Method For Producing A Monoalkene By Enzymatic Conversion Of An Alkyl Monoester Patent Application (2025)

U.S. patent application number 14/411510 was filed with the patent office on 2015-06-11 for method for producing a monoalkene by enzymatic conversion of an alkyl monoester. This patent application is currently assigned to GLOBAL BIOENERGIES. The applicant listed for this patent is Global Bioenergies, SCIENTIST OF FORTUNE S.A.. Invention is credited to Mathieu Allard, Maria Anissimova, Philippe Marliere.

METHOD FOR PRODUCING A MONOALKENE BY ENZYMATIC CONVERSION OF ANALKYL MONOESTER

Abstract

The present invention relates to a method for producing amonoalkene comprising the step of enzymatically converting an alkylmonoester. The conversion preferably makes use of an enzyme whichbelongs to the group of terpene synthases or to the family ofprenyltransferases. Moreover, the present invention relates to theuse of a terpene synthase or of a prenyltransferase forenzymatically converting an alkyl monoester into a monoalkene.

Foreign Application Data

Claims

1. A method for producing a monoalkene, the method comprising astep of converting an alkyl monoester into a monoalkene, wherein:the alkyl monoester is a compound of formula (I) ##STR00004##wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are eachindependently selected from (--H), methyl (--CH3) or ethyl(--C2H5); and wherein X is selected from: O--PO.sub.3H.sub.2monophosphate O--PO.sub.2H--O--PO.sub.3H.sub.2 diphosphateO--SO.sub.3H sulfate and wherein the monoalkene is a compound offormula (II) ##STR00005## wherein R.sup.1, R.sup.2, R.sup.3 andR.sup.4 have the same meanings as defined for the compound offormula (I), the method being characterized in that the conversionfrom the alkyl monoester into the monoalkene is achieved byenzymatic elimination of the molecule XH.

2. The method of claim 1 wherein the enzymatic elimination of themolecule XH is catalyzed by a terpene synthase.

3. The method of claim 1 wherein the terpene synthase is anisoprene synthase (EC 4.2.3.27).

4. The method of claim 1 wherein the terpene synthase is amyrcene/ocimene synthase (EC 4.2.3.15).

5. The method of claim 1 wherein the terpene synthase is afarnesene synthase (EC 4.2.3.46 or EC 4.2.3.47).

6. The method of claim 1 wherein the terpene synthase is a pinenesynthase (EC 4.2.3.14).

7. The method of claim 1 wherein the enzymatic elimination of themolecule XH is catalyzed by a prenyltransferase (EC 2.5.1).

8. The method of claim 1 wherein: (i) the alkyl monoester is ethyldiphosphate and the monoalkene is ethylene; or (ii) the alkylmonoester is propan-1-yl diphosphate (propyl diphosphate) and themonoalkene is methylethylene (propylene); or (iii) the alkylmonoester is propan-2-yl diphosphate (isopropyl diphosphate) andthe monoalkene is methylethylene (propylene); or (iv) the alkylmonoester is butan-1-yl diphosphate (1-butyl diphosphate) and themonoalkene is but-1-ene; or (v) the alkyl monoester is butan-2-yldiphosphate (2-butyl diphosphate) and the monoalkene is but-1-eneand but-2-ene; or (vi) the alkyl monoester is 2-methylpropan-1-yldiphosphate (isobutyl diphosphate) and the monoalkene is2-methylprop-1-ene (isobutene; isobutylene); or (vii) the alkylmonoester is 1,1-dimethylethyl diphosphate (tert-butyl diphosphate)and the monoalkene is 2-methylprop-1-ene (isobutene; isobutylene);or (viii) the alkyl monoester is ethyl monophosphate and themonoalkene is ethylene; or (ix) the alkyl monoester is propan-1-ylmonophosphate (propyl monophosphate) and the monoalkene ismethylethylene (propylene); or (x) the alkyl monoester ispropan-2-yl monophosphate (isopropyl monophosphate) and themonoalkene is methylethylene (propylene); or (xi) the alkylmonoester is butan-1-yl monophosphate (1-butyl monophosphate) andthe monoalkene is but-1-ene; or (xii) the alkyl monoester isbutan-2-yl monophosphate (2-butyl monophosphate) and the monoalkeneis but-1-ene and but-2-ene; or (xiii) the alkyl monoester is2-methylpropan-1-yl monophosphate (isobutyl monophosphate) and themonoalkene is 2-methylprop-1-ene (isobutene); or (xiv) the alkylmonoester is 1,1-dimethylethyl monophosphate (tert-butylmonophosphate) and the monoalkene is 2-methylprop-1-ene (isobutene;isobutylene); or (xv) the alkyl monoester is ethyl sulfate and themonoalkene is ethylene; or (xvi) the alkyl monoester is propan-1-ylsulfate (propyl sulfate) and the monoalkene is methylethylene(propylene); or (xvii) the alkyl monoester is propan-2-yl sulfate(isopropyl sulfate) and the monoalkene is methylethylene(propylene); or (xviii) the alkyl monoester is butan-1-yl sulfate(1-butyl sulfate) and the monoalkene is but-1-ene; or (xix) thealkyl monoester is butan-2-yl sulfate (2-butyl sulfate) and themonoalkene is but-1-ene and but-2-ene; or (xx) the alkyl monoesteris 2-methylpropan-1-yl sulfate (isobutyl sulfate) and themonoalkene is 2-methylprop-1-ene (isobutene; isobutylene); or (xxi)the alkyl monoester is 1,1-dimethylethyl sulfate (tent-butylsulfate) and the monoalkene is 2-methylprop-1-ene (isobutene;isobutylene).

9. The method of claim 1 wherein the step of enzymaticallyconverting an alkyl monoester into a monoalkene is realized in thepresence of an organism capable of expressing an enzyme as definedin any one of claims 2 to 7.

10. The method of claim 9 wherein the microorganism is furthermorecapable of producing the alkyl monoester to be converted.

11. Use of an enzyme as defined in claim 2 for converting an alkylmonoester into a monoalkene wherein: the alkyl monoester is acompound of formula (I) ##STR00006## wherein R.sup.1, R.sup.2,R.sup.3 and R.sup.4 are each independently selected from hydrogen,methyl or ethyl; and wherein X is selected from: O--PO.sub.3H.sub.2monophosphate O--PO.sub.2H--O--PO.sub.3H.sub.2 diphosphateO--SO.sub.3H sulfate and wherein the monoalkene is a compound offormula (II) ##STR00007## wherein R.sup.1, R.sup.2, R.sup.3 andR.sup.4 have the same meanings as defined for the compound offormula (I).

12. Use of a recombinant organism which expresses an enzyme asdefined in claim 2 for converting an alkyl monoester into amonoalkene wherein: the alkyl monoester is a compound of formula(I) ##STR00008## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 areeach independently selected from hydrogen, methyl or ethyl; andwherein X is selected from: O--PO.sub.3H.sub.2 monophosphateO--PO.sub.2H--O--PO.sub.3H.sub.2 diphosphate O--SO.sub.3H sulfateand wherein the monoalkene is a compound of formula (II)##STR00009## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 have thesame meanings as defined for the compound of formula (I).

13. A recombinant organism which is characterized by the followingfeatures: (a) it is capable of producing an alkyl monoester asdefined in claim 1; and (b) it expresses an enzyme as defined inclaim 2.

14. A composition comprising an organism of claim 13.

15. A composition comprising (i) an alkyl monoester as defined inclaim 1; and (ii) an enzyme as defined in claim 2.

Description

[0001] The present invention relates to a method for producing amonoalkene comprising the step of enzymatically converting an alkylmonoester. The conversion preferably makes use of an enzyme whichbelongs to the family of terpene synthases or to the family ofprenyltransferases. Moreover, the present invention relates to theuse of a terpene synthase or a prenyltransferase for enzymaticallyconverting an alkyl monoester into a monoalkene.

[0002] A large number of chemical compounds are currently derivedfrom petrochemicals. Alkenes (such as ethylene, propylene, thedifferent butenes, or else the pentenes, for example) are used inthe plastics industry, for example for producing polypropylene orpolyethylene, and in other areas of the chemical industry and thatof fuels. Ethylene, the simplest alkene, lies at the heart ofindustrial organic chemistry: it is the most widely producedorganic compound in the world. It is used in particular to producepolyethylene, a major plastic. Ethylene can also be converted tomany industrially useful products by reaction (e.g. by oxidation orhalogenation). Propylene plays a similarly important role: itspolymerization results in a plastic material, polypropylene. Thetechnical properties of this product in terms of resistance,density, solidity, deformability, and transparency are unequalled.The worldwide market for polypropylene has grown continuously sinceits invention in 1954. Butylene exists in four forms, one of which,isobutylene, enters into the composition of methyl-tert-butyl-ether(MTBE), an anti-knock additive for automobile fuel. Isobutylene canalso be used to produce isooctene, which in turn can be reduced toisooctane (2,2,4-trimethylpentane); the very high octane rating ofisooctane makes it the best fuel for so-called "gasoline" engines.Amylene, hexene and heptene exist in many forms according to theposition and configuration of the double bond. These products havereal industrial applications but are less important than ethylene,propylene or butenes. All these alkenes are currently produced bycatalytic cracking of petroleum products (or by a derivative of theFischer-Tropsch process in the case of hexene, from coal or gas).Their production costs are therefore tightly linked to the price ofoil. Moreover, catalytic cracking is sometimes associated withconsiderable technical difficulties which increase processcomplexity and production costs.

[0003] The production by a biological pathway of alkenes or otherorganic molecules that can be used as fuels or as precursors ofsynthetic resins is called for in the context of a sustainableindustrial operation in harmony with geochemical cycles. The firstgeneration of biofuels consisted in the fermentative production ofethanol, as fermentation and distillation processes already existedin the food processing industry. The production of secondgeneration biofuels is in an exploratory phase, encompassing inparticular the production of long chain alcohols (butanol andpentanol), terpenes, linear alkanes and fatty acids. Two recentreviews 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).

[0004] The production of ethylene by plants has long been known(Meigh et al. (Nature 186 (1960), 902)). According to the metabolicpathway elucidated, methionine is the precursor of ethylene (Adamsand Yang (PNAS 76 (1979), 170)). Conversion of 2-oxoglutarate hasalso been described (Ladygina et al. (Process Biochemistry 41(2006), 1001). Since a single ethylene molecule requires theprevious production of a four- or five-carbon chain, the equipmentand energy needs of all these pathways are unfavorable and do notbode well for their industrial application for alkenebioproduction.

[0005] Moreover, many microorganisms are capable of producingpropylene, however, with an extremely low yield

[0006] The conversion of isovalerate to isobutylene by the yeastRhodotorula 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, isfar from permitting an industrial application. The reactionmechanism was elucidated by Fukuda et al. (BBRC 201 (1994), 516)and involves a cytochrome P450 enzyme which decarboxylatesisovalerate by reduction of an oxoferryl group Fe.sup.V=0.Large-scale biosynthesis of isobutylene by this pathway seemshighly unfavorable, since it would require the synthesis anddegradation of one molecule of leucine to form one molecule ofisobutylene. Also, the enzyme catalyzing the reaction uses heme ascofactor, poorly lending itself to recombinant expression inbacteria and to improvement of enzyme parameters. For all thesereasons, it appears very unlikely that this pathway can serve as abasis for industrial exploitation. Other microorganisms have beendescribed as being marginally capable of naturally producingisobutylene from isovalerate; the yields obtained are even lowerthan those obtained with Rhodotorula minuta (Fukuda et al. (Agric.Biol. Chem. 48 (1984), 1679)).

[0007] Isoprene is produced at a significant level in higherplants, such as poplars. The production of isoprene in this contextremains however low and the pathway which leads to isopreneproduction, which is based on themevalonate-isopentenyl-pyrophosphate pathway, poorly complies withthe demands for industrial scale production.

[0008] Thus, there is still a need for efficient andenvironmentally friendly methods of producing alkenes, inparticular monoalkenes.

[0009] The present invention meets this demand by providing amethod for producing a monoalkene from an alkyl monoester byemploying an enzymatic reaction. More specifically, the presentinvention relates to a method for producing a monoalkene, themethod comprising a step of converting an alkyl monoester into amonoalkene, wherein:

the alkyl monoester is a compound of formula (I)

##STR00001##

wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are eachindependently selected from hydrogen (--H), methyl (--CH3) or ethyl(--C2H5); and wherein X is selected from: [0010] O--PO.sub.3H.sub.2monophosphate [0011] O--PO.sub.2H--O--PO.sub.3H.sub.2 diphosphate[0012] O--SO.sub.3H sulfate and wherein the monoalkene is acompound of formula (II)

##STR00002##

[0012] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 have the samemeanings as defined for the compound of formula (I), the methodbeing characterized in that the conversion from the alkyl monoesterinto the monoalkene is achieved by enzymatic elimination ofmolecule XH.

[0013] The present invention teaches for the first time that it ispossible to enzymatically convert an alkyl monoester having formula(I) as shown above into a corresponding monoalkene by eliminatingthe phosphorus or sulphur containing molecule XH with the help ofan enzyme.

[0014] In particular, it has been found that enzymes which belongto the family of terpene synthases or to the family of prenyltransferases are capable of catalyzing the conversion of an alkylmonoester into a monoalkene as described above.

[0015] The conversion of an alkyl monoester according to formula(I) into a monoalkene according to formula (II) by elimination ofmolecule XH can in principle be achieved by any enzyme which iscapable of eliminating the phosphorus or sulphur containingmolecule XH from an alkyl monoester of the formula (I). Preferably,such an enzyme is an enzyme which can be classified as belonging tothe terpene synthase family, more preferably the terpene synthaseis a plant terpene synthase. In another preferred embodiment suchan enzyme is an enzyme which can be classified as belonging to theprenyltransferase family.

[0016] The terpene synthases constitute an enzyme family whichcomprises enzymes catalyzing the formation of numerous naturalproducts always composed of carbon and hydrogen (terpenes) andsometimes also of oxygen or other elements (terpenoids). Terpenoidsare structurally diverse and widely distributed moleculescorresponding to well over 30000 defined natural compounds thathave been identified from all kingdoms of life. In plants, themembers of the terpene synthase family are responsible for thesynthesis of the various terpene molecules from two isomeric5-carbon precursor "building blocks", isoprenyl diphosphate andprenyl diphosphate, leading to 5-carbon isoprene, 10-carbonmonoterpene, 15-carbon sesquiterpene and 20-carbon diterpenes"(Chen et al.; The Plant Journal 66 (2011), 212-229).

[0017] The ability of terpene synthases to convert a prenyldiphosphate containing substrate to diverse products duringdifferent reaction cycles is one of the most unique traits of thisenzyme class. The common key step for the biosynthesis of allterpenes is the reaction of terpene synthase on correspondingdiphosphate esters. The general mechanism of this enzyme classinduces the removal of the diphosphate group and the generation ofan intermediate with carbocation as the first step. In the variousterpene synthases, such intermediates further rearrange to generatethe high number of terpene skeletons observed in nature. Inparticular, the resulting cationic intermediate undergoes a seriesof cyclizations, hydride shifts or other rearrangements until thereaction is terminated by proton loss or the addition of anucleophile, in particular water for forming terpenoid alcohols(Degenhardt et al., Phytochemistry 70 (2009), 1621-1637).

[0018] The different terpene synthases share various structuralfeatures. These include a highly conserved C-terminal domain, whichcontains their catalytic site and an aspartate-rich DDXXD motifessential for the divalent metal ion (typically Mg2+ or Mn2+)assisted substrate binding in these enzymes (Green et al. Journalof biological chemistry, 284, 13, 8661-8669). In principle, anyknown enzyme which can be classified as belonging to the EC 4.2.3enzyme superfamily can be employed.

[0019] Even more preferably the method according to the inventionmakes use of an isoprene synthase (EC 4.2.3.27), a myrcene/ocimenesynthase (EC 4.2.3.15), a farnesene synthase (EC 4.2.3.46 or EC4.2.3.47) or a pinene synthase (EC 4.2.3.14). Also enzymes whichare generally classified as monoterpene synthases can be used.

[0020] Isoprene synthase (EC 4.2.3.27) is an enzyme which naturallycatalyzes the following reaction:

Dimethylallyl diphosphateisoprene+diphosphate

[0021] This enzyme occurs in a number of organisms, in particularin plants and some bacteria. The occurrence of this enzyme has,e.g., been described for Arabidopsis thaliana, a number of Populusspecies like P. alba (UniProt accession numbers Q50L36, A9Q7C9,D8UY75 and D8UY76), P. nigra (UniProt accession number AOPFK2), P.canescence (UniProt accession number Q9AR86; see also Koksal etal., J. Mol. Biol. 402 (2010), 363-373), P. tremuloides, P.trichocarpa, in Quercus petraea, Quercus robur, Salix discolour,Pueraria montana (UniProt accession number Q6EJ97), Puerarialobata, Mucuna pruriens, Vitis vinifera, Embryophyta and Bacillussubtilis. In principle, any known isoprene synthase can be employedin the method according to the invention. In a preferredembodiment, the isoprene synthase employed in a method according tothe present invention is an isoprene synthase from a plant of thegenus Populus, more preferably from Populus trichocarpa or Populusalba. In another preferred embodiment the isoprene synthaseemployed in a method according to the present invention is anisoprene synthase from Pueraria montana, preferably from PuerariaMontana var. lobata, or from Vitis vinifera. Preferred isoprenesynthases to be used in the context of the present invention arethe isoprene synthase of Populus alba (Sasaki et al.; FEBS Letters579 (2005), 2514-2518) or the isoprene synthases from Populustrichocarpa and Populus tremuloides which show very high sequencehomology to the isoprene synthase from Populus alba. Anotherpreferred isoprene synthase is the isoprene synthase from Puerariamontana var. lobata (kudzu) (Sharkey et al.; Plant Physiol. 137(2005), 700-712). The activity of an isoprene synthase can bemeasured according to methods known in the art, e.g. as describedin Silver and Fall (Plant Physiol (1991) 97, 1588-1591). In atypical assay, the enzyme is incubated with dimethylallyldiphosphate in the presence of the required co-factors, Mg.sup.2+or Mn.sup.2+ and K.sup.+ in sealed vials. At appropriate timevolatiles compound in the headspace are collected with a gas-tightsyringe and analyzed for isoprene production by gas chromatography(GC).

[0022] Myrcene/ocimene synthases (EC 4.2.3.15) are enzymes whichnaturally catalyze the following reaction:

Geranyl diphosphate(E)-beta-ocimene+diphosphate

or

Geranyl diphosphatemyrcene+diphosphate

[0023] These enzymes occur in a number of organisms, in particularin plants and animals, for example in Lotus japanicus, Phaseoluslunatus, Abies grandis, Arabidopsis thaliana (UniProt accessionnumber Q9ZUH4), Actinidia chinensis, Perilla fructescens, Ochtodessecundiramea and in Ips pini (UniProt accession number Q58GE8. Inprinciple, any known myrcene/ocimene synthase can be employed inthe method according to the invention. In a preferred embodiment,the myrcene/ocimene synthase employed in a method according to thepresent invention is a myrcene/ocimene synthase from Lotusjapanicus (Arimura et al.; Plant Physiol. 135 (2004), 1976-1983) orfrom Phaseolus lunatus (UniProt accession number B1P189). Theactivity of an ocimene/myrcene synthase can be measured asdescribed, for example, in Arimura et al. (Plant Physiology 135(2004), 1976-1983. In a typical assay for determining the activity,the enzyme is placed in screwcapped glass test tube containingdivalent metal ions, e.g. Mg.sup.2+ and/or Mn.sup.2+, andsubstrate, i.e. geranyl diphosphate. The aqueous layer is overlaidwith pentane to trap volatile compounds. After incubation, theassay mixture is extracted with pentane a second time, both pentanefractions are pooled, concentrated and analyzed by gaschromatography to quantify ocimene/myrcene production.

[0024] Farnesene synthases are generally classified into twodifferent groups, i.e. alpha-farnesene synthases (EC 4.2.3.46) andbeta farnesene synthases (EC 4.2.3.47). Alpha-farnesene synthases(EC 4.2.3.46) naturally catalyze the following reaction:

(2E,6E)-farnesyl diphosphate(3E,6E)-alpha-farnesene+diphosphate

[0025] This enzyme occurs in a number of organisms, in particularin plants, for example in Malus.times.domestica (UniProt accessionnumbers Q84LB2, B2ZZ11, Q6Q2J2, Q6QWJ1 and Q32WI2), Populustrichocarpa, Arabidopsis thaliana (UniProt accession numbers A4FVP2and P0CJ43), Cucumis melo (UniProt accession number B2KSJ5) andActinidia deliciosa (UniProt accession number C7SHN9). Inprinciple, any known alpha-farnesene synthase can be employed inthe method according to the invention. In a preferred embodiment,the alpha-farnesene synthase employed in a method according to thepresent invention is an alpha-farnesene synthase fromMalus.times.domestica (UniProt accession numbers Q84LB2, B2ZZ11,Q6Q2J2, Q6QWJ1 and Q32WI2; see also Green et al.; Photochemistry 68(2007), 176-188).

[0026] Beta-farnesene synthases (EC 4.2.3.47) naturally catalyzethe following reaction:

(2E,6E)-farnesyl diphosphate(E)-beta-farnesene+diphosphate

[0027] This enzyme occurs in a number of organisms, in particularin plants and in bacteria, for example in Artemisia annua (UniProtaccession number Q4VM12), Citrus junos (UniProt accession numberQ94JS8), Oryza sativa (UniProt accession number Q0J7R9), Pinussylvestris (UniProt accession number D7PCH9), Zea diploperennis(UniProt accession number C7E5V9), Zea mays (UniProt accessionnumbers Q2NM15, C7E5V8 and C7E5V7), Zea perennis (UniProt accessionnumber C7E5W0) and Streptococcus coelicolor (Zhao et al., J. Biol.Chem. 284 (2009), 36711-36719). In principle, any knownbeta-farnesene synthase can be employed in the method according tothe invention. In a preferred embodiment, the beta-farnesenesynthase employed in a method according to the present invention isa beta-farnesene synthase from Mentha piperita (Crock et al.; Proc.Natl. Acad. Sci. USA 94 (1997), 12833-12838).

[0028] Methods for the determination of farnesene synthase activityare known in the art and are described, for example, in Green etal. (Phytochemistry 68 (2007), 176-188). In a typical assayfarnesene synthase is added to an assay buffer containing 50 mMBisTrisPropane (BTP) (pH 7.5), 10% (v/v) glycerol, 5 mM DTT.Tritiated farnesyl diphosphate and metal ions are added. Assayscontaining the protein are overlaid with 0.5 ml pentane andincubated for 1 h at 30.degree. C. with gentle shaking. Followingaddition of 20 mM EDTA (final concentration) to stop enzymaticactivity an aliquot of the pentane is removed for scintillationanalysis. The olefin products are also analyzed by GC-MS.

[0029] Pinene synthase (EC 4.2.3.14) is an enzyme which naturallycatalyzes the following reaction:

Geranyl diphosphatealpha-pinene+diphosphate

[0030] This enzyme occurs in a number of organisms, in particularin plants, for example in Abies grandis (UniProt accession number0244475), Artemisia annua, Chamaecyparis formosensis (UniProtaccession number C3RSF5), Salvia officinalis and Picea sitchensis(UniProt accession number Q6XDB5).

[0031] For the enzyme from Abies grandis a particular reaction wasalso observed (Schwab et al., Arch. Biochem. Biophys. 392 (2001),123-136), namely the following:

6,7-dihydrogeranyldiphosphate.revreaction.6,7-dihydromyrcene+diphosphate

[0032] In principle, any known pinene synthase can be employed inthe method according to the invention. In a preferred embodiment,the pinene synthase employed in a method according to the presentinvention is a pinene synthase from Abies grandis (UniProtaccession number 0244475; Schwab et al., Arch. Biochem. Biophys.392 (2001), 123-136).

[0033] Methods for the determination of pinene synthase activityare known in the art and are described, for example, in Schwab etal. (Archives of Biochemistry and Biophysics 392 (2001), 123-136).In a typical assay, the assay mixture for pinene synthase consistsof 2 ml assay buffer (50 mM Tris/HCl, pH 7.5, 500 mM KCl, 1 mMMnCl2, 5 mM dithiothreitol, 0.05% NaHSO3, and 10% glycerol)containing 1 mg of the purified protein. The reaction is initiatedin a Teflon-sealed screw-capped vial by the addition of 300 mMsubstrate. Following incubation at 25.degree. C. for variableperiods (0.5-24 h), the mixture is extracted with 1 ml of diethylether. The biphasic mixture is vigorously mixed and thencentrifuged to separate the phases. The organic extract is dried(MgSO4) and subjected to GC-MS and MDGC analysis.

[0034] As indicated above, it is also possible to employ othermonoterpene synthases in a method according to the invention, forexample the monoterpene synthase from Melaleuca alternifoliadescribed in Shelton et al. (Plant Physiol. Biochem. 42 (2004),875-882) or the monoterpene synthase from Eucalyptus globulus(UniProt accession number Q0PCI4).

[0035] The present inventors have shown that different types ofterpene synthases, e.g. isoprene synthases, (E,E)-alpha-farnesenesynthases and beta-ocimene synthases from different plant organismsare able to convert propan-2-yl into propylene (see Example 2).

[0036] The reactions catalyzed by the various terpene synthases, inparticular the terpene synthases mentioned above, show certaincommon features. For example, the reactions catalyzed by isoprenesynthases, by myrcene/ocimene synthases, by farnesene synthases, bypinene synthase and by other monoterpene synthases, respectively,are all believed to proceed through a common mechanism in which, ina first step a carbocation is created by elimination of thediphosphate (PP.sub.i), which is then followed by directdeprotonation so as to form the corresponding diene.

[0037] It could be shown by the present inventors that enzymeswhich belong to the family of terpene synthases are able to carryout the corresponding reaction by using an alkyl monoester asspecified in formula (I), above, so as to form a monoalkene. Thenatural reaction catalyzed by the terpene synthases is depicted ina schematic form in FIG. 1 as well as the reaction when it isapplied to an alkyl monoester as defined in formula (I), above.

[0038] As mentioned above, in another preferred embodiment theenzyme employed in a method according to the present invention isan enzyme which can be classified as belonging to theprenyltransferase family. Prenyltransferases are a class of enzymesthat transfer allylic prenyl groups to acceptor molecules.Prenyltransferases can be classified as EC 2.5.1. Theprenyltransferases and terpene synthases are mechanistically linkedby a common early step in their catalyzed reactions. The reactioncatalyzed by prenyltransferases starts with the elimination of thediphosphate ion from an allylic diphosphate to form an allyliccation. Namely, both groups of enzymes employ a divalent metal ion(coordinated by a conserved DDXXD/E motif) to facilitate cleavageof the pyrophosphate bond of an allylic diphosphate substrate(Christianson D W Chem Rev. 106 (2006), 3412-3442). In the GeneOntology database these enzymes are identified under theidentification number GO:0004659. Prenyltransferases are commonlydivided into two classes, i.e. cis (or Z) and trans (or E)depending upon the stereochemistry of the resulting products. Inthe scope of the present invention both classes can be employed.The term "prenyltransferase" as used herein comprises in particularthe following three main classes of prenyltransferases: [0039]Isoprenyl pyrophosphate synthases, which catalyze the chainelongation of allylic pyrophosphate substrates via consecutivecondensation reactions with isopentenyl pyrophosphate to generatelinear polymers with defined chain lengths; [0040] Proteinprenyltransferases, which catalyze the transfer of an isoprenylpyrophosphate to a protein or peptide; and [0041]Prenyltransferases which catalyze the cyclization of isoprenylpyrophosphate (see Liang et al., Eur. J. Biochem. 269 (2002),3339-3354, for a review). Prenyltransferases have been studied indetail as regards their structure and function and crystal data aswell as information on the reaction mechanism are available for avariety of prenyltransferases (see e.g. Chang et al., J. Biol.Chem. 278 (2003), 29298-29397; Chang et al., Protein Science 13(2004), 971-977).

[0042] In principle, any prenyltransferase can be employed in themethod according to the present invention, in particular anyprenyltransferase of the three classes mentioned above.

[0043] In a preferred embodiment the prenyltransferase employed ina method according to the present invention is adimethylallyltranstransferase (EC 2.5.1.1), a (2E,6E)-farnesyldiphosphate synthase (EC 2.5.1.10), a geranylgeranyl diphosphatesynthase (EC 2.5.1.29), a ditrans,polycis-undecaprenyl-diphosphatesynthase [(2E,6E)-farnesyl-diphosphate specific (EC 2.5.1.31) or asqualen synthase (EC 2.5.1.21).

[0044] Dimethylallyltranstransferase catalyzes the reaction:

Dimethylallyl diphosphate+isopentenyldiphosphatediphosphate+geranyl diphosphate

[0045] In principle any dimethylallyltranstransferase can beemployed in the method according to the invention. This enzyme isknown from a number of organisms, including animals, plants, fungiand bacteria and has been described, e.g., in Sacharomycescerevisiae, Rhizobium loti, Acyrthosiphon pisum, Geobacillusstearothermophilus, Ips pini, Mentha.times.piperita, Myzuspersicae, Picea abies, Gallus gallus, Homo sapiens and Susscrofa.

[0046] (2E,6E)-farnesyl diphosphate synthase catalyzes thereaction:

Geranyl diphosphate+isopentenyldiphosphatediphosphate+(2E,6E)-farnesyl diphosphate

[0047] In principle any 2E,6E)-farnesyl diphosphate synthase can beemployed in the method according to the invention. This enzyme isknown from a number of organisms, including animals, plants, fungiand bacteria and has been described, e.g., in Streptomycesargenteolus, Mycobacterium tuberculosis, E. coli, Geobacillusstearothermophilus, Abies grandis, Acyrthosiphon grandis,Anthonomus grandis, Artemisia tridentate, Bacillus subtilis, Myzuspersica, Ricinus communis, Panax ginseng, Plasmodium vivax, S.cerevisiae, Toxoplasma gondii, Trypanosoma cruzi, Rattusnorvegicus, Gallus gallus, Homo sapiens and Sus scrofa.

[0048] Geranylgeranyl diphosphate synthase catalyzes thereaction:

(2E,6E)-farnesyl diphosphate+isopentenyldiphosphatediphosphate+geranylgeranyl diphosphate

[0049] In principle any geranylgeranyl diphosphate synthase can beemployed in the method according to the invention. This enzyme isknown from a multitude of organisms, including animals, plants,fungi and bacteria and has been described, e.g., inMethanothermobacter thermautotrophicus, S. cerevisiae,Schizosaccharomyces pombe, Sulfolobus acidocaldarius, Thermustthermopilus, Toxoplasma gondii, Thermococcus kodakarensis, Ginkobiloba, Taxus.times.media, Cistus creticus, Sinapis alba, Zea mays,Solanum lycopersicum, Rattus norvegicus, Homo sapiens and Musmusculus to name just some.

[0050] Ditrans,polycis-undecaprenyl-diphosphate synthase[(2E,6E)-farnesyl-diphosphate specific] catalyzes the reaction:

(2E,6E)-farnesyl-diphosphate+8 isopentenyl diphosphate8diphosphate+ditrans,octacis-undecapernyl diphosphate

[0051] In principle any ditrans,polycis-undecaprenyl-diphosphatesynthase [(2E,6E)-farnesyl-diphosphate specific] can be employed inthe method according to the invention. This enzyme is known fromseveral organisms, including fungi and bacteria and has beendescribed, e.g., in Micrococcus luteus, E. coli, Haemophilusinfluenza, Streptococcus pneumonia, Bacillus subtilis, Helicobacterpyloris, Lactobacillus plantarum, Salmonella Newington and S.cerevisiae.

[0052] Squalen synthase catalyzes the reaction:

2 farnesyl diphosphatediphosphate+presqualen diphosphate

[0053] In principle any squalen synthase can be employed in themethod according to the invention. This enzyme is known from amultitude of organisms, including animals, plants, fungi andbacteria and has been described, e.g., in Trypanosoma cruzi, S.cerevisiae, Arabidopsis thaliana, Euphorbia tirucalli, Panaxginseng, Cavia porcellus, Macaca mulatta, Mus musculus, Rattusnorvegicus, Oryctolagus cuniculus, Cricetus cricetus and Homosapiens to name just some.

[0054] The alkyl monoester which is used as a starting material ina method according to the present invention is a compound offormula (I)

##STR00003##

wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are eachindependently selected from hydrogen, methyl or ethyl; and whereinX is selected from: [0055] O--PO.sub.3H.sub.2 monophosphate [0056]O--PO.sub.2H--O--PO.sub.3H.sub.2 diphosphate [0057] O--SO.sub.3Hsulfate

[0058] It is particularly preferred that the alkyl monoester offormula (I)) is selected from: ethyl diphosphate, propan-1-yldiphosphate (propyl diphosphate), propan-2-yl diphosphate(isopropyl diphosphate), butan-1-yl diphosphate (1-butyldiphosphate), butan-2-yl diphosphate (2-butyl diphosphate),2-methylpropan-1-yl diphosphate (isobutyl diphosphate),1,1-dimethylethyl diphosphate (tert-butyl diphosphate), ethylmonophosphate, propan-1-yl monophosphate (propyl monophosphate),propan-2-yl monophosphate (isopropyl monophosphate), butan-1-ylmonophosphate (1-butyl monophosphate), (2-butyl monophosphate),(isobutyl monophosphate), (tert-butyl monophosphate), ethylsulfate, propan-1-yl sulfate (propyl sulfate), propan-2-yl sulfate(isopropyl sulfate), butan-1-yl sulfate (1-butyl sulfate),butan-2-yl sulfate (2-butyl sulfate), 2-methylpropan-1-yl sulfate(isobutyl sulfate) and 1,1-dimethylethyl sulfate (tert-butylsulfate).

[0059] The following Table 1 gives an overview over alkylmonoesters preferably to be employed in the method according to theinvention and the resulting alkenes:

TABLE-US-00001 TABLE 1 No. Alkyl monoester Monoalkene 1 ethyldiphosphate ethene (i.e. ethylene) 2 propan-1-yl diphosphate(propyl propene (i.e. propylene; diphosphate) methylethylene) 3propan-2-yl diphosphate propene (i.e. propylene; (isopropyldiphosphate) methylethylene) 4 butan-1-yl diphosphate (1-butylbut-1-ene diphosphate) (i.e. .alpha.-butylene) 5 butan-2-yldiphosphate (2-butyl but-1-ene (i.e. .alpha.-butylene) anddiphosphate) but-2-ene (i.e. .beta.-butylene) 6 2-methylpropan-1-yl2-methylprop-1-ene diphosphate (isobutyl (isobutene); diphosphate)7 1,1-dimethylethyl diphosphate 2-methylprop-1-ene (i.e.(tert-butyl diphosphate) isobutene; isobutylene) 8 ethylmonophosphate ethene (i.e. ethylene) 9 propan-1-yl monophosphatepropene (i.e. propylene; (propyl monophosphate) methylethylene) 10propan-2-yl monophosphate propene (i.e. propylene; (isopropylmonophosphate) methylethylene) 11 butan-1-yl monophosphate (1-but-1-ene (i.e. .alpha.-butylene) butyl monophosphate) 12butan-2-yl monophosphate (2- but-1-ene (i.e. .alpha.-butylene) andbutyl monophosphate) but-2-ene (i.e. .beta.-butylene) 132-methylpropan-1-yl 2-methylprop-1-ene (i.e. monophosphate(isobutyl isobutene; isobutylene) monophosphate) 141,1-dimethylethyl 2-methylprop-1-ene (isobutene) monophosphate(tert-butyl monophosphate) 15 ethyl sulfate ethene (i.e. ethylene)16 propan-1-yl sulfate (propyl propene (i.e. propylene; sulfate)methylethylene) 17 propan-2-yl sulfate (isopropyl propene (i.e.propylene; sulfate) methylethylene) 18 butan-1-yl sulfate (1-butylbut-1-ene (i.e. .alpha.-butylene) sulfate) 19 butan-2-yl sulfate(2-butyl but-2-ene (i.e. .beta.-butylene) sulfate) 202-methylpropan-1-yl sulfate 2-methylprop-1-ene (i.e. (isobutylsulfate) isobutene; isobutylene) 21 1,1-dimethylethyl sulfate(tert- 2-methylprop-1-ene (i.e. butyl sulfate) isobutene;isobutylene)

[0060] In one preferred embodiment the alkyl monoester according toformula (I) is an alkyl monoester in which group X is diphosphateand R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independentlyselected from hydrogen, methyl or ethyl. In a particularlypreferred embodiment the alkyl monoester is selected from the groupconsisting of ethyl diphosphate, propan-1-yl diphosphate (propyldiphosphate), propan-2-yl diphosphate (isopropyl diphosphate),butan-1-yl diphosphate (1-butyl diphosphate), butan-2-yldiphosphate (2-butyl diphosphate), 2-methylpropan-1-yl diphosphate(isobutyl diphosphate) and 1,1-dimethylethyl diphosphate(tert-butyl diphosphate).

[0061] In another preferred embodiment the alkyl monoesteraccording to formula (I) is an alkyl monoester in which group X isphosphate and R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are eachindependently selected from hydrogen, methyl or ethyl. In aparticularly preferred embodiment the alkyl monoester is selectedfrom the group consisting of ethyl monophosphate, propan-1-ylmonophosphate (propyl monophosphate), propan-2-yl monophosphate(isopropyl monophosphate), butan-1-yl monophosphate (1-butylmonophosphate), (2-butyl monophosphate), (isobutyl monophosphate)and (tert-butyl monophosphate).

[0062] In another preferred embodiment the alkyl monoesteraccording to formula (I) is an alkyl monoester in which group X issulfate and R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are eachindependently selected from hydrogen, methyl or ethyl. In aparticularly preferred embodiment the alkyl monoester is selectedfrom the group consisting of ethyl sulfate, propan-1-yl sulfate(propyl sulfate), propan-2-yl sulfate (isopropyl sulfate),butan-1-yl sulfate (1-butyl sulfate), butan-2-yl sulfate (2-butylsulfate), 2-methylpropan-1-yl sulfate (isobutyl sulfate) and1,1-dimethylethyl sulfate (tert-butyl sulfate).

[0063] In a particularly preferred embodiment the monoalkene to beproduced is propylene and the alky monoester according to formula(I) is propan-1-yl diphosphate (propyl diphosphate), propan-2-yldiphosphate (isopropyl diphosphate), propan-1-yl monophosphate(propyl monophosphate), propan-2-yl monophosphate (isopropylmonophosphate), propan-1-yl sulfate (propyl sulfate) or propan-2-ylsulfate (isopropyl sulfate).

[0064] It is to be understood that the alkyl monoester to be usedin the method according to the invention may also be a mixture ofdifferent compounds of formula (I).

[0065] In a preferred embodiment of the present invention theenzyme employed in a method according to the present invention isan enzyme comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1 to 10 or a sequence which is at least n% identical to any of SEQ ID NOs: 1 to 10 and having the activityof a terpene synthase 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. The term"sequence identity" preferably means the same amino acid residuesin the same N- to C-terminal direction.

[0066] In one preferred embodiment, the enzyme employed in a methodaccording to the present invention is an enzyme comprising an aminoacid sequence as shown in SEQ ID NO: 1 or a sequence which is atleast n % identical to SEQ ID NO: 1 and having the activity of anisoprene synthase 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. SEQ ID NO: 1shows the isoprene synthase from Pueraris monotana var. lobata(Uniprot Q6EJ97).

[0067] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 2 or a sequence whichis at least n % identical to SEQ ID NO: 2 and having the activityof an (E)-beta-ocimene synthase with n being an integer between 10and 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. SEQ IDNO: 2 shows the (E)-beta-ocimene synthase from Vitis vinifera(Uniprot E5GAG5).

[0068] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 3 or a sequence whichis at least n % identical to SEQ ID NO: 3 and having the activityof an (E,E)-alpha-farnesene synthase with n being an integerbetween 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 or99. SEQ ID NO: 3 shows the (E,E)-alpha-farnesene synthase fromMalus domestica (Uniprot Q84LB2).

[0069] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 4 or a sequence whichis at least n % identical to SEQ ID NO: 4 and having the activityof an monoterpene synthase with n being an integer between 10 and100, 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. SEQ ID NO: 4shows a monoterpene synthase from Melaleuca alternifolia (UniprotQ7Y1V1).

[0070] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 5 or a sequence whichis at least n % identical to SEQ ID NO: 5 and having the activityof an beta-ocimene synthase with n being an integer between 10 and100, 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. SEQ ID NO: 5shows a beta-ocimene synthase from Phaseolus lunatus (UniprotB1P189).

[0071] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 6 or a sequence whichis at least n % identical to SEQ ID NO: 6 and having the activityof an pinene synthase 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. SEQ ID NO: 6shows a chloroplastic pinene synthase from Abies grandis (Uniprot024475).

[0072] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 7 or a sequence whichis at least n % identical to SEQ ID NO: 7 and having the activityof an pentalenene synthase with n being an integer between 10 and100, 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. SEQ ID NO: 7shows a pentalenene synthase from Streptomyces sp. (strain UC5319)(Uniprot P33247).

[0073] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 8 or a sequence whichis at least n % identical to SEQ ID NO: 8 and having the activityof an germacrene-D synthase with n being an integer between 10 and100, 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. SEQ ID NO: 8shows a germacrene-D synthase from Ocimum basilicum (UniprotQ5SBP6).

[0074] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 9 or a sequence whichis at least n % identical to SEQ ID NO: 9 and having the activityof an beta-eudesmol synthase with n being an integer between 10 and100, 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. SEQ ID NO: 9shows a beta-eudesmol synthase from Zingiber zerumbet (UniprotB1B1U4).

[0075] In another preferred embodiment, the enzyme employed in amethod according to the present invention is an enzyme comprisingan amino acid sequence as shown in SEQ ID NO: 10 or a sequencewhich is at least n % identical to SEQ ID NO: 10 and having theactivity of an squalene-hopene cyclase with n being an integerbetween 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 or99. SEQ ID NO: 10 shows a squalene-hopene cyclase fromAlicyclobacillus acidocaldarius subsp. acidocaldarius (UniprotP33247).

[0076] Preferably, the degree of identity is determined bycomparing the respective sequence with the amino acid sequence ofany one of the above-mentioned SEQ ID NOs. When the sequences whichare compared do not have the same length, the degree of identitypreferably either refers to the percentage of amino acid residuesin the shorter sequence which are identical to amino acid residuesin the longer sequence or to the percentage of amino acid residuesin the longer sequence which are identical to amino acid residuesin the shorter sequence. The degree of sequence identity can bedetermined according to methods well known in the art usingpreferably suitable computer algorithms such as CLUSTAL.

[0077] When using the Clustal analysis method to determine whethera particular sequence is, for instance, 80% identical to areference sequence default settings may be used or the settings arepreferably as follows: Matrix: blosum 30; Open gap penalty: 10.0;Extend gap penalty: 0.05; Delay divergent: 40; Gap separationdistance: 8 for comparisons of amino acid sequences. For nucleotidesequence comparisons, the Extend gap penalty is preferably set to5.0.

[0078] Other algorithms which can be used for calculating sequenceidentity are those of Needleman and Wunsch or of Smith andWatermann. For sequence comparisons the program PileUp (Feng andDoolittle, J. Mol. Evolution 25 (1987), 351-360; Higgins et al.,CABIOS 5 (1989), 151-153) or the programs Gap and Best Fit(Needleman and Wunsch, J. Mol. Biol. 48 (1970), 443-453; Smith andWaterman, Adv. Appl. Math. 2 (1981), 482-489) can be used, whichare contained in the GCG software package (Genetics Computer Group,575 Science Drive, Madison, Wis., USA). Preferably, the settingswhich are used are the standard settings for sequencecomparisons.

[0079] Preferably, the degree of identity is calculated over thecomplete length of the sequence.

[0080] The enzyme, preferably the terpene synthase orprenyltransferase, employed in the process according to theinvention can be a naturally occurring enzyme or it can be anenzyme which is derived from a naturally occurring enzyme,preferably a terpene synthase or a prenyltransferase, e.g. by theintroduction of mutations or other alterations which, e.g., alteror improve the enzymatic activity, the stability, etc. The term"terpene synthase" or "a protein/enzyme having the activity of aterpene synthase" in the context of the present application alsocovers enzymes which are derived from a terpene synthase, which arecapable of eliminating the phosphorus or sulfur containing moleculeXH from the alkyl monoester of formula (I) so as to convert it intoa monoalkene but which only have a low affinity to their naturalsubstrate or do no longer accept their natural substrate.

[0081] Similarly, the term "prenyltransferase" or "a protein/enzymehaving the activity of a prenyltransferase" in the context of thepresent application also covers enzymes which are derived from aprenyltransferase, which are capable of eliminating the phosphorusor sulfur containing molecule XH from the alkyl monoester offormula (I) so as to convert it into a monoalkene but which onlyhave a low affinity to their natural substrate or do no longeraccept their natural substrate.

[0082] Thus, the term "terpene synthase" or "a protein/enzymehaving the activity of a terpene synthase" in the context of thepresent application also covers enzymes which are derived from aterpene synthase as described herein-above, which are capable ofeliminating the phosphorus or sulfur containing molecule XH fromthe alkyl monoester of formula (I) so as to convert it into amonoalkene but which only have a low affinity to their naturalsubstrate as described herein-above in connection with thedifferent terpene synthases or do no longer accept their naturalsubstrate.

[0083] Accordingly, the term "prenyltransferase" or "aprotein/enzyme having the activity of a prenyltransferase" in thecontext of the present application also covers enzymes which arederived from a prenyltransferase as described herein-above, whichare capable of eliminating the phosphorus or sulfur containingmolecule XH from the alkyl monoester of formula (I) so as toconvert it into a monoalkene but which only have a low affinity totheir natural substrate as described herein-above in connectionwith the different prenyltransferases or do no longer accept theirnatural substrate.

[0084] Such a modification of the preferred substrate of a terpenesynthase or a prenyltransferase allows to improve the conversion ofthe alkyl monoester into the monoalkene and to reduce theproduction of unwanted by-product due to the action of the enzymeon their natural substrate(s). Methods for modifying and/orimproving the desired enzymatic activities of proteins arewell-known to the person skilled in the art and include, e.g.,random mutagenesis or site-directed mutagenesis and subsequentselection of enzymes having the desired properties or approaches ofthe so-called "directed evolution".

[0085] For example, for genetic engineering in prokaryotic cells, anucleic acid molecule encoding an enzyme, preferably a terpenesynthase or a prenyltransferase, can be introduced into plasmidswhich permit mutagenesis or sequence modification by recombinationof DNA sequences. Standard methods (see Sambrook and Russell(2001), Molecular Cloning: A Laboratory Manual, CSH Press, ColdSpring Harbor, N.Y., USA) allow base exchanges to be performed ornatural or synthetic sequences to be added. DNA fragments can beconnected to each other by applying adapters and linkers to thefragments. Moreover, engineering measures which provide suitablerestriction sites or remove surplus DNA or restriction sites can beused. In those cases, in which insertions, deletions orsubstitutions are possible, in vitro mutagenesis, "primer repair",restriction or ligation can be used. In general, a sequenceanalysis, restriction analysis and other methods of biochemistryand molecular biology are carried out as analysis methods. Theresulting enzyme, preferably terpene synthase or prenyltransferasevariants, are then tested for their enzymatic activity and inparticular for their capacity to convert an alkyl monoesteraccording to formula (I) into a monoalkene by eliminating moleculeXH and prefer an alkyl monoester according to formula (I) as asubstrate rather than their natural substrate(s) as described abovein connection with the description of the different terpenesynthases or prenyltransferases which can be used in the context ofthe present invention.

[0086] Assays for measuring the capacity of a terpene synthase or aprenyltransferase to convert an alkyl monoester according toformula (I) into a monoalkene by eliminating molecule XH aredescribe in the appended Examples.

[0087] Methods for identifying variants with improved enzymaticproperties as regards the production of monoalkenes may also becarried out in the presence of a "cofactor" which allows for asteric and/or electronic complementation in the catalytic site ofthe enzyme due to the fact that the alkyl monoester used as asubstrate may be shorter than the natural substrate of the terpenesynthase or prenyltransferase employed in the method according tothe invention. The cofactor may depend on the natural substrate ofthe enzyme to be employed in the method according to theinvention.

[0088] Moreover, it is described for terpene synthases and forprenyltransferases that they require monovalent and/or divalentcations as co-factors (Green et al., J. Biol. Chem. 284 (2009),8661-8669). Thus, in a further embodiment, a suitable amount of asuitable monovalent (e.g. K.sup.+) and/or divalent cation is addedto the reaction when carrying out the method according to theinvention. The divalent cation is preferably Mg.sup.2+ orMn.sup.2+.

[0089] The modified version of the enzyme, preferably a terpenesynthase or a prenyltransferase, accepting an alkyl monoesteraccording to formula (I), above as a substrate but having a lowaffinity to its natural substrate or no longer accepting itsnatural substrate may be derived from a naturally occurring enzyme,preferably a terpene synthase or a prenyltransferase, or from analready modified, optimized or synthetically produced enzyme,preferably a terpene synthase or a prenyltransferase.

[0090] The enzyme employed in the process according to the presentinvention can be a natural version of the protein or a syntheticprotein as well as a protein which has been chemically synthesizedor produced in a biological system or by recombinant processes. Theenzyme may also be chemically modified, for example in order toimprove its/their stability, resistance, e.g. to temperature, forfacilitating its purification or its immobilization on a support.The enzyme may be used in isolated form, purified form, inimmobilized form, as a crude or partially purified extract obtainedfrom cells synthesizing the enzyme, as chemically synthesizedenzyme, as recombinantly produced enzyme, in the form ofmicroorganisms producing them etc.

[0091] The process according to the present invention may becarried out in vitro or in vivo. An in vitro reaction is understoodto be a reaction in which no cells are employed, i.e. an acellularreaction.

[0092] For carrying out the process in vitro the substrates for thereaction and the enzyme are incubated under conditions (buffer,temperature, cofactors etc.) allowing the enzyme to be active andthe enzymatic conversion to occur. The reaction is allowed toproceed for a time sufficient to produce the monoalkene. Theproduction of the monoalkene can be detected by gas chromatography(GC) or GC/MS analysis.

[0093] The enzyme may be in any suitable form allowing theenzymatic reaction to take place. It may be purified or partiallypurified or in the form of crude cellular extracts or partiallypurified extracts. It is also possible that the enzyme isimmobilized on a suitable carrier.

[0094] Since the alkyl monoester according to formula (I), above,used as a substrate may be shorter than the natural substrate usedby the enzyme, it may be advantageous to add to the reactionmixture a "cofactor" which allows for a steric and/or electroniccomplementation in the catalytic site of the enzyme as mentionedabove.

[0095] In general, if the monoalkene product is a gaseous andscarcely soluble in water under the conditions of temperature atwhich the process is conducted, the equilibrium of the reactioncatalyzed by the enzyme employed is shifted and the reaction goesto completion in the direction of the formation of the gasousalkene, in particular if that gas is permanently removed from thereaction vessel.

[0096] In one particularly preferred embodiment, the enzyme(preferably a terpene synthase or a prenyltransferase) used in theprocess according to the invention is a thermophilic enzyme, i.e.an enzyme which is capable of catalyzing the reaction at elevatedtemperatures. The term "elevated temperatures" means temperaturesabove 37.degree. C. Such enzymes can e.g. be obtained bymutagenizing available enzyme sequences, in particular terpenesynthase sequences or prenyltransferase sequences, and testing themfor an increased enzymatic activity under increased temperatureconditions. The advantage of using an enzyme which is functional atelevated temperatures is that the produced monoalkene canimmediately go into the gaseous phase and can be constantly removedfrom the reaction thereby driving the reaction into the directionof product formation. This advantage exists for all the producedmonoalkenes which are in gaseous form at or below the temperatureat which the reaction is carried out. Accordingly, in the method ofthe present invention the step of enzymatically converting an alkylmonoester according to formula (I), above, into a monoalkene byeliminating molecule XH is preferably carried out at an elevatedtemperature (i.e. at a temperature above 37.degree. C., including atemperature above 37.degree. C. and below 100.degree. C., such as,e.g., at a temperature of 38.degree. C., 40.degree. C., 50.degree.C., 70.degree. C. or 90.degree. C.) and the enzymatic conversion iscatalyzed by a thermophilic enzyme as described herein above. Theuse of elevated temperatures also allows producing monoalkenes in amanner that they directly degas from the reaction mixture.

[0097] For carrying out the process in vivo use is made of asuitable organism/microorganism which is capable of expressing anenzyme as defined above, preferably a terpene synthase or aprenyltransferase. In a preferred embodiment, theorganism/microorganism is capable of secreting the enzyme. In suchan embodiment, the substrate for the reaction can be provided inthe culture medium and the produced monoalkene can be recoveredfrom the culture. In another preferred embodiment theorganism/microorganism is also capable of producing the substrate,i.e. the alkyl monoester according to formula (I), above, to beconverted.

[0098] Thus, in the case of this embodiment the method according tothe invention is characterised in that the conversion of the alkylmonoester according to formula (I), above, into the monoalkene isrealized in the presence of an organism/microorganism capable ofexpressing, preferably secreting, an enzyme as defined above,preferably a terpene synthase or a prenyltransferase. In anotherpreferred embodiment of such a method the organism/microorganism isalso capable of producing an alkyl monoester according to formula(I), above, which should be converted.

[0099] The term "which is capable of producing an alkyl monoesteraccording to formula (I)" in the context of the present inventionmeans that the organism/microorganism has the capacity to producesuch an alkyl monoester within the cell due to the presence ofenzymes providing enzymatic activities allowing the production ofsuch an alkyl monoester from metabolic precursors. Theorganism/microorganism can be an organism/microorganism whichnaturally has the capacity to produce the corresponding alkylmonoester or it can be an organism/microorganism which has beengenetically modified so as to be capable of producing thecorresponding alkyl monoester.

[0100] In a preferred embodiment, the organism employed in themethod according to the invention is an organism, preferably amicroorganism, which has the capacity to produce the respectivealkyl monoester according to formula (I), above, to be convertedinto the corresponding monoalkene and which is recombinant in thesense that it has further been genetically modified so as toexpress an enzyme as defined above, preferably a terpene synthaseor a prenyltransferase as described above. The term "recombinant"in one embodiment means that the organism is genetically modifiedso as to contain a foreign nucleic acid molecule encoding saidenzyme as defined above. In a preferred embodiment the organism hasbeen genetically modified so as to contain a foreign nucleic acidmolecule encoding said enzyme as defined above. The term "foreign"in this context means that the nucleic acid molecule does notnaturally occur in said organism/microorganism. This means that itdoes not occur in the same structure or at the same location in theorganism/microorganism. In one preferred embodiment, the foreignnucleic acid molecule is a recombinant molecule comprising apromoter and a coding sequence encoding the enzyme in which thepromoter driving expression of the coding sequence is heterologouswith respect to the coding sequence. Heterologous in this contextmeans that the promoter is not the promoter naturally driving theexpression of said coding sequence but is a promoter naturallydriving expression of a different coding sequence, i.e., it isderived from another gene, or is a synthetic promoter or a chimericpromoter. Preferably, the promoter is a promoter heterologous tothe organism/microorganism, i.e. a promoter which does notnaturally occur in the respective organism/microorganism. Even morepreferably, the promoter is an inducible promoter. Promoters fordriving expression in different types of organisms, in particularin microorganisms, are well known to the person skilled in theart.

[0101] In another preferred embodiment the nucleic acid molecule isforeign to the organism/microorganism in that the encoded enzyme isnot endogenous to the organism/microorganism, i.e. is naturally notexpressed by the organism/microorganism when it is not geneticallymodified. In other words, the encoded enzyme is heterologous withrespect to the organism/microorganism.

[0102] The term "recombinant" in another embodiment means that theorganism is genetically modified in the regulatory regioncontrolling the expression of an enzyme as defined above whichnaturally occurs in the organism so as to lead to an increase inexpression of the respective enzyme in comparison to acorresponding non-genetically modified organism. The meaning of theterm high "higher expression" is described further below.

[0103] Such a modification of a regulatory region can be achievedby methods known to the person skilled in the art. One example isto exchange the naturally occurring promoter by a promoter whichallows for a higher expression or to modify the naturally occurringpromoter so as to show a higher expression. Thus, in thisembodiment the organism contains in the regulatory region of thegene encoding an enzyme as defined above a foreign nucleic acidmolecule which naturally does not occur in the organism and whichleads to a higher expression of the enzyme in comparison to acorresponding non-genetically modified organism.

[0104] The foreign nucleic acid molecule may be present in theorganism/microorganism in extrachromosomal form, e.g. as a plasmid,or stably integrated in the chromosome. A stable integration ispreferred.

[0105] In another preferred embodiment the organism/microorganismis characterized in that the expression/activity of an enzyme asdefined above is higher in the organism/microorganism geneticallymodified with the foreign nucleic acid molecule in comparison tothe corresponding non-genetically modified organism/microorganism.A "higher" expression/activity means that the expression/activityof the enzyme in the genetically modified microorganism is at least10%, preferably at least 20%, more preferably at least 30% or 50%,even more preferably at least 70% or 80% and particularly preferredat least 90% or 100% higher than in the correspondingnon-genetically modified organism/microorganism. In even morepreferred embodiments the increase in expression/activity may be atleast 150%, at least 200% or at least 500%. In particularlypreferred embodiments the expression is at least 10-fold, morepreferably at least 100-fold and even more preferred at least1000-fold higher than in the corresponding non-genetically modifiedorganism/microorganism.

[0106] The term "higher" expression/activity also covers thesituation in which the corresponding non-genetically modifiedorganism/microorganism does not express a corresponding enzyme sothat the corresponding expression/activity in the non-geneticallymodified organism/microorganism is zero.

[0107] Methods for measuring the level of expression of a givenprotein in a cell are well known to the person skilled in the art.In one embodiment, the measurement of the level of expression isdone by measuring the amount of the corresponding protein.Corresponding methods are well known to the person skilled in theart and include Western Blot, ELISA etc. In another embodiment themeasurement of the level of expression is done by measuring theamount of the corresponding RNA. Corresponding methods are wellknown to the person skilled in the art and include, e.g., NorthernBlot.

[0108] Methods for measuring the enzymatic activity of thedescribed enzymes are known in the art and have already beendescribed above.

[0109] Methods for preparing an organism which is geneticallymodified so as to produce an enzyme as described above, preferablya microorganism, are well known in the art. Thus, generally, theorganism/microorganism is transformed with a DNA construct allowingexpression of the respective enzyme in the microorganism. Such aconstruct normally comprises the coding sequence in question linkedto regulatory sequences allowing transcription and translation inthe respective host cell, e.g. a promoter and/or enhancer and/ortranscription terminator and/or ribosome binding sites etc.

[0110] The term "organism" as used in the context of the presentinvention refers in general to any possible type of organism, inparticular eukaryotic organisms, prokaryotic organisms andarchaebacteria. The term includes animal, plants, fungi, bacteriaand archaebacteria. The term also includes isolated cells or cellaggregates of such organisms, like tissue or calli.

[0111] In one preferred embodiment, the organism is amicroorganism. The term "microorganism" in the context of thepresent invention refers to prokaryotic cells, in particularbacteria, as well as to fungi, such as yeasts, and also to algaeand archaebacteria. In one preferred embodiment, the microorganismis a bacterium. In principle any bacterium can be used. Preferredbacteria to be employed in the process according to the inventionare bacteria of the genus Bacillus, Clostridium, Pseudomonas,Zymomonas or Escherichia. In a particularly preferred embodimentthe bacterium belongs to the genus Escherichia and even morepreferred to the species Escherichia coli.

[0112] In another preferred embodiment the microorganism is afungus, more preferably a fungus of the genus Saccharomyces,Schizosaccharomyces, Aspergillus or Trichoderma and even morepreferably of the species Saccharomyces cerevisiae,Schizosaccharomyces pombe, Aspergillus niger or of the speciesTrichoderma reesei.

[0113] In still another preferred embodiment the microorganism is aphotosynthetically active microorganism such as bacteria which arecapable of carrying out photosynthesis or micro-algae.

[0114] In a particularly preferred embodiment the microorganism isan algae, more preferably an algae belonging to the diatomeae.

[0115] If microorganisms are used in the context of the method ofthe present invention, it is also conceivable to carry out themethod according to the invention in a manner in which two types ofmicroorganisms are employed, i.e. one type which produces the alkylmonoester according to formula (I), above, which should beconverted into a monoalkene and one type which uses the alkylmonoester produced by the first type of microorganisms to convertit with the help of an enzyme as defined herein above into therespective monoalkene.

[0116] When the process according to the invention is carried outin vivo by using microorganisms providing the respective enzymeactivity, the microorganisms are cultivated under suitable cultureconditions allowing the occurrence of the enzymatic reaction. Thespecific culture conditions depend on the specific microorganismemployed but are well known to the person skilled in the art. Theculture conditions are generally chosen in such a manner that theyallow the expression of the genes encoding the enzymes for therespective reactions. Various methods are known to the personskilled in the art in order to improve and fine-tune the expressionof certain genes at certain stages of the culture such as inductionof gene expression by chemical inducers or by a temperatureshift.

[0117] In another preferred embodiment the organism employed in themethod according to the invention is an organism which is capableof photosynthesis, such as a plant or microalgae. In principle anypossible plant can be used, i.e. a monocotyledonous plant or adicotyledonous plant. It is preferable to use a plant which can becultivated on an agriculturally meaningful scale and which allowsto produce large amounts of biomass. Examples are grasses likeLolium, cereals like rye, barley, oat, millet, maize, other starchstoring plants like potato or sugar storing plants like sugar caneor sugar beet. Conceivable is also the use of tobacco or ofvegetable plants such as tomato, pepper, cucumber, egg plant etc.Another possibility is the use of oil storing plants such as rapeseed, olives etc. Also conceivable is the use of trees, inparticular fast growing trees such as eucalyptus, poplar or rubbertree (Hevea brasiliensis).

[0118] In a particularly preferred embodiment theorganism/microorganism employed in the method according to theinvention is an organism/microorganism which is thermophilic in thesense that it can survive and catalyze the conversion of the alkylmonoester of formula (I) into a monoalkene of formula (II) atelevated temperatures. The term "elevated" temperature means atemperature over 37.degree. C. Examples for suchorganism/microorganism are bacteria of the genus Thermus, e.g.Thermus thermophilus or Thermus aquaticus, or bacteria of the genusClostridium, such as Clostridium thermocellum. Other examples aremicroorganisms which are extremely heat-tolerant, e.g.microorganisms of the genus Thermotoga, such as Thermotogamaritime, or microorganisms of the genus Aquifex, such as Aquifexaeolicus.

[0119] The present invention also relates to an organism,preferably a microorganism, which is characterized by the followingfeatures: [0120] (a) it is capable of producing an alkyl monoesteraccording to formula (I), above; and [0121] (b) it expresses anenzyme which is capable of catalyzing the conversion of said alkylmonoester into a monoalkene by elimination of molecule XH informula (I), preferably a terpene synthase or aprenyltransferase.

[0122] As regards the source, nature, properties, sequence etc. ofthe enzyme expressed in the organism according to the invention,the same applies as has been set forth above in connection with themethod according to the invention.

[0123] In one preferred embodiment, the organism according to theinvention is an organism, preferably a microorganism, whichnaturally has the capacity to produce the alkyl monoester accordingto formula (I), above, i.e., feature (a) mentioned above is afeature which the organism, preferably microorganism, showsnaturally.

[0124] In another preferred embodiment, the organism, preferablymicroorganism, according to the invention is a genetically modifiedorganism/microorganism derived from an organism/microorganism whichnaturally does not produce the respective alkyl monoester accordingto formula (I), above, but which has been genetically modified soas to produce said alkyl monoester, i.e. by introducing the gene(s)necessary for allowing the production of the alkyl monoester in theorganism/microorganism. In principle any organism/microorganism canbe genetically modified in this way. The enzymes responsible forthe synthesis of the respective alkyl monoester are generallyknown. Genes encoding corresponding enzymes are known in the artand can be used to genetically modify a given organism, preferablymicroorganism so as to produce the alkyl monoester.

[0125] In a further preferred embodiment the organism, preferably amicroorganism, according to the invention is genetically modifiedso as to express an enzyme which is capable of catalyzing theconversion of an alkyl monoester according to formula (I), above,into a monoalkene as described herein-above. In this context, theterm "recombinant" means in a first aspect that the organismcontains a foreign nucleic acid molecule encoding a correspondingenzyme. The term "foreign" in this context means that the nucleicacid molecule does not naturally occur in saidorganism/microorganism. This means that it does not occur in thesame structure or at the same location in theorganism/microorganism. In one preferred embodiment, the foreignnucleic acid molecule is a recombinant molecule comprising apromoter and a coding sequence encoding said enzyme in which thepromoter driving expression of the coding sequence is heterologouswith respect to the coding sequence. Heterologous in this contextmeans that the promoter is not the promoter naturally driving theexpression of said coding sequence but is a promoter naturallydriving expression of a different coding sequence, i.e., it isderived from another gene, or is a synthetic promoter or a chimericpromoter. Preferably, the promoter is a promoter heterologous tothe organism/microorganism, i.e. a promoter which does naturallynot occur in the respective organism/microorganism. Even morepreferably, the promoter is an inducible promoter. Promoters fordriving expression in different types of organisms, in particularmicroorganisms, are well known to the person skilled in theart.

[0126] In another preferred embodiment the nucleic acid molecule isforeign to the organism/microorganism in that the encoded enzyme isnot endogenous to the organism/microorganism, i.e. is naturally notexpressed by the organism/microorganism when it is not geneticallymodified. In other words, the encoded enzyme is heterologous withrespect to the organism/microorganism.

[0127] The term "recombinant" in another aspect means that theorganism is genetically modified in the regulatory regioncontrolling the expression of an enzyme as defined above whichnaturally occurs in the organism so as to lead to an increase inexpression of the respective enzyme in comparison to acorresponding non-genetically modified organism. The meaning of theterm high "higher expression" is described further below.

[0128] Such a modification of a regulatory region can be achievedby methods known to the person skilled in the art. One example isto exchange the naturally occurring promoter by a promoter whichallows for a higher expression or to modify the naturally occurringpromoter so as to show a higher expression. Thus, in thisembodiment the organism contains in the regulatory region of thegene encoding an enzyme as defined above a foreign nucleic acidmolecule which naturally does not occur in the organism and whichleads to a higher expression of the enzyme in comparison to acorresponding non-genetically modified organism.

[0129] In a further preferred embodiment the organism/microorganismis characterized in that the expression/activity of the enzyme ishigher in the organism/microorganism genetically modified with theforeign nucleic acid molecule in comparison to the correspondingnon-genetically modified organism/microorganism. A "higher"expression/activity means that the expression/activity of theenzyme in the genetically modified organism/microorganism is atleast 10%, preferably at least 20%, more preferably at least 30% or50%, even more preferably at least 70% or 80% and particularlypreferred at least 90% or 100% higher than in the correspondingnon-genetically modified organism/microorganism. In even morepreferred embodiments the increase in expression/activity may be atleast 150%, at least 200% or at least 500%. In particularlypreferred embodiments the expression is at least 10-fold, morepreferably at least 100-fold and even more preferred at least1000-fold higher than in the corresponding non-genetically modifiedorganism/microorganism.

[0130] The term "higher" expression/activity also covers thesituation in which the corresponding non-genetically modifiedorganism/microorganism does not express a corresponding enzyme sothat the corresponding expression/activity in the non-geneticallymodified organism/microorganism is zero.

[0131] Methods for measuring the level of expression of a givenprotein in a cell are well known to the person skilled in the art.In one embodiment, the measurement of the level of expression isdone by measuring the amount of the corresponding protein.Corresponding methods are well known to the person skilled in theart and include Western Blot, ELISA etc. In another embodiment themeasurement of the level of expression is done by measuring theamount of the corresponding RNA. Corresponding methods are wellknown to the person skilled in the art and include, e.g., NorthernBlot.

[0132] Methods for measuring the enzymatic activity of an enzyme asdescribed herein are known in the art and have already beendescribed above.

[0133] The term "organism" as used in the context of the presentinvention refers in general to any possible type of organism, inparticular eukaryotic organisms, prokaryotic organisms andarchaebacteria. The term includes animal, plants, fungi, bacteriaand archaebacteria. The term also includes isolated cells or cellaggregates of such organisms, like tissue or calli.

[0134] In one preferred embodiment, the organism is amicroorganism. The term "microorganism" in the context of thepresent invention refers to prokaryotic cells, in particularbacteria, as well as to fungi, such as yeasts, and also to algaeand archaebacteria. In one preferred embodiment, the microorganismis a bacterium. In principle any bacterium can be used. Preferredbacteria to be employed in the process according to the inventionare bacteria of the genus Bacillus, Clostridium, Pseudomonas,Zymomonas or Escherichia. In a particularly preferred embodimentthe bacterium belongs to the genus Escherichia and even morepreferred to the species Escherichia coli.

[0135] In another preferred embodiment the microorganism is afungus, more preferably a fungus of the genus Saccharomyces,Schizosaccharomyces, Aspergillus or Trichoderma and even morepreferably of the species Saccharomyces cerevisiae,Schizosaccharomyces pombe, Aspergillus niger or of the speciesTrichoderma reesei.

[0136] In still another preferred embodiment the microorganism is aphotosynthetically active microorganism such as bacteria which arecapable of carrying out photosynthesis or micro-algae.

[0137] In a particularly preferred embodiment the microorganism isan algae, more preferably an algae from the genus belonging to thediatomeae.

[0138] In another preferred embodiment the organism according tothe invention is an organism which is capable of photosynthesis,such as a plant or micro-algae. In principle, it can be anypossible plant, i.e. a monocotyledonous plant or a dicotyledonousplant. It is preferably a plant which can be cultivated on anagriculturally meaningful scale and which allows to produce largeamounts of biomass. Examples are grasses like Lolium, cereals likerye, barley, oat, millet, maize, other starch storing plants likepotato or sugar storing plants like sugar cane or sugar beet.Conceivable is also the use of tobacco or of vegetable plants suchas tomato, pepper, cucumber, egg plant etc. In another preferredembodiment the plant is an oil storing plants such as rape seed,olives etc. Also conceivable is the use of trees, in particularfast growing trees such as eucalyptus, poplar or rubber tree (Heveabrasiliensis).

[0139] In a particularly preferred embodiment theorganism/microorganism employed in the method according to theinvention is an organism/microorganism which is thermophilic in thesense that it can survive and catalyze the dehydration of the alkylmonoester of formula (I) into a monoalkene of formula (II) atelevated temperatures. The term "elevated" temperature means atemperature over 37.degree. C. Examples for suchorganism/microorganism are bacteria of the genus Thermus, e.g.Thermus thermophilus or Thermus aquaticus, or bacteria of the genusClostridium, such as Clostridium thermocellum. Other examples aremicroorganisms which are extremely heat-tolerant, e.g.microorganisms of the genus Thermotoga, such as Thermotogamaritime, or microorganisms of the genus Aquifex, such as Aquifexaeolicus.

[0140] The present invention also relates to the use of an organismwhich expresses an enzyme as described herein-above, preferably aterpene synthase or a prenyltransferase, for converting an alkylmonoester according to formula (I), above into a monoalkeneaccording to formula (II), above, by enzymatically eliminatingmolecule XH as specified in formula (I).

[0141] Preferably, in such a use, the organism is an organismaccording to the present invention, i.e. a (micro)organism, whichis characterized by the following features: [0142] (a) it iscapable of producing an alkyl monoester according to formula (I),above; and [0143] (b) it expresses an enzyme which is capable ofcatalyzing the conversion of said alkyl monoester into a monoalkeneby elimination of molecule XH in formula (I), preferably a terpenesynthase or a prenyltransferase.

[0144] I.e., the present invention also relates to the use of anorganism/microorganism according to the invention for theproduction of a monoalkene from the respective alkyl monoester.

[0145] The present invention also relates to a compositioncomprising an organism according to the present invention.

[0146] Moreover, the present invention also relates to acomposition comprising (i) an alkyl monoester according to formula(I), above; and (ii) an enzyme which is capable of catalyzing theconversion of said alkyl monoester into a monoalkene by eliminationof molecule XH in formula (I), preferably a terpene synthase or aprenyltransferase, or an organism according to the presentinvention.

[0147] For the preferred embodiments of the enzyme and theorganism, the same applies as has already been set forth above inconnection with the method and the organism according to theinvention.

[0148] Moreover, the present invention also relates to the use of aterpene synthase or of a prenyltransferase for the conversion of analkyl monoester according to formula (I), above, into a monoalkeneby elimination of molecule XH in formula (I).

[0149] For the preferred embodiments of the enzyme the same appliesas has already been set forth above in connection with the methodand the organism according to the invention.

[0150] Finally, the present invention also relates to the use of analkyl monoester according to formula (I), above, for the productionof a monoalkene, comprising the enzymatic conversion of the alkylmonoester into the monoalkene by elimination of molecule XH offormula (I).

[0151] In a preferred embodiment the enzymatic conversion isachieved by an enzyme as described above in connection with themethod according to the invention, more preferably with a terpenesynthase or a prenyltransferase and most preferably the conversionis achieved by the use of an organism according to theinvention.

[0152] FIG. 1: shows in a schematic form the natural reactioncatalyzed by the terpene synthases as well as the reaction when itis applied to an alkyl monoester as defined in formula (I),above.

[0153] FIG. 2: shows propylene production from propan-2-yldiphosphate using terpene synthases (Example 2).

[0154] FIG. 3: shows mass spectrums of commercial propylene (a) andpropylene produced from propan-2-yl diphosphate in enzymaticreaction catalyzed by isoprene synthase from Pueraria montana var.lobata (b). Characteristic ions of m/z 41 and 27, representingpropylene were observed in both spectrums.

[0155] The following Examples serve to illustrate theinvention.

EXAMPLE 1

Cloning, Expression and Purification of Enzymes

Cloning, Bacterial Cultures and Expression of Proteins.

[0156] The genes encoding the enzymes of interest were cloned inthe pET 25b(+) vector (Novagen). Nucleotide sequences encodingchloroplast transit peptides in plant terpene synthases wereremoved, resulting in a DNA sequences encoding the mature proteinsonly. A stretch of 6 histidine codons was inserted after themethionine initiation codon to provide an affinity tag forpurification. Competent E. coli BL21(DE3) cells (Novagen) weretransformed with this vector by heat shock. The transformed cellswere grown with shaking (160 rpm) on ZYM-5052 auto-induction medium(Studier F W, Prot. Exp. Pur. 41, (2005), 207-234) for 6 h at37.degree. C. and protein expression was continued at 28.degree. C.or 18.degree. C. overnight (approximately 16 h). The cells werecollected by centrifugation at 4.degree. C., 10,000 rpm for 20 minand the pellets were frozen at -80.degree. C.

Protein Purification and Concentration.

[0157] The pellets from 200 ml of culture cells were thawed on iceand resuspended in 5 ml of Na.sub.2HPO.sub.4 pH 8 containing 300 mMNaCl, 5 mM MgCl.sub.2 and 1 mM DTT. Twenty microliters of lysonase(Novagen) were added. Cells were incubated 10 minutes at roomtemperature and then returned to ice for 20 minutes. Cell lysis wascompleted by sonication for 3.times.15 seconds. The bacterialextracts were then clarified by centrifugation at 4.degree. C.,10,000 rpm for 20 min. The clarified bacterial lysates were loadedon PROTINO-1000 Ni-TED or Ni-IDA column (Macherey-Nagel) allowingadsorption of 6-His tagged proteins. Columns were washed and theenzymes of interest were eluted with 4 ml of 50 mMNa.sub.2HPO.sub.4 pH 8 containing 300 mM NaCl, 5 mM MgCl.sub.2, 1mM DTT, 250 mM imidazole. Eluates were then concentrated anddesalted on Amicon Ultra-4 10 kDa filter unit (Millipore) andresuspended in 0.25 ml 50 mM Tris-HCl pH 7.5 containing 1 mM DTTand 10 mM MgCl.sub.2. Protein concentrations were quantified bydirect UV 280 nm measurement on the NanoDrop 1000 spectrophotometer(Thermo Scientific). The purity of proteins thus purified variedfrom 60% to 90%.

EXAMPLE 2

Propylene Production from Propan-2-Yl Diphosphate with PurifiedTerpene Synthases

[0158] The enzymatic assays were carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

100 mM MgCl.sub.2

50 mM KCl

5 mM DTT

[0159] 50 mM propan-2-yl diphosphate 5 mg of the terpene synthasewas added to 0.5 ml of reaction mixture. An enzyme-free controlreaction was carried out in parallel. Assays were incubated at37.degree. C. for 60 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. One ml of the headspace phase was thencollected and injected into a gas chromatograph Varian 430-GCequipped with a flame ionization detector (FID). Nitrogen was usedas carrier gas with a flow rate of 1.5 mL/min. Volatile compoundswere chromatographically separated on RT-AluminaBond/Na.sub.2SO.sub.4 column (Restek) using an isothermal mode at130.degree. C. The enzymatic reaction product was identified bycomparison with propylene standard (Sigma). Under these GCconditions, the retention time for propylene was 2.8 min. Asignificant production of propylene was observed with severalpurified terpene synthases (FIG. 2). Gas chromatography-massspectrometry (GC-MS) was then used to confirm the identity of theproduct detected by gas chromatography with flame ionization. Thesamples were analyzed on a Varian 3400 CX gas chromatographequipped with Varian Saturn 3 mass selective detector. The massspectrum of propylene obtained by enzymatic conversion ofpropan-2-yl diphosphate was similar to the one of commercialpropylene (FIG. 3).

EXAMPLE 3

Propylene Production from Propan-2-Yl Monophosphate with PurifiedTerpene Synthases

[0160] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0161] 50 mM propan-2-yl diphosphate 5 mg of the terpene synthaseis added to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 20-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Propylene production is analyzed usingthe GC/FID procedure described in example 2.

EXAMPLE 4

Ethylene Production from Ethyl Diphosphate with Purified TerpeneSynthases

[0162] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0163] 50 mM ethyl diphosphate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 20-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. One ml of the headspace phase is thencollected and injected into a gas chromatograph Varian 430-GCequipped with a flame ionization detector (FID). Nitrogen is usedas carrier gas with a flow rate of 1.5 mL/min. Volatile compoundsare chromatographically separated on RT-AluminaBond/Na.sub.2SO.sub.4 column (Restek) using an isothermal mode at130.degree. C. The enzymatic reaction product is identified bycomparison with ethylene standard (Sigma). Under these GCconditions, the retention time for ethylene is 2.2 min

EXAMPLE 5

Propylene Production from Propan-1-Yl Diphosphate with PurifiedTerpene Synthases

[0164] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0165] 50 mM propan-1-yl diphosphate 5 mg of the terpene synthaseis added to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 20-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Propylene production is analyzed usingthe GC/FID procedure described in Example 2.

EXAMPLE 6

Isobutene Production from 2-Methylpropan-1-Yl Diphosphate withPurified Terpene Synthases

[0166] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0167] 50 mM 2-methylpropan-1-yl diphosphate 5 mg of the terpenesynthase is added to 0.5 ml of reaction mixture. An enzyme-freecontrol reaction is carried out in parallel. Assays are incubatedat 37.degree. C. for 20-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. One ml of the headspace phase is thencollected and injected into a gas chromatograph Varian 430-GCequipped with a flame ionization detector (FID). Nitrogen is usedas carrier gas with a flow rate of 1.5 mL/min. Volatile compoundsare chromatographically separated on RT-AluminaBond/Na.sub.2SO.sub.4 column (Restek) using an isothermal mode at130.degree. C. The enzymatic reaction product is identified bycomparison with isobutene standard (Sigma). Under these GCconditions, the retention time for isobutene is 4.8 min.

EXAMPLE 7

Isobutene Production from 1,1-Dimethylethyl Diphosphate withPurified Terpene Synthases

[0168] The enzymatic assays are carried out under the followingconditions

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0169] 50 mM 1,1-dimethylethyl diphosphate 5 mg of the terpenesynthase is added to 0.5 ml of reaction mixture. An enzyme-freecontrol reaction is carried out in parallel. Assays are incubatedat 37.degree. C. for 20-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Isobutene production is analyzed usingthe GC/FID procedure described in Example 6.

EXAMPLE 8

But-1-Ene Production from Butan-1-Yl Diphosphate with PurifiedTerpene Synthases

[0170] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0171] 50 mM butan-1-yl diphosphate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 20-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. One ml of the headspace phase is thencollected and injected into a gas chromatograph Varian 430-GCequipped with a flame ionization detector (FID). Nitrogen is usedas carrier gas with a flow rate of 1.5 mL/min. Volatile compoundsare chromatographically separated on RT-AluminaBond/Na.sub.2SO.sub.4 column (Restek) using an isothermal mode at130.degree. C. The enzymatic reaction product is identified bycomparison with but-1-ene standard (Sigma). Under these GCconditions, the retention time for but-1-ene is 4.3 min.

EXAMPLE 9

But-1-Ene and but-2-Ene Production from Butan-2-Yl Diphosphate withPurified Terpene Synthases

[0172] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0173] 50 mM butan-2-yl diphosphate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. But-1-ene and but-2-ene production isanalyzed using the GC/FID procedure described in Example 8. Underthese GC conditions, the retention time for trans but-2-ene and cisbut-2-ene are 4.2 min and 4.9 min, respectively.

EXAMPLE 10

Ethylene Production from Ethyl Monophosphate with Purified TerpeneSynthases

[0174] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0175] 50 mM ethyl monophosphate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Ethylene production is analyzed using theGC/FID procedure described in Example 4.

EXAMPLE 11

Propylene Production from Propan-1-Yl Monophosphate with PurifiedTerpene Synthases

[0176] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0177] 50 mM propan-1-yl monophosphate 5 mg of the terpene synthaseis added to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Propylene production is analyzed usingthe GC/FID procedure described in Example 2.

EXAMPLE 12

Isobutene Production from 2-Methylpropan-1-Yl Monophosphate withPurified Terpene Synthases

[0178] The enzymatic assays are carried out under the followingconditions:

50 mM HEPES pH 8.2

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0179] 50 mM 2-methylpropan-1-yl monophosphate 5 mg of the terpenesynthase is added to 0.5 ml of reaction mixture. An enzyme-freecontrol reaction is carried out in parallel. Assays are incubatedat 37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Isobutene production is analyzed usingthe GC/FID procedure described in example 6.

EXAMPLE 13

Isobutene Production from 1,1-Dimethylethyl Monophosphate withPurified Terpene Synthases

[0180] The enzymatic assays are carried out under the followingconditions

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0181] 50 mM 1,1-dimethylethyl monophosphate 5 mg of the terpenesynthase is added to 0.5 ml of reaction mixture. An enzyme-freecontrol reaction is carried out in parallel. Assays are incubatedat 37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Isobutene production is analyzed usingthe GC/FID procedure described in Example 6.

EXAMPLE 14

But-1-Ene Production from Butan-1-Yl Monophosphate with PurifiedTerpene Synthases

[0182] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0183] 50 mM butan-1-yl monophosphate 5 mg of the terpene synthaseis added to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. But-1-ene production is analyzed usingthe GC/FID procedure described in Example. 8

EXAMPLE 15

But-1-Ene and but-2-Ene Production from Butan-2-Yl Monophosphatewith Purified Terpene Synthases

[0184] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0185] 50 mM butan-2-yl monophosphate 5 mg of the terpene synthaseis added to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. But-1-ene and but-2-ene production isanalyzed using the GC/FID procedure described in Example 9.

EXAMPLE 16

Ethylene Production from Ethyl Sulfate with Purified TerpeneSynthases

[0186] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0187] 50 mM ethyl sulfate 5 mg of the terpene synthase is added to0.5 ml of reaction mixture. An enzyme-free control reaction iscarried out in parallel. Assays are incubated at 37.degree. C. for24-48 hours in a 1.5 ml sealed glass vial (Interchim) with shaking.Ethylene production is analyzed using the GC/FID proceduredescribed in Example 4.

EXAMPLE 17

Propylene Production from Propan-1-Yl Sulfate with Purified TerpeneSynthases

[0188] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0189] 50 mM propan-1-yl sulfate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Propylene production is analyzed usingthe GC/FID procedure described in Example 2.

EXAMPLE 18

Propylene Production from Propan-2-Yl Sulfate with Purified TerpeneSynthases

[0190] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0191] 50 mM propan-2-yl sulfate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Propylene production is analyzed usingthe GC/FID procedure described in Example 2.

EXAMPLE 19

Isobutene Production from 2-Methylpropan-1-Yl Sulfate with PurifiedTerpene Synthases

[0192] The enzymatic assays are carried out under the followingconditions:

50 mM HEPES pH 8.2

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0193] 50 mM 2-methylpropan-1-yl sulfate 5 mg of the terpenesynthase is added to 0.5 ml of reaction mixture. An enzyme-freecontrol reaction is carried out in parallel. Assays are incubatedat 37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Isobutene production is analyzed usingthe GC/FID procedure described in Example 6.

EXAMPLE 20

Isobutene Production from 1,1-Dimethylethyl Sulfate with PurifiedTerpene Synthases

[0194] The enzymatic assays are carried out under the followingconditions

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0195] 50 mM 1,1-dimethylethyl sulfate 5 mg of the terpene synthaseis added to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. Isobutene production is analyzed usingthe GC/FID procedure described in Example 6.

EXAMPLE 21

But-1-Ene Production from Butan-1-Yl Sulfate with Purified TerpeneSynthases

[0196] The enzymatic assays are carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0197] 50 mM butan-1-yl sulfate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. But-1-ene production is analyzed usingthe GC/FID procedure described in Example. 8

EXAMPLE 22

But-1-Ene and but-2-Ene Production from Butan-2-Yl Sulfate withPurified Terpene Synthases

[0198] The enzymatic assays were carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

50-100 mM MgCl.sub.2

20-50 mM KCl

2-5 mM DTT

[0199] 50 mM butan-2-yl sulfate 5 mg of the terpene synthase isadded to 0.5 ml of reaction mixture. An enzyme-free controlreaction is carried out in parallel. Assays are incubated at37.degree. C. for 24-48 hours in a 1.5 ml sealed glass vial(Interchim) with shaking. But-1-ene and but-2-ene production isanalyzed using the GC/FID procedure described in Example 9.

EXAMPLE 23

Propylene Production from Propan-2-Yl Diphosphate Using PurifiedPrenyltransferase

[0200] Enzyme catalyzed conversion of propan-2-yl diphosphate intopropylene is carried out under the following conditions:

50 mM Tris-HCl pH 7.5

[0201] 20 mM propan-2-yl diphosphate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0202] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0203] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Propyleneproduction is analyzed using GC/FID procedure described in Example2.

EXAMPLE 24

Propylene Production from Propan-2-Yl Monophosphate Using PurifiedPrenyltransferase

[0204] Enzyme catalyzed conversion of propan-2-yl monophosphateinto propylene is carried out under the following conditions:

50 mM Tris-HCl pH 7.5

[0205] 20 mM propan-2-yl monophosphate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0206] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0207] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Propyleneproduction is analyzed using GC/FID procedure described in Example2.

EXAMPLE 25

Propylene Production from Propan-2-Yl Sulfate Using PurifiedPrenyltransferase

[0208] Enzyme catalyzed conversion of propan-2-yl sulfate intopropylene is carried out under the following conditions:

50 mM Tris-HCl pH 7.5

[0209] 20 mM propan-2-yl sulfate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0210] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0211] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Propyleneproduction is analyzed using GC/FID procedure described in Example2.

EXAMPLE 26

Isobutene Production from 1,1-Dimethylethyl Diphosphate UsingPurified Prenyltransferase

[0212] Enzyme catalyzed conversion of 1,1-dimethylethyl diphosphateinto isobutene is carried out under the following conditions:

50 mM Tris-HCl pH 7.5

[0213] 20 mM 1,1-dimethylethyl diphosphate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0214] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0215] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Isobuteneproduction is analyzed using GC/FID procedure described in Example6.

EXAMPLE 27

Isobutene Production from 1,1-Dimethylethyl Monophosphate UsingPurified Prenyltransferase

[0216] Enzyme catalyzed conversion of 1,1-dimethylethylmonophosphate into isobutene is carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

[0217] 20 mM 1,1-dimethylethyl monophosphate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0218] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0219] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Isobuteneproduction is analyzed using GC/FID procedure described in Example6.

EXAMPLE 28

Isobutene Production from 1,1-Dimethylethyl Sulfate Using PurifiedPrenyltransferase

[0220] Enzyme catalyzed conversion of 1,1-dimethylethyl sulfateinto isobutene is carried out under the following conditions:

50 mM Tris-HCl pH 7.5

[0221] 20 mM 1,1-dimethylethyl sulfate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0222] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0223] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Isobuteneproduction is analyzed using GC/FID procedure described in Example6.

EXAMPLE 29

Isobutene Production from 2-Methylpropan-1-Yl Diphosphate UsingPurified Prenyltransferase

[0224] Enzyme catalyzed conversion of 2-methylpropan-1-yldiphosphate into isobutene is carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

[0225] 20 mM 2-methylpropan-1-yl diphosphate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0226] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0227] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Isobuteneproduction is analyzed using GC/FID procedure described in Example6.

EXAMPLE 30

Isobutene Production from 2-Methylpropan-1-Yl Monophosphate UsingPurified Prenyltransferase

[0228] Enzyme catalyzed conversion of 2-methylpropan-1-ylmonophosphate into isobutene is carried out under the followingconditions:

50 mM Tris-HCl pH 7.5

[0229] 20 mM 2-methylpropan-1-yl monophosphate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0230] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0231] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Isobuteneproduction is analyzed using GC/FID procedure described in Example6.

EXAMPLE 31

Isobutene Production from 2-Methylpropan-1-Yl Sulfate UsingPurified Prenyltransferase

[0232] Enzyme catalyzed conversion of 2-methylpropan-1-yl sulfateinto isobutene is carried out under the following conditions:

50 mM Tris-HCl pH 7.5

[0233] 20 mM 2-methylpropan-1-yl sulfate

33 mM KCl

33 mM MgCl.sub.2

4 mM DTT

[0234] The reaction is started by adding 3 mg of the preparation ofprenyltransferase to 0.5 ml of reaction mixture.

[0235] Assays are incubated with shaking at 37-42.degree. C. for2-72 h in 1.5 ml sealed glass vials (Interchim). Isobuteneproduction is analyzed using GC/FID procedure described in Example6.

* * * * *