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Publication Number
US-12331325-B2
Publication Date
2025-06-17
Expiration Date
Abstract
The present disclosure provides engineered transaminase polypeptides useful for the synthesis of chiral amine compounds under industrially relevant conditions. The disclosure 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 describes engineered transaminase polypeptides and engineered polynucleotides encoding the engineered transaminases. The engineered transaminases are defined by an amino acid sequence having at least 85% identity to a reference sequence, including SEQ ID NO:8, and by specified amino-acid substitutions at positions numbered with reference to the reference sequence. The substitution sets are used to obtain engineered transaminases that exhibit increased enzymatic activity relative to a corresponding engineered transaminase based on the reference sequence.
The patent further describes engineered transaminase variants within the same general framework of amino-acid residue differences relative to reference sequences, including functional fragments, deletions, insertions, fusion polypeptides, and non-encoded amino acids. The engineered transaminases are characterized as having improved properties including increased stereoselectivity, improved thermostability and/or solvent stability, and higher substrate/product tolerance.
The disclosure also describes stereoselective conversion of keto substrate compound (1) to chiral amine compound (2), including conversion of a substrate compound (1) to a product compound (2). Beyond the core engineered transaminase sequences and encoding polynucleotides, the patent includes optional host systems, expression vectors, expression/secretion signal peptides, and immobilization and binding to solid supports, including beads/resins, arrays, and kits.
Claims Coverage
The independent claim coverage centers on one engineered transaminase encoded by an engineered polynucleotide, with a sequence identity threshold to a reference sequence and defined substitutions at positions 21/163/323/408 that increase enzymatic activity. Additional claim features include operable linkage to a control sequence, expression vectors, and host-cell production.
Engineered transaminase with high identity to a reference sequence plus specified substitutions
An engineered polynucleotide encoding an engineered transaminase having an amino acid sequence of at least 85% identity to a reference sequence, including SEQ ID NO:8, or a functional fragment thereof, and a set of substitutions at positions 21/163/323/408, with positions numbered with reference to the reference sequence.
Increased enzymatic activity versus the reference-sequence-based transaminase
The engineered transaminase exhibits increased enzymatic activity relative to an engineered transaminase having the amino acid sequence set forth in the reference sequence.
Operable linkage of the polynucleotide to a control sequence
The polynucleotide sequence is operably linked to a control sequence.
Expression vector comprising the claimed polynucleotide sequence
An expression vector comprising at least one polynucleotide sequence of the claim.
Producing the engineered transaminase in a host cell by culturing
A method of producing an engineered transaminase in a host cell, comprising culturing the host cell under suitable conditions such that at least one engineered transaminase is produced.
Overall, the claim coverage focuses on engineered transaminase polypeptides and encoding polynucleotides with an identity threshold to a reference sequence and defined substitution positions that increase enzymatic activity, with additional features directed to control-sequence linkage, expression vectors, and culturing-based production in a host cell.
Stated Advantages
Increased enzymatic activity relative to an engineered transaminase having the amino acid sequence set forth in the reference sequence.
Increased stereoselectivity for stereoselective synthesis of chiral amine products.
Improved thermostability and/or solvent stability.
Higher substrate/product tolerance.
Conversion of a substrate compound (1) to a product compound (2).
Documented Applications
Industrial stereoselective synthesis of chiral amine products by conversion of keto substrate compound (1) to chiral amine compound (2) under industrially relevant conditions.
Use of engineered transaminase polynucleotides and host systems for producing the engineered transaminase in a host cell via culturing.
Immobilization and binding of the engineered transaminase to solid supports, including beads/resins, arrays, and use in kits.
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