Carboxyesterase polypeptides for amide coupling
Inventors
Voladri, Rama • Entwistle, David • Micklitsch, Christopher Michael • MITCHELL, VESNA • Dellas, Nikki • Zhang, Xiyun • Sero, Antoinette • Dorr, Brent • Fuerst, Douglas E.
Assignees
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Publication Number
US-12286653-B2
Publication Date
2025-04-29
Expiration Date
Abstract
The present invention provides engineered carboxyesterase enzymes having improved properties as compared to a naturally occurring wild-type carboxyesterase enzymes, as well as polynucleotides encoding the engineered carboxyesterase enzymes, host cells capable of expressing the engineered carboxyesterase enzymes, and methods of using the engineered carboxyesterase enzymes in amidation reactions.
Core Innovation
The disclosed subject matter relates to engineered carboxyesterases and engineered carboxyesterase variants derived from carboxyesterase sequences referenced as SEQ ID NO: 2 and other sequence identifiers. The engineered carboxyesterases are characterized by amino-acid positions numbered with reference to the corresponding SEQ ID, including an amino-acid replacement at position 282.
Polynucleotide sequences encoding the engineered carboxyesterases are defined by sequence identity thresholds to reference sequences, including at least 80% sequence identity and at least 85% sequence identity, and by the specific amino-acid residue selected at position 282, with numbering relative to the referenced SEQ ID. Additional constrained substitution sets at other numbered amino-acid positions are described as part of the engineered polypeptide definition.
The document further defines engineered carboxyesterase polypeptides and variants, including residue substitution notation, conservative amino acid substitutions, non-conservative substitutions, deletions, insertions, fragments, functional fragments, isolated and substantially pure polypeptides, and heterologous and codon-optimized polynucleotides. The embodiments further include immobilization/solid support formats, arrays, kits, and evaluation content describing carboxyesterase activity on substrate sets, including amidation activity.
Claims Coverage
The independent claims define engineered carboxyesterase polynucleotide sequences by sequence identity thresholds and engineered amino-acid substitutions at position 282, along with broad alternative SEQ ID coverage. In total, the consolidated set emphasizes three inventive features.
Position-282 replacement in identity-defined engineered carboxyesterase polynucleotides
A polynucleotide sequence encoding an engineered carboxyesterase comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein said engineered carboxyesterase comprises a replacement of leucine at position 282 with another amino acid, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.
Glutamine, serine, or threonine at position 282 in identity-defined engineered carboxyesterase polynucleotides
A polynucleotide sequence encoding an engineered carboxyesterase comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 8, wherein said engineered carboxyesterase comprises an amino acid residue selected from glutamine, serine, and threonine in said polypeptide sequence at position 282, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8.
Broad identity-defined engineered carboxyesterase sequence set coverage
A polynucleotide sequence encoding at least one engineered carboxyesterase, said polynucleotide sequence comprising at least 85% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, and/or 139.
The independent claims define engineered carboxyesterase polynucleotide sequences by sequence identity relative to specified SEQ ID NOs and by engineered amino-acid substitutions at position 282, including replacement of leucine at position 282, selection of glutamine, serine, or threonine at position 282, and a broad identity-defined engineered carboxyesterase sequence set coverage at at least 85% identity to a specified list of SEQ ID NOs.
Stated Advantages
Improved amidation activity versus wild-type carboxyesterase.
Improved solvent tolerance under highly organic co-solvent conditions.
Improved thermostability.
Improved pH stability.
Improved regiospecificity and stereoselectivity.
Reduced substrate/product inhibition.
Reduced host toxicity.
Increased activity.
Increased solvent tolerance.
Increased stereoselectivity.
Increased thermostability.
Improved conversion.
Improved DE% ranges.
Documented Applications
Amidation reaction producing amides from a carboxyester (I) and an amine (II) using the engineered carboxyesterases, with example amide products including lidocaine.
Immobilized enzyme formats including immobilization/solid support.
Arrays and 96-well plate formats.
Kits.
Amidation reaction contexts using substrate sets/products in examples.
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