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

Patent metadata

Field	Value
Publication number	US-9822346-B2
Application number	US-201414539690-A
Country	US
Kind code	B2
Filing date	Nov 12, 2014
Priority date	Nov 13, 2013
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

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. An engineered polypeptide comprising an amino acid sequence with at least 90% sequence identity to a reference sequence of SEQ ID NO:6 and comprising a residue difference at position 82, as compared to SEQ ID NO:6, wherein the polypeptide has imine reductase activity.

2. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X12M, X37P, X82T, X111A, X154S, X156N/M, X223S, X256E, X260D, X261H, X262P, X263C/E/Q, X267G, X277L, X281A, X284P/S, and X292E.

3. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X256E, X93G/Y, X94N, X96C, X111A/H, X142A, X159L, X163V, X259R, X273C, and X284P/S.

4. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least two residue differences as compared to the reference sequence of SEQ ID NO:6 selected from X82C/P/T, X141W, X143W, X153Y, X154F/Q/Y, X256V, X259I/L/M/T, X260G, X261R, X265L, X273W, X274M, X277A/I, X279L, X283V, X284L, X296N, X326V.

5. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least a combination of residue differences as compared to the reference sequence of SEQ ID NO:6 selected from:
(a) X153Y, and X283V;
(b) X141W, X153Y, and X283V;
(c) X141W, X153Y, X274L/M, and X283V;
(d) X141W, X153Y, X154F, X274L/M, and X283V;
(e) X141W, X153Y, X154F, and X283V;
(f) X141W, X153Y, X283V, and X296N/V;
(g) X141W, X153Y, X274L/M, X283V, and X296N/V:
(h) X111A, X153Y, X256E, X274M, and X283V;
(i) X111A, X141W, X153Y, X273C, X274M, X283V, and X284S;
(j) X111A, X141W, X153Y, X273C, and X283V;
(k) X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S, and X296N;
(l) X111A, X141W, X153Y, X256E, X273W, X274L, X283V, X284S, and X296N;
(m) X111H, X141W, X153Y, X273W, X274M, X284S, and X296N;
(n) X111H, X141W, X153Y, X154F, X273W, X274L, X283V, X284S, and X296N;
(o) X82P, X141W, X153Y, X256E, X274M, and X283V;
(p) X82P, X111A, X141W, X153Y, X256E, X274M, X283V, M284S, and E296V;
(q) X94N, X143W, X159L, X163V, X259M, and X279L;
(r) X141W, X153Y, X154F, and X256E; and
(s) X153Y, X256E, and X274M.

6. The engineered polypeptide of claim 5 , wherein the amino acid sequence further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X12M, X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V, X65I/V, X74W, X82C/T, X87A, X93G/Y, X94N, X96C, X108S, X111A/H, X126S, X138L, X140M, X141M/N, X142A, X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V, X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S, X197V, X200S, X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L, X253K/N, X254R, X256A/E/I/L/S/T, X257Q, X259C/R, X260A/D/N/Q/V/Y, X261E/F/H/L/P/Q/Y, X262P, X262F/G/V, X263C/D/E/H/I/K/L/M/N/P/Q/V, X264V, X267E/G/H/I/N/S, X270L, X272D, X273C, X274L/S, X276L, X277H/L, X278E/H/K/N/R/S/W, X279T, X281A, X282A/R, X284C/F/H/P/Q/S, X291E, X292E/P, X295F, and X352Q.

7. The engineered polypeptide of claim 5 , wherein the amino acid sequence further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X12M, X37P, X82T, X111A, X141W, X153Y, X154F/S, X156N/M, X223S, X256E, X259I, X260D, X261H, X262P, X263C/E/Q, X267G, X274M, X277L, X281A, X283V, X284P/S, X292E, and X296N.

8. The engineered polypeptide of claim 1 , wherein the amino acid sequence comprises the residue differences X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S, and X296N and at least residue difference or a combination of residue differences as compared to the reference sequence of SEQ ID NO:6 selected from:
(a) X156N;
(b) X37P, X82T, and X156N;
(c) X37P, X82T, X156N, and X259I;
(d) X259L/M;
(e) X82T, X156N, X223S, X259L, X267G, and X281A;
(f) X263C;
(g) X12M, X261H, X263C, X277L, and X292E;
(h) X154S; and
(i) X154S, X156M, X260D, X261H, X262P, X263E, and X284P.

9. The engineered polypeptide of claim 1 , wherein the amino acid sequence does not include a residue difference as compared to the reference sequence of SEQ ID NO:6 at a residue position selected from X29, X137, X157, X184, X197, X198, X201, X220, X232, X261, X266, X279, X280, X287, X288, X293, X295, X311, X324, X328, X332, and X353.

10. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises a residue difference as compared to the reference sequence of SEQ ID NO:6 selected from: X4H/L/R, X5T, X14P, X20T, X29R/T, X37H, X67A/D, X71C/V, X74R, X82P, X94K/R/T, X97P, X100W, X111M/Q/R/S, X124L/N, X136G, X137N, X141W, X143W, X149L, X153E/V/Y, X154F/M/Q/Y, X156G/I/Q/S/T/V, X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E, X183C, X184K/Q/R, X185V, X186K/R, X197I/P, X198A/E/H/P/S, X201L, X220D/H, X223T, X226L, X232G/A/R, X243G, X246W, X256V, X258D, X259E/H/I/L/M/S/T/V/W, X260G, X261A/G/I/K/R/S/T, X265G/L/Y, X266T, X270G, X273W, X274M, X277A/I, X279F/L/V/Y, X280L, X283M/V, X284K/L/M/Y, X287S/T, X288G/S, X292C/G/I/P/S/T/V/Y, X293H/I/K/L/N/Q/T/V, X294A/I/V, X295R/S, X296L/N/V/W, X297A, X308F, X311C/T/V, X323C/I/M/T/V, X324L/T, X326V, X328A/G/E, X332V, X353E, and X356R.

11. The engineered polypeptide of claim 1 , wherein the imine reductase activity comprises converting the following ketone and amine substrate compound paid to the listed amine product compound under suitable reaction conditions:
(c) ketone substrate compound (1j)

and amine substrate compound (2g)

to amine product compound (3q),

12. The engineered polypeptide of claim 11 , wherein the imine reductase activity in converting the ketone and amine substrate compound pair to the listed amine product compound under suitable reaction conditions is increased at least 2-fold as compared to the corresponding activity of the reference polypeptide of SEQ ID NO:6.

13. The engineered polypeptide of claim 1 , in which the amino acid sequence comprises a sequence selected from the group consisting of the even-numbered sequence identifiers of SEQ ID NOS: 22, 26, 34, 112, 182, 188, 192, 194, 198, 200-224, 228, 236, 260, 278, 290, 292, 298, 312, 318, 320, 348, 378-504, 512, 516, 584, 606-710, 722, 734, 756, 760, 762, 764, 772-780, 792, 796, 808, 814, 816, 820, 822, 828, 836-854, 858-862, 876, 880, 882, 886, 888, 890, 892, 894, 902-914, 918-924.

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12Y105/01028

  Opine dehydrogenase (1.5.1.28) · CPC title

• C12N9/0028 Primary

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

• C12P13/001

  Amines; Imines · CPC title

• C07K14/305 Primary

  from Micrococcaceae (F) · CPC title