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

Patent metadata

Field	Value
Publication number	US-11060072-B2
Application number	US-201916525834-A
Country	US
Kind code	B2
Filing date	Jul 30, 2019
Priority date	Nov 13, 2013
Publication date	Jul 13, 2021
Grant date	Jul 13, 2021

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 process for preparing an amine compound of formula (III),

wherein
R 1 and R 2 groups are independently selected from a hydrogen atom, and optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and optionally R 1 and R 2 are linked to form a 3-membered to 10-membered ring;
R 3 and R 4 groups are independently selected from a hydrogen atom, and optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl, with the proviso that both R 3 and R 4 cannot be hydrogen; and optionally R 3 and R 4 are linked to form a 3-membered to 10-membered ring; and
optionally, the carbon atom and/or the nitrogen indicated by * is chiral;

the process comprising contacting
a compound of formula (I),

wherein
R 1 , and R 2 are as defined above;

and
a compound of formula (II),

wherein
R 3 , and R 4 are as defined above;
with an engineered polypeptide, wherein the engineered polypeptide has imine reductase activity in presence of a cofactor under suitable reaction conditions and wherein the engineered polypeptide comprises an amino acid sequence with at least 90 sequence identity to a reference sequence of SEQ ID NO:6 and a residue difference at position 82, as compared to SEQ ID NO:6.

2. The process of claim 1 , in which R 3 and R 4 are linked to form a 3-membered to 10-membered ring.

3. The process of claim 1 , in which the substrate compound of formula (II) is butylamine.

4. The process of claim 1 , in which at least one of R 1 and R 2 of the compound of formula (I) is linked to at least one of R 3 and R 4 of the amine compound of formula (II), whereby the process for preparing the amine compound of formula (III) comprises an intramolecular reaction.

5. The process of claim 1 , in which the suitable reaction conditions comprise:
(a) substrate loading at about 10 g/L to 100 g/L;
(b) about 0.1 g/L to about 50 g/L of the engineered polypeptide;
(c) about 0.05 g/L (0.001 M) to about 2.5 g/L (0.050 M) of NAD(P)H;
(d) a pH of about 6 to 10;
(e) temperature of about 20° to 50° C.; and
(f) reaction time of 2-120 hrs.

6. The process of claim 1 , in which the substrate compound of formula (I) is compound (1j):

7. The process of claim 1 , wherein the process comprises contacting butylamine and the substrate compound (1j):

with an engineered polypeptide, wherein the engineered polypeptide has imine reductase activity in the presence of a cofactor under suitable reaction conditions and wherein the engineered polypeptide comprises an amino acid sequence with at least 90 sequence identity to a reference sequence of SEQ ID NO:6 and a residue difference at position 82, as compared to SEQ ID NO:6,
to produce the product of compound (3o):

Assignees

• Codexis Inc

Inventors

• Agard Nicholas J
• Alvizo Oscar
• Mayo Melissa Ann
• Minor Stefanie Ng
• Riggins James Nicholas
• Moore Jeffrey C

Classifications

• C12N9/0028 Primary

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

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12Y105/01028

  Opine dehydrogenase (1.5.1.28) · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C07K14/305 Primary

  from Micrococcaceae (F) · CPC title