IP Library › Granted Patent US 10,323,233
Granted Patent B2
US 10,323,233 · App. 15/912,892 · Granted Jun 18, 2019

Transaminase polypeptides

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Quick Facts
Patent No.
US 10,323,233
App. No.
15/912,892
Filed
Mar 6, 2018
Granted
Jun 18, 2019
Kind
B2
Art Unit
1652
USPC
435/72
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.

Claims (27)

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 the substitution X177L.

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, X9, X12, X21, X45, X86, X157, X208, X211, X253, X272, X294, X302, X316, X324, X391, X398, X418, X420, X431, X444, and X446.

4. The engineered transaminase polypeptide of claim 3 , wherein the transaminase polypeptide sequence comprises the substitution X177L and at least one further substitution selected from: X4 is R, Q, or L; X12 is K; X21 is N; X45 is H; X86 is Y; X157 is T; 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 4 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from:

X45 is H, X86 is Y, X211 is K, X294 is V, X324 is G; X391 is A, X398 is R, and X420 is N;

X9 is T, X45 is H, X86 is Y, X211 is K, X294 is V, X324 is G, and X391 is A;

X21 is N, X45 is H, X208 is I, X211 is K, X324 is G, and X391 is A; and

X9 is T, X21 is N, X86 is Y, X208 is I, and X294 is V.

6. 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 amino acceptor substrate to the corresponding chiral amino product.

7. The engineered transaminase polypeptide of claim 6 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X30 is A; X31 is A; X44 is A; X45 is N; 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; X86 is F, S, N, A, G, or H; 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; X211 is R; X228 is G or T; X233 is L, S, I, V, N, G, or T; X294 is M; 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; X324 is S; 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.

8. The engineered transaminase polypeptide of claim 7 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X30 is A; X31 is A; X56 is V; X81 is D; X82 is H; X95 is T; and X113 is C or H.

9. The engineered transaminase polypeptide of claim 6 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X12 is G and X434 is V; X44 is A and X166 is S; X57 is I and X153 is S; X81 is D and X86 is H; 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; X86 is S and X153 is S; X86 is H and X153 is A; 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.

10. The engineered transaminase polypeptide of claim 6 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X57 is A, X153 is S and X318 is G; X57 is F, X86 is H and X153 is Q; X57 is I, X86 is F and X320 is A; X57 is F, X86 is F and X153 is Q; X57 is A, X153 is C and X321 is L; X57 is C, X86 is S and X417 is T; X57 is C, X86 is A and X317 is L; X57 is F, X318 is F and X417 is S; X57 is L, X86 is S and X153 is A; X57 is F, X318 is G and X417 is I, X57 is L, X86 is F and X318 is F; 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; X57 is S, X86 is G and X417 is C; 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; X86 is H, X153 is A and X417 is C; X86 is H, X153 is S and X417 is C; X86 is F, X153 is C and X297 is A; X86 is S, X153 is T and X297 is A; X86 is H, X233 is L and X417 is A; X86 is F, X318 is R and X417 is A; X86 is N, X228 is G and X317 is L; 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.

11. The engineered transaminase polypeptide of claim 6 , wherein the transaminase polypeptide sequence further comprises at least one of the following substitutions:

X57 is L and X86 is F and X153 is S and X233 is T and X417 is T;

X86 is H and X153 is A and XA228 is G and X417 is I; and

X86 is H and X153 is S and X181 is R and X417 is T.

12. The engineered transaminase polypeptide of claim 6 , wherein the transaminase polypeptide comprises an amino acid sequence selected from SEQ ID NO: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, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, and 192.

13. The engineered transaminase polypeptide of claim 1 , wherein the transaminase polypeptide is capable of converting the amino acceptor substrate to the corresponding amino product with improved R-enantioselectivity as compared to SEQ ID NO:18.

14. The engineered transaminase polypeptide of claim 13 , 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; X86 is S,F, or H; 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.

15. The engineered transaminase polypeptide of claim 13 , wherein the transaminase polypeptide sequence further comprises at least one substitution selected from: X85 is A; X85 is A and X153 is A; and X85 is S and X153 is A.

16. The engineered transaminase polypeptide of claim 13 , wherein the transaminase polypeptide sequence is selected from SEQ ID NO:22, 30, 32, 34, 36, 40, 44, 48, 56, 58, 60, 62, 64, 66, 68, 70, 72, 76, 84, 88, 90, 92, 104, 108, 110, 120, 122, 124, 126, 128, 132, 160, and 176.

17. The engineered transaminase polypeptide of claim 1 , wherein the transaminase enzyme polypeptide is capable of converting an amino 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 or 18.

18. The engineered transaminase polypeptide of claim 17 , wherein the transaminase polypeptide sequence comprises at least one additional substitution selected from: X30 is A; X31 is A; X57 is I; X82 is H; X85 is V; X85 is C; X153 is S; 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.

19. The engineered transaminase polypeptide of claim 17 , wherein the transaminase polypeptide sequence is selected from SEQ ID NO:38, 42, 44, 52, 56, 66, 74, 76, 78, 80, 82, 84, 94, 96, 98, 100, 104, 118, 160, 164, 172, 184, and 188.

20. The engineered transaminase polypeptide of claim 17 , wherein the transaminase polypeptide is capable of converting the amino acceptor substrate 1-phenylbutan-2-one to the amino product (S)-1-phenylbutan-2-amine with improved S-enantioselectivity as compared to SEQ ID NO:2 or 18.