Engineered transaminase polypeptides for industrial biocatalysis
Inventors
Novick, Scott J. • Dellas, Nikki • Alvizo, Oscar • Garcia, Ravi David • Ching, Charlene • Entwistle, David
Assignees
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Publication Number
US-12297462-B2
Publication Date
2025-05-13
Expiration Date
Abstract
The present invention provides engineered transaminase polypeptides useful for the synthesis of chiral amine compounds under industrially relevant conditions. The invention also provides polynucleotides encoding the engineered transaminase polypeptides, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases for the production of chiral amine compounds.
Core Innovation
The invention provides engineered transaminase polypeptides and polynucleotides encoding them, together with host systems for stereoselective synthesis of chiral amine products. The engineered transaminase sequences are defined relative to reference transaminases including SEQ ID NO:4 and other SEQ IDs by sequence identity thresholds and residue-difference sets, including amino acid substitutions at positions 85 and 422, and are tied to improved enzymatic activity and stereoselectivity toward the (S) amine diastereomer.
The document further describes polynucleotides, vectors, recombinant expression constructs with control sequences, and host cells for expressing and producing the engineered transaminase. It also includes optional immobilization of the engineered transaminase on solid supports, purification and recovery concepts, and engineered variants characterized by enhanced stability and substrate/product tolerance.
The subject matter is linked to transaminase chemistry using amino group donor, amino acceptor, carbonyl by-product, and cofactors including PLP. It targets conversion of a ketone amino acceptor, including compound (2), to a chiral amine product, compound (1), with high enantiomeric excess and diastereomeric enrichment.
Claims Coverage
The claim coverage centers on polynucleotides encoding engineered transaminases defined by high sequence identity to SEQ ID NO:4 and specific substitutions at positions 85 and 422, with greater enzymatic activity and greater stereoselectivity toward the (S) amine diastereomer. Across the claim set, there are 7 inventive features.
Engineered transaminase polynucleotide relative to SEQ ID NO:4
A polynucleotide encoding an engineered transaminase comprising a polypeptide sequence having at least 93% sequence identity to SEQ ID NO:4, with amino acid substitutions at positions 85 and 422, including F85V and V422C, V422G, or V422A.
Enhanced activity and (S)-amine stereoselectivity versus SEQ ID NO:4
The engineered transaminase exhibits greater enzymatic activity and greater stereoselectivity toward the (S) amine diastereomer than the transaminase of SEQ ID NO:4.
Additional sequence-identity thresholds to multiple SEQ IDs
The engineered transaminase polynucleotide is defined with at least 94% sequence identity to SEQ ID NO:23, 29, 47, 65, 69, 87, 109, 111, 119, 137 or 155.
Improved property relative to Vibrio fluvialis JS17 wild-type
The polynucleotide encoding an engineered transaminase is characterized by at least one improved property relative to the wild-type Vibrio fluvialis transaminase.
Improved activity on a substrate
The improved property includes improved activity on a substrate.
Substrate comprises compound (1)
The substrate comprises compound (1).
Purification of the engineered transaminase
A method further defined by purifying the engineered transaminase.
Overall, the claims are directed to polynucleotides encoding engineered transaminases defined by high sequence identity to SEQ ID NO:4 and specific substitutions at positions 85 and 422, with greater enzymatic activity and greater stereoselectivity toward the (S) amine diastereomer. Dependent claims further narrow sequence identity thresholds to additional SEQ IDs, recite improved property relative to wild-type Vibrio fluvialis, specify compound (1), and include purification.
Stated Advantages
Greater enzymatic activity than the transaminase of SEQ ID NO:4.
Greater stereoselectivity toward the (S) amine diastereomer than the transaminase of SEQ ID NO:4.
At least one improved property relative to wild-type Vibrio fluvialis transaminase.
Enhanced stability and substrate/product tolerance.
Documented Applications
Industrially relevant, stereoselective synthesis of chiral amine products via transaminase conversion of a ketone amino acceptor, including compound (2), to a chiral amine product, compound (1), with extensions to other ketone substrates using formula sets (II/III to I/IV).
Transamination of substrate compound (2) to product compound (1), with activity and stereoselectivity evaluated relative to multiple parent SEQ IDs.
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