# Patent

## Title
Process for the production of isoprenol from mevalonate employing a diphospho-mevolonate decarboxylase

## Abstract

Described is a method for the enzymatic production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol as well as the use of an enzyme which is capable of catalyzing the decarboxylation of mevalonate for the production of isoprenol from mevalonate. Furthermore described is the use of mevalonate as a starting material for the production of isoprenol in an enzymatically catalyzed reaction. Also disclosed is a method for the production of isoprene comprising the method for the production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol and further comprising the step of converting the produced isoprenol into isoprene as well as a method for the production of isoamyl alcohol comprising the method for the production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol and further comprising the step of converting the produced isoprenol into isoamyl alcohol.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 23, 2012, is named 28622272.txt and is 59,418 bytes in size.
The present invention relates to a method for the production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol. The present invention also relates to the use of an enzyme which is capable of catalyzing the decarboxylation of mevalonate for the production of isoprenol from mevalonate. Furthermore, it relates to the use of mevalonate as a starting material for the production of isoprenol in an enzymatically catalysed reaction.
Moreover, the present invention relates to a method for the production of isoprene comprising the method for the production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol and further comprising the step of converting the produced isoprenol into isoprene. The present invention also relates to a method for the production of isoamyl alcohol comprising the method for the production of isoprenol using mevalonate as a substrate and enzymatically converting it by a decarboxylation step into isoprenol and further comprising the step of converting the produced isoprenol into isoamyl alcohol.
Isoprenol responds to the formula C5H10O. It can be used to produce prenol which is used in perfumes or as a building block in the pharmaceutical industry. It is produced by the chemical condensation of isobutene and formaldehyde, leading to isoprenol further isomerised into prenol.
The route which is presently used to produce isoprenol involves the mevalonate pathway: mevalonate is produced, then diphoshorylated, then decarboxylated-dehydrated into isoprenyl-pyrophosphat, and finally dephosphorylated twice into isoprenol (US patent application 20080092829).
Isoprenol can be converted into isoprene which is a key compound for the tire industry, and also has many applications in the adhesives. It is produced chemically using several routes:

    
    
        Extractive distillation from oil (C5 cut)
        Dehydrogenation of iso-amylene
        Double dehydrogenation of isopentane
        Reaction of isobutene and formaldehyde
        Reaction of acetone and acetylene
        Propylene dimerization
    
    


WO 2009/076676 reports a metabolic pathway to isoprene. The pathway is based on the dephosphorylation-dehydration of downstream intermediates in the mevalonate pathway, i.e. isoprenyl-pyrophosphate or prenyl-pyrophosphate. This process has the drawback of requiring to go through the whole mevalonate pathway: double phosphorylation of mevalonate, followed by a decarboxylation-dehydration into isoprenyl-pyrophosphate, further isomerised into prenyl-pyrophosphate, and finally double dephosphorylation/dehydration into isoprene.
Isoamyl alcohol is a very important chemical commonly used as solvents for fats, oils, resins and alkaloids. There is a demand for isoamyl alcohol in perfumery industry, for example in the manufacture of isoamyl salicylate used in soap and cosmetic fragrances. It is also used in the manufacture of phosphoric acid. Furthermore, it is used in the synthesis of pyrethroids. Commercial processes for the production of isoamyl alcohol include fractionation of fusel oils, chlorination of alkanes with subsequent hydrolysis to produce a mixture of isomers and a low pressure oxo-process or hydroformylation of n-butenes followed by hydrogenation of the resulting iso-valeraldehyde.
There is a need to provide environmentally friendly, cost efficient and simple methods for producing the above-mentioned compounds. This need is met by the subject matter as recited in the claims.
Thus, in a first aspect, the present invention relates to a method for producing isoprenol from mevalonate. In particular, the present invention relates to a method for producing isoprenol from mevalonate which is characterized by a conversion of mevalonate with an enzyme having a decarboxylase activity. Thus, the method comprises the enzymatically catalyzed decarboxylation of mevalonate. The term “decarboxylation” when used in the context of the present invention preferably refers to a dehydrative decarboxylation.
The term “mevalonate” comprises mevalonic acid as well as the anion of mevalonic acid which is the predominant form in biological media. Mevalonic acid is a precursor in the biosynthetic pathway, known as the mevalonate pathway that produces terpenes and steroids. Mevalonate is the primary precursor of isoprenyl pyrophosphate that is in turn the basis for all terpenoids. The structural formula of mevalonic acid is shown in FIG. 1.
In the context of the present invention the term isoprenol comprises compounds which respond to the formula C5H10O. The IUPAC name of isoprenol is 3-methylbut-3-en-1-ol. Synonyms of isoprenol are, for example, 2-methyl-1-buten-4-ol, 3-buten-1-ol-3-methyl, 3-isopentenyl alcohol, 3-methyl-3-buten-1-ol, isobutenylcarbinol, isopropenylethyl alcohol and methallyl carbinol.
The term “enzyme having a decarboxylase activity” in the context of the present invention refers to an enzyme which is capable of decarboxylating mevalonate, in particular according to the reaction scheme given in FIG. 2. The catalyzed reaction is a simultaneous dehydration and decarboxylation. This enzymatic activity can be measured as described in the appended Examples 1 or 7.
In a preferred embodiment the enzyme having the activity of a decarboxylase is an enzyme which is classified as a diphosphomevalonate decarboxylase or is an enzyme which is derived from such an enzyme and which has the capacity to decarboxylate mevalonate so as to produce isoprenol. Diphosphomevalonate decarboxylase is classified with the EC number EC 4.1.1.33. A diphosphomevalonate decarboxylase is able to catalyze the decarboxylation of mevalonate diphosphate. In this reaction ATP and 5-diphosphomevalonate are converted into ADP, phosphate, isoprenyl pyrophosphate and CO2. The reaction catalyzed by a diphosphomevalonate decarboxylase is shown in FIG. 2. The activity of a diphosphomevalonate decarboxylase can be measured according to methods known in the art, e.g. in Reardon et al. (Biochemistry 26 (1987), 4717-4722). Preferably, the activity is measured as described in Example 1 or 7 wherein diphosphomevalonate is used instead of mevalonate.
It has been reported that at least in some cases the reaction is divalent cation-dependent (see, e.g., Krepkiy et al., Protein Science 13 (2004), 1875-1881; Michihara et al., Biol. Pharm. Bull. 25 (2002), 302-306).
Diphosphomevalonate decarboxylase is an enzyme which, in its natural function, is part of the mevalonate pathway for isoprenoid synthesis in bacteria and of the sterol biosynthesis pathway in eukaryotes. It has been identified and isolated from various organisms such as animals, fungi, yeasts and bacteria. It is also expressed by certain plants.
The three-dimensional structure of several diphosphomevalonate decarboxylases has already been determined (see, e.g., Byres et al. (J. Mol. Biol. 371 (2007), 540-553); Bonanno et al. (Proc. Natl Acad. Sci. USA 98 (2001), 12896-12901); Voynova et al., Archives of Biochemistry and Biophysics 480 (2008), 58-67)) and considerable knowledge is available about its active site, amino acid residues crucial for the catalytic reaction and the actual enzymatic reaction (see, e.g. Byres et al. (J. Mol. Biol. 371 (2007), 540-553); Bonanno et al. (Proc. Natl Acad. Sci. USA 98 (2001), 12896-12901)). In most cases the enzyme is composed of about 300 to 400 amino acids and uses ATP as cosubstrate which is converted during the decarboxylation reaction into ADP and inorganic phosphate.
Diphosphomevalonate decarboxylases have been described for various organisms and also amino acid and nucleotide sequences encoding them are available for numerous sources.
In principle any diphosphomevalonate decarboxylase can be used in the context of the present invention, in particular from prokaryotic or eukaryotic organisms. Eukaryotic diphosphomevalonate decarboxylases are described, for example, for animals such as Rattus norvegicus, Gallus gallus, Homo sapiens, Mus musculus, Sus scrofa, D. melanogaster, C. elegans and Trypanosoma brucei, for plants such as Arabidopsis thaliana, Ginko biloba, Oryza sativa, Pisum sativum, for yeasts, such as Saccharomyces cerevisiae and Candida albicans. Also numerous prokaryotic diphosphomevalonate decarboxylases have been described, e.g. for Helicobacter, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecium, Listeria monocytgenes, Leuconostoc citreum, Lactobacillus reuteri, to name just some. Table 1 provides a list of sequences of diphosphomevalonate decarboxylases from different organisms indicating the accession numbers under which they can be retrieved from the respective databases.








TABLE 1






Genebank


Organism
Accession number







Bombyx mori
A5A7A2


Saccharomyces cerevisiae strain YJM7
A6ZSB7


Solanum lycopersicum
A8WBX7


Hevea brasiliensis
A9ZN03


Nicotiana langsdorffii × Nicotiana sanderae
B3F8H5


Saccharomyces cerevisiae (strain RM11-1a)
B3LPK0


Phaeodactylum tricornutum CCAP 1055
B7S422


Candida dubliniensis
B9W6G7


Pichia pastoris
C4QX63


Ashbya gossypii
Q751D8


Bos taurus
Q0P570


Danio rerio
Q5U403


Debaryomyces hanseni
Q6BY07


Dictyostelium discoideum
Q54YQ9


Homo sapiens
P53602


Mus musculus
Q99JF5


Rattus norvegicus
Q62967


Schizosaccharomyces pombe
O13963


Saccharomyces cerevisiae
P32377


Arnebia euchroma
Q09RL4


Aspergillus oryzae
Q2UGF4


Mus musculus
Q3UYC1


Ginkgo biloba
Q5UCT8


Rattus norvegicus
Q642E5


Oryza sativa subsp. japonica
Q6ETS8


Arabidopsis thaliana
Q8LB37


Encephalitozoon cuniculi
Q8SRR7


Hevea brasiliensis
Q944G0









Examples of diphosphomevalonate decarboxylases from different organisms are given in SEQ ID NO: 1 to 19. In a preferred embodiment of the present invention the diphosphomevalonate decarboxylase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 19 or a sequence which is at least n % identical to any of SEQ ID NO: 1 to 19 and having the activity of a diphosphomevalonate decarboxylase 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.
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.
Preferably, the degree of identity is calculated over the complete length of the sequence.
Moreover, if the term “homology” is used in the context of the present invention, this term preferably means “sequence identity”.
In a preferred embodiment the decarboxylase employed in the method according to the invention is a diphosphomevalonate decarboxylase from Picrophilus torridus or an organism which is evolutionary closely related to Picrophilus torridus. In a further preferred embodiment the decarboxylase originates from an organism of the genus Picrophilus, Thermoplasma or Ferroplasma, more preferably of the species Picrophilus torridus, Picrophilus oshimae, Thermoplasma volcanicum, Thermoplasma acidophilum, Ferroplasma acidarmanus or Ferroplasma cupricumulans. 
In a particularly preferred embodiment the decarboxylase employed in the method according to the invention is a diphosphomevalonate decarboxylase which comprises the amino acid sequence as depicted in SEQ ID NO: 6, 16, 17, 18 or 19 or which comprises an amino acid sequence which is at least n % identical to any of SEQ ID NO: 6, 16, 17, 18 or 19 and which has the activity of a diphosphomevalonate decarboxylase 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 enzyme showing the amino acid sequence as shown in SEQ ID NOs:6 and 16 originates from Picrophilus torridus. As shown in the Examples, this enzyme is particularly efficient in catalyzing the decarboxylation of mevalonate to isoprenol. Further preferred decarboxylases to be employed in the method according to the present invention are diphosphomevalonate decarboxylases which originate from organisms which are phylogenetically closely related to Picrophilus torridus, such as other bacteria of the genus Picrophilus, such as Picrophilus oshimae, bacteria of the genus Ferroplasma, e.g. Ferroplasma acidarmanus (SEQ ID NO:19), or of the genus Thermoplasma, such as Thermoplasma acidophilum (SEQ ID NO:18) and Thermoplasma volcanium (SEQ ID NO:17). The diphosphomevalonate decarboxylase of Thermoplasma acidophilum (AC number Q9HIN1) shows a homology of 38% to SEQ ID NO:6 and that of Thermoplasma volcanium (AC number Q97BY2) shows a homology of about 42% to SEQ ID NO:6.
In a further particularly preferred embodiment the decarboxylase employed in the method according to the invention is a diphosphomevalonate decarboxylase which is encoded by a nucleotide sequence as shown in SEQ ID NO: 20 or 21 or by a nucleotide sequence which is at least n % identical to any of SEQ ID NO: 20 or 21 and which encodes an enzyme having the activity of a diphosphomevalonate decarboxylase 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: 20 is the native nucleotide sequence encoding the MDP decarboxylase from P. torridus including at the N-terminus a His-tag. SEQ ID NO: 21 is a codon optimized sequence coding for the MDP decarboxylase from P. torridus including at the N-terminus a His-tag.
The decarboxylase, preferably diphosphomevalonate decarboxylase, employed in the process according to the invention can be a naturally occurring decarboxylase, preferably diphosphomevalonate decarboxylase, or it can be a decarboxylase, preferably diphosphomevalonate decarboxylase, which is derived from a naturally occurring decarboxylase, preferably diphosphomevalonate decarboxylase, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.
The term “decarboxylase”, “diphosphomevalonate decarboxylase”, “a protein/enzyme having the activity of a decarboxylase” or “a protein/enzyme having the activity of a diphosphomevalonate decarboxylase” in the context of the present application also covers enzymes which are derived from a decarboxylase, preferably a diphosphomevalonate decarboxylase, which are capable of catalyzing the decarboxylation of mevalonate but which only have a low affinity to their natural substrate, e.g. mevalonate diphosphate, or do no longer accept their natural substrate, e.g. mevalonate diphosphate. Such a modification of the preferred substrate, in particular of a diphosphomevalonate decarboxylase, allows to improve the conversion of mevalonate into isoprenol and to reduce the production of the possibly occurring by-product isoprenyl pyrophosphate. 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”, DNA shuffling or in vivo evolution.
For example, for genetic engineering in prokaryotic cells, a nucleic acid molecule encoding a decarboxylase, preferably a diphosphomevalonate decarboxylase, 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, N.Y., 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 decarboxylase, preferably diphosphomevalonate decarboxylase, variants are then tested for their enzymatic activity and in particular for their capacity to prefer mevalonate as a substrate rather than, e.g. mevalonate diphosphate.
Such methods for identifying variants with improved enzymatic properties as regards the production of isoprenol 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 substrate mevalonate is shorter than the natural substrate, e.g. mevalonate diphosphate in the case of diphosphomevalonate decarboxylase. Examples for such a cofactor would be phosphono-phosphate or phosphonamido-phosphate (see FIG. 7) or orthophosphate.
The modified version of the decarboxylase, preferably diphosphomevalonate decarboxylase, accepting or preferring mevalonate as a substrate but having a low affinity to its natural product, e.g. mevalonate diphosphate, as a substrate or no longer accepting its natural product, e.g. mevalonate diphosphate, as a substrate may be derived from a naturally occurring decarboxylase, preferably diphosphomevalonate decarboxylase, or from an already modified, optimized or synthetically synthesized decarboxylase, preferably diphosphomevalonate decarboxylase.
It has surprisingly been found that diphosphomevalonate decarboxylase is not only capable of catalyzing the decarboxylation of mevalonate diphosphate but can also accept mevalonate as a substrate and can decarboxylate it despite the absence of the diphosphate group. This is in particular surprising since Jabalquinto and Cardemil (Biochim. Biophys. Acta 996 (1989), 257-259), who investigated the substrate binding requirements of diphosphomevalonate decarboxylase, pointed out the importance of the diphosphoric moiety of mevalonate diphosphate to the binding of this substrate to the catalytic site of the enzyme (see page 259). In this context, it is important to note the substantial differences between mevalonate and diphosphomevalonate. Mevalonte only has a molecular weight of about 148 Da while diphosphomevalonate has a molecular weight of 308 Da and the phosphate groups are carrying three additional charges.
The decarboxylase, preferably diphosphomevalonate decarboxylase, 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 decarboxylase, preferably diphosphomevalonate decarboxylase, may also be chemically modified, for example in order to improve its/their stability, resistance, e.g. to temperature, for facilitating its/their purification or its immobilization on a support. The decarboxylase, preferably diphosphomevalonate decarboxylase, 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 organism/microorganisms producing them etc.
The method according to the present invention may be carried out in vitro or in vivo. An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction.
For carrying out the process in vitro the substrates for the reaction and the enzyme are incubated under conditions (buffer, temperature, cosubstrates, 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 isoprenol. The production of isoprenol can be measured by methods known in the art, such as chromatography. e.g. thin layer chromatography or liquid or gas chromatography possibly linked to mass spectrometry detection.
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.
If required, a co-substrate, a co-factor or ions are also added. It is described, for example, that some diphosphomevalonate decarboxylase enzymes use ATP as a co-substrate which is converted into ADP and inorganic phosphate during the decarboxylation reaction. Thus, in a preferred embodiment, ATP is added to the reaction when carrying out the method according to the invention. However, instead of ATP any other suitable rNTP (ribonucleoside triphosphate) or dNTP (desoxyribonucleoside triphosphate) or any mixture of these can be added to the reaction mixture. Also possible is the addition of pyrophosphate or another polyphosphate or a molecule containing a phosphoanhydride group (POP). Moreover, any mixture of any of the afore-mentioned compounds can be added. Moreover, it is described for some diphosphomevalonate decarboxylase enzymes that they require divalent cations. Thus, in a preferred embodiment, and if necessary, a suitable amount of a suitable divalent cation is added to the reaction when carrying out the method according to the invention. The divalent cation is preferably Mg2+, Mn2+ or Co2+, but it is possible to also use other divalent cations such as Ca2+. Of course, the nature of the divalent cation depends on the need of the diphosphomevalonate decarboxylase enzyme in question.
Since the substrate mevalonate is in general shorter than the natural substrate used by the enzyme, e.g. mevalonate diphosphate used by diphosphomevalonate decarboxylase, 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. Examples for such a cofactor, in the case of diphosphomevalonate decarboxylase, would be phosphono-phosphate or phosphonamido-phosphate (see FIG. 7) or orthophosphate.
For carrying out the process in vivo use is made of a suitable organism/microorganism(s) which is/are capable of providing the substrates, i.e. mevalonate, and an enzyme which is capable of catalyzing the decarboxylation of mevalonate into isoprenol. In a preferred embodiment said enzyme is a diphosphomevalonate decarboxylase. There are two alternate pathways that lead to isoprenyl-pyrophosphate. One is the mevalonate pathway, observed in eukaryotes and some prokaryotes, especially in the firmicute phylum. All these organisms thus produce mevalonate. Most of the bacteria, including E. coli, use the other pathway (DXP pathway) and are thus not producing mevalonate. However, the latter can be genetically modified so as to produce mevalonate. For example, the implementation of the mevalonate pathway in E. coli has already been done successfuly (Maury et al., FEBS Lett. 582 (2008), 4032). Overexpression of only the upstream part (thiolase, HMG-CoA synthase, HMG-CoA reductase) in organisms that have or that do not have the mevalonate pathway allows for the production of high levels of mevalonate.
In a preferred embodiment, the organism employed in the method according to the invention is an organism, preferably a microorganism, which has been genetically modified to contain a foreign nucleic acid molecule encoding an enzyme which is capable of catalyzing the decarboxylation of mevalonate to isoprenol. In a preferred embodiment the organism has been genetically modified so as to contain a foreign nucleic acid molecule encoding diphosphomevalonate decarboxylase. 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 respective enzyme, e.g. a diphosphomevalonate decarboxylase, 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 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 enzyme, e.g. the diphosphomevalonate decarboxylase, is not endogenous to the organism/microorganism, i.e. are naturally not expressed by the organism/microorganism when it is not genetically modified. In other words, the encoded decarboxylase, e.g. diphosphomevalonate decarboxylase, is heterologous with respect to the organism/microorganism.
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 a further preferred embodiment the organism/microorganism is characterized in that the expression/activity of an enzyme which is capable of catalyzing the decarboxylation of mevalonate to isoprenol, preferably a diphosphomevalonate decarboxylase, 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 enzyme, preferably the diphosphomevalonate decarboxylase, 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%.
The term “higher” expression/activity also covers the situation in which the corresponding non-genetically modified organism/microorganism does not express a corresponding enzyme, e.g. a diphosphomevalonate decarboxylase, 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, 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.
Methods for measuring the enzymatic activity of the above-mentioned enzymes, in particular diphosphomevalonate decarboxylase, are known in the art and have already been described above.
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, bacterial organisms and archae. The term includes animal, plants, fungi, bacteria and archae. 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 all classical production strains for which the engineering tools have been developed. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia or Bacillus and even more preferred to the species Escherichia coli or to the species Bacillus subtilis. 
In another preferred embodiment the microorganism is a fungus. Preferred fungi to be employed in the process according to the invention are all classical production strains for which the engineering tools have been developed. More preferably the fungus is a yeast, preferably of the genus Saccharomyces, Schizosaccharomyces, Pichia or Kluyveromyces and even more preferably of the species Saccharomyces cerevisia, Schizosaccharomyces pombe, Pichia pastoris or of the species Kluyveromyces lactis. Other preferred fungi are those of the genus Trichoderma or Aspergillus, more preferably of the species Trichoderma reesei or Aspergillus niger. 
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.
When the process according to the invention is carried out in vivo by using an organism/microorganism providing the respective enzyme activity, the organism, preferably microorganism, is cultivated under suitable culture conditions allowing the occurrence of the enzymatic reaction. The specific culture conditions depend on the specific organism/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 reaction. 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 a plant. 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, wheat, 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).
The present invention also relates to the use of an organism, preferably a microorganism, which expresses an enzyme which is capable of catalyzing the decarboxylation of mevalonate, preferably an enzyme with the activity of a diphosphomevalonate decarboxylase, for the production isoprenol by the decarboxylation of mevalonate.
I.e., the present invention also relates to the use of an organism/microorganism as described in the context of the method according to the invention for the production of isoprenol.
Moreover, the present invention also relates to a composition comprising (i) mevalonate; and (ii) an enzyme which is capable of catalyzing the decarboxylation of mevalonate.
For the preferred embodiments of the enzyme the same applies as has already been set forth above in connection with the method according to the invention.
In a particularly preferred embodiment, the composition also comprises a co-substrate (such as ATP), a co-factor and/or divalent cations (such as Mn2+, Mg2+, Co2+ or Ca2+).
Moreover, the present invention also relates to the use of an enzyme which is capable of catalyzing the decarboxylation of mevalonate, preferably a diphosphomevalonate decarboxylase, for the production of isoprenol.
For the preferred embodiments of the enzyme the same applies as has already been set forth above in connection with the method according to the invention.
The present invention also relates to the use of mevalonate for the production of isoprenol, in particular by the enzymatic conversion of mevalonate to isoprenol by a decarboxylation step. 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 enzyme having the enzymatic activity of a diphosphomevalonate decarboxylase, and most preferably the conversion is achieved by the use of an organism as described in the context of the method according to the invention.
In addition the present invention also relates to a method for producing isoprene from mevalonate comprising the method for producing isoprenol according to the invention as described above and further comprising the step of converting the produced isoprenol into isoprene. The conversion of isoprenol into isoprene can be achieved by means and methods known to the person skilled in the art. In particular, the respective reaction is a dehydration reaction.



Moreover, the present invention also relates to a method for producing isoamyl alcohol from mevalonate comprising the method for producing isoprenol according to the invention as described above and further comprising the step of converting the produced isoprenol into isoamyl alcohol. The conversion of isoprenol into isoamyl alcohol can be achieved by means and methods known to the person skilled in the art. In particular, the respective reaction is a hydrogenation reaction.
FIG. 1: shows the chemical structure of mevalonate
FIG. 2: shows the reaction of diphosphomevalonate decarboxylase on the physiological substrate diphosphomevalonate and on the precursor mevalonate
FIG. 3: shows an example of screening of enzyme library for mevalonate decarboxylase activity by following inorganic phosphate production. The control reaction was carried out with extract of E. coli BL21(DE3) transformed with pET 22b lacking MDP decarboxylase gene.
FIG. 4: (a) shows the results of optimisation of P. torridus MDP decarboxylase expression in E. coli. SDS-PAGE analysis of samples of proteins obtained from the expression of native P. torridus MDP decarboxylase DNA sequence (lanes 1 to 3) and of optimized gene (lanes 4 to 6). (b) shows Mevalonate decarboxylation activity of crude lysate of E. coli obtained from the expression of native P. torridus MDP decarboxylase DNA sequence and of optimized gene. Control reaction was carried out with extract of E. coli BL21(DE3) transformed with pET 22b lacking MDP decarboxylase gene. The enzyme activity was detected via inorganic phosphate production measurement.
FIG. 5: shows the comparison of mevalonate decarboxylation activity among MDP decarboxylases from the Picrophilus/Thermoplasma phylum. The enzyme activity was detected by means of inorganic phosphate production measurement.
FIG. 6: shows product formation as function of mevalonate concentration. The product formation was followed by permanganate assay.
FIG. 7: shows the structure of phosphono-phosphate and phosphonamido-phosphate.



The following Examples serve to illustrate the invention.
EXAMPLE 1
Screening of a Library of MDP Decarboxylase for Mevalonate Decarboxylation Activity
A library of 63 genes encoding enzymes of the MDP decarboxylase family was constructed and tested for activity on mevalonate as substrate.
Cloning, Bacterial Cultures and Expression of Proteins
The genes encoding mevalonate diphosphate (MDP) decarboxylase EC 4.1.1.33 were cloned in the pET 25b vector (Novagen) in the case of eukaryotic genes and in pET 22b (Novagen) in the case of prokaryotic genes. A stretch of 6 histidine codons (“6 histidine” disclosed as SEQ ID NO: 22) was inserted after the methionine initiation codon to provide an affinity tag for purification. Competent E. coli BL21(DE3) cells (Novagen) were transformed with these vectors according to the heat shock procedure. The transformed cells were grown with shaking (160 rpm) at 30° C. in terrific broth (TB) medium containing 0.5 M sorbitol, 5 mM betain, 100 μg/ml ampicillin until reaching an OD at 600 nm comprised between 0.8 and 1. Isopropyl-B-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM and protein expression was continued at 20° C. overnight (approximately 16 h). The cells were collected by centrifugation at 4° C., 10,000 rpm for 20 min and the pellets were frozen at −80° C.
Cell Lysis
The pellets from 12 ml of culture cells were thawed on ice and resuspended in 1 ml of 50 mM Tris/HCl pH 7.4, containing 20 mM KCl, 0.5 mM DTT, 5 mM MgCl2. One microliter of lysonase (Novagen) was added. Cells were incubated for 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 15 seconds. The bacterial extracts were then clarified by centrifugation at 4° C., 10,000 rpm for 20 min.
Enzymatic Reactions
The desired enzymatic reaction (conversion of mevalonate into isoprenol) was tested as follows.
The reaction medium contained 100 mM mevalonate, 40 mM ATP, 10 mM MgCl2, 20 mM KCl, 0.5 mM DTT and enzyme preparation varying from 0.01 to 0.05 mg/ml of protein. 50 mM sodium citrate was used in the range of pH from 4 to 6, and 50 mM Tris-HCl for pH 7 and 7.5. Enzyme-free control assays were carried out in parallel. After 72 h incubation, inorganic phosphate was quantified colorimetrically according to the ammonium molybdate method (Gawronski J D, Benson D R, Anal. Biochem. 327 (2004) 114-118). A 50 μl sample (containing not more than 0.5 μmole of phosphate) was mixed with 150 μl of ammonium molybdate reagent containing 50% v/v acetone, 1.25 N H2SO4, 2.5 mM (NH4)6Mo7O24 and then with 10 μl 1 M citric acid. The mixture was incubated for 2 minutes at room temperature. The absorbance of ammonium phosphomolybdate formed was measured at 355 nm and the quantity of inorganic phosphate estimated using a calibration curve obtained with potassium phosphate.
The results are shown in FIG. 3.
During the initial screening, only assays using the recombinant strain expressing the genetic construct inferred from Picrophilus torridus MDP decarboxylase sequence gave rise to a reproducible increase in phosphate production over the background level.
EXAMPLE 2
Optimisation of P. torridus MDP Decarboxylase Expression in E. coli 
The initial level of enzyme expression in E. coli BL21 was low, as judged from the faint band visible on SDS-PAGE gels. The Codon Optimization Index (CAI) of the native sequence for expression in E. coli measured with the “Optimizer” program available at http://genomes.urv.es/OPTIMIZER/, as based on the method of Sharp and Li (Nucl. Acids Res. 15 (1987), 1281-1295) gave a value as low as 0.23.
A gene sequence coding for an identical protein but containing codons better adapted for expression in E. coli was generated. It featured a CAI of 0.77.
The native sequence and the optimized sequence are shown in SEQ ID NO: 20 (native sequence of P. torridus (AAT43941) MDP decarboxylase including the His-tag) and SEQ ID NO: 21 (optimized sequence of P. torridus (AAT43941) MDP decarboxylase including the His-tag). The optimized sequence was synthesized by oligonucleotide concatenation and cloned in a pET25b expression vector. After transformation of E. coli strain BL21(DE3) and induction, the proteins were produced and analyzed on a gel as described according to the protocol described in Example 1. The same protocol was carried out with the native sequence for comparison.
Expression levels using either the native nucleotide sequence or the sequence optimized for expression in E. coli were compared. The results in FIG. 4a show that the protein (arrow) corresponding to the optimized gene was clearly visible on the gel in the non-purified cell lysate (lane 4), which indicates a very notable increase in expression.
The expression of the protein was improved such that the crude lysate obtained with the optimized sequence contained a higher enzyme activity with mevalonate as substrate, as shown in FIG. 4b. 
EXAMPLE 3
Characterization of the Reaction using the Optimized P. torridus MDPdecarboxylase
The recombinant enzyme was purified as follows:
Protein Purification and Concentration
The pellets from 150 ml of culture cells were thawed on ice and resuspended in 5 ml of Na2HPO4 pH 8 containing 300 mM NaCl, 5 mM MgCl2 and 1 mM DTT. Twenty microliters of lysonase (Novagen) was added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 3×15 seconds. The bacterial extracts were then clarified by centrifugation at 4° C., 10,000 rpm for 20 min. The clarified bacterial lysates were loaded on PROTINO-1000 Ni-IDA column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins (“6-His” disclosed as SEQ ID NO: 22). Columns were washed and the enzymes of interest were eluted with 4 ml of 50 mM Na2HPO4 pH 8 containing 300 mM NaCl, 5 mM MgCl2, 1 mM DTT, 250 mM imidazole. Eluates were then concentrated and desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and resuspended in 250 μl 50 mM Tris-HCl pH 7.4 containing 0.5 mM DTT and 5 mM MgCl2. Protein concentrations were quantified according to the Bradford method.
The purity of proteins thus purified was estimated as approximately 90%.
The activity of the enzyme was confirmed and further analyzed using a range substrate: The conversion rate was shown to increase with the concentration of mevalonate (FIG. 6).
EXAMPLE 4
Optimization of Reaction Conditions by Using a Cofactor
The same reaction as that described in Example 1 is carried out using purified preparations of optimized P. torridus MDP decarboxylase. In one of the samples, the phosphono-phosphate or phosphonamido-phosphate (FIG. 7) is added as cofactor at the concentration of 100 mM.
The conversion of mevalonate is observed using the colorimetric assay described in Example 1. It is found that when a cofactor is present, the amount of ATP consumed over time is markedly higher.
EXAMPLE 5
Screening of a Library of MDP Decarboxylase Homologs from the P. torridus Phylum
Sequence of MDP decarboxylase enzymes inferred from the genomes of Thermoplasma volcanium (accession number Q97BY2) and Thermoplasma acidophilum (accession number Q9HIN1) were generated as in Example 1. Proteins were purified as described in Example 3 and assayed using the assay described in Example 1. A significant increase in phosphate production was observed from these vials, indicating that these enzymes were also active toward mevalonate. Results are shown in FIG. 5.
EXAMPLE 6
Method for Synthesizing Isoprenol from Glucose
E. coli K12 is transformed with an expression plasmid, carrying the genes of thiolase, HMG-CoA synthase and HMG-CoA reductase from Saccharomyces cerevisiae in order to overproduce mevalonate.
The strain is further transformed with a second, compatible expression plasmid carrying the optimized gene encoding the His-tagged version of MDP decarboxylase from Picrophilus torridus. 
The resulting recombinant bacteria are then incubated in a fermenter in a mineral nutrient medium containing glucose, in the presence of oxygen and under moderate stirring. A significant production of isoprenol is measured using TLC or GC/MS analysis as follows:
TLC Analysis
For TLC analysis an aliquot of reaction medium is spotted on a silica-coated plate and chromatographed using as eluant ethyl acetate/heptane 1/1 v/v. Mevalonate, isoprenol, ATP. ADP are used as internal standards. After drying, plates are sprayed with alkaline KMnO4 reagent. Rf for isoprenol is found to be 0.57.
GC/MS Analysis
An aliquot of 10 μl of reaction medium is centrifuged and the supernatant is transferred to a clean vial for isoprenol detection by GC/MS. 1 μL sample is separated by GC using a DB-5 column and the presence of isoprenol is monitored by mass spectrometry.
EXAMPLE 7
Measurement of Mevalonate Decarboxylase Activity and 3-Methyl-3-Buten-1-Ol (Isoprenol) Production
Mevalonate is prepared from mevanolactone (Sigma) by hydrolysis with NaOH according to Campos et al. (Biochem. J. 2001. 353, 59-67).
The complete assay for mevalonate decarboxylation contains reaction buffer. 100 mM mevalonate, 40 mM ATP, 10 mM MgCl2, 20 mM KCl, 0.5 mM DTT and enzyme preparation at a concentration ranging from 0.01 to 0.05 mg/ml of protein. 50 mM sodium citrate is used in the range of pH from 4 to 6, and 50 mM Tris-HCl for pH 7 and 7.5. Control reactions are carried out in the absence of enzyme, substrate or co-factor.
The progress of isoprenol production is followed by analyzing aliquots taken at successive time intervals from a reaction mixture incubated at 37° C. by thin-layer chromatography (TLC), gas chromatography/mass spectrometry (GC/MS) and product determination by permanganate assay. In parallel, the release of inorganic phosphate is quantified by ammonium molybdate method.
Permanganate Assay
The formation of products containing double-bonds is followed by oxidization with alkaline potassium permanganate solution, resulting in increase of absorbance at 420 nm.
To an aliquot of reaction mixture diluted with H2O to 120 μl, 80 μl of permanganate reagent, containing 5 mM KMnO4 and 50 mM NaOH, is added. The mixture is kept at room temperature for 20 min and the absorbance at 420 nm is measured. The calibration curve is prepared using commercial isoprenol.
Inorganic Phosphate Quantification
Inorganic phosphate concentration is measured by spectroscopic colorimetry according to the ammonium molybdate method (Gawronski J D, Benson D R, Anal. Biochem. 327 (2004) 114-118). A 50 μl aliquot from the reaction assay (containing not more than 0.5 μmole of phosphate) is mixed with 150 μl ammonium molybdate reagent, containing 50% volume acetone, 1.25 N H2SO4, 2.5 mM (NH4)6Mo7O24 and then with 10 μl 1 M citric acid. The mixture is then incubated for 2 minutes at room temperature. The absorbance of ammonium phosphomolybdate formed was measured at 355 nm and the quantity of inorganic phosphate estimated using a calibration curve obtained with potassium phosphate.

## Claims

1. A method for the conversion of mevalonate to isoprenol, isoprene or isoamyl alcohol in vitro, comprising:
(a) producing isoprenol in vitro by the step consisting essentially of incubating mevalonate with diphosphomevalonate decarboxylase and NTP, rNTP, dNTP or pyrophosphate, thereby producing isoprenol, and
(b) isolating the isoprenol or further converting the isoprenol to isoprene or isoamyl alcohol,
wherein the diphosphomevalonate decarboxylase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 14 and 16.

2. The method of claim 1 wherein the enzyme comprises the amino acid sequence of SEQ ID NO: 6 or 16.

3. The method of claim 1 wherein one mole of NTP, rNTP, dNTP or pyrophosphate are consumed per mole of isoprenol produced.

4. The method of claim 1, wherein the isoprenol is converted to isoprene.

5. The method of claim 1, wherein the isoprenol is converted to isoamyl alcohol.

6. A method for producing isoprenol from mevalonate in an isolated cell,
wherein the cell is a recombinant organism expressing a heterologous diphosphomevalonate decarboxylase and lacking mevalonate kinase activity, wherein the diphosphomevalonate decarboxylase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 14 and 16,
the method consisting essentially of
(a) providing mevalonate or providing a carbon source to the cell under conditions in which mevalonate is produced; and (b) incubating the cell, thereby converting the mevalonate to isoprenol.

7. The method of claim 6, wherein the organism is a fungus.

8. The method of claim 6, wherein the heterologous diphosphomevalonate decarboxylase is from Picrophilus torridus.

9. The method of claim 6, wherein the cell is produced by transforming a unicellular organism with DNA encoding diphosphomevalonate decarboxylase.

10. A method for the production of isoprenol in a cell that does not naturally possess a mevalonate pathway consisting essentially of:
(a) transforming the cell with genes encoding thiolase, HMG-CoA synthase, HMG-CoA reductase and diphosphomevalonate decarboxylase to obtain a recombinant cell;
(b) incubating the transformed cell in the presence of a carbon source under conditions at which the cell produces mevalonate;
(c) thereby producing isoprenol.

11. The method of claim 10, wherein the cell is E. coli.