Engineered transaminase biocatalysts and methods for synthesizing chiral amines

1 . An engineered transaminase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence identity to SEQ ID NO: 6, or a functional fragment thereof, wherein said engineered transaminase comprises a 130Y substitution in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6, and wherein the engineered transaminase has an increased activity as compared to the polypeptide of SEQ ID NO: 4 in converting 1-imidazo[1,2-a]pyridin-6-ylethanone (compound (2)) to (1S)-1-imidazo[1,2-a]pyridin-6-ylethanamine (compound (3)).

2 . The engineered transaminase of claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence identity to SEQ ID NO: 6, wherein said engineered transaminase further comprises at least one substitution or substitution set in said polypeptide sequence at one or more positions selected from 13, 13/41/57/88/130/415/417, 13/41/57/89/97/417, 13/41/57/97/415/417, 13/41/57/97/415/417/443, 13/41/57/97/443, 13/41/57/417, 13/41/57/417, 13/41/88, 13/41/88/89, 13/41/88/89/97/415/443, 13/41/88/89/417, 13/41/88/97, 13/41/88/415/443, 13/41/88/443, 13/41/89/148/443, 13/41/89/417, 13/41/89/443, 13/41/97/417, 13/41/97/415, 13/41/97/415/417, 13/41/97/417, 13/41/97/417/443, 13/41/130/415/443, 13/41/415, 13/41/415/417, 13/41/415/443, 13/41/417, 13/41/417/443, 13/57/88/89/415/443, 13/57/88/97, 13/57/88/97/415/443, 13/57/88/415, 13/57/88/130/417/443, 13/57/88/415, 13/57/97/415/417/443, 13/57/97/417, 13/88/89/415/417, 13/88/89/415/417/443, 13/88/443, 13/88/415, 13/89/97/415/417, 13/89/97/417, 13/89/417, 13/97/148/415, 13/97/415, 13/97/415/417, 13/97/417, 13/415, 13/415/417, 13/417, 13/417/443, 13/415, 13/415/417, 13/415/417/443, 13/415/443, 13/417, 13/417/443, 13/443, 23/53/162/233/277/315/415/418/432, 23/53/315/417/418, 23/277/315/395/415/417/432, 23/277/395/417/418, 23/395/418, 23/418, 41, 41/57/88, 41/57/88/415/443, 41/57/148/415/417, 41/57/443, 41/57/415/417, 41/88/89/97/415, 41/88/89/415/417, 41/88/97/417, 41/88/415/417, 41/88/443, 41/97/148/415/417/443, 41/97/417, 41/97/417/443, 41/415, 41/415/417/443, 41/415/443, 41/415/443, 41/417, 41/417/443, 53/162, 53/162/395/417, 53/162/418/432, 53/233, 53/277/395, 53/277/395/417/418, 53/277/415/417, 57/88/97/415/443, 57/88/97/417, 57/88/97/417, 57/97/148/417/443, 57/417, 88, 88/89/417, 88/97/415/417/443, 88/417/443, 88/148/417/443, 88/415, 88/415/417, 88/415/417/443, 88/417, 89/97/415/417, 89/97/417, 89/443, 97, 97/148/415, 97/415, 97/415/417, 97/417, 415, 417, 443, 162/233/415/417, 162/395/415/417, 162/418, 233/315/415/417, 233/315/417, 277/395/415/418/432, 315, 315/415/418/432, 395/418, 415, 415/417, 415/417/418, 415/417/418/432, 415/417/443, 415/443, 417, and 443, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 6.

3 . The engineered polypeptide of claim 2 in which the residue differences at the residue positions 13, 41/57/415/419, 41/113/415, 53/57, 88, 88/89, 97/415, 148, 227, 260, 302, 355/415/419, 362, 417, and 443 are selected from 13A, 13E, 13G, 13K, 13S, 41V/57Y/415F/419D, 41V/113F/415F, 53M/57W, 88K, 88R, 88R/89L, 88V, 97A/415S, 148E, 148G, 260T, 302N, 355C/415S/419D, 362G, 417A, 417I, 417V, 443E, and 443M.

4 . The engineered polypeptide of claim 1 in which the amino acid sequence further comprises a combination of residue differences as compared to SEQ ID NO: 6 selected from:

T13A;

T13E;

T13G;

T13K;

T13S;

I41V, F57Y, R415F, and Q419D;

I41V, V113F, and R415F;

N53M and F57W;

L88K;

L88R;

L88R and M89L;

L88V;

S97A and R415S;

Q148E;

Q148G;

C260T;

E302N;

R355C, R415S, and Q419D;

H362G;

L417A;

L417I;

L417V;

K443E; and

K443M.

5 . The engineered polypeptide of claim 1 in which the transaminase has at least 1.2 fold increased activity as compared to the polypeptide of SEQ ID NO: 4 in converting 1-imidazo[1,2-a]pyridin-6-ylethanone (compound (2)) to (1S)-1-imidazo[1,2-a]pyridin-6-ylethanamine (compound (3)).

6 . The engineered polypeptide of claim 1 in which further the transaminase has increased enantioselectivity as compared to the polypeptide of SEQ ID NO: 4 in converting 1-imidazo[1,2-a]pyridin-6-ylethanone (compound (2)) to (1S)-1-imidazo[1,2-a]pyridin-6-ylethanamine (compound (3)).

7 . The engineered polypeptide of claim 1 in which the amino acid sequence comprises a sequence selected from SEQ ID NO: 64, 66, 68, 76, 84, 88, 94, 110, 112, 118, 120, 124, 126, 128, 132, 138, 144, 150, 170, 182, 202, 206, 224, 226, 240, 246, 274, 276, 282, 294, 296, 302, 310, 312, 332, 336, and 338.

8 . The engineered polypeptide of claim 1 in which the polypeptide is immobilized on a solid support.

9 . The engineered polypeptide of claim 8 in which the 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.