Transaminase biocatalysts
Inventors
Savile, Christopher K. • Mundorff, Emily • Moore, Jeffrey C. • Devine, Paul N. • Janey, Jacob M.
Assignees
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Publication Number
US-12344874-B2
Publication Date
2025-07-01
Expiration Date
Abstract
The present disclosure relates to polypeptides having transaminase activity, polynucleotides encoding the polypeptides, and methods of using the polypeptides.
Core Innovation
The invention provides engineered transaminase polypeptides that comprise an amino acid sequence at least 90% identical to SEQ ID NO: 2 and further include residue differences at residue position X65 and at residue position X81. The engineered polypeptides are PLP-dependent transaminases and are described with defined residue substitutions at multiple named positions and selectable sequence sets.
The invention further includes recombinant polynucleotides encoding the improved transaminases, host cells expressing the engineered transaminase polypeptide, and transaminase polypeptides with amino-acid deletions, up to about 30% of the total length, as well as optional fusion proteins and/or tags. The engineered transaminase polypeptides are also described as optionally including polypeptide non-encoded amino-acid variants.
The invention describes process embodiments using the improved, PLP-dependent transaminases for stereoselectively converting a sitagliptin ketoamide to (2R)-sitagliptin with high enantiomeric excess. The conversion uses an amino group donor, including isopropylamine, and the process schemes further describe preparation of sitagliptin and sitagliptin phosphate monohydrate, including management of carbonyl by-products and acetone removal using nitrogen and/or vacuum strategies.
Claims Coverage
The claim coverage includes independent claims directed to engineered transaminase polypeptides defined by high sequence identity to SEQ ID NO: 2 with specific residue differences at X65 and X81. The inventive features also include defined residue substitutions at positions X69, X122, X223, and X284, selected SEQ ID NO sets, and performance characteristics for conversion of a ketoamide substrate with detectable product formation, activity at or above SEQ ID NO: 4, and at least 90% enantiomeric excess.
Engineered transaminase identity with specific residue differences
A recombinant transaminase polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the amino acid sequence further comprises a residue difference as compared to SEQ ID NO: 2 at residue position X65 and at residue position X81.
Transaminase polypeptides defined by selected SEQ ID NO set
A transaminase polypeptide having an amino acid sequence that corresponds to one of specified SEQ ID NOs, including SEQ ID NOs 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.
Defined residue substitutions at multiple named positions
A transaminase polypeptide whose amino-acid sequence contains defined residue substitutions at positions X69, X122, X223, and X284.
Conversion of sitagliptin ketoamide to (2R)-sitagliptin with detectable product
The recombinant transaminase polypeptide converts a ketoamide substrate into a product in the presence of an amino group donor, where the product is detectable by HPLC-UV at 210 nm.
Activity at least that of a reference transaminase
The recombinant transaminase polypeptide converts a ketoamide substrate to a product with activity equal to or greater than that of the polypeptide in SEQ ID NO: 4 under defined reaction conditions.
High enantiomeric excess in conversion
A recombinant transaminase polypeptide converts a ketoamide substrate to a product with at least 90% enantiomeric excess.
Overall, the claim set emphasizes engineered, PLP-dependent transaminase polypeptides defined by high sequence identity to SEQ ID NO: 2 with specific residue differences and substitutions, and it further specifies conversion of a ketoamide substrate to a product with detectable formation, activity at least that of SEQ ID NO: 4, and enantiomeric excess of at least 90%.
Stated Advantages
Measurable conversion of the ketoamide substrate where wild-type or naturally occurring enzymes show no measurable activity.
High enantiomeric excess, including at least 90% enantiomeric excess.
Improved activity relative to the polypeptide of SEQ ID NO: 4, or activity equal to or greater than that of SEQ ID NO: 4.
Product detectability by HPLC-UV at 210 nm.
Documented Applications
Synthesis of sitagliptin (USAN) from a sitagliptin-related ketoamide substrate via R-selective transamination to an (2R)-amino product using engineered PLP-dependent transaminase biocatalysts.
Converting a specified ketoamide substrate to a (2R)-amine product using engineered transaminase polypeptides, with product detectability by HPLC-UV at 210 nm.
Stereoselective conversion of a sitagliptin ketoamide to (2R)-sitagliptin using PLP-dependent engineered transaminases, including embodiments addressing acetone removal and downstream preparation concepts for sitagliptin phosphate monohydrate.
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