Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
US-10370648-B2 · Aug 6, 2019 · US
US12215368B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-12215368-B2 |
| Application number | US-202017600345-A |
| Country | US |
| Kind code | B2 |
| Filing date | Apr 23, 2020 |
| Priority date | May 1, 2019 |
| Publication date | Feb 4, 2025 |
| Grant date | Feb 4, 2025 |
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Official abstract text for this publication.
The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered polypeptides, host cells capable of expressing the engineered polypeptides, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.
Opening claim text (preview).
What is claimed is: 1. An engineered polypeptide having imine reductase activity and comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein the tyrosine at the position corresponding to amino acid 222 of SEQ ID NO: 4 is substituted with another amino acid in the sequence of the engineered polypeptide. 2. The engineered polypeptide of claim 1 , further comprising at least one substitution or substitution set as compared to the amino acid sequence of SEQ ID NO: 4, wherein the at least one substitution or substitution set is at one or more positions selected from 29/94/184/223/232/288/293, 29/94/184/232/287/288/293/311, 29/94/184/232/287/288/311, 29/94/184/232/287/293/332, 29/94/184/232/288/293/311/324/353, 29/94/184/232/288/311/324/332, 29/94/184/232/293, 29/94/184/232/311, 29/94/184/287/293, 29/94/184/287/293/311/353, 29/94/184/287/311, 29/94/184/288/293/324, 29/94/184/288/293/332/353, 29/94/184/288/298/332, 29/94/184/288/353, 29/94/184/293/311, 29/94/184/324, 29/94/223/232/287, 29/94/223/232/293/311/324, 29/94/223/288/293, 29/94/232/287/288/311, 29/94/232/288/293, 29/94/287/288/293, 29/94/287/288/293/353, 29/94/287/311, 29/94/293/311/324, 29/94/293/332/353, 29/94/311, 29/94/324/353, 29/184, 29/184/223/288/293/324, 29/184/232/288, 29/184/287, 29/184/287/288/293, 29/184/287/288/293/311, 29/184/287/311/332, 29/184/288/293, 29/184/288/293/311, 29/184/293, 29/184/293/311, 29/184/293/324, 29/184/353, 29/223/287/288/293/353, 29/232/287/288/293/332/353, 29/287/288/293, 29/287/288/293/324/353, 29/288/293, 29/311, 29/311/332, 29/353, 72/94/184/288/311, 86, 94/96/223/287/288/293/311/324/332, 94/99/184/293/311/332/353, 94/184/223, 94/184/223/232/287/311, 94/184/223/288/293, 94/184/223/293, 94/184/232/287/288, 94/184/232/287/288/324/332, 94/184/232/287/293, 94/184/232/287/293/332, 94/184/287/288/293, 94/184/287/288/311, 94/184/287/293/311/324/353, 94/184/287/311, 94/184/287/353, 94/184/288/293, 94/184/288/293/311, 94/184/288/293/311/332, 94/184/293, 94/184/293/311, 94/184/293/332/353, 94/223/232/311/353/355/356/357/358/359, 94/223/287/288/293, 94/223/288/293/311, 94/232/287/288/293/353, 94/232/288/293/353, 94/232/293, 94/232/293/324/332, 94/232/311, 94/287/288/293, 94/287/288/311/324, 94/288/293, 94/288/293/324, 94/293/324, 94/311/324, 153, 184/223, 184/223/287/288/353, 184/232/287/288/293/311, 184/232/287/288/324/353, 184/232/287/293/332, 184/287/288, 184/287/288/293, 184/287/288/293/324/353, 184/287/293, 184/287/293/311, 184/288/293/311/324, 184/332/353, 205, 223/232/287/288/332/353, 223/287/288, 223/287/288/293/311, 223/287/311/353, 223/288/293, 232/287/288/293, 232/287/288/293/311, 232/287/288/293/321, 232/287/311, 232/288/293, 232/288/293/332, 234/287/288/293/311, 287/288/293, 287/288/311/324, 287/293, 287/311, 287/311/332, 287/324, 288/293, 288/293/311/324/353, 288/293/324, 293/311, and 311/332. 3. The engineered polypeptide of claim 1 , further comprising at least one substitution or substitution set as compared to the amino acid sequence of SEQ ID NO: 4, wherein the at least one substitution or substitution set is at one or more positions selected from 29/57/94/153/184/205/261/265/287, 29/57/94/153/205/265, 29/57/94/184/205/277/353, 29/57/94/205/259/261/265, 29/57/94/205/261/353, 29/57/153/184/205/259/261/265/277, 29/57/153/184/205/353, 29/57/153/205/261/265, 29/57/153/205/261/265/277, 29/57/153/205/265/277, 29/57/153/205/277/353, 29/57/184/205/259/261/265/277/287/357, 29/57/184/205/259/261/265/287, 29/57/205/259/261/265, 29/57/205/259/261/265/287, 29/57/205/261/265/353, 29/94/153/184/205, 29/94/153/205/259/261/265, 29/94/153/205/259/261/287, 29/94/153/205/259/261/287/353, 29/94/184/205/259/261, 29/94/184/205/261, 29/94/205, 29/94/205/259/261/287, 29/94/205/261, 29/94/261/265/287/353, 29/153/184/205, 29/153/205, 29/153/205/259/261/265/277/287, 29/153/261/265/287/353, 29/184/205, 29/184/205/259/261/277, 29/184/205/353, 29/184/261/265, 29/184/265, 29/205, 29/205/259/261, 29/205/259/261/265/287, 29/205/259/261/287, 29/205/261, 29/205/261/265, 29/205/261/265/353, 29/205/261/353, 29/205/277, 29/205/287, 29/205/287/353, 29/259/261/265, 29/261/353, 57/94/153/184/205/259/261, 57/94/153/205/261/265, 57/94/153/259/261/265, 57/94/153/261/265, 57/94/184/205/261/265/287, 57/94/184/261/265/287, 57/94/205/259, 57/94/205/259/261/265/277/287, 57/94/205/261/265, 57/94/205/277, 57/153/205/259/261/265/287, 57/153/205/259/265/277, 57/153/205/261, 57/153/205/261/265, 57/153/205/265, 57/153/261/265, 57/153/261/265/287/353, 57/205, 57/205/259/261, 57/205/259/261/265/277/287/353, 57/205/259/261/287/353, 57/205/259/265, 57/205/261, 57/205/261/265/277/287/353, 57/205/261/277, 57/205/261/277/353, 57/261/265/353, 94/126/184/205/259/261/265, 94/153/184/205, 94/153/184/259/261/265, 94/153/205/259/261/265/353, 94/153/205/287, 94/153/205/287/353, 94/153/205/353, 94/184/205/259/261, 94/184/205/259/261/265/287, 94/184/205/259/261/353, 94/184/205/261/265/287/353, 94/184/353, 94/205, 94/205/259/261/265, 94/205/259/261/265/277/287, 94/205/259/261/265/353, 94/205/259/261/287/353, 94/205/261/265/277, 94/205/261/265/353, 94/205/261/353, 94/205/277, 94/259/261/265, 153/184/205/261/277/287, 153/184/261/265/287, 153/205, 153/205/261, 153/205/261/265/287, 153/259/261/265, 153/259/265/287, 153/261/265, 153/261/265/287/353, 153/265, 153/265/277/287/353, 184/205, 184/205/259/261/287, 184/205/261, 184/205/261/265, 184/205/261/287, 184/205/277, 184/205/287/353, 184/261/265/287, 202, 205, 205/259/261, 205/259/261/265, 205/259/261/265/277/287/353, 205/259/261/277/287, 205/259/265, 205/261, 205/261/265, 205/261/265/274, 205/261/265/287, 205/261/265/287/353, 205/261/277, 205/261/353, 205/277/287, 205/287, 223, 225, 256, 259/261/265, 261/265/287, 265/287, 283, and 294. 4. The engineered polypeptide of claim 1 , further comprising at least one substitution or substitution set as compared to the amino acid sequence of SEQ ID NO: 4, wherein the at least one substitution or substitution set is at one or more positions selected from 197/198/201/259, 198/201/259/280, 202/205/221/223/225/261, 202/205/221/223/261/294, 202/221/225/256/261/294, 202/221/223/225/283/294, 205/221, 205/221/223/225/256/261/283, 219, 221/223/225, 221/223, 221/223/225, 221/223/225/256/261, 221/223/225/261, 221/223/225/294, 221/225, 221/225/256, 221/225/256/261, 221/225/256/261/283/294, 223/225/256/261, 225/256/261, 280, and 280/306. 5. The engineered polypeptide of claim 1 , further comprising at least one substitution or substitution set as compared to the amino acid sequence of SEQ ID NO: 4, wherein the at least one substitution or substitution set is at one or more positions selected from 17/198/259/280, 197, 197/198, 197/198/223/259, 197/223/277/280, 197/259, 197/277, 198, 198/223, 198/223/259/277/280, 198/259, 198/259/277, 198/259/277/280, 198/277/280, 223/259, 223/259/280, 258, 259, 259/268/277/280, 259/277, 259/280, 263, 277, and 280. 6. The engineered polypeptide of claim 1 , further comprising at least one substitution or substitution set as compared to the amino acid sequence of SEQ ID NO: 4, wherein the at least one substitution or substitution set is at one or more positions selected from 141, 154, 197, 197/198, and 278. 7. The engineered polypeptide of claim 1 , wherein said engineered polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 50, 306, 648 or 708. 8. The engineered polypeptide of claim 1 , wherein said polypeptide comprises an amino acid sequence with at least 90% sequence identity to an even-numbered sequence-selected from the group consisting of SEQ ID NO: 14, 16, 38-646, 650-684, 688-700, and 704-802. 9. The engineered polypeptide of claim 1 , wherein said engineered polypeptide comprises an amino acid sequence set forth in an even numbered sequence selected
with NAD or NADP as acceptor (1.5.1) · CPC title
Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title
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