Engineered purine nucleoside phosphorylase variant enzymes

Patent metadata

Field	Value
Publication number	US-2022010316-A1
Application number	US-202117490215-A
Country	US
Kind code	A1
Filing date	Sep 30, 2021
Priority date	Jul 9, 2018
Publication date	Jan 13, 2022
Grant date	—

Abstract

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

First claim

We claim:

1 . A polynucleotide sequence encoding an engineered purine nucleoside phosphorylase comprising a polypeptide sequence having at least 85% sequence identity to SEQ ID NO:2, 6, and/or 126, wherein the polypeptide sequence of said engineered purine nucleoside phosphorylase comprises a substitution at position 65 and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NOs: 2, 6, and/or 126, and wherein said engineered purine nucleoside phosphorylase comprises improved activity on the substrate of compound 3

as compared to wild-type E. coli purine nucleoside phosphorylase.

2 . A polynucleotide sequence encoding at least one engineered purine nucleoside phosphorylase, 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, 5, 125, 241, and/or 683, wherein the polynucleotide sequence of said engineered purine nucleoside phosphorylase comprises at least one substitution at one or more positions.

3 . A polynucleotide sequence encoding at least one engineered purine nucleoside phosphorylase 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, 5, 125, 241, and/or 683, or a functional fragment thereof.

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

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

6 . The polynucleotide sequence of claim 1 , wherein said polynucleotide sequence comprises a polynucleotide sequence set forth in the odd numbered sequences of SEQ ID NOs: 3-1001.

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 polynucleotide sequence of claim 1 .

10 . A method of producing an engineered purine nucleoside phosphorylase in a host cell, comprising culturing the host cell of claim 9 , under suitable conditions, such that at least one engineered purine nucleoside phosphorylase is produced.

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

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

Assignees

• Codexis Inc

Inventors

• Novick Scott J
• Dellas Nikki
• Mitchell Vesna
• Duan Da
• Nazor Jovana
• Alvizo Oscar
• Sowell-Kantz Auric Anthony
• Moore Jeffrey C
• Huffman Mark A
• Rodriguez-Granillo Agustina
• Verma Deeptak
• Marshall Nicholas M
• Russell Jay H
• Canada Keith A

Classifications

• C12Y204/02001

  Purine-nucleoside phosphorylase (2.4.2.1) · CPC title

• C12N9/1077 Primary

  Pentosyltransferases (2.4.2) · CPC title

• C12N15/63 Primary

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