Engineered glycosyltransferases and steviol glycoside glucosylation methods

Patent metadata

Field	Value
Publication number	US-11760981-B2
Application number	US-201916514578-A
Country	US
Kind code	B2
Filing date	Jul 17, 2019
Priority date	Jul 30, 2018
Publication date	Sep 19, 2023
Grant date	Sep 19, 2023

Abstract

The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides compositions comprising the GT enzymes and methods of using the engineered GT enzymes to make products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudiosides (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The present invention also provides compositions comprising the SuS enzymes and methods of using them. Methods for producing GT and SuS enzymes are also provided.

First claim

We claim:

1. An engineered glycosyltransferase comprising a polypeptide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 20, 36, 174, 406, 408, 440, 520, 626, or 678, wherein said polypeptide sequence comprises one or more mutations relative to the reference sequence of SEQ ID NO: 20, 36, 174, 406, 408, 440, 520, 626, or 678.

2. The engineered glycosyltransferase of claim 1 , wherein said polypeptide sequence of said engineered glycosyltransferase comprises at least one mutation or mutation set at one or more positions selected from 41/72/233/338, 41/72/338, 41/144/233, 41/233, 41/233/338, 61, 61/87/91/107, 61/87/91/259, 61/91/431, 61/107, 61/259/428, 61/407/428, 61/411, 72, 72/76, 72/76/163/197, 72/76/195/233, 72/76/197/204, 72/76/207/233, 72/76/207/338, 72/81, 72/81/195/233, 72/139/195/204, 72/144/338, 72/200/204/207, 72/207, 76/144/197/200, 76/195/197/204/207/233, 76/197/207/233, 76/233, 81/139/144/195/200/204/207/233, 81/144/233, 81/197/200/207/233/338, 81/233/338, 81/338, 107, 107/259, 139/144/233, 144/233, 144/233/338, 156/407, 163/233/338, 200/204/207/233, 233/338, and 259, wherein said positions are numbered with reference to SEQ ID NO: 20.

3. The engineered glycosyltransferase of claim 1 , wherein said polypeptide sequence of said engineered glycosyltransferase comprises at least one mutation or mutation set at one or more positions selected from 71, 80, 81, 81/270, 83, 85, 97, 124, 263, 286, 334, 402, 420, and 456, wherein said positions are numbered with reference to SEQ ID NO: 20.

4. The engineered glycosyltransferase of claim 1 , wherein said polypeptide sequence of said engineered glycosyltransferase comprises at least one mutation or mutation set at one or more positions selected from 41/72/233/338, 41/72/338, 61, 61/91/431, 61/259/428, 61/407/428, 81/139/144/195/200/204/207/233, and 81/197/200/207/233/338, wherein said positions are numbered with reference to SEQ ID NO: 20.

5. The engineered glycosyltransferase of claim 1 , wherein said polypeptide sequence of said engineered glycosyltransferase comprises at least one mutation or mutation set at one or more positions selected from 153, 153/326, 153/326/443, 153/326/443/455, 232, 232/273/299, 232/393/451, 299/451, 326, 404, and 451, wherein said positions are numbered with reference to SEQ ID NO: 408.

6. The engineered glycosyltransferase of claim 1 , wherein said polypeptide sequence of said engineered glycosyltransferase comprises at least one mutation or mutation set at one or more positions selected from 25, 116, 146, 170, 173, 227, 296, 300, 315, 327, 330, 361, 408, 412, 438, 448, and 449, wherein said positions are numbered with reference to SEQ ID NO: 408.

7. The engineered glycosyltransferase of claim 1 , wherein said polypeptide sequence of said engineered glycosyltransferase comprises at least one mutation or mutation set at one or more positions selected from 153, 232, 232/393/451, and 451, wherein said positions are numbered with reference to SEQ ID NO: 408.

8. The engineered glycosyltransferase of claim 1 , wherein said engineered glycosyltransferase is an NDP-glycosyltransferase selected from ADP-glucose-dependent glycosyltransferases (AGTs), CDP-glucose-dependent glycosyltransferases (CGTs), GDP-glucose-dependent glycosyltransferase (GGTs), TDP-glucose-dependent glycosyltransferases (TGTs), and IDP-glucose-dependent glycosyltransferase (IGTs).

9. The engineered glycosyltransferase of claim 8 , wherein said engineered glycosyltransferase is an ADP-glucose-dependent glycosyltransferase.

10. The engineered glycosyltransferase of claim 1 , wherein said engineered glycosyltransferase enzymatically transfers glycosyl residues from an activated sugar donor other than uracil-diphosphate-glucose.

11. A composition comprising at least one engineered glycosyltransferase of claim 1 .

12. The engineered glycosyltransferase of claim 1 , wherein said polypeptide sequence of said engineered glycosyltransferase comprises SEQ ID NO: 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, 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, 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, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, or 856.

13. An engineered polynucleotide encoding at least one engineered glycosyltransferase polypeptide provided in claim 1 .

14. A vector comprising at least one engineered polynucleotide of claim 13 .

15. The vector of claim 14 , wherein said vector further comprises at least one control sequence.

16. A host cell comprising at least one engineered polynucleotide of claim 13 .

17. A host cell comprising at least one vector of claim 14 .

18. The host cell of claim 16 , wherein said host cell is an eukaryotic or prokaryotic cell.

19. A method for producing at least one engineered glycosyltransferase, comprising culturing the host cell of claim 16 , under conditions such that said engineered glycosyltransferase is produced by said host cell.

20. The method of claim 19 , further comprising the step of recovering said engineered glycosyltransferase.

Assignees

• Codexis Inc
• Tate & Lyle Ingredients Americas Llc

Inventors

• Vroom Jonathan
• Galanie Stephanie Sue
• Liang Jack
• Liu Joyce
• Dellas Nikki
• Mayo Melissa Ann
• Entwistle David

Classifications

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• C07H15/256

  Polyterpene radicals · CPC title

• C12Y204/01013

  Sucrose synthase (2.4.1.13) · CPC title

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title