Vectors for expression of biocatalysts
US-11634695-B2 · Apr 25, 2023 · US
Engineered proline hydroxylase biocatalysts for hydroxylation of chemical compounds
US12286648B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-12286648-B2 |
| Application number | US-202318180711-A |
| Country | US |
| Kind code | B2 |
| Filing date | Mar 8, 2023 |
| Priority date | Jun 9, 2016 |
| Publication date | Apr 29, 2025 |
| Grant date | Apr 29, 2025 |
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- Title
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Abstract
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The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.
First claim
Opening claim text (preview).
What is claimed is: 1. An engineered polypeptide having proline hydroxylase activity comprises an amino acid sequence having at least 90% sequence identity to reference sequence SEQ ID NO:604 and one or more residue differences as compared to SEQ ID NO:604 at residue positions selected from: 13, 14, 24, 26, 27, 30, 57, 61, 62, 72, 76, 77, 81, 82, 86, 88, 97, 114, 127, 128, 142, 158, 161, 163, 173, 175, 176, 178, 180, 184, 185, 186, 187, 188, 189, 191, 192, 195, 198, 200, 207, 209, 210, 211, 213, 215, 217, 218, 222, 225, 230, 233, 236, 238, 240, 241, 256, 259, 263, 265, 271, and 273. 2. The engineered polypeptide of claim 1 , wherein said polypeptide has at least 90% sequence identity to SEQ ID NO: 604 or 640. 3. The engineered polypeptide of claim 1 , wherein said polypeptide comprises residue differences at positions 26, 30, 62, 82, 114, 158, 161, 271, and 273, as compared to SEQ ID NO: 604. 4. The engineered polypeptide of claim 1 , wherein said polypeptide comprises residue differences of 26A, 30N, 62D, 82K, 114S, 158N, 161P, 271W, and 273T, as compared to SEQ ID NO: 604. 5. The engineered polypeptide of claim 1 , wherein said engineered polypeptide is capable of converting(S)-pipecolic acid to (25,55)-5-hydroxypipecolic acid. 6. The engineered polypeptide of claim 5 , wherein said engineered polypeptide is capable of converting(S)-pipecolic acid to (25,55)-5-hydroxypipecolic acid with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold or more the activity of the naturally occurring enzyme. 7. The engineered polypeptide of claim 5 , wherein said engineered polypeptide is capable of converting(S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid with greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more diastereomeric excess of (2S,5R)-5-hydroxypipecolic acid. 8. A polynucleotide encoding the engineered polypeptide of claim 1 . 9. A polynucleotide encoding the engineered polypeptide of claim 3 . 10. The polynucleotide of claim 8 , wherein said polynucleotide comprises a nucleic acid sequence optimized for expression in E. coli. 11. The polynucleotide of claim 9 , wherein said polynucleotide comprises a nucleic acid sequence optimized for expression in E. coli. 12. An expression vector comprising the polynucleotide of claim 8 , optionally further comprising at least one control sequence. 13. The expression vector of claim 12 , wherein said vector comprises SEQ ID NO:1007, 1008, or 1009. 14. A host cell comprising the polynucleotide of claim 8 . 15. A method of preparing an engineered polypeptide, comprising culturing the host cell of claim 14 , under conditions suitable for expression of the polypeptide. 16. The method of claim 15 , further comprising a step of isolating the engineered polypeptide.
Assignees
Inventors
Classifications
with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11) · CPC title
Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase · CPC title
containing a six-membered hetero ring · CPC title
- C12N9/0071Primary
acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title
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- What does patent US12286648B2 cover?
- The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.
- Who is the assignee on this patent?
- Codexis Inc
- What technology area does this patent fall under?
- Primary CPC classification C12N9/0071. Mapped technology areas include Chemistry & Metallurgy.
- When was this patent published?
- Publication date Tue Apr 29 2025 00:00:00 GMT+0000 (Coordinated Universal Time) (B2). Legal status and post-grant events are not shown on this page.
- What related patents are in patentsdb?
- We list 6 related publications on this page (citations in our corpus or others sharing the same primary CPC).