeled substitutions. The engineered polypeptides retain imine reductase activity and are associated with improved enzyme properties, including activity, substrate specificity/selectivity, and thermal and solvent stability.
The polypeptides catalyze reductive amination reactions using NAD(P)H cofactor chemistry and converting compounds corresponding to formula (I) and formula (II) to compounds corresponding to formula (III). The described reactions form secondary and tertiary amines with stereoselectivity, including chiral products generated from ketone or aldehyde substrates and primary or secondary amines, and also describe reduction of preformed stable imines or enamines and intramolecular asymmetric cyclization via hydroxyamine intermediates.
The patent further describes that engineered variants obtained via directed evolution of SEQ ID NO:6 overcome limitations of wild-type CENDH activity for certain substrates and amines. It also describes polynucleotides encoding the engineered enzymes, expression vectors, host cells, preparation by culturing and optional isolation, optional cofactor regeneration systems, immobilization on solid supports, and kit/array concepts.
Claims Coverage
The claim coverage centers on one engineered imine reductase polypeptide family defined by sequence identity to SEQ ID NO:6 and two or more selected residue differences, with dependent features extending to activity improvement, substrate-to-product conversion, encoding polynucleotides, and host-cell preparation. Five inventive features are identified.
Engineered imine reductase polypeptide sequence variants
An engineered polypeptide having at least 90% sequence identity to SEQ ID NO:6 and two or more residue differences selected from X153Y, X283V, X82P, X141W, X154F, X259I/L/M, X274L/M, and X296N/V, wherein the polypeptide has imine reductase activity.
Activity improvement relative to SEQ ID NO:6
The engineered polypeptide increases imine reductase activity by at least 2-fold versus the reference polypeptide of SEQ ID NO:6 for converting a ketone and amine substrate pair to an amine product compound under suitable reaction conditions.
Ketone/amine substrate-to-amine product conversion
The imine reductase activity converts at least one specified pair of ketone substrate compound(s) and amine substrate compound(s) into the corresponding amine product compound(s) under suitable reaction conditions.
Encoding polynucleotide for the engineered imine reductase polypeptide
A polynucleotide that encodes the engineered polypeptide.
Host-cell preparation of the engineered imine reductase polypeptide
A method for preparing the engineered polypeptide with imine reductase activity by culturing the host cell under conditions suitable for expression, optionally isolating the engineered polypeptide.
Overall, the claim set centers on engineered polypeptides highly similar to SEQ ID NO:6, incorporating at least two selected residue differences and imine reductase activity. The coverage is further narrowed by improved activity, defined ketone/amine-to-amine product conversion, and additional claim forms for polynucleotide encoding and host-cell-based preparation.
Stated Advantages
Enables conversion of ketone or aldehyde substrates and primary or secondary amines to chiral secondary and tertiary amines with stereoselectivity.
Overcomes wild-type CENDH limitations for certain substrates and amines, including dimethylamine and cyclohexanone with butylamine.
Improves imine reductase activity by at least 2-fold versus SEQ ID NO:6.
Improved enzyme properties including activity and substrate specificity/selectivity.
Improved thermal and solvent stability.
Stereoselectivity and stereomeric excess in reductive amination to produce amine products.
Documented Applications
Reductive amination using engineered imine reductase (IRED) polypeptides to form chiral secondary and tertiary amines by contacting compounds corresponding to formula (I) and formula (II) to yield compounds corresponding to formula (III).
Biocatalytic conversions summarized by Table 2 and Table 8 for ketone or amine substrate pairs to amine product compounds.
Production of API or intermediate compounds, including tadalafil-related intermediates and APIs such as dexmethylphenidate, tofacitinib, lisinopril, sertraline, rotigotine, and solifenacin (Vessicare).
Expression and production of the engineered polypeptides in host cells using polynucleotides and expression vectors, including optional isolation of the engineered polypeptide.
Reduction of preformed stable imine or enamine substrates and intramolecular asymmetric cyclization via hydroxyamine intermediates.
Catalysis of reductive amination of ketones and amines to yield secondary and tertiary cyclic amines and chiral secondary and tertiary amine products.
Conversion of specified ketone and amine substrate pairs into corresponding amine product compounds using engineered imine reductase polypeptides.
Immobilization of the engineered enzyme on solid supports and use of kit/array concepts for enzyme use.
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