Biocatalysts and methods for hydroxylation of chemical compounds

Patent metadata

Field	Value
Publication number	US-12344865-B2
Application number	US-202017776900-A
Country	US
Kind code	B2
Filing date	Nov 19, 2020
Priority date	Nov 26, 2019
Publication date	Jul 1, 2025
Grant date	Jul 1, 2025

Abstract

The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

First claim

What is claimed is:

1. An engineered polypeptide having proline hydroxylase activity, comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, and further comprising one or more amino acid substitutions as compared to SEQ ID NO: 4 at positions selected from the group consisting of: 194, 123, 21, 28, 58/247, 65, 80, 85, 95, 98, 117, 120, 159, 185, 199, 200, 233, 237, 243, 250, 268, 281, 282, 287, 289, 307, 324, 326, 327, 330, 338, 343, 346, and 348.

2. The engineered polypeptide of claim 1 , comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, and further comprising a combination of amino acid substitutions as compared to SEQ ID NO: 4 at positions selected from the group consisting of: 48/66/189, 48/66, and 66/82/85/135/189/267.

3. The engineered polypeptide of claim 2 , comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 4, and further comprising one or more amino acid substitutions as compared to SEQ ID NO: 4 at positions selected from the group consisting of: 21, 29, 44, 76, 85, 117, 120, 123, 129, 135, 139, 147, 162, 175, 179, 189, 195, 199, 208, 219, 228, 233, 236, 237, 243, 262, 277, 278, 281, 282, 289, 296, 307, 324, 326, 330, 335, 343, 345, and 347.

4. The engineered polypeptide of claim 1 , wherein said polypeptide has at least 80% sequence identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 116, 118, 120, 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, 266, 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, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450,452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656 and 658.

5. A polynucleotide comprising a nucleotide sequence encoding the engineered polypeptide of claim 1 .

6. A polynucleotide comprising a nucleotide sequence encoding the engineered polypeptide of claim 4 .

7. The engineered polypeptide of claim 1 , comprising an amino acid sequence having at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 4, comprising a substitution as compared to SEQ ID NO: 4 at position 194, and wherein the substitution at position 194 is 194L.

Assignees

• Codexis Inc

Inventors

• Vroom Jonathan
• Ortega Guerra Alberto
• Liang Jack
• Cope Gregory A
• Dellas Nikki
• Prier Christopher Keltie

Classifications

• C12Y114/11002

  Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase · CPC title

• C12P13/24 Primary

  Proline; Hydroxyproline; Histidine · CPC title

• C12N2800/101

  for bacteria · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title