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

Patent metadata

Field	Value
Publication number	US-9487760-B2
Application number	US-201615048887-A
Country	US
Kind code	B2
Filing date	Feb 19, 2016
Priority date	May 11, 2012
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

The present disclosure 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. An engineered polypeptide having imine reductase activity, wherein said polypeptide has at least 90% sequence identity to the polypeptide of SEQ ID NO: 2, and comprises a substitution at a position corresponding to position 198 of SEQ ID NO: 2, wherein the amino acid at the position corresponding to position 198 of SEQ ID NO: 2 has been replaced with an aliphatic, non-polar, polar, or acidic residue.

2. The engineered polypeptide having imine reductase activity of claim 1 , wherein said polypeptide has at least 90% sequence identity to SEQ ID NO:2, wherein the amino acid at the position corresponding to position 198 of SEQ ID NO:2 has been replaced with alanine, histidine, proline, serine, or glutamic acid.

3. The engineered polypeptide of claim 1 , wherein said polypeptide is capable of converting substrate compound (1a) pyruvate,

and substrate compound (2b) butylamine

to product compound (3b), N-2-(butylamino)propanoic acid,

under suitable reaction conditions.

4. The engineered polypeptide of claim 1 , wherein said polypeptide is capable of converting substrate compound (1b) cyclohexanone,

and substrate compound (2a) L-norvaline

to product compound (3c), (S)-2-(cyclohexylamino)pentanoic acid,

under suitable reaction conditions.

5. The engineered polypeptide of claim 1 , wherein said polypeptide is capable of converting substrate compound (1b) cyclohexanone,

and substrate compound (2b) butylamine

to product compound (3d), N-butylcyclohexanamine,

under suitable reaction conditions.

6. The engineered polypeptide of claim 1 , wherein said polypeptide is capable of converting substrate compound (1i),

and substrate compound (2b)

to product compound (3n),

under suitable reaction conditions.

7. The engineered polypeptide of claim 1 , wherein said polypeptide is capable of converting substrate compound (1j),

and substrate compound (2b)

to product compound (3o),

under suitable reaction conditions.

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

wherein
(a) R 1 and R 2 are independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted alkoxy, an optionally substituted carboxy, an optionally substituted aminocarbonyl, an optionally substituted heteroalkyl, an optionally substituted heteroalkenyl, an optionally substituted heteroalkynyl, an optionally substituted carboxyalkyl, an optionally substituted aminoalkyl, an optionally substituted haloalkyl, an optionally substituted alkylthioalkyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted arylalkyl, an optionally substituted heterocycloalkyl, an optionally substituted heteroaryl, and an optionally substituted heteroarylalkyl;
(b) R 1 and R 2 are linked to form a 3-membered to 10-membered ring;
(c) R 3 and R 4 are independently selected from the group consisting of a hydrogen atom, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted alkoxy, an optionally substituted carboxy, an optionally substituted aminocarbonyl, an optionally substituted heteroalkyl, an optionally substituted heteroalkenyl, an optionally substituted heteroalkynyl, an optionally substituted carboxyalkyl, an optionally substituted aminoalkyl, an optionally substituted haloalkyl, an optionally substituted alkylthioalkyl, an optionally substituted cycloalkyl, an optionally substituted aryl, an optionally substituted arylalkyl, an optionally substituted heterocycloalkyl, an optionally substituted heteroaryl, and an optionally substituted heteroarylalkyl, with the proviso that both R 3 and R 4 cannot be hydrogen;
(d) R 3 and R 4 are linked to form a 3-membered to 10-membered ring; or
(e) at least one of R 1 and R 2 is linked to at least one of R 3 and R 4 ;
wherein 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 in (a) or (b) above; and
a compound of formula (II),

wherein R 3 and R 4 are as defined in (c) or (d) above;
with an engineered polypeptide having imine reductase activity in presence of a cofactor under suitable reaction conditions, wherein the engineered polypeptide is the engineered polypeptide of claim 1 .

9. The process of claim 8 , in which R 3 and R 4 are linked to form a 3-membered to 10-membered ring.

10. The process of claim 8 , in which the substrate compound of formula (II) is selected from methylamine, dimethylamine, isopropylamine, butylamine, isobutylaminel, L-norvaline, aniline, (S)-2-aminopent-4-enoic acid, pyrrolidine, and hydroxypyrrolidine.

11. The process of claim 8 , 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.

12. The process of claim 8 , 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.

Assignees

• Codexis Inc

Inventors

• Chen Haibin
• Collier Steven J
• Nazor Jovana
• Sukumaran Joly
• Smith Derek
• Moore Jeffrey C
• Hughes Gregory
• Janey Jacob
• Huisman Gjalt W
• Novick Scott J
• Agard Nicholas J
• Alvizo Oscar
• Cope Gregory A
• Yeo Wan Lin
• Minor Stefanie Ng

Classifications

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12Y105/01

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

• C12Y105/01024

  N5-(Carboxyethyl)ornithine synthase (1.5.1.24) · CPC title

• C12P17/12

  containing a six-membered hetero ring · CPC title

• C12P17/185

  Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system (cepam nucleus C12P35/00; penam nucleus C12P37/00) · CPC title