Engineered phosphopentomutase variant enzymes

Patent metadata

Field	Value
Publication number	US-2021261940-A1
Application number	US-202117316165-A
Country	US
Kind code	A1
Filing date	May 10, 2021
Priority date	Jul 9, 2018
Publication date	Aug 26, 2021
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 polynucleotide sequence encoding an engineered phosphopentomutase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 2, 4, 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 and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NOs: 2, 4, 118, 266, 420, 562, 656, 790, and/or 846.

2 . An engineered polynucleotide sequence encoding at least one engineered phosphopentomutase, wherein said polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1, 3 117, 265, 419, 561, 655, 789, and/or 845, and wherein the engineered polynucleotide sequence of said engineered phosphopentomutase comprises at least one substitution at one or more positions.

3 . An engineered polynucleotide sequence encoding at least one engineered phosphopentomutase comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1, 3 117, 265, 419, 561, 655, 789, and/or 845, or a functional fragment thereof.

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

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

6 . The engineered polynucleotide sequence of claim 1 , wherein said engineered polynucleotide sequence comprises a polynucleotide sequence forth in the odd numbered sequences of SEQ ID NOs: 7-1151.

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

8 . A host cell comprising at least one expression vector of claim 7 .

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

10 . A method of producing an engineered phosphopentomutase in a host cell, comprising culturing a host cell comprising at least one engineered polynucleotide of claim 1 , under suitable conditions, such that at least one engineered phosphopentomutase is produced.

11 . The method of claim 10 , further comprising recovering at least one engineered phosphopentomutase from the culture and/or host cell.

12 . The method of claim 11 , further comprising the step of purifying said at least one engineered phosphopentomutase.

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

• C12Y504/02007

  Phosphopentomutase (5.4.2.7) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N15/815

  for yeasts other than Saccharomyces · CPC title