Carboxyesterase polypeptides for amide coupling
US-11015180-B2 · May 25, 2021 · US
Carboxyesterase polypeptides for amide coupling
US12286653B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-12286653-B2 |
| Application number | US-202117236928-A |
| Country | US |
| Kind code | B2 |
| Filing date | Apr 21, 2021 |
| Priority date | Dec 13, 2017 |
| Publication date | Apr 29, 2025 |
| Grant date | Apr 29, 2025 |
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Abstract
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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.
First claim
Opening claim text (preview).
The invention claimed is: 1. 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. 2. The polynucleotide sequence of claim 1 , wherein said encoded engineered carboxyesterase further comprises at least one substitution or substitution set at positions selected from: 39, 39/323, 62, 62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263, 77, 77/184, 103, 103/147, 104, 104/429, 105, 107, 107/185, 108, 109, 109/117, 110, 111, 113, 114, 115, 117, 118, 118/269, 118/349, 119, 126, 147, 153, 153/215, 164, 164/271, 174, 174/282, 183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231, 224, 224/268/372, 231, 249, 249/284, 263, 268, 269, 270, 270/470, 271, 271/416, 276, 277, 278, 279, 279/280, 280, 281, 281/374, 283, 283/429, 284, 284/438, 285, 286, 311, 317, 320, 320/323, 320/323/372, 320/372/376, 320/376/377, 321, 323, 324, 345, 349, 372, 372/376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429, 438, and 470, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2. 3. 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. 4. The polynucleotide sequence of claim 3 , wherein said encoded engineered carboxyesterase further comprises at least one substitution or substitution set in said polypeptide sequence at positions selected from: 63, 63/65/108, 63/65/108/189, 63/65/108/377, 63/65/285/320/323, 63/65/320/323, 63/108, 63/108/285/377, 63/108/285/377, 63/108/320/323, 63/189, 63/212/215, 63/212/215/268/269/343, 63/215, 63/215/269, 63/215/270/271, 63/215/343, 63/268/269/270/429, 63/377, 65/69/70/281/372, 65/69/70/372, 65/69/372, 65/70/372, 65/320, 65/320/323, 68, 68/69/189/214, 68/69/189/214/215, 68/69/189/214/215/271, 68/69/189/214/215/271/281/343/381, 68/69/189/214/271/280, 68/69/189/214/372, 68/69/189/214/377/381, 68/69/189/271, 68/69/189/271/280/372/381, 68/69/189/280/281/372/377, 68/69/189/281/372/377/381, 68/69/189/343/381, 68/69/189/372, 68/69/189/381, 68/69/214/215/271, 68/69/214/343, 68/69/215, 68/69/271, 68/69/287, 68/69/343/372, 68/108/377, 68/184, 68/184/189, 68/189/271/372, 68/189/343, 68/214/215/271/281/372, 68/215/271/343/372/381, 68/215/377, 68/271/372, 68/377, 69, 69/70, 69/70/331/372, 69/70/372, 69/70/459, 69/108, 69/108/270/372/377, 69/108/281/285, 69/110/215/281, 69/189, 69/189/214/271/281/343, 69/189/214/343/372, 69/189/215/343, 69/189/271, 69/189/271/281, 69/189/271/343, 69/189/271/343/381, 69/189/280/343/372/381, 69/189/281/373, 69/189, 69/189/343/381, 69/189/372, 69/189/372/377, 69/189/377, 69/212/213/215/280/281, 69/214/215/271/372/377/381, 69/214/271/282, 69/214/271/343, 69/215, 69/215/269/270/377, 69/215/271/280/281, 69/215/271, 69/215/271/372, 69/215/285/317, 69/215/323, 69/215/343, 69/215/343/372/381, 69/271, 69/271/372, 69/343/372, 69/285/373, 69/372, 70, 70/212, 108, 108/189, 108/189/285/320, 108/189/320, 108/189/377, 108/215, 108/215/377, 108/269/270, 108/270, 108/285/377, 108/285, 108/320/323, 108/377, 126, 126/184/213/280/281/285/320, 126/184/213/372, 126/189/285/372, 126/215, 126/372, 181/215, 189, 189/214, 189/214/215/271, 189/215, 189/215/249/277, 189/215/271/281/377, 189/215/343/372, 189/270/285, 189/270/372, 189/280, 189/320/377, 189/343, 189/343/377, 189/372/377, 189/377, 189/381, 213/215/320, 214/215/271, 214/215/271/377, 214/271, 214/280/343/377/381, 215, 215/249/280/281/285/372, 215/271/372, 215/280/281/285/372, 215/281/285/372, 215/281/285/373, 215/281/373, 215/285, 215/285/317, 215/285/346, 215/285/445, 215/320, 215/320/372, 215/323, 215/372, 215/372/377, 215/373, 215/377, 215/381, 249/377, 269/270/281/372/377, 270/377, 271, 271/343, 271/343/372, 271/343/372/381, 280/285/372, 281/372, 285/320/323, 285/323, 320, 343/372, 372, 372/377, 372/381, 373, and 377, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 5. 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. 6. The polynucleotide sequence of claim 1 , wherein said polynucleotide sequence is operably linked to a control sequence. 7. The polynucleotide sequence of claim 1 , wherein said polynucleotide sequence is codon optimized. 8. An expression vector comprising at least one polynucleotide sequence of claim 1 . 9. A host cell comprising at least one expression vector of claim 8 . 10. A host cell comprising at least one polynucleotide sequence of claim 1 . 11. The host cell of claim 9 , wherein said host cell is E. coli. 12. A method of producing an engineered carboxyesterase in a host cell, comprising culturing a host cell comprising at least one polynucleotide of claim 1 , under suitable conditions, such that at least one engineered carboxyesterase is produced. 13. The method of claim 12 , further comprising recovering at least one engineered carboxyesterase from the culture and/or host cell. 14. The method of claim 13 , further comprising the step of purifying said at least one engineered carboxyesterase. 15. The polynucleotide sequence of claim 1 , wherein the substitution at position 282 is a polar amino acid. 16. The polynucleotide sequence of claim 1 , wherein the substitution at position 282 is selected from: L282Q, L282S, and L282T. 17. The polynucleotide sequence of claim 1 , wherein said polynucleotide sequence has at least 85% sequence identity to a sequence selected from SEQ ID NO: 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 139. 18. The polynucleotide sequence of claim 3 , wherein said polynucleotide sequence has at least 85% sequence identity to a sequence selected from SEQ ID NO: 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 139.
Assignees
Inventors
Classifications
Carboxylesterase (3.1.1.1) · CPC title
Genes encoding for enzymes or proenzymes · CPC title
Biofuels, e.g. bio-diesel · CPC title
- C12N9/18Primary
Carboxylic ester hydrolases {(3.1.1)} · CPC title
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Frequently asked questions
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- What does patent US12286653B2 cover?
- 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.
- Who is the assignee on this patent?
- Codexis Inc
- What technology area does this patent fall under?
- Primary CPC classification C12N9/18. 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 5 related publications on this page (citations in our corpus or others sharing the same primary CPC).