3-methylcrotonic acid decarboxylase (MDC) variants

Abstract

Described are 3-methylcrotonic acid decarboxylase (MDC) variants showing an improved activity in converting 3-methylcrotonic acid into isobutene as well as methods for the production of isobutene using such enzyme variants.

Claims

1. A variant of a 3-methylcrotonic acid decarboxylase (MDC) showing an improved activity in converting 3-methylcrotonic acid into isobutene relative to a parent MDC having the amino acid sequence as set forth in SEQ ID NO:1 wherein said variant comprises an amino acid sequence having at least 73% sequence identity to SEQ ID NO:1 with a substitution, a deletion or an insertion at one or more amino acid residues corresponding to positions 31, 197, 337, 351, 376, 405, 439, 441, 447 and 449 of SEQ ID NO: 1, and wherein said variant optionally further comprises a substitution, a deletion or an insertion at one or more amino acid residues corresponding to positions 2, 12, 13, 29, 33, 35, 89, 114, 195, 221, 293, 381, 388, 420, 422, 435, 436, 500, 506 and 511 of SEQ ID NO: 1.

2. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 1.

3. The method of claim 2, wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant.

4. A composition comprising the MDC variant of claim 1.

5. The composition of claim 4 further comprising 3-methylcrotonic acid.

6. The MDC variant of claim 1, wherein: (1) the amino acid residue at position 2 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine, aspartic acid, phenylalanine, lysine, leucine, asparagine, glutamine or valine; and/or (2) the amino acid residue at position 12 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine, alanine or asparagine; and/or (3) the amino acid residue at position 13 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with histidine, isoleucine, asparagine, serine, valine or tyrosine; and/or (4) the amino acid residue at position 29 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine, histidine or serine; and/or (5) the amino acid residue at position 31 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamic acid or glycine; and/or (6) the amino acid residue at position 33 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine; and/or (7) the amino acid residue at position 35 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with methionine, serine or threonine; and/or (8) the amino acid residue at position 89 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine or phenylalanine; and/or (9) the amino acid residue at position 114 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine; and/or (10) the amino acid residue at position 195 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with methionine, cysteine, phenylalanine, isoleucine, valine, tryptophan or tyrosine; and/or (11) the amino acid residue at position 197 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine or methionine; and/or (12) the amino acid residue at position 221 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (13) the amino acid residue at position 293 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (14) the amino acid residue at position 337 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with methionine or leucine; and/or (15) the amino acid residue at position 351 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine, asparagine, alanine, valine or glycine; and/or (16) the amino acid residue at position 376 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine; and/or (17) the amino acid residue at position 381 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine; and/or (18) the amino acid residue at position 388 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamic acid; and/or (19) the amino acid residue at position 405 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine, leucine, methionine, proline or glutamine; and/or (20) the amino acid residue at position 420 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (21) the amino acid residue at position 422 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with methionine; and/or (22) the amino acid residue at position 435 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with methionine; and/or (23) the amino acid residue at position 436 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (24) the amino acid residue at position 439 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (25) the amino acid residue at position 441 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with tyrosine; and/or (26) the amino acid residue at position 447 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with tryptophan, methionine or tyrosine; and/or (27) the amino acid residue at position 449 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine, methionine or valine; and/or (28) the amino acid residue at position 500 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine; and/or (29) the amino acid residue at position 506 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine or tyrosine; and/or (30) the amino acid residue at position 511 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with methionine or isoleucine.

7. The MDC variant of claim 1, wherein said variant further shows at least one modification at positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 25, 30, 34, 40, 43, 57, 60, 65, 67, 69, 70, 71, 72, 80, 84, 85, 86, 87, 90, 91, 99, 101, 102, 103, 105, 106, 108, 111, 117, 119, 120, 126, 132, 141, 146, 149, 154, 159, 160, 162, 175, 176, 187, 189, 193, 206, 211, 213, 214, 215, 216, 222, 228, 232, 244, 247, 264, 278, 284, 285, 303, 305, 306, 326, 338, 341, 342, 345, 349, 352, 375, 377, 384, 386, 392, 395, 399, 402, 404, 406, 414, 429, 440, 442, 443, 445, 448, 454, 460, 461, 462, 484, 488, 493, 494, 496, 501, 502, 509 and 512 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position.

8. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 7.

9. The method of claim 8, wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant.

10. A composition comprising the MDC variant of claim 7.

11. The composition of claim 10 further comprising 3-methylcrotonic acid.

12. The MDC variant of claim 7, wherein: (1) the amino acid residue at position 3 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine, glutamic acid, glycine, lysine, proline, tryptophan, cysteine, aspartic acid or tyrosine; and/or (2) the amino acid residue at position 4 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamic acid, leucine, methionine, alanine, serine or asparagine; and/or (3) the amino acid residue at position 5 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine; and/or (4) the amino acid residue at position 6 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with proline; and/or (5) the amino acid residue at position 7 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine; and/or (6) the amino acid residue at position 8 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (7) the amino acid residue at position 9 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with histidine, proline or tyrosine; and/or (8) the amino acid residue at position 10 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with histidine, phenylalanine, lysine, proline, threonine or leucine; and/or (9) the amino acid residue at position 11 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine, tyrosine or proline; and/or (10) the amino acid residue at position 14 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with aspartic acid; and/or (11) the amino acid residue at position 15 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with threonine; and/or (12) the amino acid residue at position 25 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine, serine or tryptophan; and/or (13) the amino acid residue at position 30 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glycine, histidine or arginine; and/or (14) the amino acid residue at position 34 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or isoleucine; and/or (15) the amino acid residue at position 40 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine or methionine; and/or (16) the amino acid residue at position 43 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine; and/or (17) the amino acid residue at position 57 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (18) the amino acid residue at position 60 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with valine; and/or (19) the amino acid residue at position 65 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine or tryptophan; and/or (20) the amino acid residue at position 67 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine or valine; and/or (21) the amino acid residue at position 69 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (22) the amino acid residue at position 70 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine or leucine; and/or (23) the amino acid residue at position 71 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glycine; and/or (24) the amino acid residue at position 72 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine; and/or (25) the amino acid residue at position 80 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (26) the amino acid residue at position 84 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (27) the amino acid residue at position 85 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or threonine; and/or (28) the amino acid residue at position 86 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine or isoleucine; and/or (29) the amino acid residue at position 87 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine, cysteine, phenylalanine, isoleucine, leucine, methionine, valine or tryptophan; and/or (30) the amino acid residue at position 90 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (31) the amino acid residue at position 91 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (32) the amino acid residue at position 99 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine or proline; and/or (33) the amino acid residue at position 101 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine or leucine; and/or (34) the amino acid residue at position 102 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (35) the amino acid residue at position 103 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine, leucine or methionine; and/or (36) the amino acid residue at position 105 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine, leucine or tryptophan; and/or (37) the amino acid residue at position 106 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with threonine; and/or (38) the amino acid residue at position 108 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with lysine, arginine or tryptophan; and/or (39) the amino acid residue at position 111 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (40) the amino acid residue at position 117 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine; and/or (41) the amino acid residue at position 120 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine or lysine; and/or (42) the amino acid residue at position 119 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with threonine; and/or (43) the amino acid residue at position 126 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with proline; and/or (44) the amino acid residue at position 132 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (45) the amino acid residue at position 141 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with aspartic acid; and/or (46) the amino acid residue at position 146 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine; and/or (47) the amino acid residue at position 149 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with valine or serine; and/or (48) the amino acid residue at position 154 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with lysine; and/or (49) the amino acid residue at position 159 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (50) the amino acid residue at position 160 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine; and/or (51) the amino acid residue at position 162 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with proline, histidine or asparagine; and/or (52) the amino acid residue at position 175 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glycine, lysine, proline, glutamine, serine, threonine or tryptophan; and/or (53) the amino acid residue at position 176 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine; and/or (54) the amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with threonine; and/or (55) the amino acid residue at position 189 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine; and/or (56) the amino acid residue at position 193 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine, threonine or valine; and/or (57) the amino acid residue at position 206 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine; and/or (58) the amino acid residue at position 211 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamic acid; and/or (59) the amino acid residue at position 213 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with proline or leucine; and/or (60) the amino acid residue at position 214 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine, threonine or valine, histidine, glutamic acid, arginine or phenylalanine; and/or (61) the amino acid residue at position 215 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (62) the amino acid residue at position 216 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (63) the amino acid residue at position 222 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (64) the amino acid residue at position 228 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine, alanine, proline, threonine or valine; and/or (65) the amino acid residue at position 232 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with valine; and/or (66) the amino acid residue at position 244 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine; and/or (67) the amino acid residue at position 247 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine; and/or (68) the amino acid residue at position 264 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with aspartic acid; and/or (69) the amino acid residue at position 278 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine; and/or (70) the amino acid residue at position 284 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with tyrosine or leucine; and/or (71) the amino acid residue at position 285 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (72) the amino acid residue at position 303 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine or proline; and/or (73) the amino acid residue at position 305 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or aspartic acid; and/or (74) the amino acid residue at position 306 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine, arginine or serine; and/or (75) the amino acid residue at position 326 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or proline; and/or (76) the amino acid residue at position 338 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with proline, alanine or serine; and/or (77) the amino acid residue at position 341 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with isoleucine; and/or (78) the amino acid residue at position 342 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glycine; and/or (79) the amino acid residue at position 345 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (80) the amino acid residue at position 349 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine; and/or (81) the amino acid residue at position 352 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glycine or leucine; and/or (82) the amino acid residue at position 375 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (83) the amino acid residue at position 377 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with histidine; and/or (84) the amino acid residue at position 384 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with tyrosine; and/or (85) the amino acid residue at position 386 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (86) the amino acid residue at position 392 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine or alanine; and/or (87) the amino acid residue at position 395 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with cysteine; and/or (88) the amino acid residue at position 399 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine or asparagine; and/or (89) the amino acid residue at position 402 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with valine or histidine; and/or (90) the amino acid residue at position 404 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with tyrosine or tryptophan; and/or (91) the amino acid residue at position 406 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamine; and/or (92) the amino acid residue at position 414 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (93) the amino acid residue at position 440 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with valine; and/or (94) the amino acid residue at position 443 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (95) the amino acid residue at position 448 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with serine, phenylalanine or trypophan; and/or (96) the amino acid residue at position 454 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glycine; and/or (97) the amino acid residue at position 460 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with phenylalanine or proline; and/or (98) the amino acid residue at position 461 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with valine, asparagine or methionine; and/or (99) the amino acid residue at position 462 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (100) the amino acid residue at position 484 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or glycine; and/or (101) the amino acid residue at position 488 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or asparagine; and/or (102) the amino acid residue at position 493 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine; and/or (103) the amino acid residue at position 494 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with arginine; and/or (104) the amino acid residue at position 496 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or phenylalanine; and/or (105) the amino acid residue at position 429 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with alanine or serine; and/or (106) the amino acid residue at position 442 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with threonine; and/or (107) the amino acid residue at position 445 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamic acid or proline; and/or (108) the amino acid residue at position 501 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamic acid, methionine, glycine or lysine; and/or (109) the amino acid residue at position 502 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with asparagine; and/or (110) the amino acid residue at position 509 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with leucine; and/or (111) the amino acid residue at position 512 in the amino acid sequence shown in SEQ ID NO:1 or at a position corresponding to this position, is deleted or substituted with glutamic acid, histidine or serine.

13. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 12.

14. The method of claim 13, wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant.

15. A composition comprising the MDC variant of claim 12.

16. The composition of claim 15 further comprising 3-methylcrotonic acid.

17. The MDC variant of claim 1, wherein said variant comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1.

18. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 1.

19. The method of claim 18, wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant.

Description

(1) In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

(2) FIG. 1A: shows a schematic reaction of the enzymatic prenylation of a flavin mononucleotide (FMN) into the corresponding modified (prenylated) flavin cofactor.

(3) FIG. 1B: Schematic reaction of the enzymatic conversion of 3-methylcrotonic acid into isobutene.

(4) FIG. 2: Multiple Sequence Alignment of 19 protein homologues of the Hypocrea atroviridis Ferulic acid decarboxylase 1 (G9NLP8), focusing on the K401-G413 segment of G9NLP8.

(5) FIG. 3: Isobutene (IBN) production from 10 mM 3-methylcrotonic acid of homologues MDC and their T.fwdarw.M variants at different temperatures (30, 40, 50 and 60° C.).

(6) FIG. 4: Isobutene (IBN) production from 30 mM 3-methylcrotonic acid (3MC) after two hours of incubation with two 3-methylcrotonic acid decarboxylase (3-MDC) enzymes. Ss5: enzyme from Streptomyces sp. 769 (Uniprot Accession Number A0A0A8EV26); Ha: enzyme from Hypocrea atroviridis (Uniprot Accession Number G9NLP8). Tests are conducted in 384 or 96 microplates (DW384 and DW96), in vitro and in vivo.

(7) FIG. 5: Isobutene (IBN) production from 30 mM 3-methylcrotonic acid (3MC) after two hours of incubation with two 3-methylcrotonic acid decarboxylase (3MDC) enzymes (Ss5 and Ha) fused with a 6-His-tag. Ss5: enzyme from Streptomyces sp. 769 (Uniprot Accession Number A0A0A8EV26); Ha: enzyme from Hypocrea atroviridis (Uniprot Accession Number G9NLP8). Tests are conducted in 384 or 96 microplates (DW384 and DW96), in vitro and in vivo.

(8) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

First Part

Example 1

Directed Evolution Strategy

(9) The enzyme Hypocrea atroviridis Ferulic acid decarboxylase 1 (SEQ ID NO:1) is capable of catalysing, amongst other reactions, the decarboxylation of 3-methylcrotonic acid (3MC) into isobutene (IBN). A directed evolution approach was used in order to specifically improve the catalytic efficiency of this reaction. This approach consisted in (1) the design of assay systems to test the activity of enzyme variants, (2) the generation of collections of single point or multiple mutants for Hypocrea atroviridis Ferulic acid decarboxylase 1, and (3) the use of the activity assays to screen the collection of mutants in order to identify variants with improved activity compared to the activity of the wild type Hypocrea atroviridis Ferulic acid decarboxylase 1.

(10) This approach led to the identification and characterization of a collection of mutants with increased activity compared to the wild type enzyme.

Example 2

Construction of Hypocrea atroviridis Ferulic Acid Decarboxylase 1 Enzyme Mutants

(11) The polynucleotide sequences coding for the different mutants identified during the evolution of the Hypocrea atroviridis Ferulic acid decarboxylase 1 enzyme were generated using a range of standard molecular biology techniques. All these techniques used a codon-optimised polynucleotide sequence for expression in Escherichia coli as template. The sequence optimisation has been done by Geneart using their GeneOptimizer software.

(12) Different PCR-based techniques known in the art were used for the construction of single-point mutants. For the generation of enzyme variants bearing multiple mutations (at least two mutations), either PCR-based techniques or other methods known in the art were used to introduce these mutations.

(13) Following mutagenesis, the mutated polynucleotide sequence was inserted into a pETDuet™-1 co-expression vector (Novagen) (used for recombinant protein production in E. coli and screening) in addition to the cDNA of the Flavin prenyltransferase UbiX protein from E. coli either using standard ligase-based subcloning techniques, whole plasmid extension by PCR or ligase-independent cloning techniques.

Example 3

Selection of the Enzyme Mutants with Increased Activity

(14) Two different screening methods were developed and used during the evolution of the Hypocrea atroviridis Ferulic acid decarboxylase 1 enzyme.

(15) 1.) In Vivo Assay in 384-Well Microplates Based on Exogenous 3MC (IN VIVO 1)

(16) This assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with the above expression vector that contain the above-described coding sequences leading to the production of the last two enzymes involved in the metabolic pathway converting 3MC into isobutene, namely the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants and the Flavin prenyltransferase UbiX protein from E. coli. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 50 μL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30° C. The LB cultures were used to inoculate 300 μL in 384 deepwell microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 30° C. in order to produce the two recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 40 μL of minimum medium (pH 7.5, Phosphate 100 mM, Glucose 10g.Math.L.sup.−1, MgSO.sub.4 1 mM) supplemented with 10 mM 3MC and incubated for a further 2 or 4 hours in a shaking incubator at 30° C., 700 rpm. During this step, the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants catalyse the decarboxylation of 3MC into IBN. After 5 min inactivation at 80° C., the IBN produced is quantified by gas chromatography as described in the following. 100 μL of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(17) 2.) In Vivo Assay in 96-Well Microplates Based on Exogenous 3MC (IN VIVO 2)

(18) This assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with the above expression vector that contains the coding sequences as described above, leading to the production of the last two enzymes involved in the metabolic pathway converting 3MC to isobutene; namely the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants and the Flavin prenyltransferase UbiX protein from E. coli. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 500 μL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 30° C. or 32° C. The LB cultures were used to inoculate 1 mL in 96 deepwell microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 30° C. or 32° C. in order to produce the two recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 400 μL of minimum medium (pH 7.5, Phosphate 100 mM, Glucose 10g.Math.L.sup.−1, MgSO.sub.4 1 mM) supplemented with 10 mM 3MC and incubated for a further 2 or 4 hours in a shaking incubator at 30° C. or 36° C., 700 rpm. During this step, the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants catalyse the decarboxylation of 3MC into IBN. After 5 mM inactivation at 80° C., the IBN produced is quantified by gas chromatography as described in the following. 100 μL of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

Example 4

Identification of Variants of Hypocrea Atroviridis Ferulic Acid Decarboxylase 1 with Further Increased Activity for the Reaction of Conversion of 3-Methylcrotonic Acid into Isobutene

(19) A collection of mutants has been created by mutagenesis, using the Hypocrea atroviridis Ferulic acid decarboxylase 1 (SEQ ID NO:1) as template. Several variants with an enhanced activity in converting 3-methylcrotonic acid into isobutene have been identified through in vivo screening assays as described above. A first round of screening with 59508 clones has been performed using the above IN VIVO 1 assay. The best 927 clones were then tested in 12-replicate using the same protocol. The plasmids of the best 95 variants were individually extracted, transformed into fresh competent BL21(DE3) cells and then tested in 8-replicate assays according to the above-described IN VIVO 2 assay.

(20) The list of improved variants is presented in the following Table 1 and the list of the individual positions presenting an increase in activity is shown in Table 2. The improvement factor shown in the below Table 1 reflects the average value of 8 replicates of the relative quantity of isobutene which has been produced and measured according to the above-described IN VIVO 2 assay compared to the wild type enzyme.

(21) TABLE-US-00002 TABLE 1 List of Hypocrea atroviridis Ferulic acid decarboxylase 1 variants presenting an increase in isobutene production from 3-methylcrotonic acid Mutations Improvement Factor/WT T405M 7.9 T405F 4.7 S2Q 4.4 S2A 4.3 L195C 4.3 L449I 4.3 S2K 4.2 S2L 4.1 S2V 4.0 S3A 3.9 S3Y 3.8 S2F 3.7 S3K L511M 3.7 T4N F91L 3.5 S2N 3.5 T4E 3.5 S3K M284Y 3.5 S3W 3.2 S3G 3.2 M293L 3.2 S3P 3.1 T4M 3.1 S3E 3.0 T405L 2.9 S2D E89S 2.8 T4L 2.7 V40M 2.6 D35M 2.5 K422M 2.4 Q448W 2.3 L195I 2.3 A285L 2.3 L195W 2.2 V34A 2.2 D12S 2.1 L500A 2.1 D351R 2.1 V34I 2.1 Q214T 2.1 E9Y 2.0 L195M 2.0 F447W 2.0 L114S 2.0 P13N 1.9 E9H 1.9 F11P 1.8 D43R 1.8 D420L 1.8 P13I 1.8 L195Y 1.8 V439L 1.8 I337M 1.8 L506I 1.8 F11L 1.7 P13Y 1.7 P120S 1.7 T429S 1.7 T436N 1.7 G435M 1.7 L195V 1.7 A149V 1.6 F447Y 1.6 P13S 1.6 Q29N 1.6 A10H 1.6 N31G 1.6 Q214A 1.5 V40I 1.5 L195F 1.5 N31E N501E 1.5 E25N 1.5 T429A 1.4 N501K 1.4 E9P 1.4 I197F 1.4 Q448S 1.4 D35T 1.4 A146S 1.4 D442T 1.4 P13H 1.3 Q214V 1.3 V445P 1.3 V445E 1.3 L33I 1.3 A381R 1.3 L221C 1.3 L449M 1.2 T405Q 1.2 T405P 1.2 T376I A388E 1.2 D35S 1.2 L449V 1.2 Q29S 1.2 N141D 1.2 F441Y 1.2

(22) TABLE-US-00003 TABLE 2 List of the positions modified in the variants of Hypocrea atroviridis Ferulic acid decarboxylase 1 with increased activity Position Wild-Type Amino Acid Mutations 2 S A, D, F, K, L, N, Q, V 3 S A, E, G, K, P, W, Y, 4 T E, L, M, N 9 E H, P, Y 10 A H 11 F L, P 12 D S 13 P H, I, N, S, Y 25 E N 29 Q N, S 31 N E, G 33 L I 34 V A, I 35 D M, S, T 40 V I, M 43 D R 89 E S 91 F L 114 L S 120 P S 141 N D 146 A S 149 A V 195 L C, F, I, M, V, W, Y 197 I F 214 Q A, T, V 221 L C 284 M Y 285 A L 293 M L 337 I M 351 D R 376 T I 381 A R 388 A E 405 T F, L, M, P, Q 420 D L 422 K M 429 T A, S 435 G M 436 T N 439 V L 441 F Y 442 D T 445 V E, P 447 F W, Y 448 Q S, W 449 L I, M, V 500 L A 501 N E, K 506 L I 511 L M

Example 5

In Vitro Activities of the Hypocrea atroviridis Ferulic Acid 1 Decarboxylase WT and T405M Mutant

(23) Gene Synthesis, Cloning, Expression and Purification of Ferulic Acid Decarboxylases

(24) The pETDuet™-1 co-expression vectors encoding Hypocrea atroviridis Ferulic acid 1 decarboxylase WT or T405M variant and the Flavin prenyltransferase UbiX protein from E. coli were obtained according to the procedure described in Example 2 and 3.

(25) The provided vector contained a stretch of 6 histidine codons after the methionine initiation codon of the ferulic acid decarboxylases in order to specifically purify the ferulic acid decarboxylase.

(26) Competent E. coli BL21 (DE3) cells (Novagen) were transformed with these vectors according to standard heat shock procedures. The transformed cells were grown with shaking (160 rpm) using ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234) at 30° C. during 24 h. The cells were collected by centrifugation at 4° C., 10,000 rpm for 20 mM and the pellets were stored at −80° C. Pellets from 500 ml of culture cells were thawed on ice and resuspended in 15 ml of 50 mM potassium phosphate buffer containing 200 mM NaCl, 10 mM MgCl.sub.2, 10 mM imidazole and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 2×15 seconds.

(27) The bacterial extracts were then clarified by centrifugation at 4° C., 4000 rpm for 40 min. The clarified bacterial lysates were loaded onto a PROTINO-2000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 6 ml of 50 mM potassium phosphate buffer containing 250 mM imidazole. Eluates were then concentrated, desalted on a Amicon Ultra-4 10 kDa filter unit (Millipore) and enzymes were resuspended in 50 mM potassium phosphate buffer containing 1 mM DTT and 20 mM NaCl. The purity of Ferulic acid decarboxylases thus purified varied from 80% to 90% as estimated by SDS-PAGE analysis. Protein concentrations were determined by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific) or by a Bradford assay (BioRad).

(28) Production of Flavin Prenyltransferases

(29) In order to perform enzymatic assays, the Flavin prenyltransferase UbiX protein from E. coli was also produced separately. Vector pCAN containing the gene coding for the Flavin prenyltransferase UbiX protein from E. coli was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection).

(30) Competent E. coli BL21 (DE3) cells (Novagen) were transformed with this vector according to standard heat shock procedures. The transformed cells were grown with shaking (160 rpm) using ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234) at 30° C. during 24 h. The cells were collected by centrifugation at 4° C., 10,000 rpm for 20 min and the pellets were stored at −80° C. Pellets from 500 ml of culture cells were thawed on ice and resuspended in 15 ml of 50 mM potassium phosphate buffer containing 200 mM NaCl, 10 mM MgCl.sub.2, 10 mM imidazole and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 2×15 seconds. The cellular lysate containing the UbiX protein was kept on ice.

(31) Enzymatic Assay

(32) Enzymatic assays were performed with Ferulic acid decarboxylase purified as described above, supplemented with a fresh cellular lysate containing the UbiX protein (see above).

(33) 0.5 M stock solution of 3-methylcrotonic acid was prepared in water and adjusted to pH 7.0 with 10 M solution of NaOH. Enzymatic assays were carried out in 2 ml glass vials (Interchim) under the following conditions: 50 mM potassium phosphate pH 7.5; 20 mM NaCl; 3 mM MgCl.sub.2; 5 mM DTT; 64 mM 3-methylcrotonic acid; 0.5 mg/ml purified of Ferulic acid decarboxylase (FDC) WT or T405M variant; 100 μl of the lysate contained the Flavin prenyltransferase UbiX protein; total volume of the assays were 300 μl.

(34) The vials were sealed and incubated for 60 minutes at 30° C. A control without Ferulic acid decarboxylase was performed in parallel. The assays were stopped by incubating for 2 minute at 80° C. and the isobutene formed in the reaction headspace was analysed by Gas Chromatography (GC) equipped with a Flame Ionization Detector (FID). For the GC headspace analysis, one ml of the headspace gas was separated in a Bruker GC-450 system equipped with a GS-alumina column (30 m×0.53 mm) (Agilent) using isothermal mode at 130° C. Nitrogen was used as carrier gas with a flow rate of 6 ml/min. The enzymatic reaction product was identified by comparison with an isobutene standard. Under these GC conditions, the retention time of isobutene was 2.42 min.

(35) Results

(36) Under these conditions, the T405M variant of Hypocrea atroviridis Ferulic acid decarboxylase 1 is about 7 time more efficient than the WT corresponding enzyme for the conversion of 3-methylcrotonic acid into isobutene; see Table 3.

(37) TABLE-US-00004 TABLE 3 Isobutene peak area, arbitrary unit FDC WT 1680 FDC T405M 11550 without FDC 2

Example 6

Identification of a Signature Sequence Around Position 405 of Hypocrea atroviridis Ferulic Acid Decarboxylase 1 (SEQ ID NO:1)

(38) The two variants of Hypocrea atroviridis Ferulic acid decarboxylase 1 (SEQ ID NO:1) having the highest increase in activity harbor a mutation at position 405.

(39) Hypocrea atroviridis belongs to the Pezizomycotina subphylum of the Ascomycota phylum. Thus, the Hypocrea atroviridis Ferulic acid decarboxylase has been compared with 1 to 19 homologue proteins belonging to the Pezizomycotina subphylum (Table 4) which are capable of catalyzing the conversion of 3-methylcrotonic acid into isobutene.

(40) These sequences have been compared in a multiple-sequence alignment using the T-Coffee software in “accurate” mode, combining sequence, structure and profile alignment (Notredame et al., JMB 302 (2000), 205-217). From this alignment, a conserved region around the T405 position of the Hypocrea atroviridis Ferulic acid decarboxylase 1 has been derived (K401-G413); (FIG. 2).

(41) This block of aligned sequences was used to derive a pattern with the PRATT software (Jonassen et al., Protein Science 4(8) (1995), 1587-1595):

(42) K-[APV]-G-x-[APT]-[FIM]-H-R-[IL]-[AILV]-[ILV]-x-G (SEQ ID NO:16) where x represents any amino acid and [XYZ] represents either amino acid X or Y or Z.

(43) This motif was then fed into the ScanProsite webtool (prosite.expasy.org/scanprosite/) to scan the UniProtKB database (Swiss-Prot including splice variants and TrEMBL). A total of 107 different proteins was found, including the original 20 from which the motif was derived (Table 5).

(44) Among these 107 proteins, 3 are annotated as 3-octaprenyl-4-hydroxybenzoate carboxylyase, one as Phenolic acid decarboxylase, one as UbiD-domain-containing protein, 3 as Uncharacterized protein and 99 as Ferulic acid decarboxylase 1. Considering that these annotations represent the decarboxylation of aromatic acids, it is assumed that this motif is representative for the UbiD family.

(45) Moreover, among these 107 proteins, 105 belong to the Dikarya sub-kingdom while 19 of these belong to the Filobasidiella/Cryptococcus neoformans species complex of the Agaricomycotina subphylum, 85 to the Pezizomycotina subphylum, one to the Saccharomycotina subphylum and two to the Phytophthora genus of the Peronosporales order.

(46) Therefore, it is assumed that this motif is not only representative for the UbiD family of the Pezizomycotina subphylum from which it was derived, but also for the UbiD family of different fungi and fungus-like eukaryotic microorganisms.

(47) TABLE-US-00005 TABLE 4 List of 19 proteins homologues to Hypocrea atroviridis Ferulic acid decarboxylase 1 (G9NLP8) presenting a 3-methylcrotonic acid decarboxylase activity Entry (UniProt) Entry name Protein names Gene names A0A094IED9 A0A094IED9_9PEZI Ferulic acid decarboxylase 1 (EC FDC1 V502_01403 4.1.1.102) (Phenacrylate decarboxylase) W9WWR1 W9WWR1_9EURO Ferulic acid decarboxylase 1 (EC FDC1 A1O5_04852 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2IKD5 A0A0D2IKD5_XYLBA Ferulic acid decarboxylase 1 (EC FDC1 Z519_02676 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2AQI6 A0A0D2AQI6_9EURO Ferulic acid decarboxylase 1 (EC FDC1 PV07_07106 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2DPQ1 A0A0D2DPQ1 _9EURO Ferulic acid decarboxylase 1 (EC FDC1 PV04_09173 4.1.1.102) (Phenacrylate decarboxylase) W9YNA8 W9YNA8_9EURO Ferulic acid decarboxylase 1 (EC FDC1 A1O1_03830 4.1.1.102) (Phenacrylate decarboxylase) M7THT1 M7THT1_BOTF1 Ferulic acid decarboxylase 1 (EC FDC1 BcDW1_8299 4.1.1.102) (Phenacrylate decarboxylase) M3DF95 M3DF95_SPHMS Ferulic acid decarboxylase 1 (EC FDC1 SEPMUDRAFT_154815 4.1.1.102) (Phenacrylate decarboxylase) F0XL98 F0XL98_GROCL Ferulic acid decarboxylase 1 (EC FDC1 CMQ_6352 4.1.1.102) (Phenacrylate decarboxylase) W9LTH3 W9LTH3_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOWG_11397 4.1.1.102) (Phenacrylate decarboxylase) A2QHE5 FDC1_ASPNC Ferulic acid decarboxylase 1 (EC fdc1 An03g06590 4.1.1.102) (Phenacrylate decarboxylase) A0A0G4P429 A0A0G4P429_PENCA Ferulic acid decarboxylase 1 (EC FDC1 4.1.1.102) (Phenacrylate decarboxylase) PCAMFM013_S005g000265 W6QKP7 W6QKP7_PENRF Ferulic acid decarboxylase 1 (EC FDC1 4.1.1.102) (Phenacrylate decarboxylase) PROQFM164_S05g000853 A1DCG7 A1DCG7_NEOFI Ferulic acid decarboxylase 1 (EC FDC1 NFIA_026010 4.1.1.102) (Phenacrylate decarboxylase) A0A0F0IHE5 A0A0F0IHE5_ASPPU Ferulic acid decarboxylase 1 (EC FDC1 P875_00128011 4.1.1.102) (Phenacrylate decarboxylase) G9MUK3 G9MUK3_HYPVG Ferulic acid decarboxylase 1 (EC FDC1 TRIVIDRAFT_53354 4.1.1.102) (Phenacrylate decarboxylase) G9NTM9 G9NTM9_HYPAI Ferulic acid decarboxylase 1 (EC FDC1 TRIATDRAFT_299540 4.1.1.102) (Phenacrylate decarboxylase) G9NLP8 G9NLP8_HYPAI Ferulic acid decarboxylase 1 (EC FDC1 TRIATDRAFT_53567 4.1.1.102) (Phenacrylate decarboxylase) G9MXT8 G9MXT8_HYPVG Ferulic acid decarboxylase 1 (EC FDC1 TRIVIDRAFT_69398 4.1.1.102) (Phenacrylate decarboxylase) A0A0G0A274 A0A0G0A274_TRIHA Ferulic acid decarboxylase 1 (EC FDC1 THAR02_01458 4.1.1.102) (Phenacrylate decarboxylase) Entry (UniProt) Organism Length A0A094IED9 Pseudogymnoascus sp. VKM F-4520 (FW-2644) 589 W9WWR1 Cladophialophora psammophila CBS 110553 503 A0A0D2IKD5 Cladophialophora bantiana CBS 173.52 503 A0A0D2AQI6 Cladophialophora immunda 505 A0A0D2DPQ1 Capronia semiimmersa 499 W9YNA8 Capronia coronata CBS 617.96 498 M7THT1 Botryotinia fuckeliana (strain BcDW1) (Noble rot 513 fungus) (Botrytis cinerea) M3DF95 Sphaerulina musiva (strain SO2202) (Poplar stem 508 canker fungus) (Septoria musiva) F0XL98 Grosmannia clavigera (strain kw1407/UAMH 11150) 500 (Blue stain fungus) (Graphiocladiella clavigera) W9LTH3 Fusarium oxysporum f. sp. lycopersici MN25 503 A2QHE5 Aspergillus niger (strain CBS 513.88/FGSC A1513) 500 A0A0G4P429 Penicillium camemberti FM 013 500 W6QKP7 Penicillium roqueforti (strain FM164) 498 A1DCG7 Neosartorya fischeri (strain ATCC 1020/DSM 3700/ 505 FGSC A1164/NRRL 181) (Aspergillus fischerianus) A0A0F0IHE5 Aspergillus parasiticus (strain ATCC 56775/NRRL 503 5862/SRRC 143/SU-1) G9MUK3 Hypocrea virens (strain Gv29-8/FGSC 10586) 507 (Gliocladium virens) (Trichoderma virens) G9NTM9 Hypocrea atroviridis (strain ATCC 20476/IMI 510 206040) (Trichoderma atroviride) G9NLP8 Hypocrea atroviridis (strain ATCC 20476/IMI 512 206040) (Trichoderma atroviride) G9MXT8 Hypocrea virens (strain Gv29-8/FGSC 10586) 511 (Gliocladium virens) (Trichoderma virens) A0A0G0A274 Trichoderma harzianum (Hypocrea lixii) 511

(48) TABLE-US-00006 TABLE 5 List of the 107 proteins found in the UniProtKB database through ScanProsite, with the K-[APV]-G-x-[APT]-[FIM]-H-R-[IL]-[AILV]-[ILV]-x-G (SEQ ID NO: 16) motif. Entry (UniProt) Entry name Protein names Gene names A0A0B4FU01 A0A0B4FU01 _9HYPO 3-octaprenyl-4-hydroxybenzoate MBR_10525 carboxylyase (Fragment) A0A0B4ENF4 A0A0B4ENF4_METAN 3-octaprenyl-4-hydroxybenzoate MAN_07756 carboxylyase (Fragment) A0A0B8N4Y5 A0A0B8N4Y5_9EURO 3-octaprenyl-4-hydroxybenzoate TCE0_047r17842 carboxylyase A2QHE5 FDC1_ASPNC Ferulic acid decarboxylase 1 (EC fdc1 An03g06590 4.1.1.102) (Phenacrylate decarboxylase) A0A0A2J5F4 A0A0A2J5F4_PENEN Ferulic acid decarboxylase 1 (EC FDC1 PEX1_019400 4.1.1.102) (Phenacrylate decarboxylase) PEX2_105470 PEXP_077520 A0A0D2YAR9 A0A0D2YAR9_FUSO4 Ferulic acid decarboxylase 1 (EC FDC1 FOXG_13395 4.1.1.102) (Phenacrylate decarboxylase) A0A0J0DBQ6 A0A0J0DBQ6_GIBFU Ferulic acid decarboxylase 1 (EC FDC1 LW94_13187 Y057_969 4.1.1.102) (Phenacrylate decarboxylase) R1EM06 R1EM06_BOTPV Ferulic acid decarboxylase 1 (EC FDC1 UCRNP2_4413 4.1.1.102) (Phenacrylate decarboxylase) A0A0G4P429 A0A0G4P429_PENCA Ferulic acid decarboxylase 1 (EC FDC1 4.1.1.102) (Phenacrylate decarboxylase) PCAMFM013_S005g000265 A0A014P6U4 A0A014P6U4_9HYPO Ferulic acid decarboxylase 1 (EC FDC1 X797_008675 4.1.1.102) (Phenacrylate decarboxylase) K9FXI0 K9FXI0_PEND1 Ferulic acid decarboxylase 1 (EC FDC1 PDIP_44190 4.1.1.102) (Phenacrylate decarboxylase) I8A854 I8A854_ASPO3 Ferulic acid decarboxylase 1 (EC FDC1 Ao3042_02507 4.1.1.102) (Phenacrylate decarboxylase) F9FQB3 F9FQB3_FUSOF Ferulic acid decarboxylase 1 (EC FDC1 FOXB_08593 4.1.1.102) (Phenacrylate decarboxylase) A0A0B7K683 A0A0B7K683_BIOOC Ferulic acid decarboxylase 1 (EC FDC1 BN869_000007130_1 4.1.1.102) (Phenacrylate decarboxylase) A0A0F9X6G5 A0A0F9X6G5_TRIHA Ferulic acid decarboxylase 1 (EC FDC1 THAR02_07744 4.1.1.102) (Phenacrylate decarboxylase) A0A064B9K3 A0A064B9K3_ASPOZ Ferulic acid decarboxylase 1 (EC FDC1 AO1008_09897 4.1.1.102) (Phenacrylate decarboxylase) A0A0G0A274 A0A0G0A274_TRIHA Ferulic acid decarboxylase 1 (EC FDC1 THAR02_01458 4.1.1.102) (Phenacrylate decarboxylase) A0A0D9QCX6 A0A0D9QCX6_METAN Ferulic acid decarboxylase 1 (EC FDC1 H633G_00051 4.1.1.102) (Phenacrylate decarboxylase) H1VUR4 H1VUR4_COLHI Ferulic acid decarboxylase 1 (EC FDC1 CH063_03208 4.1.1.102) (Phenacrylate decarboxylase) E6R9Z1 E6R9Z1_CRYGW Ferulic acid decarboxylase 1 (EC FDC1 CGB_G5620W 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0U0M0 A0A0D0U0M0_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I313_01913 4.1.1.102) (Phenacrylate decarboxylase) G9MUK3 G9MUK3_HYPVG Ferulic acid decarboxylase 1 (EC FDC1 TRIVIDRAFT_53354 4.1.1.102) (Phenacrylate decarboxylase) G9MXT8 G9MXT8_HYPVG Ferulic acid decarboxylase 1 (EC FDC1 TRIVIDRAFT_69398 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0T0X7 A0A0D0T0X7_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I309_02848 4.1.1.102) (Phenacrylate decarboxylase) W6QKP7 W6QKP7_PENRF Ferulic acid decarboxylase 1 (EC FDC1 4.1.1.102) (Phenacrylate decarboxylase) PROQFM164_S05g000853 A0A060T4A6 A0A060T4A6_BLAAD Ferulic acid decarboxylase 1 (EC FDC1 4.1.1.102) (Phenacrylate decarboxylase) GNLVRS02_ARAD1A15180g W9LTH3 W9LTH3_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOWG_11397 4.1.1.102) (Phenacrylate decarboxylase) W9LC23 W9LC23_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOWG_16734 4.1.1.102) (Phenacrylate decarboxylase) W3XQA0 W3XQA0_9PEZI Ferulic acid decarboxylase 1 (EC FDC1 PFICI_01973 4.1.1.102) (Phenacrylate decarboxylase) A0A0F0IHE5 A0A0F0IHE5_ASPPU Ferulic acid decarboxylase 1 (EC FDC1 P875_00128011 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2JJA4 A0A0D2JJA4_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I305_01613 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0Y3D9 A0A0D0Y3D9_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I306_05467 4.1.1.102) (Phenacrylate decarboxylase) W9WWR1 W9WWR1_9EURO Ferulic acid decarboxylase 1 (EC FDC1 A1O5_04852 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2DPQ1 A0A0D2DPQ1 _9EURO Ferulic acid decarboxylase 1 (EC FDC1 PV04_09173 4.1.1.102) (Phenacrylate decarboxylase) W9ZFW9 W9ZFW9_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOMG_16250 4.1.1.102) (Phenacrylate decarboxylase) X0AT48 X0AT48_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOMG_09469 4.1.1.102) (Phenacrylate decarboxylase) G7XVA2 G7XVA2_ASPKW Ferulic acid decarboxylase 1 (EC FDC1 AKAW_08975 4.1.1.102) (Phenacrylate decarboxylase) N4TMS4 N4TMS4_FUSC1 Ferulic acid decarboxylase 1 (EC FDC1 FOC1_g10005647 4.1.1.102) (Phenacrylate decarboxylase) G3Y7U5 G3Y7U5_ASPNA Ferulic acid decarboxylase 1 (EC FDC1 ASPNIDRAFT_44615 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0XAE5 A0A0D0XAE5_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I314_01406 4.1.1.102) (Phenacrylate decarboxylase) A0A0J0BVX6 A0A0J0BVX6_GIBFU Ferulic acid decarboxylase 1 (EC FDC1 LW93_9666 4.1.1.102) (Phenacrylate decarboxylase) W9YNA8 W9YNA8_9EURO Ferulic acid decarboxylase 1 (EC FDC1 A1O1_03830 4.1.1.102) (Phenacrylate decarboxylase) M7THT1 M7THT1_BOTF1 Ferulic acid decarboxylase 1 (EC FDC1 BcDW1_8299 4.1.1.102) (Phenacrylate decarboxylase) X0MQQ9 X0MQQ9_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOTG_09622 4.1.1.102) (Phenacrylate decarboxylase) X0FLR4 X0FLR4_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOCG_08056 4.1.1.102) (Phenacrylate decarboxylase) X0BC97 X0BC97_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOQG_15695 4.1.1.102) (Phenacrylate decarboxylase) X0HNC2 X0HNC2_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOPG_11348 4.1.1.102) (Phenacrylate decarboxylase) X0J4B9 X0J4B9_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOIG_11524 4.1.1.102) (Phenacrylate decarboxylase) A0A0D9NTQ8 A0A0D9NTQ8_METAN Ferulic acid decarboxylase 1 (EC FDC1 H634G_07518 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0YJS6 A0A0D0YJS6_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 L804_03800 4.1.1.102) (Phenacrylate decarboxylase) E9ENN2 E9ENN2_METRA Ferulic acid decarboxylase 1 (EC FDC1 MAA_01631 4.1.1.102) (Phenacrylate decarboxylase) G2XWX0 G2XWX0_BOTF4 Ferulic acid decarboxylase 1 (EC FDC1 BofuT4_P051470.1 4.1.1.102) (Phenacrylate decarboxylase) A0A0F7U117 A0A0F7U117_9EURO Ferulic acid decarboxylase 1 (EC FDC1 PMG11_09885 4.1.1.102) (Phenacrylate decarboxylase) A0A010QFR6 A0A010QFR6_9PEZI Ferulic acid decarboxylase 1 (EC FDC1 CFIO01_10372 4.1.1.102) (Phenacrylate decarboxylase) K9FG02 K9FG02_PEND2 Ferulic acid decarboxylase 1 (EC FDC1 PDIG_73710 4.1.1.102) (Phenacrylate decarboxylase) A0A0G2EQF2 A0A0G2EQF2_9EURO Ferulic acid decarboxylase 1 (EC FDC1 UCRPC4_g02163 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2AQI6 A0A0D2AQI6_9EURO Ferulic acid decarboxylase 1 (EC FDC1 PV07_07106 4.1.1.102) (Phenacrylate decarboxylase) S0E299 S0E299_GIBF5 Ferulic acid decarboxylase 1 (EC FDC1 FFUJ_14803 4.1.1.102) (Phenacrylate decarboxylase) B8NJ67 B8NJ67_ASPFN Ferulic acid decarboxylase 1 (EC FDC1 AFLA_064990 4.1.1.102) (Phenacrylate decarboxylase) K2RUE8 K2RUE8_MACPH Ferulic acid decarboxylase 1 (EC FDC1 MPH_04225 4.1.1.102) (Phenacrylate decarboxylase) A1DCG7 A1DCG7_NEOFI Ferulic acid decarboxylase 1 (EC FDC1 NFIA_026010 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2IKD5 A0A0D2IKD5_XYLBA Ferulic acid decarboxylase 1 (EC FDC1 Z519_02676 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0VRV5 A0A0D0VRV5_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I310_03088 4.1.1.102) (Phenacrylate decarboxylase) A0A017SAW2 A0A017SAW2_9EURO Ferulic acid decarboxylase 1 (EC FDC1 EURHEDRAFT_503163 4.1.1.102) (Phenacrylate decarboxylase) W9JNI1 W9JNI1_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOZG_15347 4.1.1.102) (Phenacrylate decarboxylase) M3DF95 M3DF95_SPHMS Ferulic acid decarboxylase 1 (EC FDC1 SEPMUDRAFT_154815 4.1.1.102) (Phenacrylate decarboxylase) W9JP63 W9JP63_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOZG_14585 4.1.1.102) (Phenacrylate decarboxylase) A0A094IED9 A0A094IED9_9PEZI Ferulic acid decarboxylase 1 (EC FDC1 V502_01403 4.1.1.102) (Phenacrylate decarboxylase) B6HRC8 B6HRC8_PENRW Ferulic acid decarboxylase 1 (EC FDC1 Pc22g03390 4.1.1.102) (Phenacrylate decarboxylase) PCH_Pc22g03390 W7MPM7 W7MPM7_GIBM7 Ferulic acid decarboxylase 1 (EC FDC1 FVEG_11829 4.1.1.102) (Phenacrylate decarboxylase) G9NLP8 G9NLP8_HYPAI Ferulic acid decarboxylase 1 (EC FDC1 TRIATDRAFT_53567 4.1.1.102) (Phenacrylate decarboxylase) G9NTM9 G9NTM9_HYPAI Ferulic acid decarboxylase 1 (EC FDC1 TRIATDRAFT_299540 4.1.1.102) (Phenacrylate decarboxylase) G9P0U1 G9P0U1_HYPAI Ferulic acid decarboxylase 1 (EC FDC1 TRIATDRAFT_35115 4.1.1.102) (Phenacrylate decarboxylase) T0K816 T0K816_COLGC Ferulic acid decarboxylase 1 (EC FDC1 CGLO_ 11546 4.1.1.102) (Phenacrylate decarboxylase) N1RLH9 N1RLH9_FUSC4 Ferulic acid decarboxylase 1 FOC4_g10005518 N1RYW4 N1RYW4_FUSC4 Ferulic acid decarboxylase 1 (EC FDC1 FOC4_g10005520 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0VVI5 A0A0D0VVI5_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I312_01590 4.1.1.102) (Phenacrylate decarboxylase) F0XL98 F0XE98_GROCL Ferulic acid decarboxylase 1 (EC FDC1 CMQ_6352 4.1.1.102) (Phenacrylate decarboxylase) F0XKQ3 F0XKQ3_GROCL Ferulic acid decarboxylase 1 (EC FDC1 CMQ_8261 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0U7G3 A0A0D0U7G3_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I315_02568 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0X8C8 A0A0D0X8C8_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I304_04581 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0X028 A0A0D0X028_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I308_01018 4.1.1.102) (Phenacrylate decarboxylase) W9HNN8 W9HNN8_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOYG_14553 4.1.1.102) (Phenacrylate decarboxylase) A0A0D9M9B3 A0A0D9M9B3_9EURO Ferulic acid decarboxylase 1 (EC FDC1 U727_00431480381 4.1.1.102) (Phenacrylate decarboxylase) W9HU82 W9HU82_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOYG_10553 4.1.1.102) (Phenacrylate decarboxylase) C7ZIA7 C7ZIA7_NECH7 Ferulic acid decarboxylase 1 (EC FDC1 NECHADRAFT_52948 4.1.1.102) (Phenacrylate decarboxylase) C7ZC09 C7ZC09_NECH7 Ferulic acid decarboxylase 1 (EC FDC1 NECHADRAFT_73218 4.1.1.102) (Phenacrylate decarboxylase) A0A0D0TEU3 A0A0D0TEU3_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I352_04272 4.1.1.102) (Phenacrylate decarboxylase) A0A095C6V3 A0A095C6V3_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 CNBG_2095 4.1.1.102) (Phenacrylate decarboxylase) L2G6I9 L2G6I9_COLGN Ferulic acid decarboxylase 1 (EC FDC1 CGGC5_6473 4.1.1.102) (Phenacrylate decarboxylase) W9P9H8 W9P9H8_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOVG_12484 4.1.1.102) (Phenacrylate decarboxylase) W9NGN5 W9NGN5_FUSOX Ferulic acid decarboxylase 1 (EC FDC1 FOVG_19288 4.1.1.102) (Phenacrylate decarboxylase) A0A086NHZ2 A0A086NHZ2_METAN Ferulic acid decarboxylase 1 (EC FDC1 MANI_006214 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2LFN2 A0A0D2LFN2_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I307_00579 4.1.1.102) (Phenacrylate decarboxylase) T2BN22 T2BN22_CRYNH Ferulic acid decarboxylase 1 (EC FDC1 CNAG_03519 4.1.1.102) (Phenacrylate decarboxylase) T2BN40 T2BN40_CRYNH Ferulic acid decarboxylase 1 (EC FDC1 CNAG_03519 4.1.1.102) (Phenacrylate decarboxylase) J9VVU7 J9VVU7_CRYNH Ferulic acid decarboxylase 1 (EC FDC1 CNAG_03519 4.1.1.102) (Phenacrylate decarboxylase) S3D5R7 S3D5R7_OPHP1 Ferulic acid decarboxylase 1 (EC FDC1 F503_04312 4.1.1.102) (Phenacrylate decarboxylase) Q2UP67 Q2UP67_ASPOR Ferulic acid decarboxylase 1 (EC FDC1 AO090001000093 4.1.1.102) (Phenacrylate decarboxylase) A0A0D2WZP7 A0A0D2WZP7_CRYGA Ferulic acid decarboxylase 1 (EC FDC1 I311_00741 4.1.1.102) (Phenacrylate decarboxylase) G4YRJ8 G4YRJ8_PHYSP Ferulic acid decarboxylase 1 (EC FDC1 PHYSODRAFT_258069 4.1.1.102) (Phenacrylate decarboxylase) H3H3G9 H3H3G9_PHYRM Ferulic acid decarboxylase 1 (EC FDC1 4.1.1.102) (Phenacrylate decarboxylase) A0A0A0WBD5 A0A0A0WBD5_9PEZI Phenolic acid decarboxylase (Fragment) padB3 Q8J0Q7 Q8J0Q7_NECHA Putative uncharacterized protein (Fragment) A0A074VCH2 A0A074VCH2_9PEZI UbiD-domain-containing protein M437DRAFT_79329 A0A0D2BJ27 A0A0D2BJ27_9EURO Uncharacterized protein PV08_02858 W9NFR4 W9NFR4_FUSOX Uncharacterized protein FOVG_17226 Entry (UniProt) Organism Length A0A0B4FU01 Metarhizium brunneum ARSEF 3297 500 A0A0B4ENF4 Metarhizium anisopliae ARSEF 549 500 A0A0B8N4Y5 Talaromyces cellulolyticus 130 A2QHE5 Aspergillus niger (strain CBS 513.88/FGSC 500 A1513) A0A0A2J5F4 Penicillium expansum (Blue mold rot fungus) 500 A0A0D2YAR9 Fusarium oxysporum f. sp. lycopersici (strain 4287/ 503 CBS 123668/FGSC 9935/NRRL 34936) (Fusarium vascular wilt of tomato) A0A0J0DBQ6 Gibberella fujikuroi (Bakanae and foot rot disease 503 fungus) (Fusarium fujikuroi) R1EM06 Botryosphaeria parva (strain UCR-NP2) (Grapevine 495 canker fungus) (Neofusicoccum parvum) A0A0G4P429 Penicillium camemberti FM 013 500 A0A014P6U4 Metarhizium robertsii 508 K9FXI0 Penicillium digitatum (strain Pd1/CECT 20795) 499 (Green mold) I8A854 Aspergillus oryzae (strain 3.042) (Yellow koji mold) 503 F9FQB3 Fusarium oxysporum (strain Fo5176) (Fusarium 506 vascular wilt) A0A0B7K683 Bionectria ochroleuca (Gliocladium roseum) 496 A0A0F9X6G5 Trichoderma harzianum (Hypocrea lixii) 495 A0A064B9K3 Aspergillus oryzae 100-8 503 A0A0G0A274 Trichoderma harzianum (Hypocrea lixii) 511 A0A0D9QCX6 Metarhizium anisopliae BRIP 53284 500 H1VUR4 Colletotrichum higginsianum (strain IMI 349063) 506 (Crucifer anthracnose fungus) E6R9Z1 Cryptococcus gattii serotype B (strain WM276/ 435 ATCC MYA-4071) (Filobasidiella gattii) (Cryptococcus bacillisporus) A0A0D0U0M0 Cryptococcus gattii Ram5 501 G9MUK3 Hypocrea virens (strain Gv29-8/FGSC 10586) 507 (Gliocladium virens) (Trichoderma virens) G9MXT8 Hypocrea virens (strain Gv29-8/FGSC 10586) 511 (Gliocladium virens) (Trichoderma virens) A0A0D0T0X7 Cryptococcus gattii LA55 501 W6QKP7 Penicillium roqueforti (strain FM164) 498 A0A060T4A6 Blastobotrys adeninivorans (Yeast) (Arxula 497 adeninivorans) W9LTH3 Fusarium oxysporum f. sp. lycopersici MN25 503 W9LC23 Fusarium oxysporum f. sp. lycopersici MN25 503 W3XQA0 Pestalotiopsis fici W106-1 501 A0A0F0IHE5 Aspergillus parasiticus (strain ATCC 56775/NRRL 503 5862/SRRC 143/SU-1) A0A0D2JJA4 Cryptococcus gattii E566 435 A0A0D0Y3D9 Cryptococcus gattii EJB2 435 W9WWR1 Cladophialophora psammophila CBS 110553 503 A0A0D2DPQ1 Capronia semiimmersa 499 W9ZFW9 Fusarium oxysporum f. sp. melonis 26406 503 X0AT48 Fusarium oxysporum f. sp. melonis 26406 503 G7XVA2 Aspergillus kawachii (strain NBRC 4308) (White 500 koji mold) (Aspergillus awamori var. kawachi) N4TMS4 Fusarium oxysporum f. sp. cubense (strain race 1) 503 (Panama disease fungus) G3Y7U5 Aspergillus niger (strain ATCC 1015/CBS 113.46/ 500 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) A0A0D0XAE5 Cryptococcus gattii CA1873 435 A0A0J0BVX6 Gibberella fujikuroi (Bakanae and foot rot disease 503 fungus) (Fusarium fujikuroi) W9YNA8 Capronia coronata CBS 617.96 498 M7THT1 Botryotinia fuckeliana (strain BcDW1) (Noble rot 513 fungus) (Botrytis cinerea) X0MQQ9 Fusarium oxysporum f. sp. vasinfectum 25433 503 X0FLR4 Fusarium oxysporum f. sp. radicis-lycopersici 26381 503 X0BC97 Fusarium oxysporum f. sp. raphani 54005 503 X0HNC2 Fusarium oxysporum f. sp. conglutinans race 2 503 54008 X0J4B9 Fusarium oxysporum f. sp. cubense tropical race 4 503 54006 A0A0D9NTQ8 Metarhizium anisopliae BRIP 53293 500 A0A0D0YJS6 Cryptococcus gattii 2001/935-1 501 E9ENN2 Metarhizium robertsii (strain ARSEF 23/ATCC 500 MYA-3075) (Metarhizium anisopliae (strain ARSEF 23)) G2XWX0 Botryotinia fuckeliana (strain T4) (Noble rot fungus) 513 (Botrytis cinerea) A0A0F7U117 Penicillium brasilianum 503 A0A010QFR6 Colletotrichum fioriniae PJ7 503 K9FG02 Penicillium digitatum (strain PHI26/CECT 20796) 499 (Green mold) A0A0G2EQF2 Phaeomoniella chlamydospora 498 A0A0D2AQI6 Cladophialophora immunda 505 S0E299 Gibberella fujikuroi (strain CBS 195.34/IMI 58289/ 503 NRRL A-6831) (Bakanae and foot rot disease fungus) (Fusarium fujikuroi) B8NJ67 Aspergillus flavus (strain ATCC 200026/FGSC 503 A1120/NRRL 3357/JCM 12722/SRRC 167) K2RUE8 Macrophomina phaseolina (strain MS6) (Charcoal 494 rot fungus) A1DCG7 Neosartorya fischeri (strain ATCC 1020/DSM 505 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) A0A0D2IKD5 Cladophialophora bantiana CBS 173.52 503 A0A0D0VRV5 Cryptococcus gattii CA1014 501 A0A017SAW2 Aspergillus ruber CBS 135680 500 W9JNI1 Fusarium oxysporum Fo47 503 M3DF95 Sphaerulina musiva (strain SO2202) (Poplar stem 508 canker fungus) (Septoria musiva) W9JP63 Fusarium oxysporum Fo47 503 A0A094IED9 Pseudogymnoascus sp. VKM F-4520 (FW-2644) 589 B6HRC8 Penicillium rubens (strain ATCC 28089/DSM 500 1075/NRRL 1951/Wisconsin 54-1255) (Penicillium chrysogenum) W7MPM7 Gibberella moniliformis (strain M3125/FGSC 503 7600) (Maize ear and stalk rot fungus) (Fusarium verticillioides) G9NLP8 Hypocrea atroviridis (strain ATCC 20476/IMI 512 206040) (Trichoderma atroviride) G9NTM9 Hypocrea atroviridis (strain ATCC 20476/IMI 510 206040) (Trichoderma atroviride) G9P0U1 Hypocrea atroviridis (strain ATCC 20476/IMI 507 206040) (Trichoderma atroviride) T0K816 Colletotrichum gloeosporioides (strain Cg-14) 506 (Anthracnose fungus) (Glomerella cingulata) N1RLH9 Fusarium oxysporum f. sp. cubense (strain race 4) 167 (Panama disease fungus) N1RYW4 Fusarium oxysporum f. sp. cubense (strain race 4) 503 (Panama disease fungus) A0A0D0VVI5 Cryptococcus gattii CA1280 435 F0XL98 Grosmannia clavigera (strain kw1407/UAMH 500 11150) (Blue stain fungus) (Graphiocladiella clavigera) F0XKQ3 Grosmannia clavigera (strain kw1407/UAMH 482 11150) (Blue stain fungus) (Graphiocladiella clavigera) A0A0D0U7G3 Cryptococcus gattii Ru294 435 A0A0D0X8C8 Cryptococcus gattii CBS 10090 501 A0A0D0X028 Cryptococcus gattii IND107 435 W9HNN8 Fusarium oxysporum FOSC 3-a 503 A0A0D9M9B3 Penicillium solitum 500 W9HU82 Fusarium oxysporum FOSC 3-a 503 C7ZIA7 Nectria haematococca (strain 77-13-4/ATCC 474 MYA-4622/FGSC 9596/MPVI) (Fusarium solani subsp. pisi) C7ZC09 Nectria haematococca (strain 77-13-4/ATCC 489 MYA-4622/FGSC 9596/MPVI) (Fusarium solani subsp. pisi) A0A0D0TEU3 Cryptococcus gattii MMRL2647 501 A0A095C6V3 Cryptococcus gattii R265 501 L2G6I9 Colletotrichum gloeosporioides (strain Nara gc5) 506 (Anthracnose fungus) (Glomerella cingulata) W9P9H8 Fusarium oxysporum f. sp. pisi HDV247 503 W9NGN5 Fusarium oxysporum f. sp. pisi HDV247 517 A0A086NHZ2 Metarhizium anisopliae (Entomophthora anisopliae) 500 A0A0D2LFN2 Cryptococcus gattii 99/473 501 T2BN22 Cryptococcus neoformans var. grubii serotype A 405 (strain H99/ATCC 208821/CBS 10515/FGSC 9487) (Filobasidiella neoformans var. grubii) T2BN40 Cryptococcus neoformans var. grubii serotype A 447 (strain H99/ATCC 208821/CBS 10515/FGSC 9487) (Filobasidiella neoformans var. grubii) J9VVU7 Cryptococcus neoformans var. grubii serotype A 532 (strain H99/ATCC 208821/CBS 10515/FGSC 9487) (Filobasidiella neoformans var. grubii) S3D5R7 Ophiostoma piceae (strain UAMH 11346) (Sap stain 500 fungus) Q2UP67 Aspergillus oryzae (strain ATCC 42149/RIB 40) 503 (Yellow koji mold) A0A0D2WZP7 Cryptococcus gattii NT-10 435 G4YRJ8 Phytophthora sojae (strain P6497) (Soybean stem 468 and root rot agent) (Phytophthora megasperma f. sp. glycines) H3H3G9 Phytophthora ramorum (Sudden oak death agent) 545 A0A0A0WBD5 Phomopsis liquidambaris 180 Q8J0Q7 Nectria haematococca 489 A0A074VCH2 Aureobasidium melanogenum CBS 110374 375 A0A0D2BJ27 Exophiala spinifera 282 W9NFR4 Fusarium oxysporum f. sp. pisi HDV247 177

Example 7

Selection of the Enzyme Mutants with Increased Activity by an In Vivo Assay in 96-Well Microplates Based on Exogenous 3MC

(49) 1.) In vivo assay in 96-well microplates based on exogenous 3MC (IN VIVO 3)

(50) This assay (IN VIVO 3) is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with the above expression vector that contains the coding sequences and lead to the production of the last two enzymes involved in the metabolic pathway converting 3-methylcrotonic acid into isobutene, i.e., the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants and the Flavin prenyltransferase UbiX protein from E. coli. This transformed strain was first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 30° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 500 μL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth was carried out with shaking for 20 hours at 30° C. or 32° C. The LB cultures were used to inoculate 1 mL in 96 deepwell microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 30° C., 32° C. or 36° C. in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes was then resuspended in 400 μL of minimum medium (MS pH 7.5, Phosphate 100 mM, Glucose 10 g.Math.L.sup.−1, MgSO.sub.4 1 mM) supplemented with 0.3 or 1 mM 3MC and incubated for a further 1 or 2 hours in a shaking incubator at 34° C. or 36° C., 700 rpm. During this step, the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants catalyse the decarboxylation of 3-methylcrotonic acid into isobutene. After 5 mM inactivation at 80° C., the isobutene produced was quantified by gas chromatography as followed. 1004 of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 column at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(51) 2.) In vivo assay in 96-well microplates based on exogenous 3MC (IN VIVO 4)

(52) This assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with an expression vector pET25b (Novagen) that contains the coding sequences and lead to the production of the last enzyme involved in the metabolic pathway converting 3MC to isobutene; namely the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants. In another variant, it can be co-transformed with an expression vector pRSFDuet (Novagen) that contains the coding sequences and lead to the production of the Flavin prenyltransferase UbiX protein from E. coli. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 32° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 500 μL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 32° C. The LB cultures were used to inoculate 1 mL in 96 deepwell microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 32° C. in order to produce the two types of recombinant enzymes. The cell pellet containing these overexpressed recombinant enzyme is then resuspended in 4004 of minimum medium (MS pH 7.5, Phosphate 100 mM, Glucose 10g.Math.L−1, MgSO.sub.4 1 mM) supplemented with 1 mM or 10 mM 3MC and incubated for a further 30 minutes or 4 hours in a shaking incubator at 34° C. or 36° C., 700 rpm. During this step, the Hypocrea atroviridis Ferulic acid decarboxylase 1 variants catalyse the decarboxylation of 3MC into IBN. After 5 mM inactivation at 80° C., the IBN produced is quantified by gas chromatography as followed. 1004 of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL mM-1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

Example 8

Identification of Variants with Multiple Mutations of Hypocrea atroviridis Ferulic Acid Decarboxylase 1 with Further Increased Activity for the Reaction of Conversion of 3-Methylcrotonic Acid into Isobutene

(53) Libraries of mutants have been created by mutagenesis, using the Hypocrea atroviridis Ferulic acid decarboxylase 1 (SEQ ID NO:1) as template. Several variants with an enhanced activity in converting 3-methylcrotonic acid into isobutene have been identified through in vivo screening assays as described above, using either the IN VIVO 2, the IN VIVO 3 or the IN VIVO 4 assay.

(54) The list showing improving variants is presented in the following Table 6. The increase in activity is described relative to the wild-type enzyme (with “+” representing a low increase in activity and “+++++” representing a high increase in activity).

(55) The mutations involved in the variants of the Hypocrea atroviridis Ferulic acid decarboxylase 1 with increased activity are summarized in Table 7.

(56) TABLE-US-00007 TABLE 6 List of Hypocrea atroviridis Ferulic acid decarboxylase 1 variants with multiple mutations presenting an increase in isobutene production from 3-methylcrotonic acid. Activity relative Mutations to WT Assay Q29N-D351N + IN VIVO 2 Q29N-Q448W + IN VIVO 2 T405M-D420L + IN VIVO 2 D351N-T405M + IN VIVO 2 Q29N-T405M + IN VIVO 2 D351R-T405M-V445P-Q448W + IN VIVO 2 D351R-T405M-V445P + IN VIVO 2 T405M-Q448W + IN VIVO 2 D351R-T405M-Q448W + IN VIVO 2 E25N-N31G-T405M + IN VIVO 2 Q29N-T405M-T429A + IN VIVO 2 Q29N-D351R-T405M-T429A-V445P-Q448W + IN VIVO 2 E25N-Q29N-T405M-G435M-V445P + IN VIVO 2 Q29N-T405M-Q448W + IN VIVO 2 E25N-Q29N-N31G-T405M-G435M-V445P + IN VIVO 2 Q29H-N31G-T405M-T429A + IN VIVO 2 E25N-Q29N-N31G-D351R-T405M-V445P + IN VIVO 2 Q29N-D351N-T405M-Q448W + IN VIVO 2 N31G-T405M-V445P + IN VIVO 2 N31G-D351R-T405M-T429A-V445P + IN VIVO 2 E25N-Q29N-N31G-T405M-T429A + IN VIVO 2 Q29N-N31G-D351R-T405M-T429A + IN VIVO 2 N31G-T405M-D420L-T429A-V445P + IN VIVO 2 E25N-Q29H-D351R-T405M-V445P-Q448W + IN VIVO 2 E25N-D351R-T405M-G435M-V445P-Q448W + IN VIVO 2 S86N-T405M-T429A-G435M-V445P-Q448W + IN VIVO 2 E25N-T405M-T429A + IN VIVO 2 E25N-Q29N-N31G-D351R-T405M-T429A + IN VIVO 2 Q29N-D351R-T405M-D414N-T429A-V445P-Q448W + IN VIVO 2 N31G-D351R-T405M-T429A-G435M-V445P + IN VIVO 2 E25N-N31G-T405M-T429A-V445P + IN VIVO 2 E25N-D351R-T405M-T429A-V445P + IN VIVO 2 E25N-T405M-T429A-Q448W + IN VIVO 2 E25N-N31G-T405M-Q448W + IN VIVO 2 E25N-Q29N-T405M-T429A-Q448W + IN VIVO 2 Q29N-N31G-D351R-T405M-T429A-G435M-V445P + IN VIVO 2 N31G-T405M-Q448W + IN VIVO 2 N31G-T405M-D420L-T429A-V445P-Q448W + IN VIVO 2 Q29N-N31G-D351G-T405M-T429A-V445P + IN VIVO 2 Q29N-T405M-T429A-G435M-V445P-Q448W + IN VIVO 2 E25N-N31G-D351R-T405M-T429A-Q448W + IN VIVO 2 Q29N-N31G-T405M-T429A-V445P + IN VIVO 2 E25N-N31G-D351R-T405M-G435M-V445P-Q448W + IN VIVO 2 N31G-D351R-T405M-T429A-Q448W + IN VIVO 2 D12N-Q29N-N31G-T405M-T429A-V445P-Q448W + IN VIVO 2 Q29N-N31G-T405M-T429A-Q448W + IN VIVO 2 E25N-T405M-T429A-V445P-Q448W + IN VIVO 2 E25N-N31G-T405M-T429A-G435M-V445P-Q448W + IN VIVO 2 E25N-Q29N-N31G-T405M-T429A-V445P-Q448W + IN VIVO 2 E25N-N31G-T405M-T429A-V445P-Q448W + IN VIVO 2 E25N-N31G-D351R-T405M-T429A-V445P-Q448W + IN VIVO 2 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-P120K ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-H303S ++ IN VIVO 3 E25N-N31G-D351G-T405M-T429A-V445P-Q448W ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-N264D ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-R392L ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-L221C ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-G305A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q214V ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-F404Y ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-P402V ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-T228L ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-I119T ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A10L ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S484A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q214E ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q214A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A211E ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-N501M ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q214F ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-T228V ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-D57N ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-D512E ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S484G ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A149V ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S8N ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A10H ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-N117A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-V132C ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-C175G ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-C175K ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-C175S ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S187T ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A193T ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-I197M ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A222C ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-T228A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-V247A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A342G ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-T399N ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-F440V ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-A460P ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-K488A ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-N501G ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S502N ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-L506Y ++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S3C-S86I-Q162P-T384Y-R392A +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q162N-T228L-R392A +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q162N-R392A +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q162N-T228L-R392A-I461V-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A-Q162N-Q214H-T228L-G338P-I461V +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-C175G-T228L-R392A-T399R-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A-Q162N-C175G-T228L-G338P-T399R-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A-Q162N-T228L-G338P-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A-Q162N-C175G-T228L-G338P-T399R-I461V-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A-Q162N-T228L-G338P-R392A-I461M-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q162N-T228L-G338P-R392A-I461V +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-Q162N-C175G-T228L-G338P-R392A-I461V-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A-C175G-T228L-G338P-R392A-T399R-I461V-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85A-Q162N-C175G-T228L-G338P-R392A-I461V-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-T228L-G338P-R392A-I461V-S494R +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-G305A-P402V +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-G305A-F404Y +++ IN VIVO 3 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-G305A-P402V-F404Y +++ IN VIVO 3 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P15T ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-D30G ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-D30H ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-D30R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-N65W ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-N65L ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-K70L ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G72R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P80L ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P87V ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P87I ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-R90L ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-T103L ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-S105W ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-S105F ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-D108R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-D108W ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-I126P ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175P-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-W176F ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G213L ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G213P ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305D ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P306R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P306S ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-C326P ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338S-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-A341I ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351V-R392A-T405M-T429A-V445P-Q448W-I461V-S494R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P402H ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-I406Q ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461N-S494R ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-T103I-D111C ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-E14D-P87A ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-W176F-L511I ++++ IN VIVO 4 E25W-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-A352L ++++ IN VIVO 4 E25S-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-A352G ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-T278I-C326P ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-A341I-G386N- ++++ IN VIVO 4 D395C E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351A-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-C349S ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-K7R-W176F ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-R84C- ++++ IN VIVO 4 K493R E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P87W- ++++ IN VIVO 4 K159C E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P87C- ++++ IN VIVO 4 K488N-Q496A E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-E89F- ++++ IN VIVO 4 Q496F E25N-N31G-G305A-D351R-K377H-P402V-F404Y-T405M-T429A-V445P-Q448W ++++ IN VIVO 3 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-F404Y ++++ IN VIVO 2 E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305A- ++++ IN VIVO 2 K377H-F404Y E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305A- ++++ IN VIVO 2 P306F-F404Y E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-K377H- ++++ IN VIVO 2 F404Y E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P306F- ++++ IN VIVO 2 F404Y E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P306F- ++++ IN VIVO 2 K377H E25N-N31G-S85A-Q162N-C175G-T228L-G338P-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-P306F- ++++ IN VIVO 2 K377H-F404Y E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-D12A ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-D35T ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A60V ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-K70I ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-K70L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P87F ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P87L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P87M ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P87V ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P87W ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A99P ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P101I ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P101L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P102L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-T103L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-S105L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-D108R ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-K189I ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A193I ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-E215C ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A244F ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-C326A ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-D375L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-D443N ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A460F ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-P102L-S484A ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-D108K-Y160F ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-F11Y-D512S ++++ IN VIVO 4 E25N-N31G-D351R-T405M-T429A-V445P-Q448W-S85T-T103M-Q162H-C175W-T228P-G338A-R392A-I461M ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-V445P-Q448W-P87F ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-S3G ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-T4A ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-T5S ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A10F ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A10P ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A10T ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-I67R ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-I67V ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-D71G ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A99N ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A149S ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-Q154K ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A193T ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A193V ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-Q206F ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-Q214R ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-A232V ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-M284L ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-H303P ++++ IN VIVO 4 E25N-N31G-G305A-D351R-P402V-F404Y-T405M-T429A-V445P-Q448W-K462N ++++ IN VIVO 4 E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-S494R-G305A-P402V-F404Y ++++ IN VIVO 2 E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305A-P402V- ++++ IN VIVO 2 F404Y E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-S494R-L228T-G305A-P402V- ++++ IN VIVO 2 F404Y E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-S494R-G305A-K377H-P402V- ++++ IN VIVO 2 F404Y E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-L228T-G305A- ++++ IN VIVO 2 F404Y E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305A-K377H- ++++ IN VIVO 2 F404Y E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305A-F404Y ++++ IN VIVO 2 E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305A-K377H- ++++ IN VIVO 2 P402V-F404Y E25N-N31G-S85A-Q162N-C175G-T228L-D351R-R392A-T405M-T429A-V445P-Q448W-I461V-S494R-G305A-K377H ++++ IN VIVO 2 E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-S2N E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-A10K E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-P13S E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-A69N E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-A106T E25N-N31G-S85A-Q162N-C175T-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R E25N-N31G-S85A-Q162N-C175K-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R E25N-N31G-S85A-Q162N-C175Q-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-E216N E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-I345L E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-S454G E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-S3D-K509L E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-T4S-P13V E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-Y6P-D512H E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405F-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405F-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-S454G E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404W-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-F447M E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448W- +++++ IN VIVO 3 I461V-S494R-I337L E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448F- +++++ IN VIVO 3 I461V-S494R-I197M E25N-N31G-G305A-D351R-K377H-P402V-F404Y-T405M-T429A-V445P-Q448W-S454G +++++ IN VIVO 3 E25N-N31G-G305A-D351R-K377H-P402V-F404Y-T405F-T429A-V445P-Q448W +++++ IN VIVO 3 E25N-N31G-G305A-D351R-K377H-P402V-F404Y-T405F-T429A-V445P-Q448W-S454G +++++ IN VIVO 3 E25N-N31G-S85A-Q162N-C175G-T228L-G305A-D351R-K377H-R392A-P402V-F404Y-T405M-T429A-V445P-Q448F- +++++ IN VIVO 3 I461V-S494R

(57) TABLE-US-00008 TABLE 7 List of mutations involved in the variants of the Hypocrea atroviridis Ferulic acid decarboxylase 1 with increased activity Wild-Type Sequence Wild-Type Sequence Mutant Amino Acid Number Mutation Mutant Amino Acid Number Mutation S2A S  2 A K189I K 189 I S2D S  2 D A193I A 193 I S2F S  2 F A193T A 193 T S2K S  2 K A193V A 193 V S2L S  2 L L195C L 195 C S2N S  2 N L195F L 195 F S2Q S  2 Q L195I L 195 I S2V S  2 V L195V L 195 V S3A S  3 A L195W L 195 W S3C S  3 C L195Y L 195 Y S3D S  3 D I197F I 197 F S3E S  3 E I197M I 197 M S3G S  3 G Q206F Q 206 F S3K S  3 K A211E A 211 E S3P S  3 P G213L G 213 L S3W S  3 W G213P G 213 P S3Y S  3 Y Q214A Q 214 A T4A T  4 A Q214E Q 214 E T4E T  4 E Q214F Q 214 F T4L T  4 L Q214H Q 214 H T4M T  4 M Q214R Q 214 R T4N T  4 N Q214T Q 214 T T4S T  4 S Q214V Q 214 V T5S T  5 S E215C E 215 C Y6P Y  6 P E216N E 216 N K7R K  7 R L221C L 221 C S8N S  8 N A222C A 222 C E9H E  9 H T228A T 228 A E9P E  9 P T228L T 228 L E9Y E  9 Y T228P T 228 P A10F A  10 F L228T L 228 T A10H A  10 H T228V T 228 V A10K A  10 K A232V A 232 V A10L A  10 L A244F A 244 F A10P A  10 P V247A V 247 A A10T A  10 T N264D N 264 D F11L F  11 L T278I T 278 I F11P F  11 P M284L M 284 L F11Y F  11 Y M284Y M 284 Y D12A D  12 A A285L A 285 L D12N D  12 N M293L M 293 L D12S D  12 S H303P H 303 P P13H P  13 H H303S H 303 S P13I P  13 I G305A G 305 A P13N P  13 N G305D G 305 D P13S P  13 S P306F P 306 F P13V P  13 V P306R P 306 R P13Y P  13 Y P306S P 306 S E14D E  14 D C326A C 326 A P15T P  15 T C326P C 326 P E25N E  25 N I337L I 337 L E25S E  25 S I337M I 337 M E25W E  25 W G338A G 338 A Q29H Q  29 H G338P G 338 P Q29N Q  29 N G338S G 338 S Q29S Q  29 S A341I A 341 I D30G D  30 G A342G A 342 G D30H D  30 H I345L I 345 L D30R D  30 R C3495 C 349 S N31E N  31 E D351A D 351 A N31G N  31 G D351G D 351 G L33I L  33 I D351N D 351 N V34A V  34 A D351R D 351 R V34I V  34 I D351V D 351 V D35M D  35 M A352G A 352 G D35S D  35 S A352L A 352 L D35T D  35 T D375L D 375 L V40I V  40 I T376I T 376 I V40M V  40 M K377H K 377 H D43R D  43 R A381R A 381 R D57N D  57 N T384Y T 384 Y A60V A  60 V G386N G 386 N N65L N  65 L A388E A 388 E N65W N  65 W R392A R 392 A I67R I  67 R R392L R 392 L I67V I  67 V D395C D 395 C A69N A  69 N T399N T 399 N K70I K  70 I T399R T 399 R K7OL K  70 L P402H P 402 H D71G D  71 G P402V P 402 V G72R G  72 R F404W F 404 W P80L P  80 L F404Y F 404 Y R84C R  84 C T405F T 405 F S85A S  85 A T405L T 405 L S85T S  85 T T405M T 405 M S86I S  86 I T405P T 405 P S86N S  86 N T405Q T 405 Q P87A P  87 A I406Q I 406 Q P87C P  87 C D414N D 414 N P87F P  87 F D420L D 420 L P87I P  87 I K422M K 422 M P87L P  87 L T429A T 429 A P87M P  87 M T429S T 429 S P87V P  87 V G435M G 435 M P87W P  87 W T436N T 436 N E89F E  89 F V439L V 439 L E89S E  89 S F440V F 440 V R90L R  90 L F441Y F 441 Y F91L F  91 L D442T D 442 T A99N A  99 N D443N D 443 N A99P A  99 P V445E V 445 E P101I P 101 I V445P V 445 P P101L P 101 L F447M F 447 M P102L P 102 L F447W F 447 W T103I T 103 I F447Y F 447 Y T103L T 103 L Q448F Q 448 F T103M T 103 M Q448S Q 448 S S105F S 105 F Q448W Q 448 W S105L S 105 L L449I L 449 I S105W S 105 W L449M L 449 M A106T A 106 T L449V L 449 V D108K D 108 K S454G S 454 G D108R D 108 R A460F A 460 F D108W D 108 W A460P A 460 P D111C D 111 C I461M I 461 M L114S L 114 S I461N I 461 N N117A N 117 A I461V I 461 V I119T I 119 T K462N K 462 N P120K P 120 K S484A S 484 A P120S P 120 S S484G S 484 G I126P I 126 P K488A K 488 A V132C V 132 C K488N K 488 N N141D N 141 D K493R K 493 R A146S A 146 S S494R S 494 R A149S A 149 S Q496A Q 496 A A149V A 149 V Q496F Q 496 F Q154K Q 154 K L500A L 500 A K159C K 159 C N501E N 501 E Y160F Y 160 F N501G N 501 G Q162H Q 162 H N501K N 501 K Q162N Q 162 N N501M N 501 M Q162P Q 162 P 5502N S 502 N C175G C 175 G L5061 L 506 I C175K C 175 K L506Y L 506 Y C175P C 175 P K509L K 509 L C175Q C 175 Q L511I L 511 I C175S C 175 S L511M L 511 M C175T C 175 T D512E D 512 E C175W C 175 W D512H D 512 H W176F W 176 F D512S D 512 S S187T S 187 T

Example 9

In Vitro Activities of Homologues of the Hypocrea atroviridis Ferulic Acid 1 Decarboxylase at Different Temperatures (Wild-Type and T→M Mutants)

(58) As described in Example 5, the T405M variant of Hypocrea atroviridis Ferulic acid 1 decarboxylase presents a very significant increase in the production of Isobutene from 3-methylcrotonic acid. To assess the general effect of this mutation on different Ferulic acid decarboxylases, we selected 4 homologues of the Hypocrea atroviridis Ferulic acid 1 decarboxylase and made the corresponding T.fwdarw.M mutation (Table 8).

(59) TABLE-US-00009 TABLE 8 List of homologues of the Hypocrea alroviridis Ferulic acid decarboxylase 1 and their T−>M mutation Uniprot SEQ Accession ID T−>M Organism Number NO Mutation Hypocrea atroviridis (Ha) G9NLP8 1 T405M Cladophialophora psammophila (Cp) W9WWR1 10 T400M Cladophialophora immunda (Ci) A0A0D2AQI6 11 T400M Sphaerulina musiva (Sm) M3DF95 12 T405M Cladophialophora bantiana (Cb) A0A0D2IKD5 13 T400M

(60) The in vitro assay was performed as described in Example 5 with 10 mM 3-methylcrotonic acid, an incubation time of 120 mM and an incubation temperature varying between 30 and 60° C. In all cases, the T.fwdarw.M mutation shows an increase in activity and in the optimal catalytic temperature (FIG. 3).

Example 10

Identification of Variants of Homologues of Hypocrea atroviridis Ferulic Acid Decarboxylase 1 with Increased Activity for the Reaction of Conversion of 3-Methylcrotonic Acid into Isobutene

(61) As was described in Example 9, we showed that the T.fwdarw.M mutation found in position 405 of the Hypocrea atroviridis Ferulic acid 1 decarboxylase increases the activity of 4 different homologues (Table 8 and FIG. 3). We decided to further evolve two of these homologues, namely the Ferulic Acid decarboxylase 1 of Cladophialophora psammophila (Uniprot Accession Number W9WWR1, SEQ ID NO: 10) and of Cladophialophora bantiana (Uniprot Accession Number A0A0D2IKD5, SEQ ID NO: 13).

(62) The assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with the above expression vector that contains the coding sequences as described above, leading to the production of the last two enzymes involved in the metabolic pathway converting 3MC to isobutene; namely the Ferulic acid decarboxylase 1 of Cladophialophora psammophila (Uniprot Accession Number W9WWR1, SEQ ID NO: 10) or the Ferulic acid decarboxylase 1 of Cladophialophora bantiana (Uniprot Accession Number A0A0D2IKD5, SEQ ID NO: 13) variants and the Flavin prenyltransferase UbiX protein from E. coli. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 32° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 500 μL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 32° C. The LB cultures were used to inoculate 1 mL in 96 deepwell microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 32° C. in order to produce the two recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 400 μL of minimum medium (pH 7.5, Phosphate 100 mM, Glucose 10g.Math.L.sup.−1, MgSO.sub.4 1 mM) supplemented with 10 mM 3MC and incubated for a further 2 or 4 hours in a shaking incubator at 36° C., 700 rpm. During this step, the Ferulic acid decarboxylase 1 variants catalyse the decarboxylation of 3MC into IBN. After 5 mM inactivation at 80° C., the IBN produced is quantified by gas chromatography as described in the following. 1004 of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(63) The list of improving variants of the Ferulic acid decarboxylase 1 of Cladophialophora psammophila (Uniprot Accession Number W9WWR1, SEQ ID NO: 10) is presented in the following Table 9 and the list of improving variants of the Ferulic acid decarboxylase 1 of Cladophialophora bantiana (Uniprot Accession Number A0A0D2IKD5, SEQ ID NO: 13) is presented in the following Table 10. The increase in activity is described relative to the wild-type enzyme (with “+” representing a low increase in activity and “++” representing a high increase in activity).

(64) The list of mutations involved in the variants of the Cladophialophora psammophila Ferulic acid decarboxylase 1 (Uniprot Accession Number W9WWR1, SEQ ID NO: 10) with increased activity is described in Table 11 and the list of mutations involved in the variants of the Cladophialophora psammophila Ferulic acid decarboxylase 1 (Uniprot Accession Number A0A0D2IKD5, SEQ ID NO: 13) with increased activity is described in Table 12.

(65) TABLE-US-00010 TABLE 9 List of Cladophialophora psammophila Ferulic acid decarboxylase 1 variants presenting an increase in isobutene production from 3-methylcrotonic acid. Activity relative Mutations to WT T400M + T400M-Y393F-G394N-Y399F ++ T400M-Y393F ++ T400M-Y393F-G394N ++ T400M-Y393F-Y399F ++ T400M-Y393F-A397V-Y399F ++ T400M-Y393F-A397V ++

(66) TABLE-US-00011 TABLE 10 List of Cladophialophora bantiana Ferulic acid decarboxylase 1 variants presenting an increase in isobutene production from 3-methylcrotonic acid. Activity relative Mutations to WT T400M + T400M-Y393F-G394N ++ T400M-Y393F-G394N-Y399F ++ T400M-Y393F-A397V ++ T400M-Y393F ++ T400M-Y393F-Y399F ++ T400M-A14T-Y393F-H395Q ++ T400M-Y393F-G394N-A397V ++ T400M-Y393F-A397V-Y399F ++ T400M-Y393F-G394N-A397V-Y399F ++

(67) TABLE-US-00012 TABLE 11 List of mutations involved in the variants of the Cladophialophora psammophila Ferulic acid decarboxylase 1 with increased activity. Wild-Type Sequence Mutant Amino Acid Number Mutation Y393F Y 393 F G394N G 394 N A397V A 397 V Y399F Y 399 F T400M T 400 M

(68) TABLE-US-00013 TABLE 12 List of mutations involved in the variants of the Cladophialophora bantiana Ferulic acid decarboxylase 1 with increased activity. Wild-Type Sequence Mutant Amino Acid Number Mutation A14T A 14 T Y393F Y 393 F G394N G 394 N H395Q H 395 Q A397V A 397 V Y399F Y 399 F T400M T 400 M

EXAMPLES

Second Part

(69) Material and Methods: Methods Used to Assess Isobutene Production Activities

(70) a) Cloning of 3-methylcrotonic acid decarboxylase (3-MDC) enzymes All the 3-methylcrotonic acid decarboxylase (3-MDC) polynucleotide sequences were codon-optimized for the expression in Escherichia coli and subsequently chemically synthesized. They were then cloned in a pET25 (Novagen) expression vector or fused with a polynucleotide tag in 5′ coding for either a 6-His purification tag before being cloned in a pET25 expression vector, resulting in 4 expression vectors for each 3-MDC sequence.

(71) b) Construction of 3-methylcrotonic acid decarboxylase (3-MDC) mutants The polynucleotide sequences coding for the different mutants identified during the evolution of the selected 3-MDC enzymes were generated using a range of standard molecular biology techniques. Different PCR-based techniques known in the art were used for the construction of single-point mutants. For the generation of enzyme variants bearing multiple mutations (at least two mutations), either PCR-based techniques or other methods known in the art were used to introduce these mutations. Following mutagenesis, the mutated polynucleotide sequence was inserted into a pET25 expression vector with or without being fused to a tag as described above either using standard ligase-based subcloning techniques, whole plasmid extension by PCR or ligase-independent cloning techniques.

(72) c) Screening of 3-methylcrotonic acid decarboxylase (3-MDC) enzymes activity A total of four different screening methods were developed and used during the evolution of the Streptomyes sp. 769 MDC enzyme.

(73) c1) In vivo assay in 384-well microplates based on exogenous 3-methylcrotonic acid (3MC) (VIVO384) This assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with two expression vectors leading to the production of the last two enzymes involved in the metabolic pathway converting 3MC to isobutene; namely the prenylated FMN Synthase UbiX protein from E. coli cloned in a pRSFDuet™ (Novagen) expression vector and the respective 3-MDC enzyme cloned in one of the above expression vectors. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotic. Cells were grown overnight at 32° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 50 μL of liquid LB medium supplemented with the appropriate antibiotics. Cell growth is carried out with shaking for 20 hours at 32° C. The LB cultures were used to inoculate 300 μL in 384 deep well microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotics and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 32° C. in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 40 μL of minimum medium (pH 7.5, Phosphate 100 mM, Glucose 10g.Math.L.sup.−1, MgSO.sub.4 1 mM) supplemented with 3, 10 or 30 mM 3MC and incubated for a further 2 or 4 hours in a shaking incubator at 36° C., 700 rpm. During this step, the 3-MDC enzyme catalyzes the decarboxylation of 3MC into IBN. After 5 min inactivation at 80° C., the IBN produced is quantified by gas chromatography as followed. 100 μL of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(74) c2) In Vivo Assay in 96-Well Microplates Based on Exogenous 3-Methylcrotonic Acid (3MC) (VIVO96) This assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with two expression vectors leading to the production of the last two enzymes involved in the metabolic pathway converting 3MC to isobutene; namely the prenylated FMN Synthase UbiX protein from E. coli cloned in a pRSFDuet™ (Novagen) expression vector and the respective 3-MDC enzyme cloned in one of the above expression vectors. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotics. Cells were grown overnight at 32° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 50 μL of liquid LB medium supplemented with the appropriate antibiotics. Cell growth is carried out with shaking for 20 hours at 32° C. The LB cultures were used to inoculate 1 mL in 96 deep well microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotics and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 32° C. in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 400 μL of minimum medium (pH 7.5, Phosphate 100 mM, Glucose 10g.Math.L.sup.−1, MgSO.sub.4 1 mM) supplemented with 3, 10 or 30 mM 3MC and incubated for a further 2 or 4 hours in a shaking incubator at 36° C., 700 rpm. During this step, the 3-MDC enzyme catalyzes the decarboxylation of 3MC into IBN. After 5 min inactivation at 80° C., the IBN produced is quantified by gas chromatography as followed. 100 μL of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(75) c3) In Vitro Assay in 384-Well Microplates Based on Exogenous 3-Methylcrotonic Acid (3MC) (VITRO384) This assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with two expression vectors leading to the production of the last two enzymes involved in the metabolic pathway converting 3MC to isobutene; namely the prenylated FMN Synthase UbiX protein from E. coli cloned in a pRSFDuet™ (Novagen) expression vector and the respective 3-MDC enzyme cloned in one of the above expression vectors. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotics. Cells were grown overnight at 32° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 50 μL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 32° C. The LB cultures were used to inoculate 300 μL in 384 deep well microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 32° C. in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 30 μL of lysis mix (pH 7.5, Phosphate 50 mM, NaCl 20 mM, MgCl.sub.2 2 mM, Lysozyme 1 mg/mL, DNAse 0.03 mg/mL) and incubated for 1 hour in a shaking incubator at 36° C., 700 rpm. The mix is then supplemented with 104 of reaction mix (final composition: pH 7.5, Phosphate 50 mM, NaCl 20 mM, MgCl.sub.2 2 mM, Lysozyme 0.75 mg/mL, DNAse 0.0225 mg/mL, KCl 100 mM) supplemented with 3, 10 or 30 mM (final) 3MC and incubated for a further 2 or 4 hours in a shaking incubator at 36° C., 700 rpm. During this step, the 3-MDC enzyme catalyzes the decarboxylation of 3MC into IBN. After 5 mM inactivation at 80° C., the IBN produced is quantified by gas chromatography as followed. 100 μL of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

(76) c4) In Vitro Assay in 96-Well Microplates Based on Exogenous 3-Methylcrotonic Acid (3MC) (VITRO96) This assay is based on the use of a bacterial strain (BL21(DE3), Novagen) transformed with two expression vectors leading to the production of the last two enzymes involved in the metabolic pathway converting 3MC to isobutene; namely the prenylated FMN Synthase UbiX protein from E. coli cloned in a pRSFDuet™ (Novagen) expression vector and the respective 3-MDC enzyme cloned in one of the above expression vectors. This strain is first plated out onto LB-agar plates supplemented with the appropriate antibiotics. Cells were grown overnight at 32° C. until individual colonies reach the desired size. Single colonies were then picked and individually transferred into 50 μL of liquid LB medium supplemented with the appropriate antibiotic. Cell growth is carried out with shaking for 20 hours at 32° C. The LB cultures were used to inoculate 1 mL in 96 deep well microplates of auto-induction medium (Studier F W, Prat.Exp.Pur. 41, (2005), 207-234) supplemented with the appropriate antibiotic and grown in a shaking incubator set at 700 rpm and 85% humidity for 24h at 32° C. in order to produce the two types of recombinant enzymes. The cell pellet containing these two overexpressed recombinant enzymes is then resuspended in 150 μL of lysis mix (pH 7.5, Phosphate 50 mM, NaCl 20 mM, MgCl.sub.2 2 mM, Lysozyme 1 mg/mL, DNAse 0.03 mg/mL) and incubated for 1 hour in a shaking incubator at 36° C., 700 rpm. The mix is then supplemented with 504 of reaction mix (final composition: pH 7.5, Phosphate 50 mM, NaCl 20 mM, MgCl.sub.2 2 mM, Lysozyme 0.75 mg/mL, DNAse 0.0225 mg/mL, KCl 100 mM) supplemented with 3, 10 or 30 mM (final) 3MC and incubated for a further 2 or 4 hours in a shaking incubator at 36° C., 700 rpm. During this step, the 3-MDC enzyme catalyzes the decarboxylation of 3MC into IBN. After 5 mM inactivation at 80° C., the IBN produced is quantified by gas chromatography as followed. 100 μL of headspace gases from each enzymatic reaction are injected in a Brucker GC-450 system equipped with a Flame Ionization Detector (FID). Compounds present in samples were separated by chromatography using a RTX-1 columns at 100° C. with a 1 mL.Math.min.sup.−1 constant flow of nitrogen as carrier gas. Upon injection, peak areas of isobutene were calculated.

Example 1

Identification of a Streptomyces sp. 769 UbiD-Like Decarboxylase Enzyme Presenting an Activity for the Reaction of Conversion of 3-Methylcrotonic Acid (3MC) into Isobutene (IBN)

(77) We tested a UbiD-like decarboxylase enzyme from Streptomyces sp. 769 (UniProt ID A0A0A8EV26, gene GZL_07100, herein called Ss5; SEQ ID NO:14) for its capacity to catalyze the reaction of conversion of 3MC into IBN. The gene was codon-optimized for expression in Escherichia Coli, synthesized, fused to a 6-His-tag or not fused to a 6-His-tag, and then subcloned into a pET25 expression vector, as described above. The expression vectors were then screened for the production of isobutene (IBN) from 3-methylcrotonic acid (3MC) in microplate 96 and 384-wells, using both an in vivo and an in vitro assay, as described above. An 3-methylcrotonic acid decarboxylase (3-MDC) enzyme from Hypocrea atroviridis (herein called Ha, Uniprot Accession Number G9NLP8) described above in the context of the first, second and third aspect of the present invention and previously described in WO2017085167 (see Example 8, Table G; example 12 Table J therein) and in WO2017191239 (SEQ ID NO:1 therein) was added as a control (FIG. 4 and FIG. 5).

Example 2

Identification of Variants of a 3-Methylcrotonic Acid Decarboxylase (3-MDC) Enzyme from Streptomyces sp. 769 with Increased Activity for the Reaction of Conversion of 3-Methylcrotonic Acid (3MC) into Isobutene (IBN)

(78) The above 3-methylcrotonic acid decarboxylase (3-MDC) gene presenting a capacity to catalyze the reaction of conversion of 3MC into IBN (UniProt ID A0A0A8EV26, gene GZL_07100 from Streptomyces sp. 769) was submitted to directed mutagenesis in order to create single point mutations or multiple mutations variants. Each of these variants was subsequently tested for their increased activity to convert 3MC into IBN, using one or more of the previously described assays. 230 variants presented an increased capacity in converting 3MC into IBN.

(79) The list of improved variants is presented in the following Table 13. The increase in activity is described relative to the wild-type enzyme (with “+” representing a low increase in activity and “+++” representing a high increase in activity).

(80) The list of mutations involved in the variants of the Streptomyces sp. 769 3-MethylCrotonate Decarboxylase with increased activity is described in Table 14.

(81) TABLE-US-00014 TABLE 13 List of Streptomyces sp. 769 3-MethylCrotonate Decarboxylase variants presenting an increase in isobutene production from 3-methylcrotonic acid. Activity relative Mutants to WT A241D + A241N + A241V + A359C + C404F + C404L + L448F + L448W + L448Y + P406A + R390S-L448W + R390S-L448Y + A241D-C404F ++ A241D-C404F-L448F ++ A241D-C404F-L448Y ++ A241D-C404F-P406A ++ A241D-C404F-P406A-L448F ++ A241D-C404F-P406A-L448Y ++ A241D-C404L ++ A241D-C404L-L448F ++ A241D-C404L-L448W ++ A241D-C404L-L448Y ++ A241D-C404L-P406A ++ A241D-C404L-P406A-L448F ++ A241D-C404L-P406A-L448Y ++ A241D-L448F ++ A241D-L448W ++ A241D-L448Y ++ A241D-P406A ++ A241D-P406A-L448F ++ A241D-P406A-L448Y ++ A241N-C404F ++ A241N-C404F-L448F ++ A241N-C404F-L448W ++ A241N-C404F-L448Y ++ A241N-C404F-P406A ++ A241N-C404F-P406A-L448F ++ A241N-C404F-P406A-L448Y ++ A241N-C404L ++ A241N-C404L-L448F ++ A241N-C404L-L448W ++ A241N-C404L-L448Y ++ A241N-C404L-P406A ++ A241N-C404L-P406A-L448F ++ A241N-C404L-P406A-L448Y ++ A241N-L448F ++ A241N-L448W ++ A241N-L448Y ++ A241N-P406A ++ A241N-P406A-L448F ++ A241N-P406A-L448W ++ A241N-P406A-L448Y ++ C404F-L446I-L448W ++ C404F-L448F ++ C404F-L448W ++ C404F-L448Y ++ C404F-P406A ++ C404F-P406A-L448F ++ C404F-P406A-L448W ++ C404F-P406A-L448Y ++ C404L-L448F ++ C404L-L448W ++ C404L-L448Y ++ C404L-P406A ++ C404L-P406A-L448F ++ C404L-P406A-L448Y ++ C404M-L446I-L448W ++ C404M-L448F ++ C404M-L448W ++ P406A-L448F ++ P406A-L448W ++ P406A-L448Y ++ V240I-C404M-L448F ++ C404M-L446N-L448W ++ C404M-P406A-L446I-L448W ++ C404M-P406S-L446I-L448W ++ S403R-C404M-L446I-L448W ++ C404M-L446I-L448W-T450A ++ C404M-L446I-L448W-T450H ++ C404M-L446I-L448W-T450M ++ C404M-L446I-L448W-T450N ++ C404M-L446I-L448W-T450S ++ C404M-P406A-L446N-L448W ++ C404M-P406A-L448W ++ C404M-P406A-L446I-L448W-T450A ++ C404M-P406S-L446N-L448W ++ C404M-P406S-L448W ++ C404M-P406S-L446I-L448W-T450A ++ C404M-P406S-L446I-L448W-T450H ++ C404M-L446N-L448W-T450A ++ C404M-L446N-L448W-T450H ++ C404M-L448W-T450A ++ C404M-L448W-T450H ++ C404M-P406A-L446N-L448W-T450A ++ C404M-P406A-L448W-T450A ++ C404M-P406A-L446N-L448W-T450H ++ C404M-P406S-L446N-L448W-T450A ++ C404M-P406S-L448W-T450A ++ C404M-P406S-L446N-L448W-T450H ++ C404M-P406A-L446I-L448W-T450H +++ C404M-P406A-L448W-T450H +++ C404M-P406S-L448W-T450H +++ A241D-C404F-L446I-L448W +++ A241D-C404F-L448W +++ A241D-C404F-P406A-L448W +++ A241D-C404F-P406S-L448W +++ A241D-C404L-P406A-G443D-L448W +++ A241D-C404L-P406A-L446V-L448W +++ A241D-C404L-P406A-L446V-L448W-T450M +++ A241D-C404L-P406A-L448W +++ A241D-C404L-P406A-L448W-T450M +++ A241D-C404M-L448W +++ A241D-C404M-P406A-L448W +++ A241D-F401Y-C404F-L446I-L448W +++ A241D-F401Y-C404F-L448W +++ A241D-F401Y-C404F-P406A-L448W +++ A241D-F401Y-C404M-L448W +++ A241D-F401Y-C404M-P406A-L448W +++ A241D-F401Y-L448W +++ A241D-F401Y-S403G-C404F-L446I-L448W +++ A241D-F401Y-S403G-C404F-L448W +++ A241D-F401Y-S403G-C404M-L448W +++ A241D-F401Y-S403P-C404F-L446I-L448W +++ A241D-F401Y-S403P-C404F-L448W +++ A241D-F401Y-S403P-C404M-P406A-L448W +++ A241D-G402A-C404L-P406A-L446V-L448W +++ A241D-G402A-C404L-P406A-L446V-L448W-T450M +++ A241D-G402A-C404L-P406A-L448W +++ A241D-G402A-C404L-P406A-L448W-T450M +++ A241D-G402A-S403C-C404L-P406A-L446V-L448W +++ A241D-G402A-S403C-C404L-P406A-L446V-L448W-T450M +++ A241D-G402A-S403C-C404L-P406A-L448W +++ A241D-G402A-S403C-C404L-P406A-L448W-T450M +++ A241D-G402A-S403V-C404L-P406A-L446V-L448W +++ A241D-G402A-S403V-C404L-P406A-L446V-L448W-T450M +++ A241D-G402A-S403V-C404L-P406A-L448W +++ A241D-G402A-S403V-C404L-P406A-L448W-T450M +++ A241D-P406A-L448W +++ A241D-P444E-L448W +++ A241D-S403C-C404L-P406A-L446V-L448W +++ A241D-S403C-C404L-P406A-L446V-L448W-T450M +++ A241D-S403C-C404L-P406A-L448W +++ A241D-S403C-C404L-P406A-L448W-T450M +++ A241D-S403G-C404F-L446I-L448W +++ A241D-S403G-C404F-L448W +++ A241D-S403G-C404F-P406A-L448W +++ A241D-S403G-C404M-P406A-L448W +++ A241D-S403G-L448W +++ A241D-S403P-C404F-L446I-L448W +++ A241D-S403P-C404F-L448W +++ A241D-S403P-C404F-P406A-L448W +++ A241D-S403P-C404M-L448W +++ A241D-S403P-C404M-P406A-L448W +++ A241D-S403P-L448W +++ A241D-S403V-C404L-P406A-L446V-L448W +++ A241D-S403V-C404L-P406A-L446V-L448W-T450M +++ A241D-S403V-C404L-P406A-L448W +++ A241D-S403V-C404L-P406A-L448W-T450M +++ A241N-C404F-L405H-P406A-L448W +++ A241N-C404F-P406A-L448W +++ A241N-C404L-P406A-L446C-L448W +++ A241N-C404L-P406A-L446V-L448W +++ A241N-C404L-P406A-L448W +++ A241N-C404L-P406A-L448W-L449I +++ A241N-C404L-P406A-L448W-T450M +++ A241N-C404L-P406A-L448W-T450Q +++ A241N-C404M-L448W +++ A241N-C404M-P406A-L448W +++ A241N-F401Y-L448W +++ A241N-L446V-L448W +++ A241N-S403C-C404L-P406A-L448W +++ A241N-S403G-L448W +++ A241N-S403N-C404L-P406A-L448W +++ A241N-S403P-L448W +++ A241N-S403V-C404L-P406A-L448W +++ C404L-P406A-L446F-L448W +++ C404L-P406A-L446S-L448W +++ C404L-P406A-L446V-L448W +++ C404L-P406A-L448W +++ C404L-P406A-L448W-T450M +++ C404L-P406A-P444H-L448W +++ P85L-A241N-C404M-L448W-T450M +++ S403V-C404L-P406A-L448W +++ A241D-S403C-C404L-P406A-G443D-L448W +++ A241D-S403C-C404L-P406A-G443H-L448W +++ A241D-S403C-C404L-P406A-G443N-L448W +++ A241D-S403C-C404L-P406A-L448W-L449I +++ A241D-S403C-C404L-P406A-P444A-L448W +++ A241D-S403C-C404L-P406A-P444H-L448W +++ A241D-S403C-C404L-P406A-P444L-L448W +++ A241D-G402A-S403C-C404L-P406A-G443H-L448W +++ A241D-G402A-S403C-C404L-P406A-G443S-L448W +++ A241D-G402A-S403C-C404L-P406A-L446M-L448W +++ A241D-G402A-S403C-C404L-P406A-P444F-L448W +++ A241D-G402A-S403C-C404L-P406A-P444H-L448W +++ A241D-S403H-C404L-P406A-L448W-T450M +++ A241D-S403C-C404L-P406A-G443A-L448W-T450M +++ A241D-S403C-C404L-P406A-G443F-L448W-T450M +++ A241D-S403C-C404L-P406A-G443Y-L448W-T450M +++ A241D-S403C-C404L-P406A-P444A-L448W-T450M +++ A241D-S403C-C404L-P406A-P444F-L448W-T450M +++ A241D-S403C-C404L-P406A-P444T-L448W-T450M +++ A241D-S403V-C404L-P406A-G443D-L446V-L448W +++ A241D-S403V-C404L-P406A-G443D-L448W +++ A241D-S403V-C404L-P406A-G443A-L448W-T450M +++ A241D-S403V-C404L-P406A-G443D-L448W-T450M +++ A241D-S403V-C404L-P406A-G443F-L448W-T450M +++ A241D-S403V-C404L-P406A-G443N-L448W-T450M +++ A241D-S403V-C404L-P406A-G443S-L448W-T450M +++ A241D-S403V-C404L-P406A-V445L-L448W-T450M +++ A241D-C404L-P406A-G443F-L446V-L448W-T450M +++ A241D-C404L-P406A-L446V-L448W-T450A +++ A241D-S403C-C404L-P406A-G443A-L446V-L448W-T450M +++ A241D-S403C-C404L-P406A-G443S-L446V-L448W-T450M +++ A241D-S403C-C404L-P406A-G443W-L446V-L448W-T450M +++ A241D-S403C-C404L-P406A-G443Y-L446V-L448W-T450M +++ H198Q-A241D-S403C-C404L-P406A-G443F-L446V-L448W- +++ T450M A241D-S403C-C404L-P406A-P444H-L446V-L448W-T450M +++ A241D-S403C-C404L-P406A-V408I-L446V-L448W-T450M +++ A241D-S403C-C404L-P406A-L446M-L448W-T450M +++ A241D-S403V-C404L-P406A-G443D-L446V-L448W-T450M +++ A241D-S403V-C404L-P406A-G443F-L446V-L448W-T450M +++ A241D-S403V-C404L-P406A-G443Y-L446V-L448W-T450M +++ A241D-S403V-C404L-P406A-L446V-L448W-T450H +++ A241D-S403A-C404L-P406A-L446V-L448W-T450M +++ A241D-S403G-C404L-P406A-L446V-L448W-T450M +++ A241D-S403V-C404L-P406A-V408I-L446V-L448W-T450M +++ A241D-S403V-C404L-P406A-L446A-L448W-T450M +++ A241D-S403V-C404L-P406A-L446M-L448W-T450M +++

(82) TABLE-US-00015 TABLE 14 List of the positions modified in the variants of Streptomyces sp. 769 3-MethylCrotonate Decarboxylase with increased activity Wild-Type Position amino acid Mutations 85 P L 198 H Q 240 V I 241 A D, N, V 359 A C 390 R S 401 F Y 402 G A 403 S A, C, G, H, N, P, R, V 404 C F, L, M 405 L H 406 P A, S 408 V I 443 G A, D, F, H, N, S, W, Y 444 P A, E, F, H, L, T 445 V L 446 L A, G, F, I, M, N, S, V 448 L F, W, Y 449 L I 450 T A, H, M, N, Q, S