METHOD FOR PRODUCING ISOBUTENE FROM 3-METHYLCROTONYL-COA

Abstract

Described is a method for the production of isobutene from 3-methylcrotonyl-CoA comprising the steps of: (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylbutyric acid; and (b) further enzymatically converting the thus produced 3-methylbutyric acid into isobutene.

The conversion of 3-methylcrotonyl-CoA into 3-methylbutyric acid can be achieved by first enzymatically converting 3-methylcrotonyl-CoA into 3-methyl butyryl-CoA and further enzymatically converting the thus produced 3-methylbutyryl-CoA into 3-methylbutyric acid. Alternatively, the conversion of 3-methylcrotonyl-CoA into 3-methylbutyric acid can be achieved by first enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid and then further enzymatically converting the thus produced 3-methylcrotonic acid into 3-methylbutyric acid.

Claims

1. A method for the production of isobutene from 3-methylcrotonyl-CoA comprising the steps of: (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylbutyric acid; and (b) further enzymatically converting the thus produced 3-methylbutyric acid into isobutene.

2. The method of claim 1, wherein step (a) is achieved by a method comprising the following steps: (a1) enzymatically converting 3-methylcrotonyl-CoA into 3-methylbutyryl-CoA; and (a2) further enzymatically converting the thus produced 3-methylbutyryl-CoA into 3-methylbutyric acid.

3. The method of claim 2, wherein step (a1) is achieved by the use of an enzyme classified in EC 1.3._._.

4. The method of claim 3, wherein the enzyme is selected from the group consisting of (i) an acyl-CoA dehydrogenase (NADP+) (EC 1.3.1.8); (ii) an enoyl-[acyl-carrier-protein] reductase (NADPH, Si-specific) (EC 1.3.1.10); (iii) a cis-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.37); (iv) a trans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38); (v) an enoyl-[acyl-carrier-protein] reductase (NADPH, Re-specific) (EC 1.3.1.39); (vi) crotonyl-CoA reductase (EC 1.3.1.86); (vii) an enoyl-[acyl-carrier-protein] reductase (NADH) (EC 1.3.1.9); (viii) a trans-2-enoyl-CoA reductase (NADH) (EC 1.3.1.44); and (ix) an isovaleryl-CoA dehydrogenase (EC 1.3.8.4).

5. The method of claim 2, wherein step (a2) is achieved by a hydrolysis reaction by use of a thioester hydrolase (EC 3.1.2).

6. The method of claim 2, wherein step (a2) is achieved by a transferase reaction by use of a CoA-transferase (EC 2.8.3).

7. The method of claim 2, wherein step (a2) is achieved by a method comprising the following steps: (i) enzymatically converting 3-methylbutyryl-CoA into 3-methylbutyryl phosphate; and (ii) further enzymatically converting the thus produced 3-methylbutyryl phosphate into 3-methylbutyric acid.

8. The method of claim 7, wherein step (i) is achieved by use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).

9. The method of claim 7, wherein step (ii) is achieved by use of a phosphotransferase (EC 2.7.2).

10. The method of claim 9, wherein the phosphotransferase (EC 2.7.2) is selected from the group consisting of a butyrate kinase (EC 2.7.2.7), a branched-chain-fatty-acid kinase (EC 2.7.2.14), a propionate kinase (EC 2.7.2.15) or an acetate kinase (EC 2.7.2.1).

11. The method of claim 1, wherein step (a) is achieved by a method comprising the following steps: (aI) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid; and (aII) further enzymatically converting the thus produced 3-methylcrotonic acid into 3-methylbutyric acid.

12. The method of claim 11, wherein step (aI) is achieved by a hydrolysis reaction by use of a thioester hydrolase (EC 3.1.2).

13. The method of claim 11, wherein step (aI) is achieved by a transferase reaction by use of a CoA-transferase (EC 2.8.3).

14. The method of claim 11, wherein step (aI) is achieved by a method comprising the following steps: (i) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and (ii) further enzymatically converting the thus produced 3-methylcrotonyl phosphate into 3-methylcrotonic acid.

15. The method of claim 14, wherein step (i) is achieved by use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).

16. The method of claim 14, wherein step (ii) is achieved by use of a phosphotransferase (EC 2.7.2).

17. The method of claim 16, wherein the phosphotransferase (EC 2.7.2) is selected from the group consisting of a butyrate kinase (EC 2.7.2.7), a branched-chain-fatty-acid kinase (EC 2.7.2.14), a propionate kinase (EC 2.7.2.15) or an acetate kinase (EC 2.7.2.1).

18. The method of claim 11, wherein the conversion of 3-methylcrotonic acid into 3-methylbutyric acid is achieved by use of a 2-enoate reductase (EC 1.3.1.31).

19. The method of claim 1, wherein step (b) is achieved by an oxidative decarboxylation catalyzed by a cytochrome P450 or by a non-heme iron oxygenase (UndA) or by a fatty acid desaturase (membrane-bound non-heme iron oxygenase or UndB).

Description

[0298] FIG. 1 shows schematically the reaction of the conversion of 3-methylcrotonyl-CoA into 3-methylbutyryl-CoA by making use of an enzyme classified as EC 1.3.1._ which uses NADPH as a co-factor.

[0299] FIG. 2 shows schematically the reaction of the conversion of 3-methylcrotonyl-CoA into 3-methylbutyryl-CoA by making use of an enzyme classified as EC 1.3.1._ which uses NADH as a co-factor.

[0300] FIG. 3 shows schematically the reaction of the conversion of 3-methylcrotonyl-CoA into 3-methylbutyryl-CoA by making use of an enzyme classified as EC 1.3.8._ which uses FADH as a co-factor.

[0301] FIG. 4 shows schematically the conversion of 3-methylbutyryl-CoA into 3-methylbutyric acid catalyzed by a thioesterase (EC 3.1.2).

[0302] FIG. 5 shows schematically the conversion of 3-methylbutyryl-CoA into 3-methylbutyric acid catalyzed by a CoA-transferase (EC 2.8.3).

[0303] FIG. 6 shows schematically the conversion of 3-methylbutyryl-CoA into 3-methylbutyryl phosphate and the subsequent conversion of 3-methylbutyryl phosphate into 3-methylbutyric acid.

[0304] FIG. 7 shows schematically the conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid catalyzed by a thioesterase (EC 3.1.2).

[0305] FIG. 8 shows schematically the conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid catalyzed by a CoA-transferase (EC 2.8.3).

[0306] FIG. 9 shows schematically the conversion of 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate and the subsequent conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid.

[0307] FIG. 10 shows schematically the conversion of 3-methylcrotonic acid into 3-methylbutyric acid by making use of an enzyme which is classified as a 2-enoate reductase (EC 1.3.1.31).

[0308] FIG. 11a shows schematically the conversion 3-methylbutyric acid into isobutene.

[0309] FIG. 11b shows schematically the conversion 3-methylbutyric acid into isobutene making use of NADPH as reducing agent and a flavodoxin/flavodoxin reductase as redox mediator protein.

[0310] FIG. 12 shows the conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA via a decarboxylation reaction.

[0311] FIG. 13 shows conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA.

[0312] FIG. 14 shows the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA.

[0313] FIG. 15 shows possible pathways for producing acetoacetyl-CoA from acetyl-CoA.

[0314] FIG. 16 shows a GC chromatogram obtained for the enzyme-catalyzed oxidative decarboxylation of isovalerate with the cytochrome P450 from Jeotgalicoccus sp. (Uniprot Accession number: E9NSU2) as outlined in Example 2.

[0315] FIG. 17 shows chromatograms of isobutene produced by an E. coli strain expressing cytochrome P450 from Jeotgalicoccus sp. and an E. coli control strain as outlined in Example 3.

[0316] FIG. 18 shows a GC chromatogram obtained for the enzyme-catalyzed oxidative decarboxylation of isovalerate with the cytochrome P450 from Macrococcus caseolyticus (Uniprot Accession number: B9EBA0) as outlined in Example 4.

[0317] FIG. 19 shows an example of a typical HPLC-chromatogram obtained for the enzymatic assay with acyl-CoA thioesterase II from Pseudomonas putida.

[0318] FIG. 20a shows an overlay of typical HPLC-chromatograms (analysis of 3-methylcrotonyl-CoA, 3-methylcrotonic acid and CoA-SH) obtained for [0319] a) enzymatic assay (assay A, Example 7) [0320] b) enzyme-free assay (assay H, Example 7). [0321] The consumption of 3-methylcrotonyl-CoA with simultaneous production of CoA-SH and 3-methylcrotonic acid was observed in the enzymatic assay combining phosphate butyryltransferase with butyrate kinase.

[0322] FIG. 20b shows an overlay of typical HPLC-chromatograms obtained for (analysis of ADP and ATP) obtained for [0323] a) enzymatic assay (assay A, Example 7) [0324] b) enzyme-free assay (assay H, Example 7). [0325] The consumption of ADP with simultaneous production of ATP was observed in the enzymatic assay combining phosphate butyryltransferase with butyrate kinase.

[0326] FIG. 21 shows the results of the production of 3-methylcrotonic acid and ATP in enzymatic assays comprising phosphate butyryltransferase from Bacillus subtilis combined with different butyrate kinases, as well as in different control assays.

[0327] FIG. 22 shows the results of the production of 3-methylcrotonic acid and ATP in enzymatic assays comprising phosphate butyryltransferase from Enterococcus faecalis combined with different butyrate kinases, as well as in different control assays.

[0328] FIG. 23 shows a GC/FID chromatogram for the conversion of isovalerate into isobutene catalyzed by fatty acid desaturase from Acinetobacter baumannii.

[0329] 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.

[0330] 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

[0331] General Methods and Materials

[0332] All reagents and materials used in the experiences were obtained from Sigma-Aldrich Company (St. Luis, Mo.) unless otherwise specified. Materials and methods suitable for growth of bacterial cultures and protein expression are well known in the art. Vector pCAN contained gene coding for flavodoxin reductase from E. coli (Uniprot P0A6L0) was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection). The provided vector contained a stretch of 6 histidine codons after the methionine initiation codon. Flavodoxin reductase thus cloned was overexpressed in E. coli BL21(DE3) strain and purified on PROTINO-2000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Fractions contained the enzyme of interest were pooled and concentrated on Amicon Ultra-4 10 kDa filter unit (Millipore). Flavodoxin reductase was then resuspended in 100 mM phosphate buffer pH 7.0, containing 100 mM NaCl to be used in subsequent enzymatic assays. Protein concentration was determined by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific).

Example 1: Cloning and Expression of Recombinant Cytochrome P450 Fatty Acid Decarboxylases

[0333] Gene Synthesis, Cloning and Expression of Recombinant Proteins

[0334] The sequences of the studied enzymes inferred from the genome of prokaryotic organisms 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).

[0335] BL21(DE3) competent cells (Novagen) were transformed with these vectors according to standard heat shock procedure and plated out onto LB agar plates supplemented with the appropriate antibiotic. Single transformants were used to inoculate 200 ml of ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234), supplemented with 0.5 mM aminolevulinic acid for cytochrome P450 expression. The cultures were incubated for 6 h at 30 C. in a shaker incubator and protein expression was continued at 18 C. overnight (approximately 16 h).

[0336] The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were stored at 80 C.

Example 2: In Vitro Oxidative Decarboxylation of Isovaleric Acid into Isobutene Catalyzed by Cytochrome P450 Fatty Acid Decarboxylase from Jeotgalicoccus sp.

[0337] The pellet from 200 ml of cultured cells was resuspended in 50 ml of lysis buffer (100 mM potassium phosphate pH 7, 100 mM KCl) supplemented with 20 l of lysonase (Merck-Novagen). Cell suspensions were then incubated for 10 minutes at room temperature followed by 20 minutes on ice. The amount of cytochrome P450 fatty acid decarboxylase in the total cell lysate was estimated on SDS-PAGE using gel densitometry.

[0338] 0.5 M stock solution of 3-methylbutyrate (isovalerate) was prepared in water and adjusted to pH 7.0 with 10 M solution of NaOH.

[0339] Enzymatic assays were set up in 2 ml glass vials (Interchim) in the following conditions:

[0340] 100 mM potassium phosphate buffer pH 7.0

[0341] 100 mM NaCl

[0342] 1 mM NADPH

[0343] 50 mM isovalerate

[0344] 0.2 mg/ml purified flavodoxin reductase from E. coli

[0345] Assays were started by adding the 30 l of cell lysate containing the recombinant P450 fatty acid decarboxylase from Jeotgalicoccus sp. (Uniprot Accession number: E9NSU2; SEQ ID NO: 1) (total volume 300 l).

[0346] A series of control assays were performed in parallel (Table 1).

[0347] The vials were sealed and incubated for 30 minutes at 30 C. The assays were stopped by incubating for 1 minute at 80 C. and the isobutene present in the reaction headspace was analysed by Gas Chromatography (GC) equipped with Flame Ionization Detector (FID).

[0348] For the GC headspace analysis, one ml of the headspace gas was separated in a Bruker GC-450 system equipped with a GS-alumina column (30 m0.53 mm) (Agilent) using isothermal mode at 130 C. Nitrogen was used as carrier gas with a flow rate of 6 ml/min.

[0349] The enzymatic reaction product was identified by comparison with an isobutene standard. Under these GC conditions, the retention time of isobutene was 2.23 min.

[0350] A significant production of isobutene from isovalerate was observed in the assay, contained cytochrome P450 fatty acid decarboxylase and redox partners (Table 1, FIG. 16).

TABLE-US-00001 TABLE 1 Isobutene peak area, Assay arbitrary units Enzymatic assay 147.9 Control assay without cytochrome P450 fatty acid 14.5 decarboxylase Control assay without flavodoxine reductase 16.8 Control assay without cytochrome P450 fatty acid 12.5 decarboxylase and without flavodoxine reductase Control assay without NADPH 8

Example 3: In Vivo Oxidative Decarboxylation of Isovaleric Acid into Isobutene Catalyzed by Cytochrome P450 Fatty Acid Decarboxylase from Jeotgalicoccus sp.

[0351] BL21(DE3) competent cells were transformed with pET-25b(+) expression vector, coding for cytochrome P450 fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456 and plated out onto LB agar plates supplemented with ampicillin (100 g/ml). BL21(DE3) strain transformed with empty pET-25b(+) vector was used as a negative control in the subsequent assays (control strain). Plates were incubated overnight at 30 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 F W (2005), local citation), supplemented with 0.5 mM aminolevulinic acid. The cultures were grown for 20 hours at 30 C. and 160 rpm shaking.

[0352] 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 E J., Cohen G N. and MarHere P, The Journal of Biological Chemistry, 268, (1993), 26827-26835), containing glucose (45 g/L) and MgSO.sub.4 (1 mM) and supplemented with 50 mM isovalerate. 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.sub.4OH.

[0353] 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.

[0354] FIG. 17 shows that a certain quantity of isobutene was produced with a control strain probably due to the spontaneous decomposition of isovalerate. The ratio of isobutene produced by E. coli strain, expressing P450 fatty acid decarboxylase, versus isobutene produced with a control strain was about 2.2 fold judging from isobutene peak areas (FIG. 17). These results clearly indicate that a production of isobutene from isovalerate can be achieved in vivo by E. coli strain expressing cytochrome P450 fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456.

Example 4: In Vitro Oxidative Decarboxylation of Isovaleric Acid into Isobutene Catalyzed by Cytochrome P450 Fatty Acid Decarboxylase from Macrococcus caseolyticus

[0355] The pellet from 200 ml of cultured cells was resuspended in 50 ml of lysis buffer (100 mM potassium phosphate pH 7, 100 mM KCl) supplemented with 20 l of lysonase (Merck-Novagen). Cell suspensions were then incubated for 10 minutes at room temperature followed by 20 minutes on ice. The amount of cytochrome P450 fatty acid decarboxylase in the total cell lysate was estimated on SDS-PAGE using gel densitometry.

[0356] 0.5 M stock solution of 3-methylbutyrate (isovalerate) was prepared in water and adjusted to pH 7.0 with 10 M solution of NaOH.

[0357] Enzymatic assays were set up in 2 ml glass vials (Interchim) in the following conditions:

[0358] 100 mM potassium phosphate buffer pH 7.0

[0359] 100 mM NaCl

[0360] 2 mM NADPH

[0361] 50 mM isovalerate

[0362] 0.2 mg/ml purified flavodoxin reductase from E. coli

[0363] Assays were started by adding 100 l of cell lysate containing the recombinant P450 fatty acid decarboxylase from Macrococcus caseolyticus (total volume 300 l).

[0364] A series of control assays were performed in parallel (Table 2).

[0365] The vials were sealed and incubated for 60 minutes at 30 C. The assays were stopped by incubating for 1 minute at 80 C. and the isobutene present in the reaction headspace was analysed by Gas Chromatography (GC) equipped with Flame Ionization Detector (FID).

[0366] For the GC headspace analysis, one ml of the headspace gas was separated in a Bruker GC-450 system equipped with a GS-alumina column (30 m0.53 mm) (Agilent) using isothermal mode at 130 C. Nitrogen was used as carrier gas with a flow rate of 6 ml/min.

[0367] The enzymatic reaction product was identified by comparison with an isobutene standard. Under these GC conditions, the retention time of isobutene was 2.45 min. A significant production of isobutene from isovalerate was observed in the assay, contained cytochrome P450 fatty acid decarboxylase and redox partners (Table 2, FIG. 18).

TABLE-US-00002 TABLE 2 Isobutene peak area, Assay arbitrary units Enzymatic assay 18 Control assay without cytochrome P450 fatty acid 2.5 decarboxylase Control assay without flavodoxine reductase 2.7 Control assay without cytochrome P450 fatty acid 2.5 decarboxylase and without flavodoxine reductase Control assay without NADPH 1.5

Example 5: Cloning and Overexpression of Recombinant Phosphate Butyryltransferases and Butyrate Kinases

[0368] Gene Synthesis, Cloning and Expression of Recombinant Proteins

[0369] The sequences of phosphate butyryltransferase genes from Bacillus subtilis (strain 168) and Enterococcus faecalis MTUP9 (Uniprot Accession number: P54530 and A0A038BNC2, respectively) and butyrate kinase from Lactobacillus casei W56 and Geobacillus sp. GHH01 (Uniprot Accession number: K0N529 and L8A0E1, respectively) were generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt). The expression of corresponding proteins was conducted following the method described in Example 1. The cells were collected by centrifugation at 4 C., 10.000 rpm for 20 min and the pellets were stored at 80 C.

[0370] Protein Purification and Concentration

[0371] The pellets from 200 ml of cultured cells were thawed on ice and resuspended in 5 ml of 50 mM potassium phosphate buffer pH 7.5 containing 100 mM NaCl, 10 mM MgCl.sub.2, 10 mM imidazole and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 230 seconds. The bacterial extracts were then clarified by centrifugation at 4 C., 4000 rpm for 40 min. The clarified bacterial lysates were loaded onto a PROTINO-2000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 6 ml of 50 mM potassium phosphate buffer pH 7.5 containing 100 mM NaCl and 250 mM imidazole. Eluates were then concentrated, desalted on an Amicon Ultra-4 10 kDa filter unit (Millipore) and enzymes were resuspended in 50 mM potassium phosphate buffer pH 7.5, containing 100 mM NaCl. The purity of proteins thus purified varied from 70% to 90% as estimated by SDS-PAGE analysis. Protein concentrations were determined by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific) or by Bradford assay (BioRad).

Example 6: Enzyme-Catalyzed Hydrolysis of 3-Methylcrotonyl-CoA into 3-Methylcrotonic Acid and Free Coenzyme-A

[0372] The gene coding for acyl-CoA thioesterase II from Pseudomonas putida was synthesized according to the procedure as described in Example 1.

[0373] The vector pCA24N which contained the gene encoding acyl-CoA thioesterase 2 (TesB) from Escherichia coli was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection). This vector rovided contained a stretch of 6 histidine codons after the methionine initiation codon.

[0374] The corresponding enzymes were produced according to the procedure described in Example 1.

[0375] The enzymatic assays were conducted in a total reaction volume of 0.2 ml.

[0376] The standard reaction mixture contained:

[0377] 50 mM HEPES pH 7.0

[0378] 10 mM 3-methylcrotonyl-CoA (Sigma-Aldrich)

[0379] 20 mM MgCl.sub.2

[0380] 20 mM NaCl

[0381] 1 mg/ml of purified recombinant thioesterase.

[0382] Control assays were performed in which either no enzyme was added or no substrate was added.

[0383] The assays were incubated for 30 min with shaking at 30 C., the reactions were stopped by the addition of 0.1 ml of acetonitrile and the samples were then analyzed by HPLC-based procedure.

[0384] HPLC Based Analysis of the Consumption of 3-Methylcrotonyl-CoA and the Formation of 3-Methylcrotonic Acid and Free Coenzyme A (CoA-SH)

[0385] HPLC analysis was performed using an 1260 Inifinity LC System (Agilent), equipped with a column heating module and a UV detector (210 nm). 5 l of samples were separated on Zorbax SB-Aq column (2504.6 mm, 5 m 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.sub.2O containing 8.4 mM sulfuric acid) and B (acetonitrile) solutions in a linear gradient (0% B at initial time 0 min.fwdarw.70% B at 8 min). Commercial 3-methylcrotonyl-CoA, 3-methylcrotonic acid (Sigma-Aldrich) and CoA-SH (Sigma-Aldrich) were used as references. In these conditions, the retention time of free coenzyme A (CoA-SH), 3-methylcrotonyl-CoA and 3-methylcrotonic acid were 4.05, 5.38 and 5.83 min, respectively.

[0386] No 3-methylcrotonic acid signal was observed in control assays.

[0387] Both studied thioesterases catalyzed the hydrolysis of 3-methylcrotonyl-CoA with the formation of 3-methylcrotonic acid. An example of a chromatogram obtained with acyl-CoA thioesterase II from Pseudomonas putida is shown in FIG. 19.

[0388] The degree of production of 3-methylcrotonic acid as observed in the enzymatic assays is shown in Table 3.

TABLE-US-00003 TABLE 3 3-METHYL- UNIPROT CROTONIC ACID GENE ACCESSION PRODUCED, NAMES ORGANISM NUMBER MM TESB ESCHERICHIA COLI P0AGG2 0.6 TESB PSEUDOMONAS Q88DR1 3.1 PUTIDA

Example 7: Conversion of 3-Methylcrotonyl-CoA and ADP into 3-Methylcrotonic Acid and ATP Catalysed by the Combined Action of Phosphate Butyryltransferase from Bacillus subtilis and Butyrate Kinase from Lactobacillus casei or Geobacillus sp.

[0389] In the assays described in the following, the following enzymes were used:

TABLE-US-00004 TABLE 4 UNIPROT GENE ACCESSION NAMES ORGANISM NUMBER PTB (YQIS) BACILLUS SUBTILIS P54530 BUK LACTOBACILLUS CASEI K0N529 BUK GEOBACILLUS SP. L8A0E1

[0390] The enzymatic assays were conducted in a total reaction volume of 0.2 ml.

[0391] The standard reaction mixture contained:

[0392] 50 mM potassium phosphate buffer pH 7.5

[0393] 4 mM 3-methylcrotonyl-CoA

[0394] 4 mM ADP

[0395] 10 mM MgCl.sub.2

[0396] 10 mM NaCl

[0397] 0.2 mg/ml of purified phosphate butyryltransferase from Bacillus subtilis

[0398] 0.2 mg/ml of purified butyrate kinase from Lactobacillus casei or Geobacillus sp.

[0399] A series of controls was performed in parallel (Assays CH as shown in Table 5).

TABLE-US-00005 TABLE 5 Assay composition A B C D E F G H 3-methylcrotonyl-CoA + + + + + + + + ADP + + + + + + phosphate butyryltransferase + + + + + from Bacillus subtilis butyrate kinase from + + + Lactobacillus casei butyrate kinase from + + + Geobacillus sp

[0400] Assays were then incubated for 20 min with shaking at 30 C.

[0401] After an incubation period, the reactions were stopped by heating the reaction medium 4 min at 90 C. The samples were centrifuged, filtered through a 0.22 m filter and the clarified supernatants were transferred into a clean vial for further analysis. The consumption of ADP and 3-methylcrotonyl-CoA, and the formation of ATP, 3-methylcrotonic acid and free coenzyme A (CoA-SH) were followed by using HPLC-based methods.

[0402] HPLC-Based Analysis of ADP and ATP

[0403] HPLC analysis was performed using the 1260 Inifinity LC System (Agilent) equipped with a column heating module and an RI detector. 2 l of samples were separated on a Polaris C18-A column (1504.6 mm, 5 m particle size, column temp. 30 C.) with a mobile phase flow rate of 1.5 ml/min. The consumption of ADP and the formation of ATP were followed by HPLC.

[0404] HPLC-Based Analysis of ADP and ATP

[0405] HPLC analysis was performed using 1260 Inifinity LC System (Agilent), equipped with column heating module and RI detector. 2 l of samples were separated on Polaris C18-A column (1504.6 mm, 5 m particle size, column temp. 30 C.) with a mobile phase flow rate of 1.5 ml/min. The separation was performed using 8.4 mM sulfuric acid in H.sub.2O/MeOH mixed solution (99/1) (V/V). In these conditions, the retention time of ADP and ATP were 2.13 min, 2.33 min, respectively (see FIG. 20b).

[0406] HPLC Based Analysis of 3-Methylcrotonyl-CoA, 3-Methylcrotonic Acid and Free Coenzyme a (CoA-SH)

[0407] HPLC analysis was performed using the 1260 Inifinity LC System (Agilent) equipped with a column heating module and a UV detector (260 nm). 1 l of samples were separated on a Zorbax SB-Aq column (2504.6 mm, 5 m particle size, column temp. 30 C.), with a mobile phase flow rate of 1.5 ml/min.

[0408] The consumption of 3-methylcrotonyl-CoA and the formation of 3-methylcrotonic acid and free coenzyme A (CoA-SH) were followed according to the procedure described in Example 6. Under these conditions, the retention time of 3-methylcrotonyl-CoA, 3-methylcrotonic acid and free coenzyme A (CoA-SH) was 5.38 min, 5.73 min and 4.07 min, respectively.

[0409] Typical chromatograms obtained for the enzymatic assay A and enzyme-free assay H are shown in FIGS. 20a and 20b. The results of the HPLC analysis are summarized in FIG. 21.

[0410] The obtained data indicate that 3-methylcrotonyl-CoA was converted into 3-methylcrotonic acid with the concomitant generation of ATP from ADP in a two-step reaction, catalyzed by two enzymes, respectively (assays A and B). Thus, the conversion occurred through the formation of the intermediate 3-methylcrotonyl phosphate followed by transfer of a phosphate group from this intermediate to ADP, thereby releasing ATP.

[0411] A significant production of 3-methylcrotonic acid without a simultaneous generation of ATP was observed when phosphate butyryltransferase was used alone (assay E). This production is due to a spontaneous hydrolysis of 3-methylcrotonyl phosphate generated by the action of phosphate butyryltransferase.

[0412] The production of 3-methylcrotonic acid was observed in the same manner for the control assays without ADP (assays C and D). This production was also due to a hydrolysis of 3-methylcrotonyl phosphate generated by the action of phosphate butyryltransferase.

Example 8: Conversion of 3-Methylcrotonyl-CoA and ADP into 3-Methylcrotonic Acid and ATP Catalysed by the Combined Action of the Phosphate Butyryltransferase from Enterococcus faecalis and Butyrate Kinase from Lactobacillus casei or Geobacillus sp.

[0413] In the assays described in the following, the following enzymes were used:

TABLE-US-00006 TABLE 6 UNIPROT GENE ACCESSION NAMES ORGANISM NUMBER PTB ENTEROCOCCUS FAECALIS A0A038BNC2 BUK LACTOBACILLUS CASEI K0N529 BUK GEOBACILLUS SP. L8A0E1

[0414] The enzymatic assays were conducted in a total reaction volume of 0.2 ml.

[0415] The standard reaction mixture contained:

[0416] 50 mM potassium phosphate buffer pH 7.5

[0417] 4 mM 3-methylcrotonyl-CoA

[0418] 4 mM ADP

[0419] 10 mM MgCl.sub.2

[0420] 10 mM NaCl

[0421] 0.2 mg/ml of purified phosphate butyryltransferase from Enterococcus faecalis

[0422] 0.2 mg/ml of purified butyrate kinase from Lactobacillus casei or Geobacillus sp.

[0423] A series of controls was performed in parallel (Assays CH Table 7).

TABLE-US-00007 TABLE 7 Assay composition A B C D E F G H 3-methylcrotonyl-CoA + + + + + + + + ADP + + + + + + phosphate butyryltransferase + + + + + from Enterococcus faecalis butyrate kinase from + + + Lactobacillus casei butyrate kinase from + + + Geobacillus sp

[0424] Assays were then incubated for 20 min with shaking at 30 C.

[0425] After an incubation period, the reactions were stopped by heating the reaction medium 4 min at 90 C. The samples were centrifuged, filtered through a 0.22 m filter and the clarified supernatants were transferred into a clean vial for further analysis. The consumption of ADP and 3-methylcrotonyl-CoA and the formation of ATP and 3-methylcrotonic acid and free coenzyme A (CoA-SH) were followed by HPLC analysis according to the methods described in Example 7 and Example 6.

[0426] The results of the HPLC analysis are summarized in FIG. 22.

[0427] The obtained data indicate that the 3-methylcrotonyl-CoA was converted into 3-methylcrotonic acid with the concomitant generation of ATP from ADP in a two-step reaction, catalyzed by two enzymes, respectively (assays A and B). Thus, the conversion occurred through the formation of the intermediate 3-methylcrotonyl phosphate followed by the transfer of a phosphate group from this intermediate on ADP, thereby releasing ATP.

[0428] A significant production of 3-methylcrotonic acid without simultaneous generation of ATP was observed when phosphate butyryltransferase was used alone (assay E). This production was due to a hydrolysis of 3-methylcrotonyl phosphate generated by the action of phosphate butyryltransferase.

[0429] The production of 3-methylcrotonic acid was observed in a similar manner for the control assays without ADP (assays C and D). This production was also due to a hydrolysis of 3-methylcrotonyl phosphate generated by the action of phosphate butyryltransferase.

Example 9: In Vivo Bioconversion of Isovalerate into Isobutene Catalyzed by Fatty Acid Desaturases (UndB)

[0430] All reagents and materials used in the experiences were obtained from Sigma-Aldrich Company (St. Luis, Mo.) unless otherwise specified. Materials and methods suitable for growth of bacterial cultures and protein expression are well known in the art.

[0431] Gene Synthesis, Cloning and Expression of Recombinant Proteins

[0432] The sequences of the studied enzymes inferred from the genome of prokaryotic organisms were generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt) (Table 8). The genes thus synthesized were cloned in a pET-25b(+) expression vector (vectors were constructed by GeneArt).

[0433] BL21(DE3) competent cells (Novagen) were transformed with these vectors according to standard heat shock procedure and plated out onto LB agar plates supplemented with the appropriate antibiotic. BL21(DE3) strain transformed with empty pET-25b(+) vector was used as a negative control in the subsequent assays (control strain). 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 F W, Prot. Exp. Pur. 41, (2005), 207-234. The cultures were grown for 20 hours at 30 C. and 160 rpm shaking.

[0434] 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 E J., Cohen G N. and MarHere P, The Journal of Biological Chemistry, 268, (1993), 26827-26835), containing glucose (45 g/L) and MgSO.sub.4 (1 mM) and supplemented with 40 mM isovalerate (3-methylbutyrate). These cultures were then incubated in 160 ml bottles, sealed with a screw cap, at 30 C. with shaking for 24 h. The pH value of the cultures was adjusted to 8.5 after 8 hours of incubation by using 30% NH.sub.4OH.

[0435] After an incubation period, the isobutene produced in the headspace was analysed by Gas Chromatography (GC) equipped Flame Ionization Detector (FID).

[0436] For the GC headspace analysis, one ml of the headspace gas was separated in a Bruker GC-450 system equipped with a GS-alumina column (30 m0.53 mm) (Agilent) using isothermal mode at 130 C. Nitrogen was used as carrier gas with a flow rate of 6 ml/min.

[0437] The enzymatic reaction product was identified by comparison with an isobutene standard. Under these GC conditions, the retention time of isobutene was 2.30 min. A significant production of isobutene from isovalerate was, e.g., observed with strains expressing fatty acid desaturases from Pseudomonas mendocina and Acinetobacter baumanii (Table 8). A typical chromatogram of isobutene production catalyzed by fatty acid decarboxylase from Acinetobacter baumanii is shown on FIG. 23.

TABLE-US-00008 TABLE 8 Uniprot isobutene peak accession number Organism area V .Math. min1 A4Y0K1 Pseudomonas mendocina 1016 A0A0D5YLX9 Acinetobacter baumannii 3637 negative control (empty vector) 0