Engineered phosphopentomutase variant enzymes

Patent metadata

Field	Value
Publication number	US-2024060064-A1
Application number	US-202318467259-A
Country	US
Kind code	A1
Filing date	Sep 14, 2023
Priority date	Jul 9, 2018
Publication date	Feb 22, 2024
Grant date	—

Abstract

The present invention provides engineered phosphopentomutase (PPM) enzymes, polypeptides having PPM activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing PPM enzymes are also provided. The present invention further provides compositions comprising the PPM enzymes and methods of using the engineered PPM enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

First claim

We claim:

1 . An engineered phosphopentomutase comprising a polypeptide sequence having at least 85% sequence identity to SEQ ID NO: 2, 76, 118, 266, 420, 562, 656, 790, and/or 846, or a functional fragment thereof, wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 64/98/99/146/179/231/261/357, 65, 77/118/147/154/231/357, 98, 99, 98/99/146/231/357, 98/99/146/179/261/357/397, 98/99/146/153/231/261/357, 98/99/146/231, 98/99/146/179/231/261/357, 98/99/231/261/357/397, 98/99/146/261/357/397, 98/99/146/153/231/357, 98/99/146/153/231/357, 98/99/146/179/231/261/357/397, 98/99/146/231/261/357/397, 98/99/146/231/261/357, 98/99/146/179/231/261/357/397, 98/99/146/261/357/397, 98/99/146/179/231/357, 98/99/146/231/357, 98/99/146/153/179/231/357, 98/99/146/153/231/261/357/397, 114, 117, 118/147/154/225/231, 118/147/154/225/233/257/357, 118/147/154/225/257/357, 118/147/154/231/309, 118/147/225/231/257/309/357, 118/147/225/231/357, 118/147/225/234/257/357, 118/147/231, 118/147/231/257/308, 118/147/231/257/309/357, 118/154/225/231/357, 118/154/225/234/257/357, 118/154/231, 118/154/231/257/309/357, 118/154/231/257/357, 118/154/231/309/338/357, 118/154/231/357, 118/231, 118/231/257/357, 145, 146, 147, 147/154/225/231/309/357, 147/231, 147/231/257/357, 147/231/309/357, 147/231/357, 153, 153/231, 154/231/309, 154/231/309/357, 154/231/354/357, 173, 225/231/257/309/357, 231, 233, 314, and 357, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2, 76, 118, 266, 420, 562, 656, 790, and/or 846.

2 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase 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: 118, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions selected from 64/99/114/231/357, 65, 67, 87/225, 99/114, 99/114/147/231/261/397, 99/114/147/231/357, 99/114/147/257, 99/114/147/261, 99/114/153, 99/114/153/231/357, 99/114/153/261, 99/114/153/261/357, 99/114/231, 99/114/231/261, 99/114/231/357, 99/114/257/261/357, 99/114/357, 99/231/257/261, 99/257, 114/231/257/261, 114/231/357/397, 114/257/357, 114/392, 154, 211, 211/316, 225, 234/310, 257, 257/261, 257/357, 263, 350, 357, and 359, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 118.

3 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85%, sequence identity to SEQ ID NO: 266, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions selected from 65/225, 65/225/263, 97, 118, 143/194, 147, 156, 160, 172, 176, 188, 192, 194, 207, 211, 217, 225, 227, 236, 238, 257, 257/261, 261, 263, 306, and 397, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 266.

4 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 266, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions selected from 116/238, 118/147/192/238, 118/147/225/257, 118/147/238/257/357, 118/156/192/194/231/257, 118/194/238, 118/231/257/357, 118/238, 118/257/357, 147/194/217/225/236/257, 147/225/238/357, 147/236/238/257, 156/192/217/236/257, 156/217/236/257/357, 156/236/238, 192/238, 192/238/357, 225/238/357, 227, 231/257, 236, 236/238/257, 238/257, and 257, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 266.

5 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 420, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions selected from 12, 94, 118/147/227/238/257/357, 118/151, 118/236/357, 121, 142, 148, 150, 151, 172, 181, 185, 186, 189, 193, 199, 206, 218, 235, 237, 239, 256, 266, 267, 281, 300, 308, and 383, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 420.

6 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85%, sequence identity to SEQ ID NO: 420, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 98/151/172/235/256/266/267, 98/256, 108/235/267, 148/150/172/235, 148/151/172/235/256, 148/151/235/256/301, 148/151/235/267, 150/151/172/235/260, 150/151/235, 150/172/235/267/301, 150/235, 150/267, 151, 151/172/235, 151/172/235/256/301, 151/235/260, 151/256, 151/267/301, 172, 172/235, 172/235/237/256/267, 172/267/301/312, 235, 235/256/267, 235/256/267/307, 235/260, 235/267, 235/267/301, 256, 260, 267, 267/301, 301, 307, and 312, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 420.

7 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 562, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 21, 118, 120, 151, 155, 220, 225, and 357, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 562.

8 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 562, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 21/66/120/155, 21/117/120/139, 21/117/120/151/155/225, 21/117/120/222, 21/117/222/225, 21/120, 21/120/139, 21/120/151, 21/120/151/155/222/225, 21/120/151/222/225, 21/120/151/225/316, 21/120/155, 21/120/222/357, 21/139/151/357, 21/139/357, 21/151/155/222/225, 21/225/357, 21/357, 50/357, 117/120/151/155, 117/120/151/222, 117/120/151/222/225, 117/120/225, 117/139/151/225, 117/151/155/222/357, 117/155/222/357, 117/220/225, 120, 120/139, 120/139/151, 120/139/151/222, 120/139/222, 120/139/357, 120/151/155, 120/151/155/222/225, 120/151/155/225, 120/151/222, 120/151/222/225, 120/151/225, 120/155, 120/155/222, 120/155/225, 120/155/357, 120/220, 120/220/222/357, 120/220/225, 120/222, 120/225, 120/225/357, 120/225/401, 120/357, 139/357, 151/155, 155/222/225/357, 220/357, 222/357, 298/357, and 357, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 562.

9 . The engineered phosphopentomutase of claim 1 , wherein the polypeptide sequence of said engineered phosphopentomutase has at least 85% sequence identity to SEQ ID NO: 656, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 6, 8, 39,

Assignees

• Codexis Inc

Inventors

• Novick Scott J
• Yi Xiang
• Dellas Nikki
• Alvizo Oscar
• Nazor Jovana
• Duan Da
• Mitchell Vesna
• Vroom Jonathan
• Sivaramakrishnan Santhosh
• Subramanian Nandhitha
• Moore Jeffrey C
• Huffman Mark
• Rodriguez-Granillo Agustina
• Verma Deeptak
• Murphy Grant S
• Marshall Nicholas
• Russell Jay
• Canada Keith A

Classifications

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/90 Primary

  Isomerases (5.) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N15/815

  for yeasts other than Saccharomyces · CPC title