Transaminase polypeptides

Patent metadata

Field	Value
Publication number	US-10995323-B2
Application number	US-201916717596-A
Country	US
Kind code	B2
Filing date	Dec 17, 2019
Priority date	Jan 8, 2009
Publication date	May 4, 2021
Grant date	May 4, 2021

Abstract

The present disclosure provides engineered transaminase enzymes having improved properties as compared to a naturally occurring wild-type transaminase enzyme. Also provided are polynucleotides encoding the engineered transaminase enzymes, host cells capable of expressing the engineered transaminase enzymes, and methods of using the engineered transaminase enzymes to synthesize a variety of chiral compounds.

First claim

What is claimed is:

1. An engineered transaminase polypeptide, wherein said transaminase polypeptide comprises a polypeptide sequence at least 90% identical to SEQ ID NO: 2, and wherein the transaminase polypeptide comprises a substitution of X86Y.

2. The engineered transaminase polypeptide of claim 1 , wherein said polypeptide has at least about 10% residual activity in the conversion of pyruvate to L-alanine in presence of amino donor isopropylamine after treatment of the polypeptide at 50° C. for 23 h.

3. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide further comprises at least one additional substitution at a residue position selected from: X4, X12, X21, X157, X208, X253, X272, X302, X316, X398, X418, X420, X431, X444, and X446.

4. The engineered transaminase polypeptide of claim 3 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X4 is R, Q, or L; X12 is K; X21 is N; X45 is H; X157 is T; X177 is L; X208 is I; X211 is K; X253 is M; X272 is A; X294 is V; X302 is K; X316 is K; X324 is G; X391 is A; X398 is R; X418 is V; X420 is N; X431 is D; X444 is V; and X446 is V.

5. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide has increased transaminase activity as compared to SEQ ID NO: 2 for conversion of an amine acceptor substrate to the corresponding chiral amine product.

6. The engineered transaminase polypeptide of claim 5 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X30 is A; X31 is A; X44 is A; X56 is V; X57 is, A, C, F, I, L, or S; X81 is D; X82 is H; X85 is A, C, S, N, T, G, or V; X95 is T; X112 is I; X113 is C or H; X127 is L; X147 is G; X153 is A, C, G, N, M, Q, S, or T; X166 is S; X181 is R; X208 is I; X228 is G or T; X233 is L, S, I, V, N, G, or T; V297 is A, S, T, I, M, Q, C, or G; X311 is V; X314 is T or V; X317 is L, M, or Y; X318 is G, F, C, K, W, or R; X319 is Q, G, M, N, or V, X320 is A or K; X321 is L, M, or I; X385 is R; X398 is R; X407 is S; X408 is A; X409 is G; X415 is M; X417 is A, C, E, F, I, N, Q, Y, S, T, or V; X420 is N; X434 is V; and X438 is L.

7. The engineered transaminase polypeptide of claim 5 , wherein the transaminase polypeptide sequence further comprises at least one substitution set selected from: X12 is G and X434 is V; X44 is A and X166 is S; X57 is I and X153 is S; X82 is H and X417 is F; X85 is A and X317 is L; X85 is S and X153 is A; X85 is A and X153 is A; X85 is S and X153 is S; X112 is I and X317 is L; X113 is H and X407 is S; X153 is S and X233 is S; X311 is V and X314 is T; X314 is V and X409 is G; and X318 is G and X408 is A.

8. The engineered transaminase polypeptide of claim 5 , wherein the transaminase polypeptide sequence further comprises at least one substitution set selected from: X57 is A, X153 is S and X318 is G; X57 is A, X153 is C and X321 is L; X57 is F, X318 is F and X417 is S; X57 is F, X318 is G and X417 is I; X57 is L, X417 is C and X438 is L; X57 is F, X127 is L and X417 is C; X57 is S, X233 is L and X417 is V; X85 is A, X147 is G and X153 is A; X85 is S, X153 is A and X233 is T; X85 is A, X153 is S and X417 is S; X95 is T, X153 is A and X417 is C; X113 is C, X385 is R and X417 is C; X153 is A, X317 is L and X318 is G; X153 is A, X233 is T and X417 is C; X153 is S, X318 is R and X417 is E; X153 is C, X233 is L and X318 is R; X153 is T, X228 is G and X321 is L; X153 is S, X317 is L and X417 is C; X153 is T, X319 is V and X417 is I; X153 is S, X228 is G and X417 is V; X153 is C, X317 is Y and X319 is Q; X153 is T, X228 is G and X417 is A; X228 is G, X317 is L and X417 is C; X228 is G, X318 is G and X417 is C; X233 is L, X321 is L and X417 is I; and X317 is L, X318 is R and X417 is T.

9. The engineered transaminase polypeptide of claim 5 , wherein the transaminase polypeptide comprises the amino acid sequence of SEQ ID NO: 178.

10. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide converts an amine acceptor substrate to a corresponding amine product with improved R-enantioselectivity at a greater rate compared to the polypeptide set forth in SEQ ID NO: 18.

11. The engineered transaminase polypeptide of claim 10 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X57 is L, X81 is D, X82 is H, X85 is A, C, N, T, or V; X95 is T; X112 is I; X147 is G; X153 is A, S, N, G, or T; X233 is S or T; X317 is L, X318 is G or R; and X319 is V.

12. The engineered transaminase polypeptide of claim 10 , wherein the transaminase polypeptide sequence further comprises at least one substitution or substitution set selected from: X85 is A; X85 is A and X153 is A; and X85 is S and X153 is A.

13. The engineered transaminase polypeptide of claim 10 , wherein the transaminase polypeptide sequence is SEQ ID NO: 104.

14. The engineered transaminase polypeptide of claim 1 , wherein the transaminase enzyme polypeptide converts an amine acceptor substrate selected from 3,4-dihydronaphthalen-1(2H)-one, 1-phenylbutan-2-one, 3,3-dimethylbutan-2-one, octan-2-one, ethyl 3-oxobutanoate, 4-phenylbutan-2-one, and 1-(4-bromophenyl)ethanone to the corresponding amine product at a rate that is greater than the polypeptide of SEQ ID NO: 2.

15. The engineered transaminase polypeptide of claim 14 , wherein the transaminase polypeptide sequence comprises at least one additional substitution or substitution set selected from: X30 is A; X31 is A; X57 is I; X82 is H; X85 is V; X85 is C; X153 is S; X177 is L; X317 is M; X317 is Y; X417 is A; X319 is Q; X320 is A; X417 is I; X113 is H and X407 is S; X44 is A and X166 is S; X314 is V and X409 is G; X153 is S and X233 is S; X311 is V and X314 is T; X153 is S, X318 is R and X417 is E; X153 is C, X233 is L and X318 is R; X153 is S, X317 is L and X417 is C; X233 is L, X321 is L, and X417 is I; and X153 is C, X317 is Y and X319 is Q.

16. The engineered transaminase polypeptide of claim 14 , wherein the transaminase polypeptide sequence is SEQ ID NO: 172.

17. The engineered transaminase polypeptide of claim 14 , wherein the transaminase polypeptide converts the amine acceptor substrate 1-phenylbutan-2-one to the amine product (S)-1-phenylbutan-2-amine with improved S-enantioselectivity as compared to SEQ ID NO: 2.

Assignees

• Codexis Inc

Inventors

• Dhawan Ish K
• Miller Gregory
• Zhang Xiyun

Classifications

• C12P13/001

  Amines; Imines · CPC title

• Y02P20/52

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

• C12P41/006

  by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12Y206/01018

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