IP Library › Granted Patent US 11,987,818
Granted Patent B2
US 11,987,818 · App. 17/330,686 · Granted May 21, 2024

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

Inventors: Margie Tabuga Borra-Garske (Palo Alto, CA); Oscar Alvizo (Fremont, CA); Melissa Ann Mayo (Foster City, CA); Stephan Jenne (Foster City, CA); Auric Anthony Sowell-Kantz (Fairfax, CA); Carmela Molinaro (Colonia, NJ)
Assignee: Codexis, Inc.
C12N9/0028C12N9/0036C12Y105/01C12Y106/00
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Quick Facts
Patent No.
US 11,987,818
App. No.
17/330,686
Filed
May 26, 2021
Granted
May 21, 2024
Kind
B2
Art Unit
1656
USPC
435/191
Abstract

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

Claims (16)

1. An engineered polynucleotide encoding an engineered imine or oxime reductase polypeptide comprising an amino acid sequence with at least 80% sequence identity to a reference sequence of SEQ ID NO: 14, and wherein said engineered polypeptide has imine reductase activity.

2. The engineered polynucleotide encoding an engineered imine or oxime reductase polypeptide of claim 1 , wherein said polypeptide comprises an arginine or serine at position 169, wherein said position is numbered with reference to SEQ ID NO:2.

3. An engineered polynucleotide comprising SEQ ID NO: 13.

4. The engineered polynucleotide encoding an engineered imine or oxime reductase polypeptide of claim 1 , wherein the engineered polynucleotides comprise substitution(s) at positions 505/506/507, wherein the positions are numbered with reference to SEQ ID NO:1.

5. The engineered polynucleotide encoding the engineered imine or oxime reductase polypeptide of claim 4 , wherein said engineered polynucleotide comprises at least one of the following substitution(s)/substitution sets selected from 505A/506A/507T and wherein the positions are numbered with reference to SEQ ID NO:1.

6. The engineered polynucleotide encoding the engineered imine or oxime reductase polypeptide of claim 4 , wherein said engineered polynucleotide comprises at least one of the following substitution(s)/substitution sets selected from G505A/C506A/A507T and G505A/C506G/A507T, and wherein the positions are numbered with reference to SEQ ID NO:1.

7. The engineered polynucleotide encoding an engineered imine or oxime reductase polypeptide of claim 1 , wherein the engineered polynucleotides comprise substitution(s) at positions 367/368/369/505/506/507/513/519/523/524/525/634/635/636/639/703/704/705, 369/370/371/372/505/506/507/513/519/638/703/704/705, 369/505/506/507/511/512/517/518/519/638/703/704/705, 369/505/506/507/513/517/518/519/523/524/525/638/703/704/705, 369/505/506/507/513/519/523/524/525/634/635/636/638/703/704/705, 369/505/506/507/513/519/635/638/703/704/705, 401/402/505/506/507/526/527/532/534/631/632/633/645/702, 401/402/505/506/507/526/527/532/534/631/633/640/645/702, 401/402/505/506/507/526/527/532/534/631/633/645/649/650/651/702, 401/402/505/506/507/526/527/534/631/632/633/645/702, 401/402/505/506/507/526/527/534/631/633/644/645/649/650/651/700/701, 401/402/505/506/507/526/532/534/631/632/633/640/645/649/650/651/702, 401/402/505/506/507/526/534/631/632/633/640/645/649/650/651/702, 401/402/505/506/507/526/534/631/632/633/645/649/650/651/702, 402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702, 402/505/506/507/526/527/532/534/702, 402/505/506/507/526/527/532/534/631/633/640/645/702, 402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702, 402/505/506/507/526/527/534/631/633/640/645/649/650/651/702, 402/505/506/507/526/527/534/631/632/633/640/643/644/645/700/701, 402/505/506/507/526/527/534/631/632/633/640/645/702, 402/505/506/507/526/527/534/631/632/633/645/649/650/651/702, 402/505/506/507/526/527/534/631/632/633/645/649/650/651/702, 402/505/506/507/526/527/534/633/640/644/645/649/650/651/702, 402/505/506/507/526/532/534/633/645/700/701, 402/505/506/507/526/532/534/645/649/650/651, 402/505/506/507/526/534/631/632/633/645/649/650/651/702, 505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702, 505/506/507/526/527/532/534/631/632/633/645/649/650/651/702, and 505/506/507/526/534/631/632/633/640/643/644/645, wherein the positions are numbered with reference to SEQ ID NO:269.

8. The engineered polynucleotide encoding the engineered imine or oxime reductase polypeptide of claim 7 , wherein said engineered polynucleotide comprises at least one of the following substitution(s)/substitution sets selected from 367G/368G/369T/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/639T/703A/704A/705G, 369G/370G/371G/372C/505A/506A/507T/513C/519C/638T/703A/704A/705G, 369G/505A/506A/507T/511G/512T/517A/518T/519T/638T/703A/704A/705G, 369G/505A/506A/507T/513C/517A/518T/519T/523C/524A/525C/638T/703A/704A/705G, 369G/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/638T/703A/704A/705G, 369G/505A/506A/507T/513C/519C/635T/638T/703A/704A/705G, 401G/402G/505A/506A/507C/526T/527C/532A/534G/631C/633A/645T/649C/650T/651A/702T, 401G/402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T, 401G/402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/702T, 401G/402G/505A/506A/507C/526T/527C/534G/631C/633A/644T/645G/649A/650T/651A/700C/701T, 401G/402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/702T, 401G/402G/505A/506A/507C/526T/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T, 401G/402G/505A/506A/507C/526T/534G/631T/632G/633G/640A/645T/649C/650T/651A/702T, 401G/402G/505A/506A/507C/526T/534T/631T/632G/633G/645T/649C/650T/651A/702T, 402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T, 402G/505A/506A/507C/526T/527C/532A/534G/702T, 402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T, 402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T, 402G/505A/506A/507C/526T/527C/534G/631C/633A/640A/645T/649A/650T/651A/702T, 402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/643T/644T/645G/700C/701T, 402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/645T/702T, 402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/645T/649C/650T/651A/702T, 402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/649A/650T/651A/702T, 402G/505A/506A/507C/526T/527C/534T/633A/640A/644T/645G/649A/650T/651A/702T, 402G/505A/506A/507C/526T/532A/534G/633A/645T/700C/701T, 402G/505A/506A/507C/526T/532A/534T/645T/649A/650T/651A, 402G/505A/506A/507C/526T/534G/631T/632G/633G/645T/649A/650T/651A/702T, 505A/506A/507C/526T/527C/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T, 505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/649C/650T/651A/702T, and 505A/506A/507C/526T/534G/631T/632G/633G/640A/643T/644T/645G, wherein the positions are numbered with reference to SEQ ID NO:269.

9. The engineered polynucleotide encoding the engineered imine or oxime reductase polypeptide of claim 7 , wherein said engineered polynucleotide comprises at least one of the following substitution(s)/substitution sets selected from A367G/C368G/C369T/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A6 36C/G639T/G703A/C704A/C705G, C369G/A370G/T371G/T372C/G505A/C506A/A507T/T513C/A519C/C638T/G703A/C704A/C705G, C369G/G505A/C506A/A507T/T511G/A512T/G517A/C518T/A519T/C638T/G703A/C704A/C705G, C369G/G505A/C506A/A507T/T513C/G517A/C518T/A519T/A523C/T524A/G525C/C638T/G703A/C704A/C705G, C369G/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A636C/C638T/G703A/C704A/C705G, C369G/G505A/C506A/A507T/T513C/A519C/A635T/C638T/G703A/C704A/C705G, C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631C/C633A/C645T/T649C/G650T/G651A/C702T, C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T, C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/C702T, C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/C644T/C645G/T649A/G650T/G651A/G700C/A701T, C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/C702T, C401G/C402G/G505A/C506A/A507C/C526T/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T, C401G/C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T, C401G/C402G/G505A/C506A/A507C/C526T/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T, C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T, C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/C702T, C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T, C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T, C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/G640A/C645T/T649A/G650T/G651A/C702T, C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G/G700C/A701T, C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/C645T/C702T, C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T, C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T, C402G/G505A/C506A/A507C/C526T/T527C/C534T/C633A/G640A/C644T/C645G/T649A/G650T/G651A/C702T, C402G/G505A/C506A/A507C/C526T/G532A/C534G/C633A/C645T/G700C/A701T, C402G/G505A/C506A/A507C/C526T/G532A/C534T/C645T/T649A/G650T/G651A, C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T, G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T, G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T, and G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G, wherein the positions are numbered with reference to SEQ ID NO:269.

10. The engineered polynucleotide encoding an engineered imine or oxime reductase polypeptide of claim 4 , wherein the engineered polynucleotide further comprises substitution(s) at positions 133, 379/381, 388/389/390, 400/401/402, 401/402, 520/521/522, 616/618, 625, 625/626/627, 634, 634/635/636, 634/636, 646, 646/647, 646, 667, 680/681, and 836/837, wherein the positions are numbered with reference to SEQ ID NO:229.

11. The engineered polynucleotide encoding the engineered imine or oxime reductase polypeptide of claim 10 , wherein said engineered polynucleotide further comprises at least one of the following substitution(s)/substitution sets selected from 133A, 379A/381G, 388C/389G/390T, 400C/401T/402T, 401G/402G, 520A/521T/522G, 616C/618T, 625A, 625A/626G/627T, 634A, 634A/635G/636G, 634C/636T, 646A, 646A/647A, 646C, 667A, 680T/681T, and 836T/837T, wherein the positions are numbered with reference to SEQ ID NO:229.

12. The engineered polynucleotide encoding the engineered imine or oxime reductase polypeptide of claim 10 , wherein said engineered polynucleotide further comprises at least one of the following substitution(s)/substitution sets selected from G133A, G379A/C381G, A388C/C389G/C390T, A400C/C401T/C402T, C401G/C402G, G520A/C521T/A522G, A616C/G618T, C625A, C625A/A626G/A627T, G634A, G634A/A635G/A636G, G634C/A636T, G646A, G646A/G647A, G646C, G667A, C680T/C681T, and A836T/C837T, wherein the positions are numbered with reference to SEQ ID NO:229.

13. A vector comprising the engineered polynucleotide of claim 1 .

14. The vector of claim 13 , further comprising at least one control sequence.

15. A host cell comprising the vector of claim 13 .

16. The host cell of claim 15 , wherein said host cell produces at least one engineered polypeptide of claim 1 .

Assignments (1)
SECURITY INTEREST Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →