# Patent

## Title
Production of alkenes by enzymatic decarboxylation of 3-hydroxyalkanoic acids

## Abstract

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

## Background
This Application is the National Phase Under 35 U.S.C. § 371 of PCT International Application No. PCT/FR2009/051332 which has an International filing date of Jul. 6, 2009, which claims priority to both U.S. Provisional Application No. 61/078,824 filed on Jul. 8, 2008 and to French Patent Application FR 08 54550 filed on Jul. 4, 2008. The entire contents of all applications listed above are hereby incorporated by reference.


INTRODUCTION
The present invention relates to a method for generating alkenes through a biological process. More specifically, the invention relates to a method for producing terminal alkenes (in particular propylene, ethylene, 1-butylene, isobutylene or isoamylene) from molecules of the 3-hydroxyalkanoate type.
BACKGROUND OF THE INVENTION
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 alkene, 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 (of oxidation, of halogenation).
Propylene holds 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-trimethylpentane); the very high combustion/explosion ratio 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 alkenes are currently produced by catalytic cracking of petroleum products (or by a derivative of the Fisher-Tropsch process in the case of hexene, from coal or gas). Their cost is therefore naturally indexed to the price of oil. Moreover, catalytic cracking is sometimes associated with considerable technical difficulties which increase process complexity and production costs.
Independently of the above considerations, the bioproduction of plastics (“bioplastics”) is a thriving field. This boom is driven by economic concerns linked to the price of oil, and by environmental considerations that are both global (carbon-neutral products) and local (waste management).
The main family of bioplastics is that of the polyhydroxyalkanoates (PHA). These are polymers obtained by condensation of molecules comprising both an acid group and an alcohol group. Condensation takes place by esterification of the acid on the alcohol of the following monomer. This ester bond is not as stable as the direct carbon-carbon bond present in the polymers of conventional plastics, which explains why PHAs have a biodegradability of a few weeks to a few months.
The PHA family includes in particular poly-3-hydroxybutyrate (PHB), a polymer of 3-hydroxybutyrate, and polyhydroxybutyrate-valerate (PHBV), an alternating polymer of 3-hydroxybutyrate and 3-hydroxyvalerate.
PHB is naturally produced by some strains of bacteria such as Alcaligenes eutrophus and Bacillus megaterium. Laboratory bacteria, like E. coli, having integrated synthetic pathways leading to PHB or to PHAs in general, have been constructed. The compound or its polymer can, in certain laboratory conditions, account for up to 80% of the bacterial mass (Wong M S et al., Biotech. Bioeng., 2008). Industrial-scale production of PHB was attempted in the 1980s, but the costs of producing the compound by fermentation were considered too high at the time. Projects involving the direct production of these compounds in genetically modified plants (having integrated the key enzymes of the PHB synthetic pathway present in producer bacteria) are in progress and might entail lower operating costs.
The production by a biological pathway of alkanes or other organic molecules that can be used as fuels 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 N et al., Process Biochemistry, 2006, 41:1001; and Wackett LP, Current Opinions in Chemical Biology, 2008, 21:187.
In the alkene chemical family, isoprene (2-methyl-1,3-butadiene) is the terpene motif which, through polymerization, leads to rubber. Other terpenes might be developed, by chemical, biological or mixed pathway, as usable products such as biofuels or to manufacture plastics. The recent literature shows that the mevalonate pathway (a key intermediate in steroid biosynthesis in many organisms) might be used in order to efficiently produce products from the terpene family at industrial yields (Withers S T et al., Appl. Environ. Microbiol., 2007, 73:6277).
The production of terminal alkenes [ethylene mono- or di-substituted at position 2: H2C═C(R1)(R2)] has apparently been less extensively investigated. The production of isobutylene from isovalerate by the yeast Rhodotorula minuta has been detected (Fujii T. et al., Appl. Environ. Microbiol., 1988, 54:583), but the efficiency of this conversion, 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 H. et al. (BBRC, 1994, 201(2):516) and involves a cytochrome P450 enzyme which decarboxylates isovalerate by reduction of an oxoferryl group FeV═O. At no point does the reaction involve hydroxylation of isovalerate. Isovalerate is also an intermediate in leucine catabolism. Large-scale biosynthesis of isobutylene by this pathway seems highly unfavorable, since it would require the synthesis and degradation of one molecule 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 of the prior art 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 H. et al, Agric. Biol. Chem., 1984, 48:1679).
These same studies have also described the natural production of propylene: many microorganisms are capable of producing propylene, once again with an extremely low yield.
The production of ethylene by plants has long been known (Meigh et al, 1960, Nature, 186:902). According to the metabolic pathway elucidated, methionine is the precursor of ethylene (Adams and Yang, PNAS, 1979, 76:170). Conversion of 2-oxoglutarate has also been described (Ladygina N. et al., Process Biochemistry 2006, 41:1001). 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.
Prior to the characterization of the enzymatic steps which, in plants, convert to ethylene its true metabolic precursor, S-adenosylmethionine (SAM) via formation of 1-amino-cyclopropane-1-carboxylate (ACC) (Adams and Yang, PNAS, 1979, 76:170), several other hypotheses had been proposed in the scientific literature to explain ethylene production, among which was the decarboxylation of acrylate (H2C═CH—CO2H) originating from the dehydration of 3-hydroxyproprionate. Several articles specifically speculated on the metabolic pathway which would convert 3-hydroxypropionate to ethylene, via acrylate, in order to interpret radiotracer studies of ethylene production in which 14C-labelled substrates were supplied to plant tissue preparations: beta-alanine-2−14C to bean cotyledon extracts (Stinson and Spencer, Plant Physiol., 1969, 44:1217; Thompson and Spencer, Nature, 1966, 210:5036), and propionate-2−14C to banana pulp homogenates (Shimokawa and Kasai, Agr. Biol. Chem., 1970, 34(11):1640). All these hypotheses of the involvement of 3-hydroxypropionate and acrylate in metabolic ethylene production, which did not lead to characterization of enzyme activities, vanished from the scientific literature once the role of methionine, SAM and ACC was discovered (Hanson and Kende, Plant Physiology, 1976, 57:528; Adams and Yang, PNAS, 1979, 76:170).
Therefore, to my knowledge, there is currently no efficient method for producing terminal alkenes such as ethylene, propylene, 1-butylene, isobutylene, 1-amylene or isoamylene by microbiological synthesis. Such method would make it possible to avoid the use of petroleum products, and to lower the costs of producing plastics and fuels. Finally, it could potentially have a considerable global environmental impact by allowing carbon to be stored in solid form.
SUMMARY OF THE INVENTION
The present invention describes a method for carrying out the synthesis of alkene compounds though a biological process.
The invention is based on the design of a novel synthetic pathway for terminal alkene compounds based on the conversion of 3-hydroxyalkanoates. The invention is also based on the demonstration that said conversion can be carried out biologically, by using an enzyme of the decarboxylase type or variants thereof. The invention can be implemented in vitro, in cell-free systems, or by using microorganisms. The invention also relates to the production of alkenes from a carbon source, and particularly a carbohydrate (in particular glucose), a polyol (in particular glycerol), a biodegradable polymer (in particular starch, cellulose, poly-3-hydroxyalkanoate); the carbon source being converted by a microorganism to a metabolic intermediate belonging to the 3-hydroxyalkanoate family, which is then converted to terminal alkene.
More specifically, it is an object of the invention to provide a method for producing a terminal alkene, characterized in that it comprises a step of converting a 3-hydroxyalkanoate in the presence of an enzyme having decarboxylase activity.
Another object of the invention is based on the use of 3-hydroxyalkanoate compounds, as precursor or substrate, for the production of terminal alkene compounds.
In particular embodiments of the invention:

    
    
        3-hydroxypropionate is converted to ethylene; or
        3-hydroxybutyrate is converted to propylene; or
        3-hydroxyvalerate is converted to 1-butylene; or
        3-hydroxy-3-methylbutyrate (or 3-hydroxyisovalerate) is converted to isobutylene; or
        3-hydroxy-3-methylvalerate is converted to isoamylene.
    
    


The invention further relates to the use of a decarboxylase enzyme, or a microorganism producing a decarboxylase, for producing terminal alkene compounds from 3-hydroxyalkanoates.
The invention also relates to a composition comprising a microorganism producing a decarboxylase, a suitable culture medium and a 3-hydroxyalkanoate compound, or a carbon source which can be converted by the microorganism to a 3-hydroxyalkanoate compound.
Another object of the invention relates to a biocatalyst comprising a decarboxylase enzyme, or a microorganism producing a decarboxylase which decarboxylates a 3-hydroxyalkanoate compound to a terminal alkene.
Another object of the invention relates to a terminal alkene compound obtained by a method such as described in the invention.
A further object of the invention is an isolated or purified enzyme having decarboxylase activity and comprising all or part of SEQ ID NO: 6 or an enzyme having at least 15% sequence homology thereto.
Another object of the invention relates to the use of an enzyme having decarboxylase activity and comprising all or part of SEQ ID NO: 6, or an enzyme having at least 15% sequence homology thereto, for producing a terminal alkene.
Another object of the invention relates to a method for producing an enzyme having decarboxylase activity and comprising all or part of SEQ ID NO: 6 or an enzyme having at least 15% sequence homology thereto, the method comprising culturing a microorganism comprising a recombinant nucleic acid coding for said sequence in conditions allowing the expression of said sequence.
Another object of the invention relates to a microorganism comprising a recombinant nucleic acid coding for an enzyme having decarboxylase activity and comprising all or part of SEQ ID NO: 6 or an enzyme having at least 15% sequence homology thereto.
Definitions
“3-hydroxyalkanoate”, as used herein, denotes any molecule comprising 3-hydroxypropionate as common motif (FIG. 1), and optionally one or two alkyl substitutions on carbon 3. Said alkyl residues or groups can be linear or branched. As used herein, the terms “alkoyl” and “alkyl” have the same meaning and are interchangeable. Likewise, the terms “residue” and “group” have the same meaning and are interchangeable. Methyl, ethyl, propyl, isopropyl, butyl, isobutyl groups are examples of said alkyl groups. Carbon 3 becomes a chiral center if the two alkyl substitutions are different. The present definition encompasses the two chiral forms, even if one of the two forms, for example the R form, is the main form produced naturally. Examples of 3-hydroxyalkanoates are presented in FIG. 3. Optionally, alkyl substituents can be added on carbon 2, which then may also become chiral (if the two substituents are different). Equally, the configurations of the 3-hydroxyalkanoate substrates in the present invention encompass all the stereoisomers. In a preferred manner, the 3-hydroxyalkanoates correspond either to 3-hydroxypropionate or to variants or derivatives of 3-hydroxypropionate in which one of the two or the two hydrogen atoms carried on carbon 3 are substituted by a motif composed solely of carbon and hydrogen atoms, the number of carbon atoms of said substituents ranging from 1 to 5, preferably from 1 to 3, such as methyl, ethyl, propyl, isopropyl, butyl or isobutyl. The suffix “oate”, as used herein, can interchangeably denote either the carboxylate ion (COO—) or carboxylic acid (COOH). It is not used to denote an ester. In a particular embodiment, the 3-hydroxyalkanoates are represented by the following formula: HO—CO—CH2—C(R1)(R2)—OH or O−—CO—CH2—C(R1)(R2)—OH.
“Terminal alkenes”, according to the present invention, denotes molecules composed solely of carbon and hydrogen (unsaturated hydrocarbons having the formula CnH2n) comprising ethylene and organic molecules derived from ethylene by mono- or di-substitution of the two hydrogen atoms bound to carbon 2 by linear or branched alkyl groups. Terminal alkenes preferably are represented by the formula H2C═C(R1)(R2) wherein R1 and R2 are selected, independently, in the group consisting of a hydrogen atom and a linear or branched alkyl group, preferably having 1 to 4 carbon atoms, more preferably from 1 to 3 carbon atoms. Preferably, at least one of the two substituents on carbon 2 of the alkene is a linear or branched alkyl group. Terminal alkenes comprise branched isoalkene compounds, such as for example isobutylene. Preferred examples of terminal alkene compounds according to the invention are in particular ethylene, propylene, isobutylene, and isoamylene (FIG. 4), or else 1-butylene and 1-amylene.
“Carbon source”, as used herein, denotes any carbon compound that can be used as substrate for the organisms according to the invention. Said term includes glucose or any other hexose, xylose or any other pentose, polyols such as glycerol, sorbitol or mannitol, or else polymers such as starch, cellulose or hemicellulose, or else poly-3-hydroxyalkanoates like poly-3-hydroxybutyrate. It may be any substrate allowing the growth of microorganisms, such as formate for example. It may also be CO2 in the case where the organisms are capable of carrying out photosynthesis.
“Recombinant”, as used herein, denotes the artificial genetic modification of an organism, either by addition, removal, or modification of a chromosomal or extra-chromosomal gene or regulatory motif such as a promoter, or by fusion of organisms, or by addition of a vector of any type, for example plasmidic. The term “recombinant expression” denotes the production of a protein involving a genetic modification, preferably in order to produce a protein of exogenous or heterologous origin with respect to its host, that is, which does not naturally occur in the production host, or in order to produce a modified or mutated endogenous protein.
“Overexpression” or “overexpressing”, as used herein, denotes the recombinant expression of a protein, preferably originating from an organism different from the one in which it is expressed, increased by at least 10% and preferably by 20%, 50%, 100%, 500% and possibly more as compared to the natural expression of said protein. This definition also encompasses the case where there is no natural expression of said protein.
A “co-substrate” is a product added to the enzymatic reaction, so as to improve certain parameters thereof, and above all the activity thereof, said product and the principal substrate being consumed in equal amounts. The co-substrate must therefore be added to the reaction at a concentration comparable to that of the principal substrate. Depending on the enzyme, the presence of a co-substrate may be required for the enzymatic reaction.
A “cofactor” is a product added to the enzymatic reaction, so as to improve certain parameters thereof and above all to improve the activity thereof, said product not being consumed during the reaction, and therefore needing only to be added at a low concentration, proportional to the amount of enzyme, said concentration therefore being referred to as “catalytic”.
A “part” of an amino acid sequence denotes a fragment comprising at least 10, preferably at least 20, 30, 40 or 50 consecutive amino acid residues of said sequence.
“Homology” denotes the existence of a similarity between two sequences as measured by the percent identity between said two sequences.
Chemical compounds are often known by several names, official or common. Herein, the common names of the molecules are preferred. Thus:

    
    
        “ethylene” is used to denote ethene
        “propylene” is used to denote propene
        “butylene” is used to denote butene
        “isobutylene” is used to denote 2-methylpropene or isobutene
        “amylene” is used to denote pentene
        “isoamylene” is used to denote 2-methyl-but-1-ene or isopentene
        “propionate” is used to denote propanoic acid or the propanoate ion
        “butyrate” is used to denote butanoic acid or the butanoate ion
        “valerate” is used to denote pentanoic acid or the pentanoate ion.
    
    


DETAILED DESCRIPTION OF THE INVENTION
In particular, the invention provides a method for producing terminal alkenes comprising a step of enzymatic decarboxylation of 3-hydroxyalkanoate compounds. The invention also relates to the use of decarboxylases to catalyze this reaction, and in particular of enzymes of the type mevalonate diphosphate decarboxylase, and of substrates such as 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxy-3-methylbutyrate (or 3-hydroxyisovalerate) and 3-hydroxypropionate.
The invention describes the use of cofactors, including ethyl diphosphate, propyl diphosphate, methyl diphosphate, analogs of said molecules, and pyrophosphate. The invention further describes the use of co-substrates, such as ATP or other compounds containing a phosphoanhydride bond.
The invention also relates to the use of carbon sources, such as glucose, for directly producing terminal alkenes from whole cells, the synthetic pathway taking place by way of 3-hydroxyalkanoates.
The invention further relates to natural or modified organisms, endogenously producing a 3-hydroxyalkanoate, and also expressing a decarboxylase converting said 3-hydroxyalkanoates to terminal alkenes.
The alkene compounds produced, in particular propylene, ethylene and isobutylene, are key molecules in the plastics and fuel industry, and their industrial production by biological pathway, from renewable resources, represents a major innovation.
Thus the invention follows from the design of a novel synthetic pathway for compounds of the terminal alkene type based on the conversion of compounds of the 3-hydroxyalkanoate type. The invention demonstrates that said conversion can be carried out biologically, by using an enzyme of the decarboxylase type, which enables the conversion of a 3-hydroxyalkanoate to a terminal alkene. As illustrated in FIG. 2, said conversion takes place via a reaction intermediate having a 3-phospho-hydroxyalkanoate structure.
The conversion step according to the invention can be carried out in vitro, in the presence of an isolated enzyme (or an enzyme system additionally comprising one or more cofactors) or in culture, in the presence of a microorganism producing the enzyme.
As described herein in example 5, a signal-to-noise ratio (measured in the absence of enzyme) of approximately 100-fold for the conversion yield could be observed in some conditions. The affinity for 3-hydroxyisovalerate (HIV) was measured at approximately 40 mM. It was not obvious that such a very significant enzymatic activity could be obtained: indeed, biochemists familiar with the theory and practice of enzymology know very well that enzyme active sites contain structural elements enabling the recognition, binding and chemical conversion of certain specific substrates. The scientific literature abounds with experimental data indicating that changes in size or electrical charge, even minor, can lead to the exclusion of substrates. Specifically, no scientific prediction allowed it to be anticipated that MDP decarboxylases could use, as substrate, molecules of the 3-hydroxyalkanoate type in general, and 3-hydroxyisovalerate in particular, the latter differing from mevalonate diphosphate not only by its size (MW 118 versus 308 for mevalonate diphosphate), but also by the electrical charges of the diphosphate group present on the natural substrate, mevalonate diphosphate.
In a particular embodiment, a cofactor is added to the reaction so as to provide steric or electronic complementation in the catalytic cleft. The cofactor is advantageously selected in the group consisting of the pyrophosphate ion, methyl diphosphate, ethyl diphosphate, or propyl diphosphate. More generally, the cofactor is a compound containing the phosphoanhydride motif, having the general formula R—O—PO2H—O—PO3H2 in which R represents in particular a hydrogen atom, a linear, branched or cyclic alkyl group, preferably having from 1 to 10 or from 1 to 5 carbon atoms, or any other monovalent organic group. The analagous motifs corresponding to the monoesters of methylene diphosphonate, represented by the general formula R—O—PO2H—CH2—PO3H2 in which phosphoanhydride is replaced by a methylene bridge having the advantage of not being hydrolyzed, are also part of the invention.
In a preferred embodiment, the conversion occurs in the presence of a co-substrate, said co-substrate preferably being a compound containing a phosphoanhydride, and preferably being ATP, an rNTP, a dNTP or a mixture of several of these molecules, a polyphosphate, or pyrophosphate. The co-substrate is generally present in the host. However, in another particular embodiment, a co-substrate can be added to the reaction, preferably selected in the group consisting of ATP, an rNTP, a dNTP, a mixture of several rNTPs or dNTPs, a polyphosphate, and preferably pyrophosphate, or a compound containing a phosphoanhydride (represented by the general formula X—PO3H2 of FIG. 2).
In a particular embodiment of the invention, a microorganism that produces the decarboxylase is used. In a preferred embodiment, the microorganism is recombinant in that it produces a heterologous decarboxylase relative to the production host. The method can thus be carried out directly in the culture medium, without the need to separate or purify the enzyme system. In an especially advantageous manner, a microorganism is used having the natural or artificial property of endogenously producing one or more 3-hydroxyalkanoates, and also expressing or overexpressing a decarboxylase, natural or modified, so as to produce terminal alkenes directly from a carbon source present in solution.
The microorganisms used in the invention can be prokaryotes or eukaryotes, and in particular bacteria, yeasts, plant cells, fungi and molds, animal cells. In a particular embodiment, the microorganisms are bacteria, in particular the strain Alcaligenes eutrophus or Bacillus megaterium. 
In another particular embodiment, the microorganisms are recombinant bacteria of an Escherichia coli strain having been modified so as to endogenously produce one or more 3-hydroxyalkanoates, and converting them to terminal alkenes.
In another particular embodiment the microorganisms are recombinant yeasts, producing 3-hydroxyalkanoates, and converting them to terminal alkenes.
In another particular embodiment, one uses a microorganism that produces one or more 3-hydroxyalkanoates on the one hand, and a decarboxylase, optionally expressed by a second microorganism, on the other hand. Optionally, one cultures and one concomitantly uses the two organisms in the method according to the invention.
In another particular embodiment, whole plants or animals, optionally modified by transgenesis, are used to produce terminal alkenes from 3-hydroxyalkanoates, whether these be produced endogenously or exogenously supplied.
In another particular embodiment, one uses a photosynthetic microorganism having the natural or artificial property of endogenously producing one or more 3-hydroxyalkanoates, and also overexpressing a decarboxylase, natural or modified, so as to produce terminal alkenes directly from CO2 present in solution. Preferably, the microorganism is a photosynthetic bacterium, or a microalgae.
The present invention further relates to the organisms described hereinabove and their use for producing terminal alkene compounds.
As described in the following, the method of the invention can be carried out in microaerophilic conditions.
Furthermore, in a preferred embodiment, the method is carried out in the presence of a system for collecting gas of terminal alkenes degassing from the reaction.
Decarboxylase, as used herein, denotes any enzyme able to convert a 3-hydroxyalkanoate with a number n of carbon atoms to a terminal alkene compound with a number n−1 of carbon atoms. As illustrated in FIG. 2, the inventive method preferably takes place via a 3-phospho-hydroxyalkanoate reaction intermediate, and the enzyme used advantageously possesses a decarboxylase activity and a phosphorylase activity.
In a particular embodiment, the decarboxylase is a member of the phylogenetic superfamily of mevalonate diphosphate (MDP) decarboxylase (enzyme nomenclature EC 4.1.1.33), that is to say, a natural or artificial enzyme, encoded by a native or synthetic gene, optionally able to catalyze the reaction illustrated in FIG. 2.
MDP decarboxylase is an enzyme involved in cholesterol biosynthesis. Said enzyme has been isolated from a variety of organisms including animals, fungi, yeasts and some bacteria. It can also be expressed by some plants (Lalitha et al., 1985). Many genes encoding this enzyme have been cloned and sequenced. These enzymes are generally composed of 300 to 400 amino acids and use ATP as co-substrate, which is converted during the reaction to ADP and inorganic phosphate. The phosphate group is transferred from the ATP molecule to the tertiary alcohol of mevalonate diphosphate, releasing ADP. The reaction intermediate phosphorylated on the 3-hydroxyl group then undergoes elimination of the phosphate group, in the physiological case releasing isopentenyl pyrophosphate (FIG. 2).
The three-dimensional structures of several enzymes from this family have been resolved. To date, relatively few studies have been carried out on the enzymes from this family, and these enzymes have only been investigated in the context of precisely describing the cholesterol biosynthetic pathway. On the other hand, to my knowledge, no studies have yet been done to divert this enzyme from its natural function and turn it into an industrial catalyst.
Several examples of MDP decarboxylases from different organisms are given in sequences SEQ ID NO: 1 to SEQ ID NO: 16.
Thus, in a preferred embodiment, the enzyme used is a decarboxylase, preferably comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 or a sequence possessing at least 15% sequence homology to one of said sequences and retaining a decarboxylase activity. Preferred enzymes advantageously have at least 50% sequence homology, preferably at least 80%, more preferably at least 85%, even more preferably, at least 90, 95, 96, 97, 98 or 99% homology to one of the primary sequences SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16. The percent of sequence homology can be determined by different methods and by means of software programs known to one of skill in the art, such as for example the CLUSTAL method or BLAST and derived software, or by using a sequence comparison algorithm such as that described by Needleman and Wunsch (J. Mol. Biol., 1970, 48:443) or Smith and Waterman (J. Mol. Biol., 1981, 147:195).
A preferred decarboxylase of the invention is represented by the enzyme having sequence SEQ ID NO: 6, as well as any enzyme having significant sequence homology thereto. Preferred enzymes advantageously have at least 50% sequence homology, preferably at least 80%, more preferably at least 85%, even more preferably at least 90, 95, 96, 97, 98 or 99% sequence homology to the primary sequence SEQ ID NO: 6. Said enzyme has been cloned from Picrophilus torridus and produced by recombinant means in the scope of the present invention. As illustrated in the examples, this enzyme is particularly efficient at producing terminal alkene compounds according to the present invention. This enzyme is also an object of the present invention, as are the preparation and the use thereof as catalyst. In particular, an object of the invention is the use of a decarboxylase enzyme comprising all or part of SEQ ID NO: 6 or an enzyme having a significant sequence homology and preferably at least 15% to SEQ ID NO: 6, for producing terminal alkene compounds. Significant sequence homology denotes a sequence homology detectable by using the aforementioned algorithms, and preferably a sequence homology greater than 15%. The organisms with the closest phylogenetic relationship to Picrophilus torridus, such as Ferroplasma acidarmanus, Thermoplasma acidophilum, Thermoplasma volcanium and Picrophilus oshimae, are able to produce MDP decarboxylases closest to that of SEQ ID NO: 6. For instance, the MDP decarboxylase of Thermoplasma acidophilum (AC number Q9HIN1) has 38% sequence homology to SEQ ID NO: 6; that of Thermoplasma volcanium (Q97BY2) has approximately 42%. The use of these MDP decarboxylases is more particularly considered in the present invention.
Other enzymes of the decarboxylase type, natural or synthetic, can be selected for their ability to produce terminal alkenes according to the invention. Thus, a selection test comprises contacting the purified enzyme, or a microorganism producing the enzyme, with the substrate of the reaction and measuring the production of the terminal alkene compound. Such tests are described in the experimental section, in which over 60 different enzymes were tested.
The enzyme that is used can be any decarboxylase that is natural or produced or artificially optimized. In particular, one advantageously uses a decarboxylase having an optimized activity with respect to one or more 3-hydroxyalkanoates.
The enzyme can be produced or selected, from a reference decarboxylase (natural or itself already synthetic or optimized), by protein engineering techniques such as random mutagenesis, massive mutagenesis, site-directed mutagenesis, DNA shuffling, synthetic shuffling, in vivo evolution, or complete synthesis of genes.
In this respect, one object of the invention also relates to a method for preparing an enzyme having decarboxylase activity towards a 3-hydroxyalkanoate substrate, the method comprising a step of treating an enzyme source and selecting an enzyme having enhanced properties towards said substrate, as compared to the untreated enzyme.
The enzyme used in the invention can thus be natural or synthetic, and produced by chemical, biological or genetic means. It can also be chemically modified, for example in order to improve its activity, resistance, specificity, purification, or to immobilize it on a support.
The invention is characterized by the use of a decarboxylase, in particular a natural or modified MDP decarboxylase, to convert 3-hydroxyalkanoates to terminal alkenes.
The natural substrate of MDP decarboxylase is mevalonate diphosphate, which does not fall under the definition of 3-hydroxyalkanoates.
The generic reaction carried out by MDP decarboxylase using various 3-hydroxyalkanoates is depicted in FIG. 2B. It is understood that these reactions lead directly and in a single step to terminal alkenes.
In a first embodiment, the native or recombinant enzyme, purified or not, is used to convert a 3-hydroxyalkanoate to terminal alkene. To do this, the enzyme preparation is incubated in the presence of the substrate in physicochemical conditions allowing the enzyme to be active, and the incubation is allowed to proceed for a sufficient period of time. At the end of the incubation, one optionally measures the presence of the terminal alkene by using any detection system known to one of skill in the art such as gas chromatography or colorimetric tests for measuring the formation of the alkene product, or of free phosphate, or else for measuring the disappearance of the 3-hydroxyalkanoate substrate or of ATP.
In a preferred embodiment, cofactors are added so as to best mimic the natural reaction. In fact, the structure of 3-hydroxyalkanoates generally corresponds to a fragment of MDP, thus leaving a large space in the catalytic cleft empty during enzyme-substrate binding. Filling this space with a cofactor to replace the missing part of the substrate has the purpose of most closely mimicking the MDP molecule. As the cofactor is not modified during the reaction, it will therefore be added only in catalytic amounts. In the case where the substrate of the reaction is 3-hydroxypropionate, the complementary cofactor will be propyl diphosphate. In the case where the substrate is 3-hydroxybutyrate or 3-hydroxy-3-methylbutyrate, the complementary cofactor will be ethyl diphosphate. In the case where the substrate is 3-hydroxyvalerate or 3-hydroxy-3-methylvalerate, the complementary cofactor will be methyl diphosphate. These different molecules are shown in FIG. 5. By chance, it may happen that the complementary cofactor of a reaction has a positive effect on the reaction of another substrate. Generally, the cofactor can be any molecule comprising a phosphoanhydride, and therefore having the general formula R—PO2H—O—PO3H2, in which R is in particular H, a linear, branched or cyclic alkyl group, or any other monovalent organic group. The analogous motifs corresponding to methylene diphosphonate monoesters, having the general formula R—O—PO2H—CH2—PO3H2 in which phosphoanhydride is replaced by a methylene bridge having the advantage of not being hydrolyzed, are also part of the invention.
More generally, the cofactors can be monophosphate, or even phosphate-free, analogs of the previous molecules, or else any other molecule that can improve the reaction yield by providing steric or electronic complementation in the enzyme catalytic site.
In a particular embodiment, a co-substrate is added to the reaction. Said cosubstrate can be either ATP, that is to say, the natural co-substrate of MDP decarboxylase, or any rNTP (ribonucleoside triphosphate) or dNTP (deoxyribonucleoside triphosphate) or any mixture of rNTP or dNTP, or else pyrophosphate, or another polyphosphate, or else any molecule containing a phosphoanhydride group (X—PO3H2 of FIG. 2).
In a preferred embodiment, for converting a 3-hydroxyalkanoate to terminal alkene one uses an enzyme having at least 15% sequence homology, preferably at least 30%, 50% and even more preferably at least 80, 90, 95, 96, 97, 98 or 99% to a natural enzyme having decarboxylase activity and in particular to one of the enzymes corresponding to sequences SEQ ID NO: 1 to 16. In particular, the enzyme can have been modified by engineering from one of the enzymes SEQ ID NO: 1 to 16, or from any other decarboxylase identified from other sources. Such enzyme may have lost its MDP decarboxylase activity in particular through genetic engineering in the laboratory, but also during natural evolution (in which case one can speak of vestige MDP decarboxylase) and retained or increased its activity towards one or more molecules of the 3-hydroxyalkanoate type. The generation of variants of these enzymes, more reactive towards said substrates, makes it possible to improve the yield of the reaction according to the invention. For instance, the reactivity of wild-type MDP decarboxylase towards 3-hydroxyalkanoates is not necessarily optimal. Any approach known to one of skill in the art by which to produce and select such variants, such as random mutagenesis, site-directed mutagenesis, massive mutagenesis, DNA shuffling, or in vivo evolution, can be used.
The invention is also characterized by the use of a totally artificial enzyme, obtained by designing and producing a synthetic gene coding for a totally new enzyme with the aim of converting a 3-hydroxyalkanoate to terminal alkene, by using or not using the known data on MDP decarboxylases to design it.
Another object of the invention is an isolated or purified enzyme having decarboxylase activity and comprising all or part of SEQ ID NO: 6.
Another object of the invention relates to the use of an enzyme having decarboxylase activity and comprising all or part of sequence SEQ ID NO: 6, or an enzyme having sequence homology such as described above, for producing a terminal alkene. In one variant, the sequence can further comprise additional residues, such as for example a Histidine tag at the N-terminal end.
Another object of the invention relates to a method for producing an enzyme having decarboxylase activity and comprising all or part of sequence SEQ ID NO: 6, or an enzyme having a sequence homology such as described above, the method comprising culturing a microorganism comprising a recombinant nucleic acid coding for said sequence in conditions allowing the expression of said sequence. In this context, the present invention describes, in addition to the native nucleic acid (SEQ ID NO: 19), a nucleic acid having a sequence that is optimized for expression of the enzyme SEQ ID NO: 6 in bacteria, in particular in E. coli (SEQ ID NO: 17). This nucleic acid, and any optimized nucleic acid (i.e. allowing at least 30% improvement in expression as compared to the wild type sequence), are an object of the present application.
Another object of the invention relates to a microorganism comprising a recombinant nucleic acid coding for an enzyme having decarboxylase activity and comprising all or part of SEQ ID NO: 6, or an enzyme having a sequence homology such as described above. The microorganism is preferably a bacterium, a yeast or a fungus. The invention also relates to any plant or non-human animal comprising a recombinant nucleic acid coding for a decarboxylase according to the invention.
In one embodiment, the MDP decarboxylase is used in purified form to convert 3-hydroxyalkanoates to terminal alkenes. However, this method is costly, since enzyme and substrate production and purification costs are high.
In another embodiment, the MDP decarboxylase is present in the reaction as a non-purified extract, or else in the form of non-lysed bacteria, so as to economize on protein purification costs. However, the costs associated with this method are still quite high due to the costs of producing and purifying the substrates.
In another embodiment of the invention, the method uses a living organism producing the enzyme by which to carry out the conversion. The invention is thus characterized by the genetically engineered modification of a bacterial strain producing one or more 3-hydroxyalkanoates [for example Alcaligenes eutrophus or Bacillus megaterium, or else an E. coli strain laboratory-modified to produce said product(s)], such that said bacterial strain overexpresses the decarboxylase, said enzyme preferably originating from an organism different from the host microorganism, and can directly generate one or more terminal alkenes. The genetic modification can consist in integrating a decarboxylase gene into the chromosome, expressing the enzyme on a plasmid containing a promoter upstream of the enzyme-coding sequence, the promoter and coding sequence preferably originating from different organisms, or any other method known to one of skill in the art. Alternatively, other bacteria or yeasts may have specific advantages and be chosen. For instance, a yeast such as Saccharomyces cerevisiae, an extremophilic bacteria such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae for example, microalgae, or photosynthetic bacteria can be used. So as to optimally produce the 3-hydroxyalkanoate(s), which will then be converted to terminal alkenes, the strains can also have been modified by genetic engineering, i.e., by in vitro recombination or by directed in vivo evolution.
In one embodiment, the inventive method is characterized by the conversion of a carbon source such as glucose, to 3-hydroxyalkanoate, followed by the conversion of said primary product into a secondary product, that is to say, terminal alkene. The different steps of said method are outlined in FIG. 6.
In a particular embodiment, the invention is characterized by the conversion of polyhydroxyalkanoates to 3-hydroxyalkanoate, by using an enzyme or a suitable physicochemical method, followed by the conversion of said primary product to secondary product, that is to say, terminal alkene. Optionally, the polyhydroxyalkanoate has been produced by a plant whose metabolic pathways have been modified in a way so that they produce high yields of polyhydroxyalkanoate.
In a particular embodiment, the invention consists in the integral method for producing products from atmospheric CO2 or from CO2 artificially added to the culture medium. The inventive method is implemented in an organism able to carry out photosynthesis, such as microalgae for example.
In these embodiments, the inventive method is further characterized by the mode of recovery of the products, which degas from the culture. As a matter of fact, short terminal alkenes, and particularly ethylene, propylene, butene isomers, adopt the gaseous state at room temperature and atmospheric pressure. The inventive method therefore does not require extraction of the product from the liquid culture medium, a step which is always very costly when performed on an industrial scale. The evacuation and storage of the gaseous hydrocarbons, and their possible subsequent physical separation and chemical conversion, can be performed according to any method known to one of skill in the art.
In a particular embodiment, the invention also comprises detecting the alkene (propylene, ethylene and isobutylene in particular) present in the gas phase of the method. The presence of the target compounds in an environment of air or another gas, even in small amounts, can be detected by using various techniques, and in particular by using gas chromatography systems with infrared or flame ionization detection, or by coupling with mass spectrometry.
In a particular embodiment, the terminal alkenes obtained are condensed, then optionally reduced, by using techniques known to one of skill in the art, so as to produce longer chain alkenes, or longer chain alkanes. In particular, isobutylene can be used to synthesize isooctane: the catalytic methods for successfully carrying out this reaction have already been described in detail.
In a particular embodiment, the method involves culturing microorganisms in standard culture conditions (30-37° C. at 1 atm, in a fermenter allowing aerobic growth of the bacteria) or non-standard conditions (higher temperature to correspond to the culture conditions of a thermophilic organism, for example).
In a particular embodiment, the microorganisms are cultured in microaerophilic conditions, the quantity of injected air being limiting so as to minimize residual oxygen concentrations in the gaseous effluents containing the alkene hydrocarbons.
Other aspects and advantages of the invention will be described in the following examples, which are given for the purpose of illustration and not by way of limitation.



LEGENDS OF DRAWINGS
FIG. 1: 3-hydroxypropionate motif.
FIG. 2: Decarboxylation of mevalonate diphosphate by MDP decarboxylase—generic activity.
FIG. 3: Examples of 3-hydroxyalkanoates.
FIG. 4: Use of MDP decarboxylase for producing terminal alkenes.
FIG. 5: Cofactors that can be used in the reaction for purposes of structural complementation in the catalytic site.
FIG. 6: Integral method for producing an alkene from glucose.
FIG. 7: Chromatogram of the enzymatic reactions carried out in condition No. 1 of example 4.
FIG. 8: SDS-PAGE of the overexpression and purification steps of the enzyme SEQ ID NO: 6.
1. Markers
2. Culture before induction
3. Lysate
4. Fraction not adsorbed on the column
5. Column wash fraction
6. Purified enzyme, MW 36.8 kDa
FIG. 9: GC/MS chromatographic analysis of the conversion of HIV to IBN.
1 and 2: Negative controls corresponding to background noise in absence of enzyme.
3 and 4: Reactions in presence of enzyme SEQ ID NO: 6.
FIG. 10: Ratio of IBN production in presence and absence of ATP.
FIG. 11: Ratio of IBN production in presence and absence of Mg2+.
FIG. 12: Enzymatic activity according to temperature. Ratio: amount of IBN formed in presence of enzyme versus background.
FIG. 13: IBN production according to concentration of HIV substrate.
FIG. 14: Measurement of optimized reaction and comparison with background. Measured by gas chromatography with flame ionization detection.
FIG. 15: Improved expression level by optimization of the nucleotide sequence coding SEQ ID NO: 6. Lane M: molecular weight markers.
Lanes 1, 2, 3: native nucleotide sequence
(1) Cell lysate, soluble fraction loaded on purification column
(2) Lysate fraction not retained on purification column
(3) Eluted fraction: 10 μg purified enzyme
Lanes 4, 5, 6: Optimized nucleotide sequence
(4) Cell lysate, soluble fraction loaded on purification column
(5) Lysate fraction not retained on purification column
(6) Eluted fraction: 10 μg purified enzyme



EXAMPLES
Example 1: Cloning and Expression of Several MDP Decarboxylases
The gene encoding MDP decarboxylase of Saccharomyces cerevisiae is synthesized from overlapping oligonucleotides and cloned in a pET plasmid (Novagen) allowing expression in bacteria. Said plasmid is then transformed by electroporation into bacterial strain BL21 (Invitrogen). The bacteria are streaked on a Petri dish containing ampicillin and incubated at 37° C. The next day, a bacterial colony is randomly selected and used to inoculate 50 ml of LB medium containing ampicillin. The culture is incubated for 24 h while shaking, after which the culture is centrifuged, the bacteria lysed by sonication, and a total protein extract prepared. An aliquot of the extract is loaded on an electrophoresis gel together with a protein extract from the same strain which has not been transformed, and with molecular weight markers. The lane corresponding to the transformed strain contains a single band of approximately 30 kD, which corresponds to the expected size of the protein, said band being absent in the lane loaded with the non-transformed bacteria.
Example 2: Measuring the Activity of the Protein Extracts Towards 3-hydroxy-3-methylbutyrate
3-hydroxy-3-methylbutyrate (Sigma, reference 55453 under the name β-hydroxyisovaleric acid), is suspended at a concentration of 10 g/l. Mevalonate diphosphate is synthesized from mevalonolactone and other reagents (Sigma) by the conventional method and resuspended at a concentration of 10 g/l.
Six chromatography vials are prepared. 50 μL buffer containing 50 mM Bistris/HCl 1 mM dithiothreitol, 10 mM MgCl2 and 5 mM ATP are added to each vial.
Vials 1 and 4: 5 μl water are added (no substrate).
Vials 2 and 5: 5 μl of the mevalonate diphosphate preparation are added (positive control).
Vials 3 and 6: 5 μl of the 3-hydroxy-3-methylbutyrate (HIV) preparation are added.
Vials 1, 2 and 3: 5 μl of water are then added (no enzyme).
Vials 4, 5 and 6: 5 μl of the enzyme preparation described in example 1 are added.
Vials are sealed with a septum and crimped. All vials are incubated at 37° C. from 4 hours to 3 days. After incubation, a gas syringe is used to collect the gas present in each vial, and the CO2 concentration in the samples is measured by gas chromatography. Vial 5 has a very high CO2 concentration, and CO2, at a lower concentration, is also detected in vial 6, which indicates a significant reaction of the enzyme preparation towards 3-hydroxy-3-methylbutyrate. The presence of isobutylene in the gas sample from vial 6 is then measured by gas chromatography with infrared or flame ionization detection.
Example 3: Optimization of Reaction Conditions by Using a Cofactor
The same reaction as that described in vial 6 of the previous example is carried out, but in one of the samples, ethyl diphosphate, synthesized to order, is added as cofactor.
In this example, three vials are used. The first contains buffers, ATP, and the enzyme extract in the amounts described in the previous example. The second vial contains the same components, but additionally contains 3-hydroxy-3-methylbutyrate in the amounts described in the previous example. The third vial contains, in addition to 3-hydroxy-3-methylbutyrate, 10 μl of 10 mg/l ethyl diphosphate.
As in the previous example, isobutylene formation is measured by gas chromatography with infrared or flame ionization detection. It is found that when ethyl diphosphate is present, the amount of isobutylene produced over time is markedly higher.
Example 4: Screening an Enzyme Library
A library of 63 genes encoding enzymes from the MDP decarboxylase family was obtained and tested for activity on HIV as substrate.
Cloning, Bacterial Cultures and Expression of Proteins.
The genes encoding the mevalonate diphosphate (MDP) decarboxylase family EC 4.1.1.33 were cloned in the pET 25b vector (Novagen) in the case of eukaryotic genes and pET 22b (Novagen) for genes of prokaryotic origin, with a 6-Histidine tag at the N-terminal end immediately after the methionine initiation codon. Competent E. coli BL21(DE3) cells (Novagen) were transformed with these vectors by heat shock. The cells were grown with shaking (160 rpm) at 30° C. in TB medium containing 0.5 M sorbitol, 5 mM betain, 100 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
1.6 g of cells were thawed on ice and resuspended in 5 ml of 50 mM Na2HPO4 pH 8 containing 300 mM NaCl, 5 mM MgCl2, 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated for 10 min at room temperature and then returned to ice for 20 min. Cell lysis was completed by sonication for 3×5 min in an ultrasound water bath at 0° C.; samples were homogenized between pulses. The bacterial extracts were then clarified by centrifugation at 4° C., 10,000 rpm for 20 min.
Protein Purification and Concentration (PROTINO Kit)
The clarified bacterial lysates were loaded on a PROTINO-1000 Ni-IDA column (Macherey-Nagel) allowing adsorption of 6-His tag proteins. Columns were washed and the enzymes of interest were eluted with 4 ml of 50 mM Na2HPO4 pH 8 containing 300 mM NaCl, 5 mM MgCl2, 1 mM DTT, 250 mM imidazole. Eluates were then concentrated in Amicon Ultra-4 10 kDa cells (Millipore) to a final volume of 250 μl. Protein was quantified by the Bradford method.
Enzymatic Reactions
The desired enzymatic reaction (conversion of 3-hydroxy-3-methylbutyrate, or 3-hydroxyisovalerate, or else HIV) was tested in two experimental conditions that differed in terms of buffer and reaction pH.
Experimental Conditions No. 1.
100 mM citrate
10 mM MgCl2 
10 mM ATP
20 mM KCl-200
mM HIV
Final pH adjusted to 5.5
Experimental Conditions No. 2.
100 mM Tris-HCl pH 7.0
10 mM MgCl2 
10 mM ATP
20 mM KCl-200
200 mM HIV
Final pH adjusted to 7.0
The enzyme was added to the reaction mixture. As the protein yield was variable, the amount of enzyme added ranged between 0.01 and 1 mg/ml from one sample to another. The enzyme-free control reactions were carried out in parallel.
The 1 ml reactions were placed in 2 ml vials (Interchim) and sealed with teflon/silica/teflon septum (Interchim). Reactions were incubated without shaking at 37° C. for 72 h.
Analysis of Reactions
The gas present above the reactions was collected with a syringe equipped with a no-return mechanism. The gas sample was analyzed by gas chromatography (GC) coupled with mass spectrometry (MS). The instrument was previously calibrated using a range of isobutylene concentrations.
Column: BPX5 (SGE)
GC/MS: MSD 5973 (HP)
For each chromatogram, three principal peaks were obtained, the first corresponding to air, the second to water, and the third to isobutylene. Out of the 63 enzymes produced and tested, eleven potential candidates were identified in the primary screening. Some of these candidates are marked with an arrow in FIG. 7. Their identities are shown below, and their sequences in SEQ ID NO: 6 to 16 (His-tag not shown).
Candidate 1: SEQ ID NO: 7
Genebank accession number: CAI97800.1
Swissprot/TrEMBL accession number: Q1GAB2
Microorganisms: Lactobacillus delbrueckii subsp. bulgaricus (strain ATCC 11842/DSM 20081)
Candidate 2: SEQ ID NO: 8
Genebank accession number: CAJ51653
Swissprot/TrEMBL accession number: Q18K00
Microorganisms: Haloquadratum walsbyi DSM 16790
Candidate 3: SEQ ID NO: 9
Genebank accession number: ABD99494.1
Swissprot/TrEMBL accession number: Q1WU41
Microorganisms: Lactobacillus salivarius subsp. salivarius (strain UCC118)
Candidate 4: SEQ ID NO: 10
Genebank accession number: ABJ57000.1
Swissprot/TrEMBL accession number: Q04EX2
Microorganisms: Oenococcus oeni (strain BAA-331/PSU-1)
Candidate 5: SEQ ID NO: 11
Genebank accession number: ABJ67984.1
Swissprot/TrEMBL accession number: Q03FN8
Microorganisms: Pediococcus pentosaceus ATCC 25745
Candidate 6: SEQ ID NO: 12
Genebank accession number: ABV09606.1
Swissprot/TrEMBL accession number: A8AUU9
Microorganisms: Streptococcus gordonii (strain Challis/ATCC 35105/CH1/DL1/V288)
Candidate 7: SEQ ID NO: 13
Genebank accession number: ABQ14154.1
Swissprot/TrEMBL accession number: A5EVP2
Microorganisms: Dichelobacter nodosus VCS1703A
Candidate 8: SEQ ID NO: 14
Genebank accession number: EDT95457.1
Swissprot/TrEMBL accession number: B2DRT0
Microorganisms: Streptococcus pneumoniae CDC0288-04
Candidate 9: SEQ ID NO: 15
Genebank accession number: AAT86835
Swissprot/TrEMBL accession number: Q5XCM8
Microorganisms: Streptococcus pyogenes serotype M6 (ATCC BAA-946/MGAS10394)
Candidate 10: SEQ ID NO: 6
Genebank accession number: AAT43941
Swissprot/TrEMBL accession number: Q6KZB1
Microorganisms: Picrophilus torridus DSM 9790
Candidate 11: SEQ ID NO: 16
Genebank accession number: AAV43007.1
Swissprot/TrEMBL accession number: Q5FJW7
Microorganisms: Lactobacillus acidophilus NCFM
The highest levels of isobutylene (IBN) production were observed with candidate 10, that is, with the purified decarboxylase enzyme of SEQ ID NO: 6 from Picrophilus torridus. This enzyme was retained for further characterization.
Example 5: Characterization of Enzyme SEQ ID NO: 6
The recombinant enzyme was purified as described in example 4. The results, presented in FIG. 8, show that enzyme purity in the final protein sample was approximately 90%.
The activity of the isolated enzyme was confirmed. The reaction was carried out in the following conditions:
100 mM Tris-HCl pH 7.0
10 mM MgCl2 
10 mM ATP
20 mM KCl
250 M HIV
Final pH adjusted to 6.0
3 mg/ml enzyme
After 72 h incubation at 30° C., the signal was measured by GC/MS. The results are shown in FIG. 9. In the presence of the enzyme, IBN production was increased here by approximately 2.3-fold over background noise. The background noise observed here is in agreement with the organic chemistry literature, showing that in aqueous solution and at a temperature of around 100° C., 3-hydroxyisovaleric acid slowly decarboxylates to tert-butanol, which is partially dehydrated to isobutylene, following an equilibrium favorable to the formation of tert-butanol (Pressman and Luca, J. Am. Chem. Soc. 1940).
Effect of ATP Co-Substrate
Test Conditions
100 mM citrate
50 mM KCl
10 mM MgCl2 
200 mM HIV (to be specified)
1 mg/ml purified enzyme
pH 5.5
Incubation 72 h at 30° C.












Conditions
ATP final concentration
Enzyme







1
 0 mM
0 mg/ml


2
 0 mM
1 mg/ml


3
10 mM
0 mg/ml


4
10 mM
1 mg/ml









The results in FIG. 10 show that enzyme activity was only observed in the presence of the co-substrate ATP. Other molecules, and in particular those containing a phosphoanhydride bond, could also be efficient co-substrates for the enzyme.
Effect of Mg2+ Cofactor
Test Conditions
100 mM citrate pH 5.5
50 mM KCl
10 mM ATP
200 mM HIV (to be specified)
pH 5.5
1 mg/ml purified enzyme
Incubation 72 h at 30° C.












Conditions
MgCl2 final concentration
Enzyme







1
0 mM
0 mg/ml


2
0 mM
1 mg/ml


3
5 mM
0 mg/ml


4
5 mM
1 mg/ml









The results in FIG. 11 show that enzyme activity was improved in the presence of Mg2+ ions. Other ions, and in particular other divalent ions, could be used as cofactor in place of or in addition to Mg2+ ions.
Enzymatic Activity According to Temperature
Test Conditions
100 mM buffer
50 mM KCl
10 mM ATP
200 mM HIV (to be specified)
1 mg/ml purified enzyme
Incubation 72 h at different temperatures.
The results in FIG. 12 show that the enzyme is moderately thermoactive with a temperature optimum of approximately 50° C.
Activity According to pH
Test Conditions
100 mM buffer
50 mM KCl
10 mM ATP
200 mM HIV (to be specified)
1 mg/ml purified enzyme
Incubation 72 h at 30° C.
Optimal conditions were obtained with a pH of 5.5 in 100 mM citrate.
Enzyme Parameters
A substrate range was tested in the previously described conditions, with incubation at 50° C. The Km of the enzyme is approximately 40 mM HIV.
Optimization of Reaction Conditions
Optimum reaction conditions were sought, and the following conditions were retained:
100 mM citrate
50 mM KCl
40 mM ATP
200 mM HIV
1 mg/ml enzyme
Incubation 48 h at 50° C.
As shown in FIG. 14, the ratio of the signal over-background noise is approximately 100.
Example 6: Optimization of P. torridus MDP Decarboxylase Expression in E. coli 
The initial level of expression in E. coli BL21 was low, as the band was difficult to see on SDS-PAGE before purification. The Codon Optimization Index (CAI) of the native sequence for expression in E. coli was measured with the “Optimizer” program available at http://genomes.urv.es/OPTIMIZER/, and based on the method of Sharp and Li (1987). The value obtained was only 0.23, reflecting the low level of expression of the protein in E. coli. 
A sequence coding for an identical protein, but containing codons better adapted for expression in E. coli, was generated. This sequence had a CAI of 0.77 which is closer to the optimum of 1. The native sequence and the optimized sequence are shown in SEQ ID NO: 17 (optimized sequence of P. torridus (AAT43941) MDP decarboxylase including the His Tag) and SEQ ID NO: 19 (native sequence of P. torridus (AAT43941) MDP decarboxylase including the His Tag). The optimized sequence was synthesized by oligonucleotide concatenation and cloned in a pET25 expression vector. After transformation of the vector into E. coli strain BL21(DE3) and induction according to the previously described protocol, the proteins were produced, purified and analyzed on a gel as described previously. The same protocol was carried out with the native sequence for purposes of comparison.
Comparison of expression levels of candidate 224 using either the native nucleotide sequence or the sequence optimized for expression in E. coli. 
The results in FIG. 15 show that the protein corresponding to the optimized gene was clearly visible on the gel in the non-purified cell lysate (lane 4), which indicates a very notable increase in expression. The level of purity of the protein after the purification step was also higher in the case of the optimized gene.
Activity was measured on the crude lysate. No activity was detected on the crude lysate corresponding to the native nucleic sequence. The expression of the protein was improved such that the crude lysate obtained with the improved sequence (optimized clone 224) now displayed this activity.
The following reaction medium was used in this test:
Reaction Medium














Products
Final concentration









Acetate reaction buffer
50 mM



(500 mM, pH 5.5)



MgCl2 (1M)
10 mM



KCl (1M)
20 mM



HIV (3M)
50 mM



ATP (100 mM)
40 mM



Protease inhibitor
1X



(100X)



H20



Enzyme
89 μg total protein




(crude lysate)










Incubation 2 days at 50° C.

Results

Condition No. 1: Lysate of optimized clone 224

Condition No. 2: Lysate of clone GB6 (empty pET plasmid)














Signal area



Conditions
surface
Ratio

















1
1083
22


2
49









Example 7: Method for Synthesizing Isobutylene from 3-Hydroxy-3-Methylbutyrate and Conversion to Isooctane
A reaction identical to that of vial 3 in example 3 was carried out in a 1 liter volume, in a fermenter equipped with a gas extraction system. The presence of the recombinant enzyme induced the conversion of 3-hydroxy-3-methylbutyrate to isobutylene, which naturally degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter. Isobutylene was then used to produce isooctene by addition catalyzed by Amberlyst 35wet or 36wet resin (Rohm and Haas). Isooctene was reduced in turn to isooctane by catalytic hydrogenation.
Example 8: Enzyme Engineering to Improve Efficacy for Substrates
Random mutagenesis technology was used to create a library containing thousands of mutants of the gene described in example 1. This mutant library was then cloned in the expression plasmid and transformed into competent bacterial strain BL21.
A thousand bacteria were then isolated and inoculated into Eppendorf tubes containing 500 μl LB medium supplemented with ampicillin. The samples were incubated on a shaker for 15 h. The next day, the amount of isobutylene produced was determined by using one or another of the experimental protocols described in the previous examples.
Clones with a significantly increased amount of isobutylene were then revalidated using the same experimental protocol. Once this improvement was validated, the plasmid was extracted from each improved clone and sequenced. Mutations responsible for the improved activity were identified and combined on a same plasmid. The plasmid containing the different improving mutations was in turn transformed into competent bacteria, and the same analysis was carried out.
The clone containing the combined mutations, which had significantly greater activity than the one containing only a single improving mutation, was then used as the basis for a new cycle of mutation/screening, to identify mutants with even further improved activity.
On completion of this protocol, the clone containing several mutations and having the best activity was selected.
Example 9: Method for Synthesizing Ethylene from 3-hydroxypropionate
The gene encoding the enzyme described in example 1 was inserted in a plasmid allowing expression of the recombinant proteins in an E. coli strain. The plasmid was transformed into the bacteria of said strain. The transformed bacteria were then incubated in a fermenter in the presence of propyl diphosphate (10 mg/l) and 3-hydroxypropionate (1 g/l). The presence of the recombinant enzyme led to the conversion of 3-hydroxypropionate to ethylene, which spontaneously degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter. Ethylene was then measured in the gas sample by gas chromatography with infrared detection in the part of the spectrum where ethylene emits strongly.
Example 10: Method for Synthesizing Propylene from 3-hydroxybutyrate
The gene encoding the enzyme described in example 1 or an enzyme described in example 4 was inserted in a plasmid allowing expression of recombinant proteins in an E. coli strain. The plasmid was transformed into the bacteria of said strain. The transformed bacteria were then incubated in a fermenter in the presence of ethyl diphosphate (10 mg/l) and 3-hydroxybutyrate (1 g/l) (Sigma, reference 166898). The presence of the recombinant enzyme led to the conversion of 3-hydroxybutyrate to propylene, which spontaneously degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter. Propylene was then measured in the gas sample by gas chromatography with infrared detection in the part of the spectrum where propylene emits strongly.
Example 11: Method for Synthesizing Propylene from Glucose
The gene encoding the enzyme described in example 1 or an enzyme described in example 4 was cloned in a plasmid allowing expression of recombinant proteins in the bacterium Alcaligenes eutrophus. The plasmid was transformed into the bacteria of said strain.
The transformed bacteria were then incubated in a fermenter in the presence of glucose and ethyl diphosphate and in microaerophilic conditions, then subjected to heat shock which induced them to produce large quantities of 3-hydroxybutyrate. The presence of the recombinant enzyme led to the simultaneous conversion of 3-hydroxybutyrate to propylene, which spontaneously degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter.
Example 12: Method for Synthesizing Propylene from Glucose
This example describes a method very similar to that of example 11. The main difference consists in the use of an E. coli strain modified so as to produce 3-hydroxybutyrate instead of a natural strain like Alcaligenes eutrophus. Said strain was obtained by the engineering of metabolic pathways so as to lead to accumulation of 3-hydroxybutyrate. Addition of an MDP decarboxylase such as described in example 1 or in example 4 enabled the conversion of 3-hydroxybutyrate to propylene.
Example 13: Method for Synthesizing Isobutylene from Glucose
The gene encoding the enzyme described in example 1 was inserted in a plasmid allowing expression of recombinant proteins in E. coli strains that had also undergone metabolic modifications so that they endogenously synthesized 3-hydroxy-3-methylbutyrate.
The bacteria were then incubated in a fermenter in the presence of glucose and in microaerophilic conditions. The presence of the recombinant enzyme induces the simultaneous conversion of 3-hydroxy-3-methylbutyrate to isobutylene, which naturally degasses, and which was recovered by a gas extraction system located in the upper part of the fermenter.

## Claims

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

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

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

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

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

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

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

8. The method according to claim 1, wherein the method further comprises adding a cofactor containing a phosphoanhydride motif to the reaction, wherein the cofactor has the general formula R—O—P02H—O—P03H2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

9. The method according to claim 1, further comprising adding a methylene diphosphonate monoester to the reaction, wherein the methylene diphosphonate monoester has the general formula R—O—P02H—CH2—P03H2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

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

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

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

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

14. The method according to claim 11, wherein the microorganism is a photosynthetic microorganism which endogenously produces one or more 3-hydroxyalkanoates, and further overexpressing the MDP decarboxylase enzyme, so as to produce terminal alkenes directly from CO2 present in solution.

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

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

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

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

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

20. The method of claim 19, wherein the method further comprises adding a cofactor containing a phosphoanhydride motif to the reaction, wherein the cofactor has the general formula R—O—P02H—O—P03H2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

21. The method according to claim 19, further comprising adding a methylene diphosphonate monoester to the reaction, wherein the methylene diphosphonate monoester has the general formula R—O—P02H—CH2—P03H2 in which R is a hydrogen atom, a methyl, ethyl or propyl group, any linear, branched or cyclic alkyl group, or any other monovalent organic group.

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

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

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

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

26. The method according to claim 23, wherein the microorganism is a photosynthetic microorganism which endogenously produces 3-hydroxy-3-methylbutyrate, and further overexpressing the MDP decarboxylase enzyme, so as to produce isobutylene directly from CO2 present in solution.

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

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

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

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

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

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

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

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