Engineered biocatalysts and methods for synthesizing chiral amines

Patent metadata

Field	Value
Publication number	US-9902943-B2
Application number	US-201314652887-A
Country	US
Kind code	B2
Filing date	Dec 16, 2013
Priority date	Dec 21, 2012
Publication date	Feb 27, 2018
Grant date	Feb 27, 2018

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 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, wherein amino acid residues at the positions corresponding to positions 57 and 316 of SEQ ID NO: 2 are mutated in said engineered polypeptide, and further wherein said residue at the position corresponding to position 316 of the amino acid sequence of SEQ ID NO: 2 is selected from cysteine, phenylalanine, glycine, asparagine, serine, and threonine.

2. The engineered polypeptide of claim 1 , further comprising amino acid residue mutations at the positions corresponding to positions selected from X19W, X34A, X53M, X73R, X155V, X165F, X171Q, X178W, X251V, X259V, X268A, X277A, X317L, X358K, X366H, X399A, X414I, X426R, and X450S of the amino acid sequence of SEQ ID NO: 2.

3. The engineered polypeptide of claim 1 , further comprising at least one additional amino acid residue mutation at the positions corresponding to positions selected from: X34A, X56A, X88H, X107G, X113L, X147H, X153C, X155V, X233V, X315G, X383I, and X450S of the amino acid sequence of SEQ ID NO: 2.

4. The engineered polypeptide of claim 1 , further comprising one or more amino acid residue mutations at the positions corresponding to positions selected from: X31M, X86N/S, X153A, X233T, X323T, X383V, and X417T of the amino acid sequence of SEQ ID NO: 2.

5. The engineered polypeptide of claim 4 , further comprising amino acid residue mutations at the positions corresponding to positions X34A, X56A, X86S, X88A, X153C, X155V, X163F, X315G, and X417T of the amino acid sequence of SEQ ID NO: 2.

6. The engineered polypeptide of claim 1 , wherein said residue at the position corresponding to position 316 of SEQ ID NO: 2 is asparagine.

7. The engineered polypeptide of claim 6 , further comprising an amino acid residue mutation at the positions corresponding to positions selected from: X31M, X57F, X323T, X383I/T, X415H, and X450S of the amino acid sequence of SEQ ID NO: 2.

8. The engineered polypeptide of claim 7 , wherein said amino acid mutation comprises a combination of amino acid residue mutations at positions corresponding to positions selected from:
X31M, X57F, X323T, and X383V;
X31M, X57F, X107G, X113L, X233T, X415H, and X450S;
X31M, X57F, X233V, X323T, X383I, X415H, and X450S; and
X31M, X57F, X147H, X323T, X383I, X415H, and X450S
of the amino acid sequence of SEQ ID NO: 2.

9. The engineered polypeptide of claim 1 , wherein said engineered polypeptide having transaminase activity 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 further comprises one or more amino acid residue mutations at positions corresponding to positions selected from: X34T, X107G, X113L, X147H, X155V, X233T/V, X323T, X383I/V, and X450S of the amino acid sequence of SEQ ID NO: 2.

10. The engineered polypeptide of claim 1 , wherein said engineered polypeptide having transaminase activity has at least 1.2 fold increased activity 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 one or more amino acid residue mutations at positions corresponding to positions selected from X56A, X86S, X88H, X153C, X415H, and X417T of the amino acid sequence of SEQ ID NO: 2.

11. The engineered polypeptide of claim 1 , wherein said engineered polypeptide having transaminase activity 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 mutation at the position corresponding to position X153C of the amino acid sequence of SEQ ID NO: 2.

12. The engineered polypeptide of claim 1 , further comprising an amino acid residue mutation at the positions corresponding to positions selected from: X18A, X19W, X21H, X31M, X34A, X53M, X56A/C, X73R, X86C/N/S/Y, X88H/Y, X107G, X113C/L/P, X146L, X147H/K/V, X153A/C/V, X155A/V, X163L, X165F, X171Q, X178W, X190K, X206K, X228G, X233T/V, X235P, X244T, X251V, X259V, X268A, X277A, X286C/H, X312N, X314N, X315G, X317L, X319N, X323T, X358K, X366H, X383C/F/I/L/M/T/V, X395P, X399A, X414I, X415A/G/H/L/V, X417T/V, X424A, X426A, X426R, X427Y, X434T, and X450S of the amino acid sequence of SEQ ID NO: 2.

13. The engineered polypeptide of claim 1 , wherein said engineered polypeptide does not comprise an amino acid residue mutation at the positions corresponding to positions X9, X45, X177, X211, X294, X324, and X291 of the amino acid sequence of SEQ ID NO: 2.

14. The engineered polypeptide of claim 1 , said engineered polypeptide comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOS: 98, 100, 012, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, 190, 192, and 202.

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

16. The engineered polypeptide of claim 15 , wherein said solid support is a bead or resin comprising polymethyacrylate with epoxide functional groups, polymethycrylate with amino epoxide functional groups, styrene/DVB copolymer or polymethyacrylate with octadecyl functional groups.

17. A polynucleotide encoding the engineered polypeptide of claim 1 .

18. A polynucleotide encoding the engineered polypeptide of claim 1 , wherein said polynucleotide comprises a nucleotide sequence selected from the nucleotide sequences set forth in SEQ ID NOS: 97, 99, 101, 135, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189, 191, and 201.

19. An expression vector comprising the polynucleotide of claim 17 .

20. The expression vector of claim 19 , wherein said expression vector comprises a control sequence.

21. A host cell comprising the polynucleotide of claim 17 .

22. A method of preparing an engineered polypeptide, comprising culturing the host cell of claim 21 under conditions suitable for expression of said engineered polypeptide.

23. The method of claim 22 , further comprising isolating said engineered polypeptide.

24. A process for preparing an amine compound of Formula (I),

wherein:
Ring A is a 6-membered carbocyclic ring, optionally including an unsaturated C—C bond between positions 2 and 3 and/or positions 5 and 6, and/or optionally substituted independently positions 2, 3, 4, 5 and 6 with a group selected from halo, hydroxy, and methyl;
Ring B is a 6-membered carbocyclic ring, optionally including an unsaturated C—C bond between positions 5 and 10, and/or optionally substituted independently at one or more of positions 9 and 10 with a group selected from halo, hydroxy, and methyl;
Ring C is a 5- or 6-membered carbocyclic ring (m=0 or 1), optionally substituted at position 10 with a group selected from halo, hydroxy, methyl, ethyl, and carbonyl;
Ring D is a 5-, 6-, or 7-membered carbocyclic ring (n=0, 1, or 2), optionally including 1, 2, or 3 unsaturated C—C bonds, and/or optionally substituted independently as follows:
at position 14 with a gro

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12P33/00

  Preparation of steroids · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · 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

• C12P15/00

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

• C12Y206/01

  Transaminases (2.6.1) · CPC title