3-methylcrotonic acid decarboxylase (MDC) variants

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: 14, wherein said variant comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 14 with a substitution, a deletion or an insertion at a position corresponding to amino acid residue 448 of SEQ ID NO: 14.

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 of claim 1 .

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

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

6. The MDP variant of claim 1 , wherein said variant further comprises a substitution, a deletion or an insertions at one or more amino acid residues corresponding to position(s) 85, 198, 240, 241, 359, 390, 401, 402, 403, 404, 405, 406, 408, 443, 444, 445, 446, 449 and 450 of SEQ ID NO: 14.

7. The MDC variant of claim 6 , wherein:

(1) the 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

(2) the 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; and/or

(3) the 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; and/or

(4) the 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

(5) the 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; and/or

(6) the 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; and/or

(7) the 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

(8) the 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

(9) the 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, asparagine, proline, arginine or valine; and/or

(10) the 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

(11) the 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; and/or

(12) the 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 alanine or serine; and/or

(13) the 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; and/or

(14) the 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, asparagine, serine, tryptophan or tyrosine; and/or

(15) the 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, leucine or threonine; and/or

(16) the 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; and/or

(17) the 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

(18) the 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

(19) the 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; and/or

(20) the 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 or serine.

8. The MDC variant of claim 1 , wherein said variant comprises an amino acid sequence at least 80% sequence identity to SEQ ID NO: 14 with a substitution, a deletion or an insertion at a position corresponding to amino acid residue 448 of SEQ ID NO: 14.

9. The MDP variant of claim 8 , wherein said variant further comprises a substitution, a deletion or an insertions at one or more amino acid residues corresponding to position(s) 85, 198, 240, 241, 359, 390, 401, 402, 403, 404, 405, 406, 408, 443, 444, 445, 446, 449 and 450 of SEQ ID NO: 14.

10. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 9 .

11. The method of claim 10 , wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant of claim 9 .

12. A composition comprising the MDC variant of claim 9 .

13. The composition of claim 12 further comprising 3-methylcrotonic acid.

14. The MDC variant of claim 1 , wherein said variant comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO: 14 with a substitution, a deletion or an insertion at a position corresponding to amino acid residue 448 of SEQ ID NO: 14.

15. The MDP variant of claim 14 , wherein said variant further comprises a substitution, a deletion or an insertions at one or more amino acid residues corresponding to position(s) 85, 198, 240, 241, 359, 390, 401, 402, 403, 404, 405, 406, 408, 443, 444, 445, 446, 449 and 450 of SEQ ID NO: 14.

16. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 15 .

17. The method of claim 16 , wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant of claim 15 .

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

19. The composition of claim 18 further comprising 3-methylcrotonic acid.

20. The MDC variant of claim 1 , wherein said variant comprises an amino acid sequence at least 95% sequence identity to SEQ ID NO: 14 with a substitution, a deletion or an insertion at a position corresponding to amino acid residue 448 of SEQ ID NO: 14.

21. The MDP variant of claim 20 , wherein said variant further comprises a substitution, a deletion or an insertions at one or more amino acid residues corresponding to position(s) 85, 198, 240, 241, 359, 390, 401, 402, 403, 404, 405, 406, 408, 443, 444, 445, 446, 449 and 450 of SEQ ID NO: 14.

22. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 21 .

23. The method of claim 22 , wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant of claim 21 .

24. A composition comprising the MDC variant of claim 21 .

25. The composition of claim 24 further comprising 3-methylcrotonic acid.

26. The MDC variant of claim 1 , wherein said variant comprises the amino acid sequence of SEQ ID NO: 14 with a substitution, a deletion or an insertion at a position corresponding to amino acid residue 448 of SEQ ID NO: 14.

27. The MDP variant of claim 26 , wherein said variant further comprises a substitution, a deletion or an insertions at one or more amino acid residues corresponding to position(s) 85, 198, 240, 241, 359, 390, 401, 402, 403, 404, 405, 406, 408, 443, 444, 445, 446, 449 and 450 of SEQ ID NO: 14.

28. A method for producing isobutene from 3-methylcrotonic acid by incubating 3-methylcrotonic acid with the MDC variant of claim 27 .

29. The method of claim 28 , wherein the enzymatic conversion is carried out in vitro or by a host cell expressing the MDC variant of claim 27 .

30. A composition comprising the MDC variant of claim 27 .

31. The composition of claim 30 further comprising 3-methylcrotonic acid.