Engineered deoxyribose-phosphate aldolases

Patent metadata

Field	Value
Publication number	US-2022162580-A1
Application number	US-202217592869-A
Country	US
Kind code	A1
Filing date	Feb 4, 2022
Priority date	Jul 9, 2018
Publication date	May 26, 2022
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 polynucleotide encoding an engineered deoxyribose-phosphate aldolase, wherein said engineered deoxyribose-phosphate aldolase comprises 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 NOs: 2, 6, and/or 466.

2 . The engineered polynucleotide encoding an engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises a 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 polynucleotide encoding an engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises 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 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 polynucleotide encoding an engineered deoxyribose-phosphate aldolase of claim 1 , wherein said engineered deoxyribose-phosphate aldolase comprises 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 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 polynucleotide encoding an 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.

6 . The engineered polynucleotide encoding an 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.

7 . 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 NOs: 1, 5, and/or 465, wherein the polynucleotide sequence of said engineered deoxyribose-phosphate aldolase comprises at least one substitution at one or more positions.

8 . An engineered polynucleotide sequence encoding at least one engineered deoxyribose-phosphate aldolase comprising 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: 1, 5, and/or 465, or a functional fragment thereof.

9 . The engineered polynucleotide sequence of claim 8 , wherein said engineered polynucleotide sequence comprises a sequence set forth in the odd numbered sequences of SEQ ID NOs: 1-477.

10 . The engineered polynucleotide sequence of claim 1 , wherein said polynucleotide sequence is operably linked to a control sequence.

11 . The engineered polynucleotide sequence of claim 1 , wherein said engineered polynucleotide sequence is codon optimized.

12 . An expression vector comprising at least one polynucleotide sequence of claim 1 .

13 . A host cell comprising at least one expression vector of claim 12 .

14 . A host cell comprising at least one polynucleotide sequence of claim 1 .

15 . A method of producing an engineered deoxyribose-phosphate aldolase in a host cell, comprising culturing the host cell of claim 14 , in a culture medium under suitable conditions, such that at least one engineered deoxyribose-phosphate aldolase is produced.

16 . The method of claim 15 , further comprising recovering at least one engineered deoxyribose-phosphate aldolase from the culture medium and/or host cell.

17 . The method of claim 16 , further comprising the step of purifying said at least one engineered deoxyribose-phosphate aldolase.

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 A
• Kolev Joshua N
• Farasat Iman
• Rodriguez-Granillo Agustina
• Verma Deeptak

Classifications

• C12Y401/02004

  Deoxyribose-phosphate aldolase (4.1.2.4) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12P21/02

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

• C12N15/63

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