Production of alkenes by enzymatic decarboxylation of 3-hydroxyalkanoic acids

Abstract

The present invention relates to a method for generating alkenes biologically. It relates more particularly to a method for producing terminal alkenes by enzymatic decarboxylation of 3-hydroxyalkanoate molecules. The invention also relates to the enzymatic systems and the microbial strains used, and also to the products obtained.

Claims

1. A method for producing a terminal alkene, comprising converting a 3-hydroxyalkanoate into a terminal alkene in a microorganism comprising a recombinantly expressed mevalonate diphosphate (MDP) decarboxylase enzyme in the presence of a co-substrate containing a phosphoanhydride bond, wherein the 3-hydroxyalkanoate is a molecule comprising 3-hydroxypropionate as a common motif and optionally one or two alkyl substitutions on carbon 3.

2. The method according to claim 1, wherein the terminal alkene comprises a linear or branched alkyl group at carbon 2.

3. The method according to claim 1, wherein the 3-hydroxyalkanoate is 3-hydroxybutyrate and the terminal alkene is propylene.

4. The method according to claim 1, wherein the 3-hydroxyalkanoate is 3-hydroxyvalerate and the terminal alkene is 1-butylene.

5. The method according to claim 1, wherein the 3-hydroxyalkanoate is 3-hydroxy-3-methylbutyrate and the terminal alkene is isobutylene.

6. The method according to claim 1, wherein the 3-hydroxyalkanoate is 3-hydroxy-3-methylvalerate and the terminal alkene is isoamylene.

7. The method according to claim 1, wherein the MDP decarboxylase enzyme comprises an amino acid sequence selected from SEQ ID NO: 1-16.

8. The method according to claim 1, wherein the method further comprises adding a cofactor containing a phosphoanhydride motif to the reaction, wherein the cofactor has the general formula ROP0.sub.2HOP0.sub.3H.sub.2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

9. The method according to claim 1, further comprising adding a methylene diphosphonate monoester to the reaction, wherein the methylene diphosphonate monoester has the general formula ROP0.sub.2HCH.sub.2P0.sub.3H.sub.2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

10. The method according to claim 1, wherein the microorganism overexpresses said MDP decarboxylase enzyme.

11. The method according to claim 1, wherein the method is carried out by a microorganism which endogenously produces one or more 3-hydroxyalkanoates, and which also expresses or overexpresses said MDP decarboxylase enzyme, so as to produce terminal alkenes directly from a carbon source.

12. The method according to claim 11, wherein the microorganism is a bacterium of strain Alcaligenes eutrophus or Bacillus megaterium, or a bacterium, yeast or fungus which recombinantly overproduces one or more 3-hydroxyalkanoates.

13. The method according to claim 11, wherein the carbon source is glucose or any other hexose, xylose or any other pentose, glycerol or any other polyol, starch, cellulose, hemicellulose, a poly-3-hydroxyalkanoate or any other polymer, the method then being carried out in the presence of a system for degrading said polymer to monomer.

14. The method according to claim 11, wherein the microorganism is a photosynthetic microorganism which endogenously produces one or more 3-hydroxyalkanoates, and further overexpressing the MDP decarboxylase enzyme, so as to produce terminal alkenes directly from CO.sub.2 present in solution.

15. The method according to claim 1, wherein the 3-hydroxyalkanoate is produced by a first microorganism that converts a carbon source to 3-hydroxyalkanoate, and the conversion is catalyzed by said MDP decarboxylase enzyme which is isolated or expressed by a second microorganism, allowing the conversion of the 3-hydroxyalkanoate to the terminal alkene.

16. A method according to claim 1, further comprising a step of collecting gas of terminal alkenes degassing from the reaction.

17. The method according to claim 1, wherein the method is carried out in microaerophilic conditions.

18. The method of claim 1, wherein the co-substrate is adenosine triphosphate (ATP), a ribonucleoside triphosphate (rNTP), a deoxyribonucleoside triphosphate (dNTP) or a mixture of several of such triphosphates, a polyphosphate, or a pyrophosphate.

19. The method according to claim 5, wherein the MDP decarboxylase enzyme comprises an amino acid sequence selected from SEQ ID NO: 1-16.

20. The method of claim 19, wherein the method further comprises adding a cofactor containing a phosphoanhydride motif to the reaction, wherein the cofactor has the general formula ROP0.sub.2HOP0.sub.3H.sub.2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

21. The method according to claim 19, further comprising adding a methylene diphosphonate monoester to the reaction, wherein the methylene diphosphonate monoester has the general formula ROP0.sub.2HCH.sub.2P0.sub.3H.sub.2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

22. The method according to claim 19, wherein the microorganism overexpresses said MDP decarboxylase enzyme.

23. The method according to claim 19, wherein the method is carried out by a microorganism which endogenously produces 3-hydroxy-3-methylbutyrate, and which also expresses or overexpresses said MDP decarboxylase enzyme, so as to produce isobutylene directly from a carbon source.

24. The method according to claim 23, wherein the microorganism is a bacterium of strain Alcaligenes eutrophus or Bacillus megaterium, or a bacterium, yeast or fungus which recombinantly overproduces 3-hydroxy-3-methylbutyrate.

25. The method according to claim 23, wherein the carbon source is glucose or any other hexose, xylose or any other pentose, glycerol or any other polyol, starch, cellulose, hemicellulose, a poly-3-hydroxyalkanoate or any other polymer, the method then being carried out in the presence of a system for degrading said polymer to monomer.

26. The method according to claim 23, wherein the microorganism is a photosynthetic microorganism which endogenously produces 3-hydroxy-3-methylbutyrate, and further overexpressing the MDP decarboxylase enzyme, so as to produce isobutylene directly from CO.sub.2 present in solution.

27. The method according to claim 19, wherein 3-hydroxy-3-methylbutyrate is produced by a first microorganism that converts a carbon source to 3-hydroxy-3-methylbutyrate, and the conversion is catalyzed by said MD P decarboxylase enzyme which is isolated or expressed by a second microorganism, allowing the conversion of the 3-hydroxy-3-methylbutyrate to isobutylene.

28. A method according to claim 19, further comprising a step of collecting gas of isobutylene degassing from the reaction.

29. The method according to claim 19, wherein the method is carried out in microaerophilic conditions.

30. The method of claim 19, wherein the co-substrate is adenosine triphosphate (ATP), a ribonucleoside triphosphate (rNTP), a deoxyribonucleoside triphosphate (dNTP) or a mixture of several of such triphosphates, a polyphosphate, or a pyrophosphate.

31. The method of claim 2, wherein the MDP decarboxylase enzyme comprises an amino acid sequence selected from SEQ ID NO: 1-16.

32. The method of claim 3, wherein the MDP decarboxylase enzyme comprises an amino acid sequence selected from SEQ ID NO: 1-16.

33. The method of claim 4, wherein the MDP decarboxylase enzyme comprises an amino acid sequence selected from SEQ ID NO: 1-16.

34. The method of claim 6, wherein the MDP decarboxylase enzyme comprises an amino acid sequence selected from SEQ ID NO: 1-16.

Description

LEGENDS OF DRAWINGS

(1) FIG. 1: 3-hydroxypropionate motif.

(2) FIG. 2: Decarboxylation of mevalonate diphosphate by MDP decarboxylasegeneric activity.

(3) FIG. 3: Examples of 3-hydroxyalkanoates.

(4) FIG. 4: Use of MDP decarboxylase for producing terminal alkenes.

(5) FIG. 5: Cofactors that can be used in the reaction for purposes of structural complementation in the catalytic site.

(6) FIG. 6: Integral method for producing an alkene from glucose.

(7) FIG. 7: Chromatogram of the enzymatic reactions carried out in condition No. 1 of example 4.

(8) FIG. 8: SDS-PAGE of the overexpression and purification steps of the enzyme SEQ ID NO: 6.

(9) 1. Markers

(10) 2. Culture before induction

(11) 3. Lysate

(12) 4. Fraction not adsorbed on the column

(13) 5. Column wash fraction

(14) 6. Purified enzyme, MW 36.8 kDa

(15) FIG. 9: GC/MS chromatographic analysis of the conversion of HIV to IBN.

(16) 1 and 2: Negative controls corresponding to background noise in absence of enzyme.

(17) 3 and 4: Reactions in presence of enzyme SEQ ID NO: 6.

(18) FIG. 10: Ratio of IBN production in presence and absence of ATP.

(19) FIG. 11: Ratio of IBN production in presence and absence of Mg.sup.2+.

(20) FIG. 12: Enzymatic activity according to temperature. Ratio: amount of IBN formed in presence of enzyme versus background.

(21) FIG. 13: IBN production according to concentration of HIV substrate.

(22) FIG. 14: Measurement of optimized reaction and comparison with background. Measured by gas chromatography with flame ionization detection.

(23) FIG. 15: Improved expression level by optimization of the nucleotide sequence coding SEQ ID NO: 6. Lane M: molecular weight markers.

(24) Lanes 1, 2, 3: native nucleotide sequence

(25) (1) Cell lysate, soluble fraction loaded on purification column

(26) (2) Lysate fraction not retained on purification column

(27) (3) Eluted fraction: 10 g purified enzyme

(28) Lanes 4, 5, 6: Optimized nucleotide sequence

(29) (4) Cell lysate, soluble fraction loaded on purification column

(30) (5) Lysate fraction not retained on purification column

(31) (6) Eluted fraction: 10 g purified enzyme

EXAMPLES

Example 1: Cloning and Expression of Several MDP Decarboxylases

(32) The gene encoding MDP decarboxylase of Saccharomyces cerevisiae is synthesized from overlapping oligonucleotides and cloned in a pET plasmid (Novagen) allowing expression in bacteria. Said plasmid is then transformed by electroporation into bacterial strain BL21 (Invitrogen). The bacteria are streaked on a Petri dish containing ampicillin and incubated at 37 C. The next day, a bacterial colony is randomly selected and used to inoculate 50 ml of LB medium containing ampicillin. The culture is incubated for 24 h while shaking, after which the culture is centrifuged, the bacteria lysed by sonication, and a total protein extract prepared. An aliquot of the extract is loaded on an electrophoresis gel together with a protein extract from the same strain which has not been transformed, and with molecular weight markers. The lane corresponding to the transformed strain contains a single band of approximately 30 kD, which corresponds to the expected size of the protein, said band being absent in the lane loaded with the non-transformed bacteria.

Example 2: Measuring the Activity of the Protein Extracts Towards 3-hydroxy-3-methylbutyrate

(33) 3-hydroxy-3-methylbutyrate (Sigma, reference 55453 under the name -hydroxyisovaleric acid), is suspended at a concentration of 10 g/l. Mevalonate diphosphate is synthesized from mevalonolactone and other reagents (Sigma) by the conventional method and resuspended at a concentration of 10 g/l.

(34) Six chromatography vials are prepared. 50 L buffer containing 50 mM Bistris/HCl 1 mM dithiothreitol, 10 mM MgCl.sub.2 and 5 mM ATP are added to each vial.

(35) Vials 1 and 4: 5 l water are added (no substrate).

(36) Vials 2 and 5: 5 l of the mevalonate diphosphate preparation are added (positive control).

(37) Vials 3 and 6: 5 l of the 3-hydroxy-3-methylbutyrate (HIV) preparation are added.

(38) Vials 1, 2 and 3: 5 l of water are then added (no enzyme).

(39) Vials 4, 5 and 6: 5 l of the enzyme preparation described in example 1 are added.

(40) Vials are sealed with a septum and crimped. All vials are incubated at 37 C. from 4 hours to 3 days. After incubation, a gas syringe is used to collect the gas present in each vial, and the CO.sub.2 concentration in the samples is measured by gas chromatography. Vial 5 has a very high CO.sub.2 concentration, and CO.sub.2, at a lower concentration, is also detected in vial 6, which indicates a significant reaction of the enzyme preparation towards 3-hydroxy-3-methylbutyrate. The presence of isobutylene in the gas sample from vial 6 is then measured by gas chromatography with infrared or flame ionization detection.

Example 3: Optimization of Reaction Conditions by Using a Cofactor

(41) The same reaction as that described in vial 6 of the previous example is carried out, but in one of the samples, ethyl diphosphate, synthesized to order, is added as cofactor.

(42) In this example, three vials are used. The first contains buffers, ATP, and the enzyme extract in the amounts described in the previous example. The second vial contains the same components, but additionally contains 3-hydroxy-3-methylbutyrate in the amounts described in the previous example. The third vial contains, in addition to 3-hydroxy-3-methylbutyrate, 10 l of 10 mg/l ethyl diphosphate.

(43) As in the previous example, isobutylene formation is measured by gas chromatography with infrared or flame ionization detection. It is found that when ethyl diphosphate is present, the amount of isobutylene produced over time is markedly higher.

Example 4: Screening an Enzyme Library

(44) A library of 63 genes encoding enzymes from the MDP decarboxylase family was obtained and tested for activity on HIV as substrate.

(45) Cloning, Bacterial Cultures and Expression of Proteins.

(46) The genes encoding the mevalonate diphosphate (MDP) decarboxylase family EC 4.1.1.33 were cloned in the pET 25b vector (Novagen) in the case of eukaryotic genes and pET 22b (Novagen) for genes of prokaryotic origin, with a 6-Histidine tag at the N-terminal end immediately after the methionine initiation codon. Competent E. coli BL21(DE3) cells (Novagen) were transformed with these vectors by heat shock. The cells were grown with shaking (160 rpm) at 30 C. in TB medium containing 0.5 M sorbitol, 5 mM betain, 100 g/ml ampicillin until reaching an OD at 600 nm comprised between 0.8 and 1. Isopropyl-B-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM and protein expression was continued at 20 C. overnight (approximately 16 h). The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were frozen at 80 C.

(47) Cell Lysis

(48) 1.6 g of cells were thawed on ice and resuspended in 5 ml of 50 mM Na.sub.2HPO.sub.4 pH 8 containing 300 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated for 10 min at room temperature and then returned to ice for 20 min. Cell lysis was completed by sonication for 35 min in an ultrasound water bath at 0 C.; samples were homogenized between pulses. The bacterial extracts were then clarified by centrifugation at 4 C., 10,000 rpm for 20 min.

(49) Protein Purification and Concentration (PROTINO Kit)

(50) The clarified bacterial lysates were loaded on a PROTINO-1000 Ni-IDA column (Macherey-Nagel) allowing adsorption of 6-His tag proteins. Columns were washed and the enzymes of interest were eluted with 4 ml of 50 mM Na.sub.2HPO.sub.4 pH 8 containing 300 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 250 mM imidazole. Eluates were then concentrated in Amicon Ultra-4 10 kDa cells (Millipore) to a final volume of 250 l. Protein was quantified by the Bradford method.

(51) Enzymatic Reactions

(52) The desired enzymatic reaction (conversion of 3-hydroxy-3-methylbutyrate, or 3-hydroxyisovalerate, or else HIV) was tested in two experimental conditions that differed in terms of buffer and reaction pH.

(53) Experimental Conditions No. 1.

(54) 100 mM citrate

(55) 10 mM MgCl.sub.2

(56) 10 mM ATP

(57) 20 mM KCl-200

(58) mM HIV

(59) Final pH adjusted to 5.5

(60) Experimental Conditions No. 2.

(61) 100 mM Tris-HCl pH 7.0

(62) 10 mM MgCl.sub.2

(63) 10 mM ATP

(64) 20 mM KCl-200

(65) 200 mM HIV

(66) Final pH adjusted to 7.0

(67) The enzyme was added to the reaction mixture. As the protein yield was variable, the amount of enzyme added ranged between 0.01 and 1 mg/ml from one sample to another. The enzyme-free control reactions were carried out in parallel.

(68) The 1 ml reactions were placed in 2 ml vials (Interchim) and sealed with teflon/silica/teflon septum (Interchim). Reactions were incubated without shaking at 37 C. for 72 h.

(69) Analysis of Reactions

(70) The gas present above the reactions was collected with a syringe equipped with a no-return mechanism. The gas sample was analyzed by gas chromatography (GC) coupled with mass spectrometry (MS). The instrument was previously calibrated using a range of isobutylene concentrations.

(71) Column: BPX5 (SGE)

(72) GC/MS: MSD 5973 (HP)

(73) For each chromatogram, three principal peaks were obtained, the first corresponding to air, the second to water, and the third to isobutylene. Out of the 63 enzymes produced and tested, eleven potential candidates were identified in the primary screening. Some of these candidates are marked with an arrow in FIG. 7. Their identities are shown below, and their sequences in SEQ ID NO: 6 to 16 (His-tag not shown).

(74) Candidate 1: SEQ ID NO: 7

(75) Genebank accession number: CAI97800.1

(76) Swissprot/TrEMBL accession number: Q1GAB2

(77) Microorganisms: Lactobacillus delbrueckii subsp. bulgaricus (strain ATCC 11842/DSM 20081)

(78) Candidate 2: SEQ ID NO: 8

(79) Genebank accession number: CAJ51653

(80) Swissprot/TrEMBL accession number: Q18K00

(81) Microorganisms: Haloquadratum walsbyi DSM 16790

(82) Candidate 3: SEQ ID NO: 9

(83) Genebank accession number: ABD99494.1

(84) Swissprot/TrEMBL accession number: Q1WU41

(85) Microorganisms: Lactobacillus salivarius subsp. salivarius (strain UCC118)

(86) Candidate 4: SEQ ID NO: 10

(87) Genebank accession number: ABJ57000.1

(88) Swissprot/TrEMBL accession number: Q04EX2

(89) Microorganisms: Oenococcus oeni (strain BAA-331/PSU-1)

(90) Candidate 5: SEQ ID NO: 11

(91) Genebank accession number: ABJ67984.1

(92) Swissprot/TrEMBL accession number: Q03FN8

(93) Microorganisms: Pediococcus pentosaceus ATCC 25745

(94) Candidate 6: SEQ ID NO: 12

(95) Genebank accession number: ABV09606.1

(96) Swissprot/TrEMBL accession number: A8AUU9

(97) Microorganisms: Streptococcus gordonii (strain Challis/ATCC 35105/CH1/DL1/V288)

(98) Candidate 7: SEQ ID NO: 13

(99) Genebank accession number: ABQ14154.1

(100) Swissprot/TrEMBL accession number: A5EVP2

(101) Microorganisms: Dichelobacter nodosus VCS1703A

(102) Candidate 8: SEQ ID NO: 14

(103) Genebank accession number: EDT95457.1

(104) Swissprot/TrEMBL accession number: B2DRT0

(105) Microorganisms: Streptococcus pneumoniae CDC0288-04

(106) Candidate 9: SEQ ID NO: 15

(107) Genebank accession number: AAT86835

(108) Swissprot/TrEMBL accession number: Q5XCM8

(109) Microorganisms: Streptococcus pyogenes serotype M6 (ATCC BAA-946/MGAS10394)

(110) Candidate 10: SEQ ID NO: 6

(111) Genebank accession number: AAT43941

(112) Swissprot/TrEMBL accession number: Q6KZB1

(113) Microorganisms: Picrophilus torridus DSM 9790

(114) Candidate 11: SEQ ID NO: 16

(115) Genebank accession number: AAV43007.1

(116) Swissprot/TrEMBL accession number: Q5FJW7

(117) Microorganisms: Lactobacillus acidophilus NCFM

(118) The highest levels of isobutylene (IBN) production were observed with candidate 10, that is, with the purified decarboxylase enzyme of SEQ ID NO: 6 from Picrophilus torridus. This enzyme was retained for further characterization.

Example 5: Characterization of Enzyme SEQ ID NO: 6

(119) The recombinant enzyme was purified as described in example 4. The results, presented in FIG. 8, show that enzyme purity in the final protein sample was approximately 90%.

(120) The activity of the isolated enzyme was confirmed. The reaction was carried out in the following conditions:

(121) 100 mM Tris-HCl pH 7.0

(122) 10 mM MgCl.sub.2

(123) 10 mM ATP

(124) 20 mM KCl

(125) 250 M HIV

(126) Final pH adjusted to 6.0

(127) 3 mg/ml enzyme

(128) After 72 h incubation at 30 C., the signal was measured by GC/MS. The results are shown in FIG. 9. In the presence of the enzyme, IBN production was increased here by approximately 2.3-fold over background noise. The background noise observed here is in agreement with the organic chemistry literature, showing that in aqueous solution and at a temperature of around 100 C., 3-hydroxyisovaleric acid slowly decarboxylates to tert-butanol, which is partially dehydrated to isobutylene, following an equilibrium favorable to the formation of tert-butanol (Pressman and Luca, J. Am. Chem. Soc. 1940).

(129) Effect of ATP Co-Substrate

(130) Test Conditions

(131) 100 mM citrate

(132) 50 mM KCl

(133) 10 mM MgCl.sub.2

(134) 200 mM HIV (to be specified)

(135) 1 mg/ml purified enzyme

(136) pH 5.5

(137) Incubation 72 h at 30 C.

(138) TABLE-US-00001 Conditions ATP final concentration Enzyme 1 0 mM 0 mg/ml 2 0 mM 1 mg/ml 3 10 mM 0 mg/ml 4 10 mM 1 mg/ml

(139) The results in FIG. 10 show that enzyme activity was only observed in the presence of the co-substrate ATP. Other molecules, and in particular those containing a phosphoanhydride bond, could also be efficient co-substrates for the enzyme.

(140) Effect of Mg.sup.2+ Cofactor

(141) Test Conditions

(142) 100 mM citrate pH 5.5

(143) 50 mM KCl

(144) 10 mM ATP

(145) 200 mM HIV (to be specified)

(146) pH 5.5

(147) 1 mg/ml purified enzyme

(148) Incubation 72 h at 30 C.

(149) TABLE-US-00002 Conditions MgCl.sub.2 final concentration Enzyme 1 0 mM 0 mg/ml 2 0 mM 1 mg/ml 3 5 mM 0 mg/ml 4 5 mM 1 mg/ml

(150) The results in FIG. 11 show that enzyme activity was improved in the presence of Mg.sup.2+ ions. Other ions, and in particular other divalent ions, could be used as cofactor in place of or in addition to Mg.sup.2+ ions.

(151) Enzymatic Activity According to Temperature

(152) Test Conditions

(153) 100 mM buffer

(154) 50 mM KCl

(155) 10 mM ATP

(156) 200 mM HIV (to be specified)

(157) 1 mg/ml purified enzyme

(158) Incubation 72 h at different temperatures.

(159) The results in FIG. 12 show that the enzyme is moderately thermoactive with a temperature optimum of approximately 50 C.

(160) Activity According to pH

(161) Test Conditions

(162) 100 mM buffer

(163) 50 mM KCl

(164) 10 mM ATP

(165) 200 mM HIV (to be specified)

(166) 1 mg/ml purified enzyme

(167) Incubation 72 h at 30 C.

(168) Optimal conditions were obtained with a pH of 5.5 in 100 mM citrate.

(169) Enzyme Parameters

(170) A substrate range was tested in the previously described conditions, with incubation at 50 C. The Km of the enzyme is approximately 40 mM HIV.

(171) Optimization of Reaction Conditions

(172) Optimum reaction conditions were sought, and the following conditions were retained:

(173) 100 mM citrate

(174) 50 mM KCl

(175) 40 mM ATP

(176) 200 mM HIV

(177) 1 mg/ml enzyme

(178) Incubation 48 h at 50 C.

(179) As shown in FIG. 14, the ratio of the signal over-background noise is approximately 100.

Example 6: Optimization of P. torridus MDP Decarboxylase Expression in E. coli

(180) The initial level of expression in E. coli BL21 was low, as the band was difficult to see on SDS-PAGE before purification. The Codon Optimization Index (CAI) of the native sequence for expression in E. coli was measured with the Optimizer program available at http://genomes.urv.es/OPTIMIZER/, and based on the method of Sharp and Li (1987). The value obtained was only 0.23, reflecting the low level of expression of the protein in E. coli.

(181) A sequence coding for an identical protein, but containing codons better adapted for expression in E. coli, was generated. This sequence had a CAI of 0.77 which is closer to the optimum of 1. The native sequence and the optimized sequence are shown in SEQ ID NO: 17 (optimized sequence of P. torridus (AAT43941) MDP decarboxylase including the His Tag) and SEQ ID NO: 19 (native sequence of P. torridus (AAT43941) MDP decarboxylase including the His Tag). The optimized sequence was synthesized by oligonucleotide concatenation and cloned in a pET25 expression vector. After transformation of the vector into E. coli strain BL21(DE3) and induction according to the previously described protocol, the proteins were produced, purified and analyzed on a gel as described previously. The same protocol was carried out with the native sequence for purposes of comparison.

(182) Comparison of expression levels of candidate 224 using either the native nucleotide sequence or the sequence optimized for expression in E. coli.

(183) The results in FIG. 15 show that the protein corresponding to the optimized gene was clearly visible on the gel in the non-purified cell lysate (lane 4), which indicates a very notable increase in expression. The level of purity of the protein after the purification step was also higher in the case of the optimized gene.

(184) Activity was measured on the crude lysate. No activity was detected on the crude lysate corresponding to the native nucleic sequence. The expression of the protein was improved such that the crude lysate obtained with the improved sequence (optimized clone 224) now displayed this activity.

(185) The following reaction medium was used in this test:

(186) Reaction Medium

(187) TABLE-US-00003 Products Final concentration Acetate reaction buffer 50 mM (500 mM, pH 5.5) MgCl.sub.2 (1M) 10 mM KCl (1M) 20 mM HIV (3M) 50 mM ATP (100 mM) 40 mM Protease inhibitor 1X (100X) H20 Enzyme 89 g total protein (crude lysate)
Incubation 2 days at 50 C.
Results
Condition No. 1: Lysate of optimized clone 224
Condition No. 2: Lysate of clone GB6 (empty pET plasmid)

(188) TABLE-US-00004 Signal area Conditions surface Ratio 1 1083 22 2 49

Example 7: Method for Synthesizing Isobutylene from 3-Hydroxy-3-Methylbutyrate and Conversion to Isooctane

(189) A reaction identical to that of vial 3 in example 3 was carried out in a 1 liter volume, in a fermenter equipped with a gas extraction system. The presence of the recombinant enzyme induced the conversion of 3-hydroxy-3-methylbutyrate to isobutylene, which naturally degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter. Isobutylene was then used to produce isooctene by addition catalyzed by Amberlyst 35wet or 36wet resin (Rohm and Haas). Isooctene was reduced in turn to isooctane by catalytic hydrogenation.

Example 8: Enzyme Engineering to Improve Efficacy for Substrates

(190) Random mutagenesis technology was used to create a library containing thousands of mutants of the gene described in example 1. This mutant library was then cloned in the expression plasmid and transformed into competent bacterial strain BL21.

(191) A thousand bacteria were then isolated and inoculated into Eppendorf tubes containing 500 l LB medium supplemented with ampicillin. The samples were incubated on a shaker for 15 h. The next day, the amount of isobutylene produced was determined by using one or another of the experimental protocols described in the previous examples.

(192) Clones with a significantly increased amount of isobutylene were then revalidated using the same experimental protocol. Once this improvement was validated, the plasmid was extracted from each improved clone and sequenced. Mutations responsible for the improved activity were identified and combined on a same plasmid. The plasmid containing the different improving mutations was in turn transformed into competent bacteria, and the same analysis was carried out.

(193) The clone containing the combined mutations, which had significantly greater activity than the one containing only a single improving mutation, was then used as the basis for a new cycle of mutation/screening, to identify mutants with even further improved activity.

(194) On completion of this protocol, the clone containing several mutations and having the best activity was selected.

Example 9: Method for Synthesizing Ethylene from 3-hydroxypropionate

(195) The gene encoding the enzyme described in example 1 was inserted in a plasmid allowing expression of the recombinant proteins in an E. coli strain. The plasmid was transformed into the bacteria of said strain. The transformed bacteria were then incubated in a fermenter in the presence of propyl diphosphate (10 mg/l) and 3-hydroxypropionate (1 g/l). The presence of the recombinant enzyme led to the conversion of 3-hydroxypropionate to ethylene, which spontaneously degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter. Ethylene was then measured in the gas sample by gas chromatography with infrared detection in the part of the spectrum where ethylene emits strongly.

Example 10: Method for Synthesizing Propylene from 3-hydroxybutyrate

(196) The gene encoding the enzyme described in example 1 or an enzyme described in example 4 was inserted in a plasmid allowing expression of recombinant proteins in an E. coli strain. The plasmid was transformed into the bacteria of said strain. The transformed bacteria were then incubated in a fermenter in the presence of ethyl diphosphate (10 mg/l) and 3-hydroxybutyrate (1 g/l) (Sigma, reference 166898). The presence of the recombinant enzyme led to the conversion of 3-hydroxybutyrate to propylene, which spontaneously degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter. Propylene was then measured in the gas sample by gas chromatography with infrared detection in the part of the spectrum where propylene emits strongly.

Example 11: Method for Synthesizing Propylene from Glucose

(197) The gene encoding the enzyme described in example 1 or an enzyme described in example 4 was cloned in a plasmid allowing expression of recombinant proteins in the bacterium Alcaligenes eutrophus. The plasmid was transformed into the bacteria of said strain.

(198) The transformed bacteria were then incubated in a fermenter in the presence of glucose and ethyl diphosphate and in microaerophilic conditions, then subjected to heat shock which induced them to produce large quantities of 3-hydroxybutyrate. The presence of the recombinant enzyme led to the simultaneous conversion of 3-hydroxybutyrate to propylene, which spontaneously degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter.

Example 12: Method for Synthesizing Propylene from Glucose

(199) This example describes a method very similar to that of example 11. The main difference consists in the use of an E. coli strain modified so as to produce 3-hydroxybutyrate instead of a natural strain like Alcaligenes eutrophus. Said strain was obtained by the engineering of metabolic pathways so as to lead to accumulation of 3-hydroxybutyrate. Addition of an MDP decarboxylase such as described in example 1 or in example 4 enabled the conversion of 3-hydroxybutyrate to propylene.

Example 13: Method for Synthesizing Isobutylene from Glucose

(200) The gene encoding the enzyme described in example 1 was inserted in a plasmid allowing expression of recombinant proteins in E. coli strains that had also undergone metabolic modifications so that they endogenously synthesized 3-hydroxy-3-methylbutyrate.

(201) The bacteria were then incubated in a fermenter in the presence of glucose and in microaerophilic conditions. The presence of the recombinant enzyme induces the simultaneous conversion of 3-hydroxy-3-methylbutyrate to isobutylene, which naturally degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter.