Production of fatty alcohols from engineered microorganisms
US-9714437-B2 · Jul 25, 2017 · US
Engineered phenylalanine ammonia lyase polypeptides
US11198861B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-11198861-B2 |
| Application number | US-201916441458-A |
| Country | US |
| Kind code | B2 |
| Filing date | Jun 14, 2019 |
| Priority date | Jul 12, 2018 |
| Publication date | Dec 14, 2021 |
| Grant date | Dec 14, 2021 |
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Abstract
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The present invention provides engineered phenylalanine ammonia lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia lyase (PAL) polypeptides. Methods for producing PAL enzymes are also provided. In some embodiments, the engineered PAL polypeptides are optimized to provide enhanced catalytic activities that are useful under industrial process conditions for the production of pharmaceutical compounds.
First claim
Opening claim text (preview).
We claim: 1. An engineered phenylalanine ammonia lyase comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 4, wherein said engineered phenylalanine ammonia lyase comprises a substitution at position 104 and at least one substitution selected from positions 220, 222, and 359 in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 2. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 20/306/564, 74/80/105/107/394/420, 74/83/102/105/107/111/222/394/416, 74/97/105/106/107, 74/102/105/106/107/175/394, 74/102/105/106/107/175/394/421, 80/84/99/104/105/107/219, 80/102/105/107/304, 83/102/105/106/107/416/420/421, 83/102/105/394/416/420, 84, 84/99, 84/99/104/105/219, 84/107, 97/102/105/106/107/111/394/420/421, 97/102/105/107/111/175/304/421/424, 97/102/111/175/222/420/421, 97/105/107/111/222/421/424, 97/105/107/111/394/416/421, 99/105/107, 102, 102/105/107/222/304/307/394/421/424, 102/105/107/222/304/394/421/424, 102/105/107/304/424, 102/105/107/394/416/424, 102/107/111/222/394, 102/107/420/424, 103, 104, 105, 105/106/107/420/421, 105/107, 105/107/111, 105/107/111/304, 105/107/111/394/420/424, 105/107/222/304/416, 105/111/219, 105/175/219, 105/219, 106, 107, 107/111/209/222/304, 107/222/304, 107/291, 107/421, 175, 216, 219, 220, 222/421/424, 304/394/416/420, 306, 359, 394, 395, 413, 416, 418, and 420, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 3. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 3, 3/550, 4, 5, 6, 7, 10, 14, 22, 22/76, 24, 25, 40, 75, 76, 76/561, 84/107/175/219, 84/107/219, 102/107/219/410, 107, 107/216/410, 107/219, 107/220, 212, 219/220, 219/220/410, 220/359, 220/410, 286, 301, 303, 410, 502, 544, 566, and 567, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 4. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 3/4/5/7/76/84/107/307, 3/5/107/307/566, 3/7/76/107/307/566, 3/7/84/307, 2 4 / 7 6/107/307, 24/84/107/307, 76, 76/84/107, 76/84/107/307, 76/84/107/307/502, 76/84/107/502, 76/107, 76/107/307, 76/307, 76/307/502, 84/107/307, 84/107/307/502, 84/301/307/566, 84/307, 107/301/502, 107/307, 107/307/566, 107/502, 107/502/566, 307, 307/502, and 307/566, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 5. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 3/4/6/7/76, 3/4/6/7/303, 3/ 4 / 7 , 3/ 4 / 7 /76, 3/6/502, 3/7/76, 7/303, 40/303, 76, 76/502, 82, 100, 102, 171, 174, 216, 218, 219, 222, 222/509, 303, 303/502, 304, and 345, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 6. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 3/4/7, 3/7, 4/7, 4/7/216/218, 7, 7/40, 7/76/82/174/222/303, 7/76/174/222, 7/76/219/303, 7/82, 7/216/218, 40/82, 47, 66, 76/82/216/218, 76/216, 76/216/219, 82, 112, 171, 174/222, 209, 216, 219, 219/345, 222, 268, 271, 331, 366, 428, 437, 443, 460, 474, 503, 524, 538, and 543, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 7. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 4, 4/304, 6, 7, 9, 16, 20, 25, 40, 40/437, 44/47, 44/47/94/509, 44/94/270/554, 44/94/554, 47, 47/76, 47/76/345, 47/94/509, 47/195/554, 47/428, 51/106, 76, 76/271, 76/345, 82, 84, 94/149, 94/195, 94/554, 98, 98/460, 109, 112/524, 271/345, 271/428, 302, 303, 304, 306, 349, 358, 410, 413, 416, and 524, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 8. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 4/47/76/82/94, 7/44/94/98, 7/47/76/82, 7/76/554, 7/94/98, 16/40/76, 16/4 4 / 7 6/98, 16/44/94/98/524, 25/54/68/72, 25/54/68/72/158/339, 25/54/68/72/209/212/339/517, 25/54/68/158/339/517, 25/5 4 / 7 2/339/517, 25/54/158/209/212/339/551, 30/68/72/207/209/212/339/495/517, 40/4 4 / 7 6/304/509, 40/44/98/304, 40/76/304/437, 40/76/554, 44/76/94/112/304, 44/76/112, 44/94/271/304/437/554, 47/76/82/94/271, 47/76/82/271/304, 47/76/94/271, 47/76/94/271/306/375/524/554, 47/76/304/306/554, 47/76/304/524/554, 47/94, 47/94/271, 47/94/271/304/554, 49/114/240/521, 54/68/158/209/212/495/517, 68/72/158/209/212/339/495/551, 68/72/158/517, 68/158/209/495/517/551, 76, 76/271/304/554, 76/304/437, 82, 82/554, 94/98, 94/98/306, 94/98/509, 94/98/524, 94/554, 98/270/304/554, 119, 294, 357, 400, 516, 527, and 565, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 9. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 25, 25/40/158/209/304/410/517, 25/54/271/517, 25/158/209/410, 25/306/339, 25/410, 40/47/68/94/98/410/517, 40/68/460/517, 47/98/339/410, 54/68/72/98/209/517, 68, 68/339/517, 72/94/158/339/410/460/517, 72/158/209/410/517, 83, 94/158/209/339/410, 100, 129, 158, 158/207/339/410, 158/209/410/517, 207/410, 207/410/460/517, 220, 317, 339, 394, 410, 410/517, 416, 460, 460/517, and 517, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 10. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 25/83/158/220/517, 25/83/220/416, 25/158/209/220/517, 25/158/220, 25/158/220/517, 25/220/339, 25/220/517, 25/410/416/517, 40, 45, 54/59, 54/285, 83, 83/209/220/410/517, 83/339/410, 119, 158/220/271/517, 209, 220, 220/410/416/517, 220/517, 244, 246, 271, 271/410/416/517, 293, 304, 339, 368, 400, 410, 410/416/517, 424, 459, 479, 520, 525, 537, 562, and 565, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 11. The engineered phenylalanine ammonia lyase of claim 1 , wherein said engineered phenylalanine ammonia lyase further comprises at least one substitution or substitution set at one or more positions selected from 25/40/45/209/424, 25/40/424, 25/45/5 4 / 7 3/246/424, 25/5 4 / 7 3/209/424/520, 40/47/54/214/503, 40/54/209/214/244/339/520, 40/54/214/244/339/503, 40/209/246/424, 54, 54/209/214/244, 54/209/214/244/339/503, 54/424, 54/424/520, 209/503, 227, 246, 246/424, 274/311, 410, 411, 413, and 424, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 8. 12.
Assignees
Inventors
Classifications
with oxygen as acceptor (1.4.3) · CPC title
- C12N9/88Primary
Lyases (4.) · CPC title
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title
Phenylalanine ammonia-lyase (4.3.1.24) · CPC title
Vectors or expression systems specially adapted for E. coli · CPC title
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- What does patent US11198861B2 cover?
- The present invention provides engineered phenylalanine ammonia lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia lyase (PAL) polypeptides. Methods for producing PAL enzymes are also provided. In some embodiments, the engineered PAL polypeptides are optimized to provide enhanced catalytic activities that are useful under …
- Who is the assignee on this patent?
- Codexis Inc
- What technology area does this patent fall under?
- Primary CPC classification C12N9/88. Mapped technology areas include Chemistry & Metallurgy.
- When was this patent published?
- Publication date Tue Dec 14 2021 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 2 related publications on this page (citations in our corpus or others sharing the same primary CPC).