Engineered biocatalysts and methods for synthesizing chiral amines

Patent metadata

Field	Value
Publication number	US-2024026401-A1
Application number	US-202118024942-A
Country	US
Kind code	A1
Filing date	Sep 17, 2021
Priority date	Sep 28, 2020
Publication date	Jan 25, 2024
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

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 NOs: 4 and/or 6, or a functional fragment thereof, wherein said engineered transaminase comprises at least one substitution or substitution set in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NOs: 4 and/or 6.

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: 4, wherein said engineered transaminase comprises at least one substitution or substitution set in said polypeptide sequence at one or more positions selected from 227, 41/227/417/443, 13, 41/57/130/415/419, 41/113/415, 53/57, 88, 88/89, 97/415, 148, 260, 302, 355/415/419, 362, 417, and 443, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4.

3 . 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 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, 13/41/57/97/130/415/417, 13/41/57/97/130/415/417/443, 13/41/57/97/443, 13/41/57/130/417, 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, 13/41/88/130/415/443, 13/41/88/443, 13/41/89/130/148/443, 13/41/89/417, 13/41/89/443, 13/41/97/130/417, 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, 13/57/88/89/130/415/443, 13/57/88/97, 13/57/88/97/415/443, 13/57/88/130/415, 13/57/88/130/417/443, 13/57/88/415, 13/57/97/130/415/417/443, 13/57/97/417, 13/88/89/415/417, 13/88/89/415/417/443, 13/88/130/443, 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, 13/130/415, 13/130/415/417, 13/130/417, 13/130/417/443, 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, 41/57/130/148/415/417, 41/57/130/443, 41/57/415/417, 41/88/89/97/130/415, 41/88/89/415/417, 41/88/97/130/417, 41/88/130/415/417, 41/88/443, 41/97/130/148/415/417/443, 41/97/417, 41/97/417/443, 41/130/415, 41/130/415/417/443, 41/130/415/443, 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, 57/88/97/130/415/443, 57/88/97/130/417, 57/88/97/417, 57/97/130/148/417/443, 57/417, 88, 88/89/130/417, 88/97/415/417/443, 88/130/417/443, 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, 97/130, 97/148/415, 97/415, 97/415/417, 97/417, 130, 130/415, 130/417, 130/443, 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.

4 . The engineered polypeptide of claim 2 in which the residue differences at the residue positions 13, 41/57/130/415/419, 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/130Y/415F/419D, 41V/113F/415F, 53M/57W, 88K, 88R, 88R/89L, 88V, 97A/415S, 148E, 148G, 227A, 227C, 260T, 302N, 355C/415S/419D, 362G, 417A, 417I, 417V, 443E, and 443M.

5 . 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: 4 selected from:
T13A;
T13E;
T13G;
T13K;
T13S;
141V, F57Y, F130Y, R415F, and Q419D;
141V, V113F, and R415F;
N53M and F57W;
L88K;
L88R;
L88R and M89L;
L88V;
S97A and R415S;
Q148E;
Q148G;
G227A;
G227C;
C260T;
E302N;
R355C, R415S, and Q419D;
H362G;
L417A;
L417I;
L417V;
K443E; and
K443M.

6 . 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 compound (2) to compound (3).

7 . The engineered polypeptide of claim 1 in which the transaminase has increased enantioselectivity as compared to the polypeptide of SEQ ID NO: 4 in converting compound (2) to compound (3).

8 . The engineered polypeptide of claim 1 in which the amino acid sequence comprises a sequence selected from SEQ ID NO: 4, 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, 154, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, and 358.

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

10 . The engineered polypeptide of claim 9 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.

11 . A polynucleotide encoding the engineered transaminase polypeptide of claim 1 .

12 . A polynucleotide encoding the engineered transaminase polypeptide of claim 1 , comprising a nucleotide sequence selected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, and 357.

13 . An expression vector comprising the polynucleotide of claim 11 .

14 . The expression vector of claim 13 comprising a control sequence.

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

16 . A method of preparing the engineered polypeptide of claim 1 , comprising culturing a host cell under conditions suitable for expression of the polypeptide.

17 .

Assignees

• Codexis Inc

Inventors

• Sivaramakrishnan Santhosh
• Bermudez Ericka
• Subramanian Nandhitha
• Entwistle David
• Forget Stephanie Marie

Classifications

• C12P17/182 Primary

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12Y206/01018

  Beta-alanine-pyruvate transaminase (2.6.1.18) · 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

• C12P13/001

  Amines; Imines · CPC title