WO2011076691A1

WO2011076691A1 - Method for producing an alkene comprising the step of converting an alcohol by an enzymatic dehydration step

Info

Publication number: WO2011076691A1
Application number: PCT/EP2010/070103
Authority: WO
Inventors: Philippe Marliere
Original assignee: Philippe Marliere
Priority date: 2009-12-21
Filing date: 2010-12-17
Publication date: 2011-06-30
Also published as: EP2336341A1

Abstract

Described is a method for producing an alkene comprising the step of converting an alcohol by an enzymatic dehydration step. The conversion preferably makes use of an unsaturated fatty acid hydratase, in particular of an oleate hydratase (EC 4.2.1.53). Moreover, the present invention describes a recombinant organism which produces an alcohol and which expresses a hydratase enzyme which is capable of catalyzing the conversion of said alcohol into an alkene.

Description

Method for producing an atkene comprising the step of converting an alcohol by an enzymatic dehydration step

The present invention relates to a method for producing an aikene comprising the step of converting an alcohol by an enzymatic dehydration step. The conversion preferably makes use of an unsaturated fatty acid hydratase, in particular of an oleate hydratase (EC 4.2.1.53). Moreover, the present invention relates to a recombinant organism which produces an alcohol and which expresses a hydratase enzyme which is capable of catalyzing the conversion of said alcohol into an aikene.

A large number of chemical compounds are currently derived from petrochemicals. Alkenes (such as ethylene, propylene, the different butenes, or else the pentenes, for example) are used in the plastics industry, for example for producing polypropylene or polyethylene, and in other areas of the chemical industry and that of fuels. Ethylene, the simplest aikene, lies at the heart of industrial organic chemistry: it is the most widely produced organic compound in the world. It is used in particular to produce polyethylene, a major plastic. Ethylene can also be converted to many industrially useful products by reaction (e.g. by oxidation or halogenation). Propylene plays a similarly important role: its polymerization results in a plastic material, polypropylene. The technical properties of this product in terms of resistance, density, solidity, deformability, and transparency are unequalled. The worldwide market for polypropylene has grown continuously since its 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 can also be used to produce isooctene, which in turn can be reduced to isooctane (2,2,4-trimethyipentane); the very high octane rating of isooctane makes it the best fuel for so-called "gasoline" engines. Amylene, hexene and heptene exist in many forms according to the position and configuration of the double bond. These products have real industrial applications but are less important than ethylene, propylene or butenes. All these a!kenes are currently produced by catalytic cracking of petroleum products (or by a derivative of the Fischer-Tropsch process in the case of hexene, from coal or gas). Their production costs are therefore tightly linked to the price of oil. Moreover, catalytic cracking is sometimes associated with considerable technical difficulties which increase process complexity and production costs.

The production by a biological pathway of alkenes or other organic molecules that can be used as fueis or as precursors of synthetic resins is called for in the context of a sustainable industrial operation in harmony with geochemical cycles. The first generation of biofuels consisted in the fermentative production of ethanol, as fermentation and distillation processes already existed in the food processing industry. The production of second generation biofuels is in an exploratory phase, encompassing in particular the production of long chain alcohols (butanol and pentanol), terpenes, linear alkanes and fatty acids. Two recent reviews provide a general overview of research in this field: Ladygina et al. (Process Biochemistry 41 (2006), 001 ) and Wackett (Current Opinions in Chemical Biology 21 (2008), 187).

The production of ethylene by plants has long been known (Meigh et al. (Nature 186 (1960), 902)). According to the metabolic pathway elucidated, methionine is the precursor of ethylene (Adams and Yang (PNAS 76 (1979), 70)). Conversion of 2- oxoglutarate has also been described (Ladygina et al. (Process Biochemistry 41 (2006), 1001 ). Since a single ethylene molecule requires the previous production of a four- or five-carbon chain, the equipment and energy needs of all these pathways are unfavorable and do not bode well for their industrial application for alkene bioproduction.

Moreover, many microorganisms are capable of producing propylene, however, with an extremely low yield

The conversion of isovalerate to isobutylene by the yeast Rhodotorula minuta has been described (Fujii et al. (Appl. Environ. Microbiol. 54 (1988), 583)), but the efficiency of this reaction, less than 1 millionth per minute, or about 1 for 1000 per day, is far from permitting an industrial application. The reaction mechanism was elucidated by Fukuda et al. (BBRC 201 (1994), 516) and involves a cytochrome P450 enzyme which decarboxylates isovalerate by reduction of an oxoferryl group Fe^v=0. Large-scale biosynthesis of isobutylene by this pathway seems highly unfavorable, since it would require the synthesis and degradation of one mofecule of leucine to form one molecule of isobutylene. Also, the enzyme catalyzing the reaction uses heme as cofactor, poorly lending itself to recombinant expression in bacteria and to improvement of enzyme parameters. For all these reasons, it appears very unlikely that this pathway can serve as a basis for industrial exploitation. Other microorganisms have been described as being marginally capable of naturally producing isobutylene from isovalerate; the yields obtained are even lower than those obtained with Rhodotorula minuta (Fukuda et al. (Agric. Biol. Chem. 48 (1984), 1679)).

Isoprene is produced at a significant level in higher plants, such as poplars. The production of isoprene in this context remains however low and the pathway which leads to isoprene production, which is based on the mevalonate-isopentenyl- pyrophosphate pathway, poorly complies with the demands for industrial scale production.

Thus, there is still a need for efficient and environmentally friendly methods of producing alkenes.

The present invention meets this demand by providing a method for producing an alkene comprising the step of converting an alcohol into an alkene by an enzymatic dehydration step.

The conversion preferably makes use of a hydratase enzyme, in particular of an oieate hydratase.

The present invention teaches that it is possible to convert an alcohol enzymatically by the use of a hydratase into an alkene. The enzyme is preferably an oieate hydratase (EC 4.2.1.53).

In particular, it has been found that an enzyme with the activity of an oieate hydratase is not only capable of catalyzing the hydration of oieate but is also capable of catalyzing the dehydration of alcohols and not only of longer alcohols but also of short chain alcohols. Thus, it was found that this enzyme can actually be used to convert alcohols into alkenes which allows the cost efficient and environmentally friendly production of alkenes in microorganism or other organisms. Figure 1 shows a scheme of the reaction catalyzed by oleate hydratase and the generic reaction of converting an alcohol into an alkene.

The alcohol to be used in the method according to the invention is a compound of formula (I):

OH H

-C- -C—

(I)

R¹, R², R³ and R⁴ are each independently selected from hydrogen or C-1-11 hydrocarbyl.

Said hydrocarbyl is preferably selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aryl, more preferably selected from alkyl, alkenyl, alkynyl, or aryl, and even more preferably selected from alkyl, alkenyl, or aryl.

Said alkyl preferably comprises 1 to 1 1 carbon atoms, more preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 (i.e. 1 , 2, 3 or 4) carbon atoms.

Said alkenyl preferably comprises 2 to 11 carbon atoms, more preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 (i.e. 2, 3 or 4) carbon atoms.

Said alkynyl preferably comprises 2 to 11 carbon atoms, more preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 (i.e. 2, 3 or 4) carbon atoms.

Said cycloalkyl preferably comprises 3 to 1 1 carbon atoms, more preferably 3 to 8 carbon atoms, and even more preferably 3 to 6 (i.e. 3, 4, 5 or 6) carbon atoms.

Said aryl is preferably selected from phenyl or naphthyl, and is more preferably phenyl. Accordingly, said d- hydrocarbyl is preferably selected from C1-11 alkyl (in particular, Ci-₆ alkyl or Ci-₄ alkyl), C₂- alkenyl (in particular, C₂-6 alkenyl or C₂-₄ alkenyl), C₂- alkynyl (in particular, C₂-₆ alkynyl or C₂.₄ alkynyl), C₃-n cycloalkyl (in particular, C₃-₆ cycloalkyl), C₃.n cycloalkenyf (in particular, C₃.₆ cycloalkenyl), phenyl or naphthyf. More preferably, said d-n hydrocarbyl is selected from C1-11 alkyl (in particular, C -6 alkyl or C1-4 alkyl), C₂- alkenyl (in particular, C₂-6 alkenyl or C₂.₄ alkenyl) or phenyl.

In a preferred embodiment, R¹ , R², R³, and R⁴ are each independently selected from hydrogen, C1-11 alkyl, C₂.n alkenyl, C2-11 alkynyl, C3.11 cycloalkyl, C3.11 cycloalkenyl, phenyl or naphthyi, and are more preferably each independently selected from hydrogen, C1-6 alkyl, C₂-6 alkenyl or phenyl, and are even more preferably each independently selected from hydrogen or linear C _-4 alkyl.

In general, it is preferred that the compound of formula (I) comprises a total of 2 to 30 carbon atoms, more preferably 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms, more preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, even more preferably 2 to 5 carbon atoms, and most preferably 2, 3 or 4 carbon atoms.

Moreover, while primary, secondary and tertiary alcohols can generally be used in the method of the invention, it is preferred to use a secondary alcohol. Accordingly, it is preferred that one of R¹ and R² comprised in the compound of formula (I) is hydrogen and the other one of R¹ and R² is d- 1 hydrocarbyl (or one of the preferred C1- 1 hydrocarbyl groups described and defined herein above).

It is further preferred that the alcohol to be used in the method according to the invention is a linear (i.e. unbranched) alcohol. Accordingly, it is preferred that one of R¹ and R² in the compound of formula (I) is hydrogen, the other one of R¹ and R² is linear -n hydrocarbyl (or one of the preferred C1-11 hydrocarbyl groups described and defined herein above which are linear, such as e.g. linear C1.4 alkyl or linear C₂-4 alkenyl), one of R³ and R⁴ in the compound of formula (I) is hydrogen, and the other one of R³ and R⁴ is linear Ci-n hydrocarbyl (or one of the preferred Ci-1 1 hydrocarbyl groups described and defined herein above which are linear, such as e.g. linear C₁-4 alky! or linear C₂.₄ alkenyl).

It is particularly preferred that the alcohol (or, accordingly, the compound of formula (I)) is selected from: ethanol; propanol, including propan-1 -ol (i.e. n-propanol) and propan-2-ol (i.e. isopropanol); butanol, including butan-1 -ol (i.e. n-butanol), 2-methylpropan-1 -ol (i.e. isobutanol), 2-methylpropan-2-ol (i.e. tert-butanol) and butan-2-ol (i.e. sec-butanol); or pentanol, including pentan-1 -ol (i.e. n-pentanol or amyl alcohol), 3-methylbutan-1 -ol (i.e. isopentanol or isoamyl alcohol), pentan-2-ol (i.e. sec-pentanol), pentan-3-ol, 2-methylbutan-1-ol, 3-methylbutan-2-ol (i.e. 1 ,2-dimethylpropanol) and 2-methylbutan-2-ol (i.e. 1 ,1 -dimethylpropanol).

The following Table 1 gives an overview over some examples of alcohols to be employed in the method according to the invention and the resulting alkenes:

Table 1

(i.e. n-pentanol) (i.e. amylene)

3-methylbutan-1 -ol 3-methylbut-1 -ene

(i.e. isopentanol)

pentan-2-ol pent-1 -ene

(i.e. sec-pentanol) (i.e. amylene)

pentan-2-ol pent-2-ene

(i.e. sec-pentanol) (i.e. β-amylene)

pentan-3-oi pent-2-ene

(i.e. β-amylene)

2-methylbutan-1 -ol 2-methylbut-1 -ene

3-methylbutan-2-ol 2-methylbut-2-ene

(i.e. 1 ,2-dimethylpropanol)

3-methylbutan-2-ol 3-methylbut-1 -ene

(i.e. 1 ,2-dimethylpropanol)

2-methylbutan-2-ol 2-methylbut-1 -ene

(i.e. 1 ,1 -dimethylpropanol)

2-methylbutan-2-ol 2-methylbut-2-ene

(i.e. 1 ,1-dimethylpropanol)

3-methylbut-3-en-1 -ol 2-methylbuta-1 ,3-diene

(i.e. isoprene)

2-methylbut-3-en-2-ol 2-methylbuta-1 ,3-diene

(i.e. isoprene)

2-methylbut-3-en-2-ol 3-methylbuta-1 ,2-diene

2-methylbut-3-en-1-ol 2-methylbuta-1 ,3-diene

(i.e. isoprene)

3-methylbut-3-en-2-ol 2-methylbuta-1 ,3-diene

(i.e. isoprene)

but-3-en-1 -ol buta-1 ,3-diene

but-3-en-2-ol buta-1 ,3-diene

1 -phenylethanol styrene

(i.e. vinylbenzene)

2-phenylethanol styrene

(i.e. vinylbenzene)

In one particularly preferred embodiment the alcohol is ethanol and the produced alkene is ethene (i.e. ethylene). In another particularly preferred embodiment the alcohol is propan~2-ol (i.e. isopropanol) and the produced alkene is propene (i.e. propylene). Yet, in a further particularly preferred embodiment the alcohol is butanol and the produced alkene is butene (i.e. butylene). in this case butanol may be any of n-butanol, tert-butanol, sec-butanol or isobutano!. The produced alkene is either but-1 -ene (i.e. a-butylene), 2-methylpropene (i.e. isobutyfene), or both but-1-ene (i.e. a-butylene) and but-2-ene (i.e. β-butylene). Most preferred in this context are n- butanol and isobutanol.

In a further particularly preferred embodiment the alcohol is any one of 3-methylbut- 3-en-1 -ol, 2-methylbut-3-en-2-ol, 2-methylbut-3-en-1 -ol, 3-methylbut-3-en-2-ol, or mixtures thereof (preferably the alcohol is any one of 3-methylbut-3-en-1 -ol, 2-methylbut-3-en-1 -ol, 3-methylbut-3-en-2-ol, or mixtures thereof) and the produced alkene is 2-methylbuta-1 ,3-diene (i.e. isoprene). In another particularly preferred embodiment the alcohol is any one of but-3-en-1-ol, but-3-en-2-ol, or a mixture thereof and the produced alkene is buta-1 ,3-diene. In another particularly preferred embodiment the alcohol is any one of 1-phenylethanoi, 2-phenylethanol, or a mixture thereof and the produced alkene is styrene (i.e. vinylbenzene).

It is to be understood that the alcohol to be used in the method according to the invention may also be a mixture of different compounds of formula (I).

With respect to the product of the method according to the invention, the term "alkene" refers to a compound comprising at least one carbon-to-carbon double bond and, in particular, refers to a compound of formula (II)

-C: :C—

(II) wherein R¹ , R², R³, and R⁴ have the same meanings and preferred meanings as described and defined herein above for the compound of formula (I).

It is to be understood that all stereoisomers of the compound of formula (I) can be used in the method according to the invention, either in admixture or in pure or substantially pure form. Furthermore, the compound of formula (II) encompasses both cis-isomers and trans-isomers.

As used herein and unless defined otherwise, the term "hydrocarbyl" refers to a monovalent hydrocarbon group, i.e. a group consisting of carbon atoms and hydrogen atoms, which group is connected to the remainder of the compound of formula (I) via a carbon-to-carbon single bond and may be saturated or unsaturated, linear, branched or cyclic, aliphatic or aromatic. A "d-n hydrocarbyl" denotes a hydrocarbyl having 1 to 1 1 carbon atoms.

As used herein and unless defined otherwise, the term "alkyl" refers to a monovalent saturated aliphatic (i.e. non-aromatic) acyclic hydrocarbon group which may be linear or branched and does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond.

As used herein and unless defined otherwise, the term "alkenyl" refers to a monovalent unsaturated aliphatic acyclic hydrocarbon group which may be linear or branched and comprises at least one carbon-to-carbon double bond while it does not comprise any carbon-to-carbon triple bond.

As used herein and unless defined otherwise, the term "alkynyl" refers to a monovalent unsaturated aliphatic acyclic hydrocarbon group which may be linear or branched and comprises at least one carbon-to-carbon triple bond and optionally one or more carbon-to-carbon double bonds.

As used herein and unless defined otherwise, the term "cycloalkyl" refers to a monovalent cyclic saturated aliphatic hydrocarbon group which does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. Non-limiting examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

As used herein and unless defined otherwise, the term "cycloalkenyl" refers to a monovalent cyclic unsaturated aliphatic hydrocarbon group which comprises at least one carbon-to-carbon double bond and does not comprise any carbon-to-carbon triple bond. Non-limiting examples of cycloalkyl groups are cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl or cyclohexadienyl.

As used herein and unless defined otherwise, the term "aryl" refers to a monovalent aromatic hydrocarbon group, including bridged ring and/or fused ring systems, containing at least one aromatic ring. "Aryl" may, for example, refer to phenyl, naphthyl or anthracenyl.

An enzyme with the activity of a hydratase is an enzyme which is capable of catalyzing the hydration-dehydration of C-0 linkages, in general, such an enzyme can also catalyze the reverse reaction, i.e. the dehydration. In the scope of dehydration such an enzyme catalyzes the elimination of oxygen and hydrogen from organic compounds in the form of water.

In general, in the context of the present invention any enzyme could be used which has hydratase activity. In a preferred embodiment the hydratase enzyme is a fatty acid hydratase. A fatty acid hydratase is understood to be an enzyme which has the capacity to catalyse the addition of a water molecule to an unsaturated fatty acid. The term "unsaturated fatty acid" refers to a fatty acid which contains at least one carbon- to-carbon double bond. In principle the unsaturated fatty acid can be any unsaturated fatty acid. Preferably the unsaturated fatty acid is a fatty acid which has more than 4, more than 6, more than 8, more than 10, more than 12 or more than 14 carbon atoms. In another preferred embodiment the unsaturated fatty acid has not more than 28, not more than 26, not more than 24, not more than 22, not more than 20 or not more than 18 carbon atoms. In a preferred embodiment the unsaturated fatty acid has a length between 10 and 22 carbon atoms. Examples for unsaturated fatty acids include obtusilic acid (C10:1), caproleic acid (C10:1), lauroleic acid (C12:1), linderic acid (C12:1 ), myristoleic acid (C14:1), physeteric acid (C14:1 ), tsuzuic acid (C14:1), palmitoleic acid (C 16:1 ), sapienic acid (C16:1 ), oleic acid (C18:1 ), linoleic acid (C18:2), alpha-linolenic acid (C18:3), petroselinic acid (C 8:1), elaidic acid (C18:1 ), vaccenic acid (C18:1), arachidonic acid (C20:4), eicosapentanoic acid (C20:5), gadoleic acid (C20:1 ), gondoic acid (C20:1 ), erucic acid (C22:1), docosahexaenoic acid (C22:6), cetoleic acid (C22:1 ), erucic acid (C22:1 ) and nervonic acid (C24: 1 ), and in particular lauroleic acid (C12:1 ), myristoleic acid (C14:1), palmitoleic acid (C 16:1), oleic acid (C18:1 ), linoleic acid (C18:2), alpha-linolenic acid (C18:3), arachidonic acid (C20:4), eicosapentanoic acid (C20:5), erucic acid (C22:1 ) and docosahexaenoic acid (C22:6). (n a preferred embodiment the fatty acid is a fatty acid with a length of between 10 and 22 carbon atoms and which has a double bond between carbon atoms 9 and 10, It is to be understood that the fatty acid may also be present in a deprotonated form; for example, oleic acid may be present in its deprotonated form as the anion oleate.

Thus, in a particularly preferred embodiment the fatty acid hydratase is an enzyme which is capable of catalysing the addition of a water molecule to an unsaturated fatty acid which has a double bond between carbon atoms 9 and 10 and which leads to the formation of a 10-hydroxy fatty acid, In an even more preferred embodiment the fatty acid is oleate. For numbering the C-atoms within the fatty acid molecules the usual numbering is used starting from the carbon of the carboxy group of the fatty acid.

The hydratase and in particular the fatty acid hydratase activity can be measured by incubating the corresponding enzyme with the corresponding substrate, in particular an unsaturated fatty acid substrate, under appropriate conditions and analysing the reaction products, e.g. by GC-MS analysis.

In a particularly preferred embodiment the fatty acid hydratase is an oleate hydratase. In the context of the present invention the term "oleate hydratase" or "an enzyme with the activity of an oleate hydratase" is understood to be an enzyme which is capable of catalyzing the hydration of a non-activated carbon-carbon bond. In particular, such an enzyme is capable of converting oleic acid (OA) into 10- hydroxystearic acid (10-HAS) via hydration. This enzymatic activity can be measured by incubating the enzyme with oleic acid and analysing the reaction products, e.g. via gas chromatography. It can in particular be assayed, e.g., by an assay as described in Bevers et al. (J. Bacterid. 191 (2009), 5010-5012). In brief, the enzyme is incubated for at least 1 h in 20 mM Tris (pH 8)-50 mM NaCI with OA at an appropriate temperature at which the enzyme shows activity (e.g., 22°C, 30°C or 37°C). The reaction is stopped by the addition of 3M HCI and the products are analysed by gas chromatography (GC). The occurrence of 10-HAS is indicative of oleate hydratase activity. Alternatively, the activity can also be tested as described in WO 2008/1 19735. In this assay 20 ^ig oleic acid is mixed with the enzyme to be tested in 1 ml 0.1 M sodium phosphate pH 7.1 , 0.1 M NaCI and incubated for at least 1 h at an appropriate temperature (e.g., 22°C, 30°C or 37°C). Subsequently the products are characterized by GC (see Example 6 of WO 2008/1 19735).

Preferably, in the context of the present application the term "oleate hydratase" or "a protein/enzyme having the activity of an oleate hydratase" refers to any enzyme which is classified in the EC number EC 4.2.1.53.

In principle any hydratase enzyme and in particular any oleate hydratase enzyme can be used in the context of the present invention, in particular any hydratase enzyme or oleate hydratase enzyme which is derived from a prokaryotic or a eukaryotic organism. Oleate hydratase has been described from different organisms, e.g. from Streptococcus pyogenes (WO 2008/119735; SEQ ID NO:1 ) and from Elizabethkingia meningoseptica (Bevers et a(., loc. cit.; GenBank Accessionnumber GQ144652; SEQ ID NO:2). It has been found that about 165 homologues to the Elizabethkingia meningoseptica sequence can be found in other organisms (see Bevers et al., loc. cit.). Originally most of these proteins were annotated as cross-myosin-reactive antigen due to their homology to the Streptococcus pyogenes 67.5 kDa protein which had later been found to have oleate hydratase activity (WO 2008/1 19735). Examples for such sequences are shown in SEQ ID NOs: 3 to 11.

Moreover, the following Table 2 lists some of the proteins found to be homologous to the above mentioned protein from Elizabethkingia meningoseptica. These proteins constitute further preferred embodiments of enzymes employed in the method according to the present invention.

Table 2

Uniprot accession Organism

number

C7DLJ6 Flavobacterium meningosepticum (Elizabethkingia

menigoseptica)

Q1VYG7 Psychroflexus torquis ATCC 700755.

Q6N2EO Rhodopseudomonas palust is

A9W1 Y8 Methylobacterium extorquens (strain PA1 ).

B2TM67 Clostridium botulinum (strain Eklund 17B / Type B).

A3I6F1 Bacillus sp. B 14905.

B9YB92 Holdemania filiformis DSM 12042.

Q5FL96 Lactobacillus acidophilus.

A6QD90 Staphylococcus aureus (strain Newman)

Q5XDG8 Streptococcus pyogenes serotype M6.

C7ZMN5 Nectria haematococca (strain 77-13-4 / FGSC 9596 /

PVI)

C3RJT3 Mollicutes bacterium D7.

In a preferred embodiment of the present invention the oleate hydratase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 1 1 or a sequence which is at least n % identical to any of SEQ ID NOs: 1 to 1 and having the activity of an oleate hydratase 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 residues in the same N- to C-terminal direction.

Preferably, the degree of identity is determined by comparing the respective sequence with the amino acid sequence of any one of the above-mentioned SEQ ID NOs. When the sequences which are compared do not have the same length, the degree of identity preferably either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. The degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.

When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

Other algorithms which can be used for calculating sequence identity are those of Needleman and Wunsch or of Smith and Watermann. For sequence comparisons the program PileUp (Feng and Doolittle, 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 and Waterman, Adv. Appl. Math. 2 (1981), 482-489) can be used, which are contained in the GCG software package (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA). Preferably, the settings which are used are the standard settings for sequence comparisons.

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

The hydratase, preferably the oleate hydratase, employed in the process according to the invention can be a naturally occurring enzyme or it can be an enzyme which is derived from a naturally occurring hydratase, preferably oleate hydratase, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.

The term "hydratase" or "a protein/enzyme having the activity of a hydratase" in the context of the present application also covers enzymes which are derived from a hydratase, which are capable of dehydrating an alcohol as defined above into an aikene but which only have a low affinity to their natural substrate or do no longer accept their natural substrate.

Accordingly, the term "oleate hydratase" or "a protein/enzyme having the activity of an oleate hydratase" in the context of the present application also covers enzymes which are derived from an oleate hydratase, which are capable of dehydrating an alcohol as defined above into an aikene but which only have a low affinity to oleate as a substrate or do no longer accept oleate as a substrate. Such a modification of the preferred substrate of an oleate hydratase allows to improve the conversion of the alcohol into the aikene and to reduce the production of unwanted by-product due to the action of the enzyme on oleate or other unsaturated fatty acids. Methods for modifying and/or improving the desired enzymatic activities of proteins are well- known to the person skilled in the art and include, e.g., random mutagenesis or site- directed mutagenesis and subsequent selection of enzymes having the desired properties or approaches of the so-called "directed evolution".

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

An assay for measuring the capacity of an oleate hydratase to dehydrate an alcohol and to convert it into an alkene is describe in Examples, in particular Examples 2 to 8.

Methods for identifying variants with improved enzymatic properties as regards the production of alkenes may also be carried out in the presence of a "cofactor" which allows for a steric and/or electronic complementation in the catalytic site of the enzyme due to the fact that the alcohol used as a substrate may be shorter than the fatty acid which functions as the natural substrate, e.g. oleate. The cofactor may depend on the natural substrate of the enzyme to be employed in the method according to the invention. The following Table 3 summarizes the substrates, products and possible cofactors. Table 3

Alcohol Alkene Possible cofactor ethanol ethene octanoic acid

(i.e. ethylene)

propan-1 -ol propene heptanoic acid

(i.e. n-propanol) (i.e. propylene)

propan-2-ol propene octanoic acid

(i.e. isopropanoi) (i.e. propylene)

butan-1 -ol but-1-ene hexanoic acid

(i.e. n-butanol) (i.e. a-butylene)

2-methylpropan-1 -ol 2-methylprop-1 -ene heptanoic acid

(i.e. isobutanol) (i.e. isobutylene)

2-methylpropan-2-ol 2-methylprop-1-ene octanoic acid

(i.e. tert-butanol) (i.e. isobutylene)

butan-2-ol but-1 -ene octanoic acid

(i.e. sec-butanol) (i.e. a-butylene)

butan-2-ol but-2-ene heptanoic acid

(i.e. sec-butanol) (i.e. β-butylene)

pentan-1-ol pent-1-ene pentanoic acid

(i.e. n-pentanol) (i.e. amylene)

3-methylbutan-1 -ol 3-methylbut-1-ene hexanoic acid

(i.e. isopentanol)

pentan-2-ol pent-1 -ene octanoic acid

(i.e. sec-pentanol) (i.e. amylene)

pentan-2-oi pent-2-ene hexanoic acid

(i.e. sec-pentanol) (i.e. β-amylene)

pentan-3-ol pent-2-ene heptanoic acid

(i.e. β-amylene)

2-methylbutan-1-ol 2-methylbut-1 -ene hexanoic acid

3-methylbutan-2-ol 2-methylbut-2-ene heptanoic acid

(i.e.

1 ,2-dimethylpropanol)

3-methylbutan-2-ol 3-methylbut-1-ene octanoic acid

(i.e.

1 ,2-dimethylpropanol)

2-methylbutan-2-ol 2-methylbut-1-ene octanoic acid

(i.e. 1 ,1 - dimethylpropanol)

2-methylbutan-2-ol 2-methylbut-2-ene heptanoic acid

(i.e. 1 ,1 - dimethylpropanol)

3-methylbut-3-en-1 -ol 2-methylbuta-1 ,3-diene hexanoic acid

(i.e. isoprene)

2-methylbut-3-en-2-o! 2-methylbuta-1 ,3-diene octanoic acid

(i.e. isoprene)

2-methylbut-3-en-2-ol 3-methylbuta-1 ,2-diene heptanoic acid

2-methy!but-3-en-1 -ol 2-methylbuta-1 ,3-diene hexanoic acid

(i.e. isoprene)

3-methyibut-3-en-2-ol 2-methylbuta-1 ,3-diene octanoic acid

(i.e. isoprene)

but-3-en-1 -ol buta-1 ,3-diene hexanoic acid

but-3-en-2-ol buta-1 ,3-diene heptanoic acid

1-phenylethanol styrene octanoic acid

(i.e. vinylbenzene)

2-phenylethanol styrene hexanoic acid or heptanoic

(i.e. vinyibenzene) acid

Preferred cofactors are linear alkanoic acids of the general formula H-(CH2)n-COOH having 2 to 8 carbon atoms, preferably 3 to 8, even more preferably 3 to 6 carbon atoms. Also conceivable as cofactors are analogs and specifically alkane sulfonic acids of the general formula H-(CH2)n-S03H.

The modified version of the hydratase, preferably oleate hydratase, accepting an alcohol as a substrate but having a low affinity to its natural substrate, e.g. a fatty acid, in particular oleate, as a substrate or no longer accepting its natural substrate, preferably a fatty acid, in particular oleate, as a substrate may be derived from a naturally occurring hydratase, preferably oleate hydratase, or from an already modified, optimized or synthetically produced hydratase, preferably oleate hydratase.

The enzyme employed in the process according to the present invention can be a natural version of the protein or a synthetic protein as well as a protein which has been chemically synthesized or produced in a biological system or by recombinant processes. The enzyme may also be chemically modified, for example in order to improve its/their stability, resistance, e.g. to temperature, for facilitating its purification or its immobilization on a support. The enzyme may be used in isolated form, purified form, in immobilized form, as a crude or partially purified extract obtained from cells synthesizing the enzyme, as chemically synthesized enzyme, as recombinantly produced enzyme, in the form of microorganisms producing them etc.

The process according to the present invention may be carried out in vitro or in vivo. An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction.

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

The enzyme may be in any suitable form allowing the enzymatic reaction to take place. It may be purified or partially purified or in the form of crude cellular extracts or partially purified extracts. It is also possible that the enzyme is immobilized on a suitable carrier.

Since the alcohol used as a substrate may be shorter than the natural substrate used by the enzyme, e.g. oleate, it may be advantageous to add to the reaction mixture a "cofactor" which allows for a steric and/or electronic complementation in the catalytic site of the enzyme as mentioned above.

In general, if the alkene product is a gaseous and scarcely soluble in water under the conditions of temperature at which the process is conducted, the equilibrium of the reaction catalyzed by the enzyme employed is shifted and the reaction goes to completion in the direction of the formation of the gasous alkene , in particular if that gas is permanently removed from the reaction vessel.

In one particularly preferred embodiment, the hydratase enzyme used in the process according to the invention is a thermophilic hydratase, i.e. a hydratase which is capable of catalyzing the reaction at elevated temperatures. The term "elevated temperatures" means temperatures above 37°C. Such enzymes can either be isolated from corresponding thermophilic organisms, in particular bacteria or archaebacteria, or they can be obtained by mutagenizing available hydratase sequences and testing them for an increased enzymatic activity under increased temperature conditions. The advantage of using a hydratase which is functional at elevated temperatures is that the produced alkene can immediately go into the gaseous phase and can be constantly removed from the reaction thereby driving the reaction into the direction of dehydration. This advantage exists for all the produced alkenes which are in gaseous form at or below the temperature at which the reaction is carried out. Accordingly, in the method of the present invention the step of converting an alcohol by an enzymatic dehydration step into an alkene is preferably carried out at an elevated temperature (i.e. at a temperature above 37°C, including a temperature above 37°C and below 100°C, such as, e.g., at a temperature of 38°C, 40°C, 50°C, 70°C or 90°C) and the enzymatic dehydration step is catalyzed by a thermophilic hydratase as described herein above. The use of elevated temperatures also allows to produce longer alkenes in a manner that they directly degas from the reaction mixture, e.g. alkenes having 6 or 7 carbon atoms. Thus, in this preferred embodiment the alkene to be produced has preferably at least 4, even more preferably at least 5 even more preferably at least 6 and most preferably at least 7 carbon atoms. Moreover, it is preferred that the alkene does not have more than 12, 10 or 8 carbon atoms.

For carrying out the process in vivo use is made of a suitable organism/microorganism which is capable of expressing a hydratase enzyme as defined above. In principle, the substrate, i.e. the alcohol to be converted into the alkene, can be provided externally to the microorganism. However, in a preferred embodiment the organism/microorganism is also capable of producing the substrate, i.e. the alcohol to be converted.

Thus, in the case of this embodiment the method according to the invention is characterised in that the conversion of the alcohol into the alkene is realized in the presence of an organism/ microorganism capable of expressing a hydratase enzyme as defined above and preferably also capable of producing an alcohol which should be converted.

The term "which is capable of producing an alcohol" in the context of the present invention means that the organism/microorganism has the capacity to produce such an alcohol within the cell due to the presence of enzymes providing enzymatic activities allowing the production of such an alcohol from metabolic precursors. The organism/microorganism can be an organism/microorganism which naturally has the capacity to produce the corresponding alcohol or it can be an organism/microorganism which has been genetically modified so as to be capable of producing the corresponding alcohol.

For example, in one preferred embodiment of the present invention the alcohol to be converted into an alkene (in particular ethylene) is ethano! and the organism/microorganism employed in the method according to the invention is an organism/microorganism which is capable of producing ethanol. Examples for microorganism which naturally produce ethanol are yeast, in particular Saccharomyces species, Klyveromyces lactis and bacteria of the genus Zymomonas, most preferably Zymomonas mobilis. However, according to this embodiment any organism can be used which has the capacity to produce ethanol. In particular, it is also possible to use an organism/microorganism which has been genetically modified so as to confer to it the capacity to produce ethanol.

In another preferred embodiment of the present invention the alcohol to be converted into an alkene (in particular a-butylene) is n-butanol and the organism/microorganism employed in the method according to the invention is an organism/microorganism which is capable of producing n-butanol. Examples for microorganism which naturally produce n-butanol are bacteria of the genus Clostridium, preferably Clostridium acetobutyiicum. However, it is also possible to use an organism/microorganism which has been genetically modified so as to confer to it the capacity to produce n-butanol.

In another preferred embodiment of the present invention the alcohol to be converted into an alkene (in particular isobutylene) is isobutanol and the organism/microorganism employed in the method according to the invention is an organism/microorganism which is capable of producing isobutanol. Examples for microorganism which produce isobutanol are bacteria of the genus E. coli which have been genetically modified so as to produce isobutanol (Connor and Liao (Curr. Opin. Biotechnol. 20 (2009), 307). Another example of a microorganism which can produce isobutanol is S. cerevisiae. In particular, S. cerevisiae is capable of producing isobutanol when fed with valine as a carbon source (Dickinson et ai. (J. Biol. Chem. 273 (1998), 25751 )). Another organism which is capable of producing isobutanol is Lactococcus lactis.

In yet a further preferred embodiment according to the present invention the alcohol to be converted into an alkene (in particular propylene) is isopropanol and the organism/microorganism employed in the method according to the invention is an organism/microorganism which is capable of producing isopropanol. Examples for microorganisms which are capable of producing isopropanol are acetone-producing bacteria which have been equipped with a secondary alcohol dehydrogenase which converts acetone into isopropanol. In particular, when a secondary alcohol dehydrogenase is produced in these bacteria the reaction (in the reverse orientation converts acetone into isopropanol that can then be further converted into isopropanol according to the method of the present invention. Corresponding E. coli cells are described in Jojima et al. (Appl. Microbiol. Biotechnol. 77 (2008), 12 9-1224) and in Hanai et al. (Appl. Environ. Microbiol. 73 (2007), 7814-7818). A source for the secondary alcohol dehydrogenase is, e.g. Clostridium beij^'erinckii.

Acetone is produced by certain microorganisms, such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa and Pseudomonas putida. The synthesis of acetone is best characterized in Clostridium acetobutylicum. It starts out with a reaction (reaction step 1 ) in which two molecules of acetyl-CoA are condensed into acetoacetyl-CoA. This reaction is catalyzed by acetyl-CoA acetyltransferase (EC 2.3.1.9). Acetoacetyl-CoA is then converted into acetoacetate by a reaction with acetic acid or butyric acid resulting also in the production of acetyl-CoA or butyryl-CoA (reaction step 2). This reaction is catalyzed e.g. by acetoacetylCoA transferase (EC 2.8.3.8). AcetoacetylCoA transferase is known from various organisms, e.g. from E. coli in which it is encoded by the atoAD gene or from Clostridium acetobutylicum in which it is encoded by the ctfAB gene. However, also other enzymes can catalyze this reaction, e.g. 3-oxoacid CoA transferase (EC 2.8.3.5) or succinate CoA ligase (EC 6.2.1.5).

Finally, acetoacetate is converted into acetone by a decarboxylation step (reaction step 3) catalyzed by acetoacetate decarboxylase (EC 4.1.1 .4). The above described reaction steps 1 and 2 and the enzymes catalyzing them are not characteristic for the acetone synthesis and can be found in various organism. In contrast, reaction step 3 which is catalyzed by acetoacetate decarboxylase (EC 4.1.1.4) is only found in those organisms which are capable of producing acetone. The prior art already describes microorganisms which have been genetically modified so as to be able to produce acetone. In particular genes from, e.g., Clostridium acetobutylicum have been introduced into E. cofi thereby allowing the synthesis of acetone in E. coli, a bacterium which naturally does not produce acetone (Bermejo et at., Appl. Environ. Microbiol. 64 (1998); 1079-1085; Hanai et al., Appl. Environ. Microbiol. 73 (2007), 7814-7818). In particular Hanai et al. (loc. cit.) shows that it is sufficient to introduce a nucleic acid sequence encoding an acetoacetate decarboxylase (such as that from Clostridium acetobutylicum) in order to achieve acetone production in E. coli indicating that the endogenous enzymes in E. coli catalyzing the above-mentioned reaction steps 1 and 2 (i.e. the expression products of the E. coli atoB and atoAD genes) are sufficient to provide substrate for the acetone production.

In one preferred embodiment the organism employed in the method according to the invention is an organism, preferably a microorganism, which naturally has the capacity to produce acetone and furthermore expresses a secondary alcohol dehydrogenase so as to convert acetone into isopropanol which can then be further converted into propylene. Thus, preferably the microorganism belongs to the genus Clostridium, Bacillus or Pseudomonas, more preferably to the species Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium celluloiyticum, Bacillus polymyxa or Pseudomonas putida. However, it is, of course, also possible to use an organism/microorganism which has been genetically modified so as to confer to it the capacity to produce acetone. The genes responsible for acetone production have been cloned and also methods for transferring these genes into other organisms, such as E. coli, to confer to them the capacity to produce acetone have already been described.

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

In another preferred embodiment the nucleic acid molecule is foreign to the organism/microorganism in that the encoded hydratase enzyme is not endogenous to the organism/microorganism, i.e. is naturally not expressed by the organism/microorganism when it is not genetically modified. In other words, the encoded hydratase enzyme is heterologous with respect to the organism/microorganism.

The term "recombinant" in another embodiment means that the organism is genetically modified in the regulatory region controlling the expression of a hydratase enzyme as defined above which naturally occurs in the organism so as to lead to an increase in expression of the respective enzyme in comparison to a corresponding non-genetically modified organism. The meaning of the term high "higher expression" is described further below. Such a modification of a regulatory region can be achieved by methods known to the person skilled in the art. One example is to exchange the naturally occurring promoter by a promoter which allows for a higher expression or to modify the naturally occurring promoter so as to show a higher expression. Thus, in this embodiment the organism contains in the regulatory region of the gene encoding an enzyme as defined above a foreign nucleic acid molecule which naturally does not occur in the organism and which leads to a higher expression of the enzyme in comparison to a corresponding non-genetically modified organism.

The foreign nucleic acid molecule may be present in the organism/microorganism in extrachromosomal form, e.g. as plasmid, or stably integrated in the chromosome. A stable integration is preferred.

In another preferred embodiment the organism/microorganism is characterized in that the expression/activity of a hydratase enzyme as defined above is higher in the organism/microorganism genetically modified with the foreign nucleic acid molecule in comparison to the corresponding non-genetically modified organism/microorganism. A "higher" expression/activity means that the expression/activity of the hydratase enzyme in the genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-genetically modified organism/microorganism. In even more preferred embodiments the increase in expression/activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-genetically modified organism/microorganism.

The term "higher" expression/activity also covers the situation in which the corresponding non-genetically modified organism/microorganism does not express a corresponding hydratase enzyme so that the corresponding expression/activity in the non-genetically modified organism/microorganism is zero.

Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art, In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, EL1SA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot.

Methods for measuring the enzymatic activity of the hydratase enzyme are known in the art and have already been described above.

Methods for preparing an organism which is genetically modified so as to produce a hydratase as described above, preferably a microorganism, are well known in the art. Thus, generally, the organism/microorganism is transformed with a DNA construct allowing expression of the respective enzyme in the microorganism. Such a construct normally comprises the coding sequence in question linked to regulatory sequences allowing transcription and translation in the respective host cell, e.g. a promoter and/or enhancer and/or transcription terminator and/or ribosome binding sites etc.

The term "organism" as used in the context of the present invention refers in general to any possible type of organism, in particular eukaryotic organisms, prokaryotic organisms and archaebacteria. The term includes animal, plants, fungi, bacteria and archaebacteria. The term also includes isolated cells or cell aggregates of such organisms, like tissue or calli.

In one preferred embodiment, the organism is a microorganism. The term "microorganism" in the context of the present invention refers to prokaryotic cells, in particular bacteria, as well as to fungi, such as yeasts, and also to algae and archaebacteria. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Pseudomonas, Zymomonas or Escherichia. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli.

In another preferred embodiment the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus or Trichoderma and even more preferably of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger or of the species Trichoderma reesei. in still another preferred embodiment the microorganism is a photosynthetically active microorganism such as bacteria which are capable of carrying out photosynthesis or micro-algae.

In a particularly preferred embodiment the microorganism is an algae, more preferably an algae belonging to the diatomeae.

If microorganisms are used in the context of the method of the present invention, it is also conceivable to carry out the method according to the invention in a manner in which two types of microorganisms are employed, i.e. one type which produces the alcohol which should be converted into an alkene and one type which uses the alcohol produced by the first type of microorganisms to convert it with the help of a hydratase enzyme as defined herein above into the respective alkene.

When the process according to the invention is carried out in vivo by using microorganisms providing the respective enzyme activity, the microorganisms are cultivated under suitable culture conditions allowing the occurrence of the enzymatic reaction. The specific culture conditions depend on the specific microorganism employed but are well known to the person skilled in the art. The culture conditions are generally chosen in such a manner that they allow the expression of the genes encoding the enzymes for the respective reactions. Various methods are known to the person skilled in the art in order to improve and fine-tune the expression of certain genes at certain stages of the culture such as induction of gene expression by chemical inducers or by a temperature shift.

In another preferred embodiment the organism employed in the method according to the invention is an organism which is capable of photosynthesis, such as a plant or microalgae. In principle any possible plant can be used, i.e. a monocotyledonous plant or a dicotyledonous plant. It is preferable to use a plant which can be cultivated on an agriculturally meaningful scale and which allows to produce large amounts of biomass. Examples are grasses like Lolium, cereals like rye, barley, oat, millet, maize, other starch storing plants like potato or sugar storing plants like sugar cane or sugar beet. Conceivable is also the use of tobacco or of vegetable plants such as tomato, pepper, cucumber, egg plant etc. Another possibility is the use of oil storing plants such as rape seed, olives etc. Also conceivable is the use of trees, in particular fast growing trees such as eucalyptus, poplar or rubber tree (Hevea brasiliensis).

In a particularly preferred embodiment the organism/microorganism employed in the method according to the invention is an organism/microorganism which is thermophilic in the sense that it can survive and catalyze the dehydration of the alcohol in question at elevated temperatures. The term "elevated" temperature means a temperature over 37°C. Examples for such organism/microorganism are bacteria of the genus Thermus, e.g. Thermus thermophilus or Thermus aquaticus, or bacteria of the genus Clostridium, such as Clostridium thermocellum. Other examples are microorganisms which are extremely heat-tolerant, e.g. microorganisms of the genus Thermotoga, such as Thermotoga maritime, or microorganisms of the genus Aquifex, such as Aquifex aeolicus.

The present invention also relates to an organism, preferably a microorganism, which is characterized by the following features;

(a) it is capable of producing an alcohol; and

(b) it expresses a hydratase enzyme which is capable of catalyzing the conversion of the alcohol into an a!kene.

As regards the source, nature, properties, sequence etc. of the hydratase enzyme expressed in the organism according to the invention, the same applies as has been set forth above in connection with the method according to the invention.

In one preferred embodiment, the organism according to the invention is an organism, preferably a microorganism, which naturally has the capacity to produce the alcohol, i.e., feature (a) mentioned above is a feature which the organism, preferably microorganism, shows naturally. In a particularly preferred embodiment the alcohol is ethanol, isopropanol, n-butanol or isobutanol.

As regards organisms/microorganisms producing such alcohols, the same as set forth above in connection with the method according to the invention applies.

In another preferred embodiment, the organism, preferably microorganism, according to the invention is a genetically modified organism/microorganism derived from an organism/microorganism which naturally does not produce the respective alcohol but which has been genetically modified so as to produce said alcohol, i.e. by introducing the gene(s) necessary for allowing the production of the alcohol in the organism/microorganism. In principle any organism/microorganism can be genetically modified in this way. The enzymes responsible for the synthesis of the respective alcohols are generally known. Genes encoding corresponding enzymes are known in the art and can be used to genetically modify a given organism, preferably microorganism so as to produce the alcohol.

In a further preferred embodiment the organism, preferably a microorganism, according to the invention is genetically modified so as to express a hydratase enzyme which is capable of catalyzing the conversion of an alcohol into an alkene. In this context, the term "recombinant" means in a first aspect that the organism contains a foreign nucleic acid molecule encoding a corresponding hydratase enzyme. The term "foreign" in this context means that the nucleic acid molecule does not naturally occur in said organism/microorganism. This means that it does not occur in the same structure or at the same location in the organism/microorganism. In one preferred embodiment, the foreign nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding said hydratase enzyme in which the promoter driving expression of the coding sequence is heterologous with respect to the coding sequence. Heterologous in this context means that the promoter is not the promoter naturally driving the expression of said coding sequence but is a promoter naturally driving expression of a different coding sequence, i.e., it is derived from another gene, or is a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter heterologous to the organism/microorganism, i.e. a promoter which does naturally not occur in the respective organism/microorganism. Even more preferably, the promoter is an inducible promoter. Promoters for driving expression in different types of organisms, in particular microorganisms, are well known to the person skilled in the art.

The term "recombinanf in another aspect means that the organism is genetically modified in the regulatory region controlling the expression of an enzyme as defined above which naturally occurs in the organism so as to lead to an increase in expression of the respective enzyme in comparison to a corresponding non- genetically modified organism. The meaning of the term high "higher expression" is described further below.

Such a modification of a regulatory region can be achieved by methods known to the person skilled in the art. One example is to exchange the naturally occurring promoter by a promoter which allows for a higher expression or to modify the naturally occurring promoter so as to show a higher expression. Thus, in this embodiment the organism contains in the regulatory region of the gene encoding an enzyme as defined above a foreign nucleic acid molecule which naturally does not occur in the organism and which leads to a higher expression of the enzyme in comparison to a corresponding non-genetically modified organism.

In a further preferred embodiment the organism/microorganism is characterized in that the expression/activity of the hydratase enzyme is higher in the organism/microorganism genetically modified with the foreign nucleic acid molecule in comparison to the corresponding non-genetically modified organism/microorganism. A "higher" expression/activity means that the expression/activity of the hydratase enzyme in the genetically modified organism/microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-genetically modified organism/microorganism. In even more preferred embodiments the increase in expression/activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-genetically modified organism/microorganism.

The term "higher" expression/activity also covers the situation in which the corresponding non-genetically modified organism/microorganism does not express a corresponding hydratase enzyme so that the corresponding expression/activity in the non-geneticaily modified organism/microorganism is zero.

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

In one preferred embodiment, the organism is a microorganism. The term "microorganism" in the context of the present invention refers to prokaryotic cells, in particular bacteria, as weil as to fungi, such as yeasts, and also to algae and archaebacteria. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Pseudomonas, Zymomonas or Escherichia. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli.

In a particularly preferred embodiment the microorganism is an algae, more preferably an algae from the genus belonging to the diatomeae.

In another preferred embodiment the organism according to the invention is an organism which is capable of photosynthesis, such as a plant or micro-algae. In principle, it can be any possible plant, i.e. a monocotyledonous plant or a dicotyledonous plant. It is preferably a plant which can be cultivated on an agriculturally meaningful scale and which allows to produce large amounts of biomass. Examples are grasses like Lolium, cereals like rye, barley, oat, millet, maize, other starch storing plants like potato or sugar storing plants like sugar cane or sugar beet. Conceivable is also the use of tobacco or of vegetable plants such as tomato, pepper, cucumber, egg plant etc. In another preferred embodiment the plant is an oil storing plants such as rape seed, olives etc. Also conceivable is the use of trees, in particular fast growing trees such as eucalyptus, poplar or rubber tree (Hevea brasiliensis).

In a particularly preferred embodiment the organism/microorganism employed in the method according to the invention is an organism/microorganism which is thermophilic in the sense that it can survive and catalyze the dehydration of the alcohol in question at elevated temperatures. The term "elevated" temperature means a temperature over 37°C. Examples for such organism/microorganism are bacteria of the genus Thermus, e.g. Thermus thermophilus or Thermus aquaticus, or bacteria of the genus Clostridium, such as Clostridium thermoce!lum. Other examples are microorganisms which are extremely heat-tolerant, e.g. microorganisms of the genus Thermotoga, such as Thermotoga maritime, or microorganisms of the genus Aquifex, such as Aquifex aeolicus.

The present invention also relates to the use of an organism, preferably a microorganism, which is characterized by the following features:

(a) it is capable of producing an alcohol; and (b) it expresses a hydratase enzyme which is capable of catalyzing the conversion of said alcohol into an a!kene

for the production of an alkene.

I.e., the present invention also relates to the use of an organism/microorganism according to the invention for the production of an alkene from the respective alcohol.

The present invention also relates to a composition comprising an organism according to the present invention.

Moreover, the present invention also relates to a composition comprising (i) an alcohol; and (ii) a hydratase enzyme which is capable of catalyzing the conversion of said alcohol into an alkene.

For the preferred embodiments of the enzyme the same applies as has already been set forth above in connection with the method and the organism according to the invention.

Moreover, the present invention also relates to the use of a hydratase enzyme which is capable of catalyzing the conversion of an alcohol into an alkene for the production of an alkene as defined herein-above from said alcohol.

Finally, the present invention also relates to the use of an alcohol for the production of an alkene, comprising the enzymatic conversion of the alcohol by a hydratase enzyme which is capable of catalyzing the conversion of the alcohol into an alkene. In a preferred embodiment the enzymatic conversion is achieved by an enzyme as described above in connection with the method according to the invention, more preferably with an oleate hydratase and most preferably the conversion is achieved by the use of an organism according to the invention.

Figure 1 : shows a scheme of the reaction catalyzed by oleate hydratase and the generic reaction of converting an alcohol into an alkene. The following Examples serve to illustrate the invention.

Example 1 : Cloning, expression and purification of a collection of oleate hydratases

A Iibrary of genes encoding enzymes homologous to the oleate hydratase (EC4.2.1.53) of Elizabethkingia meningoseptica (accession number GQ1444652) is cloned, expressed and purified.

Cloning, bacterial cultures and expression of proteins

Amino-acid sequences corresponding to proteins homologous to the oleate hydratase from Elizabethkingia meningoseptica (accession number GQ1444652) are obtained from sequence database using the BLAST algorithm. A set of 20 sequences are chosen for maximizing genetic distance. In order to obtain a high level of expression in E. coli, the DNA sequences inferred from these protein sequences are generated taking in consideration the Codon Adaptation Index, as defined by Sharp and Li (Nucl. Acids Res. 15 (1987), 1281 -1295).

The optimized sequences are synthesized by oligonucleotide concatenation (Geneart) and cloned in a pET25 expression vector, with the addition of a methionine initiation codon and a 6-histidine tag added at the N-terminal end immediately preceding the first methionine of the original protein sequence. Competent E. coli BL21 (DE3) cells (Novagen) are transformed with these vectors by heat shock. The cells are grown with shaking (160 rpm) at 30°C in TB medium containing 0.5 sorbitol, 5 mM betain, 100 /vg/ml ampicillin until reaching an OD at 600 nm comprised between 0.8 and 1. Isopropyl-B-D-thiogaiactopyranoside (!PTG) is then added to a final concentration of 1 mM and protein expression is continued at 20°C overnight (approximately 16 h). The cells are collected by centrifugation at 4°C, 10.000 rpm for 20 min and the pellets are frozen at -80°C.

Cell lysis

1 .6 g of cells are thawed on ice and resuspended in 5 ml of 50 mM Na₂HP0₄ pH 8 containing 300 mM NaCI, 5 mM MgCI₂, 1 mM DTT. Twenty microliters of lysonase (Novagen) are added. Cells are incubated for 10 min at room temperature and then returned to ice for 20 min. Cell lysis is completed by sonication for 3x 5 min in an ultrasound water bath at 0°C; samples are homogenized between pulses. The bacterial extracts are then clarified by centrifugation at 4°C, 10,000 rpm for 20 min.

Protein purification and concentration (PROTiNO kit)

The clarified bacterial lysates are loaded on a PROTINO-1000 Ni-IDA column (Macherey-Nagel) allowing adsorption of 6-His-tagged proteins. Columns are washed and the enzymes of interest are eiuted with 4 ml of 50 mM Na₂HP0₄ pH 8 containing 300 mM NaC!, 5 mM MgCI₂, 1 mM DTT, 250 mM imidazole. Eluates are then concentrated in Amicon Ultra-4 10 kDa cells (Millipore) to a final volume of 250 μ\. Proteins are quantified by the Bradford method. SDS-PAGE analyses are conducted in parallel: a main band is clearly visible around 65kD, and the purity of the protein is estimated to over 80%.

Example 2: Measuring the activity of purified oleate hydratases towards ethanol

Enzymatic reactions

Reactions are run in 1 ml of buffer containing 20mM Tris (pH 8) and 50 mM NaCI, and various concentration of ethanol, ranging from 100mM to 5M. 1 mg of enzyme is added to the sample. The enzyme-free control reactions are carried out in parallel. The 1 ml reactions are placed in 2 ml vials (Interchtm) and sealed with a teflon/silica/teflon septum (Interchim). Reactions are incubated without shaking at 37°C for 72 h.

Analysis of reactions

The gas present in the headspace is collected using a gas syringe equipped with an anti-backup mechanism. The gas sample is analyzed by gas chromatography (GC) coupled with a flame ionization detector.

Column: BPX5 (SGE)

GC: Hewlett Packard 5890 series II

For each chromatogram, the presence of a peak is analyzed precisely, and compared to negative control chromatograms and chromatograms obtained when injecting pure ethylene (Sigma). A significant production of a gas corresponding to the elution time of ethylene is observed in the case of several enzymatic preparations. The identity of the gas if further confirmed using GC/MS analysis, giving the precise physical signature of ethylene.

Column: BPX5 (SGE)

GC/MS: MSD 5973 (HP)

Example 3: Measuring the activity of purified oieate hydratases towards propanoi isomers

The same protocol as in Example 2 is followed to assay the activity of the collection of 20 enzymes towards propan-1 -ol and propan-2-ol (isopropanol) instead of ethanol. A signal indicating the significant production of a gas having the same elution time than propylene is identified in the case of several enzymes.

The identity of propylene is further confirmed in GC/MS analyses.

Example 4: Measuring the activity of purified oieate hydratases towards linear butanol isomers

The same protocol as in Example 2 is followed to assay the activity of the collection of 20 enzymes towards butan-1 -ol (/?-butanol), butan-2-ol (seobutanol) instead of ethanol. A signal indicating the significant production of a gas having the same elution time than linear butene (but-1 -ene or the cis or trans isomers of but-2-ene) is identified in the case of several enzymes.

The identity of the gas is further confirmed in GC/MS analyses. In the case of several enzymes, only one of the three linear butenes is obtained. In some other cases, a mix of two or the three compounds is obtained.

Example 5: Measuring the activity of purified oieate hydratases towards branched butanol isomers

The same protocol as in Example 2 is followed to assay the activity of the collection of 20 enzymes towards 2-methylpropan-1-ol (isobutanol) or 2-methylpropan-2-ol (fert-butanol) instead of ethanol. A signal indicating the significant production of a gas having the same elution time than 2-methylprop-1-ene (isobutene) is identified in the case of several enzymes.

The identity of the gases is further confirmed in GC/MS analyses. Example 6: Measuring the activity of purified oleate hydratases towards isopentenol

The same protocol as in Example 2 is followed to assay the activity of the collection of 20 enzymes towards isopentenol isomers (3-methylbut-3-en-1-ol, 3-methylbut-3- en-2-ol, 2-methylbut-3-en-1-ol, 2-methylbut-3-en-2-ol) instead of ethanol. A signal indicating the significant production of a gas having the same elution time than 2- methylbuta-1 ,3-diene (isoprene) is identified in the case of several enzymes.

The identity of isoprene is further confirmed in GC/MS analyses.

Example 7: Measuring the activity of purified oleate hydratases towards branched butenol isomers

The same protocol as in Example 2 is followed to assay the activity of the collection of 20 enzymes towards but-3-en-1 -ol or but-3-en-2-ol instead of ethanol. A signal indicating the significant production of a gas having the same elution time than buta- 1 ,3-diene is identified in the case of several enzymes.

The identity of buta-1 ,3-diene is further confirmed in GC/MS analyses.

Example 8: Measuring the activity of purified oleate hydratases towards phenyl-ethanol isomers

The same protocol as in Example 2 is followed to assay the activity of the collection of 20 enzymes towards 1 -phenylethanol or 2-phenylethanol instead of ethanol. As the expected product, vinylbenzene (styrene), is liquid at ambient temperature, what is harvested here is not gas from the headspace, but liquid from the reaction mix. Two microliter of such reaction mix are injected in the GC apparatus, and the signal indicating the significant production of a gas having the same elution time than styrene is identified in the case of several enzymes.

The identity of the gas is further confirmed in GC/MS analyses.

Example 9: Optimization of reaction conditions by using a cofactor

The same assays as followed in Examples 2 to 8 are carried out, but a carboxylic acid is added as cofactor to the reaction mix.

Vials containing the buffer, an enzyme preparation, an alcohol and 1 mg/mL of a carboxylic acid are prepared and tested following the same protocol as described in Example 2. Cofactors are used in accordance with the list presented in Table 3. In particular, linear octano^'ic acid is used as cofactor in the case of ethanol, propan-2- ol, 2-methyipropan-2-ol.

An increase in the rate of conversion of the alcohol into the corresponding alkene is observed in several cases.

Example 10: Bacteria synthesizing ethylene from glucose

Ethanol producing E. coli bacteria are transformed using a plasmid encoding an enzyme having shown activity in converting ethanol into ethylene, as described in Example 2. The transformed bacteria are then grown in mineral medium containing 20g/L glucose under aeration at 30°C during 24 hours. The production of ethylene in the headspace is measured by taking a sample using a gas syringe, and using a GC apparatus as described in Example 2.

Example 11 : Bacteria synthesizing propylene from glucose

E. coli bacteria engineered to produce propan-2-ol as described in Jojima et al. (2008, Appl. Microbial. Biotechnol. 77:1219-1224) are transformed by a plasmid encoding an enzyme of the oleate hydratase family showing activity in converting propan-2-ol into propylene, as described in Example 3. The transformed bacteria are then grown in mineral medium containing 20g/L glucose under aeration at 30°C during 24 hours. The production of propylene in the headspace is measured by taking a sample using a gas syringe, and using a GC apparatus as previously described.

Example 12: Bacteria synthesizing isobutene from glucose

E. coli bacteria engineered to produce 2-methylpropan-1 -o! (isobutanol), as described in Connor and Liao, Curr. Opin. Biotechnol. 20, 2009:307, are transformed using a plasmid encoding an enzyme having shown activity in converting branched butanol isomers into isobutene, as described in Example 5. The transformed bacteria are then grown in mineral medium containing 20g/L glucose under aeration at 30°C during 24 hours. The production of isobutene in the headspace is measured by taking a sample using a gas syringe, and using a GC apparatus as previously described. Example 13: Bacteria synthesizing isoprene from glucose

E. coli bacteria first engineered to produce 3-methylbut-3-en-1 -ol are further transformed using a plasmid encoding an enzyme having shown activity in converting branched methyl-butenol isomers into isoprene, as described in Example 6. The transformed bacteria are then incubated in a fermentor using 50g/l sucrose, and stirred under oxygenation at 30°C during 24 hours. The production of isoprene in the headspace is measured by taking a sample using a gas syringe, and using a GC apparatus as previously described.

Example 14: Bacteria synthesizing butadiene from glucose

E. coli bacteria first engineered to produce but-3-en-1 -ol are transformed using a plasmid encoding an enzyme having shown activity in converting butenol isomers into butadiene, as described in Example 7. The transformed bacteria are then grown in mineral medium containing 20g/L glucose under aeration at 30°C during 24 hours. The production of butadiene in the headspace is measured by taking a sample using a gas syringe, and using a GC apparatus as previously described.

Example 15: Bacteria synthesizing styrene from glucose

£ coli bacteria first engineered to produce 1-phenylethanol are transformed using a plasmid encoding an enzyme having shown activity in converting branched butenol isomers into vinylbenzene (styrene), as described in Example 8. The transformed bacteria are then grown in mineral medium containing 20g/L glucose under aeration at 30°C during 24 hours. Two microliters of the liquid fraction of the reaction mix is harvested using a syringe, and injected in a GC apparatus as previously described to identify the presence of styrene.

Claims

A method for producing an alkene, the method comprising a step of converting an alcohol by an enzymatic dehydration step into an alkene, wherein: the alcohol is a compound of formula (I)

OH H

1 I I 4

R— C C— R

R R^J

(I)

wherein R¹, R², R³ and R⁴ are each independently selected from hydrogen or Ci- hydrocarbyl; and the alkene is a compound of formula (II)

R— CC=C— R⁴

I . l ¾

R R

(ii)

wherein R¹, R², R³ and R⁴ have the same meanings as defined for the compound of formula (I),

The method of claim 1 wherein the enzymatic dehydration step is catalyzed by a hydratase.

The method of claim 1 or 2 wherein the enzymatic dehydration step is catalyzed by an unsaturated fatty acid hydratase.

The method of claim 3 wherein the fatty acid hydratase is an oleate hydratase (EC 4.2.1 .53).

5. The method of claim 4 wherein the oleate hydratase has an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 1 1 or a sequence having at least 15% sequence identity to any one of SEQ ID NO: 1 to 1 1 preferably 30%, 40%, 50%, 60%, 80% or 90%.

The method of any one of claims 1 to Swherein:

(i) the alcohol is ethanol and the alkene is ethene; or

(ii) the alcohol is propan-1-ol and the alkene is propene; or

(iii) the alcohol is propan-2-ol and the alkene is propene; or

(iv) the alcohol is butan-1 -ol and the alkene is but-1 -ene; or

(v) the alcohol is 2-methylpropan-1 -ol and the alkene is 2-methylprop-1 -ene; or

(vi) the alcohol is 2-methylpropan-2-ol and the aikene is 2-methylprop-1 -ene; or

(vii) the alcohol is butan-2-ol and the alkene is but-1 -ene; or

(viii) the alcohol is butan-2-ol and the alkene is but-2-ene; or

(ix) the alcohol is 3-methylbut-3-en-1 -ol and the aikene is 2-methyibuta-1 ,3- diene; or

(x) the alcohol is 2-methylbut-3-en-2-ol and the alkene is 2-methylbuta-1 ,3- diene; or

(xi) the alcohol is 2-methylbut-3-en-1 -ol and the alkene is 2-methylbuta-1 ,3- diene; or

(xii) the alcohol is 3-methylbut-3-en-2-ol and the alkene is 2-methy(buta-1 ,3- diene; or

(xiii) the alcohol is but-3-en-1 -ol and the alkene is buta-1 ,3-diene; or

(xiv) the alcohol is but-3-en-2-ol and the alkene is buta-1 ,3-diene; or

(xv) the alcohol is 1 -phenylethanol and the alkene is styrene; or

(xvi) the alcohol is 2-phenylethanol and the alkene is styrene.

7. The method of any one of claims 1 to 6 wherein the step of converting an alcohol by an enzymatic dehydration step into an alkene is carried out at a temperature above 37°C and the enzymatic dehydration step is catalyzed by a thermophilic hydratase.

8. The method of claim 7 wherein the alkene comprises 6 or 7 carbon atoms.

9. The method of any one of claims 1 to 8 wherein the step of converting an alcohol by an enzymatic dehydration step into an alkene is realized in the presence of an organism capable of expressing an enzyme as defined in any one of claims 2 to 5.

10. The method of claim 19 wherein the microorganism is furthermore capable of producing the alcohol to be converted. 1. A recombinant organism which is characterized by the following features:

(a) it is capable of producing an alcohol as defined in claim 1 ; and

(b) it expresses a hydratase enzyme which is capable of catalyzing the conversion of said alcohol into an alkene.

12. Use of an organism as defined in claim 1 1 for the production of an alkene.

13. A composition comprising an organism of claim 1 1 .

14. A composition comprising

(i) an alcohol as defined in claim 1 ; and

(ii) a hydratase enzyme which is capable of catalyzing the conversion of said alcohol into an alkene.

15. Use of an enzyme as defined in any one of claims 2 to 5 for the production of an alkene.