Engineered deoxyribose-phosphate aldolases

Patent metadata

Field	Value
Publication number	US-2020010868-A1
Application number	US-201916460118-A
Country	US
Kind code	A1
Filing date	Jul 2, 2019
Priority date	Jul 9, 2018
Publication date	Jan 9, 2020
Grant date	—

Abstract

The present invention provides engineered deoxyribose-phosphate aldolase polypeptides useful under industrial process conditions for the production of pharmaceutical and fine chemical compounds.

First claim

We claim:

1 . An engineered deoxyribose-phosphate aldolase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOS: 2, 6, and/or 466, or a functional fragment thereof, wherein said engineered deoxyribose-phosphate aldolase comprises at least one substitution or substitution set in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2, 6, and/or 466.

2 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2, wherein said engineered deoxyribose-phosphate aldolase comprises at least one substitution or substitution set in said polypeptide sequence at one or more positions selected from 10/47/66/141/145/156, 2, 6, 9, 10/47/88/156, 13, 31, 46, 47, 47/134/141/212, 66, 66/88/112/134/141/143/145/212, 71, 72, 88, 94, 102, 104, 112, 116, 133, 133/173/204/235/236, 134, 145, 145/173, 147, 173, 184, 189, 197, 203, 204, 207, 226, 235, 235/236, and 236, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

3 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:6, wherein said engineered deoxyribose-phosphate aldolase comprises at least one substitution or substitution set in said polypeptide sequence at one or more positions selected from 2, 2/6/10/66/88/102/184/235/236, 2/6/102/141/203/235, 2/10/102/235/236, 2/47/66/71/141/184/203/235/236, 2/47/71/88/203/235/236, 2/47/71/141/197/235/236, 2/88/141/235/236, 2/203/235/236, 5, 6/203/235/236, 9, 10/47/66/184/197/236, 10/47/66/235/236, 10/47/71/184/203/235/236, 13, 13/46, 13/46/66/94, 13/46/66/94/184/204, 13/46/66/133/134/184, 13/46/66/184, 13/46/112/134/184, 13/46/133/184, 13/66/94, 13/66/94/184, 13/66/112/133/134/184/204, 13/66/112/133/134/204, 13/66/112/184, 13/66/133/134/184/204, 13/66/184/204, 13/94/133/184, 13/94/184, 13/94/184/204, 13/133/134/184, 13/133/134/204, 13/134/204, 13/184, 27, 40, 46/66/94/112, 46/66/112/133/134/184, 46/66/133/134, 46/66/133/184/197, 46/66/133/197, 46/66/134/184/197/204, 46/66/184, 46/112/133, 46/112/133/134/204, 46/133/204, 46/197/204, 47/66/71/88/203/235/236, 47/66/71/141/184, 47/66/88/184/235, 47/66/88/203/235/236, 47/66/141/235/236, 47/66/184/197/203/235/236, 47/66/184/197/236, 47/71/88/184/203/235, 47/71/88/184/203/236, 47/71/141/184/203/235, 47/71/184, 47/71/184/197/236, 47/71/184/235, 47/71/184/236, 47/88/102/141/235/236, 47/88/203/235/236, 47/88/235/236, 47/102/184/197/235/236, 47/102/203/235/236, 47/141/184, 47/141/184/236, 47/184/236, 47/203/235, 47/203/235/236, 47/203/236, 47/235/236, 62, 66, 66/88/102/184/197/203/235/236, 66/88/197, 66/88/235, 66/88/235/236, 66/94, 66/94/112/133/184, 66/94/112/184/204, 66/102/184/235/236, 66/112/133/134/197, 66/112/133/197, 66/112/134/197, 66/112/184, 66/133/184, 66/133/197, 66/184, 66/197/203/235, 84, 88, 88/102/141/184/235/236, 88/184, 88/184/197/203/235/236, 88/184/197/235/236, 88/184/203/235, 88/184/236, 88/197/235/236, 88/236, 94/112/184/197, 94/133/184, 96, 102/141/203, 102/184/203/236, 102/184/236, 102/197/235, 112, 112/133/134, 112/133/184/204, 112/134/197, 114, 115, 120, 127, 132, 133, 133/134/184, 141/184/203/235/236, 141/184/235, 141/197, 141/203/235/236, 141/236, 146, 148, 184, 184/203, 184/203/235, 184/203/236, 184/235/236, 184/236, 197/235, 203/235/236, 203/236, 204, 218, 235, 235/236, and 236, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6.

4 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 466, wherein said engineered deoxyribose-phosphate aldolase comprises at least one substitution or substitution set in said polypeptide sequence at one or more positions selected from 71/88/94/133, 71/133, and 133, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 466.

5 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered deoxyribose-phosphate aldolase variant set forth in Table 3.1, 4.1, and/or 6.1.

6 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises a variant engineered polypeptide provided in Table 3.1, 4.1, and/or 6.1.

7 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered deoxyribose-phosphate aldolase variant set forth in SEQ ID NO: 2, 6, and/or 466.

8 . The engineered deoxyribose-phosphate aldolase of claim 6 , wherein said engineered deoxyribose-phosphate aldolase is a variant engineered polypeptide set forth in SEQ ID NO: 6 and/or 466.

9 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered deoxyribose-phosphate aldolase variant set forth in the even numbered sequences of SEQ ID NOS: 2-478.

10 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises a polypeptide sequence forth in the even numbered sequences of SEQ ID NOS: 2-478.

11 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase exhibits at least one improved property compared to wild-type Shewanella halifaxensis deoxyribose-phosphate aldolase.

12 . The engineered deoxyribose-phosphate aldolase of claim 11 , wherein said improved property comprises improved activity on a substrate.

13 . The engineered deoxyribose-phosphate aldolase of claim 12 , wherein said substrate comprises R-2-ethynyl-glyceraldehyde.

14 . The engineered deoxyribose-phosphate aldolase of claim 11 , wherein said improved property comprises improved thermostability.

15 . The engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase is purified.

16 . A composition comprising at least one engineered deoxyribose-phosphate aldolase provided in claim 1 .

17 . A polynucleotide sequence encoding at least one engineered deoxyribose-phosphate aldolase of claim 1 .

18 . An engineered polynucleotide sequence encoding at least one engineered deoxyribose-phosphate aldolase, wherein said polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1, 5, and/or 465, wherein the polynucleotide sequence of said engineered deoxyribose-phosphate aldolase comprises at least one sub

Assignees

• Codexis Inc

Inventors

• Duan Da
• Alvizo Oscar
• Nazor Jovana
• Maniar Harvinder Chagger
• Riggins James Nicholas
• Vroom Jonathan
• Sivaramakrishnan Santhosh
• Yang Hao
• Fryszkowska Anna
• Huffman Mark
• Kolev Joshua
• Farasat Iman
• Rodriguez-Granillo Agustina
• Verma Deeptak

Classifications

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y401/02004

  Deoxyribose-phosphate aldolase (4.1.2.4) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title