Engineered transaminase polypeptides for industrial biocatalysis

Patent metadata

Field	Value
Publication number	US-2022282229-A1
Application number	US-202217742560-A
Country	US
Kind code	A1
Filing date	May 12, 2022
Priority date	Jun 14, 2017
Publication date	Sep 8, 2022
Grant date	—

Abstract

The present invention provides engineered transaminase polypeptides useful for the synthesis of chiral amine compounds under industrially relevant conditions. The invention also provides polynucleotides encoding the engineered transaminase polypeptides, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases for the production of chiral amine compounds.

First claim

We claim:

1 . A polynucleotide sequence encoding an engineered transaminase 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:4, wherein said engineered transaminase comprises at least one substitution or substitution set in said polypeptide sequence at one or more positions selected from 17, 17/259/347, 20, 45/48/78/81/92, 57, 57/319, 85, 86, 157, 158, 161, 203/416, 314, 316, 319, 375/416, 404, 415, 416, 417, 420, 421, 422, and 424, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4.

2 . A polynucleotide sequence encoding an engineered transaminase 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: 120, wherein said engineered transaminase comprises at least one substitution or substitution set in said polypeptide sequence at one or more positions selected from 17, 17/21, 17/21/22, 17/22, 17/22/272, 17/22/272/320, 17/31/320/397, 17/84/164, 17/157/164, 17/157/320/397, 17/221/320, 17/272/320, 17/320, 17/397, 19/22/42/320/394, 19/42, 20/320, 25/320, 66/187/320, 66/272, 66/320, 66/394, 164/320/321, 165, 255, 272, 272/320, and 320, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 120.

3 . The polynucleotide sequence encoding an engineered transaminase of claim 2 , wherein said engineered transaminase comprises at least one improved property compared to wild-type V. fluvialis transaminase.

4 . The polynucleotide sequence encoding an engineered transaminase of claim 3 , wherein said improved property comprises improved activity on a substrate.

5 . The polynucleotide sequence encoding an engineered transaminase of claim 4 , wherein said substrate comprises compound (1).

6 . The polynucleotide sequence encoding an engineered transaminase of claim 3 , wherein said improved property comprises improved thermostability.

7 . A polynucleotide sequence encoding at least one engineered transaminase, said polynucleotide sequence 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: 3, 17, 19, 29, 47, 65, 69, 87, 109, 111, 119, 137, and/or 155, wherein the polynucleotide sequence of said engineered transaminase comprises at least one substitution at one or more positions.

8 . The polynucleotide sequence of claim 2 , wherein said polynucleotide sequence is operably linked to a control sequence.

9 . The polynucleotide sequence of claim 2 , wherein said polynucleotide sequence is codon optimized.

10 . An expression vector comprising at least one polynucleotide sequence of claim 2 .

11 . A host cell comprising at least one expression vector of claim 10 .

12 . A host cell comprising at least one polynucleotide sequence of claim 2 .

13 . A method of producing an engineered transaminase in a host cell, comprising culturing the host cell of claim 11 , under suitable conditions, such that at least one engineered transaminase is produced.

14 . The method of claim 13 , further comprising recovering at least one engineered transaminase from the culture and/or host cell.

15 . The method of claim 14 , further comprising the step of purifying said at least one engineered transaminase.

Assignees

• Codexis Inc

Inventors

• Novick Scott J
• Dellas Nikki
• Alvizo Oscar
• Garcia Ravi David
• Ching Charlene
• Entwistle David

Classifications

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12N15/63

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

• C12Y206/01

  Transaminases (2.6.1) · CPC title