3-METHYLCROTONIC ACID DECARBOXYLASE (MDC) VARIANTS

• US 20190322996A1
• Filed: 07/03/2019
• Published: 10/24/2019
• Est. Priority Date: 05/04/2016
• Status: Active Grant

• Current Assignee: Global Bioenergies
• Original Assignee: Global Bioenergies
• Inventors: Stricher, Francois, Villiers, Benoit
• Granted Patent: US 10,988,752 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 5/026 Unsaturated compounds, i.e. alkenes, alkynes or allenes
• CPC Class Codes: C12Y 401/01028 Aromatic-L-amino-acid decarboxylase (4.1.1.28), i.e. tryptophane-decarboxylase
• CPC Class Codes: C12Y 604/01004 Methylcrotonoyl-CoA carboxylase (6.4.1.4)

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 over the corresponding MDC from which it is derived and having an amino acid sequence as shown in SEQ ID NO:

1 or an amino acid sequence having at least 73% sequence identity to SEQ ID NO;

1, in which one or more amino acid residues at a position selected from the group consisting of positions 2, 12, 13, 29, 31, 33, 35, 89, 114, 195, 197, 221, 293, 337, 351, 376, 381, 388, 405, 420, 422, 435, 436, 439, 441, 447, 449, 500, 506, and 511 in the amino acid sequence shown in SEQ ID NO;

1 or at a position corresponding to any of these positions, are substituted with another amino acid residue or deleted or wherein an insertion has been effected at one or more of these positions.

2. The MDC variant of claim 1, wherein

(1) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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.

3. The MDC variant of claim 1 or 2, wherein said variant furthermore 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.

4. The MDC variant of any one of claims 1 to 3, wherein said variant furthermore 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 is an MDC variant, wherein

(1) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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) an 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 phenylalaine; and

/or

(105) an 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) an 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) an 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) an 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) an 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) an 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) an 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.

5. A nucleic acid molecule encoding the MDC variant of any one of claims 1 to 4.

6. A host cell comprising the nucleic acid molecule of claim 5.

7. A vector comprising the nucleic acid molecule of claim 5.

8. A host cell comprising the vector of claim 6.

9. Use of the MDC variant of any one of claims 1 to 4 or the host cell of claim 7 for the conversion of 3-methylcrotonic acid into isobutene.

10. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of any one of claims 1 to 4.

11. The method of claim 10, wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the nucleic acid molecule.

12. A composition comprising the MDC variant of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 7 or the host cell of claim 6 or 8.

13. A composition comprising a variant of an MDC of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 7 or the host cell of claim 6 or 8 and 3-methylcrotonic acid.

14. A variant of a 3-methylcrotonic acid decarboxylase (MDC) showing an improved activity in converting 3-methylcrotonic acid into isobutene over the corresponding MDC from which it is derived and having an amino acid sequence as shown in SEQ ID NO:

14 or an amino acid sequence having at least 63% sequence identity to SEQ ID NO;

14, in which one or more amino acid residues at a position selected from the group consisting of positions 448, 2, 3, 5, 6, 24, 25, 35, 36, 64, 74, 80, 85, 86, 126, 129, 154, 166, 175, 195, 196, 197, 198, 204, 208, 214, 236, 237, 238, 240, 277, 298, 302, 339, 343, 347, 350, 359, 366, 368, 390, 391, 394, 395, 396, 398, 401, 402, 403, 404, 405, 406, 407, 408, 426, 439, 440, 441, 442, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462 and 473 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to any of these positions, are substituted with another amino acid residue or deleted or wherein an insertion has been effected at one or more of these positions.

15. The MDC variant of claim 14, wherein

(1) an amino acid residue at position 2 in the amino acid sequence shown in SEQ ID NO:

14 or at a position corresponding to this position, is deleted or substituted with asparagine; and

/or

(2) an amino acid residue at position 3 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with leucine or methionine; and

/or

(3) an amino acid residue at position 5 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine; and

/or

(4) an amino acid residue at position 6 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with methionine; and

/or

(5) an amino acid residue at position 24 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, aspartic acid, leucine or valine; and

/or

(6) an amino acid residue at position 25 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamic acid; and

/or

(7) an amino acid residue at position 35 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine; and

/or

(8) an amino acid residue at position 36 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with tyrosine; and

/or

(9) an amino acid residue at position 64 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with histidine; and

/or

(10) an amino acid residue at position 74 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with valine; and

/or

(11) an amino acid residue at position 80 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with serine; and

/or

(12) an amino acid residue at position 85 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with leucine; and

/or

(13) an amino acid residue at position 86 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with asparagine or tyrosine; and

/or

(14) an amino acid residue at position 126 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with valine; and

/or

(15) an amino acid residue at position 129 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with valine; and

/or

(16) an amino acid residue at position 154 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with aspartic acid; and

/or

(17) an amino acid residue at position 166 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with aspartic acid; and

/or

(18) an amino acid residue at position 175 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamic acid; and

/or

(19) an amino acid residue at position 195 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine or valine; and

/or

(20) an amino acid residue at position 196 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine; and

/or

(21) an amino acid residue at position 197 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with valine; and

/or

(22) an amino acid residue at position 198 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamine or tryptophan; and

/or

(23) an amino acid residue at position 204 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with arginine or serine; and

/or

(24) an amino acid residue at position 208 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with leucine; and

/or

(25) an amino acid residue at position 214 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with threonine; and

/or

(26) an amino acid residue at position 236 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with leucine or valine; and

/or

(27) an amino acid residue at position 237 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, aspartic acid, asparagine or proline; and

/or

(28) an amino acid residue at position 238 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with histidine, arginine, serine or tryptophan; and

/or

(29) an amino acid residue at position 240 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine or asparagine; and

/or

(30) an amino acid residue at position 277 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with tyrosine; and

/or

(31) an amino acid residue at position 298 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with serine; and

/or

(32) an amino acid residue at position 302 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, cysteine, phenylalanine, lysine, methionine, glutamine, arginine or serine; and

/or

(33) an amino acid residue at position 339 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with histidine; and

/or

(34) an amino acid residue at position 343 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with serine; and

/or

(35) an amino acid residue at position 347 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine; and

/or

(36) an amino acid residue at position 350 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with serine; and

/or

(37) an amino acid residue at position 359 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine or valine; and

/or

(38) an amino acid residue at position 366 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine or phenylalanine; and

/or

(39) an amino acid residue at position 368 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine or methionine; and

/or

(40) an amino acid residue at position 390 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with serine, aspartic acid, leucine, asparagine or glutamine; and

/or

(41) an amino acid residue at position 391 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with aspartic acid or tryptophan; and

/or

(42) an amino acid residue at position 394 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine, aspartic acid, glutamine or threonine; and

/or

(43) an amino acid residue at position 395 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine, glutamic acid or valine; and

/or

(44) an amino acid residue at position 396 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine; and

/or

(45) an amino acid residue at position 398 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine or arginine; and

/or

(46) an amino acid residue at position 401 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with tyrosine; and

/or

(47) an amino acid residue at position 402 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine; and

/or

(48) an amino acid residue at position 403 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, cysteine, glycine, histidine, isoleucine, asparagine, proline, glutamine, arginine or valine; and

/or

(49) an amino acid residue at position 404 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with phenylalanine, leucine or methionine; and

/or

(50) an amino acid residue at position 405 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with histidine, isoleucine, methionine, threonine or valine; and

/or

(51) an amino acid residue at position 406 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glycine, alanine or serine; and

/or

(52) an amino acid residue at position 407 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, cysteine, isoleucine, lysine, arginine, serine or threonine; and

/or

(53) an amino acid residue at position 408 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine, alanine, cysteine, leucine, or methionine; and

/or

(54) an amino acid residue at position 426 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with valine; and

/or

(55) an amino acid residue at position 439 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine; and

/or

(56) an amino acid residue at position 440 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with leucine; and

/or

(57) an amino acid residue at position 441 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glycine, histidine, lysine, asparagine, proline, arginine, serine or threonine; and

/or

(58) an amino acid residue at position 442 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, cysteine, glutamic acid, glycine, histidine, leucine, methionine, asparagine, glutamine, arginine, serine or threonine; and

/or

(59) an amino acid residue at position 444 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, glutamic acid, phenylalanine, histidine, isoleucine, leucine, glutamine or threonine; and

/or

(60) an amino acid residue at position 446 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, cysteine, phenylalanine, isoleucine, methionine, asparagine, serine or valine; and

/or

(61) an amino acid residue at position 447 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamine; and

/or

(62) an amino acid residue at position 448 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with phenylalanine, tryptophan or tyrosine; and

/or

(63) an amino acid residue at position 449 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine, methionine or valine; and

/or

(64) an amino acid residue at position 450 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, histidine, methionine, asparagine, glutamine, serine or valine; and

/or

(65) an amino acid residue at position 451 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with asparagine or serine; and

/or

(66) an amino acid residue at position 452 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with phenylalanine; and

/or

(67) an amino acid residue at position 453 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine, glycine, leucine, asparagine, proline, arginine or serine; and

/or

(68) an amino acid residue at position 454 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, aspartic acid, glycine, histidine, lysine, leucine, methionine, glutamine, arginine, serine, threonine, valine or tryptophan; and

/or

(69) an amino acid residue at position 455 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, glutamic acid, histidine, lysine, asparagine, serine or valine; and

/or

(70) an amino acid residue at position 456 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamine; and

/or

(71) an amino acid residue at position 457 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with lysine; and

/or

(72) an amino acid residue at position 458 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamic acid, phenylalanine, glycine, leucine, methionine, asparagine, glutamine, arginine, serine or tyrosine; and

/or

(73) an amino acid residue at position 459 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with aspartic acid, glutamic acid, glycine, methionine, asparagine, glutamine, arginine, threonine or tyrosine; and

/or

(74) an amino acid residue at position 460 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamic acid, phenylalanine, glycine, leucine, methionine, asparagine, glutamine or arginine; and

/or

(75) an amino acid residue at position 461 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, glycine, leucine, methionine, glutamine, serine or valine; and

/or

(76) an amino acid residue at position 462 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine or valine; and

/or

(77) an amino acid residue at position 473 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with serine.

16. The MDC variant of claim 14 or 15, wherein in said variant one or more further amino acid residues at a position selected from the group consisting of 66, 168, 241, 296, 305, 349, 363, 443, 445, 479, and 496 in the amino acid sequence shown in SEQ ID NO:

14 or at a position corresponding to any of these positions, are substituted with another amino acid residue or deleted or wherein an insertion has been effected at one or more of these positions, and wherein said MDC variant has an improved activity in converting 3-methylcrotonic acid into isobutene over the corresponding MDC from which it is derived.

17. The MDC variant of any one of claims 14 to 16, wherein

(78) an amino acid residue at position 66 in the amino acid sequence shown in SEQ ID NO:

14 or at a position corresponding to this position, is deleted or substituted with histidine; and

/or

(79) an amino acid residue at position 168 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with valine; and

/or

(80) an amino acid residue at position 241 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with aspartic acid, asparagine or valine; and

/or

(81) an amino acid residue at position 296 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glutamine; and

/or

(82) an amino acid residue at position 305 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with isoleucine or threonine; and

/or

(83) an amino acid residue at position 349 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with methionine or serine; and

/or

(84) an amino acid residue at position 363 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with glycine; and

/or

(85) an amino acid residue at position 443 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with alanine, aspartic acid, phenylalanine, histidine, leucine, asparagine, arginine, serine, tryptophan or tyrosine; and

/or

(86) an amino acid residue at position 445 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with leucine, alanine, cysteine, serine or threonine; and

/or

(87) an amino acid residue at position 479 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with cysteine, isoleucine, leucine, methionine, serine, threonine or valine; and

/or

(88) an amino acid residue at position 496 in the amino acid sequence shown in SEQ ID NO;

14 or at a position corresponding to this position, is deleted or substituted with leucine.

18. A nucleic acid molecule encoding the MDC variant of any one of claims 14 to 17.

19. A host cell comprising the nucleic acid molecule of claim 18.

20. A vector comprising the nucleic acid molecule of claim 18.

21. A host cell comprising the vector of claim 20.

22. Use of the MDC variant of any one of claims 14 to 17 or the host cell of claim 19 or 21 or the vector of claim 20 for the conversion of 3-methylcrotonic acid into isobutene.

23. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of any one of claims 14 to 17, or the host cell of claim 19 or 21 or the vector of claim 20.

24. The method of claim 23, wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the nucleic acid molecule.

25. A composition comprising the MDC variant of any one of claims 14 to 17 or the host cell of claim 19 or 21 or the vector of claim 20.

26. A composition comprising the MDC variant of any one of claims 14 to 17 or the host cell of claim 19 or 21 or the vector of claim 20 and 3-methylcrotonic acid.