Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

Patent metadata

Field	Value
Publication number	US-10793836-B2
Application number	US-201916443250-A
Country	US
Kind code	B2
Filing date	Jun 17, 2019
Priority date	Nov 25, 2014
Publication date	Oct 6, 2020
Grant date	Oct 6, 2020

Abstract

The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

First claim

What is claimed is:

1. A polynucleotide encoding an engineered polypeptide, wherein the engineered polypeptide comprises an amino acid sequence with at least 90% sequence identity to the reference sequence of SEQ ID NO:8, wherein the alanine at the position corresponding to amino acid 239 of SEQ ID NO:8 is substituted with another amino acid in the amino acid sequence of the engineered polypeptide, and wherein the engineered polypeptide has imine reductase activity.

2. A polynucleotide encoding an engineered polypeptide, wherein the engineered polypeptide comprises an amino acid sequence with at least 90% sequence identity to the reference sequence of SEQ ID NO:8, wherein the isoleucine at the position corresponding to amino acid 330 of SEQ ID NO:8 is substituted with another amino acid in the amino acid sequence of the engineered polypeptide, wherein the amino acid sequence of the engineered polypeptide has a valine, threonine, tryptophan, glutamic acid, or tyrosine at the position corresponding to amino acid 283 of SEQ ID NO:8, and wherein the engineered polypeptide has imine reductase activity.

3. The polynucleotide of claim 2 , wherein the amino acid sequence of the engineered polypeptide comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:4, wherein the at least one residue difference is at a residue selected from the group consisting of 198, 153, 167, 265, 262, 108, 234, 284, 282, 220, 272, 256, 267, 242, 281, 197, 277, 224, and 143, and wherein the residue number corresponds to the amino acid sequence of SEQ ID NO:4.

4. The polynucleotide of claim 2 , wherein the amino acid sequence of the engineered polypeptide comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6, wherein the at least one residue difference is at a residue selected from the group consisting of 262, 9, 259, 220, 267, 153, 279, 200, 224, 256, 137, 143, 260, 261, 154, 276, and 185, and wherein the residue number corresponds to the amino acid sequence of SEQ ID NO:6.

5. The polynucleotide of claim 2 , wherein the amino acid sequence of the engineered polypeptide further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:8, wherein the at least one residue difference is at a residue selected from the group consisting of 12, 31, 37, 44, 46, 48, 55, 56, 65, 82, 93, 98, 108, 137, 138, 141, 142, 143, 153, 154, 155, 156, 159, 166, 168, 173, 177, 178, 184, 185, 195, 197, 199, 200, 201, 203, 205, 206, 210, 213, 215, 216, 218, 220, 221, 223, 226, 234, 239, 242, 245, 251, 253, 256, 257, 259, 260, 261, 262, 263, 265, 266, 267, 268, 269, 273, 274, 277, 278, 279, 280, 281, 282, 284, 285, 289, 290, 291, 292, 293, 294, and 349, and wherein the residue number corresponds to the amino acid sequence of SEQ ID NO:8.

6. The polynucleotide of claim 2 , wherein the imine reductase activity comprises converting the ketone substrate

and the amine substrate

to produce the amine product compound

under suitable reaction conditions.

7. The polynucleotide of claim 6 , wherein the imine reductase activity in converting the ketone and the amine substrate compound to the amine product compound under suitable reaction conditions is increased at least 2-fold as compared to the corresponding activity of an imine reductase polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 6, 12, 30, 126, 173, 360, or 550.

8. The polynucleotide of claim 1 , wherein the amino acid sequence of the engineered polypeptide comprises SEQ ID NO: 1188.

9. An expression vector comprising the polynucleotide of claim 1 .

10. An expression vector comprising the polynucleotide of claim 2 .

11. An isolated host cell comprising the polynucleotide of claim 1 .

12. An isolated host cell comprising the polynucleotide of claim 2 .

13. An isolated host cell comprising the expression vector of claim 9 .

14. An isolated host cell comprising the expression vector of claim 10 .

15. A method of preparing an engineered polypeptide having imine reductase activity, comprising culturing the host cell of claim 11 , under conditions suitable for expression of the engineered polypeptide, and optionally further comprising isolating the engineered polypeptide.

Assignees

• Codexis Inc

Inventors

• Alvizo Oscar
• Mayo Melissa Ann
• Moore Jeffrey C

Classifications

• C12N9/0028 Primary

  with NAD or NADP as acceptor (1.5.1) · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C07K14/195

  from bacteria · CPC title