Engineered biocatalysts and methods for synthesizing chiral amines

Patent metadata

Field	Value
Publication number	US-11732248-B2
Application number	US-202117317231-A
Country	US
Kind code	B2
Filing date	May 11, 2021
Priority date	Dec 21, 2012
Publication date	Aug 22, 2023
Grant date	Aug 22, 2023

Abstract

The present disclosure provides engineered transaminase polypeptides for the production of amines, polynucleotides encoding the engineered transaminases, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases to prepare compounds useful in the production of active pharmaceutical agents.

First claim

What is claimed is:

1. An engineered polynucleotide encoding an engineered polypeptide having transaminase activity, wherein said engineered polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 and one or more residue differences as compared to SEQ ID NO:2 at residue positions selected from: X19, X34, X53, X73, X88, X107, X155, X165, X171, X178, X251, X259, X268, X277, X286, X312, X358, X366, X383, X399, X414, X426, and X450.

2. The engineered polynucleotide of claim 1 , wherein said one or more amino acid residue mutations are selected from: X34A, X88H, X107G, X153C, X155V, X315G, X383I, and X450S of the amino acid sequence of SEQ ID NO: 2.

3. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide comprises one or more amino acid residue mutations at the positions corresponding to positions X31M, X57F/L, X153A and X383V of the amino acid sequence of SEQ ID NO: 2.

4. The engineered polynucleotide of claim 3 , wherein said engineered polypeptide comprises one or more amino acid residue mutations at the positions corresponding to positions X34A, X88A; X153C, X155V, X163F and X315G of the amino acid sequence of SEQ ID NO: 2.

5. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide comprises an amino acid residue mutation selected from cysteine, phenylalanine, glycine, asparagine, serine and threonine at the position corresponding to position 316 of the amino acid sequence of SEQ ID NO: 2.

6. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide comprises an amino acid residue mutation of threonine at the position corresponding to position 323 of the amino acid sequence of SEQ ID NO: 2.

7. The engineered polynucleotide of claim 6 , wherein said engineered polypeptide further comprises one or more amino acid residue mutations at the positions corresponding to positions X31M, X57F, X383I/T, and X450S of the amino acid sequence of SEQ ID NO: 2.

8. The engineered polynucleotide of claim 6 , wherein said amino acid mutation comprises a combination of amino acid residue mutations at positions corresponding to positions selected from:
X31M, X57F, and X383V;
X31M, X57F, X107G, and X450S;
X31M, X57F, X233V, X383I, and X450S; and
X31M, X57F, X383I, and X450S of the amino acid sequence of SEQ ID NO: 2.

9. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide has at least 1.2 fold increased stability as compared to the polypeptide of the amino acid sequence of SEQ ID NO: 4, wherein said engineered polypeptide comprises one or more amino acid residue mutations at positions corresponding to positions X34T, X107G, X155V, X383I/V, and X450S of the amino acid sequence of SEQ ID NO: 2.

10. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide has at least 1.2 fold increased stability as compared to the polypeptide of the amino acid sequence of SEQ ID NO: 4, wherein said engineered polypeptide comprises one or more amino acid residue mutations at positions corresponding to positions selected from X88H and X153C of the amino acid sequence of SEQ ID NO: 2.

11. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide has increased enantioselectivity as compared to the polypeptide of the amino acid sequence of SEQ ID NO: 4 in converting compound (2)

to compound (1)

wherein said engineered polypeptide further comprises the amino acid residue mutations at the positions corresponding to positions X57F and X153C of the amino acid sequence of SEQ ID NO:2.

12. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide comprises at least one amino acid residue mutation at the positions corresponding to positions selected from: X18A, X19W, X31M, X34A, X53M, X57C/F/L, X73R, X88H/Y, X107G, X146L, X153A/C/V, X155A/V, X163L, X165F, X171Q, X178W, X190K, X206K, X228G, X235P, X244T, X251V, X259V, X268A, X277A, X286C/H, X312N, X314N, X315G, X319N, X358K, X366H, X383C/F/I/L/M/T/V, X395P, X399A, X424A, X426R, X427Y, and X450S of the amino acid sequence of SEQ ID NO:2.

13. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide does not comprise an amino acid residue mutation at positions corresponding to positions X9, X45, X177, X211, X294, X324, and X391 of the amino acid sequence of SEQ ID NO: 2.

14. The engineered polynucleotide of claim 1 , wherein said engineered polypeptide is immobilized on a solid support.

15. The engineered polynucleotide of claim 14 , wherein said solid support is a bead or resin comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene/DVB copolymer or polymethacrylate with octadecyl functional groups.

16. An expression vector comprising the engineered polynucleotide of claim 1 .

17. The expression vector of claim 16 , wherein said expression vector further comprises a control sequence.

18. A host cell comprising the engineered polynucleotide of claim 1 .

19. A host cell comprising the expression vector of claim 17 .

20. A method of preparing an engineered polypeptide, comprising culturing the host cell of claim 19 , under conditions suitable for expression of said engineered polypeptide.

21. The method of claim 20 , further comprising isolating the engineered polypeptide.

Assignees

• Codexis Inc

Inventors

• Tang Weng Lin
• Hsieh Helen
• Pham Son
• Smith Derek
• Collier Steven J

Classifications

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12P15/00

  Preparation of compounds containing at least three condensed carbocyclic rings {(gibbanes C12P27/00; naphthacenes C12P29/00)} · CPC title

• C12P17/12

  containing a six-membered hetero ring · CPC title

• C12P17/188

  Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms (ergot-alcaloids C12P17/183) · CPC title