Biocatalysts and methods for synthesizing derivatives of tryptamine and tryptamine analogs

Patent metadata

Field	Value
Publication number	US-2016289649-A1
Application number	US-201615180355-A
Country	US
Kind code	A1
Filing date	Jun 13, 2016
Priority date	Mar 23, 2012
Publication date	Oct 6, 2016
Grant date	—

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 a polypeptide having transaminase activity, wherein said polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 and one or more residue differences as compared to SEQ ID NO:4 at residue positions selected from: X14; X26; X31; X33; X41; X47; X57; X70; X86; X88; X107; X132; X148; X163; X168; X173; X203; X250; X284; X314; X315; X324; X346; X395; X398; X400; X417; X419; X423; X448; and X451, wherein the residue differences at residue positions X31; X57; X86; X163; X168; X314; X324; X398; and X417 are selected from: X31S; X57Y; X86D; X163I; X163L; X163R; X163V; X168S; X314N; X324H; X398L; X398V; X398W; and X417M.

2 . The engineered polynucleotide of claim 1 , in which the residue differences in said encoded polypeptide having transaminase activity further comprises substitutions at the residue positions X14; X26; X33; X41; X47; X70; X88; X107; X132; X148; X173; X203; X250; X284; X315; X346; X395; X400; X419; X423; X448; and X451, wherein said substitutions are selected from X14V; X26R; X33T; X41L; X47N; X70A; X88A; X88L; X107P; X132F; X148Q; X148R; X173A; X203S; X250A; X284A; X315G; X346L; X395P; X400G; X419S; X423I; X448E; and X451D.

3 . The engineered polynucleotide of claim 2 , in which the amino acid sequence of said encoded polypeptide having transaminase activity further comprises one or more residue differences selected from: X57F; X113L; X113V; X168K; X420N; and X424V.

4 . The engineered polynucleotide of claim 1 , in which the amino acid sequence of said encoded polypeptide having transaminase activity comprises at least one or more residue differences selected from: X14V; X26R; X31S/D; X86D; X163I/L/R/V; X284A; X315G; X398L/V/W; and X400G.

5 . The engineered polynucleotide of claim 1 , in which the amino acid sequence of said encoded polypeptide having transaminase activity comprises a combination of residue differences selected from:
X14V and X163I/L/R/V; X86D and X400G; X57F/Y and X163I/L/R/V; X57F/Y and X398L/V/W; X14V, X113L/V, X163I/L/R/V, X284A, and X424V; X31S, X57F/Y, X163I/L/R/V, X315G, X346L, and X398L/V/W X14V, X113L, X163L, X284A, and X424V; X14V, X26R, X163L, X284A, and X400G; X14V, X26R, X88L, and X113L; X57F, X163L, X168K, X314N, X315G, X346L, and X398V; X14V, X163L, X173A, X400G, and X420N; X14V, X113L, X163L, and X284A; X14V, X26R, X163L, X284A, and X400G; and X14V, X33T, X57F, X113L, and X163L.

6 . The engineered polynucleotide of claim 1 , in which the amino acid sequence of said encoded polypeptide having transaminase activity has at least 1.2 fold increased stability as compared to the polypeptide of SEQ ID NO:4, wherein the amino acid sequence comprises one or more residue differences selected from: X14V; X26R; X31S; X33T; X41L; X70A; X86D; X88A/L; X163I/L/R/V; X284A; X324H; X419S; and X423I.

7 . The engineered polynucleotide of claim 1 , in which the amino acid sequence of said encoded polypeptide having transaminase activity comprises a sequence selected from: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, and 154.

8 . An expression vector comprising the polynucleotide of claim 1 .

9 . The expression vector of claim 8 , further comprising at least one control sequence.

10 . The expression vector of claim 9 , wherein said control sequence comprises a promoter.

11 . A host cell comprising the polynucleotide of claim 1 .

12 . A host cell comprising the expression vector of claim 8 .

13 . A host cell comprising the expression vector of claim 9 .

14 . A host cell comprising the expression vector of claim 10 .

15 . The host cell of claim 11 , wherein said host cell is E. coli.

16 . The host cell of claim 12 , wherein said host cell is E. coli.

17 . The host cell of claim 13 , wherein said host cell is E. coli.

18 . The host cell of claim 14 , wherein said host cell is E. coli.

19 . A method of preparing an engineered polypeptide having transaminase activity, comprising culturing the host cell of claim 11 under conditions suitable for expression of the polypeptide, optionally further comprising isolating the engineered polypeptide.

20 . A method of preparing an engineered polypeptide having transaminase 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

• Nazor Jovana
• Smith Derek J
• Crowe Michael A
• Song Shiwei
• Collier Steven J

Classifications

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12Y206/01018

  Beta-alanine-pyruvate transaminase (2.6.1.18) · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title