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Title:
IMPROVED METHODS FOR PRODUCING ISOBUTENE FROM 3-METHYLCROTONIC ACID
Document Type and Number:
WIPO Patent Application WO/2018/206262
Kind Code:
A1
Abstract:
Described are methods for the production of isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) into a flavin-derived cofactor, wherein said method further comprises providing said DMAP enzymatically. Moreover, described are methods for the production of isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl pyrophosphate (DMAPP), wherein said method further comprises providing said DMAPP enzymatically.

Inventors:
CHAYOT ROMAIN (FR)
ALLARD MATHIEU (FR)
ANISSIMOVA MARIA (FR)
Application Number:
PCT/EP2018/060051
Publication Date:
November 15, 2018
Filing Date:
April 19, 2018
Export Citation:
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Assignee:
GLOBAL BIOENERGIES (FR)
International Classes:
C12P5/02; C12N1/21
Domestic Patent References:
WO2017085167A22017-05-26
WO2010001078A22010-01-07
WO2012018624A22012-02-09
WO2013028519A12013-02-28
WO2013186215A12013-12-19
WO2013053824A12013-04-18
WO2009006429A12009-01-08
WO2013173437A12013-11-21
WO2009006429A12009-01-08
WO2013040383A12013-03-21
WO2011032934A12011-03-24
Foreign References:
US20090092975A12009-04-09
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Attorney, Agent or Firm:
VOSSIUS & PARTNER (NO 31) (DE)
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Claims:
Claims

1. A method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3- methylcrotonic acid into isobutene is achieved by making use of an FMN- dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) into a flavin- derived cofactor,

wherein said method further comprises providing said DMAP enzymatically by:

(i) the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into said DMAP; or

(ii) a single enzymatic step in which prenol is directly enzymatically converted into said DMAP; or

(iii) two enzymatic steps comprising:

first enzymatically converting DMAPP into prenol; and

then enzymatically converting the thus obtained prenol into said

DMAP; or

(iv) the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP,

or by a combination of any one of (i) to (iv); and/or

wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl pyrophosphate (DMAPP), wherein said method further comprises providing said DMAPP enzymatically by:

(v) the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP; or

(vi) the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP; or

(vii) the enzymatic conversion of prenol into said DMAPP;

(viii) or by a combination of any one of (v) to (vii).

2. The method of claim 1 (i), wherein the enzymatic conversion of DMAPP into said DMAP is achieved by making use of a phosphatase.

3. The method of claim 2, wherein the phosphatase is:

an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), preferably an ADP-ribose pyrophosphatase (EC 3.6.1 .13), an 8-oxo-dGTP diphosphatase (EC 3.6.1 .55), a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1.45), exopolyphosphatase (EC 3.6.1.1 1 ), a guanosine-5'-triphosphate/3'-diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1 .22), a nucleotide diphosphatase (EC 3.6.1 .9) or an acylphosphatase (EC 3.6.1 .7); or

a phosphoric-monoester hydrolase (EC 3.1 .3.-), preferably a 3'(2'),5'- bisphosphate nucleotidase (EC 3.1 .3.7), a 5-amino-6-(5-phospho-D- ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ).

4. The method of claim 1 (i), wherein the enzymatic conversion of DMAPP into DMAP is achieved by making use of an isopentenyl phosphate kinase (EC 2.7.4.26).

5. The method of any one of claims 2 to 4, further comprising providing the DMAPP by the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP.

6. The method of claim 5, wherein the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP is achieved by making use of an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

7. The method of claim 1 (ii), wherein the enzymatic conversion of prenol into DMAP is achieved by making use of a kinase.

8. The method of claim 7, wherein the kinase is a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), preferably a hydroxyethylthiazole kinase (EC 2.7.1 .50).

9. The method of claim 1 (iii), wherein the enzymatic conversion of DMAPP into prenol is achieved by making use of a phosphatase or pyrophosphatase and/or the enzymatic conversion of the thus obtained prenol into said DMAP is achieved by making use of a kinase.

10. The method of claim 9, wherein the phosphatase or pyrophosphatase is:

an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC 3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1.3.5), a pyridoxal phosphate phosphatase (EC 3.1.3.74) or a fructose-1 6- bisphosphatase (EC 3.1.3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and/or

wherein the kinase is a phosphotransferase with an alcohol group as acceptor

(EC 2.7.1.-), preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

11. The method of claim 1 (iv), wherein the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP is achieved by making use of an isomerase.

12. The method of claim 11 , wherein the isomerase is an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

13. The method of any one of claims 1 to 12, further comprising providing said flavin cofactor enzymatically by the enzymatic conversion of riboflavin into flavin mononucleotide (FMN).

14. The method of claim 13, wherein the enzymatic conversion of riboflavin into FMN is achieved by making use of:

a kinase, preferably an archaeal riboflavin kinase (EC 2.7.1.161 ),

a flavokinase derived from S. cerevisiae or from Rattus norvegicus,

a flavokinase derived from Megasphaera elsdenii,

a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 ), preferably an erythritol kinase (2.7.1.27) or a glycerol kinase (2.7.1.30),

a phosphotransferase with a phosphate group as acceptor (EC 2.7.4), preferably an isopentenyl phosphate kinase (EC 2.7.4.26); or

a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF); or

a variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived.

15. The method of claim 14, wherein said variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived is a variant having an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence having at least 30% sequence identity to SEQ ID NO:34, in which one or more amino acid residues at a position selected from the group consisting of positions 29 and 32 in the amino acid sequence shown in SEQ ID NO:34 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.

16. The method of claim 15, wherein

(1 ) an amino acid residue at position 29 in the amino acid sequence shown in SEQ ID NO:34 or at a position corresponding to this position, is deleted or substituted with alanine; and/or

(2) an amino acid residue at position 32 in the amino acid sequence shown in SEQ ID NO:34 or at a position corresponding to this position, is deleted or substituted with serine or alanine.

17. The method of claim 1 (v), wherein the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP is achieved by making use of an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

18. The method of claim 1 (vi), wherein the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP is achieved by making use of a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26).

19. The method of claim 18, further comprising providing the DMAP by the enzymatic conversion of prenol into DMAP or by the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP.

20. The method of claim 19, wherein the enzymatic conversion of prenol into said DMAP is achieved by making use of a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

21. The method of claim 19, wherein the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP is achieved by making use of an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

22. The method of claim 1 (vii), wherein the enzymatic conversion of prenol into DMAPP is achieved by making use of a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3).

23. A recombinant organism or microorganism which recombinantly expresses an FMN-dependent decarboxylase associated with an FMN prenyl transferase; wherein said recombinant organism or microorganism further recombinantly expresses at least one of the following (i) to (iv):

(i) an enzyme catalyzing the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into dimethylallyl phosphate (DMAP), wherein said enzyme is a phosphatase,

preferably an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), more preferably an ADP-ribose pyrophosphatase (EC 3.6.1 .13), an 8-oxo-dGTP diphosphatase (EC 3.6.1.55), a bis(5'- nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1 .45), exopolyphosphatase (EC 3.6.1 .1 1 ), a guanosine-5'-triphosphate/3'-diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1.22), a nucleotide diphosphatase (EC 3.6.1.9) or an acylphosphatase (EC 3.6.1.7); or preferably a phosphoric-monoester hydrolase (EC 3.1 .3.-), more preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1 .3.7), a 5- amino-6-(5-phospho-D-ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1 .3.1 1 ); or

preferably an isopentenyl phosphate kinase (EC 2.7.4.26);

an enzyme catalyzing the direct enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 .-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

an enzyme catalyzing the enzymatic conversion of DMAPP into prenol, wherein said enzyme is a phosphatase or pyrophosphatase, preferably an alkaline phosphatase (EC 3.1 .3.1 ), a sugar phosphatase (EC 3.1 .3.23), a phosphatidylglycerophosphatase (EC 3.1 .3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1 .3.81 ), a phosphatidate phosphatase (EC 3.1 .3.4), a phosphoserine phosphatase (EC 3.1 .3.3), a phosphoglycolate phosphatase (EC 3.1 .3.18), a pyrimidine 5'-nucleotidase (EC 3.1 .3.5), a pyridoxal phosphate phosphatase (EC 3.1 .3.74) or a fructose-1 6-bisphosphatase (EC 3.1 .3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1 .45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1 .27); or

a prenyl-diphosphatase (EC 3.1 .7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and

an enzyme catalyzing the thus obtained prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 .-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50); and (iv) an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

The recombinant organism or microorganism of claim 23 (i), further recombinantly expressing an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

A recombinant organism or microorganism which recombinantly expresses an FMN-dependent decarboxylase associated with an FMN prenyl transferase; wherein said recombinant organism or microorganism further recombinantly expresses at least one of the following (v) to (vii):

(v) an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2);

(vi) an enzyme catalyzing the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP, wherein said enzyme is a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26); and

(vii) an enzyme catalyzing the enzymatic conversion of prenol into said DMAPP, wherein said enzyme is a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4- hydroxy-6-hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3).

The recombinant organism or microorganism of claim 25, further recombinantly expressing an enzyme catalyzing the enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

27. The recombinant organism or microorganism of claim 25 or 26, further recombinantly expressing an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

28. The recombinant organism or microorganism of any one of claims 23 to 27, further recombinantly expressing an enzyme catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN).

29. Use of a recombinant organism or microorganism as defined in any one of claims 23 to 28 for the production of isobutene.

30. Use of a combination comprising an FMN-dependent decarboxylase associated with an FMN prenyl transferase and an enzyme or enzymes of any one of the following (i) to (iv):

(i) an enzyme catalyzing the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into dimethylallyl phosphate (DMAP), wherein said enzyme is a phosphatase,

preferably an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), more preferably an ADP-ribose pyrophosphatase (EC 3.6.1 .13), an 8-oxo-dGTP diphosphatase (EC 3.6.1 .55), a bis(5'- nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1 .45), exopolyphosphatase (EC 3.6.1 .1 1 ), a guanosine-5'-triphosphate/3'-diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1 .22), a nucleotide diphosphatase (EC 3.6.1 .9) or an acylphosphatase (EC 3.6.1.7); or preferably a phosphoric-monoester hydrolase (EC 3.1.3.-), more preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1 .3.7), a 5- amino-6-(5-phospho-D-ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1 .3.1 1 ); or

preferably an isopentenyl phosphate kinase (EC 2.7.4.26);

(ii) an enzyme catalyzing the direct enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iii) an enzyme catalyzing the enzymatic conversion of DMAPP into prenol, wherein said enzyme is a phosphatase or pyrophosphatase, preferably an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC 3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1.3.5), a pyridoxal phosphate phosphatase (EC 3.1.3.74) or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and

an enzyme catalyzing the thus obtained prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50); and

(iv) an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2)

for the production of isobutene from 3-methylcrotonic acid.

31. The use of claim 30, further comprising an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

32. Use of a combination comprising an FMN-dependent decarboxylase associated with an FMN prenyl transferase and an enzyme or enzymes of at least one of the following (v) to (vii): (v) an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2);

(vi) an enzyme catalyzing the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP, wherein said enzyme is a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26); and

(vii) an enzyme catalyzing the enzymatic conversion of prenol into said DMAPP, wherein said enzyme is a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4- hydroxy-6-hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3).

33. The use of claim 32, further comprising an enzyme catalyzing the enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

34. The use of claim 32 or 33, further comprising an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

35. The use of any one of claims 30 to 34, further comprising an enzyme catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN).

36. A composition comprising DMAPP, IMP or prenol and a recombinant organism or microorganism as defined in any one of claims 23, 24 or 28; or DMAPP, IMP or prenol and an enzyme or enzymes as defined in any one of claims 1 to 22.

37. A composition comprising DMAP, IPP or prenol and a recombinant organism or microorganism as defined in any one of claims 25 to 28; or DMAP, IPP or prenol and an enzyme or enzymes as defined in any one of claims 1 to 22.

Description:
Improved methods for producing isobutene from 3-methylcrotonic acid

The present invention relates to methods for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) into a flavin-derived cofactor, wherein said method further comprises providing said DMAP enzymatically by: (i) the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into said DMAP; or (ii) a single enzymatic step in which prenol is directly enzymatically converted into said DMAP; or (iii) two enzymatic steps comprising: first enzymatically converting DMAPP into prenol; and then enzymatically converting the thus obtained prenol into said DMAP; or (iv) the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP, or by a combination of any one of (i) to (iv). Moreover, the present invention relates to methods for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl pyrophosphate (DMAPP), wherein said method further comprises providing said DMAPP enzymatically by: (v) the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP; or (vi) the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP; or (vii) the enzymatic conversion of prenol into said DMAPP; or (viii) by a combination of any one of (v) to (vii). Moreover, the present invention relates to methods for providing said flavin cofactor enzymatically by the enzymatic conversion of riboflavin into flavin mononucleotide (FMN).

A large number of chemical compounds are currently derived from petrochemicals. Alkenes (such as ethylene, propylene, the different butenes, or else the pentenes, for example) are used in the plastics industry, for example for producing polypropylene or polyethylene, and in other areas of the chemical industry and that of fuels.

Butylene exists in four forms, one of which, isobutene (also referred to as isobutylene), enters into the composition of methyl-tert-butyl-ether (MTBE), an antiknock additive for automobile fuel. Isobutene can also be used to produce isooctene, which in turn can be reduced to isooctane (2,2,4-trimethylpentane); the very high octane rating of isooctane makes it the best fuel for so-called "gasoline" engines. Alkenes such as isobutene are currently produced by catalytic cracking of petroleum products (or by a derivative of the Fischer-Tropsch process in the case of hexene, from coal or gas). The production costs are therefore tightly linked to the price of oil. Moreover, catalytic cracking is sometimes associated with considerable technical difficulties which increase process complexity and production costs.

The production by a biological pathway of alkenes such as isobutene is called for in the context of a sustainable industrial operation in harmony with geochemical cycles. The first generation of biofuels consisted in the fermentative production of ethanol, as fermentation and distillation processes already existed in the food processing industry. The production of second generation biofuels is in an exploratory phase, encompassing in particular the production of long chain alcohols (butanol and pentanol), terpenes, linear alkanes and fatty acids. Two recent reviews provide a general overview of research in this field: Ladygina et al. (Process Biochemistry 41 (2006), 1001 ) and Wackett (Current Opinions in Chemical Biology 21 (2008), 187). The conversion of isovalerate to isobutene by the yeast Rhodotoruia minuta has been described (Fujii et al. (Appl. Environ. Microbiol. 54 (1988), 583)), but the efficiency of this reaction, less than 1 millionth per minute, or about 1 for 1000 per day, is far from permitting an industrial application. The reaction mechanism was elucidated by Fukuda et al. (BBRC 201 (1994), 516) and involves a cytochrome P450 enzyme which decarboxylates isovalerate by reduction of an oxoferryl group Fe v =O. Large-scale biosynthesis of isobutene by this pathway seems highly unfavourable, since it would require the synthesis and degradation of one molecule of leucine to form one molecule of isobutene. Also, the enzyme catalyzing the reaction uses heme as cofactor, poorly lending itself to recombinant expression in bacteria and to improvement of enzyme parameters. For all these reasons, it appears very unlikely that this pathway can serve as a basis for industrial exploitation. Other microorganisms have been described as being marginally capable of naturally producing isobutene from isovalerate; the yields obtained are even lower than those obtained with Rhodotorula minuta (Fukuda et al. (Agric. Biol. Chem. 48 (1984), 1679)).

Gogerty et al. (Appl. Environm. Microbiol. 76 (2010), 8004-8010) and van Leeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387) describe the production of isobutene from acetoacetyl-CoA by enzymatic conversions wherein the last step of the proposed pathway is the conversion of 3-hydroxy-3-methylbutyric acid (also referred to as 3-hydroxyisovalerate (HIV)) by making use of a mevalonate diphosphate decarboxylase. This reaction for the production of isobutene from 3- hydroxy-3-methylbutyric acid is also described in WO2010/001078. In Gogerty et al. (loc. cit.) and in van Leeuwen et al. (loc. cit.) the production of 3-hydroxy-3- methylbutyric acid is proposed to be achieved by the conversion of 3-methylcrotonyl- CoA via 3-hydroxy-3-methylbutyryl-CoA. In order to further improve the efficiency and variability of methods for producing isobutene from renewable resources, alternative routes for the provision of isobutene and its precursors have been developed by providing methods for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid (also termed 3-methyl-2-butenoic acid, 3,3-dimethylacrylic acid or senecioic acid) into isubutene.

The enzymatic conversion of 3-methylcrotonic acid into isobutene is a decarboxylation reaction. A decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO 2 ).

The decarboxylation of 3-methylcrotonic acid has already been suggested in US-A1- 2009/0092975 while there is no experimental evidence for this conversion. In US-A1 - 2009/0092975, a nucleic acid sequence called PAD1 derived from Saccharomyces cerevisiae is described and is disclosed to encode a decarboxylation enzyme. This enzyme is suggested to be useful as a selectable marker in a recombinant organism while it is described that a "weak acid" may be used as the selecting agent. 3- methylcrotonic acid is mentioned, among many others, as a potential "weak acid". However, it was only later found that the above PAD1 , in reality, does not provide for the decarboxylase activity.

In fact, the bacterial ubiD and ubiX or the homologous eukaryotic fdd and padl genes have been implicated in the non-oxidative reversible decarboxylation. The combined action of phenylacrylic acid decarboxylase (PAD) and ferulic acid decarboxylase (FDC) is considered to be essential for the decarboxylation of phenylacrylic acid in Saccharomyces cerevisiae (J. Biosci. Bioeng. 109, (2010), 564- 569; AMB Express, 5:12 (2015) 1 -5; ACS Chem. Biol. 10 (2015), 1137-1 144).

Recently, the above enzyme family described as phenylacrylic acid decarboxylase (PAD) was characterized as an FMN prenyl-transferase and no longer as a decarboxylase. It has been shown that Fdc1 (but not PAD) is solely responsible for the reversible decarboxylase activity and that it requires a new type of cofactor, namely a prenylated flavin synthesized by the associated UbiX (or Pad1 ) protein. Thus, the real enzymatic activity of this PAD enzyme has been identified as the transformation of a flavin mononucleotide (FMN) cofactor with a prenyl moiety (from di-methyl-allyl-phosphate or pyrophosphate called DMAP or DMAPP).

Accordingly, in contrast to the prior art's belief, the real decarboxylase is the ferulic acid decarboxylase (FDC) in association with the modified FMN (prenylated-FMN). This mechanism of the ferulic acid decarboxylase (FDC) in association with the modified FMN (prenylated-FMN) (the latter provided by the PAD enzyme) was recently described and involves a surprising enzymatic mechanism, i.e., an α,β- unsaturated acid decarboxylation via a 1 ,3-dipolar cyclo-addition. Moreover, the structure of this FDC decarboxylase has recently been elucidated (Nature 522 (2015), 497-501 ; Nature, 522 (2015), 502-505; Appl. Environ. Microbiol. 81 (2015), 4216- 4223).

The use of the above family of enzymes has previously been described for the conversion of α-β unsaturated carboxylic acid into terminal alkenes in US-A1- 2009/0092975 as mentioned above while WO2012/018624 is directed to microorganisms and methods for the biosynthesis of aromatics, 2,4-pentadienoate and 1 ,3-butadiene and WO2013/028519 is directed to microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1 ,3-butanediol and related alcohols.

Moreover, WO2013/186215 describes a method for preparing a mono-unsaturated alkene comprising contacting an aliphatic mono-unsaturated carboxylic acid with an Fdc1 polypeptide and a Pad1 polypeptide. However, in WO2013/186215, both, the Fdc1 polypeptide and the Pad1 polypeptide are classified as enzymes having a decarboxylase activity. In contrast, in light of this background, methods have been developed wherein the above enzymes are artificially implemented in a pathway which ultimately leads to the production of isobutene. Thus, methods for the production of isobutene have been developed comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene (step I as shown in Figure 1), wherein the enzymatic conversion of 3- methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) into a flavin-derived cofactor while it has only been speculated that said FMN prenyl transferase also catalyzes the prenylation of a flavin cofactor (FMN or FAD) into a flavin-derived cofactor when utilizing dimethylallyl pyrophosphate (DMAPP).

Moreover, methods have been developed, wherein such a method further comprises

(a) providing the 3-methylcrotonic acid by the enzymatic conversion of 3- methylcrotonyl-CoA into 3-methylcrotonic acid (steps Via, Vlb or Vic as shown in Figure 1), or

(b) providing the 3-methylcrotonic acid by the enzymatic conversion of 3- hydroxyisovalerate (HIV) into 3-methylcrotonic acid (step II as shown in Figure

1).

The above method which has been developed for the production of isobutene from 3- methylcrotonyl-CoA via 3-methylcrotonic acid or from 3-hydroxyisovalerate (HIV) via 3-methylcrotonic acid may be embedded in a pathway for the production of isobutene starting from acetyl-CoA which is a central component and an important key molecule in metabolism used in many biochemical reactions. The corresponding reactions are schematically shown in Figure 1.

As outlined in more detail further below, the present invention has also found that 3- methylcrotonic acid is enzymatically converted into isobutene by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase when dimethylallyl pyrophosphate (DMAPP) instead of DMAP is used.

In the above described methods, the enzymatic conversion of 3-methylcrotonic acid into isobutene which is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) into a flavin-derived cofactor is a key step of the above overall metabolic pathway. In this key step, the availability of dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) as well as the availability of the flavin cofactor FMN are limiting factors. Therefore, there is a need for improved methods by increasing the pool/amount of dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) in order to ensure the efficient biosynthesis of the prenylated flavin cofactor (FMN or FAD). In addition, in order to ensure the efficient biosynthesis of the prenylated flavin cofactor (FMN or FAD) there is, therefore, also a need for the provision of an increased pool of the flavin cofactor FMN.

The present invention meets this demand by providing a method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase,

wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) into a flavin-derived cofactor, wherein said method further comprises providing said DMAP enzymatically by:

(i) the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into said DMAP; or

(ii) a single enzymatic step in which prenol is directly enzymatically converted into said DMAP; or

(iii) two enzymatic steps comprising: first enzymatically converting DMAPP into prenol; and then enzymatically converting the thus obtained prenol into said DMAP; or

(iv) the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP, or by a combination of any one of (i) to (iv).

Moreover, the present invention has found that the enzymatic conversion of 3- methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) into a flavin- derived cofactor, also when utilizing dimethylallyl pyrophosphate (DMAPP). Therefore, the present invention also meets the above demand by providing a method for the production of isobutene comprising the enzymatic conversion of 3- methylcrotonic acid into isobutene wherein the enzymatic conversion of 3- methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase,

wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor

(FMN or FAD) utilizing dimethylallyl pyrophosphate (DMAPP),

wherein said method further comprises providing said DMAPP enzymatically by:

(v) the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP; or

(vi) the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP; or

(vii) the enzymatic conversion of prenol into said DMAPP; or

(viii) by a combination of any one of (v) to (vii).

Moreover, the present invention provides a method for providing said flavin cofactor enzymatically by the enzymatic conversion of riboflavin into flavin mononucleotide (FMN).

The method according to the present invention is in particular useful for large scale production of isobutene in vitro or in vivo, in particular for a commercial production. Thus, the present invention relates to a method for large scale production, in particular the commercial production of isobutene wherein said method comprises the steps as described above.

The enzymatic conversion of 3-methylcrotonic acid into isobutene

The enzymatic conversion of 3-methylcrotonic acid into isobutene (step I as shown in Figure 1) is schematically shown in Figure 2B.

According to the present invention, the enzymatic conversion of 3-methylcrotonic acid (also termed 3-methyl-2-butenoic acid or 3,3-dimethyl-acrylic acid) into isobutene (also termed isobutylene or 2-methyl-propene) can be achieved by a decarboxylation by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase. "Decarboxylation" is generally a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO 2 ).

The enzymatic conversion of 3-methylcrotonic acid into isobutene utilizing an FMN- dependent decarboxylase associated with an FMN prenyl transferase relies on a reaction of two consecutive steps catalyzed by the two enzymes, i.e., the FMN- dependent decarboxylase (catalyzing the actual decarboxylation of 3-methylcrotonic acid into isobutene) with an associated FMN prenyl transferase which provides the modified flavin cofactor.

The flavin cofactor may preferably be FMN or FAD. FMN (flavin mononucleotide; also termed riboflavin-5'-phosphate) is a biomolecule produced from riboflavin (vitamin B2) by the enzyme riboflavin kinase and functions as prosthetic group of various reactions. FAD (flavin adenine dinucleotide) is a redox cofactor, more specifically a prosthetic group, involved in several important reactions in metabolism.

Thus, in the conversion of 3-methylcrotonic acid into isobutene, in a first step, a flavin cofactor (FMN or FAD) is modified into a (modified) flavin-derived cofactor. This modification is catalyzed by said FMN prenyl transferase. FMN prenyl transferase prenylates the flavin ring of the flavin cofactor (FMN or FAD) into a (modified) prenylated flavin cofactor. This reaction is schematically illustrated in Figure 2A. In a second step, the actual conversion of 3-methylcrotonic acid into isobutene is catalyzed by said FMN-dependent decarboxylase via a 1 ,3-dipolar cycloaddition based mechanism wherein said FMN-dependent decarboxylase uses the prenylated flavin cofactor (FMN or FAD) provided by the associated FMN prenyl transferase. This reaction is schematically illustrated in Figure 2B.

In a preferred embodiment, said FMN prenyl transferase which modifies the flavin cofactor (FMN or FAD) into a (modified) flavin-derived cofactor is a phenylacrylic acid decarboxylase (PAD)-type protein, or the closely related prokaryotic enzyme UbiX, an enzyme which is involved in ubiquinone biosynthesis in prokaryotes.

In Escherichia coli, the protein UbiX (also termed 3-octaprenyl-4-hydroxybenzoate carboxy-lyase) has been shown to be involved in the third step of ubiquinone biosynthesis.

It catalyses the reaction 3-octaprenyl-4-hydroxybenzoate 2-octaprenylphenol + C0 2 .

Moreover, the knockout of the homologous protein in yeast (Pad1 ) has been shown to confer sensitivity to phenylacrylic acid, showing that this enzyme functions as a phenylacrylic acid decarboxylase. E. coli strains also contain, in addition to UbiX, a second paralogue named Pad1. Its amino acid sequence shows 52% identity to UbiX and slightly higher sequence identity to Saccharomyces cerevisiae phenylacrylic acid decarboxylase Pad1. Despite its higher sequence similarity with yeast Pad1 , E. coli Pad1 does not seem to have phenylacrylic acid decarboxylase activity. Its function is unknown, Pad1 may remove the carboxylate group from derivatives of benzoic acid but not from substituted phenolic acids.

Thus, in a preferred embodiment, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by the FMN- containing protein phenylacrylic acid decarboxylase (PAD). The enzymes involved in the modification of the flavin cofactor (FMN or FAD) into the corresponding modified flavin-derived cofactor were initially annotated as decarboxylases ( EC 4.1.1.-). Some phenylacrylic acid decarboxylases (PAD) are now annotated as flavin prenyl transferases as EC 2.5.1.-.

Moreover, enzymes capable of catalyzing the enzymatic reaction described herein for flavin prenyl transferases have rectently also been annotated as flavin prenyl transferases as EC 2.5.2.129.

In a more preferred embodiment, the conversion of 3-methylcrotonic acid into isobutene makes use of a phenylacrylic acid decarboxylase (PAD)-type protein as the FMN prenyl transferase which modifies a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor wherein said phenylacrylic acid decarboxylase (PAD)-type protein is derived from Candida albicans (Uniprot accession number Q5A8L8), Aspergillus niger (Uniprot accession number A3F715), Saccharomyces cerevisiae (Uniprot accession number P33751 ) or Cryptococcus gattii (Uniprot accession number E6R9Z0).

In a preferred embodiment, the phenylacrylic acid decarboxylase (PAD)-type protein employed in the method of the present invention is a phenylacrylic acid decarboxylase (PAD)-type protein derived from Candida albicans (Uniprot accession number Q5A8L8; SEQ ID NO:1 ), Aspergillus niger (Uniprot accession number A3F715; SEQ ID NO:2), Saccharomyces cerevisiae (Uniprot accession number P33751 ; SEQ ID NO:3), Cryptococcus gattii (Uniprot accession number E6R9Z0; SEQ ID NO:4) or Hypocrea atroviridis (also termed Trichoderma atroviride; Uniprot accession number G9NTN1 ) having the amino acid sequence as shown in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:71 , respectively. In a preferred embodiment of the present invention the phenylacrylic acid decarboxylase (PAD)-type protein is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and 71 or a sequence which is at least n % identical to any of SEQ ID NOs: 1 to 4 and 71 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of modifying a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor.

As regards the determination of sequence identity, the following should apply: When the sequences which are compared do not have the same length, the degree of identity either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. Preferably, it refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence. The degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.

When using the Clustal analysis method to determine whether a particular sequence is, for instance, at least 60% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiple comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.

Preferably, the degree of identity is calculated over the complete length of the sequence.

Amino acid residues located at a position corresponding to a position as indicated herein-below in the amino acid sequence shown in any one of SEQ ID NOs:1 to 4 and 71 can be identified by the skilled person by methods known in the art. For example, such amino acid residues can be identified by aligning the sequence in question with the sequence shown in any one of SEQ ID NOs:1 to 4 and 71 and by identifying the positions which correspond to the above indicated positions of any one of SEQ ID NOs:1 to 4 and 71. The alignment can be done with means and methods known to the skilled person, e.g. by using a known computer algorithm such as the Lipman-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. It is preferred that in such an alignment maximum homology is assigned to conserved amino acid residues present in the amino acid sequences.

In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiple comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.

Preferably, the degree of identity is calculated over the complete length of the sequence.

In another preferred embodiment, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by the FMN- containing protein 3-octaprenyl-4-hydroxybenzoate carboxy-lyase also termed UbiX (initially annotated EC 4.1.1.-). As mentioned above, the enzymes involved in the modification of the flavin cofactor (FMN or FAD) into the corresponding modified flavin-derived cofactor were initially annotated as decarboxylases. Some phenylacrylic acid decarboxylases (PAD) are now annotated as flavin prenyl transferases as EC 2.5.1.-.

As mentioned above, enzymes capable of catalyzing the enzymatic reaction described herein for flavin prenyl transferases have rectently also been annotated as flavin prenyl transferases as EC 2.5.2.129.

In a more preferred embodiment, the conversion of 3-methylcrotonic acid into isobutene makes use of a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also termed UbiX) as the FMN prenyl transferase which modifies the flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor wherein said 3- octaprenyl-4-hydroxybenzoate carboxy-lyase (also termed UbiX) is derived from Escherichia coli (Uniprot accession number P0AG03), Bacillus subtilis (Uniprot accession, number A0A086WXG4), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1 ).

In an even more preferred embodiment, the 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also termed UbiX) employed in the method of the present invention is a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also termed UbiX) derived from Escherichia coli (Uniprot accession number P0AG03; SEQ ID NO:5), Bacillus subtilis (Uniprot accession, number A0A086WXG4; SEQ ID NO:6), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8; SEQ ID NO:7) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1 ; SEQ ID NO:8) having the amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

In a preferred embodiment of the present invention the 3-octaprenyl-4- hydroxybenzoate carboxy-lyase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8 or a sequence which is at least n % identical to any of SEQ ID NOs: 5 to 8 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of modifying a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by an Ubx-like flavin prenyl transferase derived from E. coli encoded by kpdB and ecdB, respectively (UniProt accession number A0A023LDW3 and UniProt accession number P69772, respectively; SEQ ID NO: 66), and an Ubx-like flavin prenyl transferase derived from Klebsiella pneumoniae encoded by kpdB (UniProt accession number Q462H4; SEQ ID NO:70).

In a preferred embodiment of the present invention the Ubx-like flavin prenyl transferase is an enzyme comprising an amino acid sequence of selected from the group consisting of SEQ ID NO: 66 and SEQ ID NO: 70 or a sequence which is at least n % identical to SEQ ID NO: 66 or SEQ ID NO: 70 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of modifying a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by a flavin prenyl transferase.

As mentioned above, the actual decarboxylation, i.e., the conversion of 3- methylcrotonic acid into isobutene is catalyzed by an FMN-dependent decarboxylase via a 1 ,3-dipolar cycloaddition based mechanism wherein said FMN-dependent decarboxylase uses the prenylated flavin cofactor (FMN or FAD) provided by any of the above described associated FMN prenyl transferases.

In a preferred embodiment, said FMN-dependent decarboxylase catalyzing the decarboxylation of 3-methylcrotonic acid into isobutene is catalyzed by a ferulic acid decarboxylase (FDC). Ferulic acid decarboxylases (FDC) belong to the enzyme class EC 4.1.1.-.

In an even more preferred embodiment, the conversion of 3-methylcrotonic acid into isobutene makes use of a ferulic acid decarboxylases (FDC) which is derived from Saccharomyces cerevisiae (Uniprot accession number Q03034), Enterobacter sp. (Uniprot accession number V3P7U0), Bacillus pumilus (Uniprot accession number Q45361 ), Aspergillus niger (Uniprot accession number A2R0P7) or Candida dubliniensis (Uniprot accession number B9WJ66).

In a preferred embodiment, the ferulic acid decarboxylases (FDC) employed in the method of the present invention is a ferulic acid decarboxylases (FDC) derived from Saccharomyces cerevisiae (Uniprot accession number Q03034; SEQ ID NO:9), Enterobacter sp. (Uniprot accession number V3P7U0; SEQ ID NO: 10), Bacillus pumilus (Uniprot accession number Q45361 ; SEQ ID NO:11 ), Aspergillus niger (Uniprot accession number A2R0P7; SEQ ID NO:12) or Candida dubliniensis (Uniprot accession number B9WJ66; SEQ ID NO:13) having the amino acid sequence as shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12 and SEQ ID NO:13, respectively.

In another more preferred embodiment, the conversion of 3-methylcrotonic acid into isobutene makes use of a protocatechuate decarboxylase (EC 4.1 .1.63).

Thus, in one preferred embodiment, the conversion of 3-methylcrotonic acid into isobutene is catalyzed by a protocatechuate (PCA) decarboxylase (EC 4.1.1.63). PCA decarboxylases (also termed AroY) are known to catalyze the following reaction, i.e., the enzymatic conversion of protocatechuate (PCA) into catechol (Johnson et al., Metabolic Engineering Communications 3 (2016), 1 1 1 ):

3,4-dihydroxybenzoate ^ " ^" catechol + CO 2

This enzyme occurs in a variety of organisms and has, e.g., been described in Enterobacter aerogenes, Enterobacter cloacae, Rhodopseudomonas sp. and Sedimentibacter hydroxybenzoicus.

In a preferred embodiment of the present invention, the PCA decarboxylase employed in the method of the present invention is a PCA decarboxylase which is derived from Klebsiella pneumoniae (Uniprot accession number B9AM6), Leptolyngbya sp. (Uniprot accession number A0A0S3U6D8), or Phascolarctobacterium sp. (Uniprot accession number R6IIV6).

In a preferred embodiment, the PCA decarboxylase employed in the method of the present invention is an enzyme derived from Klebsiella pneumonia (SEQ ID NO:14), Leptolyngbya sp. (SEQ ID NO:15), or Phascolarctobacterium sp. (SEQ ID NO:16). In a preferred embodiment of the present invention the PCA decarboxylase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 16 or a sequence which is at least n % identical to any of SEQ ID NOs: 14 to 16 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting 3-methylcrotonic acid into isobutene. As regards the determination of the sequence identity, the same applies as has been set forth above.

In a preferred embodiment of the present invention the ferulic acid decarboxylase (FDC) is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 13 or a sequence which is at least n % identical to any of SEQ ID NOs: 9 to 13 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting 3- methylcrotonic acid into isobutene. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, said FMN-dependent decarboxylase catalyzing the decarboxylation of 3-methylcrotonic acid into isobutene is an enzyme which is closely related to the above ferulic acid decarboxylase (FDC), namely a 3-polyprenyl-4- hydroxybenzoate decarboxylase (also termed UbiD). 3-polyprenyl-4- hydroxybenzoate decarboxylase belongs to the UbiD decarboxylase family classified as EC 4.1.1.-.

In a more preferred embodiment, the conversion of 3-methylcrotonic acid into isobutene makes use of a 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) which is derived from Hypocrea atroviridis (UniProt Accession number G9NLP8), Sphaerulina musiva (UniProt Accession number M3DF95), Penecillinum requeforti (UniProt Accession number W6QKP7), Fusarium oxysporum f. sp. lycopersici (UniProt Accession number W9LTH3), Saccharomyces kudriavzevii (UniProt Accession number J8TRN5), Saccaromyces cerevisiae, Aspergillus parasiticus, Candida albicans, Grosmannia clavigera, Escherichia coli (Uniprot accession number P0AAB4), Bacillus megaterium (Uniprot accession number D5DTL4), Methanothermobacter sp. CaT2 (Uniprot accession number T2GKK5), Mycobacterium chelonae 1518 (Uniprot accession number X8EX86) or Enterobacter cloacae (Uniprot accessin number V3DX94).

In an even more preferred embodiment, the 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) employed in the method of the present invention is a 3- polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) derived from Escherichia coli (Uniprot accession number P0AAB4; SEQ ID NO: 17), Bacillus megaterium (Uniprot accession number D5DTL4; SEQ ID NO: 18), Methanothermobacter sp. CaT2 (Uniprot accession number T2GKK5; SEQ ID NO:19) Mycobacterium chelonae 1518 (Uniprot accession number X8EX86; SEQ ID NO:20), Hypocrea atroviridis (SEQ ID NO:21 ), Sphaerulina musiva (SEQ ID NO:22), Penecillinum requeforti (SEQ ID NO:23), Fusarium oxysporum f. sp. lycopersici (SEQ ID NO:24), Saccharomyces kudriavzevii (SEQ ID NO:25), Saccaromyces cerevisiae (SEQ ID NO:26), Aspergillus parasiticus (SEQ ID NO:27), Candida albicans (SEQ ID NO:28), Grosmannia clavigera (SEQ ID NO:29) or Enterobacter cloacae (SEQ ID NO:30) having the amino acid sequence as shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively.

In a preferred embodiment of the present invention the 3-polyprenyl-4- hydroxybenzoate decarboxylase (UbiD) is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 30 or a sequence which is at least n % identical to any of SEQ ID NOs: 17 to 30 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting 3-methylcrotonic acid into isobutene. As regards the determination of the sequence identity, the same applies as has been set forth above.

The provision of D AP

As mentioned above, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP) into a flavin-derived cofactor, the availability of DMAP is one limiting factor. The chemical structure of dimethylallyl phosphate (DMAP) (also termed 3-methylbut- 2-en-1-yl phosphate, 3,3-dimethylallyl phosphate and prenyl phosphate) is shown in Figure 3. DMAPP contains one additional phosphate as compared to DMAP and its chemical structure is also shown in Figure 3.

As mentioned above, the mechanism of the ferulic acid decarboxylase (FDC) in association with the modified FMN (prenylated-FMN) (the latter provided by the PAD enzyme) was recently described (Nature 522 (2015), 497-501 ; Nature, 522 (2015), 502-505). However, the metabolic route for the provision of DMAP (required for the prenylation of the flavin cofactor by the FMN prenyl transferase) remained unclear while the metabolic routes leading to the formation of dimethylallyl pyrophosphate (DMAPP) via the mevalonate (MEVA) and 1-deoxy-D-xylulose-5-phosphate (DXP) pathways are known in the art. In fact, it has only previously been described by Wang et al. (Cell Chem Biol. 25 (2018) 1-11 ) that E. coli produces DMAP by phosphorylation of prenol and dephosphorylation of DMAPP.

As the exogenous supplementation of DMAP and/or DMAPP in a culture medium is not feasible since DMAP and/or DMAPP is assumed to not enter the cell, the present invention provides methods for endogenously generating DMAP and/or DMAPP and, preferably, to increase the pool of DMAP and/or DMAPP.

While the provision of DMAPP is described further below, according to the present invention, DMAP can be provided via different routes (in the following referred to as route (i), (ii), (iii) and (iv), respectively) which are schematically shown in Figure 4. Accordingly, the above described method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene further comprises providing said DMAP enzymatically by:

(i) the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into said DMAP; or

(ii) a single enzymatic step in which prenol is directly enzymatically converted into said DMAP; or

(iii) two enzymatic steps comprising: first enzymatically converting DMAPP into prenol; and then enzymatically converting the thus obtained prenol into said DMAP; or

(iv) the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP, or by a combination of any one of (i) to (iv).

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene, the enzymatic provision of said DMAP is enhanced/increased over naturally occurring (enzymatic) reactions/conversions leading to the production of DMAP, preferably by overexpressing corresponding enzymes capable of catalyzing any of the above reactions (i) to (iv). Means and methods for increasing/enhancing the expression of an enzyme are described in more detail further below.

These different routes (i), (ii), (iii) and (iv) for the provision of DMAP are illustrated in . _

18

Figure 4 while each of the above conversions is described in more detail in the following:

Route (i): The provision of DMAP by the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into said DMAP

According to the present invention, DMAP can be provided enzymatically by the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into said DMAP.

In a preferred embodiment, the enzymatic conversion of DMAPP into said DMAP is achieved by making use of a phosphatase. Phosphatases are known in the art and are generally known as enzymes capable of removing a phosphate group (PO 4 3~ ) from its substrate by hydroiysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group in a reaction called dephosphorylation. The term "dephosphorylation" refers to the removal of a phosphate group from an organic compound by hydrolysis.

Enzymes catalyzing the conversion (i.e., the dephosphorylation) of dimethylallyl pyrophosphate (DMAPP) into DMAP are enzymes which catalyze the reaction as shown in Figure 5.

In case the above conversion is performed in a cell, said DMAPP is preferably metabolically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of dimethylallyl pyrophosphate (DMAPP), e.g., via the mevalonate (MEVA) and/or 1-deoxy-D-xylulose-5-phosphate (DXP) pathways which are known in the art.

In case the above conversion is performed in vitro, said DMAPP is preferably added to the reaction.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of DMAPP into said DMAP by making use of a phosphatase, the expression of said phosphatase is increased/enhanced. Preferably, said phosphatase is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below. In a preferred embodiment, the phosphatase is:

an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-); or

a phosphoric-monoester hydrolase (EC 3.1.3.-).

Thus, in one preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of an enzyme belonging to the family of enzymes acting on phosphorous containing anhydrides (EC 3.6.1.-).

Preferred examples of such enzymes which are classified as EC 3.6.1.- (i.e., enzymes acting on phosphorous containing anhydrides) are:

ADP-ribose pyrophosphatase (EC 3.6.1.13),

8-oxo-dGTP diphosphatase (EC 3.6.1.55),

bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ),

UDP-sugar diphosphatase (EC 3.6.1.45),

exopolyphosphatase (EC 3.6.1.11 ),

guanosine-5'-triphosphate/3'-diphosphate pyrophosphatase (EC 3.6.1.40),

NADH pyrophosphatase (EC 3.6.1.22),

nucleotide diphosphatase (EC 3.6.1.9), and

acylphosphatase (EC 3.6.1.7).

Thus, in one preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of an ADP-ribose pyrophosphatase (EC 3.6.1.13).

ADP-ribose pyrophosphatases (EC 3.6.1.13) are enzymes which catalyze the following reaction:

ADP-D-ribose + H 2 0 ^f*" AMP + D-ribose 5-phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Arabidopsis sp., Arabidopsis thaliana, Artemia sp, Autographa californica multiple nucleopolyhedrovirus, Bacillus subtilis (Uniprot accession number P54570), Danio rerio (Uniprot accession number Q7T291 ), Deinococcus radiourans (Uniprot accession number Q9RSC1 ), E. coli, Francisella tularensis (Uniprot accession number Q5NHR1 ), Haemophilus influencae (Uniprot accession number P44684), Homo sapiens, Methanocaldococcus jannaschii, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus, Rhodobacter spaeroides, Saccharomyces cerevisiae, Synechococcus sp. (SwissProt accession number Q83ZD0), Synechocystis sp., Thermus thermophilus and Thermus thermophilus DSM 579 (Uniprot accession number Q5SHB0).

In a preferred embodiment, the ADP-ribose pyrophosphatase (EC 3.6.1 .13) is the E. coli-derived enzyme encoded by nudF (SEQ ID NO:39).

Thus, in a preferred embodiment of the present invention, the ADP-ribose pyrophosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO:39 or a sequence which is at least n % identical to SEQ ID NO: 39 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of an 8-oxo-dGTP diphosphatase (EC 3.6.1 .55).

8-oxo-dGTP diphosphatases (EC 3.6.1.55) are enzymes which catalyze the following reaction:

8-oxo-dGTP + H 2 0 8-oxo-dGMP + diphosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Bartonella henselae (Uniprot accession number Q6G5F4), Ciona intestinalis, E. coli, Homo sapiens (SwissProt accession number P36639) and Hordeum vulgare subsp. vulgare (Uniprot accession number F2DYN1 ).

In a preferred embodiment, the 8-oxo-dGTP diphosphatases (EC 3.6.1 .55) is the E. coli-derived enzyme encoded by mutT (SEQ ID NO:40).

Thus, in a preferred embodiment of the present invention, the 8-oxo-dGTP diphosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO:40 or a sequence which is at least n % identical to SEQ ID NO: 40 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above. In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ).

bis(5'-nucleosyl)-tetraphosphatases (EC 3.6.1.41 ) are enzymes which catalyze the following reaction:

P1 ,P4-bis(5'-adenosyl) tetraphosphate + H 2 0 →^ 2 ADP

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as fungi and bacteria. The enzyme has, e.g., been described in Acidaminococcus fermentans, E. coli, Myxococcus xanthus (Uniprot accession number Q1CWE7 and Q1 DC62), Physarum polycephalum, Pyrodictium occultum, Salmonella enterica and Shigella flexneri.

In a preferred embodiment, the bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ) is the E. coli-derived enzyme encoded by apaH (SEQ ID NO:41 ).

Thus, in a preferred embodiment of the present invention, the bis(5'-nucleosyl)- tetraphosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO:41 or a sequence which is at least n % identical to SEQ ID NO: 41 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of a UDP-sugar diphosphatase (EC 3.6.1.45).

UDP-sugar diphosphatases (EC 3.6.1.45) are enzymes which catalyze the following reaction:

UDP-sugar + H 2 0 UMP + alpha-D-aldose 1 -phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, fungi and bacteria. The enzyme has, e.g., been described in Corynebacterium glutamicum, Enterobacter aerogenes (Uniprot accession number Q9RQT7), E. coli (Uniprot accession number P07024), Homo sapiens (Uniprot accession number 095848), Mus musculus (Uniprot accession number Q9D142), Peptoclostridium difficile, Saccharomyces cerevisiae, Salmonella enterica, Salmonella sp., Sus scrofa and Yersinia intermedia (Uniprot accession number A4URQ8).

In a preferred embodiment, the UDP-sugar diphosphatases (EC 3.6.1.45) is the E. coli-derived enzyme encoded by ushA (SEQ ID NO:42).

Thus, in a preferred embodiment of the present invention, the UDP-sugar diphosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO:42 or a sequence which is at least n % identical to SEQ ID NO: 42 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of an exopolyphosphatase (EC 3.6.1.11 ).

Exopolyphosphatase (EC 3.6.1.11 ) are enzymes which catalyze the following reaction:

(polyphosphate)n + H 2 0 ■ (polyphosphate)n-l + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, plants, fungi and bacteria. The enzyme has, e.g., been described in Campylobacter jejuni (Uniprot accession number A0A0H3PAT6 and A0A0H3PES2), Chlorobaculum tepidum (Uniprot accession number Q8KBS0 and Q8KG69), Corynebacterium glutamicum (Uniprot accession number Q8NRR8 and Q8NT99), Cyberlindnera jadinii, Escherichia coli, Euglena gracilis, Funneliformis mosseae, Homo sapiens, Leishmania major, Lemna gibba, Lemna minor, Lemna trisulca, Magnusiomyces magnusii, Microlunatus phosphovorus, Mycobacterium tuberculosis (Uniprot accession number P9WHV4 and P9WHV5), Neisseria meningitidis, Pseudomonas aeruginosa (SwissProt accession number Q9S605), Pseudomonas sp., Rhipicephalus microplus, Riccia fluitans, Saccharomyces cerevisiae, Solanum tuberosum, Streptomyces aureofaciens, Sulfolobus metallicus, Sulfolobus solfataricus, Tethya aurantium (SwissProt accession number Q97YV9), Trypanosoma brucei (Uniprot accession number Q7Z032), Trypanosoma cruzi (SwissProt accession number Q6Y656) and Wolffia arrhiza.

In a preferred embodiment, the exopolyphosphatase (EC 3.6.1.11 ) is the E. coli- derived enzyme encoded by ppX (SEQ ID NO:43) or by gpp.

Thus, in a preferred embodiment of the present invention, the exopolyphosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO:43 or a sequence which is at least n % identical to SEQ ID NO: 43 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of a guanosine-5'-triphosphate/3'-diphosphate pyrophosphatase (EC 3.6.1.40).

Guanosine-5'-triphosphate/3'-diphosphate pyrophosphatases (EC 3.6.1.40) are enzymes which catalyze the following reaction:

guanosine 5'-triphosphate 3'-diphosphate + H 2 O guanosine 3', 5'- bis(diphosphate) + phosphate

This enzyme is known from a variety of organisms, including prokaryotic organisms such as bacteria. The enzyme has, e.g., been described in Aquifex aeolicus, Campylobacter jejuni (Uniprot accession number A0A0H3PAT6 and A0A0H3PES2), and E. coli.

In a preferred embodiment, the guanosine-5'-triphosphate/3'-diphosphate pyrophosphatase (EC 3.6.1.40) is the E. coli-derived enzyme encoded by gppA (SEQ ID NO:44).

Thus, in a preferred embodiment of the present invention, the guanosine-5'- triphosphate/3'-diphosphate pyrophosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO:44 or a sequence which is at least n % identical to SEQ ID NO: 44 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of an NADH pyrophosphatase (EC 3.6.1.22).

NADH pyrophosphatases (EC 3.6.1.22) are enzymes which catalyze the following reaction:

NAD + + H 2 O →" AMP + NMN

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, plants, fungi and bacteria. The enzyme has, e.g., been described in Aedes aegypti, Arabidopsis sp., Caenorhabditis elegans, E. coli (Uniprot accession number P07024), Haemophilus influencae, Homo sapiens, Mycobacterium bovis (Uniprot accession number C1AGW8), Mycobacterium tuberculosis (Uniprot accession number P9WIX5), Nicotiana tabacum, Proteus vulgaris, Rattus norvegicus, Saccharomyces cerevisiae, Salmonella enterica and Solanum tuberosum.

In a preferred embodiment, the NADH pyrophosphatase (EC 3.6.1.22) is the E. coli- derived enzyme encoded by nudC (SEQ ID NO: 45).

Thus, in a preferred embodiment of the present invention, the NADH pyrophosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 45 or a sequence which is at least n % identical to SEQ ID NO: 45 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of a nucleotide diphosphatase (EC 3.6.1.9).

Nucleotide diphosphatases (EC 3.6.1.9) are enzymes which catalyze the following reaction:

a dinucleotide + H 2 O 2 mononucleotides

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, plants fungi and bacteria. The enzyme has, e.g., been described in Amycolatopsis mediterranei, Bos taurus, Bothrops jararaca, Brassica oleracea, Clostridium perfringens, Columba livia, Crotalus adamanteus, Crotalus durissus, Dictyostelium discoideum, Escherichia coli, Glycine max, Haemophilus influenzae, Haemophilus parasuis, Homo sapiens, Hordeum vulgare, Lens culinaris, Mus musculus, Opuntia ficus-indica, Oryza sativa, Ovis aries aries, Proteus vulgaris, Rattus norvegicus, Saccharomyces cerevisiae, Solarium tuberosum, Sus scrofa, Triticum aestivum (UniProt accession number D9YT79), Vigna radiata var. radiata, Xanthomonas citri, and Xanthomonas citri 306.

In a preferred embodiment, the nucleotide diphosphatase (EC 3.6.1.9) is the E. coli- derived enzyme encoded by yhdE (SEQ ID NO: 46).

Thus, in a preferred embodiment of the present invention, the nucleotide diphosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO:46 or a sequence which is at least n % identical to SEQ ID NO: 46 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of an acylphosphatase (EC 3.6.1.7).

Acylphosphatases (EC 3.6.1.7) are enzymes which catalyze the following reaction: an acylphosphate + H 2 O ^ " ^" a carboxylate + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, plants, fungi and bacteria. The enzyme has, e.g., been described in Anas platyrhynchos, Bacillus subtilis, Bos taurus, Cavia porcellus, Chondrichthyes, Drosophila mauritiana, Drosophila melanogaster, Drosophila simulans, Equus caballus, Escherichia coli, Gallus gallus, Homo sapiens, Meleagris gallopavo, Oryctolagus cuniculus, Pyrococcus horikoshii (Uniprot accession number P84142), Rattus norvegicus, Saccharomyces cerevisiae, Sulfolobus solfataricus (Uniprot accession number Q97ZL0), Sus scrofa, Thermus thermophilus (Uniprot accession number Q5SKS6), Vibrio cholerae (Uniprot accession number A5F8G9) and Vigna unguiculata.

In a preferred embodiment, the acylphosphatase (EC 3.6.1.7) is the E. coli-derived enzyme encoded by yccX (SEQ ID NO: 67).

Thus, in a preferred embodiment of the present invention the acylphosphatase (EC 3.6.1.7) is an enzyme comprising the amino acid sequence of SEQ ID NO: 67 or a sequence which is at least n % identical to SEQ ID NO: 67 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

As mentioned above, in other preferred embodiments, the enzymatic conversion of DMAPP into DMAP is achieved by the use of an enzyme belonging to the family of phosphoric-monoester hydrolases (EC 3.1.3.-).

Preferred examples of such enzymes which are classified as EC 3.1.3.- (i.e., phosphoric-monoester hydrolases) are:

3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7);

5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatase (belonging to the family of phosphoric-monoester hydrolases (EC 3.1.3.-); and

fructose-1 6-bisphosphatase (EC 3.1.3.11 ).

Thus, in one preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7).

3'(2'),5'-bisphosphate nucleotidases (EC 3.1.3.7) are enzymes which catalyze the following reaction:

adenosine 3',5'-bisphosphate + H 2 0 AMP + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, plants, fungi and bacteria. The enzyme has, e.g., been described in Arabidopsis thaliana, Arthrospira platensis (Uniprot accession number Q3LS17), Chlorella pyrenoidosa, Chromobacterium violaceum (Uniprot accession number Q7NXD4), Debaryomyces hansenii, Drosophila melanogaster (SwissProt accession number Q9VHS0), Escherichia coli, Gossypium hirsutum (Uniprot accession number Q8VWZ6), Homo sapiens, Mus musculus, Mycobacterium tuberculosis (Uniprot accession number P9WKJ1 ), Oryctolagus cuniculus, Oryza sativa (Uniprot accession number P0C5A3), Rattus norvegicus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Solanum lycopersicum and Zea mays (SwissProt accession number Q94FY6 and Q94G04).

In a preferred embodiment, the 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7) is the E. coli-derived enzyme encoded by cysQ (SEQ ID NO: 47). Thus, in a preferred embodiment of the present invention, the 3'(2'),5'-bisphosphate nucleotidase is an enzyme comprising the amino acid sequence of SEQ ID NO:47 or a sequence which is at least n % identical to SEQ ID NO: 47 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of a 5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatase (). 5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatases are enzymes which belong to the family of phosphoric-monoester hydrolases (EC 3.1.3.-) and catalyze the following reaction:

5-amino-6-(5-phospho-D-ribitylamino)uracil + H 2 O >" 5-amino-6-(D- ribitylamino)uracil + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants and bacteria. The enzyme has, e.g., been described in E. coli or Bacillus subtilis.

In a preferred embodiment, the 5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatase is encoded by yigB, ybjl, ywtE, yitU or ycsE.

In another preferred embodiment, the 5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatase is the Bacillus subtilis-derived enzyme encoded by yitU (Uniprot P70947), ywtE (UniProt P96741 ) or by ycsE (Uniprot P42962).

In a preferred embodiment, the 5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatase is the E. coli-derived enzyme encoded by yigB (Uniprot P0ADP0; SEQ ID NO: 48) or ybjl (Uniprot P75809; SEQ ID NO: 49).

Thus, in a preferred embodiment of the present invention, the 3'(2'),5'-bisphosphate nucleotidase is an enzyme comprising the amino acid sequence of SEQ ID NO:48 or SEQ ID NO: 49 or a sequence which is at least n % identical to SEQ ID NO: 48 or SEQ ID NO: 49 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the use of a fructose-1 ,6-bisphosphatase (EC 3.1.3.11 ).

Fructose-1 ,6-bisphosphatases (EC 3.1.3.11 ) are enzymes which catalyze the following reaction:

D-fructose 1 ,6-bisphosphate + H2O D-fructose 6-phosphate + phosphate

This reaction is a key step of gluconeogenesis and in the Calvin cycle which are both anabolic pathways found in most organisms. Thus, fructose-1 ,6-bisphosphatase is an ubiquituous enzyme which occurs in basically all organisms, including eukaryotic and prokaryotic organisms such as animals, plants, fungi and bacteria. The enzyme has, e.g., been described in Anabaena sp., Arabidopsis thaliana, Archaeoglobus fulgidus, Bacillus licheniformis, Bacillus methanolicus, Bacillus subtilis, Beta vulgaris, Bombus terrestris, Bos taurus, Bothriocephalus scorpii, Brassica napus, Canis lupus familiaris, Cenarchaeum symbiosum, Citrus x paradisi, Clonorchis sinensis (Uniprot accession number G7YVB4), Coreus marginatus, Corynebacterium glutamicum, Cyberlindnera jadinii, Cyprinus carpio, Dactylis glomerata, Escherichia coli, Festuca rupicola, Filipendula vulgaris, Galdieria sulphuraria (SwissProt accession number Q95AJ2), Gallus gallus, Glycine max, Hominoidea, Homo sapiens, Ignicoccus hospitalis, llyocoris cimicoides, Kluyveromyces marxianus, Lactobacillus delbrueckii subsp. lactis, Leishmania major, Leptolyngbya boryana, Lygus pratensis, Malus domestica, Meleagris gallopavo, Methanococcus maripaludis, Mus musculus, Mycobacterium tuberculosis, Mytilus galloprovincialis, Neisseria meningitidis, Notostira elongata, Ogataea angusta, Oryctolagus cuniculus, Oryza coarctata, Oryza sativa, Ovis aries, Pelophylax esculentus, Peltigera rufescens, Phagocata sibirica, Phocidae, Pisum sativum, Polysphondylium pallidum, Ptyas dhumnades, Pyrobaculum neutrophilum (Uniprot accession number B1YAL1 ), Pyrococcus furiosus (SwissProt accession number Q8TZH9), Rattus norvegicus, Rhodococcus opacus, Rhodopseudomonas palustris, Ricinus communis, Saccharomyces cerevisiae, Salmonella enterica, Salvia nemorosa, Schizosaccharomyces pombe, Solanum lycopersicum, Solanum tuberosum, Sparus aurata (Uniprot accession number Q8AYI5), Spinacia oleracea, Struthio camelus, Sulfolobus tokodaii, Sulfolobus tokodaii 7, Sus scrofa, Sus scrofa domesticus, Synechococcus elongatus PCC 7942 (Uniprot accession number Q59943), Synechococcus sp., Synechocystis sp., Thermococcus kodakarensis, Thermococcus onnurineus, Thermotoga maritima, Thermus thermophilus (Uniprot accession number Q5SJM8), Triticum aestivum, Yarrowia lipolytica (Uniprot accession number Q7Z8Q0) and Zea mays.

In a preferred embodiment, the fructose-1 ,6-bisphosphatase (EC 3.1.3.11 ) is the E. coli-derived enzyme encoded by fbp (SEQ ID NO: 50).

Thus, in a preferred embodiment of the present invention, the fructose-1 ,6- bisphosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 50 or a sequence which is at least n % identical to SEQ ID NO: 50 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

According to the present invention, DMAP can also be provided enzymatically by the enzymatic conversion of dimethylailyl pyrophosphate (DMAPP) into said DMAP by the concomitant formation of ATP. Thus, the dephosphorylation of DMAPP into DMAPP can also be achieved by kinases capable of forming ATP from ADP having the promiscuous activity to catalyze the formation of DMAP from DMAPP.

Kinases catalyzing the conversion (i.e., the dephosphorylation) of dimethylailyl pyrophosphate (DMAPP) into DMAP by concomitantly forming ATP from ADP are enzymes which catalyze the reaction as shown in Figure 6.

In case the above conversion is performed in a cell, said DMAPP is preferably metabolically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of dimethylailyl pyrophosphate (DMAPP), e.g., via the mevalonate (MEVA) and/or 1 -deoxy-D-xylulose-5-phosphate (DXP) pathways which are known in the art.

In case the above conversion is performed in vitro, said DMAPP is preferably added to the reaction.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of DMAPP into said DMAP by making use of a kinase, the expression of said kinase is increased/enhanced. Preferably, said kinase is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

In a preferred embodiment, the kinase is an isopentenyl phosphate kinase (EC 2.7.4.26).

Isopentenyl phosphate kinases (EC 2.7.4.26) are enzymes which catalyze the following reaction:

ATP + isopentenyl phosphate ADP + isopentenyl diphosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants and bacteria. The enzyme has, e.g., been described in Haloferax volcanii (Uniprot accession number D4GWT7), Mentha x piperita (SwissProt accession number P56848), Methanocaldococcus jannaschii (SwissProt accession number Q60352), Methanothermobacter thermautotrophicum (Uniprot accession number 026153), and Thermoplasma acidophilum (Uniprot accession number Q9HLX1 ).

In a preferred embodiment, the isopentenyl phosphate kinase (EC 2.7.4.26) is the enzyme derived from Haloferax volcanii (Uniprot accession number D4GWT7; SEQ ID NO:51 ), Methanocaldococcus jannaschii (SwissProt accession number Q60352; SEQ ID NO:53), Methanothermobacter thermautotrophicum (Uniprot accession number 026153; SEQ ID NO:52) or Thermoplasma acidophilum (Uniprot accession number Q9HLX1 ; SEQ ID NO:54).

As demonstrated in the appended examples, the isopentenyl phosphate kinase (EC 2.7.4.26) derived from Methanocaldococcus jannaschii (strain ATCC 43067; SwissProt Q60352) is capable of catalyzing the above conversion.

Thus, in a preferred embodiment, the enzymatic conversion of DMAPP into DMAP is achieved by the isopentenyl phosphate kinase (EC 2.7.4.26) of Methanocaldococcus jannaschii (strain ATCC 43067; SwissProt Q60352). In other preferred embodiments, the enzymatic conversion of DMAPP into DMAP is achieved by the isopentenyl phosphate kinase (EC 2.7.4.26) of Thermoplasma acidophilum (strain ATCC 25905; Uniprot accession number Q9HLX1 ) or of Methanothermobacter thermautotrophicus (strain ATCC 29096; Uniprot accession number 026153). The isopentenyl phosphate kinases derived from these organisms are described by Chen M and Poulter CD (Biochemistry 49 (2010), 207-210).

Thus, in a preferred embodiment of the present invention, the isopentenyl phosphate kinase (EC 2.7.4.26) is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51 to SEQ ID NO:54 or a sequence which is at least n % identical to any one of SEQ ID NO: 51 to SEQ ID NO:54 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into DMAP by the concomitant formation of ATP. As regards the determination of the sequence identity, the same applies as has been set forth above.

The provision of DMAPP (corresponding to the isomerisation step preceding the dephosphorylation step of route 1 )

The DMAPP which is converted into DMAP according to the method of the present invention may itself be provided by an enzymatic reaction.

According to the present invention, DMAPP can be provided enzymatically by the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said dimethylallyl pyrophosphate (DMAPP).

In a preferred embodiment, the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said dimethylallyl pyrophosphate (DMAPP) is achieved by making use of an isomerase. Isomerases are known in the art and are generally known as enzymes which convert a molecule from one isomer to another, meaning that the end product has the same molecular formula but a different physical structure.

Enzymes catalyzing the isomerisation, i.e., the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said dimethylallyl pyrophosphate (DMAPP) are enzymes which catalyze the reaction as shown in the upper part of Figure 4.

In case the above conversion is performed in a cell, said isopentenyl pyrophosphate is preferably metabolically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of isopentenyl pyrophosphate, e.g., via the mevalonate (MEVA) and/or 1-deoxy-D-xylulose-5-phosphate (DXP) pathways which are known in the art.

In case the above conversion is performed in vitro, said isopentenyl pyrophosphate is preferably added to the reaction.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of DMAPP into said DMAP wherein said DMAPP is itself provided enzymatically by the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said dimethylallyl pyrophosphate (DMAPP) by making use of an isomerase, the expression of said isomerase is increased/enhanced. Preferably, said isomerase is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

In a preferred embodiment, the isomerase is an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2)

Thus, in one preferred embodiment, the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said dimethylallyl pyrophosphate (DMAPP) is achieved by the use of an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

Isopentenyl-diphosphate DELTA isomerases (EC 5.3.3.2) are enzymes which catalyze the following reaction:

Isopentenyl diphosphate dimethylallyl diphosphate

The occurrence of this enzyme has been described for a large number of organisms, e.g. for E. coli, Staphylococcus aureus, Sulfolobus shibatae, Bacillus subtilis, Thermococcus kodakarensis, Solanum lycopersicum, Arabidopsis thaliana, Bombyx mori, Camptotheca acuminata, Capsicum annuum, Catharanthus roseus, Cinchona robusta, Citrus sp., Claviceps purpurea, Curcubita sp., Gallus gallus and Homo sapiens, to name just some. In a preferred embodiment, the enzyme originating from E. coli or an enzyme derived therefrom and which still shows the activity as the enzyme from E. coli is employed in the methods according to the present invention. Route (ii): The provision of DMAP by a single enzymatic step in which prenol is directly enzymatically converted into said DMAP

According to the present invention, DMAP can be provided enzymatically by the enzymatic conversion of prenol into said DMAP.

Prenol (also termed or 3-methyl-2-buten-1-ol or 3,3-dimethylallyl alcohol) is an alcohol and occurs naturally in citrus fruits, cranberry, bilberry, currants, grapes, raspberry, blackberry, tomato, white bread, hop oil, coffee, arctic bramble, cloudberry and passion fruit.

In case the above conversion of prenol into DMAP is performed in a cell (i.e., in vivo), said prenol is preferably metaboiicaily provided by naturally occurring or artificially introduced metabolic routes leading to the formation of prenol.

Alternatively or in addition to the above, said prenol may preferably be supplemented/added to the culture medium.

In case the above conversion is performed in vitro, said prenol is preferably added to the in vitro reaction.

There are organisms known in the art which are capable of naturally producing prenol or by artificially introduced metabolic routes. Corresponding organisms may preferentially be used in the methods of the present invention for the conversion of prenol into DMAP.

WO2013/053824 describes a possible artificial route for the production of prenol. WO2009006429A1 and WO2013173437 describe the provision of prenol by the dephosphorylation of DMAPP.

A new oxido-reductase called 321 -MB dehydrogenase derived from Pseudomonas putida was recently identified as being capable of catalyzing the reversible oxidation of 3-methylbuten-1-ol or prenol into 3-methylbutenal or prenal (Appl. Envir. Microbiol. 65(6) (1999), 2622).

Ginger et al. (J. Biol. Chem. 276(15) (2001 ), 1 1674) describe the involvement of leucine catabolism in sterol biosynthesis in the trypanosomatid Leishmania mexicana. A metabolic pathway composed of, in a first part, the degradation of leucine into 3-methylcrotonyl-CoA is described. In a second part, 3-methylcrotonyl- CoA is converted into hydroxyl-methylglutaryl-CoA (HMG-CoA) via 3- methylglutaconyl-CoA. The descried pathway corresponds to the reverse metabolic pathway described in WO2013/053824 mentioned above. In addition, the authors suggest a possible hypothetical pathway involving the enzymatic reduction of 3- methylcrotonyl-CoA into 3-methylbuten-1-ol (i.e., prenol).

The possibility of the enzymatic reduction of 3-methylcrotonyl-CoA into 3- methylbuten-1 -ol (i.e., prenol) is also proposed by Mahmud at al. (ChemBioChem. 6 (2005), 322). They describe the biosynthetic shunt pathway of mevalonate towards branched carboxylic acids in Myxobacteria, such as yxococcus xanthus. This pathway involves the conversion of hydroxyl-methylglutaryl-CoA (HMG-CoA) into 3- methylcrotonyl-CoA via 3-methylglutaconyl-CoA.

In a preferred embodiment, the enzymatic conversion of prenol into said DMAP is achieved by making use of a kinase. Kinases are known in the art and are generally known as enzymes capable of catalyzing the transfer of phosphate groups from high- energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the substrate gains a phosphate group and the high- energy ATP molecule donates a phosphate group. This reaction is a transesterification and produces a phosphorylated substrate and ADP.

Enzymes catalyzing the enzymatic conversion (i.e., the phosphorylation) of prenol into said DMAP are enzymes which catalyze the reaction as shown in Figure 7.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of prenol into said DMAP by making use of a kinase, the expression of said kinase is increased/enhanced. Preferably, said kinase is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

In a preferred embodiment, the kinase is a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 .-).

Preferably, ATP is the donor of the phospho group.

A preferred example of enzymes which are classified as EC 2.7.1.- (i.e., phosphotransferases with an alcohol group as acceptor) is hydroxyethylthiazole kinase (EC 2.7.1 .50). Hydroxyethylthiazole kinases (EC 2.7.1.50) are enzymes which catalyze the following reaction:

ATP + 4-methyl-5-(2-hydroxyethyl)thiazole

ADP + 4-methyl-5-(2-phosphoethyl)thiazole

The occurrence of this enzyme has been described for several organisms, e.g. for E. coli, Bacillus subtilis, Rhizobium leguminosarum, Pyrococcus horikoshii OT3, Saccharomyces cerevisiae.

In principle, any known hydroxyethylthiazole kinase can be employed in the method according to the invention. In one aspect of the present invention, a hydroxyethylthiazole kinase of bacterial origin is used, such as a hydroxyethylthiazole kinase from a bacterium belonging to the genus Escherichia, Bacillus or Rhizobium, preferably of E. coli, B. subtilis or of R. leguminosarum. Amino acid and nucleotide sequences for these enzymes are available. Examples are provided in SEQ ID NOs: 31 to 33.

In a preferred embodiment of the present invention the hydroxyethylthiazole kinase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 33 or a sequence which is at least n % identical to any of SEQ ID NOs: 31 to 33 with n being an integer between 0 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting prenol into DMAP. As regards the determination of the sequence identity, the same applies as has been set forth above.

Route fiii): The provision of DMAP by two enzymatic steps comprising: first enzymatically converting DMAPP into prenol; and then enzymaticallv converting the thus obtained prenol into said DMAP

According to the present invention, DMAP can be provided enzymatically by two enzymatic steps comprising:

first enzymatically converting DMAPP into prenol; and

then enzymatically converting the thus produced prenol into said DMAP. In a preferred embodiment, the enzymatic conversion of DMAPP into said prenol is achieved by making use of a phosphatase or pyrophosphatase. In another preferred embodiment, the enzymatic conversion of the thus produced prenol into said DMAP is achieved by making use of a kinase.

As regards the enzymatic conversion of prenol into DMAP and the preferred embodiments for the enzymes capable of converting prenol into DMAP, preferably the kinases (particularly preferred the phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), preferably the hydroxyethylthiazole kinase (EC 2.7.1 .50)), the same applies as has been set forth above in connection with the enzymatic conversion of route (ii) according to the invention.

Regarding the conversion of DMAPP into prenol, this conversion is preferably achieved by making use of a phosphatase or pyrophosphatase. Pyrophosphatases are known in the art and are generally known as acid anhydride hydrolases that act upon diphosphate bonds. Pyrophosphatases have, e.g., been described in WO2009/006429, WO2013173437 and in Biotechnology for biofuels 6 (2013), 1-13. As already defined above, phosphatases are known in the art and are generally known as enzymes capable of removing a phosphate group (PO 3~ ) from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group in a reaction called dephosphorylation.

Enzymes catalyzing the conversion of dimethylallyl pyrophosphate (DMAPP) into prenol (by dephosphorylating DMAPP twice) are enzymes which catalyze the reaction as shown in the middle of Figure 8.

In case the above conversion is performed in a cell, said DMAPP is preferably metabolically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of dimethylallyl pyrophosphate (DMAPP), e.g., via the mevalonate (MEVA) and/or 1 -deoxy-D-xylulose-5-phosphate (DXP) pathways which are known in the art.

In case the above conversion is performed in vitro, said DMAPP is preferably added to the reaction.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of DMAPP into said prenol by making use of a phosphatase or pyrophosphatase, the expression of said phosphatase or pyrophosphatase is increased/enhanced. Preferably, said phosphatase or pyrophosphatase is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

The pathway for the enzymatic provision of DMAP by two enzymatic steps comprising first enzymatically converting DMAPP into prenol and then enzymatically converting the thus produced prenol into said DMAP wherein said DMAPP may be provided by the enzymatic conversion of isopentenyl pyrophosphate (IPP; a product of the mevalonate (MEVA) and 1-deoxy-D-xylulose-5-phosphate (DXP) pathways) is shown in Figure 8.

Preferably, in the methods of the present invention, the production of the DMAPP can be increased by overexpressing one or more of the genes encoding enzymes of the mevalonate (MEVA) and/or the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway.

In a preferred embodiment, the phosphatase or pyrophosphatase for converting DMAPP into prenol is:

an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylgl cerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC

3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1.3.5), a pyridoxal phosphate phosphatase (EC 3.1.3.74) or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ); or an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26)

Thus, in one preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of an alkaline phosphatase (EC 3.1.3.1 ).

Alkaline phosphatases (EC 3.1.3.1 ) are enzymes which catalyze the following reaction:

a phosphate monoester + H 2 0 an alcohol + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Aeropyrum pernix, Alexandrium catenella, Anabaena sp., Antarctic bacterium TAB5 (UniProt accession number Q9KWY4), Aspergillus caespitosus, Aspergillus oryzae, Aspergillus terricola, Bacillus licheniformis, Bacillus subtilis (UniProt accession number P19406), Bos taurus, Burkholderia cenocepacia (UniProt accession number B4EKR2), Callithrix jacchus, Camelus bactrianus, Campylobacter jejuni (UniProt accession number A3ZF85), Candida tropicalis, Canis lupus familiaris, Cavia porcellus, Chlorocebus sabaeus, Cobetia marina, Cricetulus griseus, Syrian hamster, Cyberlindnera jadinii, Cyrtograpsus angulatus, Daphnia magna, Debaryomyces hansenii, Dictyostelium sp., Drosophila melanogaster, Drosophila virilis, Echinococcus multilocularis, Eledone cirrhosa, Enterococcus faecalis, Equus caballus, Escherichia coli, Felis catus, Gadus morhua, Gallus gallus, Geobacillus caldoxylosilyticus (UniProt accession number C1 K6P2), Geobacillus stearothermophilus, Geobacillus thermodenitrificans (UniProt accession number A8WEG4), Glomus etunicatum, Haliotis diversicolor, Haloarcula marismortui, Halobacterium salinarum, Halomonas sp., Helicoverpa armigera, Heliothis virescens, Homo sapiens, Klebsiella pneumoniae, Lepus townsendii, Lysobacter enzymogenes, Macaca mulatta, Meretrix lusoria, Meriones unguiculatus, Mesocricetus auratus, Micrococcus sodonensis, Mus musculus, Neohelice granulata, Neurospora crassa, Nilaparvata lugens, Onchocerca ochengi, Ophicephalus punctatus Bloch, Oreochromis mossambicus, Oryctolagus cuniculus, Oryctolagus sp., Ovis aries, Oxybasis rubra, Pandalus borealis, Papio cynocephalus, Paramecium tetraurelia, Parawixia bistriata, Pasteurella multocida (UniProt accession number A1C3J6), Penaeus monodon, Penicillium chrysogenum, Phaeodactylum tricornutum, Phoca groenlandica, Physarum polycephalum, Pinctada fucata, Porphyromonas gingivalis, Prevotella intermedia, Prorocentrum donghaiense, Pseudomonas aeruginosa, Pyrococcus abyssi, Pyrococcus furiosus, Rattus norvegicus, Rhizopus microsporus, Roseobacter denitrificans, Saccharomyces cerevisiae, Saccharomyces pombe, Schistosoma mansoni (UniProt accession number A8TKU6), Scrobicularia plana, Scytalidium thermophilum, Serratia marcescens, Shewanella sp., Skeletonema costatum, Sphingomonas sp. BSAR-1 , Sus scrofa, Synechococcus elongatus PCC 7942, Terfezia claveryi, Thermotoga maritima, Thermotoga neapolitana, Thermus sp. (Swissprot accession number O86025), Thermus thermophilus (Swissprot accession number Q153J0), Thermus yunnanensis, Ulva pertusa, Vibrio sp. (UniProt accession number Q93P54) and Walterinnesia aegyptia.

In a preferred embodiment, the alkaline phosphatase (EC 3.1.3.1 ) is the E. coli- derived enzyme encoded by phoA (SEQ ID NO: 55).

Thus, in a preferred embodiment of the present invention, the alkaline phosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 55 or a sequence which is at least n % identical to SEQ ID NO: 55 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a sugar phosphatase (EC 3.1.3.23).

Sugar phosphatases (EC 3.1.3.23) are enzymes which catalyze the following reaction:

sugar phosphate + H 2 O sugar + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, fungi and bacteria. The enzyme has, e.g., been described in Arabidopsis thaliana, Bacillus subtilis (Swissprot accession number Q9ZVJV), Enterobacter aerogenes, Enterococcus faecalis, Escherichia acidilactici, Escherichia coli, Lactococcus lactis, Neisseria meningitidis, Plasmodium falciparum (UniProt accession number Q8IJ74), Saccharomyces cerevisiae, Streptococcus equinus and Streptococcus pyogenes.

In a preferred embodiment, the sugar phosphatase (EC 3.1.3.23) is the E. coli- derived enzyme encoded by ybiV (SEQ ID NO: 56) or yidA (SEQ ID NO: 57).

Thus, in a preferred embodiment of the present invention, the sugar phosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57 or a sequence which is at least n % identical to SEQ ID NO: 56 or SEQ ID NO: 57 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a phosphatidylglycerophosphatase (EC 3.1.3.27).

Phosphatidylglycerophosphatases (EC 3.1 .3.27) are enzymes which catalyze the following reaction:

phosphatidylglycerophosphate + H 2 O phosphatidylglycerol + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, fungi and bacteria. The enzyme has, e.g., been described in Anabaena sp., Bacillus licheniformis, Enterobacter aerogenes, Escherichia coli, Listeria monocytogenes, Mesocricetus auratus, Syrian hamster, Micrococcus cerificans, Rattus sp., Rhodopirellula baltica, Saccharomyces cerevisiae, Salmonella enterica subsp. enterica serovar Typhimurium, Serratia marcescens, Streptococcus sanguinis and Vigna radiata.

In a preferred embodiment, the phosphatidylglycerophosphatase (EC 3.1.3.27) is the E. coli-derived enzyme encoded by pgpA (SEQ ID NO: 58), pgpC (SEQ ID NO: 59) or pgpB (SEQ ID NO: 60).

Thus, in a preferred embodiment of the present invention, the phosphatidylglycerophosphatase (EC 3.1 .3.27) is an enzyme comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NO: 58 to SEQ ID NO: 60 or a sequence which is at least n % identical to SEQ ID NO: 58 to SEQ ID NO: 60 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ). Diacylglycerol pyrophosphate phosphatases (EC 3.1 .3.81 ) are enzymes which catalyze the following reaction:

,2-diacyl-sn-glycerol 3-diphosphate + H 2 O ^ 1 ,2-diacyl-sn-glycerol 3-phosphate + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, fungi and bacteria. The enzyme has, e.g., been described in Catharanthus roseus, Escherichia coli, Homo sapiens, Mus musculus (Swissprot accession number Q61469) and Saccharomyces cerevisiae. In a preferred embodiment, the pyrophosphate phosphatase (EC 3.1.3.81 ) is the E. coli-derived enzyme encoded by pgpB (SEQ ID NO: 60) already described above. It is of note that pgpB has not only been classified under EC 3.1.3.81 but also under EC 3.1.3.27 as phosphatidylglycerophosphatase B enzymes (EC 3.1.3.27). Phosphatidylglycerophosphatase B enzymes (EC 3.1.3.27) are also termed diacylglycerol pyrophosphate phosphatases (EC 3.1.3.81 ), DGPP phosphatases, phosphatidate phosphatases (EC 3.1.3.4), undecaprenyl pyrophosphate phosphatases (EC 3.6.1.27) and undecaprenyl-diphosphatases.

Thus, in a preferred embodiment of the present invention, the pyrophosphate phosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 60 or a sequence which is at least n % identical to SEQ ID NO: 60 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting D APP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a phosphatidate phosphatase (EC 3.1.3.4).

Phosphatidate phosphatases (EC 3.1.3.4) are enzymes which catalyze the following reaction:

a 1 ,2-diacylglycerol 3-phosphate + H 2 O a 1 ,2-diacyl-sn-glycerol + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Acholeplasma laidlawii, Arabidopsis thaliana, Arachis hypogaea, Bos taurus, Caenorhabditis elegans, Canis lupus familiaris, Cavia porcellus, Cricetulus griseus, Drosophila melanogaster, Escherichia coli, Geobacillus toebii (UniProt accession number A5HKK6), Homo sapiens, Mesocricetus auratus, Momordica charantia, Mus musculus, Rattus norvegicus, Rhodococcus jostii (UniProt accession number Q0SKM5), Saccharomyces cerevisiae, Spinacia oleracea, Streptomyces coelicolor, Sus scrofa, Vicia faba, Vigna radiata and Vigna unguiculata. In a preferred embodiment, the phosphatidate phosphatase (EC 3.1.3.4) is the S. cerevisiae-derived enzyme encoded by pahl (SEQ ID NO: 68).

Thus, in a preferred embodiment of the present invention, the phosphatidate phosphatase (EC 3.1.3.4) is an enzyme comprising the amino acid sequence of SEQ ID NO: 68 or a sequence which is at least n % identical to SEQ ID NO: 68 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a phosphoserine phosphatase (EC 3.1.3.3).

Phosphoserine phosphatases (EC 3.1.3.3) are enzymes which catalyze the following reaction:

O-phospho-L(or D)-serine + H 2 O ^ "" ^" L(or D)-serine + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Arabidopsis thaliana, Bos taurus, Desulfovibrio desulfuricans, Escherichia coli, Gallus gallus, Homo sapiens, Hydrogenobacter thermophilus (UniProt accession number D3DFG8), Methanocaldococcus jannaschii (Swissprot accession number Q58989), Methylophilus methylotrophus, Mus musculus (UniProt accession number Q99LS3), Mycobacterium tuberculosis (UniProt accession number Ο53289), Pisum sativum, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rattus norvegicus, Rhodobacter capsulatus, Saccharomyces cerevisiae (Swissprot accession number P42941 ), Streptomyces azureus and Thermococcus onnurineus (UniProt accession number B6YX36). In a preferred embodiment, the phosphoserine phosphatase (EC 3.1.3.3) is the E. coli-derived enzyme encoded by serB (SEQ ID NO: 61 ).

Thus, in a preferred embodiment of the present invention, the phosphoserine phosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 61 or a sequence which is at least n % identical to SEQ ID NO: 61 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a phosphoglycolate phosphatase (EC 3.1.3.18).

Phosphoglycolate phosphatases (EC 3.1.3.18) are enzymes which catalyze the following reaction:

2-Phosphoglycolate + H 2 O j^T*" glycolate + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Agrobacterium tumefaciens, in the alpha proteobacterium endosymbiont of Amoeba proteus (Swissprot accession number B3VBH3), in Amaranthus caudatus, Anabaena variabilis, Aquifex aeolicus (UniProt accession number 067359), Arabidopsis thaliana, Chlamydomonas reinhardtii, Chlorella vulgaris, Cupriavidus necator, Enterobacter aerogenes (UniProt accession number Q9Eyy5), Escherichia coli, Glycine max, Haemophilus influenzae, Homo sapiens, Hordeum vulgare, Megathyrsus maximus, Nicotiana tabacum, Panicum miliaceum, Panicum milioides, Phaseolus vulgaris, Pisum sativum, Rattus norvegicus, Saccharomyces cerevisiae (Swissprot accession number P19881 ), Salmonella enterica, Shigella flexneri, Sorghum bicolor, Spinacia oleracea, Synechococcus elongatus PCC 7942, Synechocystis sp. (Swissprot accession number Q8XC69), Thermoplasma acidophilum (UniProt accession number Q9HLQ2), Triticum aestivum and Zea mays.

In a preferred embodiment, the phosphoglycolate phosphatase (EC 3.1.3.18) is the E. coli-derived enzyme encoded by gph (SEQ ID NO: 62). Thus, in a preferred embodiment of the present invention, the phosphoglycolate phosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 62 or a sequence which is at least n % identical to SEQ ID NO: 62 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a pyrimidine 5'-nucleotidase (EC 3.1.3.5).

Pyrimidine 5'-nucleotidases (EC 3.1.3.5) are enzymes which catalyze the following reaction:

a 5'-ribonucleotide + H 2 O a ribonucleoside + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Aliivibrio fischeri, Arachis hypogaea, Bacillus sp., Bos taurus, Bothrops sp., Cajanus cajan, Candida parapsilosis, Cavia porcellus, Columba sp., Corynebacterium glutamicum, Crocodylus siamensis, Crotalus sp., Daboia russelii, Danio rerio, Dictyostelium sp., Dosidicus gigas, Escherichia coli, Gadus macrocephalus, Gallus gallus, Giardia intestinalis, Gloydius brevicaudus (UniProt accession number B6EWW8), Haemophilus influenzae, Helicobacter pylori (Swissprot accession number Q6UC93), Hemachatus haemachatus, Homo sapiens, Kocuria varians, Lachesis muta muta, Legionella pneumophila (UniProt accession number Q5ZZB6), Leishmania chagasi, Loxosceles gaucho, Lutzomyia longipalpis, Micrurus frontalis, Mus musculus, Mycoplasma sp., Naja naja, Neurospora crassa, Oncorhynchus sp., Ovis aries, Photobacterium sp., Proteus vulgaris, Pseudomonas aeruginosa (Swissprot accession number Q9I767), Rattus norvegicus, Rhipicephalus microplus, Saccharomyces cerevisiae, Salinivibrio costicola, Salmonella enterica, Salvator rufescens, Sebastes inermis, Shigella sonnei, Solanum tuberosum, Sturnus vulgaris, Sus scrofa, Torpedo marmorata, Trachurus japonicus, Triakis scyllium, Trichinella spiralis (Swissprot accession number Q8MQS9), Trichomonas sp., Tritrichomonas suis, Ureaplasma urealyticum, Varanus gouldii, Vibrio sp., Xylella fastidiosa (UniProt accession number Q9PBQ1 ) and Zea mays. In a preferred embodiment, the pyrimidine 5 -nucleotidase (EC 3.1.3.5) is the E. coli- derived enzyme encoded by yjjG (SEQ ID NO: 63).

Thus, in a preferred embodiment of the present invention, the pyrimidine 5'- nucleotidase is an enzyme comprising the amino acid sequence of SEQ ID NO: 63 or a sequence which is at least n % identical to SEQ ID NO: 63 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a pyridoxal phosphate phosphatase (EC 3.1.3.74).

Pyridoxal phosphate phosphatases (EC 3.1.3.74) are enzymes which catalyze the following reaction:

pyridoxal 5'-phosphate + H 2 O ^ "" * * pyridoxal + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, fungi and bacteria. The enzyme has, e.g., been described in Bos taurus, Brachylagus idahoensis, Canis lupus familiaris, Escherichia coli, Felis catus, Gallus gallus, Homo sapiens, Meriones unguiculatus, Mus musculus, Paenibacillus thiaminolyticus, Rattus norvegicus, Sinorhizobium meliloti and Sus scrofa.

In a preferred embodiment, the pyridoxal phosphate phosphatase (EC 3.1.3.74) is the E. coli-derived enzyme encoded by yigL (SEQ ID NO: 64).

Thus, in a preferred embodiment of the present invention, the pyridoxal phosphate phosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 64 or a sequence which is at least n % identical to SEQ ID NO: 64 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a fructose-1 , 6-bisphosphatase (EC 3.1.3.11).

Fructose-1 , 6-bisphosphatases (EC 3.1.3.11 ) are enzymes which catalyze the following reaction:

D-fructose 1 ,6-bisphosphate + H 2 O D-fructose 6-phosphate + phosphate

As regards the preferred embodiments of said fructose-1 , 6-bisphosphatase (EC 3.1.3.11 ) for the enzymatic conversion of DMAPP into prenol, the same applies, mutatis mutandis, as has been set forth above with respect to the fructose-1 , 6- bisphosphatases (EC 3.1.3.11 ) in the enzymatic conversion of DMAPP into DMAP according to route (i) of the present invention.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of an UDP-sugar diphosphatase (EC 3.6.1.45).

UDP-sugar diphosphatases (EC 3.6.1.45) are enzymes which catalyze the following reaction:

UDP-sugar + H 2 O UMP + alpha-D-aldose 1 -phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, fungi and bacteria. The enzyme has, e.g., been described in Corynebacterium glutamicum, Enterobacter aerogenes (UniProt accession number Q9RQT7), Escherichia coli (UniProt accession number P07024), Homo sapiens (UniProt accession number 095848), Mus musculus (UniProt accession number Q9D142), Peptoclostridium difficile, Saccharomyces cerevisiae, Salmonella sp., Sus scrofa and Yersinia intermedia (UniProt accession number A4URQ8).

In a preferred embodiment, the UDP-sugar diphosphatase (EC 3.6.1.45) is the E. coli-derived enzyme encoded by ushA (SEQ ID NO: 65).

Thus, in a preferred embodiment of the present invention, the UDP-sugar diphosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 65 or a sequence which is at least n % identical to SEQ ID NO: 65 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27). Undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27) are enzymes which catalyze the following reaction:

ditrans.octacis-undecaprenyl diphosphate + H 2 O ^_^ditrans,octacis-undecaprenyl phosphate + phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Bacillus subtilis, Cupriavidus metallidurans, Enterococcus faecalis, Escherichia coli and Micrococcus luteus.

In a preferred embodiment, the undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27) is the E. coli-derived enzyme encoded by pgpB (SEQ ID NO: 60) already described above.

It is of note that pgpB has not only been classified under EC 3.1.3.81 but also under EC 3.1.3.27 as phosphatidylglycerophosphatase B enzymes (EC 3.1.3.27). Phosphatidylglycerophosphatase B enzymes (EC 3.1.3.27) are also termed diacylglycerol pyrophosphate phosphatases (EC 3.1.3.81 ), DGPP phosphatases, phosphatidate phosphatases (EC 3.1.3.4), undecaprenyl pyrophosphate phosphatases (EC 3.6.1.27) and undecaprenyl-diphosphatases.

Thus, in a preferred embodiment of the present invention, the undecaprenyl pyrophosphate phosphatase is an enzyme comprising the amino acid sequence of SEQ ID NO: 60 or a sequence which is at least n % identical to SEQ ID NO: 60 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting DMAPP into prenol. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of a prenyl-diphosphatase (EC 3.1.7.1 ).

Prenyl-diphosphatases (EC 3.1.7.1 ) are enzymes which catalyze the following reaction: Prenyl diphosphate + H 2 O prenol + diphosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants and animals. The enzyme has, e.g., been described in Citrus sinensis, Datura stramonium, Oryza sativa and Rattus norvegicus.

In another preferred embodiment, the enzymatic conversion of DMAPP into prenol is achieved by the use of an isopentenyl phosphate kinase (EC 2.7.4.26).

As regards the preferred embodiments for isopentenyl phosphate kinase (EC 2.7.4.26) for the enzymatic conversion of DMAPP into prenol, the same applies as has been set forth above in connection with the enzymatic conversion of DMAPP into DMAP according to the invention.

Route (iv): The provision of DMAP by the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP

According to the present invention, DMAP can be provided enzymatically by the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP.

In a preferred embodiment, the enzymatic conversion of isopentenyl monophosphate

(IMP) into said DMAP is achieved by making use of an isomerase.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of IMP into said DMAP by making use of a isomerase, the expression of said isomerase is increased/enhanced.

Preferably, said isomerase is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

As regards the preferred embodiments for enzymes catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into said DMAP and the isomerases, the same applies as has been set forth above in connection with the conversion of IPP into DMAPP according to the invention.

In a preferred embodiment, the isomerase is an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2). In case the above conversion is performed in a cell, said isopentenyl monophosphate (IMP) may be metabolically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of IMP from mevalonate-5-phosphate. Vinokur et al. (Biochemistry 53 (2014), 4161-4168) describes the existence of an alternative mevalonate pathway in Archaea wherein IMP is produced from mevalonate-5-phosphate.

Alternatively, in organisms which do not naturally have the metabolic routes leading to the formation of IMP from mevalonate-5-phosphate, the genes encoding the enzymes for the production of IMP can artificially be introduced (and preferably overexpressed) in a host cell.

Genes encoding the enzymes for the production of IMP are known in the art and can be derived from the different reactions known for the mevalonate pathway shown in Figure 44. These enzymes are termed acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate-5-kinase, mevalonate-3-kinase, mevalonate-3-phosphate-5-kinase, and mevalonate-5-phosphate-decarboxylase. IMP (termed isopentenyl phosphate in Figure 44) is known to be produed either by a pathway known as "Archaea I" or "Archaea II".

In the pathway known as "Archaea Tmevalonate is converted into mevalonate-5- phosphate by a mevalonate-5-kinase and the thus produced mevalonate-5- phosphate is converted into IMP by a mevalonate-5-phosphate decarboxylase.

In the pathway known as "Archaea II" mevalonate is converted into mevalonate-3- phosphate by a mevalonate-3-kinase wherein said mevalonate-3-phosphate is then futher converted into mevalonate-3,5-bisphosphate by a mevalonate-3-phosphate-5- kinase wherein said mevalonate-3,5-bisphosphate is then further converted into said IMP by a mevalonate-5-phosphate-decarboxylase.

Thus, in one embodiment, in (micro-)organisms which naturally have the metabolic routes leading to the formation of mevalonate, the genes encoding the enzymes for the production of IMP from mevalonate can artificially be introduced (and preferably overexpressed) in a host cell. These genes are preferably the genes encoding mevalonate-3-kinase, mevalonate-3-phosphate-5-kinase, and mevalonate-5- phosphate-decarboxylase (in accordance with the above known pathway known as "Archaea II"). Alternatively (or additionally), these genes are preferably the genes encoding mevalonate-5-kinase and mevalonate-5-phosphate decarboxylase (in accordance with the above known pathway known as "Archaea I").

In this embodiment, the microorganism is preferably yeast, more preferably S. cerevisiae which is known to have the metabolic routes ieading to the formation of mevalonate.

In another embodiment, in (micro-)organisms which do not naturally have the metabolic routes Ieading to the formation of mevalonate, mevalonate can be produced from the central metabolite acetyl-CoA by artificially introducing (and preferably overexpressing) in a host cell the genes encoding the enzymes for the production of mevalonate from acetyl-CoA. These genes are preferably the genes encoding acetoacetyl-CoA thiolase (converting acetyl-CoA into acetoacetyl-CoA), HMG-CoA synthase (converting acetoacetyl-CoA into HMG-CoA) and HMG-CoA reductase (converting HMG-CoA into mevalonate). In this host cell, the genes encoding the enzymes for the production of IMP from mevalonate can additionally artificially be introduced (and preferably overexpressed). These genes are preferably the genes encoding mevalonate-3-kinase, mevalonate-3-phosphate-5-kinase, and mevalonate-5-phosphate-decarboxylase (in accordance with the above known pathway known as "Archaea II"). Alternatively (or additionally), these genes are preferably the genes encoding mevalonate-5-kinase and mevalonate-5-phosphate decarboxylase (in accordance with the above known pathway known as "Archaea I"). In this embodiment, the microorganism is preferably E. coli, which is known to lack the metabolic routes Ieading to the formation of mevalonate.

In case the above conversion is performed in vitro, said IMP is preferably added to the reaction.

Increasing the pool of DMAP by reducing the activity of endogenous phosphatases, thereby reducing the leakage of DMAP

When implementing the method of the present invention for providing DMAP enzymatically according to any one of steps (i) to (iv) in vivo, DMAP may be hydrolyzed into prenol by the activity of endogenous phosphatases, thereby reducing the pool of DMAP. In order to reduce/prevent this leakage of DMAP, in a preferred embodiment of the present invention, the above methods for the provision of DMAP may further comprise a method wherein the activity/activities of enzymes capable of dephosphorylating DMAP into prenol is/are reduced, or lost/inactivated.

The term "dephosphorylation" refers to the removal of a phosphate group from an organic compound by hydrolysis as it, e.g., occurs in the conversion of DMAP into prenol.

Enzymes capable of dephosphorylating DMAP into prenol are preferably phosphatases.

Preferably, this reduction (or complete loss) of the activity of enzymes capable of dephosphorylating DMAP into prenol, preferably of phosphatases, is achieved by a genetic modification which leads to said inactivation or reduction. This can be achieved e.g., by random mutagenesis or site-directed mutagenesis of the promoter and/or the enzyme and subsequent selection of promoters and/or enzymes having the desired properties or by complementary nucleotide sequences or RNAi effect. In the context of the present invention, a "reduced activity" means that the expression and/or the activity of an enzyme capable of dephosphorylating DMAP into prenol, preferably of a phosphatase, is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% lower than the expression in the corresponding non- modified cell and than the activity of the non-modified enzyme respectively. Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In short, these methods may, e.g., employ methods of measuring the expression on the RNA-level (by, e.g., RT-PCR technologies) or on the protein level (by, e.g., Western blot methods).

Assays for measuring the reduced enzyme activity of dephosporylation are known in the art.

A genetic modification of the cell which leads to said inactivation or reduction of the dephosphorylation activity/activities is preferably achieved by inactivation of the gene(s) encoding said enzymes (preferably phosphatases) capable of dephosphorylating DMAP into prenol.

The inactivation of the gene(s) encoding an enzyme (preferably a phosphatase) capable of dephosphorylating DMAP into prenol in the context of the present invention means that the gene(s) coding for (an) enzyme(s) (preferably (a) phosphatase(s)) capable of dephosphorylating DMAP into prenol which is (are) present in the cell is (are) inactivated so that they are no longer expressed and/or do not lead to the synthesis of a functional enzyme having dephosphorylation activity. Inactivation can be achieved by many different ways known in the art. The inactivation can, e.g., be achieved by the disruption of the gene(s) encoding the corresponding enzyme or by clean deletion of said gene(s) through the introduction of a selection marker. Alternatively, the promoter of the gene(s) encoding the corresponding enzyme can be mutated in a way that the gene(s) is/are no longer transcribed into mRNA. Other ways to inactivate the gene(s) encoding the corresponding enzyme known in the art are: to express a polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of the gene encoding the enzyme having dephosphorylation activity so that the mRNA can no longer be translated into a protein, to express a polynucleotide encoding RNA that suppresses the expression of said gene(s) through RNAi effect; to express a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of said gene(s); or to express a polynucleotide encoding RNA that suppresses expression of said gene(s) through a co-suppression effect. These polynucleotides can be incorporated into a vector, which can be introduced into the cell by transformation to achieve the inactivation of the gene(s) encoding enzyme having dephosphorylation activity.

The term "inactivation" in the context of the present invention preferably means complete inactivation, i.e. that the cell does not show an enzyme having dephosporylation activity.

Preferably, "inactivation" means that the gene(s) encoding the enzyme having dephosporylation activity which are present in the cell are genetically modified so as to prevent the expression of the enzyme. This can be achieved, e.g., by deletion of the gene or parts thereof wherein the deletion of parts thereof prevents expression of the enzyme, or by disruption of the gene either in the coding region or in the promoter region wherein the disruption has the effect that no protein is expressed or a dysfunctional protein is expressed.

The provision of DMAPP

As mentioned above, the metabolic routes leading to the formation of dimethylallyl pyrophosphate (DMAPP) via the mevalonate (MEVA) and 1-deoxy-D-xylulose-5- phosphate (DXP) pathways are known in the art.

However, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN- dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) into a flavin-derived cofactor (utilizing dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP)), also the availability of DMAPP is another limiting factor.

The chemical structure of dimethylallyl pyrophosphate (DMAPP) is shown in Figure 3.

As mentioned above, the mechanism of the ferulic acid decarboxylase (FDC) in association with the modified FMN (prenylated-FMN) (the latter provided by the PAD enzyme) was recently described (Nature 522 (2015), 497-501 ; Nature, 522 (2015), 502-505). Moreover, the metabolic routes leading to the formation of dimethylallyl pyrophosphate (DMAPP) via the mevalonate (MEVA) and 1-deoxy-D-xylulose-5- phosphate (DXP) pathways are known in the art. However, it remained unclear whether said FMN prenyl transferase (catalyzing the prenylation of a flavin cofactor (FMN or FAD) into a flavin-derived cofactor), in the context of the production of isobutene from 3-methylcrotonic acid, is also capable of utilizing dimethylallyl pyrophosphate (DMAPP). Indeed, it has only previously been described by Arunrattanamook and Marsh (Biochemistry 57(5) (2018), 696-700) that a prenyl transferase from S. cerevisiae uses DMAPP as a co-substrate.

Only the present invention has shown that it is possible to use a FMN prenyl transferase (catalyzing the prenylation of a flavin cofactor (FMN or FAD) into a flavin- derived cofactor), by utilizing dimethylallyl pyrophosphate (DMAPP), in a method for the production of isobutene in accordance with the present invention.

The exogenous supplementation of DMAPP in a culture medium is not feasible since DMAPP is assumed to not enter the cell. Moreover, although the metabolic routes leading to the formation of dimethylallyl pyrophosphate (DMAPP) via the mevalonate (MEVA) and 1-deoxy-D-xylulose-5-phosphate (DXP) pathways are known in the art, there is a need to increase the intracellular pool of DMAPP as the availability of DMAPP is limiting for the production of isobutene from 3-methylcrotonic acid in accordance with the present invention. Therefore, the present invention also provides methods for endogenously generating DMAPP and, preferably, to increase the pool of DMAPP.

According to the present invention, DMAPP can be provided via different routes (in the following referred to as route (v), (vi) and (vii), respectively) which are schematically shown in Figure 45 (and designated in said Figure with the names "Isomerase", "Kinase 2" and "Diphosphokinase", respectively).

Accordingly, the above described method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene further comprises providing said DMAPP enzymatically by:

(v) the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP; or

(vi) the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP; or

(vii) the enzymatic conversion of prenol into said DMAPP; or by a combination of any one of (v) to (vii).

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene, the enzymatic provision of said DMAPP is enhanced/increased over naturally occurring (enzymatic) reactions/conversions leading to the production of DMAPP, preferably by overexpressing corresponding enzymes capable of catalyzing any of the above reactions (v) to (vii). Means and methods for increasing/enhancing the expression of an enzyme are described in more detail further below.

These different routes (v), (vi) and (vii) for the provision of DMAPP are illustrated in Figure 45 while each of the above conversions is described in more detail in the following:

Route (v): The provision of DMAPP by the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP

According to the present invention, DMAPP can be provided enzymatically by the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP.

In a preferred embodiment, the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP is achieved by making use of an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

As regards said isomerase and said isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2) for the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP, the same applies, mutatis mutandis, as has already been set forth above.

In case the above conversion is performed in a cell, said isopentenyl pyrophosphate is preferably metabolically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of isopentenyl pyrophosphate, e.g., via the mevalonate (MEVA) and/or 1 -deoxy-D-xylulose-5-phosphate (DXP) pathways which are known in the art.

In case the above conversion is performed in vitro, said isopentenyl pyrophosphate is preferably added to the reaction.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of IPP into said DMAPP by making use of an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2), the expression of said isomerase, preferably said isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2) is increased/enhanced. Preferably, said isomerase, more preferably said isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2), is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

Route (vi): The provision of DMAPP by the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP

According to the present invention, DMAPP can be provided enzymatically by the enzymatic conversion dimethylallyl phosphate (DMAP) into said DMAPP.

In a preferred embodiment, the enzymatic conversion of DMAP into DMAPP is achieved by making use of a kinase. Kinases are known in the art and are generally known as enzymes capable of catalyzing the transfer of phosphate groups from high- energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the substrate gains a phosphate group and the high- energy ATP molecule donates a phosphate group. This reaction is a transesterification and produces a phosphorylated substrate and ADP.

Enzymes catalyzing the enzymatic conversion (i.e., the phosphorylation) of DMAP into said DMAPP are enzymes which catalyze the reaction as shown in Figure 46.

In case the above conversion is performed in a cell, said DMAP is preferably metabolically provided as described herein-above and below.

In case the above conversion is performed in vitro, said DMAP is preferably added to the reaction.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of DMAP into said DMAPP by making use of a kinase, the expression of said kinase is increased/enhanced. Preferably, said kinase is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

In a preferred embodiment, the kinase is an isopentenyl monophosphate kinase (EC 2.7.4.26).

Isopentenyl phosphate kinases (EC 2.7.4.26) are enzymes which catalyze the following reaction:

ATP + isopentenyl phosphate ADP + isopentenyl diphosphate

This enzyme is known in the art and has, e.g., been described by Chen and Poulter (Biochemistry 49 (2010), 207-210). This enzyme has, e.g., been described in Haloferax volcanii (UniProt accession number D4GWT7), Mentha x piperita (SwissProt accession number P56848), Methanocaldococcus jannaschii (SwissProt accession number Q60352), Methanothermobacter thermautotrophicus (UniProt 026153), and Thermoplasma acidophilum (UniProt accession number Q9HLX1 ). The provision of DMAP

The DMAP which is converted into DMAPP according to the method of the present invention may itself be provided by an enzymatic conversion as described herein above and below.

Preferably, according to the present invention, DMAP can be provided enzymatically by the enzymatic conversion of prenol into DMAP or by the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP.

In case the above conversion of prenol into DMAP is performed in a cell (i.e., in vivo), said prenol is preferably metaboiically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of prenol.

Alternatively or in addition to the above, said prenol may preferably be supplemented/added to the culture medium.

In case the above conversion is performed in vitro, said prenol is preferably added to the in vitro reaction.

As described above, there are organisms known in the art which are capable of naturally producing prenol or by artificially introduced metabolic routes. Thus, as described above, corresponding organisms may preferentially be used in the methods of the present invention for the conversion of prenol into DMAP.

As described above, in case the above conversion is performed in a cell, said isopentenyl monophosphate (IMP) may be metaboiically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of IMP from mevalonate-5-phosphate. Vinokur et al. (Biochemistry 53 (2014), 4161-4168) describes the existence of an alternative mevalonate pathway in Archaea wherein IMP is produced from mevalonate-5-phosphate.

Alternatively, in organisms which do not naturally have the metabolic routes leading to the formation of IMP from mevalonate-5-phosphate, the genes encoding the enzymes for the production of IMP can artificially be introduced (and preferably overexpressed) in a host cell as described above.

In a preferred embodiment, the enzymatic conversion of prenol into said DMAP is achieved by making use of a kinase, more preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-) and even more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

In another preferred embodiment, the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP is achieved by making use of an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of prenol into DMAP or the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP by making use of a kinase and isomerase, respectively, the expression of said kinase and isomerase, respectively, is increased/enhanced. Preferably, said kinase and isomerase, respectively, is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

As regards said kinase, said phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), said hydroxyethylthiazole kinase (EC 2.7.1.50), said isomerase and said isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2), the same applies, mutatis mutandis, as has been set forth above.

Route (vii): The provision of DMAPP by the enzymatic conversion of prenol into said DMAPP

According to the present invention, DMAPP can be provided enzymatically by the direct enzymatic conversion of prenol into said DMAPP. The direct enzymatic conversion of prenol into said DMAPP in one step can, e.g., be achieved by the use of an enzyme which is able to catalyze the transfer of a diphosphate group, such as a diphosphotransferase, for example enzymes which are classified as EC 2.7.6.- (diphosphotransferases). Examples are 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3) and thiamine diphosphokinase (EC 2.7.6.2). Preferably, ATP is the donor of the diphosphate group in such a reaction. Previously, the use of a diphosphokinase EC 2.7.6.- has been described in WO 2013/040383 for the phosphorylation of dimethylallyl alcohol (i.e., prenol) to then produce isoprene.

In case the above conversion of prenol into DMAPP is performed in a cell (i.e., in vivo), said prenol is preferably metabolically provided by naturally occurring or artificially introduced metabolic routes leading to the formation of prenol.

Alternatively or in addition to the above, said prenol may preferably be supplemented/added to the culture medium.

In case the above conversion is performed in vitro, said prenol is preferably added to the in vitro reaction.

As described above, there are organisms known in the art which are capable of naturally producing prenol or by artificially introduced metabolic routes. Thus, as described above, corresponding organisms may preferentially be used in the methods of the present invention for the conversion of prenol into DMAPP.

In a preferred embodiment, the enzymatic conversion of prenol into DMAPP is achieved by making use of a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3).

Thus, in one embodiment, the direct enzymatic conversion of prenol into DMAPP can be achieved by the use of a 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3). This enzyme is an enzyme which catalyzes the following reaction:

2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine + ATP 2-amino-7,8- dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine + AMP

The occurrence of this enzyme has been described for several organisms, e.g. for E. coli, Plasmodium falciparum, Plasmodium chabaudi, Streptococcus pneumoniae, Toxoplasma gondii, Yersinia pestis, Pneumocystis carinii, Haemophilus influenzae, S. cerevisiae, Arabidopsis thaliana and Pisum sativum. In principle, any known 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase can be employed in the method according to the invention.

In another embodiment the direct enzymatic conversion of prenol into DMAPP can be achieved by the use of a thiamine diphosphokinase (EC 2.7.6.2). This enzyme is an enzyme which catalyzes the following reaction:

ATP + thiamine AMP + thiamine diphosphate

The occurrence of this enzyme has been described for several organisms, e.g. for Salmonella enterica, Plasmodium falciparum, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Arabidopsis thaliana, Caenorhabditis elegans, Rattus norvegicus, Mus musculus and Homo sapiens. In principle, any known thiamine diphosphokinase can be employed in the method according to the invention.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the enzymatic conversion of prenol into said DMAPP by making use of a diphosphotransferase (EC 2.7.6.-) (preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3)), the expression of said diphosphotransferase (EC 2.7.6.-) (preferably of said thiamine diphosphokinase (EC 2.7.6.2) or of said 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase (EC 2.7.6.3)) is increased/enhanced. Preferably, said enzyme is overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

The provision of the flavin cofactor

As mentioned above, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) into a flavin-derived cofactor, the availability of DMAP and/or DMAPP is one limiting factor. Another limiting factor may be the availability of the flavin cofactor FMN.

Flavin mononucleotide (FMN), also termed riboflavin-5'-phosphate, is a biomolecule produced from riboflavin (vitamin B 2 ). FMN is known to be a co-factor for several enzymatic reactions. The pathway for its biosynthesis is known and has, e.g., been described in E. coli. The pathway for its biosynthesis starting from GTP is illustrated in Figure 9.

As FMN is a co-factor for several enzymatic reactions, it is known to occur in many organisms. Because the availability of FMN in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene according to the present invention may be a limiting factor, the present invention provides, in accordance with the above described methods, a method increasing the intracellular pool of FMN, thereby increasing the availability of FMN. Accordingly, a method for providing said flavin cofactor enzymatically by the enzymatic conversion of riboflavin into flavin mononucleotide (FMN) is provided.

Accordingly, the present invention also relates to a method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) into a flavin-derived cofactor as described herein above, wherein said method further comprises providing said flavin cofactor enzymatically by the enzymatic conversion of riboflavin into flavin mononucleotide (FMN), thereby increasing the pool of FMN.

In case the above conversion is performed in a cell (i.e., in vivo), said riboflavin (i.e., the precursor of FMN) may be provided by naturally occurring metabolic routes leading to the formation of riboflavin by the pathway for its biosynthesis known to occur in many organisms or by artificially introduced metabolic routes. Alternatively, or in addition to the above, riboflavin may also be added to the culture medium which enters the (host) cell and is then enzymatically converted into FMN according to the above and below described methods.

In case the above conversion is performed in vitro, said riboflavin is preferably added to the reaction.

Preferably, in the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene of the present invention wherein the method further comprises the provision of the flavin cofactor by the enzymatic conversion of riboflavin into flavin mononucleotide (FMN), the enzymatic conversion of riboflavin into FMN is achieved by making use of:

a kinase, preferably:

an archaeal riboflavin kinase (EC 2.7.1.161 ),

flavokinases derived from S. cerevisiae or from Rattus norvegicus,

a flavokinase derived from Megasphaera elsdenii,

phosphotransferases with an alcohol group as acceptor (EC 2.7.1 ), preferably erythritol kinases (2.7.1 .27) or glycerol kinases (2.7.1.30),

phosphotransferases with a phosphate group as acceptor (EC 2.7.4), preferably isopentenyl phosphate kinases (EC 2.7.4.26); or

a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF); or

a variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived.

In a preferred embodiment, in the enzymatic conversion of riboflavin into FMN, the expression of said kinase, preferably said archaeal riboflavin kinase (EC 2.7.1.161 ), said flavokinase derived from S. cerevisiae or from Rattus norvegicus, said flavokinase derived from Megasphaera elsdenii, said phosphotransferase with an alcohol group as acceptor (EC 2.7.1 ), said erythritol kinase (2.7.1.27), said glycerol kinase (2.7.1 .30), said phosphotransferase with a phosphate group as acceptor (EC 2.7.4), said isopentenyl phosphate kinase (EC 2.7.4.26), said bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) or said variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived is increased/enhanced. Preferably, said enzyme(s) is/are overexpressed. Means and methods for increasing/enhancing/overexpressing the expression of an enzyme are described in more detail further below.

Thus, in a preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of a kinase, preferably an archaeal riboflavin kinase (EC 2.7.1 .161 ), a flavokinase derived from S. cerevisiae or from Rattus norvegicus, or a flavokinase derived from Megasphaera elsdenii, a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 ), preferably an erythritol kinase (2.7.1.27) or a glycerol kinase (2.7.1.30) or a phosphotransferase with a phosphate group as acceptor (EC 2.7.4), preferably an isopentenyl phosphate kinases (EC 2.7.4.26).

Thus, in a preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of an archaeal riboflavin kinase (EC 2.7.1.161 ). Archaeal riboflavin kinases (EC 2.7.1 .161 ) are enzymes which catalyze the following reaction: CTP + riboflavin CDP + FMN

This enzyme is, e.g., known from Methanocaldococcus jannaschii and Trichophyton rubrum.

In a more preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of the archaeal riboflavin kinase derived from Methanocaldococcus jannaschii (UniProt accession number Q60365; SEQ ID NO: 69). This enzyme is described by Mashhadi et al. (Journal of Bacteriology 190 (7) (2008), 2615) to be monofunctional (only converting riboflavin into FMN).

Thus, in a preferred embodiment of the present invention, the archaeal riboflavin kinase (EC 2.7.1.161 ) is an enzyme comprising the amino acid sequence of SEQ ID NO: 69 or a sequence which is at least n % identical to SEQ ID NO: 69 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting riboflavin into FMN. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of an eukaryotic flavokinase enzyme derived from Saccharomyces cerevisiae or from Rattus norvegicus. Santos ef al. (JBC 275 (2000), 28618) and Kasi er a/. (J. Biochem. 107 (1990), 298) describe eukaryotic flavokinase enzymes derived from Saccharomyces cerevisiae and Rattus norvegicus, respectively, which may be used, in a preferred embodiment, for the enzymatic conversion of riboflavin into FMN.

In another preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 .-).

In a preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of an erythritol kinase (2.7.1.27) or a glycerol kinase (2.7.1 .30).

Erythritol kinases (2.7.1.27) are enzymes which catalyze the following reaction:

ATP + erythritol * * ADP + D-erythritol 4-phosphate

This enzyme has been described, e.g., in Brucella abortus and Propionibacterium acidipropionici.

Glycerol kinases (2.7.1.30) are enzymes which catalyze the following reaction:

ATP + glycerol ■ ADP + sn-glycerol 3-phosphate

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Avena sativa, Bacillus subtilis, Bombus sp., Bombyx mori, Bos taurus, Candida mycoderma, Candida tropicalis, Cavia porcellus, Cellulomonas sp., Clostridium novyi, Columba sp., Cucumis sativus, Culex quinquefasciatus, Cyberlindnera jadinii, Debaryomyces hansenii, Drosophila melanogaster, Elizabethkingia meningoseptica, Enterobacter aerogenes, Enterococcus casseliflavus, Enterococcus faecalis, Epidermophyton floccosum, Escherichia coli, Felis catus, Gallus gallus, Geobacillus stearothermophilus, Geotrichum candidum, Gluconobacter oxydans, Haemophilus influenzae, Halobacterium salinarum, Haloferax volcanii, Homo sapiens, Mesocricetus auratus, Microsporum gypseum, Mus musculus, Mycobacterium butyricum, Mycobacterium smegmatis, Mycobacterium sp., Mycobacterium tuberculosis, Neurospora crassa, Nocardia asteroides, Oryctolagus cuniculus, Osmerus mordax, Pediococcus pentosaceus, Phaseolus vulgaris, Pisum sativum, Plasmodium falciparum (UniProt accession number Q8IDI4), Pseudomonas aeruginosa, Rattus norvegicus, Saccharomyces cerevisiae, Shigella sonnei, Staphylococcus aureus, Sus scrofa, Thermococcus kodakarensis, Thermus aquaticus, Thermus thermophilus, Trypanosoma brucei (UniProt accession number D3KVM3 and Q9NJP9), Trypanosoma congolense (UniProt accession number Q75T26), Trypanosoma vivax (UniProt accession number B0I530), Vicia faba, Vigna radiata var. radiata, Wickerhamomyces anomalus and Zea mays.

In another preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of a phosphotransferase with a phosphate group as acceptor (EC 2.7.4-).

In a preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of isopentenyl phosphate kinase (EC 2.7.4.26).

Isopentenyl phosphate kinases (EC 2.7.4.26) are enzymes which catalyze the following reaction:

ATP + isopentenyl phosphate ADP + isopentenyl diphosphate

This enzyme has, e.g., been described in Haloferax volcanii (UniProt accession number D4GWT7), Mentha x piperita (SwissProt accession number P56848), Methanocaldococcus jannaschii (SwissProt accession number Q60352), Methanothermobacter thermautotrophicus (UniProt 026153), and Thermoplasma acidophilum (UniProt accession number Q9HLX1 ).

In another preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF).

Generally, riboflavin is converted into catalytically active cofactors (FAD and FMN) by the actions of riboflavin kinase EC 2.7.1 .26, which converts it into FMN, and FAD synthetase EC 2.7.7.2, which adenylates FMN to FAD. Eukaryotes usually have two separate enzymes, while most prokaryotes have a single bifunctional protein that can carry out both catalyses.

ribF is a bifunctional enzyme having a riboflavin kinase activity and an FMN adenylyltransferase activity.

Generally, enyzmes having a riboflavin kinase activity are enzymes which are classified as riboflavin kinases (EC 2.7.1.26) while enzymes having an FMN adenylyltransferase activity are enzymes which are classified as FMN adenylyltransferases (EC 2.7.7.2).

Riboflavin kinases (EC 2.7.1.26) are enzymes which catalyze the following reaction: ATP + riboflavin ADP + FMN

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Arabidopsis thaliana, Bacillus subtilis, Bos taurus, Corynebacterium ammoniagenes, Homo sapiens, Megasphaera elsdenii, Mus musculus, Neurospora crassa, Nicotiana tabacum, Rattus norvegicus, Saccharomyces cerevisiae, Schizosaccharomyces pombe (UniProt accession number 074866), Streptomyces davawensis (Swissprot accession number A3FM23) and Vigna radiata.

FMN adenylyltransferases (EC 2.7.7.2) are enzymes which catalyze the following reaction:

ATP + FMN ^~*~ diphosphate + FAD

This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. The enzyme has, e.g., been described in Arabidopsis thaliana, Bacillus subtilis (UniProt accession number P54575), Bos taurus, Candida glabrata (UniProt accession number Q6FNA9), Corynebacterium ammoniagenes, Homo sapiens, Methanocaldococcus jannaschii (UniProt accession number Q58579), Nicotiana tabacum, Rattus norvegicus, Saccharomyces cerevisiae, Streptomyces davawensis and Thermotoga maritima.

In a preferred embodiment, the bifunctional enzyme having a riboflavin kinase activity and an FMN adenylyltransferase activity is the enzyme encoded by the E. coli's ribF gene (SEQ ID NO: 34). This enzyme catalzyzes the following reactions:

ATP + riboflavin ^-*" ADP + FMN; and

ATP + FMN ^ " ^ * diphosphate + FAD

In a preferred embodiment of the present invention the bifunctional enzyme having a riboflavin kinase activity and an FMN adenylyltransferase activity is an enzyme comprising an amino acid sequence of SEQ ID NO: 34 or a sequence which is at least n % identical to SEQ ID NO: 34 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity of converting riboflavin into FMN. As regards the determination of the sequence identity, the same applies as has been set forth above.

In another preferred embodiment, the enzymatic conversion of riboflavin into FMN is achieved by making use of a variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived.

Preferably, such a variant is a variant wherein the activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived is improved while the FMN adenylyltransferase activity is not increased. In another preferred embodiment, the latter activity may be reduced over the corresponding activity of a bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived.

Serrano et al. (Int. J. Mol. Sci. 13 (2012), 14492-14517) recently identified two positions in the Corynebacterium ammoniagenes bifunctional riboflavin kinase/FMN adenylyltransferase, i.e., H28 and H31 , which, when mutated, lead to an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived while the FMN adenylyltransferase activity of converting FMN into FAD was not affected or even reduced.

Based on this knowledge, it is possible for the skilled person to provide variants of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) from bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) enzymes from other organisms which show an improved activity in converting riboflavin into FMN (while, preferably, the FMN adenylyltransferase activity of converting FMN into FAD is not affected or even reduced).

The enzymatic activity of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) to convert riboflavin into FMN and to convert FMN into FAD may be determined by methods known to the person skilled in the art.

In a preferred embodiment, the variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived is a variant having an amino acid sequence as shown in SEQ ID NO: 34 or an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 34, in which one or more amino acid residues at a position selected from the group consisting of positions 29 and 32 in the amino acid sequence shown in SEQ ID NO: 34 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.

As regards the determination of the sequence identity, the same applies as has been set forth above.

Such variants can be produced by starting out from any known bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) enzyme, e.g. any known naturally occurring bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) enzyme, and by effecting the amino acid substitution(s) at the position(s) indicated above according to routine measures, such as site directed mutagenesis.

In a more preferred embodiment, the variant is a variant wherein

(1 ) an amino acid residue at position 29 in the amino acid sequence shown in SEQ ID NO: 34 or at a position corresponding to this position, is deleted or substituted with alanine; and/or

(2) an amino acid residue at position 32 in the amino acid sequence shown in SEQ ID NO: 34 or at a position corresponding to this position, is deleted or substituted with serine or alanine. The pathways for the provision of 3-methylcrotonic acid which is then further converted into isobutene

As mentioned above, the method for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase according to the invention as defined above may be embedded in a pathway for the production of isobutene starting from acetyl-CoA via 3-methylcrotonyl-CoA and 3-methylcrotonic acid or via 3-hydroxyisovalerate (HIV) and 3-methylcrotonic acid. The corresponding reactions are schematically shown in Figure 1 and will be described in more detail in the following.

The enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid: step II as shown in Figure 1

The 3-methylcrotonic acid which is converted according to the method of the present invention into isobutene may itself be provided by an enzymatic reaction.

According to the present invention, the 3-methylcrotonic acid can be provided via different routes which are schematically shown in Figure 1.

Thus, according to one option, the 3-methylcrotonic acid may itself be provided by the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid. The enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid (step II as shown in Figure 1 ) is schematically illustrated in Figure 10.

According to the present invention, the enzymatic conversion of 3-hydroxyisovalerate (HIV) into said 3-methylcrotonic acid preferably makes use of an enzyme catalyzing the dehydration of a β-hydroxy acid (i.e., e.g., 3-hydroxyisovalerate (HIV)) into an α,β-unsaturated acid (i.e., e.g., 3-methylcrotonic acid). The term "dehydration" generally refers to a reaction involving the removal of H 2 O. Enzymes catalyzing 3- hydroxyisovalerate (HIV) dehydration are enzymes which catalyze the reaction as shown in Figure 10. Preferably, such an enzyme belongs to the family of hydrolases (EC 4.2.-.-).

Preferred examples of such enzymes which are classified as EC 4.2.-.- (i.e., hydro- lyases) are:

aconitase (EC 4.2.1.3);

fumarase (EC 4.2.1 .2); and

enoyl-CoA hydratase/dehydratease (EC 4.2.1.17).

The enzymatic condensation of acetone and acetyl-CoA into 3- hydroxyisovalerate (HIV): step III as shown in Figure 1

The 3-hydroxyisovalerate (HIV) which is converted according to the method of the present invention into 3-methylcrotonic acid may itself be provided by an enzymatic reaction, namely the enzymatic condensation of acetone and acetyl-CoA into said 3- hydroxyisovalerate (HIV). The condensation of acetone and acetyl-CoA into said 3- hydroxyisovalerate (HIV) (step III as shown in Figure 1 ) is schematically illustrated in Figure 11.

Thus, the present invention also relates to a method for producing isobutene from acetone in which acetone is first condensed with acetyl-CoA into 3- hydroxyisovalerate (HIV) which is then converted into 3-methylcrotonic acid. Further, 3-methylcrotonic acid is then converted into isobutene as described herein above.

According to the present invention, the condensation of acetone and acetyl-CoA into 3-hydroxyisovalerate (HIV) preferably makes use of an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e., the C=0) group of acetone and acetyl-CoA, in particular the methyl group of acetyl- CoA. According to this reaction scheme, the oxo group of acetone reacts as an electrophile and the methyl group of acetyl-CoA reacts as a nucleophile. The general reaction of the conversion of acetone and acetyl-CoA is shown in Figure 11. Enyzmes which are capable of enzymatically condensing acetone and acetyl-CoA into 3-hydroxyisovalerate (HIV) are known in the art and have, e.g., been described in WO 201 1/032934.

Preferably, the enzyme employed in the enzymatic condensation of acetone and acetyl-CoA into 3-hydroxyisovalerate (HIV) is an enzyme with the activity of a HMG CoA synthase (EC 2.3.3.10) and/or a PksG protein and/or an enzyme with the activity of a C-C bond cleavage/condensation lyase (preferably enzymes classified as isopropylmalate synthase (EC 2.3.3.13), as homocitrate synthase (EC 2.3.3.14) or as 4-hydroxy-2-ketovalerate aldolase (EC 4.1 .3.39)), such as a HMG CoA lyase (EC 4.1.3.4).

The enzymatic conversion of acetoacetate into acetone: step IV as shown in Figure 1

The acetone which is converted according to the method of the present invention into 3-hydroxyisovalerate (HIV) may itself be provided by an enzymatic reaction, namely the enzymatic conversion of acetoacetate into acetone. The conversion of acetoacetate into acetone (step IV as shown in Figure 1 ) is schematically illustrated in Figure 12. This reaction is a decarboxylation reaction and is a natural occurring reaction in organisms capable of producing acetone, i.e., organisms of the genus Clostridia.

Thus, the present invention also relates to a method for producing isobutene from acetoacetate in which acetoacetate is first converted into acetone which is then condensed with acetyl-CoA into 3-hydroxyisovalerate (HIV) which is then converted into 3-methylcrotonic acid as described herein above. Further, said 3-methylcrotonic acid is then converted into isobutene as described herein above.

According to the present invention, the conversion of acetoacetate into said acetone preferably makes use of an acetoacetate decarboxylase (EC 4.1.1.4).

The enzymatic conversion of acetoacetyl-CoA into acetoacetate: step Va and step Vb as shown in Figure 1

The acetoacetate which is converted according to the method of the present invention into acetone may itself be provided by an enzymatic reaction, namely the enzymatic conversion of acetoacetyl-CoA into acetoacetate. The conversion of acetoacetyl-CoA into acetoacetate can be achieved by two different routes. One possibility is the conversion of acetoacetyl-CoA into acetoacetate by hydrolysing the CoA thioester of acetoacetyl-CoA into acetoacetate. This reaction (step Va as shown in Figure 1 ) is schematically illustrated in Figure 13. In another, more preferred, aspect the CoA group of acetoacetyl-CoA is transferred on acetate, resulting in the formation of acetoacetate and acetyl-CoA. This reaction (step Vb as shown in Figure 1 ) is schematically illustrated in Figure 14.

Thus, the present invention also relates to a method for producing isobutene from acetoacetyl-CoA in which acetoacetyl-CoA is first converted into acetoacetate which is then converted into acetone which is then condensed with acetyl-CoA into 3- hydroxyisovalerate (HIV) which is then converted into 3-methylcrotonic acid as described herein above. Further, said 3-methylcrotonic acid is then converted into isobutene as described herein above.

As mentioned, in one aspect, the CoA thioester of acetoacetyl-CoA is hydrolyzed to result in acetoacetate. According to this aspect of the present invention, the enzymatic conversion of acetoacetyl-CoA into acetoacetate is achieved by preferably making use of an acetoacetyl-CoA hydrolase (EC 3.1.2.1 1 ) which naturally catalyzes this reaction.

As mentioned, in another, more preferred, possibility, the CoA group of acetoacetyl- CoA is transferred on acetate, resulting in the formation of acetoacetate and acetyl- CoA. According to this possibility of the present invention, the enzymatic conversion of acetoacetyl-CoA into acetoacetate is achieved by preferably making use of an enzyme which is capable of transferring the CoA group of acetoacetyl-CoA on acetate.

Preferably, such an enzyme capable of transferring the CoA group of acetoacetyl- CoA on acetate belongs to the family of CoA transferases (EC 2.8.3.-).

Thus, the present invention relates to a method for the enzymatic conversion of acetoacetyl-CoA into acetoacetate by making use of an enzyme capable of transferring the CoA group of acetoacetyl-CoA on acetate, preferably a CoA transferase (EC 2.8.3.-). A preferred example of an enzyme catalysing the conversion of acetoacetyl-CoA into acetoacetate which can be employed in the method of the present invention is an enzyme classified as an acetate CoA transferase (EC 2.8.3.8). The enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid: step VI as shown in Figure 1

The 3-methylcrotonic acid can be provided by another possible route which is described in the following.

Thus, in another embodiment, the 3-methylcrotonic acid which is converted into isobutene may itself be provided by another enzymatic reaction, namely the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid. The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid (step VI as shown in Figure 1) is schematically illustrated in Figure 15.

The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid can, e.g., be achieved in different ways, e.g., by three alternative enzymatic routes described in the following and as shown in Figure 1 (step Via, step VIb or step Vic as shown in Figure 1).

Thus, the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid may be achieved by

(a) a single enzymatic reaction in which 3-methylcrotonyl-CoA is directly converted into 3-methylcrotonic acid, preferably by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18) (step Via as shown in Figure 1);

(b) a single enzymatic reaction in which 3-methylcrotonyl-CoA is directly converted into 3-methylcrotonic acid, preferably by making use of a thioester hydrolase (EC 3.1.2.-), preferably an acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20) (step VIb as shown in Figure 1); or

(c) two enzymatic steps comprising

(i) first enzymatically converting 3-methylcrotonyl-CoA into 3- methylcrotonyl phosphate; and

(ii) then enzymatically converting the thus obtained 3-methylcrotonyl phosphate into said 3-methylcrotonic acid (step Vic as shown in Figure 1 )·

As regards (c), i.e., the enzymatic conversion of 3-methylcrotonyl-CoA into 3- methylcrotonic acid which is achieved by two enzymatic steps comprising (i) first enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and (ii) then enzymatically converting the thus obtained 3-methylcrotonyl phosphate into said 3-methylcrotonic acid, the corresponding reaction is schematically shown in Figure 16.

The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).

The conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase. Such enzymes use a carboxy group as acceptor. Thus, the conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-). In a preferred embodiment, the conversion of 3-methylcrotonyl phosphate into 3- methylcrotonic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty- acid kinase (EC 2.7.2.14).

As mentioned above, the conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid can also be achieved by two alternative conversions wherein 3-methylcrotonyl- CoA is directly converted into 3-methylcrotonic acid.

Preferably, in one embodiment, 3-methylcrotonyl-CoA is directly converted into 3- methylcrotonic acid by hydrolyzing the thioester bond of 3-methylcrotonyl-CoA into 3- methylcrotonic acid by making use of an enzyme which belongs to the family of thioester hydrolases (in the following referred to as thioesterases (EC 3.1 .2.-)). This reaction is schematically shown in Figure 17.

Examples for preferred thioester hydrolases (EC 3.1 .2.-) are an acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) and an acyl-CoA hydrolase (EC 3.1.2.20) (step Vlb as shown in Figure 1 ). In an alternative embodiment, 3-methylcrotonyl-CoA is directly converted into 3- methylcrotonic acid, preferably by making use of an enzyme which belongs to the family of CoA-transferases (EC 2.8.3.-). This reaction is schematically shown in Figure 18.

Examples for preferred CoA transferases (EC 2.8.3.-) are a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) and a succinyl- CoA:acetate CoA-transferase (EC 2.8.3.18) (step Via as shown in Figure 1).

Thioesterases (TEs; also referred to as thioester hydrolases) are enzymes which are classified as EC 3.1.2. Presently thioesterases are classified as EC 3.1.2.1 through EC 3.1.2.30 while TEs which are not yet classified/unclassified are grouped as enzymes belonging to EC 3.1.2.-. Cantu et al. (Protein Science 19 (2010), 1281- 1295) describe that there are 23 families of thioesterases which are unrelated to each other as regards the primary structure. However, it is assumed that all members of the same family have essentially the same tertiary structure. Thioesterases hydrolyze the thioester bond between a carbonyl group and a sulfur atom.

In a preferred embodiment, a thioesterase employed in a method according to the present invention for converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid is selected from the group consisting of:

acetyl-CoA hydrolase (EC 3.1.2.1 );

palmitoyl-CoA hydrolase (EC 3.1.2.2);

3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4);

oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);

ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);

ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19); and acyl-CoA hydrolase (EC 3.1.2.20).

As described above, the direct conversion of 3-methylcrotonyl-CoA into 3- methylcrotonic acid can also be achieved by making use of an enzyme which is classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group of 3-methylcrotonyl-CoA to a carboxylic acid.

CoA-transferases are found in organisms from all lines of descent. Most of the CoA- transferases belong to two well-known enzyme families (referred to in the following as families I and II) and there exists a third family which had been identified in anaerobic metabolic pathways of bacteria. A review describing the different families can be found in Heider (FEBS Letters 509 (2001 ), 345-349).

Family I contains, e.g., the following CoA-transferases:

For 3-oxo acids: enzymes classified in EC 2.8.3.5 or EC 2.8.3.6;

For short chain fatty acids: enzymes classified in EC 2.8.3.8 or EC 2.8.3.9;

For succinate: succinyl-CoA:acetate CoA-transferases, i.e. enzymes classified in EC

2.8.3.18 (see also Mullins et al., Biochemistry 51 (2012), 8422-34; Mullins et al., J.

Bacteriol. 190 (2006), 4933-4940).

Most enzymes of family I naturally use succinyl-CoA or acetyl-CoA as CoA donors.

These enzymes contain two dissimilar subunits in different aggregation states. Two conserved amino acid sequence motives have been identified:

Prosites entry PS01273 (http://prosite.expasy.org/cgi-bin/prosite/prosite-search- ac?PDOC00980)

COA_TRANSF_1 , PS01273; Coenzyme A transferases signature 1 (PATTERN) Consensus pattern:

[DN]-[GN]-x(2)-[LIVMFA](3)-G-G-F-x(3)-G-x-P

and

Prosites entries PS01273 (http://prosite.expasy.org/cgi-bin/prosite/prosite-search- ac?PDOC00980)

COA_TRANSF_2, PS01274; Coenzyme A transferases signature 2 (PATTERN)

Consensus pattern:

[LF]-[HQ]-S-E-N-G-[LIVF](2)-[GA]

E (glutamic acid) is an active site residue.

The family II of CoA-transferases consists of the homodimeric a-subunits of citrate lyase (EC 2.8.3.10) and citramalate lyase (EC 2.8.3.1 1 ). These enzymes catalyse the transfer of acyl carrier protein (ACP) which contains a covalently bound CoA- derivative. It was shown that such enzymes also accept free CoA-thioester in vitro, such as acetyl-CoA, propionyl-CoA, butyryl-CoA in the case of citrate lyase (Dimroth et al., Eur. J. Biochem. 80 (1977), 479-488) and acetyl-CoA and succinyl-CoA in the case of citramalate lyase (Dimroth et al., Eur. J. Biochem. 80 (1977), 469-477).

According to Heider (loc. cit.) family III of CoA-transferases consists of formyl-CoA: oxalate CoA-transferase, succinyl-CoA:(R)-benzylsuccinate CoA-transferase, (E)- cinnamoyl-CoA:(R)-phenyllactate CoA-transferase and butyrobetainyl-CoA:(R)- carnitine CoA-transferase. A further member of family III is succinyl-CoA:L-malate CoA-transferase whose function in autrophic CO 2 fixation of Chloroflexus aurantiacus is to activate L-malate to its CoA thioester with succinyl-CoA as the CoA donor (Friedman S. et al. J. Bacteriol. 188 (2006), 2646-2655). The amino acid sequences of the CoA-tranferase of this family show only a low degree of sequence identity to those of families I and II. These CoA-transferases occur in prokaryotes and eukaryotes.

In a preferred embodiment the CoA-transferase employed in a method according to the present invention is a CoA-transferase which belongs to family I or II as described herein-above.

Preferably, the CoA-transferase employed in a method according to the present invention for the direct conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid is selected from the group consisting of:

propionate:acetate-CoA transferase (EC 2.8.3.1 );

acetate CoA-transferase (EC 2.8.3.8); and

butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

The enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid: an alternative route to the above-described step VI

In another preferred embodiment, the conversion of 3-methylcrotonyl-CoA into 3- methylcrotonic acid is achieved by an alternative route wherein 3-methylcrotonyl-CoA is first enzymatically converted into 3-methylbutyryl-CoA which is then enzymatically converted into 3-methylbutyric acid which is then ultimately converted into 3- methylcrotonic acid. This alternative conversion of 3-methylcrotonyl-CoA into 3- methylcrotonic acid via 3-methylbutyryl-CoA and 3-methylbutyric acid is schematically illustrated in Figure 19.

Accordingly, the present invention relates to a method for producing isobutene from 3-methylcrotonyl-CoA in which 3-methylcrotonyl-CoA is first enzymatically converted into 3-methylbutyryl-CoA which is then enzymatically converted into 3-methylbutyric acid which is then converted into 3-methylcrotonic acid which is then further converted into isobutene as described herein above.

The first enzymatic conversion, i.e., the conversion of 3-methylcrotonyl-CoA into 3- methylbutyryl-CoA, is a desaturation reaction, i.e., reduction of the double bond C=C of 3-methylcrotonyl-CoA into 3-methylbutyryl-CoA. The enzymatic conversion of 3- methylcrotonyl-CoA into 3-methylbutyryl-CoA, i.e. the reduction of the double bond in 3-methylcrotonyl-CoA, can, for example, be achieved by employing an enzyme classified as EC 1.3._._. Enzymes classified as EC 1.3._._ are oxidoreductases acting on the CH-CH group of a donor molecule. This subclass contains enzymes that reversibly catalyze the conversion of a carbon-carbon single bond to a carbon- carbon double bond between two carbon atoms. Sub-classes of EC 1.3 are classified depending on the acceptor. In one preferred embodiment the enzyme is an enzyme which is classified as EC 1.3._._ and which uses NADH or NADPH as co-factor. In one particularly preferred embodiment the enzyme is an enzyme which uses NADPH as a co-factor. In a preferred embodiment the enzyme is selected from the group consisting of:

- acyl-CoA dehydrogenase (NADP+) (EC 1.3.1.8);

- enoyl-[acyl-carrier-protein] reductase (NADPH, Si-specific) (EC 1.3.1.10);

- cis-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.37);

- trans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38);

- enoyl-[acyl-carrier-protein] reductase (NADPH, Re-specific) (EC 1.3.1.39); and

- crotonyl-CoA reductase (EC .3.1.86).

The second enzymatic conversion, i.e., the conversion of 3-methylbutyryl-CoA into 3-methylbutyric acid, can be achieved by different enzymatic conversions. One possibility is the direct conversion via a hydrolysis reaction. Another possibility is the direct conversion via a reaction catalyzed by a CoA-transferase and a third possibility is a two-step conversion via 3-methylbutyryl phosphate.

Thus, according to the present invention, the enzymatic conversion of 3- methylbutyryl-CoA into 3-methylbutyric acid is achieved by

(a) a single enzymatic reaction in which 3-methylbutyryl-CoA is directly converted into 3-methylbutyric acid, preferably by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18);

(b) a single enzymatic reaction in which 3-methylbutyryl-CoA is directly converted into 3-methylbutyric acid, preferably by making use of a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20); or

(c) two enzymatic steps comprising

(i) first enzymatically converting 3-methylbutyryl-CoA into 3-methylbutyryl phosphate; and

(ii) then enzymatically converting the thus obtained 3-methylbutyryl phosphate into said 3-methylbutyric acid.

As regards the enzyme capable of converting 3-methylbutyryl-CoA into 3- methylbutyryl phosphate and the enzyme capable of converting 3-methylbutyryl phosphate into said 3-methylbutyric acid, the same applies as has been set forth above in connection with the enzymatic conversion of step Via, step VIb and step Vic according to the invention.

The third enzymatic conversion, i.e., the conversion of 3-methylbutyric acid into 3- methylcrotonic acid can, e.g., be achieved by a 2-enoate reductase (EC 1.3.1.31 ).

The enzymatic conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl- CoA: step VII as shown in Figure 1

The 3-methylcrotonyl-CoA which is converted according to the method of the present invention into 3-methylcrotonic acid according to any of the above described methods (and further converted according to the method of the present invention into isobutene according to any of the above described methods) may itself be provided by an enzymatic reaction, namely the enzymatic conversion of 3-methylglutaconyl- CoA into 3-methylcrotonyl-CoA. The conversion of 3-methylglutaconyl-CoA into 3- methylcrotonyl-CoA is schematically illustrated in Figure 20.

Accordingly, the present invention relates to a method for producing isobutene from 3-methylglutaconyl-CoA in which 3-methylglutaconyl-CoA is first converted into 3- methylcrotonyl-CoA which is then further converted into 3-methylcrotonic acid which is then further converted into isobutene as described herein above.

The conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA may be catalyzed by different enzymes. According to the present invention, the conversion of 3-methylglutaconyl-CoA into said 3-methylcrotonyl-CoA preferably makes use of (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1 .4); or (ii) a geranoyl-CoA carboxylase (EC 6.4.1.5) (as shown in step VII of Figure 1 ).

In another preferred embodiment the conversion of 3-methylglutaconyl-CoA via decarboxylation into 3-methylcrotonyl-CoA is catalyzed by a 3-methylglutaconyl-CoA decarboxylase, e.g. a 3-methylglutaconyl-CoA decarboxylase of Myxococcus xanthus encoded by the liuB gene. This gene codes for an enzyme having the two subunits AibA and AibB (Li et al., Angew. Chem. Int. Ed. 52 (2013), 1304-1308).

The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3- methylglutaconyl-CoA: step VIII as shown in Figure 1

The 3-methylglutaconyl-CoA which is converted into 3-methylcrotonyl-CoA may itself be provided by an enzymatic reaction, namely the enzymatic conversion of 3- hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA; see Figure 21.

Accordingly, the present invention also relates to a method for producing isobutene from 3-hydroxy-3-methylglutaryl-CoA in which 3-hydroxy-3-methylglutaryl-CoA is first converted into 3-methylglutaconyl-CoA which is then converted into 3-methylcrotonyl- CoA which is then further converted into 3-methylcrotonic acid which is then further converted into isobutene as described herein above.

According to the present invention, the enzymatic conversion of 3-hydroxy-3- methylglutaryl-CoA into 3-methylglutaconyl-CoA is an enzymatic dehydration reaction which occurs naturally, and which is catalyzed, e.g., by enzymes classified as 3- methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18). Accordingly, the enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA preferably makes use of a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18) (as shown in step VIII of Figure 1).

The conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA can also be achieved by making use of a 3-hydroxy-3-methylglutaryl-coenzyme A dehydratase activity which has been identified, e.g., in Myxococcus xanthus and which is encoded by the liuC gene (Li et al., Angew. Chem. Int. Ed. 52 (2013), 1304- 1308). The 3-hydroxy-3-methylglutaryl-coenzyme A dehydratase derived from Myxococcus xanthus has the Uniprot Accession number Q1 D5Y4.

The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3- methylglutaconyl-CoA can also be achieved by making use of a 3-hydroxyacyl-CoA dehydratase or an enoyl-CoA hydratase. 3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases catalyze the same reaction while the name of one of these enzymes denotes one direction of the corresponding reaction while the other name denotes the reverse reaction. As the reaction is reversible, both enzyme names can be used.

3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases belong to enzymes classified as EC 4.2.1.-.

The enzymatic conversion of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl- CoA: step IX as shown in Figure 1

The 3-hydroxy-3-methylglutaryl-CoA which is converted into 3-methylglutaconyl-CoA may itself be provided by an enzymatic reaction, namely the enzymatic condensation of acetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA; see Figure 22.

Accordingly, the present invention also relates to a method for producing isobutene from acetoacetyl-CoA and acetyl-CoA in which acetoacetyl-CoA and acetyl-CoA are first condensed into 3-hydroxy-3-methylglutaryl-CoA which is then converted into 3- methylglutaconyl-CoA which is then converted into 3-methylcrotonyl-CoA which is then further converted into 3-methylcrotonic acid which is then further converted into isobutene as described herein above.

According to the present invention, the enzymatic condensation of acetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA makes preferably use of a 3- hydroxy-3-methylglutaryl-CoA synthase (see step IX of Figure 1 ).

The condensation of acetyl-CoA and acetoacetyl-CoA is a reaction which is naturally catalyzed by the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (also referred to as HMG-CoA synthase). Thus, preferably, the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA makes use of a 3-hydroxy-3- methylglutaryl-CoA synthase (also referred to as HMG-CoA synthase). HMG-CoA synthases are classified in EC 2.3.3.10 (formerly, HMG-CoA synthase has been classified as EC 4.1 .3.5 but has been transferred to EC 2.3.3.10). The term "HMG- CoA synthase" refers to any enzyme which is able to catalyze the reaction where acetyl-CoA condenses with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) (see Figure 22). HMG-CoA synthase is part of the mevalonate pathway. Two pathways have been identified for the synthesis of isopentenyl pyrophosphate (IPP), i.e. the mevalonate pathway and the glyceraldehyde 3-phosphate-pyruvate pathway. HMG-CoA synthase catalyzes the biological Claisen condensation of acetyl-CoA with acetoacetyl-CoA and is a member of a superfamily of acyl- condensing enzymes that includes beta-ketothiolases, fatty acid synthases (beta- ketoacyl carrier protein synthase) and polyketide synthases.

The enzymatic conversion of acetyl-CoA into acetoacetyl-CoA: steps XIII, XIV and XV as shown in Figure 1

The acetoacetyl-CoA which is either converted into 3-hydroxy-3-methylglutaryl-CoA or which is converted into acetoacetate may itself be provided by an enzymatic reaction, namely the enzymatic conversion of acetyl-CoA into acetoacetyl-CoA.

According to the present invention, the conversion of acetyl-CoA into said acetoacetyl-CoA can be achieved by different routes. One possibility is to first convert acetyl-CoA into malonyl-CoA (step XIV as shown in Figure 1 ) and then to further condense said malonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV as shown in Figure 1 ). Another possibility is to directly condense in a single enzymatic reaction two molecules of acetyl-CoA into acetoacetyl-CoA (step XIII as shown in Figure 1 ). These reactions are schematically shown in Figure 23 (step XIII), Figure 24 (step XIV) and Figure 25 (step XV), respectively. Thus, the present invention also relates to a method for producing isobutene from acetyl-CoA in which acetyl-CoA is first converted into acetoacetyl-CoA by any of the above-mentioned routes which is then condensed with acetyl-CoA into 3-hydroxy-3- methylglutaryl-CoA which is then converted into 3-methylglutaconyl-CoA which is then converted into 3-methylcrotonyl-CoA which is then further converted into 3- methylcrotonic acid which is then further converted into isobutene as described herein above.

Moreover, the present invention also relates to a method for producing isobutene from acetyl-CoA in which acetyl-CoA is first converted into acetoacetyl-CoA by any of the above-mentioned routes by any of the above-mentioned routes which is then converted into acetoacetate which is then converted into acetone which is then condensed with acetyl-CoA into 3-hydroxyisovalerate (HIV) which is then converted into 3-methylcrotonic acid as described herein above. Further, said 3-methylcrotonic acid is then further converted into isobutene as described herein above.

According to the present invention, the enzymatic conversion of acetyl-CoA into malonyl-CoA preferably makes use of an acetyl-CoA carboxylase (EC 6.4.1.2) (step XIV as shown in Figure 1 ). This naturally occurring reaction fixes CO 2 on acetyl-CoA utilizing ATP resulting in malonyl-CoA.

Moreover, according to the present invention, the enzymatic condensation of malonyl-CoA and acetyl-CoA into said acetoacetyl-CoA preferably makes use of an acetoacetyl-CoA synthase (EC 2.3.1.194) (step XV as shown in Figure 1 ). This is a natural occurring reaction and condenses malonyl-CoA and acetyl-CoA in a decarboxylation reaction.

Alternatively, the enzymatic conversion of acetyl-CoA into said acetoacetyl-CoA consists of a single enzymatic reaction in which acetyl-CoA is directly converted into acetoacetyl-CoA by the enzymatic condensation of two molecules of acetyl-CoA into acetoacetyl-CoA. Preferably, the enzymatic conversion of acetyl-CoA into acetoacetyl-CoA is achieved by making use of an acetyl-CoA acetyltransferase (EC 2.3.1.9). This reaction is a naturally occurring reaction. The enzymatic recycling of metabolites occurring in the pathway of the present invention: steps Xa, Xb, XI and XII as shown in Figure 1

The above-described method of the present invention for producing isobutene from acetyl-CoA may be supplemented by one or more of the following reactions as shown in step Xa, step Xb, step XI and step XII of Figure 1.

These steps relate to alternative bioconversions which may occur concomitantly to any of the above-described methods for producing isobutene.

Thus, the present invention relates to any of the above-described methods for producing isobutene from 3-methylcrotonic acid (or from any of the above-described intermediates in the described pathways from acetyl-CoA into isobutene) wherein additionally

a) 3-hydroxyisovalerate (HIV) is enzymatically converted into 3-methylcrotonic acid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA on 3- hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA (step Xa as schematically shown in Figure 27); and/or

b) 3-hydroxyisovalerate (HIV) is enzymatically converted into 3-hydroxyisovaleryl- CoA (step Xb as schematically shown in Figure 28); and/or

c) 3-hydroxyisovaleryl-CoA is enzymatically converted into 3-methylcrotonyl-CoA (step XI as schematically shown in Figure 29); and/or

d) 3-hydroxyisovalerate (HIV) is enzymatically converted into 3- hydroxyisovaleryl-CoA (step XII as schematically shown in Figure 30).

These reactions which which will be described in more detail in the following, may occur concomitantly to any of the above-described methods for producing isobutene are beneficial for several reasons. First, it is known that the hydration of an enoyl- CoA (such as, e.g., 3-methylcrotonyl-CoA) is a favoured reaction in vivo in an aqueous medium. Accordingly, the above reactions represent possibilities which allow to drive the metabolic flux toward the precursor of isobutene, i.e., 3- methylcrotonic acid, even in case the pathway "leaks" into the direction of 3- hydroxyisovalerate (HIV) and/or 3-hydroxyisvaleryl-CoA. Second, the above conversions beneficially involve the conservation of energy into a thioester CoA bond via a transfer of a thioester group. The enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA on 3- hvdroxyisovalerate (HIV) to result in 3-hvdroxyisovaleryl-CoA as shown in step Xa of Figure 26

Thus, in a first aspect, the 3-methylcrotonic acid which is converted into isobutene may be provided by an enzymatic reaction wherein 3-hydroxyisovalerate (HIV) is enzymaticaliy converted into 3-methylcrotonic acid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA to 3-hydroxyisovalerate (HIV) to result in 3- hydroxyisovaleryl-CoA (step Xa as shown in Figure 18). This reaction is schematically illustrated in Figure 27.

Thus, the present invention also relates to a method for producing isobutene from 3- hydroxyisovalerate (HIV) wherein 3-hydroxyisovalerate (HIV) is enzymaticaliy converted into 3-methylcrotonic acid with a concomitant transfer of CoA from 3- methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl- CoA. Further, the thus produced 3-methylcrotonic acid is then enzymaticaliy converted into isobutene as described herein above.

Moreover, the present invention also relates to a method for producing 3- methylcrotonic acid and 3-hydroxyisovaleryl-CoA from 3-hydroxyisovalerate (HIV) and from 3-methylcrotonyl-CoA wherein 3-hydroxyisovalerate (HIV) is enzymaticaliy converted into 3-methylcrotonic acid with a concomitant transfer of CoA from 3- methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl- CoA.

According to the present invention, the conversion of 3-hydroxyisovalerate (HIV) and 3-methylcrotonyl-CoA into 3-methylcrotonic acid and 3-hydroxyisovaleryl-CoA wherein 3-hydroxyisovalerate (HIV) is enzymaticaliy converted into 3-methylcrotonic acid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA on 3- hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA preferably makes use of an enzyme which is classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group of 3-methylcrotonyl-CoA to a carboxylic acid, i.e., 3- hydroxyisovalerate (HIV). CoA-transferases (EC 2.8.3.-) have already been described above. Accordingly, as regards these enzymes, the same applies to the conversion of 3-hydroxyisovalerate (HIV) and 3-methylcrotonyl-CoA into 3-methylcrotonic acid and 3-hydroxyisovaleryl- CoA as has been set forth above.

Preferably, the CoA-transferase employed in a method according to the present invention in the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3- methylcrotonic acid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA is a CoA-transferase selected from the group consisting of:

propionate:acetate-CoA transferase (EC 2.8.3.1 );

acetate CoA-transferase (EC 2.8.3.8); and

butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

The enzymatic conversion of 3-hvdroxyisovalerate (HIV) into 3-hvdroxyisovaleryl- CoA as shown in step Xb of Figure 26

In addition or in the alternative to the above-described methods (step Xa), the 3- hydroxyisovaleryl-CoA may also be provided by an enzymatic conversion of 3- hydroxyisovalerate into said 3-hydroxyisovaleryl-CoA (step Xb as shown in Figure 26). In this reaction, 3-hydroxyisovalerate reacts with an acyl-CoA to result in 3- hydroxyisovaleryl-CoA and an acid. This reaction is schematically illustrated in Figure 27.

Preferably, said acyl-CoA is acetyl-CoA.

Thus, the present invention also relates to a method for producing 3- hydroxyisovaleryl-CoA from 3-hydroxyisovalerate (HIV) wherein 3-hydroxyisovalerate reacts with an acyl-CoA, preferably with acetyl-CoA, to result in 3-hydroxyisovaleryl- CoA and a respective acid.

Preferably, this conversion is achieved by making use of an enzyme which is classified as a CoA-transferase (EC 2.8.3.-). As regards the preferred embodiments of said CoA-transferase (EC 2.8.3.-) in the context of step Xb, the same applies, mutatis mutandis, as has been set forth above with respect to the CoA-transferases (EC 2.8.3.-) in the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3- methylcrotonic acid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA (step Xa as shown in Figure 26).

The enzymatic conversion of 3-hvdroxyisovaleryl-CoA into 3-methylcrotonyl-CoA as shown in step XI of Figure 26

In addition or in the alternative to the above-described methods (step VII), the 3- methylcrotonyl-CoA may be provided by an enzymatic reaction wherein 3- hydroxyisovaleryl-CoA is enzymatically converted into 3-methylcrotonyl-CoA (step XI as shown in Figure 26). This reversible reaction is a dehydration reaction wherein 3- hydroxyisovaleryl-CoA is dehydrated into 3-methylcrotonyl-CoA and is schematically illustrated in Figure 29.

Thus, the present invention also relates to a method for producing isobutene from 3- hydroxyisovaleryl-CoA wherein 3-hydroxyisovaleryl-CoA is first enzymatically converted into 3-methylcrotonyl-CoA wherein 3-methylcrotonyl-CoA is further enzymatically converted into 3-methylcrotonic acid according to any of the above- described methods. Further, the thus produced 3-methylcrotonic acid is then enzymatically converted into isobutene as described herein above.

According to the present invention, the enzymatic conversion of 3-hydroxyisovaleryl- CoA into 3-methylcrotonyl-CoA preferably makes use of

(i) an enoyl-CoA hydratase (EC 4.2.1.17);

(ii) a long-chain-enoyl-CoA hydratase (EC 4.2.1.74);

(iii) a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116);

(iv) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55);

(v) a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59);

(vi) a crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58);

(vii) a 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60);

(viii) a 3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61); or

(ix) a 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18). The enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-hvdroxyisovaleryl- CoA as shown in step XII of Figure 26

In addition or in the alternative to the above-described methods (step Xa or step Xb), the 3-hydroxyisovaleryl-CoA may also be provided by an enzymatic conversion of 3- hydroxyisovalerate (HIV) into said 3-hydroxyisovaleryl-CoA (step XII as shown in Figure 26). This general reaction wherein coenzyme A (CoASH) is fixed is schematically illustrated in Figure 30.

Thus, the present invention also relates to a method for producing isobutene from 3- hydroxyiso valerate (HIV) in which 3-hydroxyisovalerate (HIV) is first converted into 3- hydroxyisovaleryl-CoA wherein 3-hydroxyisovaleryl-CoA is then enzymatically converted into 3-methylcrotonyl-CoA wherein 3-methylcrotonyl-CoA is further enzymatically converted into 3-methylcrotonic acid according to any of the above- described methods. Further, the thus produced 3-methylcrotonic acid is then enzymatically converted into isobutene as described herein above.

According to the present invention, the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA preferably makes use of an enzyme belonging to the family of ligases forming a carbon-sulfur bond (EC 6.2.1.-). The general reaction of the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl- CoA wherein coenzyme A (CoASH) is fixed can be catalyzed by an enzyme belonging to the family of ligases forming a carbon-sulfur bond (EC 6.2.1.-) via two alternative mechanisms. In a first alternative reaction, an acyl-AMP is generated as an intermediate before coenzyme A is fixed as schematically illustrated in Figure 31. In a second alternative reaction, an acyl phosphate is generated as an intermediate before coenzyme A is fixed as schematically illustrated in Figure 32.

Enzymes which belong to the family of ligases forming a carbon-sulfur bond (EC 6.2.1.-) which are capable of enzymatically converting 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA wherein an acyl-AMP intermediate (i.e., the acyl adenylate intermediate 3-hydroxyisovaleryl-adenosine monophosphate) is generated before coenzyme A is fixed coenzyme A (CoASH) share common structural motifs which are referenced in the InterPro (lnterPro44.0; Release September 25, 2013) as InterPro IPR020845, AMP-binding, conserved site

(http://www.ebi.ac.uk/interpro/entry/IPR020845) and IPR000873

(http://www.ebi.ac.uk/interpro/entry/IPR000873). The accession number for these enzymes in the Pfam database is PF00501.

As regards the first alternative reaction (wherein an acyl-AMP is generated as an intermediate before coenzyme A is fixed as schematically illustrated in Figure 23), examples of enzymes which belong to the above family of ligases forming a carbon- sulfur bond (EC 6.2.1.-) which are capable of enzymatically converting 3- hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA wherein an acyl-AMP intermediate (i.e., the acyl adenylate intermediate 3-hydroxyisovaleryl-adenosine monophosphate) is generated before coenzyme A is fixed coenzyme A (CoASH) and which may be used in the method for producing 3-hydroxyisovaleryl-CoA from 3- hydroxyisovalerate (HIV) are summarized in the following Table A:

Table A: CoA ligases (EC 6.2.1 .-) capable of enzymatically converting 3- hydroxyiso valerate (HIV) into 3-hydroxyisovaleryl-CoA involving an acyl-adenylate as an intermediate

Enzyme name EC number

Acetate-CoA ligase 6.2.1 .1

Butyrate-CoA ligase 6.2.1.2

Long chain fatty-acid-CoA ligase 6.2.1.3

4-coumarate-CoA ligase 6.2.1.12

Arachidonate-CoA ligase 6.2.1.15

Propionate-CoA ligase 6.2.1 .17

Phytanate-CoA ligase 6.2.1 .24

o-succinylbenzoate-CoA ligase 6.2.1.26

3-alpha,7-alpha-dihydroxy-5-beta-

6.2.1.28

cholestanate-CoA ligase

2-furoate-CoA ligase 6.2.1 .31

4-chlorobenzoate-CoA ligase 6.2.1 .33

3-hydroxybenzoate-CoA ligase 6.2.1.37

4-hydroxybutyrate-CoA ligase 6.2.1 .40

3-oxocholest-4-en-26-oate-CoA ligase 6.2.1 .42

3-(methylthio)propionyl-CoA ligase 6.2.1.44

Cholate-CoA ligase 6.2.1.7

Oxalate-CoA ligase 6.2.1 .8

Biotin-CoA ligase 6.2.1 .11

6-carboxyhexanoate-CoA ligase 6.2.1 .14 Acetoacetate~CoA ligase 6.2.1 .16

Dicarboxylate-CoA ligase 6.2.1.23

Benzoate-CoA ligase 6.2.1.25

4-hydroxybenzoate-CoA ligase 6.2.1.27

Phenylacetate-CoA ligase 6.2.1.30

Anthranilate-CoA ligase 6.2.1.32

3-hydroxypropionyl-CoA synthase 6.2.1.36

(2,2, 3-tri methyl-5-oxocyclo pent-3-

6.2.1.38

enyl)acetyl-CoA synthase

3-((3aS,4S,7aS)-7a-methyl-1 ,5-dioxo- octahydro-1 H-inden-4-yl)propanoate- 6.2.1.41

CoA ligase

2-hydroxy-7-methoxy-5-methyl-1 -

6.2.1.43

naphthoate--CoA ligase

Malonate-CoA ligase 6.2.1.n3

In a preferred embodiment, the enzymatic conversion of 3-hydroxyiso valerate (HIV) into 3-hydroxyiso valeryl-CoA via an acyl adenylate intermediate can, e.g., be achieved by the use of a butanoate:CoA ligase (AMP forming) (EC 6.2.1 .2).

As regards the second alternative reaction (wherein an acyl phosphate is generated as an intermediate before coenzyme A is fixed as schematically illustrated in Figure 32), examples of enzymes which belong to the above family of ligases forming a carbon-sulfur bond (EC 6.2.1.-) which are capable of enzymatically converting 3- hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA wherein an acyl phosphate intermediate (i.e., the acyl phosphate intermediate 3-hydroxyisovaleryl phosphate) is generated before coenzyme A is fixed coenzyme A (CoASH) and which may be used in the method for producing 3-hydroxyisovaleryl-CoA from 3-hydroxyiso valerate (HIV) are summarized in the following Table B.

Table B: CoA ligases (EC 6.2.1.-) capable of enzymatically converting 3- hydroxyiso valerate (HIV) into 3-hydroxyisovaleryl-CoA involving an acyl phosphate as an intermediate

Enzyme name EC number

Succinate-CoA ligase (GDP-forming) 6.2.1.4

Glutarate-CoA ligase 6.2.1.6

Acid-CoA ligase (GDP-forming) 6.2.1 .10

Citrate-CoA ligase 6.2.1.18

enzyme name EC number

Succinate-CoA ligase (ADP-forming) 6.2.1.5

Malate-CoA ligase 6.2.1 .9

Acetate-CoA ligase (ADP-forming) 6.2.1 .13 The alternative route for the enzymatic conversion from acetyl-CoA into isobutene via 3-methyl-3-butenoyl-CoA and 3-methyl-3-butenoic acid

In an alternative to the above, the present invention also relates to a method for the production of isobutene via an alternative route as also shown in Figure 1 wherein isobutene is produced by the enzymatic conversion of 3-methyl-3-butenoic acid into isobutene. Thus, the present invention provides a method for the production of isobutene comprising the enzymatic conversion of 3-methyl-3-butenoic acid into isobutene. Preferably, the enzymatic conversion of 3-methyl-3-butenoic acid into isobutene is achieved by making use of an 3-methyl-3-butenoic acid decarboxylase.

In accordance with this alternative route, the present invention not only relates to a method for the production of isobutene from 3-methyl-3-butenoic acid. Rather, as will be outlined in more detail further below, this conversion is preferably embedded in a pathway for the production of isobutene starting from acetyl-CoA which is a central component and an important key molecule in metabolism used in many biochemical reactions.

Therefore, the present invention also relates to a pathway starting from acetyl-CoA wherein two acetyl-CoA molecules are enzymatically condensed into acetoacetyl- CoA. Alternatively, acetyl-CoA is enzymatically converted into malonyl-CoA which may then be converted into said acetoacetyl-CoA by the enzymatic condensation of malonyl-CoA and acetyl-CoA into said acetoacetyl-CoA.

Further, the thus produced acetoacetyl-CoA can enzymatically be converted into 3- methyl-3-butenoic acid (which is then ultimately converted into isobutene) via the following briefly summarized pathway.

In this pathway, the thus produced acetoacetyl-CoA can further enzymatically be converted into 3-hydroxy-3-methylglutaryl-CoA. Moreover, the thus produced 3- hydroxy-3-methylglutaryl-CoA can further enzymatically be converted into 3- methylglutaconyl-CoA. Further, the thus produced 3-methylglutaconyl-CoA can enzymatically be converted into 3-methyl-3-butenoyl-CoA. Further, the thus produced 3-methyl-3-butenoyl-CoA can further be converted in a subsequent enzymatic reaction into 3-methyl-3-butenoic acid (which can then ultimately be converted into isobutene as described above and further below). g2

The enzymatic conversion of 3-methyl-3-butenoic acid into isobutene: step XVI as shown in Figure 1

According to the present invention, the enzymatic conversion of 3-methyl-3-butenoic acid into isobutene can be achieved by a decarboxylation. "Decarboxylation" is generally a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO 2 ); see Figure 33.

The enzymatic conversion of 3-methyl-3-butenoic acid into isobutene can preferably be achieved by making use of an 3-methyl-3-butenoic acid decarboxylase. In accordance with the present invention, an 3-methyl-3-butenoic acid decarboxylase is an enzyme which is capable of converting 3-methyl-3-butenoic acid into isobutene. In preferred embodiments, the 3-methyl-3-butenoic acid decarboxylase is selected from the group consisting of:

(i) an FMN-dependent decarboxylase associated with an FMN prenyl transferase; or

(ii) an aconitate decarboxylase (EC 4.1.1.6); or

(iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or

(iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or

(v) a protocatechuate (PCA) decarboxylase (EC 4.1.1 .63).

In other preferred embodiments, the 3-methyl-3-butenoic acid decarboxylase is selected from the group consisting of: 6-methylsalicylate decarboxylase (EC 4.1 .1.52), 2-oxo-3-hexenedioate decarboxylase (EC 4.1 .1 .77) and 5-oxopent-3-ene- 1 ,2,5-tricarboxylate decarboxylase (EC 4.1.1.68).

The enzymatic conversion of 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid: steps XVHa, XVIlb or XVIIc as shown in Figure 1

The 3-methyl-3-butenoic acid may itself be provided by an enzymatic reaction, namely the enzymatic conversion of 3-methyl-3-butenoyl-CoA into 3-methyl-3- butenoic acid; see Figure 34.

Accordingly, the present invention relates to a method for producing isobutene from 3-methyl-3-butenoyl-CoA in which 3-methyl-3-butenoyl-CoA is first converted into 3- methyl-3-butenoic acid which is then further converted into isobutene as described herein above.

According to the present invention, the conversion of 3-methyl-3-butenoyl-CoA into 3- methyl-3-butenoic acid can, e.g., be achieved by three different alternative enzymatic routes, i.e., by:

(a) a single enzymatic reaction (see Figure 35) in which 3-methyl-3-butenoyl-CoA is directly converted into 3-methyl-3-butenoic acid, preferably by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18);

(b) a single enzymatic reaction(see Figure 36) in which 3-methyl-3-butenoyl-CoA is directly converted into 3-methyl-3-butenoic acid, preferably by making use of a thioester hydrolase (EC 3.1 .2.-), preferably acetyl-CoA hydrolase (EC 3.1 .2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1 .2.20); or

(c) two enzymatic steps (see Figure 37) comprising

(i) first enzymatically converting 3-methyl-3-butenoyl-CoA into 3-methyl-3- butenoyl phosphate, preferably by making use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1 .8); and

(ii) then enzymatically converting the thus obtained 3-methyl-3-butenoyl phosphate into said 3-methyl-3-butenoic acid, preferably by making use of a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).

The enzymatic conversion of 3-methylglutaconyl-CoA into 3-methyl-3-butenoyl- CoA: step XVIH as shown in Figure 1

The 3-methyl-3-butenoyl-CoA may itself be provided by an enzymatic reaction, namely the enzymatic conversion of 3-methylglutaconyl-CoA into 3-methyl-3- butenoyl-CoA; see Figure 38.

Accordingly, the present invention relates to a method for producing isobutene from 3-methyl-3-butenoyl-CoA in which 3-methylglutaconyl-CoA is first converted into 3- methyl-3-butenoyl-CoA which is then further converted into 3-methyl-3-butenoic acid which is then further converted into isobutene as described herein above.

Moreover, the present invention relates to a method for producing 3-methyl-3- butenoyl-CoA by converting 3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA. According to the present invention, the conversion of 3-methylglutaconyl-CoA into 3- methyl-3-butenoyl-CoA can preferably be achieved by making use of

(a) (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) a geranoyl-CoA carboxylase (EC 6.4.1.5),

(b) an N-terminal domain of CurF from Lynbya majuscula multifunctional protein or a 3-methylglutaconyl-CoA decarboxylase, preferably a 3-methylglutaconyl- CoA decarboxylase of Myxococcus xanthus encoded by the liuB gene; or

(c) an enzyme of the 4-oxalocrotonate decarboxylase family.

As regards the aforementioned embodiments, for the methylcrotonyl-CoA carboxylase (EC 6.4.1.4), the geranoyl-CoA carboxylase (EC 6.4.1.5) and the 3- methylglutaconyl-CoA decarboxylase, preferably the 3-methylglutaconyl-CoA decarboxylase of Myxococcus xanthus encoded by the liuB gene, the same applies as has been set forth above in connection with the other methods of the present invention.

In a preferred embodiment the conversion of 3-methylglutaconyl-CoA via decarboxylation into 3-methyl-3-butenoyl-CoA is catalyzed by an N-terminal domain of CurF from Lynbya majuscula multifunctional protein. The N-terminal domain of CurF from Lynbya majuscula multifunctional protein is a domain of a polyketide synthase (PKS)/non ribosomale peptide synthase (NRPS) of the CurF multifunctional protein from Lyngbya majuscula. This N-terminal domain of CurF has been classified as a protein belonging to the crotonase superfamily by studying the crystal structure and it naturally catalyzes the decarboxylation of 3-methylglutaconyl-ACP (Acyl Carrier Protein) into 3-methyl-crotonyl-ACP. ACP is similar to CoA as both molecules have a phosphopantetheine moiety in common (as shown in Figure 39). Moreover, both ACP and CoA can form a thioester with a biological acid (J. Biol. Chem. 289: 35957-35963 (2007) and Chemistry & Biology 11 :817-833 (2004)).

In another preferred embodiment the conversion of 3-methylglutaconyl-CoA via decarboxylation into 3-methyl-3-butenoyl-CoA is catalyzed by an enzyme of the 4- oxalocrotonate decarboxylase family (EC 4.1.1.77).

The enzymatic conversion of 3-hvdroxy-3-methylglutaryl-CoA into 3- methylglutaconyl-CoA: step VIII as shown in Figure 1

The 3-methylglutaconyl-CoA which can be converted into 3-methyl-3-butenoyl-CoA according to any of the above described methods may itself be provided by an enzymatic reaction, namely the enzymatic conversion of 3-hydroxy-3-methylglutaryl- CoA into 3-methylglutaconyl-CoA.

Accordingly, the present invention also relates to a method for producing isobutene from 3-hydroxy-3-methylglutaryl-CoA in which 3-hydroxy-3-methylglutaryl-CoA is first converted into 3-methylglutaconyl-CoA which is then converted into 3-methyl-3- butenoyl-CoA which is then further converted into 3-methyl-3-butenoic acid which is then further converted into isobutene as described herein above.

According to the present invention, the enzymatic conversion of 3-hydroxy-3- methylglutaryl-CoA into 3-methylglutaconyl-CoA is an enzymatic dehydration reaction which occurs naturally, and which is catalyzed, e.g., by enzymes classified as 3- methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18). Accordingly, the enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA preferably makes use of a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18).

As regards the afore-mentioned embodiment, for the enzymes classified as 3- methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18), the same applies as has been set forth above in connection with the other methods of the present invention. The enzymatic conversion of acetoacetyl-CoA into 3-hvdroxy-3-methylglutaryl- CoA: step IX as shown in Figure 1

The 3-hydroxy-3-methylglutaryl-CoA may itself be provided by an enzymatic reaction, namely the enzymatic condensation of acetoacetyl-CoA and acetyl-CoA into 3- hydroxy-3-methylglutaryl-CoA which has already been described in detail above.

Accordingly, the present invention also relates to a method for producing isobutene from acetoacetyl-CoA and acetyl-CoA in which acetoacetyl-CoA and acetyl-CoA are first condensed into 3-hydroxy-3-methylglutaryl-CoA which is then converted into 3- methylglutaconyl-CoA which is then converted into 3-methyl-3-butenoyl-CoA which is then further converted into 3-methyl-3-butenoic acid which is then further converted into isobutene as described herein above.

The enzymatic conversion of acetyl-CoA into acetoacetyl-CoA: step XIII, step XIV and step XV as shown in Figure 1

The acetoacetyl-CoA may itself be provided by an enzymatic reaction, namely the enzymatic conversion of acetyl-CoA into acetoacetyl-CoA via several different routes which have already been described in detail above.

Thus, the present invention also relates to a method for producing isobutene from acetyl-CoA in which acetyl-CoA is first converted into acetoacetyl-CoA by any of the above-mentioned routes which is then condensed with acetyl-CoA into 3-hydroxy-3- methylglutaryl-CoA which is then converted into 3-methylglutaconyl-CoA which is then converted into 3-methyl-3-butenoyl-CoA which is then further converted into 3- methyl-3-butenoic acid which is then further converted into isobutene as described herein above.

A method according to the present invention may be carried out in vitro or in vivo. An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction. Thus, in vitro preferably means in a cell-free system. The term "in vitro" in one embodiment means in the presence of isolated enzymes (or enzyme systems optionally comprising possibly required cofactors). In one embodiment, the enzymes employed in the method are used in purified form.

For carrying out the method in vitro the substrates for the reaction and the enzymes are incubated under conditions (buffer, temperature, cosubstrates, cofactors etc.) allowing the enzymes to be active and the enzymatic conversion to occur. The reaction is allowed to proceed for a time sufficient to produce the respective product. The production of the respective products can be measured by methods known in the art, such as gas chromatography possibly linked to mass spectrometry detection. The enzymes may be in any suitable form allowing the enzymatic reaction to take place. They may be purified or partially purified or in the form of crude cellular extracts or partially purified extracts. It is also possible that the enzymes are immobilized on a suitable carrier.

In another embodiment the method according to the invention is carried out in culture, in the presence of an organism, preferably a microorganism, producing the enzymes described above for the conversions of the methods according to the present invention as described herein above. A method which employs a microorganism for carrying out a method according to the invention is referred to as an "in vivo" method. It is possible to use a microorganism which naturally produces the enzymes described above for the conversions of the methods according to the present invention or a microorganism which had been genetically modified so that it expresses (including overexpresses) one or more of such enzymes. Thus, the microorganism can be an engineered microorganism which expresses enzymes described above for the conversions of the methods according to the present invention, i.e. which has in its genome a nucleotide sequence encoding such enzymes and which has been modified to overexpress them. The expression may occur constitutively or in an induced or regulated manner.

In another embodiment the microorganism can be a microorganism which has been genetically modified by the introduction of one or more nucleic acid molecules containing nucleotide sequences encoding one or more enzymes described above for the conversions of the methods according to the present invention. The nucleic acid molecule can be stably integrated into the genome of the microorganism or may be present in an extrachromosomal manner, e.g. on a plasmid.

Such a genetically modified microorganism can, e.g., be a microorganism that does not naturally express enzymes described above for the conversions of the methods according to the present invention and which has been genetically modified to express such enzymes or a microorganism which naturally expresses such enzymes and which has been genetically modified, e.g. transformed with a nucleic acid, e.g. a vector, encoding the respective enzyme(s), and/or insertion of a promoter in front of the endogenous nucleotide sequence encoding the enzyme in order to increase the respective activity in said microorganism.

However, the invention preferably excludes naturally occurring microorganisms as found in nature expressing an enzyme as described above at levels as they exist in nature. Instead, the microorganism of the present invention and employed in a method of the present invention is preferably a non-naturally occurring microorganism, whether it has been genetically modified to express (including overexpression) an exogenous enzyme of the invention not normally existing in its genome or whether it has been engineered to overexpress an exogenous enzyme. Thus, the enzymes and (micro)organisms employed in connection with the present invention are preferably non-naturally occurring enzymes or (micro)organisms, i.e. they are enzymes or (micro)organisms which differ significantly from naturally occurring enzymes or microorganism and which do not occur in nature. As regards the enzymes, they are preferably variants of naturally occurring enzymes which do not as such occur in nature. Such variants include, for example, mutants, in particular prepared by molecular biological methods, which show improved properties, such as a higher enzyme activity, higher substrate specificity, higher temperature resistance and the like. As regards the (micro)organisms, they are preferably genetically modified organisms as described herein above which differ from naturally occurring organisms due to a genetic modification. Genetically modified organisms are organisms which do not naturally occur, i.e., which cannot be found in nature, and which differ substantially from naturally occurring organisms due to the introduction of a foreign nucleic acid molecule.

By overexpressing an exogenous or endogenous enzyme as described herein above, the concentration of the enzyme is substantially higher than what is found in nature, which can then unexpectedly force the reaction of the present invention which uses a non-natural for the respective enzyme. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.

A "non-natural" substrate is understood to be a molecule that is not acted upon by the respective enzyme in nature, even though it may actually coexist in the microorganism along with the endogenous enzyme. This "non-natural" substrate is not converted by the microorganism in nature as other substrates are preferred (e.g. the "natural substrate"). Thus, the present invention contemplates utilizing a non- natural substrate with the enzymes described above in an environment not found in nature.

Thus, it is also possible in the context of the present invention that the microorganism is a microorganism which naturally does not have the respective enzyme activity but which is genetically modified so as to comprise a nucleotide sequence allowing the expression of a corresponding enzyme. Similarly, the microorganism may also be a microorganism which naturally has the respective enzyme activity but which is genetically modified so as to enhance such an activity, e.g. by the introduction of an exogenous nucleotide sequence encoding a corresponding enzyme or by the introduction of a promoter for the endogenous gene encoding the enzyme to increase endogenous production to overexpressed (non-natural) levels.

If a microorganism is used which naturally expresses a corresponding enzyme, it is possible to modify such a microorganism so that the respective activity is overexpressed in the microorganism. This can, e.g., be achieved by effecting mutations in the promoter region of the corresponding gene or introduction of a high expressing promoter so as to lead to a promoter which ensures a higher expression of the gene. Alternatively, it is also possible to mutate the gene as such so as to lead to an enzyme showing a higher activity.

By using microorganisms which express enzymes described above for the conversions of the methods according to the present invention, it is possible to carry out the methods according to the invention directly in the culture medium, without the need to separate or purify the enzymes.

In one embodiment the organism employed in a method according to the invention is a microorganism which has been genetically modified to contain a foreign nucleic acid molecule encoding at least one enzyme described above for the conversions of the methods according to the present invention. The term "foreign" or "exogenous" in this context means that the nucleic acid molecule does not naturally occur in said microorganism. This means that it does not occur in the same structure or at the same location in the microorganism. In one preferred embodiment, the foreign nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding the respective enzyme in which the promoter driving expression of the coding sequence is heterologous with respect to the coding sequence. "Heterologous" in this context means that the promoter is not the promoter naturally driving the expression of said coding sequence but is a promoter naturally driving expression of a different coding sequence, i.e., it is derived from another gene, or is a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter heterologous to the microorganism, i.e. a promoter which does naturally not occur in the respective microorganism. Even more preferably, the promoter is an inducible promoter. Promoters for driving expression in different types of organisms, in particular in microorganisms, are well known to the person skilled in the art.

In a further embodiment the nucleic acid molecule is foreign to the microorganism in that the encoded enzyme is not endogenous to the microorganism, i.e. is naturally not expressed by the microorganism when it is not genetically modified. In other words, the encoded enzyme is heterologous with respect to the microorganism. The foreign nucleic acid molecule may be present in the microorganism in extrachromosomal form, e.g. as a plasmid, or stably integrated in the chromosome. A stable integration is preferred. Thus, the genetic modification can consist, e.g. in integrating the corresponding gene(s) encoding the enzyme(s) into the chromosome, or in expressing the enzyme(s) from a plasmid containing a promoter upstream of the enzyme-coding sequence, the promoter and coding sequence preferably originating from different organisms, or any other method known to one of skill in the art.

The term "microorganism" in the context of the present invention refers to bacteria, as well as to fungi, such as yeasts, and also to algae and archaea. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas or Escherichia. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli. In another preferred embodiment the bacterium belongs to the species Pseudomonas putida or to the species Zymomonas mobilis or to the species Corynebacterium glutamicum or to the species Bacillus subtilis. It is also possible to employ an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae.

In another preferred embodiment the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus, Trichoderma, Kluyveromyces or Pichia and even more preferably of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger, Trichoderma reesei, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris, Pichia torula or Pichia utilis.

In another embodiment, the method according to the invention makes use of a photosynthetic microorganism expressing at least one enzyme for the conversion according to the invention as described above. Preferably, the microorganism is a photosynthetic bacterium, or a microalgae. In a further embodiment the microorganism is an algae, more preferably an algae belonging to the diatomeae. It is also conceivable to use in the method according to the invention a combination of microorganisms wherein different microorganisms express different enzymes as described above. The genetic modification of microorganisms to express an enzyme of interest will also be further described in detail below.

In a preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism, which is capable of consuming glucose.

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism, which is capable of consuming fructose. In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism, which is capable of consuming xylose. In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism, which is capable of consuming mannose. In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism, which is capable of consuming more than one sugar. Preferably, said more than one sugar comprises sucrose, glucose, mannose and/or xylose. In a more preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism, which is capable of consuming two or more sugars selected from the group consisiting of sucrose, glucose, mannose and xylose. Organisms and/or microorganisms which are capable of consuming glucose, fructose, xylose and/or mannose do naturally occur and are known in the art.

In another embodiment, said organism and/or microorganism is genetically modified in order to be capable of consuming glucose, fructose, xylose and/or mannose and/or genetically modified in order to increase the organism's and/or microorganism's capability of consuming glucose, fructose, xylose and/or mannose. Corresponding genetic modifications are known in the art.

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism which is capable of consuming sugar through a Phosphotransferase Transport System (PTS).

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism which is capable of consuming sugar through a non-Phosphotransferase Transport System (non-PTS).

Organisms and/or microorganisms which are capable of consuming sugar through a Phosphotransferase Transport System (PTS) and/or through a non- Phosphotransferase Transport System (non-PTS) are known in the art.

In another embodiment, said organism and/or microorganism is genetically modified in order to be capable of consuming sugar through a Phosphotransferase Transport System (PTS) or through a non-Phosphotransferase Transport System (non-PTS). In another preferred embodiment, said organism and/or microorganism is genetically modified in order to increase the organism's and/or microorganism's capability of consuming sugar through a Phosphotransferase Transport System (PTS) or through a non-Phosphotransferase Transport System (non-PTS). Corresponding genetic modifications are known in the art.

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism having a diminished or inactivated Phosphotransferase Transport System (PTS).

Without being bound to theory, such an organism, preferably a microorganism, may preferably be genetically modified by deleting or inactivating (a) gene(s) of said Phosphotransferase Transport System (PTS).

Corresponding genetic modifications are known in the art.

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism having an enhanced non- Phosphotransferase Transport System (non-PTS) for sugar uptake.

Without being bound to theory, such an organism, preferably a microorganism, may preferably be genetically modified by overexpressing (a) gene(s) of said non- Phosphotransferase Transport System (non-PTS) for sugar uptake.

Corresponding genetic modifications are known in the art.

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism having a diminished or inactivated Phosphotransferase Transport System (PTS) and an enhanced non Phosphotransferase Transport System (non-PTS) for sugar uptake.

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism which is capable of consuming sucrose through a non-Phosphotransferase Transport System (non-PTS).

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism consuming sucrose, wherein said organism, preferably said microorganism, has genetically been modified by the introduction of at least one gene of a non-Phosphotransferase Transport System (non-PTS). Without being bound to theory, such an organism and/or microorganism has genetically been modified by introducing a gene selected from the group consisting of cscA, cscB, and cscK from Escherichia coli W (M. Bruschi et al., Biotechnology Advances 30 (2012) 1001-1010).

In another preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism which has genetically been modified to have a diminished or inactivated Phosphotransferase Transport System (PTS) and an overexpression of at least one gene selected from the group consisting of galP, glk and glf.

In a preferred embodiment, the method of the present invention makes use of an organism, preferably a microorganism, which is genetically modified in order to avoid the leakage of acetyl-CoA, thereby increasing the intracellular concentration of acetyl-CoA. Genetic modifications leading to an increase in the intracellular concentration of acetyl-CoA are known in the art. Without being bound to theory, such an organism, preferably a microorganism, may preferably be genetically modified by deleting or inactivating the following genes:

AackA (acetate kinase), Aldh (lactate dehydrogenase), AadhE (alcohol dehydrogenase), AfrdB and/or AfrdC (fumarate reductase and fumarate dehydrogenase).

Alternatively, or in addition to any of the above deletions, the organism or microorganism may genetically be modified by overexpressing the gene panK/coaA encoding Pantothenate kinase, thereby increasing the CoA/acetyl-CoA intracellular pool.

These modifications which avoid the leakage of acetyl-CoA are known in the art and corresponding modified organisms have been used in methods for the bioconversion of exogenous isoamyl alcohol into isoamyl acetate by an E. coli strain expressing ATF2 (Metab. Eng. 6 (2004), 294-309).

In another embodiment, the method of the invention comprises the step of providing the organism, preferably the microorganism carrying the respective enzyme activity or activities in the form of a (cell) culture, preferably in the form of a liquid cell culture, a subsequent step of cultivating the organism, preferably the microorganism in a fermenter (often also referred to a bioreactor) under suitable conditions allowing the expression of the respective enzyme and further comprising the step of effecting an enzymatic conversion of a method of the invention as described herein above. Suitable fermenter or bioreactor devices and fermentation conditions are known to the person skilled in the art. A bioreactor or a fermenter refers to any manufactured or engineered device or system known in the art that supports a biologically active environment. Thus, a bioreactor or a fermenter may be a vessel in which a chemical/biochemical like the method of the present invention is carried out which involves organisms, preferably microorganisms and/or biochemically active substances, i.e., the enzyme(s) described above derived from such organisms or organisms harbouring the above described enzyme(s). In a bioreactor or a fermenter, this process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, and may range in size from litres to cubic metres, and are often made of stainless steel. In this respect, without being bound by theory, the fermenter or bioreactor may be designed in a way that it is suitable to cultivate the organisms, preferably microorganisms, in, e.g., a batch-culture, feed-batch-culture, perfusion culture or chemostate-culture, all of which are generally known in the art.

The culture medium can be any culture medium suitable for cultivating the respective organism or microorganism.

When carried out by making use of a microorganism, the method according to the present invention may, e.g. be designed as a continuous fermentation culturing method or as a batch culture or any suitable culture method known to the person skilled in the art.

In a preferred embodiment the method according to the present invention also comprises the step of recovering the isobutene produced by the method. For example, if the method according to the present invention is carried out in vivo by fermenting a corresponding microorganism expressing the necessary enzymes, the isobutene can be recovered from the fermentation off-gas by methods known to the person skilled in the art.

In a preferred embodiment, the present invention relates to a method as described herein above in which a microorganism as described herein above is employed, wherein the microorganism is capable of enzymatically converting 3-methylcrotonic acid into isobutene, wherein said method comprises culturing the microorganism in a culture medium.

The enzymes used in the method according to the invention can be naturally occurring enzymes or enzymes which are derived from a naturally occurring enzymes, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.

Methods for modifying and/or improving the desired enzymatic activities of proteins are well-known to the person skilled in the art and include, e.g., random mutagenesis or site-directed mutagenesis and subsequent selection of enzymes having the desired properties or approaches of the so-called "directed evolution".

For example, for genetic modification in prokaryotic cells, a nucleic acid molecule encoding a corresponding enzyme can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be ligated by using adapters and linkers complementary to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods. The resulting enzyme variants are then tested for the desired activity, e.g., enzymatic activity, with an assay as described above and in particular for their increased enzyme activity.

As described above, the microorganism employed in a method of the invention or contained in the composition of the invention may be a microorganism which has been genetically modified by the introduction of a nucleic acid molecule encoding a corresponding enzyme. Thus, in a preferred embodiment, the microorganism is a recombinant microorganism which has been genetically modified to have an increased activity of at least one enzyme described above for the conversions of the method according to the present invention. This can be achieved e.g. by transforming the microorganism with a nucleic acid encoding a corresponding enzyme. A detailed description of genetic modification of microorganisms will be given further below. Preferably, the nucleic acid molecule introduced into the microorganism is a nucleic acid molecule which is heterologous with respect to the microorganism, i.e. it does not naturally occur in said microorganism.

In the context of the present invention, an "increased activity" or "improved activity" means that the expression and/or the activity of an enzyme in the genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-modified microorganism. In even more preferred embodiments the increase in expression and/or activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 0-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-modified microorganism. The term "increased improved" expression/activity also covers the situation in which the corresponding non-modified microorganism does not express a corresponding enzyme so that the corresponding expression/activity in the non-modified microorganism is zero. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30%, or 40% of the total host cell protein.

Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot.

In the context of the present invention the term "recombinant" means that the microorganism is genetically modified so as to contain a nucleic acid molecule encoding an enzyme as defined above as compared to a wild-type or non-modified microorganism. A nucleic acid molecule encoding an enzyme as defined above can be used alone or as part of a vector.

The nucleic acid molecules can further comprise expression control sequences operably linked to the polynucleotide comprised in the nucleic acid molecule. The term "operatively linked" or "operably linked", as used throughout the present description, refers to a linkage between one or more expression control sequences and the coding region in the polynucleotide to be expressed in such a way that expression is achieved under conditions compatible with the expression control sequence.

Expression comprises transcription of the heterologous DNA sequence, preferably into a translatable mRNA. Regulatory elements ensuring expression in fungi as well as in bacteria, are well known to those skilled in the art. They encompass promoters, enhancers, termination signals, targeting signals and the like. Examples are given further below in connection with explanations concerning vectors.

Promoters for use in connection with the nucleic acid molecule may be homologous or heterologous with regard to its origin and/or with regard to the gene to be expressed. Suitable promoters are for instance promoters which lend themselves to constitutive expression. However, promoters which are only activated at a point in time determined by external influences can also be used. Artificial and/or chemically inducible promoters may be used in this context.

The vectors can further comprise expression control sequences operably linked to said polynucleotides contained in the vectors. These expression control sequences may be suited to ensure transcription and synthesis of a translatable RNA in bacteria or fungi.

In addition, it is possible to insert different mutations into the polynucleotides by methods usual in molecular biology (see for instance Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), leading to the synthesis of polypeptides possibly having modified biological properties. The introduction of point mutations is conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide.

Moreover, mutants possessing a modified substrate or product specificity can be prepared. Preferably, such mutants show an increased activity. Alternatively, mutants can be prepared the catalytic activity of which is abolished without losing substrate binding activity.

Furthermore, the introduction of mutations into the polynucleotides encoding an enzyme as defined above allows the gene expression rate and/or the activity of the enzymes encoded by said polynucleotides to be reduced or increased.

For genetically modifying bacteria or fungi, the polynucleotides encoding an enzyme as defined above or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods. Thus, in accordance with the present invention a recombinant microorganism can be produced by genetically modifying fungi or bacteria comprising introducing the above-described polynucleotides, nucleic acid molecules or vectors into a fungus or bacterium.

The polynucleotide encoding the respective enzyme is expressed so as to lead to the production of a polypeptide having any of the activities described above. An overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440). An overview of yeast expression systems is for instance given by Hensing et al. (Antonie van Leuwenhoek 67 (1995), 261 -279), Bussineau et al. (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991 ), 742-745) and Buckholz (Bio/Technology 9 (1991 ), 1067-1072).

Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence. The DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481 ; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), Ip1 , rac (Boras et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters. In order to obtain an optimum amount of polypeptide, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl^-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21 -25). Termination signals for transcription are also described in the literature.

The transformation of the host cell with a polynucleotide or vector as described above can be carried out by standard methods, as for instance described in Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.

As mentioned above, the method according to the present invention is in particular useful for large scale production of isobutene in vivo, in particular for a commercial production. The present invention describes novel ways to commercially and cost- effectively produce large quantities of isobutene which has not been obtainable to date. The generated large quantities of isobutene can then be further converted, in a commercial setting, to produce large quantities of, e.g., drop-in gasoline (e.g. isooctane, ETBE, MTBE), jet-fuel, , cosmetics, chemicals, such as methacrylic acid, polyisobutene, or butyl rubber. As used herein, "large scale production", "commercial production" and "bioprocessing" of isobutene in a fermentation reactor or in vitro is carried out at a capacity greater than at least 100 liters, and preferably greater than at least 400 liters, or more preferably production of 1 ,000 liters of scale or more, even more preferably production of 5,000 liters of scale or more. As used herein, "large quantities" specifically excludes trace amounts that may be produced inherently in a microorganism. For example, in preferred embodiments, the yields for continuous cultures according to methods described herein are at least about 0.2 grams of isobutene per liter per day, at least about 0.3 grams of isobutene per liter per day, at least about 0.4 grams of isobutene per liter per day, at least about 0.5 grams of isobutene per liter per day, at least about 0.6 grams of isobutene per liter per day, at least about 0.7 grams of isobutene per liter per day, at least about 0.8 grams of isobutene per liter per day, or at least about 1.0 grams of isobutene per liter per day. In further embodiments, the yields for continuous cultures according to methods described herein are between about 0.3 grams and about 1.0 grams of isobutene per liter per day, between about 0.4 grams to about 1.0 grams of isobutene per liter per day, and between about 0.5 grams and about 1 .0 grams of isobutene per liter per day. In other specific embodiments, the yields for continuous cultures according to methods described herein are between about 0.5 grams to about 0.75 grams of isobutene per liter per day. In other specific embodiments, the yields for continuous cultures according to methods described herein are between about 0.5 grams and about 1.5 grams of isobutene per liter per day.

In further embodiments, the yields for batch cultures according to methods described herein are at least about 2 grams per liter in batch culture, at least about 5 grams per liter in batch culture, at least about 10 grams per liter in batch culture, and at least about 15 grams per liter in batch culture. In some embodiments, the yields for batch cultures according to methods described herein are between about 2 grams and about 5 grams per liter in batch culture, between about 5 grams and about 10 grams per liter in batch culture, and still more preferably between about 10 grams and about 20 grams per liter in batch culture. In other specific embodiments, the yields for batch cultures according to methods described herein are between about 2.4 grams and about 4.8 grams per liter, and preferably between about 4.8 grams and about 9.4 grams per liter in batch culture, and still more preferably between about 9.4 grams and about 18.6 grams per liter in batch culture.

In additional embodiments, the concentration of the 3-methylcrotonic acid in the in vitro composition used to commercially produce isobutene is at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8% 9%, 10% or 20% as compared to all components, preferably soluble components, of the in vitro composition. Alternatively, the concentration of the FMN-dependent decarboxylase associated with an FMN prenyl transferase in the in vitro composition used to commercially produce isobutene is at least 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% 0.9%, 1.0% or 2.0% as compared to all components, preferably soluble components, of the in vitro composition.

In additional embodiments, the concentration of the 3-methylcrotonic acid in the microorganism or organism used to commercially produce isobutene is at least 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% 0.9%, 1.0% or 2.0% as compared to all other molecules found in the microoganism or organism. Alternatively, the concentration of the FMN-dependent decarboxylase associated with an FMN prenyl transferase in the microorganism or organism used to commercially produce isobutene is at least 0.1 mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM 0.9mM, 1.0mM or 2.0mM as compared to all proteins and/or enzymes found in the microorganism or organism.

As used herein, the term "about" is used to refer to an amount that is approximately, nearly, almost, or in the vicinity of being equal to or is equal to a stated amount, e.g., the state amount plus/minus about 5%, about 4%, about 3%, about 2% or about 1 %.

Recombinant organisms or microorganisms expressing enzymes of step I and any one of route (i), (ii), (iii) and/or (iv) for the provision of DMAP

The present invention also relates to a recombinant organism or microorganism which recombinantiy expresses an FMN-dependent decarboxylase associated with an FMN prenyl transferase (step I of Figure 1);

wherein said recombinant organism or microorganism further recombinantiy expresses at least one of the following (i) to (iv):

(i) an enzyme catalyzing the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into dimethylallyl phosphate (DMAP), wherein said enzyme is a phosphatase,

preferably an enzyme acting on phosphorous containing anhydrides (EC 3.6.1 .-), more preferably an ADP-ribose pyrophosphatase (EC 3.6.1.13), an 8- oxo-dGTP diphosphatase (EC 3.6.1.55), a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1.45), exopolyphosphatase (EC 3.6.1.1 1 ), a guanosine-5'-triphosphate/3'- diphosphate pyrophosphatase (EC 3.6.1 .40), an NADH pyrophosphatase (EC 3.6.1 .22), a nucleotide diphosphatase (EC 3.6.1 .9) or an acylphosphatase (EC 3.6.1.7); or

preferably a phosphoric-monoester hydrolase (EC 3.1.3.-), more preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7), a 5-amino-6-(5-phospho-D- ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ); or

preferably an isopentenyl phosphate kinase (EC 2.7.4.26);

(ii) an enzyme catalyzing the direct enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iii) an enzyme catalyzing the enzymatic conversion of D APP into prenol, wherein said enzyme is a phosphatase or pyrophosphatase, preferably an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC 3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1.3.5), a pyridoxal phosphate phosphatase (EC 3.1.3.74) or a fructose-1 6- bisphosphatase (EC 3.1.3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and

an enzyme catalyzing the thus obtained prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iv) an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

In another preferred embodiment, this recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme capable of enzymatically converting DMAPP into said DMAP of (i) is a phosphatase. In a more preferred embodiment, said phosphatase is:

an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), preferably an ADP-ribose pyrophosphatase (EC 3.6.1.13), an 8-oxo-dGTP diphosphatase (EC 3.6.1.55), a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1.45), exopolyphosphatase (EC 3.6.1.11 ), a guanosine-5'- triphosphate/3'-diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1.22), a nucleotide diphosphatase (EC 3.6.1.9) or an acylphosphatase (EC 3.6.1.7); or

a phosphoric-monoester hydrolase (EC 3.1.3.-), preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7), a 5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ).

In another preferred embodiment, this recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme capable of enzymatically converting DMAPP into said DMAP of (i) is an isopentenyl phosphate kinase (EC 2.7.4.26).

In another preferred embodiment, this recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme capable of enzymatically converting prenol into DMAP of (ii) is a kinase. In a more preferred embodiment, said kinase is a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

In another preferred embodiment, this recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme capable of enzymatically converting DMAPP into prenol of (iii) is a phosphatase or pyrophosphatase and/or the enzyme capable of enzymatically converting prenol into DMAP of (iii) is a kinase. In a more preferred embodiment, said phosphatase or pyrophosphatase is:

an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC 3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1.3.5), a pyridoxal phosphate phosphatase (EC 3.1.3.74) or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ); or an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and/or

said kinase is a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

In another preferred embodiment, this recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme capable of enzymatically converting isopentenyl monophosphate (IMP) into said DMAP of (iv) is an isomerase. In a more preferred embodiment, said isomerase is an isopentenyl- diphosphate DELTA isomerase (EC 5.3.3.2).

As regards the enzymes mentioned herein above in connection with the organisms/microorganisms of the present invention and as regards preferred embodiments of these enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further recombinantly expresses an enzyme capable of enzymatically providing said DMAPP by the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP. In a more preferred embodiment, said enzyme capable of enzymatically providing said DMAPP by the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

As regards the isomerase and the isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2), the same applies to the recombinant organism or microorganism as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further recombinantly expresses an enzyme capable of catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN). In preferred embodiment, the enzyme capable of catalyzing the enzymatic conversion of riboflavin into FMN is a kinase, preferably an archaeal riboflavin kinase (EC 2.7.1.161 ),

a flavokinase derived from S. cerevisiae or from Rattus norvegicus,

a flavokinase derived from Megasphaera elsdenii,

a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 ), preferably an erythritol kinase (2.7.1.27) or a glycerol kinase (2.7.1.30),

a phosphotransferase with a phosphate group as acceptor (EC 2.7.4), preferably an isopentenyl phosphate kinase (EC 2.7.4.26); or

a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF); or

a variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived.

In a more preferred embodiment, said variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived is a variant having an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence having at least

60% sequence identity to SEQ ID NO:34, in which one or more amino acid residues at a position selected from the group consisting of positions 29 and 32 in the amino acid sequence shown in SEQ ID NO:34 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.

More preferably, said variant is a variant wherein

(1 ) an amino acid residue at position 29 in the amino acid sequence shown in SEQ ID NO:34 or at a position corresponding to this position, is deleted or substituted with alanine; and/or

(2) an amino acid residue at position 32 in the amino acid sequence shown in SEQ ID NO:34 or at a position corresponding to this position, is deleted or substituted with serine or alanine.

As regards the enzymes mentioned herein above in connection with this aspect and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention. Recombinant organisms or microorganisms expressing enzymes of step I and any one of route (v), (vi), and/or (vii) for the provision of DMAPP

The present invention also relates to a recombinant organism or microorganism which recombinantly expresses an FMN-dependent decarboxylase associated with an FMN prenyl transferase (step I of Figure 1 );

wherein said recombinant organism or microorganism further recombinantly expresses at least one of the following (v) to (vii):

(v) an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2);

(vi) an enzyme catalyzing the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP, wherein said enzyme is a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26); and

(vii) an enzyme catalyzing the enzymatic conversion of prenol into said DMAPP, wherein said enzyme is a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase.

As regards the enzymes mentioned herein above in connection with the organisms/microorganisms of the present invention and as regards preferred embodiments of these enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further recombinantly expresses an enzyme capable of catalyzing the enzymatic conversion of prenol into DMAP.

In a preferred embodiment, this recombinant organism or microorganism is a recombinant organism or microorganism, wherein said enzyme capable of catalyzing the enzymatic conversion of prenol into DMAP is a kinase, more preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 .-), and even more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further recombinantly expresses an enzyme capable of catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP. In a preferred embodiment, this recombinant organism or microorganism is a recombinant organism or microorganism, wherein said enzyme capable of catalyzing the enzymatic conversion of IMP into DMAP is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

As regards the enzymes mentioned herein above in connection with the organisms/microorganisms of the present invention and as regards preferred embodiments of these enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further recombinantly expresses an enzyme capable of catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN). In preferred embodiment, the enzyme capable of catalyzing the enzymatic conversion of riboflavin into FMN is a kinase, preferably

an archaeal riboflavin kinase (EC 2.7.1.161 ),

a flavokinase derived from S. cerevisiae or from Rattus norvegicus,

a flavokinase derived from Megasphaera elsdenii,

a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 ), preferably an erythritol kinase (2.7.1.27) or a glycerol kinase (2.7.1.30),

a phosphotransferase with a phosphate group as acceptor (EC 2.7.4), preferably an isopentenyl phosphate kinase (EC 2.7.4.26); or

a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF); or

a variant of a bifunctional riboflavin kinase/FMN adenylyltransferase (ribF) which shows an improved activity in converting riboflavin into FMN over the corresponding bifunctional riboflavin kinase/FMN adenylyltransferase from which it is derived.

As regards the enzymes mentioned herein above in connection with this aspect and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention. Recombinant organisms or microorganisms expressing enzymes of step I and any one of route (i), (it), (iii) and/or (iv) for the provision of DMAP and/or any one of route (v), (vi) and/or (vii) for the provision of DMAPP and/or F N and optionally further expressing enzymes of step II, step 111, step IV and step V as well as optionally further expressing enzymes of steps XIII, XIV and XV

The present invention also relates to a recombinant organism or microorganism which expresses an enzyme capable of enzymatically converting 3-methylcrotonic acid into isobutene (step I as shown in Figure 1) and an enzyme capable of providing DMAP of any one of routes (i) to (iv) and/or an enzyme capable of providing DMAPP of any one of routes (v) to (vii) and/or an enzyme capable of catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN) as well as an enzyme capable of enzymatically converting 3-hydroxyisovalerate (HIV) into 3- methylcrotonic acid (step II as shown in Figure 1).

In a preferred embodiment, the enzyme capable of enzymatically converting 3- hydroxyisovalerate (HIV) into 3-methylcrotonic acid is a hydro-lyase (EC 4.2.-.-) as defined herein above, preferably an aconitase (EC 4.2.1.3), a fumarase (EC 4.2.1.2) or an enoyl-CoA hydratase/dehydratease (EC 4.2.1.17) as defined herein above. As regards these enzymes and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically condensing acetone and acetyl-CoA into 3-hydroxyisovalerate (HIV) (step III as shown in Figure 1). In a preferred embodiment, the enzyme capable of enzymatically condensing acetone and acetyl-CoA into 3-hydroxyisovalerate (HIV) is a HMG CoA synthase (EC 2.3.3.10) or a PksG protein or an enzyme with the activity of a C-C bond cleavage/condensation lyase, such as a HMG CoA lyase (EC 4.1.3.4) as defined herein above.

As regards these enzymes and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically converting acetoacetate into acetone (step IV as shown in Figure 1 ), preferably an acetoacetate decarboxylase (EC 4.1.1.4) as described herein above. As regards these enzymes and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of converting acetoacetyl-CoA into acetoacetate (step Va or Vb as shown in Figure 1), preferably

(i) an acetoacetyl-CoA hydrolase (EC 3.1.2.11 ); or

(ii) an enzyme which is capable of transferring the CoA group of acetoacetyl-CoA on acetate

as described herein above.

In a preferred embodiment, the enzyme capable of transferring the CoA group of acetoacetyl-CoA on acetate is a CoA transferase (EC 2.8.3.-), preferably an acetate CoA transferase (EC 2.8.3.8) as described herein above.

As regards these enzymes and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically converting acetyl-CoA into acetoacetyl-CoA comprising

(a) (i) an enzyme capable of converting acetyl-CoA into malonyl-CoA (step XIV as shown in Figure 1 ); and

(ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV as shown in Figure 1 ); or

(b) an enzyme capable of directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA (step XIII as shown in Figure 1 ).

In a preferred embodiment, the enzyme capable of converting acetyl-CoA into malonyl-CoA is an acetyl-CoA carboxylase (EC 6.4.1.2) as described herein above. In another preferred embodiment, the enzyme capable of condensing malonyl-CoA and acetyl-CoA into acetoacetyl-CoA is an acetoacetyl-CoA synthetase (EC 2.3.1.194) as described herein above.

In a preferred embodiment, the enzyme capable of directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA is an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) as described herein above.

As regards the enzyme which is capable of converting acetyl-CoA into malonyl-CoA, these enzymes mentioned in connection with this aspect and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention.

Recombinant organisms or microorganisms expressing enzymes of step I and any one of route (i), (ii), (iii) and/or (iv) for the provision of DMAP and/or any one of route (v), (vi) and/or (vii) for the provision of DMAPP and/or FMN and optionally further expressing enzymes of step step VI, step VII, step VIII and step IX as well as optionally further expressing enzymes of steps XIII, XIV and XV

The present invention also relates to a recombinant organism or microorganism which expresses an enzyme capable of enzymatically converting 3-methylcrotonic acid into isobutene (step I as shown in Figure 1) and an enzyme capable of providing DMAP of any one of routes (i) to (iv) and/or an enzyme capable of providing DMAPP of any one of routes (v) to (vii) and/or an enzyme capable of catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN) as well as an enzyme capable of enzymatically converting 3-methylcrotonyl-CoA into 3- methylcrotonic acid (step Via, Vlb or Vic as shown in Figure 1).

In a preferred embodiment, the enzyme capable of converting 3-methylcrotonic acid into isobutene is a 3-methylcrotonic acid decarboxylase, preferably

(i) an FMN-dependent decarboxylase associated with an FMN prenyl transferase; or

(ii) an aconitate decarboxylase (EC 4.1.1.6); or

(iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or

(iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or

(v) a protocatechuate (PCA) decarboxylase (EC 4.1.1.63)

as defined herein above.

As regards these enzymes and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention. In a preferred embodiment, the enzyme capable of enzymatically converting 3- methylcrotonyl-CoA into 3-methylcrotonic acid is

(a) an enzyme capable of directly converting 3-methylcrotonyl-CoA into 3- methylcrotonic acid wherein said enzyme capable of directly converting 3- methylcrotonyl-CoA into 3-methylcrotonic acid is a CoA transferase (EC 2.8.3.- ), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18) (step Via as shown in Figure 1 ) as described herein above; or

(b) an enzyme capable of directly converting 3-methylcrotonyl-CoA into 3- methylcrotonic acid wherein said enzyme capable of directly converting 3- methylcrotonyl-CoA into 3-methylcrotonic acid is a thioester hydrolase (EC 3.1 .2.-), preferably acetyl-CoA hydrolase (EC 3.1 .2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1 .2.18) or an acyl-CoA hydrolase (EC 3.1.2.20) (step Vlb as shown in Figure 1 ) as described herein above.

In another preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism which expresses the following two enzymes, namely

(c) (i) an enzyme capable of enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate as described herein above; and

(ii) an enzyme capable of converting 3-methylcrotonyl phosphate into 3- methylcrotonic acid (step Vic as shown in Figure 1 ) as described herein above.

In a preferred embodiment, the enzyme capable of converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate is a phosphate butyryltransferase (EC 2.3.1 .19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme capable of converting 3- methylcrotonyl phosphate into 3-methylcrotonic acid is a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain- fatty-acid kinase (EC 2.7.2.14) as described herein above.

As regards the above-mentioned enzymes and preferred embodiments thereof, the same applies as has been set forth above for the methods according to the present invention. In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically converting 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA (step VII as shown in Figure 1), preferably (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) a geranoyl-CoA carboxylase (EC 6.4.1.5) as described herein above.

As regards said enzymes as well as preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically converting 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl- CoA (step VIII as shown in Figure 1), preferably a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18), a 3-hydroxyacyl-CoA dehydratase (EC 4.2.1.-) or an enoyl- CoA hydratase (EC 4.2.1.-).

As regards said enzymes as well as preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically condensing acetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3- methylglutaryl-CoA (step IX as shown in Figure 1), preferably a 3-hydroxy-3- methylglutaryl-CoA synthase.

As regards said enzyme as well as preferred embodiments of said enzyme, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism which expresses an enzyme capable of enzymatically converting 3-methylcrotonic acid into isobutene (step I as shown in Figure 1 ) and an enzyme capable of providing DMAP of any one of routes (i) to (iv) and an enzyme capable of enzymatically converting 3- methylcrotonyl-CoA into 3-methylcrotonic acid (step Via, Vlb or Vic as shown in Figure 1) (and optionally further expressing an enzyme capable of enzymatically converting 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA and optionally further expressing an enzyme capable of enzymatically converting 3-hydroxy-3- methylglutaryl-CoA into 3-methylgutaconyl-CoA and optionally further expressing an enzyme capable of enzymatically condensing acetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA) is preferably an organism or microorganism which further expresses an enzyme capable of enzymatically converting acetyl-CoA into acetoacetyl-CoA, more preferably an enzyme capable of directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA (step XIII as shown in Figure 1).

In another preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism which expresses the following two enzymes, namely

(i) an enzyme capable of converting acetyl-CoA into malonyl-CoA (step XIV as shown in Figure 1); and

(ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV as shown in Figure 1).

In a preferred embodiment, the enzyme capable of converting acetyl-CoA into malonyl-CoA is an acetyl-CoA carboxylase (EC 6.4.1.2) as described herein above. In another preferred embodiment, the enzyme capable of condensing malonyl-CoA and acetyl-CoA into acetoacetyl-CoA is an acetoacetyl-CoA synthetase (EC 2.3.1.194) as described herein above.

In a preferred embodiment, the enzyme capable of directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA is an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) as described herein above.

As regards the above-mentioned enzymes as well as the preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention. Recombinant organisms or microorganisms expressing enzymes of any one of route (0, (ii), (iii) and/or (iv) for the provision of D AP and/or any one of route (v), (vi) and/or (vii) for the provision of DMAPP and/or FMN, one or more enzymes of the alternative route for the enzymatic conversion from acetyl-CoA into isobutene via 3-methyl-3-butenoyl-CoA and 3-methyl-3-butenoic acid: recombinant organisms or microorganisms expressing enzymes of step XVI and step XVII, and optionally further expressing enzymes of step XVIII, step VIII and step IX as well as optionally further expressing enzymes of steps XIII, XIV and XV

As mentioned above, in an alternative to the above first route for the production of isobutene via 3-methylcrotonic acid, the present invention also relates to a method for the production of isobutene via an alternative route wherein isobutene is produced by the enzymatic conversion of 3-methyl-3-butenoic acid into isobutene. In the following, the recombinant organisms or microorganisms expressing enzymes of any one of route (i), (ii), (iii) and/or (iv) for the provision of DMAP and/or an enzyme capable of providing DMAPP of any one of routes (v) to (vii) and/or enzymes for the provision of FMN as well as enzymes of this alternative route for the enzymatic conversion from acetyl-CoA into isobutene via 3-methyl-3-butenoyl-CoA and 3- methyl-3-butenoic acid are described.

The present invention also relates to a recombinant organism or microorganism which expresses an enzyme capable of enzymatically converting 3-methyl-3-butenoic acid into isobutene (step XVI as shown in Figure 1) and an enzyme capable of enzymatically converting 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (step XVII as shown in Figure 1 ).

In a preferred embodiment, the enzyme capable of enzymatically converting 3- methyl-3-butenoic acid into isobutene is a 3-methyl-3-butenoic acid decarboxylase as described herein above, more preferably

(i) an FMN-dependent decarboxylase associated with an FMN prenyl transferase; or

(ii) an aconitate decarboxylase (EC 4.1 .1.6); or

(iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or

(v) a protocatechuate (PCA) decarboxylase (EC 4.1 .1 .63)

as described herein above.

In another preferred embodiment, the 3-methyl-3-butenoic acid decarboxylase is selected from the group consisting of 6-methylsalicylate decarboxylase (EC 4.1.1.52), 2-oxo-3-hexenedioate decarboxylase (EC 4.1.1.77) and 5-oxopent-3-ene-1 ,2,5- tricarboxylate decarboxylase (EC 4.1.1.68) as described herein above.

As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a preferred embodiment, the enzyme capable of enzymatically converting 3- methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid is

(a) an enzyme capable of directely converting 3-methyl-3-butenoyl-CoA into 3- methyl-3-butenoic acid, wherein said enzyme capable of directely converting 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid is a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18) (step XVIIa as shown in Figure 1 ) as described herein above.

In another preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism which expresses the following two enzymes, namely

(b) an enzyme capable of directely converting 3-methyl-3-butenoyl-CoA into 3- methyl-3-butenoic acid, wherein said enzyme capable of directely converting 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid is a thioester hydrolase (EC 3.1 .2.-), preferably acetyl-CoA hydrolase (EC 3.1 .2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1 .2.18) or an acyl-CoA hydrolase (EC 3.1.2.20) (step XVIIb as shown in Figure 1 ) as described herein above; or

(c) (i) an enzyme capable of enzymatically converting 3-methyl-3-butenoyl- CoA into 3-methyl-3-butenoyl phosphate; and

(ii) an enzyme capable of enzymatically converting 3-methyl-3-butenoyl phosphate into said 3-methyl-3-butenoic acid(step XVIIc as shown in Figure 1) as described herein above.

In a preferred embodiment, the enzyme capable of enzymatically converting said 3- methyl-3-butenoyl-CoA into 3-methyl-3-butenoyl phosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme capable of enzymatically converting 3-methyl-3-butenoyl phosphate into 3-methyl-3-butenoic acid is a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14) as described herein above.

As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically converting 3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shown in Figure 1 ), preferably

(a) (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) a geranoyl-CoA carboxylase (EC 6.4.1.5), or

(b) an N-terminal domain of CurF from Lynbya majuscula multifunctional protein or a 3-methylglutaconyl-CoA decarboxylase, preferably a 3-methylglutaconyl- CoA decarboxylase of Myxococcus xanthus encoded by the liuB gene; or

(c) an enzyme of the 4-oxalocrotonate decarboxylase family,

as described herein above.

As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically converting 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl- CoA (step VIII as shown in Figure 1), preferably a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18), a 3-hydroxyacyl-CoA dehydratase (EC 4.2.1.-) or an enoyl- CoA hydratase (EC 4.2.1.-).

As regards the above-mentioned enzyme as well as preferred embodiments of said enzyme, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme capable of enzymatically condensing acetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3- methylglutaryl-CoA (step IX as shown in Figure 1 ).

In a preferred embodiment, the enzyme capable of enzymatically condensing acetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA is a 3-hydroxy- 3-methylglutaryl-CoA synthase.

As regards the afore-mentioned enzyme as well as preferred embodiments of said enzyme, the same applies as has been set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism is an organism or microorganism which further expresses an enzyme or several enzymes capable of enzymatically converting acetyl-CoA into acetoacetyl-CoA.

In one preferred embodiment, the recombinant organism or microorganism expresses a combination of enzymes, namely

(i) an enzyme capable of converting acetyl-CoA into malonyl-CoA (step XIV as shown in Figure 1 ); and

(ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV as shown in Figure 1 ).

In an alternative embodiment, the recombinant organism or microorganism expresses an enzyme capable of directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA (step XIII as shown in Figure 1 ).

As regards the first above-mentioned embodiment, the enzyme capable of converting acetyl-CoA into malonyl-CoA is preferably an acetyl-CoA carboxylase (EC 6.4.1.2) as described herein above.

Moreover, the enzyme capable of condensing malonyl-CoA and acetyl-CoA into acetoacetyl-CoA is an acetoacetyl-CoA synthetase (EC 2.3.1 .194) as described herein above. As regards the second above-mentioned embodiment, the enzyme capable of directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA is preferably an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) as described herein above.

As regards the above-mentioned enzymes as well as the preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

Recombinant organisms or microorganisms expressing enzymes of the additional/supplemental pathways of steps Xa, Xb. XI and XII

As mentioned above, the above-described methods of the present invention for producing isobutene from acetyl-CoA may be supplemented by one or more of the reactions as shown in step Xa, step Xb, step XI and step XII of Figure 1 (also summarized in Figure 26) and as described in detail herein above.

Thus, in a further aspect, the present invention relates to any of the above-described recombinant organism or microorganism wherein the organism or microorganism additionally further expresses

a) an enzyme capable of enzymatically converting 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a concomitant transfer of CoA from 3- methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in 3- hydroxyisovaleryl-CoA (step Xa as schematically shown in Figure 19); and/or b) an enzyme capable of enzymatically converting 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step Xb as schematically shown in Figure 20); and/or

c) an enzyme capable of enzymatically converting 3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA (step XI as schematically shown in Figure 21 ); and/or

d) an enzyme capable of enzymatically converting 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step XII as schematically shown in Figure 22) as described herein above.

As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

The above microorganism is preferably a bacterium, a yeast or a fungus. In another preferred embodiment, the organism is a plant or a non-human animal. As regards other preferred embodiments of the bacterium, recombinant organism or microorganism, the same applies as has been set forth above in connection with the methods according to the present invention.

The present invention also relates to the use of any of the above-described recombinant organisms or microorganisms for the production of isobutene from 3- methylcrotonic acid. Thus, the present invention furthermore relates to the use of a recombinant organism or microorganism for the production of isobutene wherein said recombinant organism or microorganism recombinantly expresses an FMN- dependent decarboxylase associated with an FMN prenyl transferase;

wherein said recombinant organism or microorganism further recombinantly expresses at least one of the following (i) to (iv):

(i) an enzyme catalyzing the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into dimethylallyl phosphate (DMAP), wherein said enzyme is a phosphatase,

preferably an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), more preferably an ADP-ribose pyrophosphatase (EC 3.6.1.13), an 8- oxo-dGTP diphosphatase (EC 3.6.1.55), a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1.45), exopolyphosphatase (EC 3.6.1.11 ), a guanosine-5'-triphosphate/3'- diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1.22), a nucleotide diphosphatase (EC 3.6.1.9) or an acylphosphatase (EC 3.6.1.7); or

preferably a phosphoric-monoester hydrolase (EC 3.1.3.-), more preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7), a 5-amino-6-(5-phospho-D- ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ); or

preferably an isopentenyl phosphate kinase (EC 2.7.4.26);

(ii) an enzyme catalyzing the direct enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iii) an enzyme catalyzing the enzymatic conversion of DMAPP into prenol, wherein said enzyme is a phosphatase or pyrophosphatase, preferably an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC 3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1.3.5), a pyridoxal phosphate phosphatase (EC 3.1.3.74) or a fructose-1 6- bisphosphatase (EC 3.1.3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and

an enzyme catalyzing the thus obtained prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iv) an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

The present invention furthermore relates to the use of any of the above-described recombinant organisms or microorganisms for the production of isobutene which further recombinantly expresses an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

The present invention furthermore relates to the use of a recombinant organism or microorganism for the production of isobutene wherein said recombinant organism or microorganism recombinantly expresses an FMN-dependent decarboxylase associated with an FMN prenyl transferase;

wherein said recombinant organism or microorganism further recombinantly expresses at least one of the following (v) to (vii):

(v) an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2);

(vi) an enzyme catalyzing the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP, wherein said enzyme is a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26); and

(vii) an enzyme catalyzing the enzymatic conversion of prenol into said DMAPP, wherein said enzyme is a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase.

The present invention furthermore relates to the use of any of the above-described recombinant organisms or microorganisms which further recombinantly expresses at least one of the above (v) to (vii) for the production of isobutene, wherein said recombinant organism or microorganism further recombinantly expresses an enzyme catalyzing the enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

The present invention furthermore relates to the use of any of the above-described recombinant organisms or microorganisms which further recombinantly expresses at least one of the above (v) to (vii) for the production of isobutene, wherein said recombinant organism or microorganism further recombinantly expresses an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

The present invention furthermore relates to the use of any of the above-described recombinant organisms or microorganisms for the production of isobutene which further recombinantly expresses an enzyme catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN).

The present invention furthermore relates to the use of any of the above-described recombinant organisms or microorganisms for the production of isobutene which additionally recombinantly expresses one or more of the enzymes described above for the method steps preceding the production of 3-methylcrotonic acid or 3-methyl- butenoic acid.

As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies to the use of the recombinant organism or microorganism for the production of isobutene as has been set forth above for the methods and recombinant organisms or microorganisms according to the present invention.

The present invention furthermore relates to the use a combination comprising an FMN-dependent decarboxylase associated with an FMN prenyl transferase and an enzyme or enzymes of any one of the following (i) to (iv):

(i) an enzyme catalyzing the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into dimethylallyl phosphate (DMAP), wherein said enzyme is a phosphatase,

preferably an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), more preferably an ADP-ribose pyrophosphatase (EC 3.6.1.13), an 8- oxo-dGTP diphosphatase (EC 3.6.1.55), a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1.45), exopolyphosphatase (EC 3.6.1.11 ), a guanosine-5'-triphosphate/3'- diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1.22), a nucleotide diphosphatase (EC 3.6.1.9) or an acylphosphatase (EC 3.6.1.7); or

preferably a phosphoric-monoester hydrolase (EC 3.1.3.-), more preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7), a 5-amino-6-(5-phospho-D- ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ); or

preferably an isopentenyl phosphate kinase (EC 2.7.4.26);

(ii) an enzyme catalyzing the direct enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iii) an enzyme catalyzing the enzymatic conversion of DMAPP into prenol, wherein said enzyme is a phosphatase or pyrophosphatase, preferably an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1 .3.3), a phosphoglycolate phosphatase (EC 3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1 .3.5), a pyridoxal phosphate phosphatase (EC 3.1 .3.74) or a fructose-1 6- bisphosphatase (EC 3.1.3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and

an enzyme catalyzing the thus obtained prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50); and

(iv) an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2) for the production of isobutene from 3-methylcrotonic acid.

In a further aspect, the present invention relates to any of the above uses of enzymes for the production of isobutene from 3-methylcrotonic acid, wherein additionally an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl- diphosphate DELTA isomerase (EC 5.3.3.2) as described herein above is used.

The present invention furthermore relates to the use of a combination comprising an FMN-dependent decarboxylase associated with an FMN prenyl transferase and an enzyme or enzymes of at least one of the following (v) to (vii):

(v) an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2);

(vi) an enzyme catalyzing the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP, wherein said enzyme is a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26); and (vii) an enzyme catalyzing the enzymatic conversion of prenol into said DMAPP, wherein said enzyme is a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase,

for the production of isobutene from 3-methylcrotonic acid.

The present invention furthermore relates to any of the above-described uses for the production of isobutene from 3-methylcrotonic acid wherein at least one of the above (v) to (vii) enyzymes is additionally used, wherein additionally an enzyme catalyzing the enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50) as described herein above is used.

The present invention furthermore relates to any of the above-described uses for the production of isobutene from 3-methylcrotonic acid wherein at least one of the above (v) to (vii) enyzymes is further used, wherein, additionally, an enzyme catalyzing catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2) as described herein above is used.

In a further aspect, the present invention relates to any of the above uses of enzymes for the production of isobutene from 3-methylcrotonic acid, wherein additionally an enzyme catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN) as described herein above is used.

In a further aspect, the present invention relates to any of the above uses of enzymes for the production of isobutene from 3-methylcrotonic acid, wherein additionally one or more of the enzymes described above for the method steps preceding the production of 3-methylcrotonic acid are used.

As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies to the use for the production of isobutene as has been set forth above for the methods and recombinant organisms or microorganisms according to the present invention. Furthermore, the present invention relates to a composition comprising DMAPP, IMP and/or prenol and a recombinant organism or microorganism, wherein said recombinant organism or microorganism recombinantly expresses an FMN- dependent decarboxylase associated with an FMN prenyl transferase;

wherein said recombinant organism or microorganism further recombinantly expresses at least one of the following (i) to (iv):

(i) an enzyme catalyzing the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into dimethylallyl phosphate (DMAP), wherein said enzyme is a phosphatase,

preferably an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), more preferably an ADP-ribose pyrophosphatase (EC 3.6.1.13), an 8- oxo-dGTP diphosphatase (EC 3.6.1.55), a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1.45), exopolyphosphatase (EC 3.6.1 .11 ), a guanosine-5'-triphosphate/3'- diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1.22), a nucleotide diphosphatase (EC 3.6.1 .9) or an acylphosphatase (EC 3.6.1.7); or

preferably a phosphoric-monoester hydrolase (EC 3.1.3.-), more preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7), a 5-amino-6-(5-phospho-D- ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1 .3.11 ); or

preferably an isopentenyl phosphate kinase (EC 2.7.4.26);

(ii) an enzyme catalyzing the direct enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1 .-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iii) an enzyme catalyzing the enzymatic conversion of DMAPP into prenol, wherein said enzyme is a phosphatase or pyrophosphatase, preferably an alkaline phosphatase (EC 3.1 .3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1 .3.81 ), a phosphatidate phosphatase (EC 3.1 .3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC 3.1 .3.18), a pyrimidine 5'-nucleotidase (EC 3.1 .3.5), a pyridoxal phosphate phosphatase (EC 3.1 .3.74) or a fructose-1 6- bisphosphatase (EC 3.1.3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and

an enzyme catalyzing the thus obtained prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

(iv) an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

The present invention furthermore relates to any of the above-described compositions wherein said recombinant organisms or microorganisms further recombinantly expresses an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

Furthermore, the present invention relates to a composition comprising DMAP, IPP and/or prenol and a recombinant organism or microorganism, wherein said recombinant organism or microorganism recombinantly expresses an FMN- dependent decarboxylase associated with an FMN prenyl transferase;

wherein said recombinant organism or microorganism further recombinantly expresses at least one of the following (v) to (vii):

(v) an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2);

(vi) an enzyme catalyzing the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP, wherein said enzyme is a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26); and

(vii) an enzyme catalyzing the enzymatic conversion of prenol into said DMAPP, wherein said enzyme is a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase.

The present invention furthermore relates to any of the above-described compositions wherein said recombinant organism or microorganism further recombinantly expresses at least one of the above (v) to (vii), wherein additionally, said recombinant organism or microorganism further recombinantly expresses an enzyme catalyzing the enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50). The present invention furthermore relates to any of the above-described compositions wherein said recombinant organism or microorganism further recombinantly expresses at least one of the above (v) to (vii), wherein additionally, said recombinant organism or microorganism further recombinantly expresses an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl- diphosphate DELTA isomerase (EC 5.3.3.2).

The present invention furthermore relates to any of the above-described compositions wherein said recombinant organisms or microorganisms further recombinantly expresses an enzyme catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN).

The present invention furthermore relates to any of the above-described compositions wherein said recombinant organisms or microorganisms additionally recombinantly express one or more of the enzymes described above for the method steps preceding the production of 3-methylcrotonic acid or 3-methyl-butenoic acid.

In a further aspect, the present invention relates to a composition comprising DMAPP, IMP and/or prenol and an FMN-dependent decarboxylase associated with an FMN prenyl transferase and an enzyme or enzymes of any one of the following (i) to (iv):

(i) an enzyme catalyzing the enzymatic conversion of dimethylallyl pyrophosphate (DMAPP) into dimethylallyl phosphate (DMAP), wherein said enzyme is a phosphatase,

preferably an enzyme acting on phosphorous containing anhydrides (EC 3.6.1.-), more preferably an ADP-ribose pyrophosphatase (EC 3.6.1.13), an 8- oxo-dGTP diphosphatase (EC 3.6.1.55), a bis(5'-nucleosyl)-tetraphosphatase (EC 3.6.1.41 ), an UDP-sugar diphosphatase (EC 3.6.1.45), exopolyphosphatase (EC 3.6.1.11 ), a guanosine-5'-triphosphate/3'- diphosphate pyrophosphatase (EC 3.6.1.40), an NADH pyrophosphatase (EC 3.6.1.22), a nucleotide diphosphatase (EC 3.6.1.9) or an acylphosphatase (EC 3.6.1.7); or

preferably a phosphoric-monoester hydrolase (EC 3.1.3.-), more preferably a 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7), a 5-amino-6-(5-phospho-D- ribitylamino) uracil phosphatase or a fructose-1 6-bisphosphatase (EC 3.1.3.11 ); or

preferably an isopentenyl phosphate kinase (EC 2.7.4.26);

> an enzyme catalyzing the direct enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50);

) an enzyme catalyzing the enzymatic conversion of DMAPP into prenol, wherein said enzyme is a phosphatase or pyrophosphatase, preferably an alkaline phosphatase (EC 3.1.3.1 ), a sugar phosphatase (EC 3.1.3.23), a phosphatidylglycerophosphatase (EC 3.1.3.27), a diacylglycerol pyrophosphate phosphatase (EC 3.1.3.81 ), a phosphatidate phosphatase (EC 3.1.3.4), a phosphoserine phosphatase (EC 3.1.3.3), a phosphoglycolate phosphatase (EC 3.1.3.18), a pyrimidine 5'-nucleotidase (EC 3.1.3.5), a pyridoxal phosphate phosphatase (EC 3.1.3.74) or a fructose-1 6- bisphosphatase (EC 3.1.3.11 ); or

an UDP-sugar diphosphatase (EC 3.6.1.45) or an undecaprenyl pyrophosphate phosphatase (EC 3.6.1.27); or

a prenyl-diphosphatase (EC 3.1.7.1 ); or

an isopentenyl phosphate kinase (EC 2.7.4.26); and

an enzyme catalyzing the thus obtained prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50); and

) an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

In a further aspect, the present invention relates to any of the above compositions which further additionally comprises

an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl- diphosphate DELTA isomerase (EC 5.3.3.2) as described herein above.

In a further aspect, the present invention relates to a composition comprising DMAP, IPP and/or prenol and an FMN-dependent decarboxylase associated with an FMN prenyl transferase and an enzyme or enzymes of at least one of the following (v) to (vii):

(v) an enzyme catalyzing the enzymatic conversion of isopentenyl pyrophosphate (IPP) into said DMAPP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2);

(vi) an enzyme catalyzing the enzymatic conversion of dimethylallyl phosphate (DMAP) into said DMAPP, wherein said enzyme is a kinase, preferably an isopentenyl monophosphate kinase (EC 2.7.4.26); and

(vii) an enzyme catalyzing the enzymatic conversion of prenol into said DMAPP, wherein said enzyme is a diphosphotransferase (EC 2.7.6.-), preferably a thiamine diphosphokinase (EC 2.7.6.2) or a 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine diphosphokinase.

The present invention furthermore relates to any of the above-described compositions further comprising at least one of the above (v) to (vii), wherein additionally, said composition further comprises an enzyme catalyzing the enzymatic conversion of prenol into DMAP, wherein said enzyme is a kinase, preferably a phosphotransferase with an alcohol group as acceptor (EC 2.7.1.-), more preferably a hydroxyethylthiazole kinase (EC 2.7.1.50).

The present invention furthermore relates to any of the above-described compositions further comprising at least one of the above (v) to (vii), wherein additionally, said composition further comprises an enzyme catalyzing the enzymatic conversion of isopentenyl monophosphate (IMP) into DMAP, wherein said enzyme is an isomerase, preferably an isopentenyl-diphosphate DELTA isomerase (EC 5.3.3.2).

In a further aspect, the present invention relates to any of the above compositions which additionally further comprises an enzyme catalyzing the enzymatic conversion of riboflavin into flavin mononucleotide (FMN) as described herein above .

In a further aspect, the present invention relates to any of the above compositions which additionally comprises one or more of the enzymes described above for the method steps preceding the production of 3-methylcrotonic acid.

As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies to the compositions as has been set forth above for the methods according to the present invention.

Figure 1 : shows an artificial pathway for isobutene production from acetyl-CoA via

3-methylcrotonic acid. Moreover, enzymatic recycling of metabolites which may occur during the pathway are shown in steps Xa, Xb, XI and XII.

Figure 2A: Schematic reaction of the enzymatic prenylation of a flavin mononucleotide (FMN) into the corresponding modified (prenylated) flavin cofactor.

Figure 2B: Schematic reaction of the enzymatic conversion of 3-methylcrotonic acid into isobutene.

Figure 3: Chemical structure of DMAP and DMAPP.

Figure 4: Schematic reactions for the different routes for the provision of DMAP and to increase the DMAP pool.

Figure 5: Schematic reaction of the enzymatic conversion/dephosphorylation of

DMAPP into DMAP. Figure 6: Schematic reaction of the enzymatic conversion/dephosphorylation of DMAPP into DMAP by the formation ATP from ADP.

Figure 7: Schematic reaction of the enzymatic conversion/phosphorylation of prenol into DMAP.

Figure 8: Schematic reaction for the enzymatic conversion of DMAPP into prenol, the enzymatic conversion of prenol into DMAP as well as a preceding step of the enzymatic conversion of isopentenyl pyrophosphate into DMAPP.

Figure 9: illustrates the pathway for the biosynthesis of flavin mononucleotide

(FMN) starting from GTP.

Figure 10: Schematic reaction of the enzymatic conversion of 3-hydroxyisovalerate

(HIV) into 3-methylcrotonic acid.

Figure 11 : Schematic reaction of the enzymatic condensation of acetyl-CoA and acetone into 3-hydroxyisovalerate.

Figure 12: Schematic reaction of the enzymatic conversion of acetoacetate into acetone.

Figure 13: Schematic reaction of the enzymatic conversion of acetoacetyl-CoA into acetoacetate by hydrolysing the CoA thioester of acetoacetyl-CoA resulting in acetoacetate.

Figure 14: Schematic reaction of the enzymatic conversion of acetoacetyl-CoA into acetoacetate by transferring the CoA group of acetoacetyl-CoA on acetate, resulting in the formation of acetoacetate and acetyl-CoA.

Figure 15: Schematic reaction of the enzymatic conversion of 3-methylcrotonyl- CoA into 3-methylcrotonic acid.

Figure 16: Schematic reaction of the enzymatic conversion of 3-methylcrotonyl- CoA into 3-methylcrotonic acid via step Vic as shown in Figure 1.

Figure 17: Schematic reaction of the enzymatic conversion of 3-methylcrotonyl- CoA into 3-methylcrotonic acid via step Vlb as shown in Figure 1.

Figure 18: Schematic reaction of the enzymatic conversion of 3-methylcrotonyl- CoA into 3-methylcrotonic acid via step Via as shown in Figure 1.

Figure 19: Schematic illustration for the conversion of 3-methylcrotonyl-CoA into 3- methylcrotonic acid via 3-methylbutyryl-CoA and 3-methylbutyric acid.

Figure 20: Schematic reaction of the enzymatic conversion of 3-methylglutaconyl- CoA into 3-methylcrotonyl-CoA.

Figure 21 : Schematic reaction of the enzymatic conversion of 3-hydroxy-3- methylglutaryl-CoA into 3-methylglutaconyl-CoA.

Figure 22: Schematic reaction of the enzymatic condensation of acetylCoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA.

Figure 23: Schematic reaction of the enzymatic condensation of two molecules of acetyl-CoA into acetoacetyl-CoA.

Figure 24: Schematic reaction of the enzymatic conversion of acetyl-CoA into malonyl-CoA.

Figure 25: Schematic reaction of the enzymatic condensation of malonyl-CoA and acetyl-CoA into acetoacetyl-CoA.

Figure 26: shows enzymatic recycling steps of metabolites (steps Xa, Xb, XI and

XII as also shown in Figure 1 ) which may occur during the pathway of isobutene production from acetyl-CoA via 3-methylcrotonic acid.

Figure 27: Schematic reaction of the enzymatic conversion of 3-hydroxyisovalerate

(HIV) into 3-methylcrotonic acid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in 3- hydroxyisovaleryl-CoA.

Figure 28: Schematic reaction of the enzymatic conversion of 3-hydroxyisovalerate

(HIV) into 3-hydroxyisovaleryl-CoA.

Figure 29: Schematic reaction of the enzymatic conversion of 3-hydroxyisovaleryl- CoA into 3-methylcrotonyl-CoA.

Figure 30: Schematic reaction of the general enzymatic conversion of 3- hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA.

Figure 31 : Schematic reaction of the enzymatic conversion of 3-hydroxyisovalerate

(HIV) into 3-hydroxyisovaleryl-CoA via 3-hydroxyisovaleryl-adenosine monophosphate.

Figure 32: Schematic reaction of the enzymatic conversion of 3-hydroxyisovalerate

(HIV) into 3-hydroxyisovaleryl-CoA via 3-hydroxyisovaleryl phosphate.

Figure 33: Schematic reaction of the enzymatic conversion of 3-methyl-3-butenoic acid into isobutene.

Figure 34: Schematic reaction of the enzymatic conversion of 3-methyl-3-butenoyl- CoA into 3-methyl-3-butenoic acid.

Figure 35: Schematic reaction of the enzymatic conversion of 3-methyl-3-butenoyl- CoA into 3-methyl-3-butenoic acid by making use of a CoA-transferase.

Figure 36: Schematic reaction of the enzymatic conversion of 3-methyl-3-butenoyl- CoA into 3-methyl-3-butenoic acid by making use of a thioester hydrolase.

Figure 37: Schematic reaction of the enzymatic conversion of 3-methyl-3-butenoyl- CoA into 3-methyl-3-butenoic acid in a two-step reaction via 3-methyl-3- butenoyl phosphate.

Figure 38: Schematic reaction of the enzymatic conversion of 3-methylglutaconyl- CoA into 3-methyl-3-butenoyl-CoA.

Figure 39: Structure of a phosphopantetheine moiety.

Figure 40: shows an overlay of typical GC-chromatograms obtained for the catalytic assay of UbiD protein from Saccharomyces cerevisiae with the corresponding controls as outlined in Example 2.

Figure 41 : shows an overlay of typical chromatograms obtained for the production of isobutene from 3-methylcrotonic in a recombinant E. coli strain overexpressing UbiD protein from Saccharomyces cerevisiae and UbiX protein from Escherichia coli (strain A) or overexpressing UbiD protein from Saccharomyces cerevisiae alone (strain B) or carrying an empty vector (negative control, strain C).

Figure 42: shows bacterial growth and isobutene production without addition of external prenol.

a) Bacterial growth of the constructed E.coli strains.

b) Specific isobutene productivity obtained with the constructed E.coli strains.

Figure 43: shows bacterial growth and isobutene production with addition of external prenol.

a) Bacterial growth of the constructed E.coli strains.

b) Specific isobutene productivity obtained with the constructed E.coli strains.

Figure 44: shows the schematic reactions of the mevalonate pathway.

Figure 45: Schematic reactions for the different routes for the provision of DMAPP and to increase the DMAPP pool. Figure 46: Schematic reaction of the enzymatic conversion of DMAP into DMAPP.

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.

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

GENERAL METHODS AND MATERIALS

All reagents and materials used in the experiences were obtained from Sigma-Aldrich Company (St. Louis, MO) unless otherwise specified. Materials and methods suitable for growth of bacterial cultures and protein expression are well known in the art.

Example 1 : Gene synthesis, cloning and expression of recombinant proteins as used in the below Examples 2 to 5

The sequences of the studied enzymes were generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt®). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The gene thus synthesized was cloned in a pET-25b (+) expression vector (vectors were constructed by GeneArt®). Vector pCAN contained gene coding for UbiX protein (3-octaprenyl-4- hydroxybenzoate carboxy-lyase partner protein) from Escherichia coli (Uniprot Accession Number: P0AG03) was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection). Provided vector contained a stretch of 6 histidine codons after the methionine initiation codon.

Competent E. coli BL21 (DE3) cells (Novagen) were transformed with these vectors according to standard heat shock procedure. The transformed cells were grown with shaking (160 rpm) using ZYM-5052 auto-induction medium (Studier FW, Prot. Exp. Pur. 41 , (2005), 207-234) for 6h at 30°C and protein expression was continued at 18°C overnight (approximately 16 h). For the recombinant strain over-expressing UbiX from E. coli, 500 μΜ of Flavin Mononucleotide (FMN) were added to the growth medium. The cells were collected by centrifugation at 4°C, 10,000 rpm for 20 min and the pellets were stored at -80°C.

Protein purification and concentration

The pellets from 200 ml of cultured cells were thawed on ice and resuspended in 6 ml of 50 mM Tris-HCI buffer pH 7.5 containing 100 mM NaCI in the case of the recombinant strain overexpressing UbiX protein and in 6 ml of 50 mM Tris-HCI buffer pH 7.5, 10 mM MgCI 2 , 10 mM imidazole and 5 mM DTT in the case of the recombinant strain overexpressing UbiD protein. Twenty microliters of lysonase (Novagen) were added. Cells were then incubated 10 min at room temperature, returned to ice for 20 min and the lysis was completed by sonication 3 x15 seconds. The cellular lysate contained UbiX protein was reserved on ice. The bacterial extracts contained UbiD proteins 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 100 mM Tris-HCI buffer pH 7.5 containing 100 mM NaCI and 250 mM imidazole. Eluates were then concentrated, desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and enzymes were resuspended in 50 mM Tris-HCI buffer pH 7.5, containing 50 mM NaCI and 5 mM DTT.

The purity of proteins thus purified varied from 80% to 90 % as estimated by SDS- PAGE analysis. Protein concentration was determined by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific) and by Bradford assay (BioRad). Example 2: In vitro decarboxylation of 3-methylcrotonic acid into isobutene catalyzed by an association of lysate, containing UbiX protein, with purified UbiD protein.

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.

Two UbiD proteins (Table C) were purified according to the procedure described in Example 1.

Enzymatic assays were carried out in 2 ml glass vials (Interchim) under the following conditions:

50 mM Tris-HCI buffer pH 7.5

20 mM NaCI

10 mM MgCI 2

5 mM DTT

50 mM 3-methylcrotonic acid

1 mg/ml purified UbiD protein

50 μΙ lysate contained UbiX protein

Total volume of the assays were 300 μΙ.

A series of control assays were performed in parallel (Table C).

The vials were sealed and incubated for 120 min at 30°C. The assays were stopped by incubating for 2 min at 80°C and the isobutene formed in the reaction headspace was analysed by Gas Chromatography (GC) equipped with Flame Ionization Detector

(FID).

For the GC analysis, one ml of the headspace gas was separated in a Bruker GC- 450 system equipped with a GS-alumina column (30 m x 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. A significant production of isobutene from 3-methylcrotonic acid was observed in the combined assays (UbiD protein + UbiX protein). Incubation of lysate containing UbiX protein alone did not result in isobutene production. These data indicate that the two enzymes present in the assays cooperated to perform the decarboxylation of 3- methylcrotonic acid into isobutene. A typical chromatogram obtained in the assay with UbiD protein from Saccharomyces cerevisiae is shown on Figure 40.

Table C.

Example 3: In vivo decarboxylation of 3-methylcrotonic acid into isobutene catalyzed by an association of UbiX protein from Escherichia coli and UbiD protein from Saccharomyces cerevisiae.

The gene coding for UbiD protein from S. cerevisiae (Uniprot Accession Number: Q03034) was codon optimized for expression in E.coli and synthesized by GeneArt® (Life Technologies). This studied gene was then PCR amplified from the pMK-RQ vector (master plasmid provided by GeneArt) using forward primer with Ncol restriction site and a reverse primer, containing BamHI restriction site. The gene coding for UbiX protein from E.coli (Uniprot Accession Number: P0AG03) was amplified by PCR with a forward primer, containing Ndel restriction site and a reverse primer containing Kpnl restriction site. The previously described pCAN vector (Example 1 ) served as template for this PCR step. These two obtained PCR products (UbiD protein and UbiX protein) were cloned into pETDuet™-1 co- expression vector (Novagen). The constructed recombinant plasmid was verified by sequencing. Competent E. coli BL21 (DE3) cells (Novagen) were transformed with this vector according to standard heat shock procedure and plated out onto LB agar plates supplemented with ampicillin (0.1 mg/ml) (termed "strain A").

BL21(DE3) strain transformed with pET-25b(+) vector, carrying only the gene of UbiD protein from S. cerevisiae was also used in this study (termed "strain B"). BL21(DE3) strain transformed with an empty pET-25b(+) vector was used as a negative control in the subsequent assays (termed "strain C").

Single transformants were used to inoculate LB medium, supplemented with ampicillin, followed by incubation at 30°C overnight. 1 ml of this overnight culture was used to inoculate 300 ml of ZYM-5052 auto-inducing media (Studier FW (2005), local citation). The cultures were grown for 20 hours at 30°C and 160 rpm shaking.

A volume of cultures corresponding to OD600 of 30 was removed and centrifuged. The pellet was resuspended in 30 ml of MS medium (Richaud C, Mengin-Leucreulx D., Pochet S., Johnson EJ., Cohen GN. and Marliere P, The Journal of Biological Chemistry, 268, (1993), 26827-26835), containing glucose (45 g/L) and MgSO4 (1 mM) and supplemented with 10 mM 3-methylcrotonic acid. These cultures were then incubated in 160 ml bottles, sealed with a screw cap, at 30°C with shaking for 22 h. The pH value of the cultures was adjusted to 8.5 after 8 hours of incubation by using 30 % NH 4 OH.

After an incubation period, the isobutene produced in the headspace was analysed by Gas Chromatography (GC) equipped Flame Ionization Detector (FID). One ml of the headspace gas phase was separated and analysed according to the method described in Example 2.

No isobutene was formed with the control strain C carrying an empty vector. The highest production of isobutene was observed for the strain A over-expressing the both genes, UbiD protein from S. cerevisiae and UbiX protein from E.coli. A significant production of isobutene was observed for the strain B over-expressing UbiD protein alone. Thus, endogenous UbiX of E.coli can probably contribute to activate UbiD protein from S. cerivisiae (Figure 41 ). Example 4: In vitro screening of the UbiD proteins for the decarboxylation of 3- methylcrotonic acid into isobutene.

Several genes coding for UbiD protein were codon optimized for the expression in E.coli and synthesized by GeneArt® (Thermofisher). The corresponding enzymes were purified according to the procedure described in Example 1. The list of the studied enzymes is shown in Table D.

Enzymatic assays were carried out in 2 ml glass vials (Interchim) under the following conditions:

50 mM Tris-HCI buffer pH 7.5

20 mM NaCI

10 mM MgCI2

1 mM DTT

50 mM 3-methylcrotonic acid

1 mg/ml purified UbiD protein

100 μΙ lysate contained UbiX protein from E. coli

Total volume of the assays were 300 μΙ.

A series of control assays were performed in parallel, in which either no UbiD protein was added, or no enzymes were added (Table D).

The vials were sealed and incubated for 60 min at 30°C. The assays were stopped by incubating for 2 min at 80°C and the isobutene formed in the reaction headspace was analysed by Gas Chromatography (GC) equipped with Flame Ionization Detector (FID), according to the procedure described in Example 2.

The results of the GC analysis are shown in Table D. No isobutene production was observed in control reactions. These results show that all the UbiD proteins, studied under the conditions of this screening assay, were able to perform the decarboxylation of 3-methylcrotonic acid into isobutene in presence of E. coli cell lysate contained UbiX protein.

Table D.

Isobutene

Candidate UbiD protein Assay composition produced, arbitrary units

Saccharomyces cerevisiae

UbiD protein alone 9

(Uniprot Accession Number: Q03034) UbiD protein + Cell lysate

945 contained UbiX protein

UbiD protein alone 70

Sphaerulina musiva (Uniprot

Accession Number: M3DF95) UbiD protein + Cell lysate

3430 contained UbiX protein

UbiD protein alone 34

Penicillium roqueforti (Uniprot

Accession Number: W6QKP7) UbiD protein + Cell lysate

1890 contained UbiX protein

UbiD protein alone 60

Hypocrea atroviridis (Uniprot

Accession Number: G9NLP8) UbiD protein + Cell lysate

5200 contained UbiX protein

Fusarium oxysporum sp. UbiD protein alone 13 lycopersici (Uniprot Accession

Number: W9LTH3) UbiD protein + Cell lysate

1390 contained UbiX protein

Saccharomyces kudriavzevii UbiD protein alone 10

(Uniprot Accession Number:

J8TRN5) UbiD protein + Cell lysate

920 contained UbiX protein

«No UbiD control»: Cell lysate contained UbiX protein alone 0

Control without any enzymes 0

Example 5: Enzymatic decarboxylation of 3-methylcrotonic acid into isobutene catalyzed in the presence of a lysate containing UbiX protein and with purified decarboxylase.

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.

Proteins encoded by the aroY gene and one protein annotated as UbiD protein were produced according to the procedure described in Example 1.

Enzymatic assays were carried out in 2 ml glass vials (Interchim) under the following conditions:

50 mM potassium phosphate buffer pH 7.5

20 mM NaCI 10 mM gCI 2

5 mM DTT

50 mM 3-methylcrotonic acid

1 mg/ml purified AroY or UbiD protein

50 μΙ lysate contained UbiX protein

Total volume of the assays were 300 μΙ.

A series of control assays were performed in parallel (Table E).

The vials were sealed and incubated for 120 min at 30°C. The assays were stopped by incubating for 2 min at 80°C and the isobutene formed in the reaction headspace was analysed by Gas Chromatography (GC) equipped with Flame Ionization Detector

(FID).

For the GC analysis, one ml of the headspace gas was separated in a Bruker GC- 450 system equipped with a GS-alumina column (30 m x 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. A significant production of isobutene from 3-methylcrotonic acid was observed in the combined assays (AroY or UbiD protein + UbiX protein). Incubation of lysate containing UbiX protein alone did not result in isobutene production. These data indicate that the proteins encoded by aroY gene in association with UbiX protein can catalyze the decarboxylation of 3-methylcrotonic acid into isobutene.

Table E.

Assay composition Isobutene production, arbitrary units

AroY protein from K. pneumoniae

(Uniprot Acession Number : B9A9M6) +

10.5

lysate contained UbiX protein from E. coli

+ substrate

AroY protein from K. pneumoniae

(Uniprot Acession Number : B9A9M6) + 0

substrate

UbiD protein from E. cloacae (Uniprot

Acession Number : V3DX94) + lysate, 8

contained UbiX protein from E. coli + substrate

UbiD protein from E. cloacae (Uniprot

0

Acession Number : V3DX94) + substrate

AroY protein from Leptolyngbya sp.

(Uniprot Acession Number :

5.5

A0A0S3U6D8) + lysate, contained UbiX

protein from E. coli + substrate

AroY protein from Leptolyngbya sp.

(Uniprot Acession Number : 0

A0A0S3U6D8) + substrate

AroY protein from Phascolarctobacterium

sp. (Uniprot Acession Number :R6IIV6) +

5.5

lysate, contained UbiX protein from E.

coli + substrate

AroY protein from Phascolarctobacterium

sp. (Uniprot Acession Number :R6IIV6) + 0

substrate

Lysate contained UbiX protein from E.

0

coli + substrate

Example 6: Gene synthesis, cloning and expression of recombinant proteins as used in the below Examples 7 to 8.

Gene synthesis, cloning and expression of recombinant proteins

The sequences of the studied enzymes inferred from the genomes of microorganisms were generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt®). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The genes thus synthesized were cloned in a pET-25b(+) expression vector (vectors were constructed by GeneArt®), Competent E. coli BL21 (DE3) cells (Novagen) were transformed with these vectors according to standard heat shock procedure. The transformed cells were grown with shaking (160 rpm) using ZYM-5052 auto-induction medium (Studier FW, Prot. Exp. Pur. 41 , (2005), 207-234) for 20h at 30°C. The cells were then collected by centrifugation at 4°C, 10,000 rpm for 20 min and the pellets were stored at -80°C.

Protein purification and concentration

The pellets from 500 ml of culture cells were thawed on ice and resuspended in 5 ml of 50 mM Tris-HCI buffer pH 7.5 containing 500 mM NaCI, 10 mM MgCI 2 , 10 mM imidazole and 1 mM DTT. Fifty 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 x 30 seconds. The bacterial extracts were then clarified by centrifugation at 4°C, 10,000 rpm for 20 min. The clarified bacterial lysates were loaded onto a PROTINO-2000 Ni-NTA 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 Tris-HCI buffer pH 7.5 containing 300 mM NaCI, 200 mM imidazole. Eluates were then concentrated, desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and enzymes were resuspended in solution containing 50 mM Tris-HCI pH 7.5, containing 100 mM NaCI. Protein concentrations were quantified by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific). The purity of proteins was estimated by SDS-PAGE analysis.

Example 7: Conversion of D APP into DMAP catalyzed by isopentenyl phosphate kinases.

The genes coding for isopentenyl phosphate kinases were synthesized and the corresponding enzymes were further produced according to the procedure described in Example 6. The enzymatic assays were conducted in total reaction volume of 0.2 ml.

Standard reaction mixture contained:

50 mM Tris-HCI pH 7.5

20 mM dimethylallyl pyrophosphate (DMAPP) (Sigma-Aldrich)

20 mM ATP (Sigma-Aldrich)

5 mM MgCI 2

100 mM NaCI 1 mg/ml of purified isopentenyl phosphate kinases

The enzyme free control was performed in parallel. The assays were incubated for 16h hours at 34°C with shaking and stopped by adding half volume of acetonitrile (ice cold). Assays were then centrifuged and an aliquot of the clarified supernatant were transferred into a clean vial for LC/MS analysis.

HPLC analyses were performed using a 1260 Inifinity LC System (Agilent), equipped with column heating module and UV detector. 5 pi of samples were separated on Zorbax SB-Aq column (250 x 4.6 mm, 5 pm particle size, column temp. 30°C) with a mobile phase flow rate of 1.5 ml/min. The separation was performed using mixed A (H 2 0 containing 8.4 mM sulfuric acid) and B (acetonitrile) solutions in a linear gradient (0% B at initial time 0 min→70% B at 8 min). Commercial dimethylallyl phosphate (DMAP) (Sigma-Aldrich) was used as reference. In these conditions, the retention time of DMAP was 4.32 min.

Al the tested isopentenyl phosphate kinases (EC 2.7.4.26) were able to catalyze this conversion (Table F).

Table F

Example 8: Microorganisms with improved production of isobutene from 3- methylcrotonic acid.

This working example shows the production of isobutene by recombinant E. coli, expressing: (i) recombinant proteins, associated with isobutene production from 3- methylcrotonic acid (ii) different combinations of recombinant enzymes, associated with isobutene production from 3-methylcrotonic acid and enzymes to increase the pool of DMAP. Recombinant protein expression

The sequences of the studied enzymes inferred from the genomes of the corresponding microorganisms were generated by oligonucleotide concatenation to fit the codon usage of E. coli (Table G). All the genes were commercially synthesized by GeneArt® (Thermofisher), except the gene encoding for UbiX protein, which was directly amplified from the genomic DNA of E.coli MG1655.

Table G

A pETDuet™-271 co-expression vector (Novagen) was used for the expression of the different combinations of ubiX, FDC1V4, thiM, MJ0044. The following constructions were created (Table H, Table I).

Table H

Vector Strain number

pGB6346 Strain 1 , expressing recombinant pETDuet PT7 FDC1V4 PT7 UbiX FDC1V4 and UbiX proteins

pGB6580 Strain 2, , expressing recombinant pETDuet PT7 UbiDv4 PT7 UbiX- FDC1V4 and UbiX proteins and a

MJ0044 recombinant Isopentenyl phosphate

kinase MJ0044

pGB6389 Strain 3, expressing recombinant pETDuet PT7 UbiDv4 PT7 UbiX-thiM FDC1V4, and UbiX proteins and a

recombinant 4-methyl-5-(2-hydroxethyl) thiazole kinase thiM Λ _ Λ

Table I: Plasmids sequences used in this study

Plasmid Sequences

pGB6346 ctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgag caccgccgccgcaagga atggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatac ccacgccgaaacaa gcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatatag gcgccagcaaccgcac ctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatcgatctcg atcccgcgaaattaata cgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgttt aactttaagaaggagatatac catgagcagcaccacctataaaagtgaagcatttgatccggaaccgcctcatctgagctt tcgtagctttgttaatgcac tgcgtcaggatggggatctggtggatattaatgaaccggttgatccggatctggaagcag cagcaattacccgtctggt ttgtgaaaccgatgataaagcaccgctgtttaataacgtgattggtgcaaaagatggtct gtggcgtattctgggtgcac cggcaagcctgcgtgcgagcccgaaagaacgttttggtcgtctggcacgtcatctggcac tgcctccgaccgcaagc gcaaaagatattctggataaaatgctgagcgccaatagcattccgcctattgaaccggtt attgttccgaccggtccggt taaagaaaatagcattgaaggcgaaaacattgatctggaagccctgcctgcaccgatggt tcatcagagtgatggtg gcaagtatatcaatacctatggtatgcatgttatccagagtccggatggtgggtggacca attggagcattgcccgtgc aatggttagcggtaaacgtaccctggcaggtctggttattagtccgcagcatattcgtaa aattcaggatcagtggcgtg caattggccaagaagaaattccttgggcactggcatttggtgttccgcctctggcaatta tggcaagcagtatgccgatt ccggatggtgttagcgaagcaggttatgttggtgcaattgccggtgaaccgattaaactg gttaaatgcgataccaaca atctgtatgttccggcaaatagcgaaattgttctggaaggcaccctgagcaccaccaaaa tggcaccggaaggtccg tttggtgaaatgcatggttatgtttatccgggtgaaagccatccgggtccggtttatacc gttaacaaaattacctatcgca acaatgcaattctgccgatgagcgcatgtggtcgtctgaccgatgaaacccagaccatga ttccgaccctggcagca gcagaaattcgtcagctgtgtcagagggcaggtctgccgattaccgatgcatttgcaccg tttgttggtcaggcaacctg ggttgcactgaaagttgataccaaacgtctgcgtgcaatgaaaaccaatggtaaagcatt tgcaaaagcggttggtga tgttgtgtttacccagaaaccgggttttatgattcatcgtctgattctggttggtgatga tattgatgtgtatgacgataaagat gtgatgtgggcatttgctacccgttgtcgtccgggtacagatgaagttttttttgatgat gttcctggcttttggctgatcccgt atatgagtcatggtaatgccgaagcagtgaaaggtggtaaagttgttagtgatgcactgc tgaccgcagaatatacca ccggtaaagattgggaaagcgcagatttcaaaaacagctatccgaaacgtatccaggata aagttctgaatagctgg gaacgcctgggtttcaaaaaactggattaataaggatccgaattcgagctcggcgcgcct gcaggtcgacaagcttg cggccgcataatgcttaagtcgaacagaaagtaatcgtattgtacacggccgcataatcg aaattaatacgactcact ataggggaattgtgagcggataacaattccccatcttagtatattagttaagtataagaa ggagatatacatatgaaac gactcattgtaggcatcagcggtgccagcggcgcgatttatggcgtgcgcttattacagg ttctgcgcgatgtcacagat atcgaaacgcatctggtgatgagccaggcagcgcgccagaccttatccctcgaaacggat ttttctctgcgcgaagtg caggcattagccgatgtcacgcacgatgcgcgcgatattgccgccagcatctcttccggt tctttccagacgctgggga tggtgattttaccctgttcaatcaaaaccctttccggcattgtccatagctatactgatg gcttactgacccgtgcggcagat gtggtgctgaaagagcgtcgcccgttggtgctctgcgtgcgtgaaacaccattgcactta ggccatctgcgtttaatgac tcaggcggcagaaatcggtgcggtgattatgcctcccgttccggcgttttatcatcgccc gcaatcccttgatgatgtgat aaatcagacggttaatcgtgttcttgaccagtttgcgataacccttcctgaagatctctt tgcccgctggcagggcgcata ataaggtaccctcgagtctggtaaagaaaccgctgctgcgaaatttgaacgccagcacat ggactcgtctactagcg cagcttaattaacctaggctgctgccaccgctgagcaataactagcataaccccttgggg cctctaaacgggtcttgag gggttttttgctgaaaggaggaactatatccggattggcgaatgggacgcgccctgtagc ggcgcattaagcgcggc gggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcc tttcgctttcttcccttccttt ctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacc tcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgataga cggtttttcgccctttgacgt tggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaacccta tctcggtctattcttttgatttata agggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaa cgcgaattttaacaaaatatta acgtttacaatttctggcggcacgatggcatgagattatcaaaaaggatcttcacctaga tccttttaaattaaaaatgaa gttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaa tcagtgaggcacctatctcagc gatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgat acgggagggcttaccatctggc cccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaata aaccagccagccggaa gggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgtt gccgggaagctagagtaag tagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtc acgctcgtcgtttggtatggcttc attcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaa agcggttagctccttcggtc ctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcac tgcataattctcttactgtcatg ccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatag tgtatgcggcgaccgagttgc tcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctc atcattggaaaacgttctt cggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactc gtgcacccaactgatcttc agcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgc aaaaaagggaataagg gcgacacggaaatgttgaatactcatactcttcctttttcaatcatgattgaagcattta tcagggttattgtctcatgagcg gatacatatttgaatgtatttagaaaaataaacaaataggtcatgaccaaaatcccttaa cgtgagttttcgttccactga gcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgta atctgctgcttgcaaacaaaa aaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccg aaggtaactggcttcagcag agcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaa ctctgtagcaccgcctaca tacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctt accgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagc ttggagcgaacgacc tacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaaggg agaaaggcggacag gtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaa cgcctggtatcttta tagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg ggggcggagcctatggaaaaa cgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtt ctttcctgcgttatcccctgattctgt ggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccga gcgcagcgagtcagtg agcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatt tcacaccgcatatatggtgc actctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgc tacgtgactgggtcatggctg cgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcat ccgcttacagacaagc tgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgc gaggcagctgcggtaaa gctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagct cgttgagtttctccagaagcg ttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtca ctgatgcctccgtgtaaggggga tttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggtt actgatgatgaacatgccc ggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaa aaatcactcagggtca atgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgc gatgcagatccggaacat aatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagac cattcatgttgttgctcag gtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattc tgctaaccagtaaggcaacc ccgccagcctagccgggtcctcaacgacaggagcacgatcatgctagtcatgccccgcgc ccaccggaaggagct gactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagcta acttacattaattgcgttgc gctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggcc aacgcgcggggagaggc ggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctg attgcccttcaccgcctggcc ctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgttt gatggtggttaacggcgg gatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatgtccgcaccaac gcgcagcccggactcggt aatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaac gatgccctcattcagca tttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcg gctgaatttgattgcgagtgag atatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaa cagcgcgatttgctggt gacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataa tactgttgatgggtgtct ggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatgg catcctggtcatccag cggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgcttt acaggcttcgacgccgctt cgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgcc gcgacaatttgcgacggc gcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagt tgttgtgccacgcggtt gggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaac gtggctggcctggttcaccac gcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactgg tttcacattcaccaccctg aattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatg gtgtccgggatctcgacg

(SEQ ID NO:36)

pGB6580 caccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccac ggggcctgccacc atacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcg gtgatgtcggcgatat aggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtaga ggatcgagatcgatc tcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattccc ctctagaaataattttgttta actttaagaaggagatataccatgagcagcaccacctataaaagtgaagcatttgatccg gaaccgcctcatctgag ctttcgtagctttgttaatgcactgcgtcaggatggggatctggtggatattaatgaacc ggttgatccggatctggaagc agcagcaattacccgtctggtttgtgaaaccgatgataaagcaccgctgtttaataacgt gattggtgcaaaagatggt ctgtggcgtattctgggtgcaccggcaagcctgcgtgcgagcccgaaagaacgttttggt cgtctggcacgtcatctgg cactgcctccgaccgcaagcgcaaaagatattctggataaaatgctgagcgccaatagca ttccgcctattgaaccg gttattgttccgaccggtccggttaaagaaaatagcattgaaggcgaaaacattgatctg gaagccctgcctgcaccg atggttcatcagagtgatggtggcaagtatatcaatacctatggtatgcatgttatccag agtccggatggtgggtggac caattggagcattgcccgtgcaatggttagcggtaaacgtaccctggcaggtctggttat tagtccgcagcatattcgta aaattcaggatcagtggcgtgcaattggccaagaagaaattccttgggcactggcatttg gtgttccgcctctggcaatt atggcaagcagtatgccgattccggatggtgttagcgaagcaggttatgttggtgcaatt gccggtgaaccgattaaac tggttaaatgcgataccaacaatctgtatgttccggcaaatagcgaaattgttctggaag gcaccctgagcaccacca aaatggcaccggaaggtccgtttggtgaaatgcatggttatgtttatccgggtgaaagcc atccgggtccggtttatacc gttaacaaaattacctatcgcaacaatgcaattctgccgatgagcgcatgtggtcgtctg accgatgaaacccagacc atgattccgaccctggcagcagcagaaattcgtcagctgtgtcagagggcaggtctgccg attaccgatgcatttgca ccgtttgttggtcaggcaacctgggttgcactgaaagttgataccaaacgtctgcgtgca atgaaaaccaatggtaaa gcatttgcaaaagcggttggtgatgttgtgtttacccagaaaccgggttttatgattcat cgtctgattctggttggtgatgat attgatgtgtatgacgataaagatgtgatgtgggcatttgctacccgttgtcgtccgggt acagatgaagttttttttgatgat gttcctggcttttggctgatcccgtatatgagtcatggtaatgccgaagcagtgaaaggt ggtaaagttgttagtgatgca ctgctgaccgcagaatataccaccggtaaagattgggaaagcgcagatttcaaaaacagc tatccgaaacgtatcc aggataaagttctgaatagctgggaacgcctgggtttcaaaaaactggattaataaggat ccgaattcgagctcggcg cgcctgcaggtcgacaagcttgcggccgcataatgcttaagtcgaacagaaagtaatcgt attgtacacggccgcat aatcgaaattaatacgactcactataggggaattgtgagcggataacaattccccatctt agtatattagttaagtataag aaggagatatacatatgaaacgactcattgtaggcatcagcggtgccagcggcgcgattt atggcgtgcgcttattac aggttctgcgcgatgtcacagatatcgaaacgcatctggtgatgagccaggcagcgcgcc agaccttatccctcgaa acggatttttctctgcgcgaagtgcaggcattagccgatgtcacgcacgatgcgcgcgat attgccgccagcatctcttc cggttctttccagacgctggggatggtgattttaccctgttcaatcaaaaccctttccgg cattgtccatagctatactgatg gcttactgacccgtgcggcagatgtggtgctgaaagagcgtcgcccgttggtgctctgcg tgcgtgaaacaccattgc acttaggccatctgcgtttaatgactcaggcggcagaaatcggtgcggtgattatgcctc ccgttccggcgttttatcatc gcccgcaatcccttgatgatgtgataaatcagacggttaatcgtgttcttgaccagtttg cgataacccttcctgaagatct ctttgcccgctggcagggcgcataataaggtaccGAAGGAGATATACATATGCTGACCAT TCTGA

AACTGGGTGGTAGCATTCTGAGCGATAAAAATGTTCCGTATAGCATTAAATGGG

ACAACCTGGAACGTATCGCAATGGAAATCAAAAATGCCCTGGACTACTACAAAA

ATCAGAATAAAGAAATTAAACTGATTCTGGTGCATGGTGGTGGTGCATTTGGTCA

TCCGGTTGCCAAAAAATACCTGAAAATTGAGGACGGCAAAAAAATCTTTATTAAC

ATGGAAAAAGGCTTTTGGGAAATCCAGCGTGCAATGCGTCGTTTTAACAACATT

ATCATTGATACCCTGCAGAGCTATGATATTCCGGCAGTTAGCATTCAGCCGAGC

AGCTTTGTTGTTTTTGGTGATAAACTGATCTTTGACACCAGCGCCATTAAAGAAA

TGCTGAAACGTAATCTGGTTCCGGTGATTCATGGTGATATTGTGATTGATGATAA

AAATGGCTACCGCATCATTAGCGGTGATGATATTGTTCCGTATCTGGCCAATGA

ACTGAAAGCAGATCTGATTCTGTATGCCACCGATGTTGATGGTGTTCTGATTGAT

AACAAACCGATTAAACGCATTGATAAAAACAATATCTATAAAATCCTGAATTATCT

GAGCGGCAGCAACAGCATTGATGTTACCGGTGGTATGAAATACAAAATCGACAT

GATTCGCAAAAACAAATGCCGTGGCTTTGTGTTCAATGGCAATAAAGCCAACAA

C ATCT AT AAAG C ACTG CTG GGTG AAGTTG AAGG C ACCG AAATTG ATTTT AG CG A

ATAATAATTAATTAAcctaggctgctgccaccgctgagcaataactagcataacccc ttggggcctctaaac gggtcttgaggggttttttgctgaaaggaggaactatatccggattggcgaatgggacgc gccctgtagcggcgcatta agcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcg cccgctcctttcgctttctt cccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccc tttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgcc ctgatagacggtttttcgc cctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaaca ctcaaccctatctcggtctattct tttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaa caaaaatttaacgcgaattttaac aaaatattaacgtttacaatttctggcggcacgatggcatgagattatcaaaaaggatct tcacctagatccttttaaatta aaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacca atgcttaatcagtgaggcacc tatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagat aactacgatacgggagggcttac catctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttat cagcaataaaccagcca gccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatt aattgttgccgggaagcta gagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg tggtgtcacgctcgtcgtttggt atggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttg tgcaaaaaagcggttagctc cttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttat ggcagcactgcataattctctta ctgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct gagaatagtgtatgcggcgac cgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaa aagtgctcatcattggaa aacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgt aacccactcgtgcacccaa ctgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca aaatgccgcaaaaaaggg aataagggcgacacggaaatgttgaatactcatactcttcctttttcaatcatgattgaa gcatttatcagggttattgtctc atgagcggatacatatttgaatgtatttagaaaaataaacaaataggtcatgaccaaaat cccttaacgtgagttttcgtt ccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct gcgcgtaatctgctgcttgca aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactct ttttccgaaggtaactgg cttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccacca cttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataag tcgtgtcttaccgggttggac tcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcaca cagcccagcttggag cgaacgacctacaccgaactgagatacctacagcgtgagciatgagaaagcgccacgctt cccgaagggagaaa ggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcc agggggaaacgc ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtg atgctcgtcaggggggcggagcc tatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttg ctcacatgttctttcctgcgttatc ccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcag ccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatct gtgcggtatttcacaccgc atatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacac tccgctatcgctacgtgactg ggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtct gctcccggcatccgctt acagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcac cgaaacgcgcgaggcag ctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatcc gcgtccagctcgttgagtttct ccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcct gtttggtcactgatgcctccgtg taagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacg atacgggttactgatgat gaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgg gaccagagaaaaatc actcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccag cagcatcctgcgatgcag atccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacgg aaaccgaagaccattca tgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtat cggtgattcattctgctaaccag taaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgctagtca tgccccgcgcccacc ggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaat gagtgagctaacttaca ttaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcat taatgaatcggccaacgcgc ggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgg gcaacagctgattgcccttc accgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcga aaatcctgtttgatggtg gttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatg tccgcaccaacgcgcagc ccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatc gcagtgggaacgatgc cctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttccc gttccgctatcggctgaatttg attgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaa tgggcccgctaacag cgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttc atgggagaaaataatact gttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagc ttccacagcaatggcat cctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgt gcaccgccgctttacagg cttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgc gagatttaatcgccgcga caatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgact gtttgcccgccagttgt tgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgc gttttcgcagaaacgtggctg gcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcg tataacgttactggtttc acattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggtt ttgcgccattcgatggtgtcc gggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggtt gaggccgttgag

(SEQ ID NO:37)

pGB6389 caccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccac ggggcctgccacc atacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcg gtgatgtcggcgatat aggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtaga ggatcgagatcgatc tcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattccc ctctagaaataattttgttta actttaagaaggagatataccatgagcagcaccacctataaaagtgaagcatttgatccg gaaccgcctcatctgag ctttcgtagctttgttaatgcactgcgtcaggatggggatctggtggatattaatgaacc ggttgatccggatctggaagc agcagcaattacccgtctggtttgtgaaaccgatgataaagcaccgctgtttaataacgt gattggtgcaaaagatggt ctgtggcgtattctgggtgcaccggcaagcctgcgtgcgagcccgaaagaacgttttggt cgtctggcacgtcatctgg cactgcctccgaccgcaagcgcaaaagatattctggataaaatgctgagcgccaatagca ttccgcctattgaaccg gttattgttccgaccggtccggttaaagaaaatagcattgaaggcgaaaacattgatctg gaagccctgcctgcaccg atggttcatcagagtgatggtggcaagtatatcaatacctatggtatgcatgttatccag agtccggatggtgggtggac caattggagcattgcccgtgcaatggttagcggtaaacgtaccctggcaggtctggttat tagtccgcagcatattcgta aaattcaggatcagtggcgtgcaattggccaagaagaaattccttgggcactggcatttg gtgttccgcctctggcaatt atggcaagcagtatgccgattccggatggtgttagcgaagcaggttatgttggtgcaatt gccggtgaaccgattaaac tggttaaatgcgataccaacaatctgtatgttccggcaaatagcgaaattgttctggaag gcaccctgagcaccacca aaatggcaccggaaggtccgtttggtgaaatgcatggttatgtttatccgggtgaaagcc atccgggtccggtttatacc gttaacaaaattacctatcgcaacaatgcaattctgccgatgagcgcatgtggtcgtctg accgatgaaacccagacc atgattccgaccctggcagcagcagaaattcgtcagctgtgtcagagggcaggtctgccg attaccgatgcatttgca ccgtttgttggtcaggcaacctgggttgcactgaaagttgataccaaacgtctgcgtgca atgaaaaccaatggtaaa gcatttgcaaaagcggttggtgatgttgtgtttacccagaaaccgggttttatgattcat cgtctgattctggttggtgatgat attgatgtgtatgacgataaagatgtgatgtgggcatttgctacccgttgtcgtccgggt acagatgaagttttttttgatgat gttcctggcttttggctgatcccgtatatgagtcatggtaatgccgaagcagtgaaaggt ggtaaagttgttagtgatgca ctgctgaccgcagaatataccaccggtaaagattgggaaagcgcagatttcaaaaacagc tatccgaaacgtatcc aggataaagttctgaatagctgggaacgcctgggtttcaaaaaactggattaataaggat ccgaattcgagctcggcg cgcctgcaggtcgacaagcttgcggccgcataatgcttaagtcgaacagaaagtaatcgt attgtacacggccgcat aatcgaaattaatacgactcactataggggaattgtgagcggataacaattccccatctt agtatattagttaagtataag aaggagatatacatatgaaacgactcattgtaggcatcagcggtgccagcggcgcgattt atggcgtgcgcttattac aggttctgcgcgatgtcacagatatcgaaacgcatctggtgatgagccaggcagcgcgcc agaccttatccctcgaa acggatttttctctgcgcgaagtgcaggcattagccgatgtcacgcacgatgcgcgcgat attgccgccagcatctcttc cggttctttccagacgctggggatggtgattttaccctgttcaatcaaaaccctttccgg cattgtccatagctatactgatg gcttactgacccgtgcggcagatgtggtgctgaaagagcgtcgcccgttggtgctctgcg tgcgtgaaacaccattgc acttaggccatctgcgtttaatgactcaggcggcagaaatcggtgcggtgattatgcctc ccgttccggcgttttatcatc gcccgcaatcccttgatgatgtgataaatcagacggttaatcgtgttcttgaccagtttg cgataacccttcctgaagatct ctttgcccgctggcagggcgcataataaggtaccGAAGGAGATATACATATGCAGGTTGA TCTG

CTGGGTAGCGCACAGAGCGCACATGCACTGCACCTGTTTCATCAGCATAGTCC

GCTGGTTCATTGTATGACCAATGATGTTGTTCAGACCTTTACCGCAAATACCCTG

CTGGCACTGGGTGCAAGTCCGGCAATGGTTATTGAAACCGAAGAAGCAAGCCA

GTTTGCAGCAATTGCAAGCGCACTGCTGATTAATGTTGGCACCCTGACCCAGCC TCGTGCACAGGCAATGCGTGCAGCAGTTGAACAGGCAAAAAGCAGCCAGACCC

CGTGGACCCTGGACCCGGTTGCAGTTGGTGCACTGGATTATCGTCGTCATTTTT

GTCATGAACTGCTGAGCTTTAAACCGGCAGCAATTCGTGGTAATGCAAGCGAAA

TTATGGCACTGGCAGGTATTGCAAATGGTGGTCGTGGTGTTGATACCACCGATG

CAGCAGCAAATGCAATTCCGGCAGCACAGACCCTGGCACGTGAAACCGGTGCA

ATTGTTGTTGTTACCGGTGAAATGGATTATGTTACCGATGGTCATCGTATTATTG

GTATTCATGGTGGTGATCCGCTGATGACCAAAGTTGTTGGCACCGGTTGTGCAC

TGAGCGCAGTTGTTGCAGCATGTTGTGCACTGCCTGGTGATACCCTGGAAAATG

TTGCAAGCGCATGTCATTGGATGAAACAGGCAGGCGAACGTGCAGTTGCACGT

AGCGAAGGTCCGGGTAGCTTTGTTCCGCATTTTCTGGATGCACTGTGGCAGCT

GACCCAGGAAGTTCAGGCATAATAATTAATTAAcctaggctgctgccaccgctgagc aataact agcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaac tatatccggattggcgaat gggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtga ccgctacacttgccag cgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctt tccccgtcaagctctaaatcggg ggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatt agggtgatggttcacgtagtg ggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata gtggactcttgttccaaactgga acaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcg gcctattggttaaaaaatgagctg atttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttctggcggc acgatggcatgagattatcaa aaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagta tatatgagtaaacttggtctga cagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatc catagttgcctgactccccgtcg tgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgc gagacccacgctcaccg gctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcct gcaactttatccgcctc catccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagttt gcgcaacgttgttgccattgct acaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaa cgatcaaggcgagttacatg atcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaag taagttggccgcagtgttatc actcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgctt ttctgtgactggtgagtactcaa ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatac gggataataccgcgccac atagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa ggatcttaccgctgttgag atccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcac cagcgtttctgggtgagcaaaa acaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactc atactcttcctttttca atcatgattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgta tttagaaaaataaacaaatagg tcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa agatcaaaggatcttcttga gatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg gtggtttgtttgccggatcaa gagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatact gtccttctagtgtagccgta gttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcct gttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgc agcggtcgggctgaacg gggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagataccta cagcgtgagctatga gaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtc ggaacaggagag cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgc cacctctgacttgagcgtcg atttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctt tttacggttcctggccttttg ctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtat taccgcctttgagtgagctgatacc gctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgc ctgatgcggtattt tctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatc tgctctgatgccgcatagttaag ccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgcca acacccgctgacgcgcc ctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggag ctgcatgtgtcagaggtttt caccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaa gcgattcacagatgtct gcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctg ataaagcgggccatgttaagg gcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggg gtaatgataccgatgaaacgag agaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtg agggtaaacaactggcg gtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaat acagatgtaggtgttcc acagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctga cttccgcgtttccagac tttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgc agcagcagtcgcttcacgtt cgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgg gtcctcaacgacaggag cacgatcatgctagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctct caagggcatcggtcga gatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgct ttccagtcgggaaacctgtcg tgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgc cagggtggtttttcttttca ccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagca agcggtccacgctggtt tgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctg tcttcggtatcgtcgtatcc cactaccgagatgtccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcc cagcgccatctgatcg ttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttga aaaccggacatggcactcc agtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagc cagccagacgcagacgcgc cgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccag atgctccacgcccagt cgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatca agaaataacgccggaac attagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgat cagcccactgacgcgttgc gcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgac accaccacgctggcaccc agttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccaga ctggaggtggcaacgc caatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattca gctccgccatcgccgcttcc actttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtc tgataagagacaccggcat actctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctctt ccgggcgctatcatgccatacc gcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgact cctgcattaggaagcagccc agtagtaggttgaggccgttgag

I (SEQ ID NO:38)

A BL21(DE3) strain was transformed with the constructed vectors. The single transformants were used to inoculate LB medium, supplemented with ampicillin, followed by incubation at 30°C overnight. This overnight pre-cultures were then used to inoculate 0.5 L of batch medium in 1 L bioreactor so to obtain an initial OD 6 oo around of 0.05.

Bioreactor fermentation conditions

The fermentation assays were performed in 1 liter bioreactors (Multifors). The culture medium was composed of ZYM auto-induction medium (Studier FW, Prot. Exp. Pur. 41 , (2005), 207-234) complemented with 0,5 mM riboflavin, 10 g/L Glycerol, 2.5 g/L Glucose, 4 g/L lactose and ampicillin (0.1 g/L). During the phase of bacterial growth, the operational fermentation parameters were temperature 30°C, medium pH 6.8 (adjusting by NH 4 OH and Η 3 Ρ0 4 ), p0 2 20%. The phase of bacterial growth was conducted until OD 6 oo around of 20-30. The isobutene production was then initiated by modifying the fermentation parameters as:

- Temperature was increased to reach 34°C

- Glucose concentration was increased to 3 g/L and then maintained beyond 1 g/L during isobutene (IBN) production phase.

- 3-methylcrotonic acid was added to the culture medium at initial concentration of 25 mM and then maintained beyond 20 mM during IBN production phase.

- When the external prenol was added to the culture medium, initial concentration was 8 mM through a pulse addition. There was no further addition of prenol during IBN production phase.

The isobutene (IBN) production was analyzed continuously using a Prima Pro Process mass spectrometer (Thermo Scientific) calibrated with 0.5%mol isobutene in argon.

The results are shown in Figure 42 and Figure 43. As can be derived from the results, the over-expression of enzymes capable of increasing the pool of DMAP led to an increase in the production of isobutene.

Example 9: Assay for the formation of prenylated FMN by using either DMAP or DMAPP as co-substrate by different FMN prenyl transferases.

The following enzymes were used in this study (Table J).

Table J

Enzyme expression and production

The sequences of the studied enzymes were generated and cloned in a pET-25b (+) expression vector as described in Example 1. The enzymes were then expressed and purified according to the procedure from Example 1 , with the following modifications. The transformed cells were grown without added Flavin Mononucleotide. 50 mM phosphate pH7.5, containing 100 mM NaCI and 10% glycerol was used during protein purification instead of a Tris-HCI buffer. The purity of proteins was estimated to be around 90-95 % according to SDS-PAGE analysis.

Enzymatic biosynthesis of prenylated FMN

Standard assay mixture contained:

50 mM phosphate buffer pH 7.5 containing 100 mM NaCI.

10 mM dimethylallyl pyrophosphate (DMAPP) or 10 mM dimethylallyl phosphate (DMAP) (Sigma-Aldrich)

5 mM Flavin Mononucleotide (FMN)

10 mM sodium dithionite

All the components of the assay (buffer, FMN, DMAP or DMAPP, sodium dithionite) were made up as as stock solution, transferred into the anaerobic chamber (Whitley DG250 anaerobic workstation) and incubated for at least one hour. Enzymatic assays were typically performed in 1.5 mL Eppendorf opaque black microtubes (Dutscher with a total assay volume of 0.25 mL. Reactions were initiated by the addition of prenyl transferase (200 μΜ final concentration). The enzyme free controls were performed in parallel. The assays were incubated for 1 hour at 30°C. Then, the enzymes were removed from the incubation mixture by ultrafiltration using 10 kDa Amicon filter while being in the anaerobic chamber.

The supernatant containing prenylated FMN thus synthesized was diluted by adding half a volume of acetonitrile (ice cold). Assays were then centrifuged and an aliquot of the clarified supernatant were transferred into a clean vial for HPLC analysis.

HPLC analysis of prenylated FMN

The amount of prenylated FMN was determined by alkyl reverse phase using a 1260 Inifinity LC System (Agilent), equipped with a column heating module and a UV detector. 5 μΙ of samples were separated on Zorbax SB-Aq column (250 x 4.6 mm, 5 pm particle size, column temp. 30°C with a mobile phase flow rate of 1.5 ml/min. The separation was performed using mixed A (H 2 0 containing 8.4 mM sulfuric acid) and B (acetonitrile) solutions in a linear gradient (0% B at initial time 0 min→70% B at 8 min). FMN was used as reference to estimate the amount of produced prenylated FMN.

The consumption of DMAP or DMAPP as well as FMN was followed in parallel. In the described conditions, the retention time of FMN and prenylated FMN were 4.8 min and 5.7 min, respectively and the retention time of DMAPP and DMAP were 3.5 min and 4.4 min, respectively.

The amount of prenylated FMN formed in the enzymatic assays with DMAP and DMAPP are shown in the Table K. Tabel K

No prenylated FMN was observed in the control assays without enzymes either with DMAP or DMAPP as co-substrate.