Engineered sucrose phosphorylase variant enzymes

Patent metadata

Field	Value
Publication number	US-2022243183-A1
Application number	US-202017617533-A
Country	US
Kind code	A1
Filing date	Jun 30, 2020
Priority date	Jul 2, 2019
Publication date	Aug 4, 2022
Grant date	—

Abstract

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

First claim

1 . An engineered sucrose phosphorylase 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 NO: 2 and/or 4, or a functional fragment thereof, wherein the polypeptide sequence of said engineered sucrose phosphorylase 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 NO: 2 and/or 4.

2 . The engineered sucrose phosphorylase 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, and wherein the polypeptide sequence of said engineered sucrose phosphorylase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 397, 7, 10, 48, 136, 158, 205, 207, 211, 215, 301, 333, 378, and 400, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

3 . The engineered sucrose phosphorylase of claim 1 , wherein said polypeptide sequence of said engineered sucrose phosphorylase 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: 4, and wherein said polypeptide sequence of said engineered sucrose phosphorylase comprises at least one substitution or substitution set at one or more positions selected from 10/215/400, 158, 158/207/215, 158/207/215/301/400, 158/207/215/400, 158/207/400, 158/211/400, 158/215/301/400, 158/215/400, 158/301/400, 158/400, 205, 207, 207/215, 207/215/400, 207/400, 215/301, 215/400, 242/400, 301, 301/400, and 400, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4.

4 .- 5 . (canceled)

6 . The engineered sucrose phosphorylase of claim 1 , wherein said engineered sucrose phosphorylase comprises a variant engineered sucrose phosphorylase set forth in SEQ ID NO: 4.

7 . The engineered sucrose phosphorylase of claim 1 , wherein said engineered sucrose phosphorylase 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 sucrose phosphorylase variant set forth in the even numbered sequences of SEQ ID NOS: 4-84.

8 . The engineered sucrose phosphorylase of claim 1 , wherein said engineered sucrose phosphorylase comprises a polypeptide sequence forth in at least one of the even numbered sequences of SEQ ID NOS: 4-84.

9 . The engineered sucrose phosphorylase of claim 1 , wherein said engineered sucrose phosphorylase comprises at least one improved property compared to wild-type Alloscardovia omnicolens sucrose phosphorylase.

10 . The engineered sucrose phosphorylase of claim 9 , wherein said improved property comprises improved activity on a substrate.

11 . The engineered sucrose phosphorylase of claim 10 , wherein said substrate comprises sucrose and/or inorganic phosphate.

12 . The engineered sucrose phosphorylase of claim 9 , wherein said improved property comprises improved production of compound (1) and/or compound (3).

13 . The engineered sucrose phosphorylase of claim 1 , wherein said engineered sucrose phosphorylase is purified.

14 . The engineered sucrose phosphorylase of claim 1 , wherein said engineered sucrose phosphorylase is part of a multi-enzyme system for producing a nucleoside analogue.

15 . A composition comprising at least one engineered sucrose phosphorylase of claim 1 .

16 . A polynucleotide sequence encoding at least one engineered sucrose phosphorylase of claim 1 .

17 . A polynucleotide sequence encoding at least one engineered sucrose phosphorylase, 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 and/or 3, wherein the polynucleotide sequence of said engineered sucrose phosphorylase comprises at least one substitution at one or more positions.

18 . (canceled)

19 . The polynucleotide sequence of claim 16 , wherein said polynucleotide sequence is operably linked to a control sequence.

20 . The polynucleotide sequence of claim 16 , wherein said polynucleotide sequence is codon optimized.

21 . The polynucleotide sequence of claim 16 , wherein said polynucleotide sequence comprises a polynucleotide sequence set forth in the odd numbered sequences of SEQ ID NOS: 3-83.

22 . An expression vector comprising at least one polynucleotide sequence of claim 16 .

23 . A host cell comprising at least one expression vector of claim 22 .

24 . A host cell comprising at least one polynucleotide sequence of claim 16 .

25 . A method of producing an engineered sucrose phosphorylase in a host cell, comprising culturing the host cell of claim 23 , under suitable conditions, such that at least one engineered sucrose phosphorylase is produced.

26 . The method of claim 25 , further comprising recovering at least one engineered sucrose phosphorylase from the culture and/or host cell.

27 . The method of claim 25 , further comprising the step of purifying said at least one engineered sucrose phosphorylase.

Assignees

• Codexis Inc

Inventors

• Vroom Jonathan
• Sivaramakrishnan Santhosh
• Subramanian Nandhitha

Classifications

• C12P19/40

  having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y204/01007

  Sucrose phosphorylase (2.4.1.7) · CPC title

• C12N15/72

  Expression systems using regulatory sequences derived from the lac-operon · CPC title