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

Patent metadata

Field	Value
Publication number	US-10407668-B2
Application number	US-201715783657-A
Country	US
Kind code	B2
Filing date	Oct 13, 2017
Priority date	Nov 13, 2013
Publication date	Sep 10, 2019
Grant date	Sep 10, 2019

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

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

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

5. The engineered polynucleotide of claim 1 , wherein the amino acid sequence of said engineered polypeptide 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) X141W, X153Y, X256E, X274M, and X283V;
(p) 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 polynucleotide of claim 1 , wherein the amino acid sequence of said engineered polypeptide 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, 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 polynucleotide of claim 1 , wherein the amino acid sequence of said engineered polypeptide further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X12M, X37P, X111A, X141W, X153Y, X154 F/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 polynucleotide of claim 1 , wherein the amino acid sequence of said engineered polypeptide further comprises the residue differences X111A, X141W, X153Y, X256E, X274M, X283V, 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 and X156N;
(c) X37P, X156N, and X259I;
(d) X259L/M;
(e) 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 polynucleotide of claim 1 , wherein the amino acid sequence of said engineered polypeptide 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 polynucleotide of claim 1 , wherein the amino acid sequence of said engineered polypeptide 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, 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/TN, 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/TN/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. An expression vector comprising the engineered polynucleotide of claim 1 .

12. A host cell comprising the expression vector of claim 11 .

13. A method of preparing an engineered polypeptide having imine reductase activity, comprising culturing the host cell of claim 12 , under conditions suitable for expression of the polypeptide, optionally further comprising isolating the engineered polypeptide.

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12P13/001

  Amines; Imines · CPC title

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12N9/0028 Primary

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

• C12Y105/01028

  Opine dehydrogenase (1.5.1.28) · CPC title

• C07K14/305 Primary

  from Micrococcaceae (F) · CPC title