Engineered galactose oxidase variant enzymes

Abstract

The present invention provides engineered galactose oxidase (GOase) enzymes, polypeptides having GOase activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing GOase enzymes are also provided. The present invention further provides compositions comprising the GOase enzymes and methods of using the engineered GOase enzymes. The present invention finds particular use in the production of pharmaceutical and other compounds.

Claims

1. An engineered galactose oxidase comprising a polypeptide sequence having at least 95%, sequence identity to SEQ ID NO: 2, or a functional fragment thereof, wherein said engineered galactose oxidase comprises substitutions in said polypeptide sequence at positions 331, 406, and 465, and wherein said engineered galactose oxidase does not comprise substitutions at positions 11 and 71, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2, and wherein said engineered galactose oxidase comprises production of 2-ethynylglyceraldehyde with increased enantiomeric excess of the R-stereoisomer as compared to wild-type F. graminearium galactose oxidase.

2. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 170, 171, 173, 191, 192, 193, 194, 197, 198, 199, 202, 204, 205, 220, 227, 243, 247, 248, 252, 269, 294, 296, 324, 332, 407, 463, 466, 493, 515, 517, 520, 521, and 522, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

3. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 171, 173, 188, 192, 197, 199, 203, 220, 223, 243, 252, 294, 295, 296, 332, 407, 466, 493, 515, 517, 520, and 521, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

4. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 171/220/295/296/407, 171/220/296/407/520, 171/220/332/407, 171/220/407, 171/243/296/332, 171/295/332, 171/296/407, 171/332, 171/407, 171/407/520, 173, 192/220/295/296/332/521, 192/220/295/520/521, 192/220/296/332/520, 192/220/407, 192/295/296, 192/295/296/332, 192/295/296/520/521, 192/296, 198/294/296, 198/295, 198/295/296, 204, 220, 220/243, 220/243/295/296/332/407/521, 220/243/295/296/407, 220/243/407, 220/243/407/520/521, 220/252/332/407, 220/295/296, 220/295/296/332, 220/295/296/332/407/520, 220/295/296/332/407/521, 220/295/296/407, 220/295/407/520/521, 220/295/407/521, 220/296, 220/296/332/407, 220/296/332/407/520/521, 220/296/407, 220/407, 220/407/520, 227, 243/295, 243/295/407, 243/515/517, 277/296/407/520/521, 284/295/296, 294/296/407 294/521, 295/296, 295/296/332/407, 295/296/407, 295/296/407/521, 295/296/521, 295/332/407/521, 295/332/520/521, 295/407, 296, 296/332/407/520, 296/332/407/521, 296/407, 296/520/521, 332/407, 332/407/520/521, 332/407/521, 407, 407/520, and 517, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

5. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 4, 8, 8/29/92/196/258/426, 8/46/92/279/296/426/549/553/597, 8/56/192/194/460/571/598, 8/56/243/560/598, 8/92/258/363/549/553/567, 8/192/194/243/460/553/560, 8/192/460/560/598, 8/258/363/426/553/597, 16, 16/24/43/56/103/220/295/296/499/549, 16/43/56/103/148/220/295/296/499/549, 16/43/63/103/295/304/499/549, 16/43/63/148/295/499, 16/43/103/148/220/295/426/549, 16/43/148/220/295/499, 16/43/295/296/549, 16/43/426/549, 16/56/63/148/295/296/304/426, 16/56/296, 16/56/426/499, 16/63/103/220/295/426/549, 16/148/220/295/296/304/499, 16/148/220/295/426/499/549, 16/148/220/296/549, 16/148/295/426/549, 16/220/499, 16/295/296/426/499/549, 24, 24/36, 24/36/43/148/319/560/637, 24/36/92/148, 24/36/92/222/560/637, 24/36/92/279/319/363/637, 24/36/148/222/279/560/637, 24/43/92/279/363/560/637, 24/43/92/279/499, 24/43/148/295/560, 24/43/148/363, 24/43/148/560, 24/43/222, 24/92/148/279/363, 24/148/637, 24/637, 29, 29/46/92/196/258/279/363/426/481/567/597, 36, 36/43/92/148/222/279/295/499/560/637, 36/92/148/279/319/363/560/637, 36/92/148/499/637, 36/92/319/363/637, 36/92/560, 36/222/279/319/363/560, 43, 43/56/220/296/426/499/549, 43/56/220/426/549, 43/92/148/222/279/499/560, 43/148/279/295/560, 43/148/549, 43/222/279, 43/295/499, 46, 56, 63, 92, 103, 134, 148, 192/243, 194, 196, 220/295/520, 220/295/521, 220/296/304/426/549, 222, 257, 258, 279, 279/560/637, 295, 296, 304, 319, 363, 363/426/481/553, 426, 499, 549, 553, 560, 567, 597, and 637, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

6. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 4/8/56/598, 4/56/192/194/257/571/598, 4/56/194/329/598, 4/194/243/549/598, 8/29/46/196/363/481/549/553, 8/56, 8/56/192/194/243/329, 8/56/192/194/243/329/460/560, 8/56/192/194/243/460/560/598, 8/56/192/243/460, 8/56/192/243/598, 8/56/194/257/460/549/560/598, 8/92/196/258/426/597, 8/92/196/481/597, 8/192/194/243/329/460/560, 8/196/258/279/481/549/553, 8/243/460/560/571/598, 8/257/460/560/598, 8/258/363/426/549, 8/279/363/426/481/549/553, 16/43/63/103/295/296/499, 16/43/103/148/295/426/499/549, 16/43/103/304/499/549, 16/43/148/295/296/304/499/549, 16/43/148/296/426/499, 16/43/295/296/499, 16/56/103/220/295, 16/56/103/220/295/296, 16/56/103/295/549, 16/56/148/295/296/304/426/549, 16/56/220/295, 16/56/220/499/549, 16/56/295, 16/56/295/296/549, 16/56/295/499, 16/56/499/549, 16/63/103/148/426/499/549, 16/63/103/426/499/549, 16/63/148/220/295/296/426/499, 16/63/148/499/549, 16/103, 16/103/148, 16/103/148/220/499, 16/103/148/295/426/499/549, 16/103/220/295, 16/148/220/295/426/499, 16/148/295/426, 16/148/295/549, 16/148/426/549, 16/220/295/296, 16/304/426/499, 16/304/499/549, 24/36/92/279/295/363/499, 24/43/92/148/279/295/319/637, 24/43/92/222/279, 24/43/222/319, 24/43/363/637, 24/92, 24/92/148/279, 24/92/222/279/319/637, 24/222/637, 24/279/319, 29/46/92/196/426/481/549/597, 29/46/481/549/553/597, 29/426/549, 29/549/553, 36/43/222/279/363/560, 36/92/148/222/279/319/363/499/560/637, 36/92/222/637, 36/148/279/319/499, 43/148/222/279/560/637, 43/220/295/549, 56/148/220/295/499, 56/194/243/257/329/460, 56/243, 63/103/148/220/295/549, 63/220/295/304/426/499/549, 63/220/295/304/549, 92, 92/222/279/499/560/637, 92/258/363/426/481/549/597, 103/295/499/549, 148/220/304, 148/222, 148/222/560/637, 148/279/319/499, 194/243/329/460, 194/243/329/560/571/598, 220/295/549, 279/296/481/549/553/567/597, 279/560/637, 295/296/426/549, 295/296/549, 295/499/560/637, 296/363/426/481/549, 319/560, 319/637, and 363/560, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

7. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 4/43/46/56/63/279/295/319/567/598, 4/43/46/295/319/549/560, 4/43/46/426/549/560, 4/43/148/196/279/319/363/560, 16, 16/24/29, 16/24/29/92/220/279/597/598, 16/24/29/279/549, 16/24/29/279/637, 16/24/43/92/549, 16/24/43/192/220/279/549, 16/29/36/192/319/549/597/598/637, 16/29/36/279/549, 16/29/43/92/192/319, 16/29/43/192/222/319/549/637, 16/29/43/222/279/549, 16/29/92/192/549/637, 16/29/92/220/222/319/549/598, 16/29/92/279/549, 16/29/92/319/549, 16/29/92/549/637, 16/29/192/220/549, 16/29/192/220/549/597, 16/29/192/222/279/549, 16/29/192/222/637, 16/29/192/549, 16/29/220/222/279/549/637, 16/29/220/222/597/598, 16/29/222/549/598/637, 16/29/549/637, 16/36/43/192/597/637, 16/36/92/220/222/279/549, 16/36/192/549/597/598/637, 16/36/319/549/597/598/637, 16/43/56/192/549/597/598/637, 16/43/92/222/597/598, 16/43/192/549, 16/43/220/549/637, 16/43/279/319/597, 16/43/279/549/597, 16/43/319/549/598, 16/43/597, 16/92/192/279/319/549/637, 16/92/192/279/637, 16/92/220/549, 16/92/319/597/637, 16/192/319/549/637, 16/192/549, 16/220/222/279/549/598/637, 16/220/279/549, 16/220/319/549/597/598, 16/222/319/597/598, 16/222/637, 16/279/319/549/597/637, 16/279/549/597/598/637, 16/279/597, 16/319/597, 16/319/597/598, 16/549, 16/549/598, 16/597, 29/63/134/520/597/598, 29/63/520/537/538/598, 29/134/237/537/538/567/571, 29/237/520, 29/237/520/538, 29/237/567/598, 29/237/597, 29/597/598, 36/92/549, 36/134/237/520/537/538/571, 36/134/237/567/571/597/598, 36/520/537/538/597, 43/46/56/148/258/279/363/549/571, 43/46/63/258/295/426/560/567/571, 43/46/196/319/549/560/567, 43/279/549/560/567, 46/295/319/426, 46/560, 95, 134/237/520/597, 134/520/597/598, 220/222/597/637, 224, 237/520, 237/520/537/538/598, 237/520/537/598, 237/520/538/597, 237/520/567/571/597, 237/520/597/598, 237/538/597/598, 237/571, 237/597/598, 279/319/560, 294, 295/549/560, 343, 433, 483, 486, 520/571/598, 520/597/598, 549/598, 556, 564, 567/571/597, 568, and 609, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

8. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 4/43/46/426/549/560, 36/63/520, 95, 394, 483, 520/597, 556, 562, 568, and 598, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

9. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 63/196, 173, 189, 194, 196, 197, 198, 198/447, 220/294/296/332, 290, 292, 327, and 638, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

10. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 16/43/220/258/538/637, 18, 24/222/237/520/538, 24/222/520, 43/222/237/258/426/597, 63, 63/95/173/343/564/568/609, 95/173/258/426/556/564, 95/173/556/609, 173/556, 194, 222/237, 222/520/597, 237/258/549/597, 237/265/279, 258/267, 258/426, and 258/538/549/637, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

11. The engineered galactose oxidase of claim 1, and wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 18/95/327/548, 28, 36, 43, 43/46/56/63/191, 43/237/279/538/597/598, 43/237/294/538, 43/237/520, 43/237/520/549/598, 43/237/520/597, 43/279/294, 43/538, 43/549/597, 51/55/111/150/367/564, 55, 61, 95/327/548, 99, 183, 198, 224, 229, 237/520/538/597, 243, 252, 258, 291, 295, 312, 335, 342, 343, 367/371/564/594, 371, 384, 468, 485, 520, 544, 549, 564/604, 567, 568, 570, 594, 596, 604, 635, and 637, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

12. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 28, 28/99/520/637, 55/295, 55/342, 55/568, 55/568/594, 55/568/637, 61/224/343/520/637, 99/343/637, 99/520/637, 99/637, 224/520/637, 295/342, 295/342/568, 342/568, 342/594, 343/520/637, 403/520/637, 520/637, 568/637, 594, and 637, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

13. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 13, 13/26/156/274/359/429, 13/156/262/274/315, 13/156/262/315/429/437, 13/156/274/437/568/606, 13/262, 13/262/274/380, 13/262/274/595, 13/262/437/488, 13/274, 13/274/315/437, 13/274/373/437, 13/328/437, 13/373, 13/437, 13/437/541, 26/262/274/315/437, 35, 37, 37/89, 37/89/274, 37/263/274/380/559/561, 37/380, 45, 45/262/274/373/437, 89, 89/263/274/380, 89/263/559, 89/274/380, 105, 154, 156, 156/274/315, 200, 217, 217/274/380/561, 217/274/478, 217/354/380, 217/380, 224, 239, 241, 253, 262, 262/274, 262/274/315, 262/274/437, 262/373/595, 262/380, 262/437, 262/541, 263, 263/274, 263/274/380, 263/354/380/559, 263/380, 263/380/441, 274, 274/328, 274/354, 274/359, 274/373/437, 274/380, 274/380/441, 274/380/559, 274/393/437, 274/437, 274/437/541, 274/437/568, 315, 328, 336, 354, 354/380, 359, 366, 373, 373/595, 375, 380, 380/437, 380/559/561, 393, 429, 437, 438, 439, 441, 478, 478/561, 488, 541, 550, 559, 561, 568, 595, 605, 627, and 641, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

14. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 13/99/156/262/437/559/641, 13/99/156/380/437, 13/99/156/380/437/559/563/641, 13/99/257/262/263/559, 13/99/262/263/380/437/563/641, 13/99/263, 13/99/263/380, 13/99/263/380/437, 13/99/263/380/437/641, 13/99/380/437/559, 13/99/437/563, 13/99/563/641, 13/156/262/263/437/559, 13/156/263/437, 13/156/380, 13/262, 13/262/263, 13/262/263/380/437/559, 13/380/437, 13/380/437/559, 13/437, 13/470/559/563, 29, 30, 43/46/56/63/99/156/262/263/403/559/563, 62, 99, 99/156/262, 99/156/262/263/380/437/559, 99/156/262/263/437, 99/156/262/263/559, 99/156/263/559, 99/156/380, 99/156/380/437, 99/156/437, 99/262/263/437/559/641, 99/262/437/559, 99/263/437/563, 99/380/437/559/641, 99/380/563, 99/437, 108, 149, 175, 177, 184, 194, 197, 208, 234, 251, 254, 262, 262/263, 262/263/437/559, 262/263/559/563, 262/437/641, 263/380, 263/437/559/563, 278, 280, 287, 356, 373, 380, 407, 409, 463, 466, 489, 559/641, 565, 569, 592, 596, 601, 610, and 615, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

15. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 29/149/463/601, 29/177/197/592, 29/177/463, 29/197/592, 29/463, 62/208/417/615, 62/286/615, 62/373/466, 62/466, 62/466/597, 149, 149/208/615, 149/463, 177/194/197/463/565, 177/197/463/565, 177/280/463/594/601, 177/463/565, 177/463/592, 184, 197, 197/280/463, 197/463/592, 197/466/569/596, 208/251/259/278, 234, 234/384, 251, 251/399/615, 278, 373/466, 384/569, 399/615, 417/615, 463/565, 466, 546, 569, and 569/597, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

16. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 3, 4, 9, 18, 26, 29, 30, 38, 40, 42, 43, 44, 48, 50, 75, 79, 135, 136, 142, 156, 159, 161, 197, 486, and 601, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

17. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 29, 29/30/50/79/136/197, 29/30/50/79/197/407, 29/30/79/136/156/197, 29/30/79/197, 29/30/79/197/407, 29/30/136/197/407/486, 29/30/136/407, 29/30/197, 29/30/197/407, 29/50/197/407/486, 29/197/407, 29/197/407/486, 30, 30/50/79/136/156/197, 43/197/407, 50/136/197/486, 65, 79, 79/136/197/407, 79/156/197/407, 136, 136/197/407, 136/197/486, 156/161/486, 197, 197/407, 197/486, 486, and 615, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

18. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 24, 47, 63, 78, 95, 119, 121, 197, 207, 214, 219, 220, 249, 294, 324, 365, 408, 414, 437, 480, 485, 520, 556, 571, 598, 600, and 626, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

19. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 14/130/257/472, 14/257, 24, 29, 29/136, 29/136/197/436, 29/136/436, 29/136/436/453, 29/197, 29/197/342/436, 29/197/436, 29/197/436/453, 29/197/453, 29/436, 29/436/472, 29/453, 29/472, 43, 63, 95, 119, 130/421, 136, 136/197/436, 136/197/436/453, 136/436, 144, 197, 197/436, 197/436/453, 197/436/472, 197/453, 214, 219, 249, 257, 257/472, 297, 359, 436, 437, 460, 485, 495, 520, 556, 560, 567, and 592, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

20. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 24, 24/51/63/197/359, 24/119/197, 24/197/249/437, 43/197/359, 43/249, 63, 63/67/197/571, 63/67/214/556, 63/119/197, 63/119/197/207/214, 63/119/197/339/341, 63/119/197/556, 63/119/197/556/571, 63/119/556, 63/197, 63/197/207/556, 63/197/207/556/571, 63/197/214/571, 63/197/249/495, 63/197/556/571, 95/197, 95/219/359, 119, 119/197, 119/197/207/571, 119/197/214, 119/197/214/556, 119/197/214/571, 119/197/339, 119/197/556, 119/197/556/571, 119/197/571, 119/207/556/571, 197, 197/207, 197/207/214/471, 197/214, 197/219, 197/339/556/571, 197/556, 197/556/571, 197/571, 214/249/359, 219, and 556, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

21. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 194/330/495, 196, 246/408/442/462, 246/442, 292, 327, 327/329, 330, 407, 442, 442/462/515, 462/583, 498, and 583, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

22. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 14, 14/24/36/96, 14/24/36/296/424/560, 14/24/78/120/258, 14/24/92/96/99/408, 14/24/96/258/626, 14/24/258/560, 14/78/120/258/488/560/626, 14/92/96/99, 14/92/96/99/120/537, 14/92/96/120/376, 14/92/99/120/537, 14/95/120/296/480/560, 14/120/480/626, 14/258, 14/258/296/560, 14/376/560, 14/408, 23/36/92/95/96, 23/36/92/95/99/408/596, 23/36/408/428, 23/36/537/596, 23/218/537, 23/408/596, 24, 24/36/46/99/426/532/549, 24/36/95/96, 24/36/95/99/404/426/485, 24/36/96/99/532/549, 24/36/99/404/426/532/549/600, 24/36/120/296/480/560, 24/36/404/426/532, 24/36/404/480/485/532/560/600, 24/46/92/404/426/532, 24/46/92/426/532, 24/46/92/426/549, 24/46/95/99/426/532, 24/46/99/426/549/600, 24/46/404/426/485/532, 24/96/404/426, 24/99, 24/99/404/485/532/600, 24/296, 24/296/324/480, 24/404/426/532, 24/404/480/485, 24/404/480/532/549/560, 36, 36/92/95/99/404/426/560, 36/92/95/428/596, 36/92/96/408/428/540/596, 36/92/485, 36/258/296, 36/258/296/324/433/626, 36/404, 36/404/426/549/600, 36/408/537/596, 36/408/596, 36/426/485/600, 46, 46/92/560, 92, 92/95/485/532/549/560, 92/99/218/560, 95, 95/120/296/626, 95/404/426/532, 96, 96/99, 96/258/560/626, 99, 99/404/426/560, 99/426/480/485, 120, 120/324/480/560, 218, 218/408, 296/324, 296/324/560, 324/560, 404, 404/485/600, 408, 480, 485, 532, 537, 537/640, 549, 549/560, 560, 596, and 600, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

23. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 14, 14/24/36/96, 14/24/36/296/424/560, 14/24/78/120/258, 14/24/99/218/408/537/560, 14/24/258/560, 14/46/47/376, 14/46/96/99/560, 14/92/96/99/376/560, 14/92/96/120/376, 14/92/96/376, 14/92/99/120/218/408, 14/92/99/120/537, 14/92/99/218/408, 14/92/218/408, 14/95/120/296/480/560, 14/376, 14/376/537, 14/376/560, 14/408, 14/537, 23/36, 23/36/92/95/96, 23/36/92/95/99/408/596, 23/36/96/408/596/640, 23/36/408/428, 23/36/537/540/640, 23/36/537/596, 23/218/596/640, 24, 24/36/46/99/426/532/549, 24/36/95/96, 24/36/95/99/404/426/485, 24/36/96/99/532/549, 24/36/120/296/480/560, 24/36/404/426/532, 24/36/404/480/485/532/560/600, 24/46/92/404/426/532, 24/46/92/426/532, 24/46/92/426/549, 24/46/95/99/426/532, 24/46/99/426/549/600, 24/46/404/426/485/532, 24/96/404/426, 24/96/404/426/560, 24/99, 24/296, 24/404/426/532, 24/404/480/485, 24/532, 36, 36/92/95/428/596, 36/95/96, 36/99/426/485/600, 36/258/296, 36/258/296/324/433/626, 36/404, 36/404/426/549/600, 36/408, 36/408/537/596, 36/426/485/600, 46/92/560, 78, 92, 92/95/485/532/549/560, 92/96, 92/99/120, 92/99/218/560, 92/218, 92/404, 95, 96, 96/99, 99, 99/426/480/485, 99/640, 120, 120/324/480/560, 120/376, 218/537/596, 218/596, 258, 296, 296/324, 296/324/560, 296/480/560, 324, 361, 404, 404/426/485, 408/596, 424, 426/485, 426/532/549, 480, 532, 537/640, 549, 549/560, 560, 600, 626, and 640, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

24. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase is a variant engineered polypeptide set forth in SEQ ID NOS: 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300, or 2424.

25. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase comprises a polypeptide sequence that is at least 95% identical to the sequence of an engineered galactose oxidase variant set forth in the even numbered sequences of SEQ ID NOS: 4-2860.

26. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase comprises a polypeptide sequence set forth in one of the even numbered sequences of SEQ ID NOS: 4-2860.

27. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase has at least 1.2 fold improved enzymatic activity on a primary alcohol as compared to the galactose oxidase of SEQ ID NO: 2.

28. The engineered galactose oxidase of claim 1, wherein said engineered galactose oxidase is purified.

29. A composition comprising at least one engineered galactose oxidase of claim 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention provides engineered galactose oxidase (GOase) enzymes, polypeptides having GOase activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing GOase enzymes are also provided. The present invention further provides compositions comprising the GOase enzymes and methods of using the engineered GOase enzymes. The present invention finds particular use in the production of pharmaceutical and other compounds.

(2) Galactose oxidase (GOase) from F. graminearium is a naturally-occurring copper-dependent enzyme capable of performing oxidations on primary alcohol-containing substrates under mild reaction conditions. In addition to copper, the enzyme relies on a post-translationally formed cofactor, which is the result of the bound copper and molecular oxygen-mediated cross-linking of the active site residues tyrosine and cysteine. The enzyme is then active and capable of catalyzing the oxidation of primary alcohols by reducing oxygen and producing an aldehyde and hydrogen peroxide via a radical mechanism.

(3) ##STR00001##

(4) Early directed evolution efforts were performed which focused on evolving a GOase variant with improved selectivity and activity on 3-ethynylglycerol (EGO) for generating the corresponding aldehyde. An initial evolved variant demonstrated only slight enrichment for the S-enantiomer of 3-ethynylglyceraldehyde (EGA). This enzyme was further evolved to a variant that possessed enantioselectivity which favored formation of the R-enantiomer (See, Scheme 2). Additional directed evolution was needed in order to further enhance the R-enantioselectivity and improve oxidation activity at process conditions.

(5) ##STR00002##

(6) Further directed evolution efforts were performed which focused on evolving a GOase variant with improved activity on the ethynyl glycerol phosphate (EGP) for generating the corresponding phosphorylated aldehyde (Compound P) (See, Scheme 3).

(7) ##STR00003##
Engineered GOase Polypeptides

(8) The present invention provides engineered GOase polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods for using the polypeptides. Where the description relates to polypeptides, it is to be understood that it also describes the polynucleotides encoding the polypeptides. In some embodiments, the present invention provides engineered, non-naturally occurring GOase enzymes with improved properties as compared to wild-type GOase enzymes. Any suitable reaction conditions find use in the present invention. In some embodiments, methods are used to analyze the improved properties of the engineered polypeptides to carry out the oxidation reaction. In some embodiments, the reaction conditions are modified with regard to concentrations or amounts of engineered GOase, substrate(s), buffer(s), solvent(s), co-factors, pH, conditions including temperature and reaction time, and/or conditions with the engineered GOase polypeptide immobilized on a solid support, as further described below and in the Examples.

(9) In some embodiments, additional reaction components or additional techniques are utilized to supplement the reaction conditions. In some embodiments, these include taking measures to stabilize or prevent inactivation of the enzyme, reduce product inhibition, shift reaction equilibrium to desired product formation.

(10) In some further embodiments, any of the above described processes for the conversion of substrate compound to product compound can further comprise one or more steps selected from: extraction, isolation, purification, crystallization, filtration, and/or lyophilization of product compound(s). Methods, techniques, and protocols for extracting, isolating, purifying, and/or crystallizing the product(s) from biocatalytic reaction mixtures produced by the processes provided herein are known to the ordinary artisan and/or accessed through routine experimentation. Additionally, illustrative methods are provided in the Examples below.

(11) Engineered GOase Polynucleotides Encoding Engineered Polypeptides,

(12) Expression Vectors and Host Cells

(13) The present invention provides polynucleotides encoding the engineered enzyme polypeptides described herein. In some embodiments, the polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. In some embodiments, expression constructs containing at least one heterologous polynucleotide encoding the engineered enzyme polypeptide(s) is introduced into appropriate host cells to express the corresponding enzyme polypeptide(s).

(14) As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode an engineered enzyme (e.g., GOase) polypeptide. Thus, the present invention provides methods and compositions for the production of each and every possible variation of enzyme polynucleotides that could be made that encode the enzyme polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in the various Tables).

(15) In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. Consequently, codon optimized polynucleotides encoding the engineered enzyme polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region.

(16) In some embodiments, the enzyme polynucleotide encodes an engineered polypeptide having enzyme activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from the SEQ ID NOS provided herein, or the amino acid sequence of any variant (e.g., those provided in the Examples), and one or more residue differences as compared to the reference polynucleotide(s), or the amino acid sequence of any variant as disclosed in the Examples (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). In some embodiments, the reference polypeptide sequence is selected from SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300, and/or 2424.

(17) In some embodiments, the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from any polynucleotide sequence provided herein, or a complement thereof, or a polynucleotide sequence encoding any of the variant enzyme polypeptides provided herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an enzyme polypeptide comprising an amino acid sequence that has one or more residue differences as compared to a reference sequence.

(18) In some embodiments, an isolated polynucleotide encoding any of the engineered enzyme polypeptides herein is manipulated in a variety of ways to facilitate expression of the enzyme polypeptide. In some embodiments, the polynucleotides encoding the enzyme polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the enzyme polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, suitable promoters are selected based on the host cells selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25 [1983]). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]).

(19) In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra).

(20) In some embodiments, the control sequence is also a suitable leader sequence (i.e., a non-translated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

(21) In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

(22) In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway). In some embodiments, the 5′ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5′ end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

(23) In some embodiments, the control sequence is also a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

(24) In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

(25) In another aspect, the present invention is directed to a recombinant expression vector comprising a polynucleotide encoding an engineered enzyme polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described herein are joined together to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the enzyme polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

(26) The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the enzyme polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

(27) In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.

(28) In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

(29) In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the present invention, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the engineered enzyme enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21). Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, and or tetracycline resistance.

(30) In some embodiments, the expression vectors of the present invention contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vectors rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.

(31) In some alternative embodiments, the expression vectors contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements preferably contain a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

(32) For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A ori or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), or pACYC184 permitting replication in E. coli, and pUB110, pE194, or pTA1060 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes it's functioning temperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).

(33) In some embodiments, more than one copy of a nucleic acid sequence of the present invention is inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

(34) Many of the expression vectors for use in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to the p3×FLAGTM™ expression vectors (Sigma-Aldrich Chemicals), which include a CMV promoter and hGH polyadenylation site for expression in mammalian host cells and a pBR322 origin of replication and ampicillin resistance markers for amplification in E. coli. Other suitable expression vectors include, but are not limited to pBluescriptII SK(−) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (See e.g., Lathe et al., Gene 57:193-201 [1987]).

(35) Thus, in some embodiments, a vector comprising a sequence encoding at least one variant galactose oxidase is transformed into a host cell in order to allow propagation of the vector and expression of the variant galactose oxidase(s). In some embodiments, the variant galactose oxidases are post-translationally modified to remove the signal peptide and in some cases may be cleaved after secretion. In some embodiments, the transformed host cell described above is cultured in a suitable nutrient medium under conditions permitting the expression of the variant galactose oxidase(s). Any suitable medium useful for culturing the host cells finds use in the present invention, including, but not limited to minimal or complex media containing appropriate supplements. In some embodiments, host cells are grown in HTP media. Suitable media are available from various commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection).

(36) In another aspect, the present invention provides host cells comprising a polynucleotide encoding an improved galactose oxidase polypeptide provided herein, the polynucleotide being operatively linked to one or more control sequences for expression of the galactose oxidase enzyme in the host cell. Host cells for use in expressing the galactose oxidase polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Bacillus megaterium, Lactobacillus kefir, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culture media and growth conditions for the above-described host cells are well known in the art.

(37) Polynucleotides for expression of the galactose oxidase may be introduced into cells by various methods known in the art. Techniques include among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

(38) In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, the fungal host cells are yeast cells and filamentous fungal cells. The filamentous fungal host cells of the present invention include all filamentous forms of the subdivision Eumycotina and Oomycota. Filamentous fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other complex polysaccharides. The filamentous fungal host cells of the present invention are morphologically distinct from yeast.

(39) In some embodiments of the present invention, the filamentous fungal host cells are of any suitable genus and species, including, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/or teleomorphs, or anamorphs, and synonyms, basionyms, or taxonomic equivalents thereof.

(40) In some embodiments of the present invention, the host cell is a yeast cell, including but not limited to cells of Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the present invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

(41) In some embodiments of the invention, the host cell is an algal cell such as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp. ATCC29409).

(42) In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to Gram-positive, Gram-negative and Gram-variable bacterial cells. Any suitable bacterial organism finds use in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the present invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A. ureafaciens). In some embodiments of the present invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens). In some embodiments, the host cell is an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and/or B. amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (e.g., C. glutamicum and C. acetoacidophilum). In some embodiments the bacterial host cell is an Escherichia species (e.g., E. coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments, the bacterial host cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (e.g., P. citrea, and P. agglomerans). In some embodiments the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Z. mobilis, and Z. lipolytica).

(43) Many prokaryotic and eukaryotic strains that find use in the present invention are readily available to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

(44) In some embodiments, host cells are genetically modified to have characteristics that improve protein secretion, protein stability and/or other properties desirable for expression and/or secretion of a protein. Genetic modification can be achieved by genetic engineering techniques and/or classical microbiological techniques (e.g., chemical or UV mutagenesis and subsequent selection). Indeed, in some embodiments, combinations of recombinant modification and classical selection techniques are used to produce the host cells. Using recombinant technology, nucleic acid molecules can be introduced, deleted, inhibited or modified, in a manner that results in increased yields of galactose oxidase variant(s) within the host cell and/or in the culture medium. For example, knockout of Alp1 function results in a cell that is protease deficient, and knockout of pyr5 function results in a cell with a pyrimidine deficient phenotype. In one genetic engineering approach, homologous recombination is used to induce targeted gene modifications by specifically targeting a gene in vivo to suppress expression of the encoded protein. In alternative approaches, siRNA, antisense and/or ribozyme technology find use in inhibiting gene expression. A variety of methods are known in the art for reducing expression of protein in cells, including, but not limited to deletion of all or part of the gene encoding the protein and site-specific mutagenesis to disrupt expression or activity of the gene product. (See e.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant Microbe Interact., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352 [2004]; and You et al., Arch. Microbiol., 191:615-622 [2009], all of which are incorporated by reference herein). Random mutagenesis, followed by screening for desired mutations also finds use (See e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon et al., Eukary. Cell 2:247-55 [2003], both of which are incorporated by reference).

(45) Introduction of a vector or DNA construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, PEG-mediated transformation, electroporation, or other common techniques known in the art. In some embodiments, the Escherichia coli expression vector pCK100900i (See, U.S. Pat. No. 9,714,437, which is hereby incorporated by reference) finds use.

(46) In some embodiments, the engineered host cells (i.e., “recombinant host cells”) of the present invention are cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the galactose oxidase polynucleotide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and are well-known to those skilled in the art. As noted, many standard references and texts are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin.

(47) In some embodiments, cells expressing the variant galactose oxidase polypeptides of the invention are grown under batch or continuous fermentations conditions. Classical “batch fermentation” is a closed system, wherein the compositions of the medium is set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a “fed-batch fermentation” which also finds use in the present invention. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. “Continuous fermentation” is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

(48) In some embodiments of the present invention, cell-free transcription/translation systems find use in producing variant galactose oxidase(s). Several systems are commercially available and the methods are well-known to those skilled in the art.

(49) The present invention provides methods of making variant galactose oxidase polypeptides or biologically active fragments thereof. In some embodiments, the method comprises: providing a host cell transformed with a polynucleotide encoding an amino acid sequence that comprises at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to SEQ ID NO: 2, 70, 122, 514, 426, and comprising at least one mutation as provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded variant galactose oxidase polypeptide; and optionally recovering or isolating the expressed variant galactose oxidase polypeptide, and/or recovering or isolating the culture medium containing the expressed variant galactose oxidase polypeptide. In some embodiments, the methods further provide optionally lysing the transformed host cells after expressing the encoded galactose oxidase polypeptide and optionally recovering and/or isolating the expressed variant galactose oxidase polypeptide from the cell lysate. The present invention further provides methods of making a variant galactose oxidase polypeptide comprising cultivating a host cell transformed with a variant galactose oxidase polypeptide under conditions suitable for the production of the variant galactose oxidase polypeptide and recovering the variant galactose oxidase polypeptide. Typically, recovery or isolation of the galactose oxidase polypeptide is from the host cell culture medium, the host cell or both, using protein recovery techniques that are well known in the art, including those described herein. In some embodiments, host cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including, but not limited to freeze-thaw cycling, sonication, mechanical disruption, and/or use of cell lysing agents, as well as many other suitable methods well known to those skilled in the art.

(50) Engineered galactose oxidase enzymes expressed in a host cell can be recovered from the cells and/or the culture medium using any one or more of the techniques known in the art for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available under the trade name CelLytic B™ (Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide is recovered/isolated and optionally purified by any of a number of methods known in the art. For example, in some embodiments, the polypeptide is isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing, and size exclusion), or precipitation. In some embodiments, protein refolding steps are used, as desired, in completing the configuration of the mature protein. In addition, in some embodiments, high performance liquid chromatography (HPLC) is employed in the final purification steps. For example, in some embodiments, methods known in the art, find use in the present invention (See e.g., Parry et al., Biochem. J., 353:117 [2001]; and Hong et al., Appl. Microbiol. Biotechnol., 73:1331 [2007], both of which are incorporated herein by reference). Indeed, any suitable purification methods known in the art find use in the present invention.

(51) Chromatographic techniques for isolation of the galactose oxidase polypeptide include, but are not limited to reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., are known to those skilled in the art.

(52) In some embodiments, affinity techniques find use in isolating the improved galactose oxidase enzymes. For affinity chromatography purification, any antibody which specifically binds the galactose oxidase polypeptide may be used. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with the galactose oxidase. The galactose oxidase polypeptide may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

(53) In some embodiments, the galactose oxidase variants are prepared and used in the form of cells expressing the enzymes, as crude extracts, or as isolated or purified preparations. In some embodiments, the galactose oxidase variants are prepared as lyophilisates, in powder form (e.g., acetone powders), or prepared as enzyme solutions. In some embodiments, the galactose oxidase variants are in the form of substantially pure preparations.

(54) In some embodiments, the galactose oxidase polypeptides are attached to any suitable solid substrate. Solid substrates include but are not limited to a solid phase, surface, and/or membrane. Solid supports include, but are not limited to organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of the substrate can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous, and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location. A plurality of supports can be configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.

(55) In some embodiments, immunological methods are used to purify galactose oxidase variants. In one approach, antibody raised against a variant galactose oxidase polypeptide (e.g., against a polypeptide comprising any of SEQ ID NO: 2, 70, 122, 514 and/or 426, and/or an immunogenic fragment thereof) using conventional methods is immobilized on beads, mixed with cell culture media under conditions in which the variant galactose oxidase is bound, and precipitated. In a related approach, immunochromatography finds use.

(56) In some embodiments, the variant galactose oxidases are expressed as a fusion protein including a non-enzyme portion. In some embodiments, the variant galactose oxidase sequence is fused to a purification facilitating domain. As used herein, the term “purification facilitating domain” refers to a domain that mediates purification of the polypeptide to which it is fused. Suitable purification domains include, but are not limited to metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; See e.g., Wilson et al., Cell 37:767 [1984]), maltose binding protein sequences, the FLAG epitope utilized in the FLAGS extension/affinity purification system (e.g., the system available from Immunex Corp), and the like. One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography; See e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) while the enterokinase cleavage site provides a means for separating the variant galactose oxidase polypeptide from the fusion protein. pGEX vectors (Promega) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.

(57) Accordingly, in another aspect, the present invention provides methods of producing the engineered enzyme polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered enzyme polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise the steps of isolating and/or purifying the enzyme polypeptides, as described herein.

(58) Appropriate culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing polynucleotides for expression of the enzyme polypeptides into cells will find use in the present invention. Suitable techniques include, but are not limited to electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

(59) Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative, and not limiting.

EXPERIMENTAL

(60) The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention. Indeed, there are various suitable sources for many of the reagents and equipment described below. It is not intended that the present invention be limited to any particular source for any reagent or equipment item.

(61) In the experimental disclosure below, the following abbreviations apply: M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); psi and PSI (pounds per square inch); ° C. (degrees Centigrade); RT and rt (room temperature); RH (relative humidity); CV (coefficient of variability); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (Luria broth); TB (terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, Conn.); HTP (high throughput); HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-Ultraviolet Visible Detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold improvements over positive control); Sigma and Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, Mich.); Microfluidics (Microfluidics, Westwood, Mass.); Life Technologies (Life Technologies, a part of Fisher Scientific, Waltham, Mass.); Amresco (Amresco, LLC, Solon, Ohio); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto, Calif.); Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); Infors (Infors USA Inc., Annapolis Junction, Md.); and Thermotron (Thermotron, Inc., Holland, Mich.).

Example 1

GOA Improvements Over SEQ ID NO: 2 for Enantioselective Production of EGA

(62) The parent genes for the GOA (SEQ ID NO: 2) enzyme used to produce the variants of the present invention were codon optimized for expression in E. coli and synthesized and cloned into the pET-30a vector. BL21(DE3) E. coli cells were transformed with the respective plasmid containing the GOA-encoding genes and plated on Luria broth (LB) agar plates containing 1% glucose and 50 μg/mL kanamycin (KAN), and grown overnight at 37° C. Monoclonal colonies were picked and inoculated into 180 μL LB containing 1% glucose and 50 μg/mL KAN 96-well shallow-well microtiter plates. The plates were sealed with O.sub.2-permeable seals and cultures were grown overnight at 30° C., 200 rpm and 85% relative humidity (RH). Then, 10 μL of each of the cell cultures were transferred into the wells of 96-well deep-well plates containing 390 μL TB, 50 μg/mL KAN and 0.5 mM CuSO.sub.4. The deep-well plates were sealed with O.sub.2-permeable seals and incubated at 30° C., 250 rpm and 85% RH until OD.sub.600 0.6-0.8 was reached. The cell cultures were then induced by isopropyl thioglycoside (IPTG) to a final concentration of 1 mM and incubated overnight at 25° C., 270 rpm. The cells were then pelleted using centrifugation at 4000 rpm for 10 min. The supernatants were discarded and the pellets frozen at −80° C. prior to lysis.

(63) Frozen pellets were lysed with 200 μL lysis buffer containing 50 mM sodium phosphate (NaPi) buffer, pH 7.4, 1 mg/mL lysozyme, 0.5 mg/mL PMBS, 17 mg/ml horseradish peroxidase (HRP) and 17 mg/ml catalase. The lysis mixture was shaken at room temperature (RT) for 2.5 hours. The plate was then centrifuged for 10 min at 4000 rpm and 4° C. The supernatants were then used in biocatalytic reactions as clarified lysate to determine the activity levels.

(64) Libraries of SEQ ID NO: 2 were produced using well-established techniques (e.g., recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP, the clarified lysate was generated as described above.

(65) Each 100 μL reaction was carried out in 96-well deep-well (2 mL vol.) plates with 50 μL of the clarified lysate solution, 50 g/L Compound X (2-ethynylglycerol, EGO), 50 mM NaPi buffer, 50 μM CuSO.sub.4 at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Infors shaker overnight maintained at 85% RH for 20 hours.

(66) Plate wells were derivatized by taking 50 μL aliquots and adding 10 μL (R)-(+)-1-Amino-2-(methoxymethyl)pyrrolidine (R-AMP) and incubating with shaking in a 96-well round bottom (0.3 mL vol.) plate for ˜30 minutes at RT. The samples were quenched by adding 200 μL acetonitrile (MeCN), shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for 5 minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 8.1.

(67) Variant activity relative to that of SEQ ID NO: 2 was determined by 2-ethynylglyceraldehyde (EGA) peak abundance in SIM MS (227.3 m/z) for data samples relative to the SIM MS product peak abundance of the corresponding SEQ ID NO: 2. The amount of EGA was quantified by multiplying the area of the MS product peak by a factor calculated using a standard dose-response curve of EGA on the MS using Analytical Method 8.1. The 10 samples with the highest improvement in product production were selected to be analyzed using Analytical Method 9.1 in order to identify improved enantioselectivity variants.

(68) Enantioselectivity relative to SEQ ID NO: 2 was calculated as the enantiomeric excess with respect to the R-enantiomer of EGA (% ee R—Compound Y) formed relative to that of the corresponding SEQ ID NO: 2% ee R. Enantioselectivity was quantified by subtracting the Compound Y from the S-enantiomer of EGA (Compound Z) and dividing that difference by the sum of the R-enantiomer and S-enantiomer product peaks as determined by HPLC analysis.

(69) TABLE-US-00001 TABLE 4.1 Activity and Selectivity of GOase Variants Relative to SEQ ID NO: 2 Percent Conversion Amino Acid Differences Improvement SEQ ID NO: (Relative to SEQ (Relative to SEQ Selectivity (nt/aa) ID NO: 2) ID NO: 2) * (% ee R) † 1/2 + + 3/4 K331R/F406Y/F465A +++ ++ 5/6 K331R/F406Y/F465Q +++ ++

(70) TABLE-US-00002 TABLE 4.1 Activity and Selectivity of GOase Variants Relative to SEQ ID NO: 2 Percent Conversion Amino Acid Differences Improvement SEQ ID NO: (Relative to SEQ (Relative to SEQ Selectivity (nt/aa) ID NO: 2) ID NO: 2) * (% ee R) † 7/8 K331R/F406Y/E407Q/ +++ ++ F465A  9/10 K331R/F406Y/E407Q/ +++ ++ F465A 11/12 K331R/F406Y/F465A +++ ++ * Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 2, and defined as follows: “+” = 1.0 to 1.2; “++” >1.20 to 10.0; and “+++” >10.0. † Selectivity (% ee R) was defined as follows “+” = −50.0 to 0.0; and “++” = 0.0 to 20.0.

Example 2

Preparation of Galactose Oxidase (GOA) Wet Cell Pellets

(71) The parent genes for the GOA (SEQ ID NO: 2) enzyme used to produce the variants of the present invention were codon optimized for expression in E. coli and synthesized and cloned into a pCK900 vector (See e.g., U.S. Pat. No. 9,714,437, which is hereby incorporated by reference herein). W3110 E. coli cells were transformed with the respective plasmid containing the GOA-encoding genes and plated on LB) agar plates containing 1% glucose and 30 μg/mL CAM, and grown overnight at 37° C. Monoclonal colonies were picked and inoculated into 180 μL LB containing 1% glucose and 30 μg/mL CAM in 96-well shallow-well microtiter plates. The plates were sealed with O.sub.2-permeable seals and cultures were grown overnight at 30° C., 200 rpm and 85% RH. Then, 10 μL of each of the cell cultures were transferred into the wells of 96-well deep-well plates containing 390 μL TB and 30 μg/mL CAM. The deep-well plates were sealed with 02-permeable seals and incubated at 30° C., 250 rpm and 85% RH until OD.sub.600 0.6-0.8 was reached. The cell cultures were then induced by IPTG to a final concentration of 1 mM and incubated overnight at 30° C., 250 rpm. The cells were then pelleted using centrifugation at 4000 rpm for 10 mM. The supernatants were discarded and the pellets frozen at −80° C. prior to lysis.

Example 3

Preparation of HTP GOA-Containing Cell Lysates

(72) Frozen pellets prepared as specified in Example 2 were lysed with 400 μL lysis buffer containing 50 mM NaPi buffer, pH 7.4, 1 mg/mL lysozyme, 0.5 mg/mL PMBS. The lysis mixture was shaken at RT for 2 hours. The plate was then centrifuged for 15 min at 4000 rpm and 4° C. The supernatants were then used in biocatalytic reactions as clarified lysates, in experiments described below to determine the activity levels.

Example 4

Preparation of Retest GOA-Containing Cell Lysates

(73) Frozen pellets prepared as specified in Example 2 were lysed with 150 μL lysis buffer containing 50 mM NaPi buffer, pH 7.4, 1 mg/mL lysozyme, 0.5 mg/mL PMBS. The lysis mixture was shaken at RT for 2 hours. The plate was then centrifuged for 15 min at 4000 rpm and 4° C. The supernatants were then used in biocatalytic reactions as clarified lysates in experiments described below to determine the activity levels.

Example 5

GOA Improvements Over SEQ ID NO: 4 for Enantioselective Production of Compound Y

(74) SEQ ID NO: 4 was selected as the parent enzyme for the next round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 3.

(75) Each 100 μL reaction was carried out in 96-well deep-well (2 mL vol.) plates with 50 μL clarified lysate, 20 g/L Compound X, 50 mM NaPi buffer, 25 μM CuSO.sub.4, 0.25 g/L horseradish peroxidase (HRP), 0.25 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(76) The variants in the plate wells were derivatized by taking 50 μL aliquots and adding 10 μL (R)-(+)-1-Amino-2-(methoxymethyl)pyrrolidine (R-AMP) and incubating with shaking in a 96-well half-deep-well (1 mL vol.) plate for ˜30 minutes at RT. The samples were quenched by adding 200 μL acetonitrile (MeCN), shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 8.1, as described in Example 8.

(77) The activity of each variant relative to that of SEQ ID NO: 4 was calculated as the % conversion of the product (Compound Y+Z) formed per percent conversion of the corresponding SEQ ID NO: 4. Percent conversion was quantified by multiplying the area of the HPLC product peak by a factor calculated using a standard dose-response curve of Compound Y on the HPLC using Analytical Method 8.1. Variants with activity having a fold-improvement over positive control (HOP) of greater than 0.7 were selected to be analyzed using Analytical Method 9.1, as described in Example 9, in order to identify improved enantioselectivity variants.

(78) Enantioselectivity of each variant relative to SEQ ID NO: 4 was calculated as the enantiomeric excess with respect to the Compound Y product formed relative to that of the corresponding SEQ ID NO: 4% ee R. Enantioselectivity was quantified by subtracting Compound Y from Compound Z and dividing that difference by the sum of Y and Z product peaks as determined by HPLC analysis. The results are provided below.

(79) TABLE-US-00003 TABLE 5.1 Activity and Selectivity of GOase Variants Relative to SEQ ID NO: 4 Percent Conversion Amino Acid Differences Improvement SEQ ID NO: (Relative to SEQ (Relative to SEQ Selectivity (nt/aa) ID NO: 4) ID NO: 4)* (% ee R) † 13/14 I463K +++ 15/16 E407V +++ 17/18 L204S + +++ 19/20 G197K +++ 21/22 T520V ++ +++ 23/24 I202T +++ 25/26 R191A + +++ 27/28 G517S +++ 29/30 P199T ++ +++ 31/32 I463V +++ 33/34 T520L +++ 35/36 G517L ++ +++ 37/38 F296A ++ +++ 39/40 P199G +++ 41/42 E466R +++ 43/44 V220P +++ 45/46 G517D + +++ 47/48 A324G +++ 49/50 V220E +++ ++ 51/52 T243V +++ ++ 53/54 N192I ++ ++ 55/56 A248T ++ ++ 57/58 T205A + ++ 59/60 G294N +++ ++ 61/62 G517E ++ ++ 63/64 L515T ++ ++ 65/66 D247G + ++ 67/68 L204Q + ++ 69/70 F296W + ++ 71/72 N522S + ++ 73/74 T243C + ++ 75/76 A173S + ++ 77/78 A173C + ++ 79/80 A248E ++ ++ 81/82 D193T +++ ++ 83/84 T521S + ++ 85/86 P199R + ++ 87/88 V269Q + ++ 89/90 A465G ++ ++ 91/92 I170L ++ ++ 93/94 V220R + ++ 95/96 S198A + ++ 97/98 N192Q + +  99/100 V171A + + 101/102 T521V + + 103/104 T520P ++ + 105/106 A324S + + 107/108 F296S + + 109/110 I463R + + 111/112 S252T + + 113/114 S198T ++ + 115/116 T520G + + 117/118 F296L + + 119/120 T520S + + 121/122 N192M ++ + 123/124 V171L ++ + 125/126 T521G ++ + 127/128 G294K + + 129/130 V493G + + 131/132 G294S +++ + 133/134 V171C +++ + 135/136 S198G +++ + 137/138 M227L ++ + 139/140 G517M ++ + 141/142 L204V + + 143/144 I202C + + 145/146 A194V + + 147/148 V269Y + + 149/150 T521P + + 151/152 S332R + + 153/154 G197S ++ + *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 4, and defined as follows: “+” >1.2 but less than 1.8; “++” >1.8 but less than 2.5; and “+++” >2.5. † Selectivity (% ee R) was defined as follows: “+” = 0.0 but less than 15.0; “++” = 15.0 but less than 20.0; and “+++” = 20.0 but less than 40.0.

Example 6

GOA Improvements Over SEQ ID NO: 4 for Enantioselective Production of Compound Y

(80) SEQ ID NO: 4 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(81) Each 100 μL reaction was carried out in 96-well deep-well plates with 50 μL clarified lysate, 20 g/L Compound X, 50 mM NaPi buffer, 25 μM CuSO.sub.4, 0.25 g/L HRP, 0.25 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(82) The enzyme variants in the plate wells were derivatized by taking 50 μL aliquots and adding 10 μL R-AMP and incubating with shaking in a 96-well half-deep-well plate for ˜30 minutes at RT. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 8.1.

(83) The activity of each enzyme variant relative to that of SEQ ID NO: 4 was calculated as the % conversion of the product (Compound Y+Z)) formed per percent conversion of the corresponding SEQ ID NO: 4. Percent conversion was quantified by multiplying the area of the HPLC product peak by a factor calculated using a standard dose-response curve of Compound Y on the HPLC using Analytical Method 8.1. Variants with activity having a FIOP of greater than 0.7 were selected to be analyzed using Analytical Method 9.1 in order to identify improved enantioselectivity variants.

(84) Enantioselectivity of each variant relative to SEQ ID NO: 4 was calculated as the % ee R EGA with respect to the % ee R EGA formed of the corresponding SEQ ID NO: 4 as in Example 5. The results are provided below.

(85) TABLE-US-00004 TABLE 6.1 Variant GOase Activity and Selectivity Relative to SEQ ID NO: 4 Percent Conversion Amino Acid Differences Improvement SEQ ID NO: (Relative to SEQ (Relative to SEQ Selectivity (nt/aa) ID NO: 4) ID NO: 4)* (% ee R) † 155/156 V295N +++ 157/158 V295G +++ 159/160 A465T +++ 161/162 E407M +++ 163/164 S252R +++ 165/166 V295R +++ 167/168 V295E +++ 169/170 E407I +++ 171/172 V295S +++ 173/174 T223N +++ 175/176 E407F +++ 177/178 S252V +++ 179/180 T223L +++ 181/182 A465M +++ 183/184 V220P +++ 185/186 G517K +++ 187/188 S252M +++ 189/190 S332Q +++ 191/192 T223H ++ +++ 193/194 V220C + +++ 195/196 T223M +++ 197/198 A173S + +++ 199/200 S252T + ++ 201/202 T521Y + ++ 203/204 V220S + ++ 205/206 A465G + ++ 207/208 T203V + ++ 209/210 P199S + ++ 211/212 T243A + ++ 213/214 G197S + ++ 215/216 G294E + ++ 217/218 T521A + ++ 219/220 V220E ++ + 221/222 T521V + + 223/224 T243S + + 225/226 F296S + + 227/228 V171A + + 229/230 G197T + + 231/232 L515V + + 233/234 P199N + + 235/236 G294Q + + 237/238 G294S + + 239/240 V220M + + 241/242 E466R + + 243/244 P199A + + 245/246 T521Q + + 247/248 T521G ++ + 249/250 E466G + + 251/252 T520A + + 253/254 G517S + + 255/256 S188T + + 257/258 G197K + + 259/260 N192Q + + 261/262 G517D + + 263/264 T520S + + 265/266 V493T + + *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 4, and defined as follows: “+” = 1.2 but less than 1.8; “++” = 1.8 but less than 2.5“; and +++” >2.5. † Selectivity (% ee R) was defined as follows: “+” = 0.0 but less than 15.0; “++” = 15.0 but less than 20.0; and “+++” = 20.0 but less than 40.0.

Example 7

GOA Improvements Over SEQ ID NO: 166 for Enantioselective Production of Compound Y

(86) SEQ ID NO: 166 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(87) Each 100 μL reaction was carried out in 96-well deep-well plates with 45 μL clarified lysate, 20 g/L Compound X, 50 mM NaPi buffer, 50 μM CuSO.sub.4, 0.25 g/L HRP, 0.25 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(88) The enzyme variants in the plate wells were derivatized by taking 50 μL aliquots and adding 10 μL R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜30 minutes at RT. In the case of samples analyzed by Analytical Method 10.1 (as described in Example 10), the samples were quenched by adding 60 μL of ethanol followed by mixing, and further followed by transferring 20 μL of the diluted samples into a 96-well shallow-well plate containing 120 μL of water. The plates were briefly agitated and were then analyzed.

(89) In the case of samples analyzed by Analytical Method 8.1, the samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis.

(90) The activity of each variant relative to that of SEQ ID NO: 166 was calculated as ultraviolet (UV) absorbance at 247 nm of the RAMP-derived product formed per UV.sub.247 absorbance of the corresponding SEQ ID NO: 166 using Analytical Method 10.1. Variants with activity having a FIOP of greater than 0.75 were selected to be analyzed using Analytical Method 9.1 (Example 9), in order to identify improved enantioselectivity variants.

(91) Enantioselectivity of each variant relative to SEQ ID NO: 166 was calculated as the % ee R EGA with respect to the % ee R EGA formed of the corresponding SEQ ID NO: 166 as in Example 5.

(92) TABLE-US-00005 TABLE 7.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 166 Percent Conversion Amino Acid Differences Improvement SEQ ID NO: (Relative to SEQ (Relative to SEQ Selectivity (nt/aa) ID NO: 166) ID NO: 166)* (% ee R) † 267/268 V220E/S252V/S332Q/ +++ E407I 269/270 V220S/F296V/S332Q/ +++ E407V 271/272 V220S/F296V/A465T +++ 273/274 V220E/E407I +++ 275/276 V171C/V220S/S332Q/ +++ E407V 277/278 V220S/F296V/S332Q/ +++ E407V/T520A/T521G 279/280 V171L/R295E/F296V/ +++ S332Q/A465T 281/282 V220E/E407I +++ 283/284 F296V/S332Q/E407V/ +++ T520A 285/286 V220E/F296V/E407V +++ 287/288 S332Q/E407I +++ 289/290 S332Q/E407V +++ 291/292 E407Q/A465T +++ 293/294 V220E/R295E/F296V/ + +++ A465T/T520A 295/296 V171L/F296V/E407I +++ 297/298 V220E/R295E/F296V/ + +++ S332Q/E407V/T520A 299/300 V171C/F296V/E407V/ +++ A465T 301/302 S332Q/E407V/T521G +++ 303/304 V220E/E407V +++ 305/306 V220E/T243V/E407I +++ 307/308 V220E/T243V/R295E/ +++ F296V/S332Q/E407V/ T521G 309/310 V220E/T243V/E407V/ +++ A465T 311/312 V220S/R295E/E407I/ ++ T521G 313/314 S332Q/E407V/T520A/ ++ T521G 315/316 V220S/E407I ++ 317/318 F296V/S332Q/E407V/ ++ T521G 319/320 V171C/V220S/T243V/ ++ E407Q/A465T 321/322 V220C/A465G/G517R ++ 323/324 R295E/S332Q/E407V/ ++ T521G 325/326 E407I ++ 327/328 V220S/R295G/E407I/ ++ T520A/T521G 329/330 V220S/R295G/F296V/ ++ S332Q 331/332 L204A/T243A/A465G/ ++ G517R/T521Q 333/334 A277T/F296V/E407I/ ++ T520A/T521G 335/336 V220S/R295E/F296V/ ++ S332Q/E407Q/T521G 337/338 V220S/R295G/F296V/ ++ E407V 339/340 R295G/F296V/E407I/ ++ T521G 341/342 V220E/A465T/T520A + ++ 343/344 V220E/E407V/T520A ++ 345/346 V220S/R295G/F296V/ ++ S332Q/E407I/A465T/ T521G 347/348 E407V/T520A ++ 349/350 A173S/A465G ++ 351/352 R295E/S332Q/E407V/ ++ A465T 353/354 V171C/V220E/E407V ++ 355/356 G294E/F296S/E407M ++ 357/358 E407I/T520A ++ 359/360 A173S/N192Q/T243A/ + ++ A465G 361/362 V220E/R295G/E407V/ ++ A465T 363/364 V220M/R295E/A465T/ + ++ T520A 365/366 E407V ++ 367/368 V220S/F296V/A465T/ ++ T521G 369/370 V220S/R295E/S332Q/ ++ A465T/T520A/T521G 371/372 V220E/S332Q/E407Q/ ++ A465T/T521G 373/374 V220M/T243V/E407V/ ++ T520A/T521G 375/376 R295E/E407I ++ 377/378 V171L/V220E/A465T/ ++ T520A 379/380 T243V/R295E/E407I ++ 381/382 R295E/F296V/E407Q ++ 383/384 V171C/V220M/F296V/ ++ E407V/T520A 385/386 V220E/T243V/R295G/ ++ F296V/E407V 387/388 N192T/V220E/E407V ++ 389/390 V171C/E407V/T520A ++ 391/392 R295E/F296V/S332Q/ ++ E407I 393/394 T221I/M227N/T243A/ ++ A465G 395/396 V220C/A465G ++ 397/398 V220S/T243V/E407Q/ ++ T520A/T521G 399/400 V220S/R295E/F296V + ++ 401/402 G294S/A465G/L515V ++ 403/404 V171C/S332Q ++ 405/406 R295E/S332Q/T520A/ + ++ T521G 407/408 V220S/F296V + ++ 409/410 N192T/V220E/R295E/ ++ F296V/S332Q/T521G 411/412 A465G/L515V + ++ 413/414 V171C/E407V ++ 415/416 V220M/R295E/S332Q ++ 417/418 V284I/R295E/F296V + ++ 419/420 V171C/T243V/F296V/ ++ S332Q 421/422 N192T/V220M/F296V/ + ++ S332Q/T520A 423/424 A465G/G517H/T521Q ++ 425/426 S198R/R295S/F296S ++ 427/428 V220E/R295E/A465T/ + ++ T521G 429/430 V171C/V220E/R295E/ ++ F296V/E407Q 431/432 A465G/L515V/G517R/ ++ T521Q 433/434 R295E/F296V ++ 435/436 R295E/F296V/T521G + ++ 437/438 F296V/T520A/T521G + ++ 439/440 A465M ++ 441/442 G294S/A465G ++ 443/444 G294S/T521Q ++ 445/446 V171C/R295E/S332Q ++ 447/448 F296S/E407M ++ 449/450 A173S ++ 451/452 N192T/R295G/F296V/ ++ S332Q 453/454 V220E ++ 455/456 M227N ++ 457/458 N192Q/G294S/A465G/ ++ L515V 459/460 T243A/L515V/G517R ++ 461/462 F296V ++ 463/464 S198R/R295N ++ 465/466 S198A/G294E/F296S ++ 467/468 V220S/T243V + + 469/470 L204S + + 471/472 G517N + + 473/474 N192T/R295E/F296V + + 475/476 N192T/F296V + + 477/478 T243V/R295E + + 479/480 N192T/R295G/F296V/ + + T520A/T521G 481/482 N192T/V220E/R295E/ + + T520A/T521G *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 166, and defined as follows: “+” = 1.2 but less than 1.8; “++” = 1.8 but less than 2.5; and “+++” >2.5. † Selectivity (% ee R) is defined as follows: “+” = 20.0 but less than 40.0; “++” = 40.0 but less than 60.0; and “+++” = 60.0 but less than 80.0.

Example 8

Analytical Detection of R-AMP-Derived 2-Ethynylglyceraldehyde

(93) Data described in Examples 1, 5, 6, 7, 11, 14 and 16 were collected using the analytical method in Table 8.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

(94) TABLE-US-00006 TABLE 8.1 Analytical Method Instrument Thermo Vanquish UPLC with UV and/or MS Detection Column Waters Cortecs C18, 2.1 × 50 mm, 1.6 μm Mobile Phase A: water, 0.01% formic acid; B: MeCN. Gradient 2-40% B over 1.0 min; 40-80% B over 0.7 min. Flow Rate 0.8 mL/min Run Time ~2.3 min Product Elution 2-ethynylgluteraldehyde: 0.9 min Column Temperature 50° C. Injection Volume 0.5 μL Detection UV 247 nm; SIM MS 227.3 m/z

Example 9

Analytical Detection of Enantiomers of R-AMP-Derived 2-Ethynylglyceraldehyde

(95) Data described in Examples 1, 5, 6, 7, 11, 14, 15, 17 and 18 were collected using the analytical method provided in Table 9.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

(96) TABLE-US-00007 TABLE 9.1 Analytical Method Instrument Agilent 1290 - UPLC Column ChiralPak OZ-3, 4.6 × 150 mm (PN# 42524) Mobile Phase A: heptane, 0.1% diethylamine (v/v); B: ethanol. Gradient Isocratic at 27% B. Flow Rate 1.7 mL/min Run Time 4.0 min Product Elution order R-2-ethynylglyceraldehyde: ~2.6 min S-2-ethynylglyceraldehyde: ~3.1 min Column Temperature 30° C. Injection Volume 1.2 μL Detection UV 247 nm

Example 10

Spectrophotometric Analytical Detection of R-AMP-Derived 2-Ethynylglyceraldehyde

(97) Data described in Example 7 were collected using the analytical method provided in Table 10.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

(98) TABLE-US-00008 TABLE 10.1 Analytical Method Instrument Molecular Devices Spectramax M2 Analysis Plate Greiner Bio-one, “UV-Star” 96-well Plate, Microplate, COC, F-Bottom, Chimney Well. Sample Volume 200 μL Detection UV: 247 nm Temperature Room Temperature

Example 11

GOA Improvements Over SEQ ID NO: 272 for Enantioselective Production of Compound Y

(99) SEQ ID NO: 272 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(100) Each 100 μL reaction was carried out in 96-well deep-well plates with 50 μL clarified lysate, 20 g/L Compound X, 50 mM NaPi buffer, 25 μM CuSO.sub.4, 0.25 g/L HRP, 0.25 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(101) The enzyme variants in the plate wells were derivatized by taking 50 μL aliquots and adding 10 μL of 100 g/L solution in water of R-AMP and incubating with shaking in a 96-well half-deep-well plate for ˜30 minutes at RT. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 8.1.

(102) The activity of each enzyme variant relative to that of SEQ ID NO: 272 was calculated as the % conversion of the product (Compound Y+Z)) formed per percent conversion of the corresponding SEQ ID NO: 272. Percent conversion was quantified by multiplying the area of the HPLC product peak by a factor calculated using a standard dose-response curve of Compound Y on the HPLC using Analytical Method 8.1. Variants with activity having a FIOP of greater than 0.7 were selected to be analyzed using Analytical Method 9.1 in order to identify improved enantioselectivity variants.

(103) Enantioselectivity of each variant relative to SEQ ID NO: 272 was calculated as the % ee R EGA with respect to the % ee R EGA formed of the corresponding SEQ ID NO: 272 as in Example 5. The results are provided below.

(104) TABLE-US-00009 TABLE 11.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 272 Percent Conversion Improvement SEQ ID NO: Amino Acid Differences (Relative to SEQ Selectivity (nt/aa) (Relative to SEQ ID NO: 272) ID NO: 272)* (% ee R) † 483/484 S220E; R295E; T520A + +++ 485/486 S220E; R295E; T521G + +++ 487/488 N192Q; T243S ++ +++ 489/490 T465G + +++ 491/492 A16S ++ +++ 493/494 R295T ++ +++ 495/496 Q553S + +++ 497/498 V222D ++ +++ 499/500 N29Y + +++ 501/502 M279T + +++ 503/504 S426L + +++ 505/506 A16E ++ +++ 507/508 S426A + +++ 509/510 V222T + +++ 511/512 S220E + +++ 513/514 R295E ++ +++ 515/516 S24E + +++ 517/518 Y56I ++ +++ 519/520 Q148A ++ +++ 521/522 S92V + +++ 523/524 S24A ++ +++ 525/526 E196D + +++ 527/528 S304C + +++ 529/530 V296T + +++ 531/532 S24P ++ +++ 533/534 T63V ++ +++ 535/536 V222Y ++ +++ 537/538 S257D + +++ 539/540 R560W ++ +++ 541/542 R637L ++ +++ 543/544 I499F ++ +++ 545/546 S24Q + +++ 547/548 A46V + +++ 549/550 Q148R + +++ 551/552 I499V + +++ 553/554 K36P ++ +++ 555/556 R549G + +++ 557/558 S8V + +++ 559/560 S258L + +++ 561/562 V296E ++ +++ 563/564 S363E + +++ 565/566 N134H + +++ 567/568 F43Q ++ +++ 569/570 N597D + +++ 571/572 A4Q + +++ 573/574 S567M + +++ 575/576 S103I + +++ 577/578 A194E + +++ 579/580 R549Q + +++ 581/582 R295S + +++ 583/584 R295Q + +++ 585/586 F43A ++ +++ 587/588 N319S ++ +++ 589/590 S24P; F43A; Q148A; S363L + 591/592 S24P; Q148A; N319S; T465F; R637L + 593/594 S24P; K36P; S92D; V222D; R560W; R637L + 595/596 S24P; F43A; S92D; M279L; I499F + 597/598 S24P; R637L + 599/600 S24P; K36P; S363L; T465F; R637L + 601/602 K36P; S92D; Q148A; M279L; N319S; S363L; R560W; R637L + 603/604 F43A; S92D; Q148A; V222D; M279L; I499F; R560W + 605/606 S24P; K36P; F43A; Q148A; N319R; R560W; R637L + 607/608 S24P; Q148A; R637L + 609/610 S24P; F43A; Q148A; R295E; R560W + 611/612 K36P; S92D; R560W + 613/614 K36P; V222D; M279L; N319S; S363L; R560W + 615/616 S24P; K36P; Q148A; V222D; M279L; R560W; R637L + 617/618 K36P; S92D; Q148A; I499F; R637L + 619/620 K36P; F43Q; S92D; Q148A; V222D; M279L; R295D; I499F; + R560W; R637L 621/622 F43A; V222D; M279L + 623/624 S24P; K36P; S92D; M279L; N319R; S363L; R637L + 625/626 M279L; R560W; R637L + 627/628 K36P; Q148A; V222D; N319S; T465F; I499F + 629/630 S24P; S92D; Q148A; M279L; S363L + 631/632 S24P; K36P; S92D; Q148A + 633/634 K36P; S92D; N319R; S363L; R637L + 635/636 F43Q; S92D; V222Y; T465F; I499F; R637L + 637/638 S24P; S92D; M279L; S363L; T465F; I499F; R560W; R637L + 639/640 S92D; Q148A; R295E; N319S; T465F; R637L + 641/642 S92D; M279L; T465F; R637L + 643/644 S24P; F43A; Q148A; R560W + 645/646 S24P; K36P + 647/648 F43A; Q148A; M279L; R295D; R560W + 649/650 S24P; F43A; V222D + 651/652 S24P; F43Q; S92D; M279L; S363L; R560W; R637L + 653/654 A16S; S24A; F43E; Y56F; S103I; S220E; R295T; V296L; + I499V; R549W 655/656 F43E; Q148R; R549W + 657/658 A16S; F43E; Y56F; S103I; Q148R; S220E; R295S; V296E; + I499V; R549W 659/660 A16S; R295Q; V296E; S426L; I499V; R549W + 661/662 A16S; T63V; S103I; S220E; R295Q; S426L; R549W + 663/664 Q148R; R295Q; T465F + 665/666 A16S; Q148R; S220E; V296E; R549W + 667/668 S220E; V296E; S304C; S426L; R549W + 669/670 A16S; F43E; S103I; Q148R; S220E; R295Q; S426L; R549W + 671/672 A16E; Q148R; S220E; R295T; S426L; I499V; R549W + 673/674 A16S; Q148R; S220E; R295Q; V296E; S304C; I499V + 675/676 A16S; Y56F; V296E + 677/678 A16S; Y56F; T63V; Q148R; R295T; V296E; S304C; S426L + 679/680 A16E; S220E; I499V + 681/682 A16S; F43E; Q148R; S220E; R295S; I499V + 683/684 A16E; S103I; S220E; T465F + 685/686 A16S; F43E; R295T; V296E; R549W + 687/688 A16E; R295Q; S426L; T465F; I499V; R549W + 689/690 F43E; Y56F; S220E; V296E; S426L; I499V; R549W + 691/692 A16S; F43E; T63V; S103I; R295Q; S304C; I499V; R549W + 693/694 A16E; S103I; S220E; V296E; T465F; R549W + 695/696 F43E; R295Q; I499V + 697/698 A16E; Y56F; S426L; I499V + 699/700 F43E; Y56F; S220E; S426L; R549W + 701/702 A16S; F43E; T63V; Q148R; R295Q; I499V + 703/704 A16E; F43E; S426L; R549W + 705/706 A16S; Q148R; R295Q; S426L; R549W + 707/708 S8I; S258L; S363E; S426P; Q553S; N597D + 709/710 S8I; N29T; S92V; E196D; S258L; S426P + 711/712 S8I; A46V; S92V; M279T; V296T; S426P; R549G; Q553S; + N597D 713/714 S363E; S426P; Q481D; Q553S + 715/716 S8I; S92V; S258L; S363E; R549G; Q553S; S567M + 717/718 E196D; S258L; S363E; S426P; T465F; R549G; N597D + 719/720 N29T; A46V; S92V; E196D; S258L; M279T; S363E; S426P; + Q481D; S567M; N597D 721/722 A46V; E196D; F228W; M279T; V296T; T465F; Q553S + 723/724 S8V; N192Q; R460A; R560T; N598E + 725/726 S8V; Y56I; N192Q; A194G; R460Q; K571A; N598E + 727/728 S8V; N192Q; A194E; T243S; R460Q; Q553S; R560T + 729/730 S8V; Y56I; T243S; R560T; N598E + *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 272 and defined as follows: “+” 1.2 to 1.8, “++” 1.8 to 2.5, “+++” >2.5 † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0

Example 12

GOA Improvements Over SEQ ID NO: 272 for Enantioselective Production of Compound Y

(105) SEQ ID NO: 272 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(106) Each 100 μL reaction was carried out in 96-well deep-well plates with 50 μL clarified lysate, 20 g/L Compound X, 50 mM NaPi buffer, 25 μM CuSO.sub.4, 0.25 g/L HRP, 0.25 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(107) The enzyme variants in the plate wells were derivatized by taking 50 μL aliquots and adding 10 μL of 100 mg/mL solution in water of R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜30 minutes at RT. In the case of samples analyzed by Analytical Method 20.1 (as described in Example 20), the samples were quenched by adding 60 μL of ethanol followed by mixing, and further followed by transferring 20 μL of the diluted samples into a 96-well shallow-well plate containing 120 μL of water. The plates were briefly agitated and were then analyzed.

(108) The activity of each variant relative to that of SEQ ID NO: 272 was calculated as ultraviolet (UV) absorbance at 340 nm of the RAMP-derived product formed per UV.sub.340 absorbance of the corresponding SEQ ID NO: 272 using Analytical Method 20.1.

(109) TABLE-US-00010 TABLE 12.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 272 Percent Conversion Improvement SEQ ID NO: Amino Acid Differences (Relative to SEQ (nt/aa) (Relative to SEQ ID NO: 272) ID NO: 272)* 731/732 S24P; S92D; V222D; M279L; N319S; R637L ++ 731/732 S24P; S92D; V222D; M279L; N319S; R637L ++ 733/734 F43Q; Q148A; V222D; M279L; R560W; R637L + 735/736 N319R; R637L + 737/738 Q148A; M279L; N319S; I499F + 739/740 Q148A; V222D + 741/742 S24P; K36P; S92D; M279L; R295E; S363L; I499F + 743/744 S24P; S92D + 745/746 S24P; F43A; V222Y; N319R ++ 747/748 S24P; F43A; S92D; Q148A; M279L; R295D; N319S; R637L + 749/750 N319S; R560W + 751/752 M279L; R560W; R637L ++ 753/754 R295D; I499F; R560W; R637L ++ 755/756 S24P; F43A; S92D; V222Y; M279L + 757/758 S92D; V222D; M279L; I499F; R560W; R637L ++ 759/760 S24P; F43A; S363L; R637L ++ 761/762 K36P; S92D; V222D; R637L ++ 763/764 K36P; F43Q; V222Y; M279L; S363L; R560W + 765/766 S24P; M279L; N319R + 767/768 Q148A; V222D; R560W; R637L ++ 769/770 K36P; Q148A; M279L; N319S; I499F + 771/772 S363L; R560W + 773/774 K36P; S92D; Q148A; V222D; M279L; N319S; S363L; I499F; R560W; ++ R637L 775/776 S24P; S92D; Q148A; M279L + 777/778 S24P; V222D; R637L ++ 779/780 S92D + 781/782 Q148R; S220E; S304C + 783/784 A16E; T63V; Q148R; S220E; R295Q; V296E; S426L; I499V + 785/786 A16E; Q148R; R295Q; R549W + 787/788 A16S; F43E; Q148R; V296E; S426L; I499V ++ 789/790 T63V; S103I; Q148R; S220E; R295S; R549W + 791/792 A16S; T63V; S103I; Q148R; S426L; I499V; R549W + 793/794 A16E; Y56F; Q148R; R295Q; V296E; S304C; S426L; R549W + 795/796 A16S; S103I; Q148R; S220E; I499V + 797/798 F43E; S220E; R295S; R549W ++ 799/800 A16S; T63V; S103I; S426L; I499V; R549W + 801/802 T63V; S220E; R295T; S304C; S426L; I499V; R549W ++ 803/804 A16S; T63V; Q148R; I499V; R549W + 805/806 A16E; F43E; S103I; S304C; I499V; R549W + 807/808 R295S; V296E; R549W + 809/810 S103I; R295T; I499V; R549W + 811/812 A16S; Y56F; R295T; I499V + 813/814 A16E; Q148R; S426L; R549W ++ 815/816 A16S; Y56F; S103I; S220E; R295S ++ 817/818 A16S; S103I; Q148R; R295S; S426L; I499V; R549W + 819/820 A16S; S103I; Q148R + 821/822 A16E; S103I; S220E; R295Q ++ 823/824 T63V; S220E; R295T; S304C; R549W ++ 825/826 A16S; F43E; Q148R; R295Q; V296E; S304C; I499V; R549W ++ 827/828 A16E; Y56F; S103I; R295T; R549W + 829/830 A16S; S103I + 831/832 A16E; Q148R; R295Q; S426L + 833/834 A16E; S304C; I499V; R549W + 835/836 A16S; S304C; S426L; I499V + 837/838 Y56F; Q148R; S220E; R295Q; I499V + 839/840 A16E; Y56F; S103I; S220E; R295T; V296E + 841/842 A16E; F43E; S103I; Q148R; R295Q; S426L; I499V; R549W + 843/844 S220E; R295T; R549W + 845/846 A16E; Q148R; S220E; R295S; S426L; I499V + 847/848 A16S; F43E; T63V; S103I; R295Q; V296E; I499V + 849/850 A16S; Y56F; S220E; I499V; R549W ++ 851/852 A16E; S220E; R295Q; V296E + 853/854 R295Q; V296E; S426L; R549W + 855/856 A16E; F43E; R295T; V296E; I499V + 857/858 A16S; Y56F; S220E; R295S ++ 859/860 A16E; Y56F; R295Q; V296E; R549W + 861/862 A16S; Y56F; R295Q + 863/864 A16S; Y56F; I499V; R549W + 865/866 N29Y; S426P; R549G + 867/868 S8I; N29Y; A46V; E196D; S363E; Q481D; R549G; Q553S ++ 869/870 S8I; S92V; E196D; Q481D; N597D ++ 871/872 S92V; S258L; S363E; S426P; Q481D; R549G; N597D + 873/874 V296T; S363E; S426P; Q481D; R549G + 875/876 N29T; R549G; Q553S + 877/878 S8I; S258L; S363E; S426P; R549G + 879/880 N29T; A46V; Q481D; R549G; Q553S; N597D + 881/882 M279T; V296T; Q481D; R549G; Q553S; S567M; N597D + 883/884 S8I; S92V; E196D; S258L; S426P; N597D + 885/886 N29T; A46V; S92V; E196D; S426P; Q481D; R549G; N597D + 887/888 S8I; E196D; S258L; M279T; Q481D; R549G; Q553S + 889/890 S8I; M279T; S363E; S426P; Q481D; R549G; Q553S ++ 891/892 A194E; T243S; L329A; R560T; K571A; N598E + 893/894 S8V; Y56I; N192Q; T243S; N598E + 895/896 S8V; Y56I; N192Q; A194E; T243S; R460A; R560T; N598E + 897/898 A194G; T243S; L329A; R460Q + 899/900 S8V; S257D; R460Q; R560T; N598E + 901/902 S8V; Y56I; N192Q; A194E; T243S; L329A; R460A; R560T ++ 903/904 S8V; Y56I + 905/906 A4Q; Y56I; N192Q; A194G; S257N; K571A; N598E + 907/908 S8V; Y56I; N192Q; T243S; R460Q ++ 909/910 Y56I; T243S ++ 911/912 S8V; Y56I; N192Q; A194G; T243S; L329A + 913/914 S8V; N192Q; A194G; T243S; L329A; R460Q; R560T + 915/916 Y56I; A194G; T243S; S257N; L329A; R460A + 917/918 S8V; T243S; R460Q; R560T; K571A; N598L ++ 919/920 A4Q; Y56I; A194E; L329A; N598E + 921/922 A4Q; A194G; T243S; R549G; N598E + 923/924 A4Q; S8V; Y56I; N598L ++ 925/926 S8V; Y56I; A194G; S257D; R460A; R549G; R560T; N598L ++ *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 272 and defined as follows: “+” 1.2 to 1.8, “++” 1.8 to 2.5,“+++” >2.5

Example 13

GOA Improvements Over SEQ ID NO: 928 for Enantioselective Production of Compound Y

(110) In this round of directed evolution the strep tag was removed from the C-terminus of SEQ ID NO: 908 and a His-tag added. The resulting sequence SEQ ID NO: 928 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(111) Each 100 μL reaction was carried out in 96-well deep-well plates with 10 μL clarified lysate, 30 g/L Compound X, 50 mM MES buffer, 200 μM CuSO.sub.4, 0.20 g/L HRP, 0.20 g/L catalase, at pH 7.4. The plates were sealed with 02-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(112) The enzyme variants in the plate wells were derivatized by taking 50 μL aliquots and adding 10 μL of 100 mg/mL solution in water of R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜30 minutes at RT. In the case of samples analyzed by Analytical Method 20.1 (as described in Example 20), the samples were quenched by adding 60 μL of ethanol followed by mixing, and further followed by transferring 20 μL of the diluted samples into a 96-well shallow-well plate containing 120 μL of water. The samples were further diluted to make a 100-fold final dilution in the analysis plate. The plates were briefly agitated and were then analyzed.

(113) The activity of each variant relative to that of SEQ ID NO: 928 was calculated as ultraviolet (UV) absorbance at 340 nm of the RAMP-derived product formed per UV.sub.340 absorbance of the corresponding SEQ ID NO: 928 using Analytical Method 20.1.

(114) TABLE-US-00011 TABLE 13.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 928 Percent Conversion Improvement SEQ ID NO: Amino Acid Differences (Relative to SEQ (nt/aa) (Relative to SEQ ID NO: 928) ID NO: 928)* 927/928 / / 929/930 A4Q; F43Q; A46V; S426L; R549G; R560W + 931/932 F43Q; A46V; T63V; S258L; R295Q; S426L; R560W; S567M; K571A + 933/934 F43Q; A46V; E196D; N319S; R549G; R560W; S567M + 935/936 F43Q; A46V; I56F; Q148A; S258L; M279L; S363L; R549G; K571A + 937/938 F43Q; M279L; R549G; R560W; S567M + 939/940 R295Q; R549G; R560W + 941/942 A4Q; F43Q; A46V; I56F; T63V; M279L; R295T; N319S; S567M; N598E + 943/944 A4Q; F43Q; Q148A; E196D; M279L; N319S; S363L; R560W + 945/946 A46V; R560W + 947/948 R549G; N598E + 949/950 M279L; N319S; R560W + 951/952 A4Q; F43Q; A46V; R295Q; N319S; R549G; R560W + 953/954 A46V; R295T; N319S; S426L + 955/956 A16S; S92D; Q192N; M279T; R637L + 957/958 A16E; K36P; Q192N; R549W; N597D; N598L; R637L + 959/960 A16E; F43E; I56V; Q192N; R549W; N597D; N598L; R637L + 961/962 A16S; M279T; R549W; N597D; N598L; R637L + 963/964 A16E; M279T; N319R; R549W; N597D; R637L + 965/966 A16E; N29T; S92D; S220E; V222D; N319R; R549W; N598L + 967/968 A16E; F43E; N597D ++ 969/970 A16E; N597D + 971/972 A16E; Q192N; R549W + 973/974 A16E; Q192N; N319R; R549W; R637L + 975/976 A16E + 977/978 A16E; K36P; F43A; Q192N; N597D; R637L ++ 979/980 K36P; S92D; R549W + 981/982 A16E; N29Y; S92D; N319R; R549W + 983/984 A16E; S220E; V222D; M279T; R549W; N598L; R637L + 985/986 A16E; F43E; Q192N; R549W ++ 987/988 A16E; N29Y; F43E; Q192N; V222D; N319R; R549W; R637L + 989/990 A16E; F43E; M279T; N319R; N597D ++ 991/992 A16E; N29T; S220E; V222D; M279T; R549W; R637L + 993/994 A16E; S24P; F43E; S92D; R549W ++ 995/996 A16E; N29Y; S220E; V222D; N597D; N598L + 997/998 A16E; N29Y; Q192N; R549W ++  999/1000 A16E; N29T; Q192N; S220E; R549W; N597D + 1001/1002 A16E; S24P; N29Y + 1003/1004 A16E; F43A; M279T; R549W; N597D ++ 1005/1006 A16E; N29Y; Q192N; V222D; M279T; R549W + 1007/1008 A16S; S92D; N319R; N597D; R637L + 1009/1010 A16E; F43A; S220E; R549W; R637L ++ 1011/1012 A16E; S220E; M279T; R549W + 1013/1014 S220E; V222D; N597D; R637L + 1015/1016 A16E; N319R; N597D + 1017/1018 A16E; N29T; Q192N; R549W + 1019/1020 A16E; M279T; N597D + 1021/1022 A16E; S92D; S220E; R549W + 1023/1024 A16E; S92D; Q192N; M279T; N319R; R549W; R637L + 1025/1026 A16S; N29Y; S92D; Q192N; R549W; R637L + 1027/1028 A16E; S24P; F43E; Q192N; S220E; M279T; R549W ++ 1029/1030 A16E; N29T; F43A; S92D; Q192N; N319R + 1031/1032 A16E; N29T; K36P; Q192N; N319R; R549W; N597D; N598L; R637L + 1033/1034 A16E; N29T; K36P; M279T; R549W + 1035/1036 A16E; N29Y; R549W; R637L + 1037/1038 A16E; N29T; S92D; M279T; R549W + 1039/1040 A16E; S24P; N29T; M279T; R549W + 1041/1042 A16E; R549W + 1043/1044 A16E; S220E; N319R; R549W; N597D; N598L + 1045/1046 A16E; N319R; N597D; N598L + 1047/1048 A16E; K36P; N319R; R549W; N597D; N598L; R637L + 1049/1050 A16E; S24P; N29T; S92D; S220E; M279T; N597D; N598L + 1051/1052 A16E; N29T; F43E; V222D; M279T; R549W ++ 1053/1054 A16E; R549W; N598L + 1055/1056 A16E; V222D; N319R; N597D; N598L + 1057/1058 A16E; V222D; R637L + 1059/1060 A16S; N29T; S92D; R549W; R637L + 1061/1062 A16E; F43A; S92D; V222D; N597D; N598L + 1063/1064 A16E; K36P; S92D; S220E; V222D; M279T; R549W + 1065/1066 A16E; N29T; V222D; R549W; N598L; R637L + 1067/1068 A16E; N29T; Q192N; S220E; R549W + 1069/1070 A16E; S24P; N29T; M279T; R637L + 1071/1072 A16E; N29T; Q192N; V222D; R637L + 1073/1074 A16S; F43E; N319R; R549W; N598L ++ 1075/1076 S220E; V296S ; E407I; T465G + 1077/1078 G294E + 1079/1080 T520A; N597D; N598E + 1081/1082 N29H; N597D; N598E + 1083/1084 N237D; T520A; N538D; N597D + 1085/1086 N29H; T63V; T520A; S537G; N538D; N598E + 1087/1088 N237D; T520A; S567M; K571A; N597D ++ 1089/1090 N237D; T520A; N597D; N598E ++ 1091/1092 N237D; T520A; S537G; N598E + 1093/1094 S567M; K571A; N597D + 1095/1096 N237D; N538D; N597D; N598E + 1097/1098 N29H; N237D; S567M; N598E + 1099/1100 N29H; N237D; T520A; N538D + 1101/1102 N134A; T520A; N597D; N598E + 1103/1104 N29H; N237D; N597D + 1105/1106 K36V; T520A; S537G; N538D; N597D + 1107/1108 K36V; N134A; N237D; S567M; K571A; N597D; N598E + 1109/1110 N29H; N134A; N237D; S537G; N538D; S567M; K571A + 1111/1112 N29H; N237D; T520A + 1113/1114 K36V; N134A; N237D; T520A; S537G; N538D; K571A + 1115/1116 N237D; K571A + 1117/1118 N237D; T520A + 1119/1120 N237D; N597D; N598E + 1121/1122 N134A; N237D; T520A; N597D + 1123/1124 T520A; K571A; N598E + 1125/1126 N29H; T63V; N134A; T520A; N597D; N598E + 1127/1128 N237D; T520A; S537G; N538D; N598E + 1129/1130 T483R ++ 1131/1132 S568E + 1133/1134 K486P + 1135/1136 K556A + 1137/1138 K556V + 1139/1140 S568P + 1141/1142 S564W + 1143/1144 T95E + 1145/1146 T465G + 1147/1148 S564D + 1149/1150 S564E + 1151/1152 S609D + 1153/1154 S433G ++ 1155/1156 K224D + 1157/1158 S564T + 1159/1160 K343G + 1161/1162 K556S + *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 928 and defined as follows: “+” 1.2 to 1.8, “++” 1.8 to 2.5, “+++” >2.5

Example 14

GOA Improvements Over SEQ ID NO: 928 for Enantioselective Production of Compound Y

(115) SEQ ID NO: 928 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(116) Each 100 μL reaction was carried out in 96-well deep-well plates with 15 μL clarified lysate 21 g/L Compound X, 9 g/L Compound Y, 50 mM MES buffer, 200 μM CuSO.sub.4, 0.20 g/L HRP, 0.20 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(117) The enzyme variants in the plate wells were diluted 3-fold with acetonitrile by adding 20 uL of the reaction to a 96-well shallow-well plate containing 40 uL of acetonitrile. 20 uL of this 3-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 170 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 8.1 (Table 14.1) or 9.1 (Table 14.2).

(118) TABLE-US-00012 TABLE 14.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 928 Percent Conversion Improvement SEQ ID NO: Amino Acid Differences (Relative to SEQ (nt/aa) (Relative to SEQ ID NO: 928) ID NO: 928)* 1163/1164 A4Q; F43Q; A46V; S426L; R549G; +++ R560W 1165/1166 T520A; N597D + 1167/1168 K36V; T63V; T520A + 1169/1170 N598E + 1171/1172 K556 ++ 1173/1174 T562D + 1175/1176 T483R ++ 1177/1178 S568D + 1179/1180 T95V + 1181/1182 S568P + 1183/1184 K394A + *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 928 and defined as follows: “+” 1.2 to 1.8, “++” 1.8 to 2.5, “+++” >2.5

(119) TABLE-US-00013 TABLE 14.2 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 928 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 928) (% ee R) † 1185/1186 G294E; E407M; T465G +++ 1187/1188 S220M; G294E; T465G +++ 1189/1190 S220E; G294E; V296S; S332Q +++ 1191/1192 E407V; T465G +++ 1193/1194 V638A +++ 1195/1196 T63A; E196L +++ 1197/1198 G197R +++ 1199/1200 A194R +++ 1201/1202 E196A +++ 1203/1204 S198T; V447I +++ 1205/1206 G197Q +++ 1207/1208 G197P +++ 1209/1210 E196R +++ 1211/1212 E196L +++ 1213/1214 A194W +++ 1215/1216 S290G +++ 1217/1218 G197A +++ 1219/1220 Q327R +++ 1221/1222 S198G +++ 1223/1224 A173V +++ 1225/1226 E196G +++ 1227/1228 S189A +++ 1229/1230 S290A +++ 1231/1232 S292G +++ 1233/1234 E196Q +++ 1235/1236 E196V +++ 1237/1238 E196I +++ 1239/1240 A194V +++ 1241/1242 S198T +++ 1243/1244 A173S +++ 1245/1246 G197E +++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0

Example 15

GOA Improvements Over SEQ ID NO: 932 for Enantioselective Production of Compound Y

(120) SEQ ID NO: 932 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(121) Each 100 μL reaction was carried out in 96-well deep-well plates with 7.5 μL clarified lysate from total lysate volume of 200 uL, 30 g/L Compound X, 50 mM MES buffer, 200 μM CuSO.sub.4, 5 g/L HRP, 0.2 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH

(122) The enzyme variants in the plate wells were diluted 3-fold with acetonitrile by adding 20 uL of the reaction to a 96-well shallow-well plate containing 40 uL of acetonitrile. 20 uL of this 3-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 1704 MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 9.1.

(123) Enantioselectivity of each variant relative to SEQ ID NO: 932 was calculated as the % ee R EGA with respect to the % ee R EGA formed of the corresponding SEQ ID NO: 932 as in Example 5. The results are provided below.

(124) TABLE-US-00014 TABLE 15.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 932 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 932) (% ee R) † 1247/1248 A16E; Q43A; S220M; L258S; N538D; +++ R637L 1249/1250 A16E; L258S; L426S; T465G; N538D; +++ R549W; R637L 1251/1252 Q43A; L258S; E407I; L426S; T465G; ++++ N538D; R549W; R637L 1253/1254 S220M; G294E; Q295S; N319S; E407I; ++++ L426S; T465G; N538D; R549W; R637L 1255/1256 L258S; N538D; R549W; R637L ++++ 1257/1258 L258S; M267T ++++ 1259/1260 L258S; N319S; L426S; T465G; R549W; +++ R637L 1261/1262 V63T; T95E; A173S; K343G; S564D; +++ S568P; S609D 1263/1264 T95V; A173S; L258S; L426S; K556V; ++++ S564W 1265/1266 L258S; L426S +++ 1267/1268 A173S; K556V ++++ 1269/1270 T95E; A173S; K556V; S609D ++++ 1271/1272 V63T +++ 1273/1274 T18K ++++ 1275/1276 S24P; V222D; T520A +++ 1277/1278 V222D; T520A; N597D +++ 1279/1280 Q43E; V222D; N237D; L258S; L426S; +++ N597D 1281/1282 V222D; N237D +++ 1283/1284 N237D; L258S; R549G; N597D ++++ 1285/1286 N237D; P265S; M279L ++++ 1287/1288 S24P; V222D; N237D; T520A; N538D +++ 1289/1290 A194R ++++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0, “++++” 80.0 to 90.0

Example 16

GOA Improvements Over SEQ ID NO: 1264 for Enantioselective Production of Compound Y

(125) SEQ ID NO: 1264 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4, except a 300 ul per well lysis volume was used.

(126) Each 100 μL reaction was carried out in 96-well deep-well plates with 50 μL clarified lysate from a 300 uL lysis total volume, 18 g/L Compound X, 12 g/L Compound Y, ˜80 mM NaPi buffer, 200 μM CuSO.sub.4, 0.20 g/L HRP, 0.20 g/L catalase, at pH 7.4. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(127) The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35 uL of the reaction to a 96-well shallow-well plate containing 35 uL of acetonitrile. 20 uL of this 2-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 8.1.

(128) TABLE-US-00015 TABLE 16.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 1264 Percent Conversion Improvement SEQ ID NO: Amino Acid Differences (Relative to SEQ (nt/aa) (Relative to SEQ ID NO: 1264) ID NO: 1264)* 1291/1292 Q43E; N237D; T520A; N597D + 1293/1294 Q43E + 1295/1296 Q43E; M279L; G294E + 1297/1298 R549G + 1299/1300 Q43E; N237D; M279L; N538D; + N597D; N598E 1301/1302 Q43E; N237D; G294E; N538D + 1303/1304 Q43E; R549G; N597D + 1305/1306 Q43E; N237D; T520A; R549G; + N598E 1307/1308 Q43E; N538D + 1309/1310 Q43E; N237D; T520A + 1311/1312 N237D; T520A; N538D; N597D + 1313/1314 K51P; T55W; T111Q; S150P; + K367I; W564D 1315/1316 K367I; K371D; W564D; T594Q + 1317/1318 T18K; V95E; Q327R; T548M + 1319/1320 V95E; Q327R; T548M + 1321/1322 S258H + 1323/1324 C229S + 1325/1326 K371A + 1327/1328 S243K + 1329/1330 K342R ++ 1331/1332 T635K + 1333/1334 S468N + 1335/1336 S604M + 1337/1338 R549E + 1339/1340 S568A + 1341/1342 K371P + 1343/1344 K342S + 1345/1346 F291V + 1347/1348 K36N + 1349/1350 S312T + 1351/1352 R183D + 1353/1354 K224G + 1355/1356 T520N + 1357/1358 C384G + 1359/1360 K61E + 1361/1362 T55M + 1363/1364 S570K + 1365/1366 T520E + 1367/1368 W564K; S604G + 1369/1370 R637N + 1371/1372 Q295T + 1373/1374 S99H + 1375/1376 M567G + 1377/1378 C28S + 1379/1380 R637W + 1381/1382 Q43F; V46A; I56Y; V63T; + R191V 1383/1384 R544P + 1385/1386 K343S + 1387/1388 T55R + 1389/1390 C28P + 1391/1392 S243L + 1393/1394 Y485L + 1395/1396 H335R + 1397/1398 T594C + 1399/1400 S252G + 1401/1402 S198R + 1403/1404 T596G + *Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 1264 and defined as follows: “+” 1.2 to 1.8, “++” 1.8 to 2.5, “+++” >2.5

Example 17

GOA Improvements Over SEQ ID NO: 1264 for Enantioselective Production of Compound Y

(129) SEQ ID NO: 1264 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4, except a 200 uL per well lysis volume was used.

(130) Each 100 μL reaction was carried out in 96-well deep-well plates with 10 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜100 mM NaPi buffer, 200 μM CuSO.sub.4, 5 g/L HRP, 0.20 g/L catalase, at pH 7.4. The plates were sealed with 02-permeable seals and incubated at 30° C. and agitated at 300 rpm in a 50 mm throw Kuhner shaker overnight maintained at 85% RH.

(131) The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35 uL of the reaction to a 96-well shallow-well plate containing 35 uL of acetonitrile. 20 uL of this 2-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 9.1.

(132) TABLE-US-00016 TABLE 17.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 1264 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 1264) (% ee R) † 1405/1406 C28P ++++ 1407/1408 S99H; T520E; R637N ++++ 1409/1410 C28P; S99H; T520E; R637N ++++ 1411/1412 S403P; T520E; R637N ++++ 1413/1414 S99H; R637N ++++ 1415/1416 K224G; T520E; R637N ++++ 1417/1418 K61E; K224G; K343S; T520E; R637N ++++ 1419/1420 S99H; K343S; R637N ++++ 1421/1422 T520E; R637N ++++ 1423/1424 K343S; T520E; R637N ++++ 1425/1426 R637W ++++ 1427/1428 Q295T; K342S; S568A ++++ 1429/1430 T594C ++++ 1431/1432 T55R; S568A ++++ 1433/1434 K342S; S568A ++++ 1435/1436 K342S; T594C ++++ 1437/1438 T55R; S568A; R637W ++++ 1439/1440 T55R; S568A; T594C ++++ 1441/1442 S568A; R637W ++++ 1443/1444 T55R; K342S ++++ 1445/1446 Q295T; K342S ++++ 1447/1448 T55R; Q295T +++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0, “++++” 80.0 to 99.0

Example 18

GOA Improvements Over SEQ ID NO: 1416 for Enantioselective Production of Compound Y

(133) SEQ ID NO: 1416 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4, except a 200 uL per well lysis volume was used.

(134) Each 100 μL reaction was carried out in 96-well deep-well plates with 40 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were heat sealed and incubated at 30° C. and agitated at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(135) The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35 uL of the reaction to a 96-well shallow-well plate containing 35 uL of acetonitrile. 20 uL of this 2-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of R-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 9.1.

(136) TABLE-US-00017 TABLE 18.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 1416 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 1416) (% ee R) † 1449/1450 Q274G ++++ 1451/1452 A439G ++++ 1453/1454 Y437 ++++ 1455/1456 Y437C ++++ 1457/1458 Q156V ++++ 1459/1460 T429V ++++ 1461/1462 Y437V ++++ 1463/1464 I262V ++++ 1465/1466 A336P ++++ 1467/1468 N375L ++++ 1469/1470 F605L ++++ 1471/1472 N35D ++++ 1473/1474 L253V ++++ 1475/1476 V393T ++++ 1477/1478 I561S ++++ 1479/1480 N488L ++++ 1481/1482 A354D ++++ 1483/1484 G200 ++++ 1485/1486 S105R ++++ 1487/1488 V478L ++++ 1489/1490 A627R ++++ 1491/1492 G45V ++++ 1493/1494 S568K ++++ 1495/1496 V393G ++++ 1497/1498 P380H ++++ 1499/1500 A154H ++++ 1501/1502 Q156L ++++ 1503/1504 N13H ++++ 1505/1506 Q239M ++++ 1507/1508 L595W ++++ 1509/1510 N315G ++++ 1511/1512 Y359F +++ 1513/1514 G328K ++++ 1515/1516 Q274N ++++ 1517/1518 V393D ++++ 1519/1520 V366T +++ 1521/1522 Q373T +++ 1523/1524 Y437R +++ 1525/1526 P380R +++ 1527/1528 V241I ++++ 1529/1530 V393P +++ 1531/1532 P380L ++++ 1533/1534 D37M ++++ 1535/1536 D37Y ++++ 1537/1538 A354T ++++ 1539/1540 V478M ++++ 1541/1542 P263S ++++ 1543/1544 G559S ++++ 1545/1546 D37I ++++ 1547/1548 Y89R ++++ 1549/1550 F438S ++++ 1551/1552 I561T ++++ 1553/1554 L541R ++++ 1555/1556 G328R ++++ 1557/1558 Y437G ++++ 1559/1560 N488T ++++ 1561/1562 T550S ++++ 1563/1564 N13K ++++ 1565/1566 G328L ++++ 1567/1568 D217P ++++ 1569/1570 T441I ++++ 1571/1572 P380K ++++ 1573/1574 N13A; Q156V; I262V; N315G; T429V; ++++ Y437V 1575/1576 Q274G; Y359F ++++ 1577/1578 I262V; Q274G; Y437R ++++ 1579/1580 Q274G; Y437V; S568K ++++ 1581/1582 N13A; Q274G ++++ 1583/1584 N26M; I262V; Q274G; N315G; Y437R ++++ 1585/1586 Q156V; Q274G; N315G ++++ 1587/1588 N13A; N26M; Q156V; Q274G; Y359F; ++++ T429V 1589/1590 I262V; Q274G; N315G ++++ 1591/1592 N13A; Q156L; I262V; Q274G; N315G ++++ 1593/1594 N13A; Q156L; Q274G; Y437K; S568K; ++++ Q606S 1595/1596 N13A; Q274G; N315G; Y437V ++++ 1597/1598 Q274N; Y437L ++++ 1599/1600 Q274N; Q373T; Y437L ++++ 1601/1602 N13K; Q373T ++++ 1603/1604 N13K; I262V ++++ 1605/1606 N13K; Y437L; L541R ++++ 1607/1608 N13K; I262V; Q274N; L595W ++++ 1609/1610 I262V; P380H ++++ 1611/1612 I262V; Q274N ++++ 1613/1614 N13K; G328R; Y437C +++ 1615/1616 I262V; L541R ++++ 1617/1618 I262V; Q373T; L595W +++ 1619/1620 N13K; I262V; Y437L; N488L +++ 1621/1622 N13K; Y437L +++ 1623/1624 Q373T; L595W ++++ 1625/1626 P380H; Y437C ++++ 1627/1628 G641D +++ 1629/1630 N13K; Q274N; Q373T; Y437C ++++ 1631/1632 G45V; I262V; Q274N; Q373T; Y437C ++++ 1633/1634 N13K; I262V; Q274N; P380H ++++ 1635/1636 Q274N; V393P; Y437L ++++ 1637/1638 Q274N; P380H ++++ 1639/1640 I262V; Y437C +++ 1641/1642 Q274N; Y437L; L541R ++++ 1643/1644 G224W ++++ 1645/1646 P263S; Q274N ++++ 1647/1648 P263S; Q274N; P380L ++++ 1649/1650 P263S; P380K ++++ 1651/1652 Q274N; G328L ++++ 1653/1654 A354T; P380K ++++ 1655/1656 D217P; Q274N; V478M ++++ 1657/1658 V478M; I561T ++++ 1659/1660 D217P; P380L ++++ 1661/1662 D37M; Y89R; Q274N ++++ 1663/1664 Y89R; Q274N; P380R ++++ 1665/1666 D37Y; P263S; Q274N; P380K; G559S; ++++ I561T 1667/1668 D37V; P380K +++ 1669/1670 Q274N; A354T ++++ 1671/1672 D217P; Q274N; P380K; I561T ++++ 1673/1674 D217P; A354T; P380K ++++ 1675/1676 P380K; G559S; I561T +++ 1677/1678 P263S; P380K; T441I ++++ 1679/1680 Q274N; P380R; T441I ++++ 1681/1682 D37M; Y89R ++++ 1683/1684 Q274N; P380K ++++ 1685/1686 D217P; P380R ++++ 1687/1688 P263S; A354T; P380K; G559S ++++ 1689/1690 Q274N; P380R; G559S +++ 1691/1692 Y89R; P263S; G559S +++ 1693/1694 Y89R; P263S; Q274N; P380R ++++ 1695/1696 Y89R; P263S; Q274N; P380K ++++ 1697/1698 P263S; Q274N; P380R ++++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0, “++++” 80.0 to 99.0

Example 19

GOA Improvements Over SEQ ID NO: 1598 for Enantioselective Production of Compound Y

(137) SEQ ID NO: 1598 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(138) Each 100 μL reaction was carried out in 96-well deep-well plates with 40 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(139) The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35 uL of the reaction to a 96-well shallow-well plate containing 35 uL of acetonitrile. 20 uL of this 2-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of S-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 21.1.

(140) TABLE-US-00018 TABLE 19.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 1598 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 1598) (% ee R) † 1699/1700 N13K; P470L; G559S; I563A +++ 1701/1702 I262V; P263S; G559S; I563A ++++ 1703/1704 Q43F; V46A; I56Y; V63T; S99H; ++++ Q156L; I262V; P263S; S403P; G559S; I563A 1705/1706 G559S; G641D ++++ 1707/1708 S99H; L437V ++++ 1709/1710 S99H; Q156L; I262V; P263S; P380K; ++++ L437C; G559S 1711/1712 I262V; P263S ++++ 1713/1714 I262V; L437V; G641D ++++ 1715/1716 N13K; P380K; L437V; G559S ++++ 1717/1718 S99H; P380K; L437C; G559S; G641D ++++ 1719/1720 N13K; Q156L; P380K ++++ 1721/1722 N13K; Q156L; P263S; L437C ++++ 1723/1724 S99H; Q156L; P380K ++++ 1725/1726 N13K; I262V; P263S ++++ 1727/1728 N13K; S99H; P263S; P380K; L437V; ++++ G641D 1729/1730 S99H; Q156L; P380K; L437V ++++ 1731/1732 P263S; L437V; G559S; I563A ++++ 1733/1734 N13K; S99H; Q156L; I262V; L437C; ++++ G559S; G641D 1735/1736 S99H; P380K; I563A ++++ 1737/1738 N13K; Q156L; I262V; P263S; L437C; ++++ G559S 1739/1740 S99H; I262V; L437V; G559S ++++ 1741/1742 N13K; L437V ++++ 1743/1744 N13K; S99H; P263S; P380K; L437V ++++ 1745/1746 S99H; I262V; P263S; L437V; G559S; ++++ G641D 1747/1748 N13K; S99H; P380K; L437R; G559S ++++ 1749/1750 N13K; S99H; S257R; I262V; P263S; ++++ G559S 1751/1752 N13K; S99H; I262V; P263S; P380K; ++++ L437R; I563A; G641D 1753/1754 S99H; Q156L; I262V; P263S; L437C ++++ 1755/1756 S99H; Q156L; P263S; G559S ++++ 1757/1758 N13K; S99H; I563A; G641D ++++ 1759/1760 N13K; P380K; L437C ++++ 1761/1762 N13K; I262V; P263S; P380K; L437V; ++++ G559S 1763/1764 S99H; P263S; L437R; I563A ++++ 1765/1766 N13K; L437R ++++ 1767/1768 S99H ++++ 1769/1770 S99H; Q156L; I262V ++++ 1771/1772 S99H; Q156L; I262V; P263S; G559S ++++ 1773/1774 N13K; S99H; Q156L; P380K; L437C ++++ 1775/1776 N13K; S99H; P263S ++++ 1777/1778 N13K; S99H; L437V; I563A ++++ 1779/1780 N13K; I262V ++++ 1781/1782 P263S; P380K ++++ 1783/1784 N13K; S99H; P263S; P380K ++++ 1785/1786 S99H; Q156L; I262V; P263S; P380K; ++++ L437V; G559S 1787/1788 S99H; Q156L; L437V ++++ 1789/1790 I262V ++++ 1791/1792 P380K ++++ 1793/1794 K30E ++++ 1795/1796 N13K; S99H; Q156L; P380K; L437V; ++++ G559S; I563A; G641D 1797/1798 I262V; P263S; L437C; G559S ++++ 1799/1800 T565S ++++ 1801/1802 L615I ++++ 1803/1804 Y254L ++++ 1805/1806 A175G ++++ 1807/1808 I287L ++++ 1809/1810 I177L ++++ 1811/1812 S409R ++++ 1813/1814 P592K ++++ 1815/1816 S409H ++++ 1817/1818 W208F ++++ 1819/1820 N601G ++++ 1821/1822 A149R ++++ 1823/1824 S280M ++++ 1825/1826 N356S ++++ 1827/1828 D610V ++++ 1829/1830 T62D ++++ 1831/1832 N29V ++++ 1833/1834 W208L ++++ 1835/1836 T251V ++++ 1837/1838 T278L ++++ 1839/1840 P489I ++++ 1841/1842 I569L ++++ 1843/1844 A149N ++++ 1845/1846 V184L ++++ 1847/1848 M234L ++++ 1849/1850 T62G ++++ 1851/1852 T62Q ++++ 1853/1854 T596S ++++ 1855/1856 P489L ++++ 1857/1858 P592G ++++ 1859/1860 S280N ++++ 1861/1862 N601L ++++ 1863/1864 Q373K ++++ 1865/1866 E407Q ++++ 1867/1868 Q373D ++++ 1869/1870 E466V ++++ 1871/1872 G197A ++++ 1873/1874 A108F ++++ 1875/1876 A194Q ++++ 1877/1878 G197P ++++ 1879/1880 I463V ++++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0, “++++” 80.0 to 99.0

Example 20

Spectrophotometric Analytical Detection of R-AMP-Derived 2-Ethynylglyceraldehyde

(141) Data described in Example 12 and 13 were collected using the analytical method provided in Table 20.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

(142) TABLE-US-00019 TABLE 20.1 Analytical Method Instrument Molecular Devices Spectramax M2 Analysis Greiner Bio-one, “UV-Star” 96-well Plate, Microplate, Plate COC, F-Bottom, Chimney Well. Sample Volume 200 μL Detection UV: 340 nm Temperature Room Temperature

Example 21

Analytical Detection of Enantiomers of R-AMP-Derived 2-Ethynylglyceraldehyde

(143) Data described in Example 19, 22, 28 and 30 were collected using the analytical method provided in Table 21.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

(144) TABLE-US-00020 TABLE 21.1 Analytical Method Instrument Agilent 1100 - HPLC Column ChiralPak IA column, 2.1 × 150 mm, 5 uM Mobile Phase A: heptane, 0.1% diethylamine (v/v); B: ethanol, 0.1% diethylamine (v/v) Gradient Isocratic at 7% B. Flow Rate 1.0 mL/min Run Time 3.0 min Product Elution order R-2-ethynylglyceraldehyde: ~1.7 min S-2-ethynylglyceraldehyde: ~2.2 min Column Temperature 40° C. Injection Volume 2 μL Detection UV 260 nm

Example 22

GOA Improvements Over SEQ ID NO: 1866 for Enantioselective Production of Compound Y

(145) SEQ ID NO: 1866 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(146) Each 100 μL reaction was carried out in 96-well deep-well plates with 40 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(147) The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35 uL of the reaction to a 96-well shallow-well plate containing 35 uL of acetonitrile. 20 uL of this 2-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of S-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 2004 MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 21.1.

(148) TABLE-US-00021 TABLE 22.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 1866 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 1866) (% ee) † 1881/1882 M234L ++++ 1883/1884 Q373D; E466V ++++ 1885/1886 T62Q; E466V ++++ 1887/1888 E466V ++++ 1889/1890 I569L ++++ 1891/1892 G197A; E466V; I569L; T596S ++++ 1893/1894 T62D; E466V; N597A ++++ 1895/1896 T62D; E466V ++++ 1897/1898 C384N; I569L ++++ 1899/1900 T62Q; Q373D; E466V ++++ 1901/1902 G197A ++++ 1903/1904 I569L; N597A ++++ 1905/1906 V184L ++++ 1907/1908 T62Q; E466V; N597A ++++ 1909/1910 M234L; C384N ++++ 1911/1912 G197P; I463V; P592G ++++ 1913/1914 I463V; T565S ++++ 1915/1916 N29V; G197P; P592G ++++ 1917/1918 I177L; S280N; I463V; T594M; N601L ++++ 1919/1920 I177L; A194Q; G197P; I463V; T565S ++++ 1921/1922 I177L; G197P; I463V; T565S ++++ 1923/1924 I177L; I463V; T565S ++++ 1925/1926 I177L; I463V; P592G ++++ 1927/1928 A149N; I463V ++++ 1929/1930 N29V; I177L; I463V ++++ 1931/1932 N29V; I177L; G197P; P592G ++++ 1933/1934 N29V; I463V ++++ 1935/1936 G197P; S280N; I463V ++++ 1937/1938 K546E ++++ 1939/1940 T251V ++++ 1941/1942 A149R ++++ 1943/1944 T251V; T399V; L615I ++++ 1945/1946 T62G; T286C; L615I ++++ 1947/1948 T62G; W208F; I417L; L615I ++++ 1949/1950 I417L; L615I ++++ 1951/1952 A149R; W208F; L615I ++++ 1953/1954 W208F; T251V; D259N; T278L ++++ 1955/1956 T278L ++++ 1957/1958 T399V; L615I ++++ 1959/1960 T251V ++++ 1961/1962 N29V; A149N; I463V; N601L ++++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0, “++++” 80.0 to 99.0

Example 23

GOA Improvements Over SEQ ID NO: 1912 for Enantioselective Production of Compound Y

(149) SEQ ID NO: 1912 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(150) Each 100 μL reaction was carried out in 96-well deep-well plates with 204 clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(151) The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20 uL of the reaction to a 96-deep-well plate containing 180 uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000 rpm at 4° C. for five minutes. The supernatants were diluted 4-fold with deinonized (DI) water by adding 50 uL of the supernatant to a 96-shallow-well plates containing 150 uL of DI water. The plates were heat sealed for analysis by Analytical Method 24.1.

(152) TABLE-US-00022 TABLE 23.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 1912 SEQ ID NO: Amino Acid Differences Activity (nt/aa) (Relative to SEQ ID NO: 1912) (% conversion) † 1963/1964 P50T ++ 1965/1966 Q43G ++ 1967/1968 Q43T ++ 1969/1970 P197L ++ 1971/1972 K486I ++ 1973/1974 S3K + 1975/1976 T42F + 1977/1978 W40P + 1979/1980 P50V + 1981/1982 K30N + 1983/1984 Q43D + 1985/1986 A142H + 1987/1988 K486P + 1989/1990 T38M + 1991/1992 A142C + 1993/1994 Q156T + 1995/1996 Y44H + 1997/1998 P50I + 1999/2000 Q156L + 2001/2002 R161V + 2003/2004 P50H + 2005/2006 K486L + 2007/2008 A9L + 2009/2010 N26T + 2011/2012 K486V + 2013/2014 A142V + 2015/2016 K486R + 2017/2018 Q79P + 2019/2020 T18S + 2021/2022 A142S + 2023/2024 N601L + 2025/2026 R161Q + 2027/2028 N29T + 2029/2030 L159G + 2031/2032 Q75N + 2033/2034 G135D + 2035/2036 N29M + 2037/2038 P197D + 2039/2040 K30L + 2041/2042 N29V + 2043/2044 L159S + 2045/2046 P50D + 2047/2048 Q136A + 2049/2050 G48P + 2051/2052 A142G + 2053/2054 K486A + 2055/2056 N29A + 2057/2058 K30R + 2059/2060 Q136G + 2061/2062 Q43P + 2063/2064 N29Y + 2065/2066 N26H + 2067/2068 N26C + 2069/2070 A4K + 2071/2072 L159K + 2073/2074 G48C + 2075/2076 Q79S + 2077/2078 Q79A + † Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 1912 and defined as follows: “+” 1.2 to 1.8, “++” 1.8 to 2.5, “+++” >2.5

Example 24

Analytical Detection of Conversion of Compound X to Compound Y

(153) Data described in Example 23, 25, 26, 27 and 31 were collected using the analytical method provided in Table 24.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

(154) TABLE-US-00023 TABLE 24.1 Analytical Method Instrument Thermo Scientific Dionex Ultimate 3000 Column Benson BP-800 Ca Mobile Phase A: DI water Gradient Isocratic at 100% A. Flow Rate 1.0 mL/min Run Time 2.5 min Product Elution order 2-ethynylglyceraldehyde: ~1.9 min 2-ethynylglycerol: ~2.1 min Column Temperature 80° C. Injection Volume 10 μL Detection UV 190 nm

Example 25

GOA Improvements Over SEQ ID NO: 1912 for Enantioselective Production of Compound Y

(155) SEQ ID NO: 1912 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(156) Each 100 μL reaction was carried out in 96-well deep-well plates with 60 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(157) The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20 uL of the reaction to a 96-deep-well plate containing 180 uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000 rpm at 4° C. for five minutes. The supernatants were diluted 4-fold with DI water by adding 50 uL of the supernatant to a 96-shallow-well plates containing 150 uL of DI water. The plates were heat sealed for analysis by Analytical Method 24.1.

(158) TABLE-US-00024 TABLE 25.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 1912 SEQ ID NO: Amino Acid Differences Activity (nt/aa) (Relative to SEQ ID NO: 1912) (% conversion) † 2037/2038 P197D ++ 2075/2076 Q79S + 2079/2080 F291Y; N375D; T453V; T465L +++ 2081/2082 M279L; F291Y; T465L +++ 2083/2084 M279L; F291Y; N375D; T465L; +++ D536N; N538D 2085/2086 F291Y; T465L +++ 2087/2088 F291Y; T429V; T465L +++ 2089/2090 F291Y; T465L; N538D ++ 2091/2092 F291Y; T453V; T465L; D536N; ++ N538D 2093/2094 F291Y; L420I; T465L; Q481T; ++ N538D 2095/2096 M279L; N375D; L420I; T429V; ++ T453V; T465L; F472L; N538D 2097/2098 T453V; T465L ++ 2099/2100 F291Y; N375D; L436M; T465G; ++ N538D 2101/2102 N375D; T465G ++ 2103/2104 N29S; P197D; Q407D; K486S ++ 2105/2106 N29I; K30N; Q79S; P197D; Q407D ++ 2107/2108 Q136G; P197D; Q407D ++ 2109/2110 Q79S; Q136G; P197D; Q407D ++ 2111/2112 N29S; K30N; P50V; Q79S; P197D; ++ Q407D 2113/2114 N65A ++ 2115/2116 N29A; K30R; P50V; Q79S; Q136G; + P197D 2117/2118 N375D; L420I; T465L + 2119/2120 Q79S; Q156C; P197D; Q407D + 2121/2122 N29I; K30N; Q79S; P197D + 2123/2124 N29S; K30R; Q136G; P197D; Q407D; + K486S 2125/2126 N29I; K30R; P197D; Q407D + 2127/2128 M279L; T465L + 2129/2130 M279L; F291Y; T465L; D536N + 2131/2132 T465G + 2133/2134 L420I; L436M; T465L + 2135/2136 N29S; K30R; Q79S; P197D; Q407D + 2137/2138 T453V; T465G; V478F; Q481T + 2139/2140 P197D; Q407D + 2141/2142 N29S; P50V; P197D; Q407D; K486S + 2143/2144 N375D; L420I; T465G + 2145/2146 N29S; P197D; Q407D + 2147/2148 N29S; K30R; P197D + 2149/2150 K30N; P50T; Q79S; Q136G; Q156C; + P197D 2151/2152 N29A; K30N; Q79E; Q136G; Q156C; + P197D 2153/2154 T453V; T465G + 2155/2156 F291Y; N375D; L420I; V430I; + T465G; N538D 2157/2158 N375D; T465L + 2159/2160 M279L; F291Y; N375D; L420I; + T429V; L436M; T453V; T465G 2161/2162 N29I + 2163/2164 Q136G + 2165/2166 K30R + 2167/2168 P50V; Q136G; P197D; K486I + 2169/2170 N29T + 2171/2172 N29A + 2173/2174 Q43D; P197D; Q407D + 2175/2176 N375D; T429V; T453V; T465G + 2177/2178 N29S; K30R; Q136G; Q407D + 2179/2180 N29S + 2181/2182 M279L; T465G + 2183/2184 K486S + 2185/2186 P197D; K486S + 2187/2188 M279L; N375D; L420I; T465G + 2189/2190 T429V; T465G + 2191/2192 T465G; D536N; N538D + 2193/2194 Q136G + 2195/2196 N29S; K30R; Q79S; P197D + 2197/2198 Q136G; P197D; K486I + 2199/2200 L615I + 2201/2202 Q156M; R161A; K486A + † Activity (% conversion) was defined as follows: “+” 60.0 to 70.0, “++” 70.0 to 80.0, “+++” 80.0 to 90.0, “++++” 90.0 to 99.0

Example 26

GOA Improvements Over SEQ ID NO: 2080 for Enantioselective Production of Compound Y

(159) SEQ ID NO: 2080 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(160) Each 100 μL reaction was carried out in 96-well deep-well plates with 30 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm for 2 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(161) The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20 uL of the reaction to a 96-deep-well plate containing 180 uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000 rpm at 4° C. for five minutes. The supernatants were diluted 4-fold with DI water by adding 50 uL of the supernatant to a 96-shallow-well plates containing 150 uL of DI water. The plates were heat sealed for analysis by Analytical Method 24.1.

(162) TABLE-US-00025 TABLE 26.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 2080 SEQ ID NO: Amino Acid Differences Activity (nt/aa) (Relative to SEQ ID NO: 2080) (% conversion) † 2203/2204 N47L ++ 2205/2206 A324G ++ 2207/2208 L437R ++ 2209/2210 I214L ++ 2211/2212 D408A ++ 2213/2214 S626W ++ 2215/2216 T119Q ++ 2217/2218 E480L ++ 2219/2220 N414L ++ 2221/2222 N598T ++ 2223/2224 P121G ++ 2225/2226 P197G + 2227/2228 P197M + 2229/2230 P197L + 2231/2232 P197R + 2233/2234 P197E + 2235/2236 G600D + 2237/2238 P197S + 2239/2240 N78L + 2241/2242 V556S + 2243/2244 D365H + 2245/2246 P197H + 2247/2248 S220Q + 2249/2250 Y485L + 2251/2252 S24R + 2253/2254 P197Q + 2255/2256 V95R + 2257/2258 S207Q + 2259/2260 E520L + 2261/2262 N47D + 2263/2264 P197W + 2265/2266 A571S + 2267/2268 T219V + 2269/2270 K249N + 2271/2272 V63T + 2273/2274 S220R + 2275/2276 G294K + 2277/2278 L437N + † Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 2080 and defined as follows: “+” 1.2 to 1.8, “++” 1.8 to 2.5, “+++” >2.5

Example 27

GOA Improvements Over SEQ ID NO: 2080 for Enantioselective Production of Compound Y

(163) SEQ ID NO: 2080 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(164) Each 100 μL reaction was carried out in 96-well deep-well plates with 60 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with 02-permeable seals and incubated at 30° C. and agitated at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(165) The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in Acetonitrile by adding 20 uL of the reaction to a 96-deep-well plate containing 180 uL of 0.04% TFA in Acetonitrile. The samples were centrifuged at 4000 rpm at 4° C. for five minutes. The supernatants were diluted 4-fold with DI water by adding 50 uL of the supernatant to a 96-shallow-well plates containing 150 uL of DI water. The plates were heat sealed for analysis by Analytical Method 24.1.

(166) TABLE-US-00026 TABLE 27.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 2080 SEQ ID NO: Amino Acid Differences Activity (nt/aa) (Relative to SEQ ID NO: 2080) (% conversion) † 2225/2226 P197G ++++ 2233/2234 P197E ++++ 2237/2238 P197S ++++ 2241/2242 V556S +++ 2253/2254 P197Q ++++ 2255/2256 V95R +++ 2267/2268 T219V +++ 2271/2272 V63T +++ 2279/2280 P197R ++++ 2281/2282 P197H ++++ 2283/2284 N29I; P197D; K342R; L436M ++++ 2285/2286 Q136G; P197D; L436M; V453T ++++ 2287/2288 N29I; P197D; V453T ++++ 2289/2290 P197D ++++ 2291/2292 N29S; P197D ++++ 2293/2294 N29S; Q136G; P197D; L436M ++++ 2295/2296 N29I; P197D; L436M; V453T ++++ 2297/2298 P197D; L436M; F472L ++++ 2299/2300 N29I; P197D ++++ 2301/2302 P197D; L436M; V453T ++++ 2303/2304 N29I; P197D; L436M ++++ 2305/2306 N29S; P197D; L436M ++++ 2307/2308 P197D; V453T ++++ 2309/2310 N29I; Q136G; P197D; L436M ++++ 2311/2312 P197D; L436M ++++ 2313/2314 Q136G; P197D; L436M ++++ 2315/2316 Y359L ++++ 2317/2318 N29S; P197D; L436M; V453T ++++ 2319/2320 P197L ++++ 2321/2322 N29I; L436M ++++ 2323/2324 I144V ++++ 2325/2326 L436M ++++ 2327/2328 N29S; Q136G; L436M; V453T ++++ 2329/2330 P197M ++++ 2331/2332 N29S; V453T ++++ 2333/2334 N29I ++++ 2335/2336 Q43G ++++ 2337/2338 E520Y ++++ 2339/2340 N14T; I130M; S257Q; F472L ++++ 2341/2342 Y485R ++++ 2343/2344 K249N ++++ 2345/2346 N14T; S257Q +++ 2347/2348 A495T +++ 2349/2350 N29S; L436M +++ 2351/2352 S257A +++ 2353/2354 P197W +++ 2355/2356 N29S; L436M; F472L +++ 2357/2358 G592H +++ 2359/2360 N29I; Q136G +++ 2361/2362 L437R +++ 2363/2364 N29S; F472L +++ 2365/2366 T119M +++ 2367/2368 S24Q +++ 2369/2370 L437G +++ 2371/2372 I214A +++ 2373/2374 L437Y +++ 2375/2376 V63E +++ 2377/2378 M567S +++ 2379/2380 N29I; Q136G; L436M +++ 2381/2382 Q136G; L436M +++ 2383/2384 E297T +++ 2385/2386 W560I +++ 2387/2388 W560G +++ 2389/2390 N14K; S257E +++ 2391/2392 Q460G +++ 2393/2394 Q136G +++ 2395/2396 I130V; G421N +++ 2397/2398 S257A; F472L +++ † Activity (% conversion) was defined as follows: “+” 60.0 to 70.0, “++” 70.0 to 80.0, “+++” 80.0 to 90.0, “++++” 90.0 to 99.0

Example 28

GOA Improvements Over SEQ ID NO: 2300 for Enantioselective Production of Compound Y

(167) SEQ ID NO: 2300 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(168) Each 100 μL reaction was carried out in 96-well deep-well plates with 60 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with 02-permeable seals and incubated at 30° C. and agitated at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(169) The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35 uL of the reaction to a 96-well shallow-well plate containing 35 uL of acetonitrile. 20 uL of this 2-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of S-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 2004 MeCN, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 21.1.

(170) TABLE-US-00027 TABLE 28.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 2300 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 2300) (% ee) † 2399/2400 D197L; S207D ++++ 2401/2402 T119M; D197M; F339V ++++ 2403/2404 T219V ++++ 2405/2406 V63T; T119M; V556G ++++ 2407/2408 D197S; T219I ++++ 2409/2410 T119M; D197M; V556G ++++ 2411/2412 V63T; D197M; V556G; A571S ++++ 2413/2414 I214A; K249N; Y359L ++++ 2415/2416 T119M; D197M ++++ 2417/2418 V63T; N67K; I214A; V556G ++++ 2419/2420 T119M; D197S; V556G ++++ 2421/2422 V63T; D197R; K249N; A495T ++++ 2423/2424 D197R; T219V ++++ 2425/2426 D197M ++++ 2427/2428 D197H; I214A ++++ 2429/2430 V63T; D197G ++++ 2431/2432 T119M; D197S ++++ 2433/2434 D197L; V556G ++++ 2435/2436 S24Q; T119M; D197R ++++ 2437/2438 V63T ++++ 2439/2440 V63T; T119M; D197S ++++ 2441/2442 D197M; V556G; A571S ++++ 2443/2444 V63T; D197S; I214A; A571S ++++ 2445/2446 D197S; I214A ++++ 2447/2448 T119M; D197L; A571S ++++ 2449/2450 T119M; D197G; I214A; V556G ++++ 2451/2452 D197M; I214A ++++ 2453/2454 Q43G; K249N ++++ 2455/2456 V63T; T119M; D197L; F339V; W341R ++++ 2457/2458 D197S ++++ 2459/2460 D197L ++++ 2461/2462 V95R; D197R ++++ 2463/2464 V63T; D197S ++++ 2465/2466 T119M; D197M; I214A; A571S ++++ 2467/2468 D197S; V556G ++++ 2469/2470 T119M; D197S; I214A ++++ 2471/2472 V63T; N67K; D197S; A571S ++++ 2473/2474 V63T; T119M; D197L; S207D; I214A ++++ 2475/2476 S24Q ++++ 2477/2478 S24Q; D197Q; K249N; L437R ++++ 2479/2480 T119M; D197S; A571S ++++ 2481/2482 V63T; T119M; D197S; V556G; A571S ++++ 2483/2484 T119M ++++ 2485/2486 V95R; T219V; Y359L ++++ 2487/2488 V63T; T119M; D197G; V556G ++++ 2489/2490 V556G ++++ 2491/2492 V63T; D197S; S207D; V556G ++++ 2493/2494 Q43G; D197R; Y359L ++++ 2495/2496 T119M; S207D; V556G; A571S ++++ 2497/2498 D197S; V556G; A571S ++++ 2499/2500 D197M; F339V; V556G; A571S ++++ 2501/2502 T119M; D197S; V556G; A571S ++++ 2503/2504 D197S; A571S ++++ 2505/2506 V63T; D197S; S207D; V556G; A571S ++++ 2507/2508 T119M; D197G; S207D; A571S ++++ 2509/2510 D197G; S207D; I214A; V471I ++++ 2511/2512 S24Q; K51Q; V63T; D197R; Y359L ++++ 2513/2514 T119M; D197L; I214A; V556G ++++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0, “++++” 80.0 to 99.0

Example 29

GOA Improvements Over SEQ ID NO: 2300 for Improved Production of Compound P

(171) Directed evolution efforts were performed which focused on evolving a GOase variant with improved activity on the ethynyl glycerol phosphate (EGP) for generating the corresponding phosphorylated aldehyde (Compound P) (See, Scheme 3, above).

(172) SEQ ID NO: 2300 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(173) Each 100 μL reaction was carried out in 96-well deep-well plates with 20 μL of 2-fold diluted clarified lysate from a 200 uL lysis total volume, 10 g/L of ethynyl glycerol phosphate, 200 μM CuSO.sub.4, 1 g/L HRP, 0.20 g/L catalase, 50 mM PIPES buffer, at pH 7.0. The plates were heat-sealed and shaken at 400 rpm for 3 hours at 30° C.

(174) After 3 hours, the samples were diluted with 200 μL of 50 mM potassium phosphate, pH 7.5. In separate plates, 50 μL of the diluted samples were transferred and mixed with 150 μL of 10 g/L solution O-benzylhydrozylamine in methanol. The plates were sealed and shaken at 400 rpm, at 25° C. for 20-30 minutes. Derivatized samples were diluted 2× in methanol prior to UPLC analysis, as described in the Table 29.1.

(175) TABLE-US-00028 TABLE 29.1 Analytical Method Instrument Thermo Fisher UltiMate 3000 Column Waters Acquity HSS T3, 2.1 × 50 mm Mobile Phase Gradient (A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile Time(min) % B 0.00 8 0.10 8 1.00 70 1.10 100 1.50 8 2.1 8 Flow Rate 1.0 mL/min Run time 2.1 min Peak Retention Time derivatized product at 1.28 min Column Temperature 40° C. Injection Volume 5 μL UV Detection 254 nm

(176) TABLE-US-00029 TABLE 29.2 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 2300 SEQ ID NO: Amino Acid Differences Activity (nt/aa) (Relative to SEQ ID NO: 2300) (FIOP Product) † 2515/2516 E196R +++ 2517/2518 Q327R +++ 2519/2520 Q407R +++ 2521/2522 L465R ++ 2523/2524 Y330H ++ 2525/2526 F442Y ++ 2527/2528 N246Q; F442Y + 2529/2530 T583S + 2531/2532 Q327K + 2533/2534 N246Q; D408N; F442Y; G462A + 2535/2536 S292R + 2537/2538 T583G + 2539/2540 G462A; T583A + 2541/2542 Q407K + 2543/2544 S498C + 2545/2546 E196Q + 2547/2548 Q327R; L329W + 2549/2550 N246S; F442Y + 2551/2552 F442Y; G462A; L515M + 2553/2554 A194G; Y330H; A495S + † Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 2300 and defined as follows: “+” 1.50 to 2.50, “++” >2.50 “+++” >5.00

Example 30

GOA Improvements Over SEQ ID NO: 2424 for Enantioselective Production of Compound Y

(177) SEQ ID NO: 2424 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(178) Each 100 μL reaction was carried out in 96-well deep-well plates with 60 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, 50 mM PIPES buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(179) The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35 uL of the reaction to a 96-well shallow-well plate containing 35 uL of acetonitrile. 20 uL of this 2-fold diluted reaction was derivatized by adding 10 uL of 100 g/L solution in acetonitrile of S-AMP and incubating with shaking in a 96-well half-deepwell plate for ˜45 minutes at 30° C. The samples were quenched by adding 200 μL of MeCN:heptane (1:1) mixture, shaking briefly to mix, and centrifuged at 4000 rpm at 4° C. for five minutes. The supernatant was transferred to a 96-well shallow-well plate and heat sealed for analysis by Analytical Method 21.1.

(180) TABLE-US-00030 TABLE 30.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 2424 SEQ ID NO: Amino Acid Differences Selectivity (nt/aa) (Relative to SEQ ID NO: 2424) (% ee) † 2555/2556 D408Q ++++ 2557/2558 E480L ++++ 2559/2560 G600N ++++ 2561/2562 K36L ++++ 2563/2564 N14L ++++ 2565/2566 N14R ++++ 2567/2568 N532G ++++ 2569/2570 N96M ++++ 2571/2572 N96S ++++ 2573/2574 P404A ++++ 2575/2576 R120S ++++ 2577/2578 R218M ++++ 2579/2580 R549L ++++ 2581/2582 S24P ++++ 2583/2584 S537C ++++ 2585/2586 S92C ++++ 2587/2588 S92G ++++ 2589/2590 S92V ++++ 2591/2592 S99L ++++ 2593/2594 S99V ++++ 2595/2596 V46P ++++ 2597/2598 V95A ++++ 2599/2600 W560I ++++ 2601/2602 Y485C ++++ 2603/2604 S24P; V46E; S92G; S426W; R549L ++++ 2605/2606 K36L; S426W; Y485C; G600N ++++ 2607/2608 S24P; V46E; S92G; P404A; S426W; N532G ++++ 2609/2610 S99V; P404A; S426W; W560E ++++ 2611/2612 S24P; V46E; S92G; S426W; N532G ++++ 2613/2614 S24P; K36L; P404A; E480L; Y485C; N532G; ++++ W560E; G600N 2615/2616 S24P; S99V ++++ 2617/2618 S24P; V46E; P404A; S426W; Y485C; N532G ++++ 2619/2620 S24P; K36L; S99V; P404A; S426W; N532G; ++++ R549L; G600N 2621/2622 S24P; V46E; S99V; S426W; R549L; G600N ++++ 2623/2624 S92G; V95S; Y485C; N532G; R549L; W560E ++++ 2625/2626 S24P; K36L; V46E; S99V; S426W; N532G; ++++ R549L 2627/2628 S99V; S426W; E480L; Y485C ++++ 2629/2630 K36L; S92G; Y485C ++++ 2631/2632 S24P; S99V; P404A; Y485C; N532G; G600N ++++ 2633/2634 S24P; K36L; P404A; S426W; N532G ++++ 2635/2636 K36L; P404A ++++ 2637/2638 S24P; V46E; V95S; S99V; S426W; N532G ++++ 2639/2640 S24P; P404A; E480L; N532G; R549L; W560E ++++ 2641/2642 S24P; N96M; P404A; S426W ++++ 2643/2644 K36L; S92G; V95S; S99V; P404A; S426W; ++++ W560E 2645/2646 S24P; P404A; E480L; Y485C ++++ 2647/2648 S24P; K36L; N96M; S99V; N532G; R549L ++++ 2649/2650 R549L; W560E ++++ 2651/2652 S24P; K36L; V95S; S99V; P404A; S426W; ++++ Y485C 2653/2654 K36L; P404A; S426W; R549L; G600N ++++ 2655/2656 V95S; P404A; S426W; N532G ++++ 2657/2658 P404A; Y485C; G600N ++++ 2659/2660 S24P; P404A; S426W; N532G ++++ 2661/2662 S92V; S99L; R218M; W560I ++++ 2663/2664 V46P; S92V; W560I ++++ 2665/2666 N14L; S92V; N96S; R120S; R376M ++++ 2667/2668 N14R; D408Q ++++ 2669/2670 N14R; R376M; W560I ++++ 2671/2672 N96S; S99L ++++ 2673/2674 N14L; S92V; N96S; S99L ++++ 2675/2676 N14R; S92V; S99L; R120S; S537C ++++ 2677/2678 N14R; S24P; S92C; N96S; S99L; D408R ++++ 2679/2680 V95R; R120L; V296R; S626G ++++ 2681/2682 N14L; S92V; N96S; S99L; R120S; S537C ++++ 2683/2684 N14A; S24V; N78I; R120L; S258V ++++ 2685/2686 N14A; S24V; K36P; N96G ++++ 2687/2688 S24V; V296R ++++ 2689/2690 N14A; S24V; N96G; S258V; S626G ++++ 2691/2692 R120L; A324F; E480R; W560M ++++ 2693/2694 N14A; N78I; R120L; S258V; N488T; W560M; ++++ S626G 2695/2696 S24V; K36P; V95R; N96G ++++ 2697/2698 S24V; K36P; R120L; V296R; E480R; W560M ++++ 2699/2700 K36P; S258V; V296R ++++ 2701/2702 N96G ++++ 2703/2704 N96L; S258V; W560M; S626G ++++ 2705/2706 N14A; S24V; S258V; W560M ++++ 2707/2708 S24V; V296R; A324F; E480R ++++ 2709/2710 V296R; A324F ++++ 2711/2712 V296R; A324F; W560M ++++ 2713/2714 A324F; W560M ++++ 2715/2716 N14A; S258V; V296R; W560M ++++ 2717/2718 N14A; R120L; E480R; S626G ++++ 2719/2720 E480R ++++ 2721/2722 N14A; S24V; K36P; V296R; G424W; W560M ++++ 2723/2724 K36P; S258V; V296R; A324F; S433G; S626G ++++ 2725/2726 N14A; V95R; R120L; V296R; E480R; W560M ++++ 2727/2728 N14A; S258V ++++ 2729/2730 K36L; D408L; S537W; T596Q ++++ 2731/2732 Q23A; K36L; S92C; V95F; N96G ++++ 2733/2734 Q23A; K36L; S537W; T596Q ++++ 2735/2736 K36L; S92C; V95F; N428H; T596Q ++++ 2737/2738 S537W; Q640R ++++ 2739/2740 K36L; D408L; T596Q ++++ 2741/2742 R218G; D408L ++++ 2743/2744 Q23A; R218G; S537W ++++ 2745/2746 T596V ++++ 2747/2748 K36L; S92C; N96G; D408L; N428H; N540R; ++++ T596V 2749/2750 Q23A; D408L; T596V ++++ 2751/2752 Q23A; K36L; D408L; N428H ++++ 2753/2754 Q23A; K36L; S92C; V95F; S99F; D408L; ++++ T596V 2755/2756 V95F ++++ 2757/2758 D408L ++++ † Selectivity (% ee) was defined as follows: “+” 20.0 to 40.0, “++” 40.0 to 60.0, “+++” 60.0 to 80.0, “++++” 80.0 to 99.0

Example 31

GOA Improvements Over SEQ ID NO: 2424 for Enantioselective Production of Compound Y

(181) SEQ ID NO: 2424 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the clarified lysates were generated as described in Example 4.

(182) Each 100 μL reaction was carried out in 96-well deep-well plates with 60 μL clarified lysate from a 200 uL lysis total volume, 30 g/L Compound X, 50 mM PIPES buffer, 200 μM CuSO.sub.4, 0.2 g/L HRP, 0.20 g/L catalase, at pH 6.5. The plates were sealed with O.sub.2-permeable seals and incubated at 30° C. and agitated at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

(183) The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20 uL of the reaction to a 96-deep-well plate containing 180 uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000 rpm at 4° C. for five minutes. The supernatants were diluted 4-fold with DI water by adding 50 uL of the supernatant to a 96-shallow-well plates containing 150 uL of DI water. The plates were heat sealed for analysis by Analytical Method 24.1.

(184) TABLE-US-00031 TABLE 31.1 Enzyme Variant Activity and Selectivity Relative to SEQ ID NO: 2424 SEQ ID NO: Amino Acid Differences Activity (nt/aa) (Relative to SEQ ID NO: 2424) (% conversion) † 2603/2604 S24P; V46E; S92G; S426W; R549L ++++ 2625/2626 S24P; K36L; V46E; S99V; S426W; ++++ N532G; R549L 2623/2624 S92G; V95S; Y485C; N532G; R549L; ++++ W560E 2613/2614 S24P; K36L; P404A; E480L; Y485C; ++++ N532G; W560E; G600N 2641/2642 S24P; N96M; P404A; S426W ++++ 2759/2760 S24P; N96M; P404A; S426W; W560E ++++ 2635/2636 K36L; P404A ++++ 2651/2652 S24P; K36L; V95S; S99V; P404A; ++++ S426W; Y485C 2579/2580 R549L +++ 2581/2582 S24P +++ 2557/2558 E480L +++ 2761/2762 V296R +++ 2763/2764 E480R +++ 2597/2598 V95A +++ 2765/2766 S24V +++ 2767/2768 R120L +++ 2769/2770 W560M +++ 2771/2772 N78I +++ 2773/2774 S92G +++ 2567/2568 N532G +++ 2775/2776 N96G +++ 2777/2778 S258V +++ 2779/2780 S361P +++ 2781/2782 S626G +++ 2647/2648 S24P; K36L; N96M; S99V; N532G; +++ R549L 2621/2622 S24P; V46E; S99V; S426W; R549L; +++ G600N 2637/2638 S24P; V46E; V95S; S99V; S426W; +++ N532G 2607/2608 S24P; V46E; S92G; P404A; S426W; +++ N532G 2611/2612 S24P; V46E; S92G; S426W; N532G ++ 2617/2618 S24P; V46E; P404A; S426W; Y485C; ++ N532G 2783/2784 S426W; Y485C ++ 2785/2786 P404A ++ 2633/2634 S24P; K36L; P404A; S426W; N532G ++ 2615/2616 S24P; S99V ++ 2787/2788 S426W; N532G; R549L ++ 2627/2628 S99V; S426W; E480L; Y485C ++ 2789/2790 S24P; N532G ++ 2791/2792 R549L; W560E ++ 2645/2646 S24P; P404A; E480L; Y485C ++ 2653/2654 K36L; P404A; S426W; R549L; G600N ++ 2659/2660 S24P; P404A; S426W; N532G ++ 2605/2606 K36L; S426W; Y485C; G600N ++ 2793/2794 P404A; S426W; Y485C ++ 2795/2796 K36L; S99V; S426W; Y485C; G600N ++ 2797/2798 S92G; P404A ++ 2799/2800 A324F ++ 2593/2594 S99V ++ 2565/2566 N14R ++ 2801/2802 N96M ++ 2561/2562 K36L ++ 2803/2804 N14R; S24P; S99L; R218M; D408Q; ++ S537C; W560I 2559/2560 G600N ++ 2683/2684 N14A; S24V; N78I; R120L; S258V ++ 2805/2806 N14R; S537C ++ 2807/2808 N14R; V46P; N96S; S99L; W560I ++ 2809/2810 N14R; S92V; S99L; R218M; D408Q ++ 2663/2664 V46P; S92V; W560I ++ 2665/2666 N14L; S92V; N96S; R120S; R376M ++ 2671/2672 N96S; S99L ++ 2811/2812 N14L; R376M; S537C ++ 2725/2726 N14A; V95R; R120L; V296R; E480R; ++ W560M 2661/2662 S92V; S99L; R218M; W560I ++ 2813/2814 N14L; S92V; S99L; R120S; R218M; ++ D408R 2815/2816 N14R; S92C; N96S; R376M ++ 2817/2818 N14L; R376M ++ 2685/2686 N14A; S24V; K36P; N96G ++ 2819/2820 S92V; R218M ++ 2705/2706 N14A; S24V; S258V; W560M ++ 2821/2822 N14R; S92V; N96S; S99L; R376M; ++ W560I 2823/2824 N14L; S92C; R218M; D408Q ++ 2825/2826 Q23A; K36L; N96G; D408L; T596V; ++ Q640R 2827/2828 N14R; V46P; N47P; R376M ++ 2829/2830 K36L; D408L ++ 2831/2832 S92V; N96S ++ 2667/2668 N14R; D408Q ++ 2833/2834 N14L; R376M; W560I ++ 2699/2700 K36P; S258V; V296R ++ 2687/2688 S24V; V296R ++ 2721/2722 N14A; S24V; K36P; V296R; G424W; ++ W560M 2835/2836 V296R; E480R; W560M ++ 2675/2676 N14R; S92V; S99L; R120S; S537C ++ 2729/2730 K36L; D408L; S537W; T596Q ++ 2837/2838 R120S; R376M ++ 2839/2840 Q23A; K36L; S537W; N540R; Q640R ++ 2841/2842 G424W ++ 2843/2844 S92V; S99L; R120S ++ 2691/2692 R120L; A324F; E480R; W560M ++ 2695/2696 S24V; K36P; V95R; N96G ++ 2753/2754 Q23A; K36L; S92C; V95F; S99F; ++ D408L; T596V 2709/2710 V296R; A324F ++ 2845/2846 K36L; V95F; N96G ++ 2711/2712 V296R; A324F; W560M ++ 2755/2756 V95F ++ 2697/2698 S24V; K36P; R120L; V296R; E480R; ++ W560M 2723/2724 K36P; S258V; V296R; A324F; S433G; ++ S626G 2847/2848 Q23A; K36L + 2733/2734 Q23A; K36L; S537W; T596Q + 2849/2850 S99L; Q640R + 2731/2732 Q23A; K36L; S92C; V95F; N96G + 2751/2752 Q23A; K36L; D408L; N428H + 2851/2852 R218G; S537W; T596V + 2735/2736 K36L; S92C; V95F; N428H; T596Q + 2737/2738 S537W; Q640R + 2853/2854 Q640R + 2855/2856 Q23A; R218G; T596Q; Q640R + 2857/2858 D408L; T596Q + 2859/2860 R218G; T596Q + † Activity (% conversion) was defined as follows: “+” 60.0 to 70.0, “++” 70.0 to 80.0, “+++” 80.0 to 90.0, “++++” 90.0 to 99.0

(185) All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

(186) While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).