BRPI0914215B1 - PRODUCTION OF ALKENES BY ENZYMATIC DECARBOXYLATION OF 3-HYDROXY-ALKANOIC ACIDS - Google Patents

Abstract

PRODUCTION OF ALKENES BY ENZYMATIC DECARBOXYLATION OF 3-HYDROXY-ALKANOIC ACIDS. The present invention relates to a process for producing alkenes biologically. It refers more particularly to a process of producing terminal alkenes by enzymatic decarboxylation of 3-hydroxy-alkanoate molecules. The invention also refers to the enzyme systems and microbial strains used, as well as the products obtained.

Description

Introduction

The present invention relates to a process for generating alkenes biologically. It refers more particularly to a process for producing terminal alkenes (notably propylene, ethylene, 1-butylene, iso-butylene or iso-amylene) from molecules of the 3-hydroxy-alkanoate type.

Invention Planning

From the petrochemical probe currently derived a large number of chemical compounds. Alkenes (such as ethylene, propylene, different butenes, or even pentenes, for example) are used in the plastics industry, for example, to produce polypropylene or polyethylene, as well as in other areas of the chemical and fuel industry. .

Ethylene, the simplest alkene, has a central place in industrial organic chemistry: it is the most produced organic compound in the world. It notably allows the generation of polyethylene, a leading plastic. By reaction (oxidation, halogenation), ethylene can also be transformed into numerous products useful for industry.

Propylene plays a role of comparable importance: a plastic material, polypropylene, is derived from it by polymerization. The technical characteristics of this product, in terms of resistance, density, solidity, deformability and transparency, have no equivalent. The global polypropylene market has been developing steadily since its invention in 1954.

Butylene exists in four forms, one of which, isobutylene, is part of methyl tert-butyl ether (MTBE), an anti-knock additive for automobile fuels. Iso-butylene can also be used to generate iso-octene, which can then be reduced to iso-octane (2,2,4-trimethyl-pentane); The very high combustion/explosion ratio of iso-octane makes it the best fuel for so-called "gasoline" engines.

Amylene, hexene and heptene exist in numerous forms, depending on the location and configuration of the double chain. These products have real industrial applications, but are of less importance than ethylene, propylene or butenes.

All of 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). Its cost is, therefore, naturally indexed to that of oil. On the other hand, catalytic cracking sometimes presents important technical difficulties, which translate into process complexity and production costs.

Regardless of the foregoing considerations, the bioproduction of plastic materials ("bioplastics") is experiencing important progress. The motivation comes from economic considerations linked to the cost of oil, and from global (carbon neutral products) or local (waste management) environmental considerations.

The main family of bioplastics is polyhydroxyalkanoates (PHA). It is a polymer obtained by condensation of molecules comprising, at the same time, an acid group and an alcohol group. Condensation occurs by esterification of the acid over the alcohol of the following monomer. This ester bond is also not stable than the direct carbon-carbon bond found in classic plastic polymers, which explains the biodegradability, in a few weeks to a few months, of PHAs.

In the PHA family, one can notably mention poly-3-hydroxybutyrate (PHB), a polymer of 3-hydroxy-butyrate, or polyhydroxy-butyrate-valerate (PHBV), a polymer that alternates the 3 -hydroxy-butyrate and 3-hydroxy-valerate.

PHB is produced naturally by particular bacterial strains, such as Alcaligenes eutrophus or Bacillus megaterium. Laboratory bacteria, such as Escherichia coli, having integrated synthetic pathways that lead to PHB or PHA, in general, were produced. The compound or its polymer can represent, under certain laboratory conditions, up to 80% of the mass of the bacteria. (Wong MS et al., Biotech. Bioeng 2008). Attempts at industrial production of PHB were made in the 1980s, but the production cost per fermentation was then considered to be very high. Projects for the direct production of these compounds in genetically modified plants (having integrated the key enzymes of the PHB synthesis pathway of the producing bacteria) are underway. They could be associated with better exploration costs.

The biological generation of alkanes or other organic molecules that can be used as fuels or as precursors for synthetic resins is necessary in the context of durable industrial exploitation in harmony with geochemical cycles. The first generation of biofuels consisted of the fermentative production of ethanol, the fermentation and distillation processes already being in place in the agri-food industry. The production of second-generation biofuels is in an exploratory phase, notably covering the production of long-chain alcohols (butanol and pentanol), terpenes, linear alkanes and fatty acids. Two recent analyzes provide a general table of research in this field: Ladygina N et a/., Process Biochemistry 2006 41: 1001, et Wackett LP Current Opinion in Chemical Biology, 2008, 21: 187.

In the chemical family of alkenes, isoprene (2-methyl-1,3-butadiene) is the terpene motif that leads to rubber polymerization. Other terpenes could be developed, chemically, biologically or mixed, into 25 products that can be used as biofuels or to make plastics. Recent publications show that the mevalonate pathway (a key intermediate in steroid biosynthesis in numerous organisms) could be utilized to effectively produce products from the terpene family with industrial yields (Withers ST et al., Appl. Environ Microbiol 2007 73: 30 6277).

The production of terminal alkenes (mono- or bissubstituted ethylene in position 2, H2C = C(R1) (R2) was apparently less explored: the production of iso-butylene from iso-valerate by the yeast Rhodotorula minuta was detected (Fujii T. et al., Appl. Environ. Microbiol. 1988 54: 583), but the effectiveness of this conversion, less than 1 millionth per minute, be it approximately 1 to 1000 per day, is far from allowing an industrial application. The reaction mechanism was elucidated in Fukuda H et al., BBRC 1994 201 : 2 : 516: it involves an enzyme from the cytochrome P450 family that carries out a decarboxylation reaction of iso-valerate, by reducing an oxoferryl group Fev = O . At no point does the reaction involve the hydroxylation of iso-valerate. Iso-valerate is, on the other hand, an intermediate compound in the catabolism of leucine. The massive biosynthesis of iso-butylene via this route seems very unfavorable, as it would be It is necessary to synthesize and degrade a leucine molecule to form an iso-butylene molecule. Finally, the enzyme that catalyzes the reaction uses heme as a cofactor, making it difficult for recombinant expression in bacteria and for improving enzymatic parameters. For all these reasons, it seems unlikely that this prior art route could serve as the basis for an industrial development. Other microorganisms have been mentioned as marginally capable of naturally generating iso-butylene from isovalerate. The yields obtained are even lower than those obtained using Rhodotorula minuta (Fukuda H. et al., Agric. Biol. Chem. 1984 48: 1679).

These same works also constitute a state of natural propylene production: numerous microorganisms can produce propylene, with an extremely low yield.

The production of ethylene by plants has long been described (Meigh et al., 1960 Nature 186: 902). Methionine constitutes the precursor of ethylene according to the elucidated metabolic pathway (Adams et Yang, PNAS 1979 76: 170). A conversion from 2-oxoglutarate has also been reported (Ladygina N et al., Process Biochemistry 2006 41:1001). Since a single ethylene molecule requires the prior production of a chain of four or five carbon atoms, the material and energy balances of all these pathways are unfavorable and negatively predict their industrial application for the bioproduction of alkenes.

Prior to the characterization of the enzymatic steps that, in plants, convert its true metabolic precursor, S-adenosyl-methionine (SAM), into ethylene via the formation of 1-amino-cycle propane-1-carboxylate (ACC) (Adams et Yang , PNAS 1979 76: 170), several other hypotheses have been proposed in the scientific literature to explain the production of ethylene, including the decarboxylation of acrylate (H2C = CH - CO2H), which would come from the dehydration of 3-hydroxy-propionate. Several articles explicitly speculate about the metabolic pathway that would convert ethylene, via acrylate, to 3-hydroxy-propionate, to interpret radioactive ethylene tracing experiments, providing 14C-labeled substrates to plant tissue preparations, beta-alanine-2 -14C to pod coltyledon 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 et Kasai, Agr. Biol. Chem. 1970 34: 11: 1640). All these speculations about the implication of 3-hydroxy propionate and acrylate in the metabolic production of ethylene, which did not lead to the characterization of enzymatic activities, have disappeared from the scientific literature since the role of methionine, SAM and ACC was discovered (Hanson et al. Kende, Plant Physiology 1976 57: 528; Adams et Yang, PNAS 1979 76: 170).

Therefore, the applicant does not currently know of an effective process for producing terminal alkenes, such as ethylene, propylene, 1-butylene, iso-butylene, 1-amylene or iso-amylene, by microbiological synthesis. This process would make it possible to avoid consuming petroleum products and reduce the costs of producing plastic and fuel. Ultimately, it could eventually have an important global effect on the environment, allowing carbon to be stored in solid form.

Summary of the Invention

The present invention describes a process that allows the synthesis of alkene compounds biologically.

The invention arises from the conception of a new route for the synthesis of terminal alkene compounds based on the conversion of 3-hydroxyalkanoates. The invention is also based on the demonstration that this conversion can be carried out biologically, using a decarboxylase-type enzyme or variants thereof. The invention can be used in vitro in acellular systems, or using microorganisms. The invention also aims at the production of alkenes from a carbon source, and notably a carbohydrate (notably glucose), a polyol (notably glycerol), a biodegradable polymer (notably starch, cellulose , poly-3-hydroxyalkanoate); the carbon source being converted by a microorganism into a metabolic intermediate of the 3-hydroxyalkanoate family, which will then be converted into a terminal alkene.

More precisely, an object of the invention resides in a process for producing a terminal alkene, characterized by the fact that it comprises a step of converting a 3-hydroxy-alkanoate in the presence of an enzyme that has decarboxylase activity.

Another objective of the invention lies in the use of 3-hydroxy-alkanoate compounds, as a precursor compound or substrate, for the production of terminal alkene compounds.

In particular embodiments of the invention: - 3-hydroxypropionate is converted into ethylene; or - 3-hydroxybutyrate is converted into propylene; or - 3-hydroxy-valerate is converted into 1-butylene; or - converts 3-hydroxy-3-methyl-butyrate (or 3-hydroxy-isovalerate) into iso-butylene; or - 3-hydroxy-methyl-valerate is converted into iso-amylene.

The invention further relates to the use of a decarboxylase enzyme, or a microorganism, producing a decarboxylase, for the production of terminal alkene compounds from 3-hydroxyalkanoates.

The invention also relates to a composition comprising a microorganism that produces a decarboxylase, an appropriate culture medium and a 3-hydroxyalkanoate compound, or a carbon source convertible by the microorganism into a 3-hydroxyalkanoate compound. .

Another object of the invention relates to a biocatalyst comprising a decarboxylase enzyme, or a microorganism that produces a decarboxylase, decarboxylating a 3-hydroxy-alkanoate compound in the terminal alkene.

Another object of the invention relates to a terminal alkene compound obtained by a process as described in the invention.

Another object of the invention resides in an isolated or purified enzyme having a decarboxylase activity and comprising all or part of the sequence SEQ ID NO: 6 or an enzyme that has a sequence homology of at least 15% thereto.

Another object of the invention relates to the use of an enzyme that has a decarboxylase activity and that comprises all or part of the sequence SEQ ID NO: 6, or an enzyme that has a sequence homology of at least 15% with it, to produce a terminal alkene.

Another object of the invention relates to a process for producing an enzyme that has a decarboxylase activity and that comprises all or part of the sequence SEQ ID NO: 6 or an enzyme that has a sequence homology of at least 15% with this, the process comprising culturing a microorganism comprising a recombinant nucleic acid encoding that sequence under conditions that allow expression of that sequence.

Another object of the invention relates to a microorganism that comprises a recombinant nucleic acid encoding an enzyme that has a decarboxylase activity and comprising all or part of the sequence SEQ ID NO: 6 or an enzyme that presents a sequence homology to at least 15% with this one.

Definitions

"3-hydroxy-alkanoate" is defined in this case as any molecule that comprises 3-hydroxy-propionate as a common motif (figure 1), and possibly one or two alcoholic substitutions on carbon 3. These residues or alcoholic radicals can be linear or branched. In the present application the terms "alcohol" and "alkyl" have the same meaning and are interchangeable. Likewise, the terms "residue" and "radical" have the same meaning and are interchangeable. Methyl, ethyl, propyl, isopropyl, butyl, iso-butyl residues are examples of these alcoholic residues. Carbon 3 becomes a chiral center if the two alcohol substitutions are different. The present definition encompasses both chiral forms, even if one of the two forms, for example the R form, is the main naturally produced form. Examples of 3-hydroxyalkanoates are shown in figure 3. Eventually, alkyl substitutions can be added on carbon 2, which is then also capable of becoming chiral (if the two substituents are different). I ndifferently the configurations of the 3-hydroxyalkanoate substrates in the present invention cover the set of stereoisomers. Preferably, 3-hydroxyalkanoates correspond either to 3-hydroxypropionate or to variants or derivatives of 3-hydroxypropionate in which one of the two hydrogen atoms carried by carbon 3 is replaced by a composed only of carbon and hydrogen atoms, the carbon number of these substituents varying from 1 to 5, preferably from 1 to 3, such as methyl, ethyl, propyl, isopropyl, butyl or isobutyl. The suffix "oate" can mean either the carboxylate ion (COO-) or the carboxylic acid (COOH). It is not used to designate an ester. In a particular way, 3-hydroxyalkanoates have the following formula: HO-CO-CH2-C(R1)(R2) -OH, or O-CO-CH2-C(R1)(R2)-OH.

By "terminal alkenes", in the sense of the present invention we mean molecules composed only of carbon and hydrogen (unsaturated hydrocarbons of the formula CnH2n) comprising ethylene and organic molecules that derive from ethylene by mono- or bisubstitution of the two hydrogen atoms linked to carbon 2 by linear or branched alcoholic radicals. Terminal alkenes preferably respond to the formula H2C =C(R1)(R2), in which R1 and R2 are chosen, independently, from a hydrogen atom and an alcoholic radical, linear or branched, preferably having from 1 to 4 carbon atoms. carbon, even more preferably 1 to 3 carbon atoms. Preferably, at least one of the two substituents on carbon 2 of the alkene is an alkyl radical, linear or branched. Terminal alkenes comprise branched iso-alkene compounds, such as iso-butylene. Preferred examples of terminal alkene compounds according to the invention are notably ethylene, propylene, iso-butylene, and iso-amylene (figure 4) or even 1-butylene and 1-amylene. "Carbon source means in this case any carbon compound usable as a substrate by organisms, according to the invention. This term includes glucose or any other hexose, xylose or any other pentose, polyols such as glycerol, sorbitol or mannitol, or also polymers, such as starch, cellulose or hemicellulose, or even poly-3-hydroxyalkanoates, such as poly-3-hydroxybutyrate. This can be any substrate that allows the growth of microorganisms, such as the shape, for example. It can also be CO2 in the case where the organism is capable of carrying out photosynthesis.

By "recombinant", we mean the artificial genetic modification of an organism, whether by addition, subtraction or modification of a chromosomal or extrachromosomal gene or a regulatory motif such as a promoter, whether by fusion of organisms, or by addition of a vector of any nature, for example, plasmid. "Recombinant expression" designates the production of a protein, implying a genetic modification, preferably to produce a protein of foreign or heterologous origin compared to its host, that is, one that does not naturally occur in the production host, or to produce an endogenous protein modified or mutated.

By "overexpression" or "overexpressing", we mean the recombinant expression of a protein, preferably originating from an organism other than that in which it is expressed, increased by at least 10% and, preferably, by 20%, 50%, 100%, 500% or possibly more in relation to the natural expression of that protein. The case in which there is no natural expression of this protein also falls within the scope of this definition.

A "cosubstrate" is a product added to the enzymatic reaction, in order to improve certain parameters and, firstly, its activity, this product and the main substrate being consumed in equal quantities. The co-substrate must therefore be added in a concentration comparable to that of the main substrate. According to the enzyme, the presence of a cosubstrate may be necessary for the enzymatic reaction.

A "cofactor" is a product added to the enzymatic reaction, in order to improve certain parameters and, firstly, its activity, this product is not consumed during the reaction, and therefore needs to be added only in concentration low, proportional to the amount of enzyme, this concentration being, therefore, said to be "catalytic".

A "part" of an amino acid sequence means a fragment comprising at least 10, preferably at least 20, 30, 40 or 50 consecutive amino acid residues of that sequence.

By homology, we mean the existence of a similarity between two sequences as measured by the percentage of identity between these two sequences.

Chemical compounds are often known by various names, official or used. In this case, the usual names of the molecules were preferred. Thus: - ethylene is used to designate ethylene; - propylene is used to designate propylene; - butylene is used to designate butene; - iso-butylene is used to designate 2-methyl-propene or iso-butene; - amylene is used to designate pentene; - isoamylene is used to designate 2-methyl-but-1-ene or iso-pentene; - propionate is used to designate propanoic acid or propanoate ion; - butyrate is used to designate butanoic acid or butanoate ion; - valerate is used to designate pentanoic acid or pentanoate ion.

Detailed Description of the Invention

The invention notably describes a process for producing terminal alkenes, comprising an enzymatic decarbonylation step of 3-hydroxyalkanoate compounds. The invention also arises from the use of decarboxylases to catalyze this reaction, and, in particular, enzymes of the mevalonate diphosphate decarboxylase type, and substrate, such as 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxy -3-methyl-butyrate (or 3-hydroxy-isovalerate) and 3-hydroxy-propionate. The invention describes the use of cofactors, including ethyl diphosphate, propyl diphosphate, methyl diphosphate, analogues of these molecules, and pyrophosphate. The invention further describes the use of cosubstrates, such as ATP or other compounds that bear a phospho-anhydrous bond.

The invention also relates to the use of carbon sources, such as glucose, to directly produce terminal alkenes, from whole cells, the synthesis route going through 3-hydroxy-alkanoates.

The invention also refers to organisms, natural or modified, endogenously producing a 3-hydroxyalkanoate and expressing, on the other hand, a decarboxylase that transforms these 3-hydroxyalkanoates into terminal alkenes.

The alkene compounds produced, notably propylene, ethylene and iso-butylene, are key molecules in the plastics and fuel industry, and their industrial manufacture by biological means, from renewable resources, constitutes a major innovation.

The invention thus arises from the conception of a new route for the synthesis of terminal alkene-type compounds based on the conversion of 3-hydroxy-alkanoate-type compounds. The invention demonstrates that this conversion can be carried out biologically, using a decarboxylase-type enzyme, which allows a 3-hydroxyalkanoate to be transformed into a terminal alkene. As illustrated in figure 2, this conversion is carried out via a reaction intermediate with a 3-phospho-hydroxy-alkanoate 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, further comprising one or more cofactors) or in culture, in the presence of a microorganism producing the enzyme.

As described herein in example 5, a ratio of signal over background noise (measured in the absence of enzyme) to the conversion rate of a factor of 100 approximately can be observed under certain conditions. The affinity towards 3-hydroxyisovalerate (HIV) was measured at around 40 mM. It was not evident that this very significant enzymatic activity could be obtained: in fact, it is well-known to biochemists who consider the theory and practice of enzymology that the active sites of enzymes contain structural elements, allowing recognition, binding and the chemical conversion of certain specific substrates. The scientific literature presents experimental data, indicating that changes in size or electrical charge, even minor ones, are exclusion criteria for substrates. Specifically, no scientific precision could allow predicting that MDP-decarboxylases could use molecules of the 3-hydroxy-alkanoate type as a substrate in general, and, in particular, 3-hydroxy-isovalerate, the latter differing from mevalonate-diphosphate not only in that size (MW of 118 versus 308 for mevalonate diphosphate), but also by the electrical charges of the diphosphate group present on the natural substrate, mavelonate-diphosphate.

In a particular way, a cofactor is added to the reaction, in order to carry out spherical or electronic complementation in the catalytic channel. The cofactor is advantageously chosen from the pyrophosphate ion, methyl diphosphate, ethyl diphosphate, or propyl diphosphate. More generally, the cofactor is a compound that contains the phosphoanhydride motif, with the general formula R-O-PO2H-O-PO3H2, in which R is notably a hydrogen atom, an alcoholic residue, linear or branched or cyclic, preferably from 1 to 10 or 1 to 5 carbon atoms, or any other monovalent organic radical. The analogous motifs, corresponding to methylene diphosphonate monoesters, with the general formula R-O-PO2H-CH2-PO3H2, in which the phosphoanhydride is replaced by a methylene bridge that has the advantage of not being hydrolyzed, make also part of the invention.

In a preferred mode, the conversion is carried out in the presence of a cosubstrate, said cosubstrate being, preferably, 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 cosubstrate is generally present in the host. However, in another particular way, a cosubstrate can be added to the reaction, preferably chosen from ATP, rNTP, dNTP, a mixture of several rNTPs or dNTPs, a polyphosphate, and, preferably, pyrophosphate, or a compound containing a phosphoanhydride (corresponding to the general formula X-PO3H2 in figure 2).

In a particular mode of application of the invention, a microorganism that produces decarboxylase is used. In a preferred embodiment, the microorganism is recombinant in the sense that it produces a decarboxylase that is heterologous to the production host. The process can thus be carried out directly in the culture medium, without the need to separate or purify the enzyme system. In a particularly advantageous way, a microorganism is used that has the natural or artificial property of producing one or more 3-hydroxyalkanoates endogenously, and expressing or overexpressing, on the other hand, a decarboxylase, natural or modified, the in order to produce terminal alkenes directly from a carbon source present in solution.

The microorganisms used in the invention can be prokaryotes or eukaryotes, and notably bacteria, yeast, plant cells, mushrooms and molds, animal cells. In a particular way, the microorganisms are bacteria, notably of the Alcaligenes eutrophus or Bacillus megaterium strain.

In another particular way, the microorganisms are recombinant bacteria of the Escherichia coli strain having been modified to endogenously produce one or more 3-hydroxyalkanoates and converting these into terminal alkenes.

In another particular way, microorganisms are recombinant yeasts, producing 3-hydroxyalkanoates and converting these into terminal alkenes.

In another particular way, a microorganism producing one or more 3-hydroxyalkanoates is used, on the one hand, and a decarboxylase, possibly expressed by a second microorganism, on the other hand. Eventually, the two organisms are cultivated and used concomitantly in the process, according to the invention.

In another particular way, whole plants or animals, eventually modified by transgenesis, are used to produce terminal alkenes from 3-hydroxyalkanoates, whether they are produced endogenously or supplied exogenously.

In another particular mode of use, a photosynthetic microorganism is used that has the natural or artificial property of producing one or more 3-hydroxyalkanoates, endogenously, and overexpressing, on the other hand, a decarboxylase, natural or modified, in order to produce terminal alkenes directly from CO2 present in solution. Preferably, the microorganism is a photosynthetic bacterium, or microalgae

The organisms described above represent other objects of the present invention, as well as their use for the production of terminal alkene compounds.

As will be described below, the process of the invention can be carried out in microaerophilia.

On the other hand, in a preferred embodiment, the process is carried out in the presence of a gas collection system for the terminal alkenes, degassing the reaction.

By decarboxylase is meant any enzyme capable of converting a 3-hydroxyalkanoate with n number of carbon atoms into a terminal alkene compound with n-1 number of carbon atoms. As illustrated in figure 2, the process of the invention is preferably carried out via a 3-phospho-hydroxy-alkanoate reaction intermediate, and the enzyme used advantageously presents a decarboxylase activity and a phosphorylase activity.

In a particular mode of use, decarboxylase is a representative of the phylogenetic superfamily of mevalonate diphosphate (MDP) decarboxylase (enzyme nomenclature EC 4.1.133), that is, an enzyme, natural or artificial, specified by a gene native or synthetic, eventually capable of catalyzing the reaction shown in figure 2.

MDP decarboxylase is an enzyme involved in the biosynthesis of cholesterol. This enzyme was isolated from different organisms, such as animals, mushrooms, yeast and certain bacteria. It could also be expressed by certain plants (Lalitha et al., 1985). Numerous genes that specify this enzyme have been cloned and sequenced. These enzymes are generally composed of 300 to 400 amino acids and involve ATP as a cosubstrate, which is transformed during the reaction into ADP and inorganic phosphate. The phosphate group is transferred from the ATP molecule to the tertiary alcohol of mevalonate diphosphate, releasing ADP. The phosphonylated reaction intermediate on the 3-hydroxyl group then undergoes elimination of the phosphate group, releasing isopentenyl-pyrophosphate in the physiological case (figure 2).

The three-dimensional structure of several enzymes in this family has been solved. Relatively little work has been done to date on the enzymes of this family, which have only been studied within the scope of the precise description of the cholesterol biosynthesis pathway. On the contrary, no study has yet been done to divert this enzyme from its natural function and make it an industrial catalyst.

Several examples of MDP decarboxylases from different organisms are given in the 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 chosen from SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence that shows at least 15% sequence homology with one of these and retains a decarboxylase activity. Preferred enzymes advantageously comprise at least 50% sequence homology, preferably at least 80%, more preferably at least 85%, even more preferably, preferably at least 80%, more preferably at least 85%, even more preferably at least least 90, 95, 96, 97, 98 or 99% sequence 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 degree of sequence homology can be determined by different methods and by means of programs known to the person skilled in the art, such as, for example, according to the CLUSTAL method or BLAST programs or derivatives, or using a sequence comparison, such as that of Needleman et Wunsch (J. Mol. Biol. 1970 48: 443) or that of Smith et Waterman (J. Mol. Biol. 1981 147: 195).

A preferred decarboxylase of the invention is represented by the enzyme of the sequence SEQ ID NO: 6, as well as any enzyme that has significant sequence homology thereto. Preferred enzymes advantageously comprise 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. This enzyme was cloned from Picrophilus tomidus and produced recombinantly within the scope of the present invention. As illustrated in the examples, this enzyme is particularly effective for producing terminal alkene compounds according to the present invention. This enzyme also represents an object of the present invention, as does its preparation and use as a catalyst. Notably, an object of the invention resides in the use of a decarboxylase enzyme, comprising all or part of SEQ ID NO: 6 or an enzyme that presents a significant sequence homology and, preferably, of at least 15% with SEQ ID NO: : 6, for the production of terminal alkene compounds. By significant sequence homology, we mean a detectable sequence homology, using the aforementioned algorithms, and, preferably, a sequence homology greater than 15%. The organisms phylogenetically closest to Picrophilus tornidus, such as Ferroplasma acidamanus, Thermoplasma acidophilum, Thermoplasma volcanium and Picrophilus oshimae are capable of producing the MDP decarboxylases closest to that of SEQ ID NO 6. Sequence homology to SEQ ID NO 6 of MDP decarboxylase from Thermoplasma acidophilum (AC number Q9HIN1) is thus 38%; that of Thermoplasma volcanium (Q97BY2) is approximately 42% homologous to SEQ ID NO 6. The use of these MDP decarboxylases is particularly considered in the present invention.

Other decarboxylase-type enzymes, natural or synthetic, can be selected for their ability to produce terminal alkenes, according to the invention. Thus, a selection test involves contacting the purified enzyme, or a microorganism that produces the enzyme, as a substrate for the reaction and measuring the production of the terminal alkene compound. Examples of these tests are provided in the experimental part, which presents tests on more than 60 different enzymes.

The enzyme used can be any natural decarboxylase or one produced or optimized artificially. In particular, a decarboxylase is advantageously used which has optimized activity against one or more 3-hydroxyalkanoates.

The enzyme can be produced or selected, from a reference decarboxylase (natural or already symmetric or optimized), by protein engineering techniques, such as random mutagenesis, massive mutagenesis, directed mutagenesis, DNA " shuffling", synthetic "shuffling", in vivo evolution, or complete gene synthesis.

In this regard, an object of the invention also relates to a process for preparing an enzyme that has decarboxylase activity towards a 3-hydroxy-alkanoate substrate, the process comprising a step of treating an enzyme source and selecting of an enzyme that has increased properties compared to the untreated enzyme.

The enzyme used in the invention can therefore be natural or synthetic, and produced chemically, biologically, or genetically. It can also be chemically modified, for example, to improve its activity, resistance, specificity, purification, or to immobilize it on support.

The invention is characterized by the use of a decarboxylase, in particular a natural or modified MDP decarboxylase, to convert 3-hydroxyalkanoates into terminal alkenes.

The natural substrate of MDP decarboxylase is mevalonate diphosphate, which is not included in the definition of 3-hydroxyalkanoates.

The generic reaction carried out by MDP carboxylase, using different 3-hydroxy-alkanoates, is represented in figure 2B. It is assumed 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 transform a 3-hydroxyalkanoate into a terminal alkene. To do this, the enzyme preparation is incubated in the presence of a substrate under physicochemical conditions, allowing the enzyme to be active and incubation is allowed to occur for a sufficient time. At the end of the incubation, the presence of the terminal alkene is eventually measured, using any detection system known to the technician, such as gas chromatography or colorimetric tests, allowing the appearance of the Alkene product, free phosphate, to be measured, or even allowing the measurement of the disappearance of the substrate 3-hydroxyalkanoate or ATP.

In a preferred embodiment, cofactors are added in order to imitate the natural reaction as much as possible. In fact, 3-hydroxyalkanoates have a structure generally corresponding to an MDP fragment, and an important space in the catalytic channel is left empty during the enzyme/substrate association. The purpose of filling this space with a cofactor that replaces the missing part of the substrate is to imitate the MDP molecule as closely as possible. Since it is not modified by the reaction, the cofactor must, therefore, be added to the reaction only in catalytic quantities. In the case where the reaction substrate is 3-hydroxypropionate, the complementary cofactor will be propyl diphosphate. In the case where the substrate is 3-hydroxy-butyrate or 3-hydroxy-3-methyl-butyrate, the complementary cofactor will be ethyl diphosphate. In the case where the substrate is 3-hydroxy-valerate or 3-hydroxy-3-methyl-valerate, the complementary cofactor will be methyl-diphosphate. These different molecules are shown in figure 5. Incidentally, it may happen that the complementary cofactor of a reaction has a positive effect on the reaction of another substrate. Generally, the cofactor may be no matter which molecule, comprising a phosphoanhydride and therefore having the general formula R-PO2H-O-PO3H2, in which R is notably H, an alcoholic, linear, branched or cyclic residue, or any other monovalent organic radical. Analogous motifs corresponding to methylene diphosphanate monoesters, with the general formula R-O-PO2H-CH2-PO3H2, in which the phosphoanhydride is replaced by a methylene bridge that has the advantage of not being hydrolyzed, are also part of the invention.

More generally, cofactors can be monophosphate analogues, even without phosphate, preceding molecules, or any molecule capable of improving the reaction yield, carrying out steric or electronic complementation in the catalytic site of the enzyme.

In a particular embodiment, a cosubstrate is added to the reaction. This cosubstrate can be either ATP, that is, the natural cosubstrate of MDP decarboxylase, or any dNTP (ribonucleoside triphosphate) or dNTP (deoxyribonucleoside triphosphate) or any mixture of rNTP or NTP, or even pyrophosphate, or another polyphosphate, or also any molecule containing a phosphoanhydride group (X-PO3H2 in figure 2).

In a preferred embodiment, an enzyme is used to transform a 3-hydroxyalkanoate into a terminal alkene that has a sequence homology of at least 15%, and preferably at least 30%, 50%, and even more preferably at least 80, 90, 95, 96, 97, 98 or 99% with a natural enzyme that exhibits decarboxylase activity and, in particular, with one of the enzymes corresponding to the SEQ ID sequences: 1 to 16. Notably, the enzyme may have been engineered from one of the enzymes SEQ ID: 1 to 16, or from any other decarboxylase identified from other sources. This enzyme may have lost its MDP decarboxylase activity notably through gene engineering in the laboratory, but also through natural evolution (one can then speak of a trace of MDP decarboxylase) and retained or increased its activity on one or more molecules of type 3- hydroxyalkanoate. The generation of variants of these enzymes, more reactive towards these substrates, makes it possible to improve the reaction yield, in accordance with the invention. Thus, the reactivity of wild-type MDP decarboxylase towards 3-hydroxyalkanoates is not necessarily optimal. All approaches known to the person skilled in the art to realize and select these variants, such as random mutagenesis, directed mutagenesis, massive mutagenesis, gene shuffling (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 creating a synthetic gene that specifies a new enzyme for the purpose of converting a 3-hydroxy-alkanoate into a terminal alkene, using or not the known data of MDP decarboxylase. to conceive it.

Another object of the invention resides in an isolated or purified enzyme having a decarboxylase activity and comprising all or part of the sequence SEQ ID NO: 6.

Another object of the invention relates to the use of an enzyme that has a decarboxylase activity and that comprises all or part of the sequence SEQ ID NO: 6, or of an enzyme that presents a sequence homology as described above, for produce a terminal alkene. In a variant, the sequence may further comprise additional residues, such as, for example, an N-terminal Histidine tag.

Another object of the invention relates to a process for producing an enzyme that has a decarboxylase activity and comprising all or part of the sequence SEQ ID NO: 6, or an enzyme that presents a sequence homology as described above , the process comprising culturing a microorganism comprising a recombinant nucleic acid encoding that sequence under conditions that allow expression of that sequence. In this context, the present invention describes, in addition to the native nucleic acid (SEQ ID NO: 19), a nucleic acid that presents a sequence optimized by the expression of the enzyme SEQ ID NO: 6 in bacteria, notably in E.coli (SEQ ID NO : 17). This nucleic acid, in the same way as any optimized nucleic acid (that is, allowing an improved expression of at least 30% in relation to the wild-type sequence) represents an object of the present application.

Another object of the invention relates to a microorganism that comprises a recombinant nucleic acid encoding an enzyme that has a decarboxylase activity and comprises all or part of the sequence SEQ ID NO: 6, or an enzyme that presents a sequence homology such as described above. The microorganism is preferably a bacterium, a yeast or a mushroom. The invention also relates to any non-human plant or animal, which comprises a recombinant nucleic acid encoding a decarboxylase, according to the invention.

In one embodiment, MDO decarboxylase is used in a purified form to transform 3-hydroxyalkanoates into terminal alkenes. However, the costs associated with this process are high, with the production and purification costs of the enzyme and substrates being important.

In another embodiment, MDP decarboxylase is present in the reaction in the form of a non-purified extract, or even in the form of non-lysed bacteria, in order to save the costs of purifying the protein. However, the costs associated with this process are still quite high due to the production and purification costs of the substrates.

In another embodiment of the invention, the process uses a living organism that produces the enzyme that allows the conversion to be carried out. The invention is thus also characterized by the modification by genetic engineering of a bacterial strain producing one or more 3-hydroxyalkanoate (for example, Alcaligenes eutrophus or Bacillus megaterium or even a strain of E. coli modified in the laboratory, so as to produce this product(s)), so that this bacterial strain overexpresses decarboxylase, this enzyme preferably originating from an organism other than the host microorganism, and can directly generate one or more alkene(s) terminal(s). Genetic modification may consist of chromosomal integration of a decarboxylase gene, expression of the enzyme by a plasmid containing a promoter upstream of the enzyme coding sequence, the promoter and coding sequence preferably originating from different organisms, or by any other means known to the technician. Alternately, other bacteria or yeast may present particular advantages and be retained. Thus, a yeast such as Saccharomyces cerevisiae, an extremophile bacterium such as Thermus thermophilus, or anaerobic bacteria from the Clostridiae family, e.g. microalgae, photosynthetic bacteria can be used. In order to optimally produce the 3-hydroxyalkanoate(s), which will then be converted into terminal alkenes, the strains may also have been modified by genetic engineering, i.e., by in vitro recombination or by in vitro directed evolution. alive.

In one embodiment, the process of the invention is characterized by transforming a carbon source, such as glucose, into 3-hydroxyalkanoate, then transforming this primary product into a secondary product, that is, into a terminal alkene. The different stages of this process are explained in figure 6.

In a particular embodiment, the invention is characterized by the transformation of polyhydroxyalkanoates into 3-hydroxyalkanoate, using an enzyme or an adapted physicochemical process, then by transforming this primary product into a secondary product, that is, into an alkene. terminal. Eventually, polyhydroxyalkanoate was produced by a plant having modified its metabolic pathways in order to produce high yields of polyhydroxyalkanoate.

In a particular embodiment, the invention consists of the integrated process of producing products from atmospheric CO2 or artificially added to the culture medium. The process of the invention is carried out in an organism capable of carrying out photosynthesis, such as microalgae, for example.

In this embodiment, the process of the invention is further characterized by the method of recovering the products, which degas the culture. The short terminal alkenes, and notably ethylene, propylene, butene isomers, have, in effect, a gaseous state at room temperature and atmospheric pressure. The process of the invention does not, therefore, require extraction of the product from the liquid culture medium, a step that always constitutes an important source of costs at an industrial level. The evacuation and storage of gaseous hydrocarbons, and their eventual subsequent physical separation and chemical conversion, can be operated according to any process known to the person skilled in the art.

In a particular embodiment, the invention also comprises the detection of alkene (propylene, ethylene and iso-butylene in particular) present in the gas phase of the process. The presence of targeted compounds in an environment of air or another gas, even in small quantities, can be determined using various technologies, notably using gas chromatography systems and infrared detection, by flame ionization, or by coupling with a mass spectrometer.

In a particular embodiment, the terminal alkenes obtained are condensed, then eventually reduced, using techniques known to the art, in order to produce longer chain alkenes, or longer chain alkanes. In particular, iso-butylene can be used to synthesize iso-octane: the catalytic processes to conduct this reaction well have been widely described.

In a particular embodiment, the process involves culturing the microorganisms under usual culture conditions (30 to 37 °C under 1 atmosphere, in a fermenter that allows the anaerobic growth of bacteria) or unusual (more high to match the culture conditions of a thermophilic organism, for example).

In a particular embodiment, the microorganisms are cultivated in microaerophilia, the amount of air injected being limiting, in order to minimize the concentration of residual dioxygen in the gaseous effluents loaded with alkene hydrocarbons.

Other aspects and advantages of the invention will be described in the following examples, which should be considered as illustrative and not limiting. Figure Captions Figure 1: 3-hydroxy-propionate motif Figure 2: decarboxylation of mevalonate di-phosphate by MDP-decarboxylase - generic activity. Figure 3: Examples of 3-hydroxyalkanoates. Figure 4: Use of MDP decarboxylase to generate terminal alkenes. Figure 5: Cofactors usable for the reaction, in order to carry out structural complementation at the catalytic site. Figure 6: Integrated process for producing an alkene from glucose. Figure 7: Chromatogram performed on enzymatic reactions conducted under conditions no. 1 of example 4. Figure 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 Unabsorbed fraction on column 5. Wash fraction from column 6. Purified enzyme, Mw = 36.8 kDa. Figure 9: Chromatogram of an analysis of the HIV to IBN conversion reaction by CPG/MS. 1 and 2: negative controls corresponding to the background noise observed without enzyme. 3 and 4: reactions in the presence of the enzyme SEQ ID NO: 6. Figure 10: ratio of IBN production in the presence and absence of ATP. Figure 11: ratio of IBN production in the presence and absence of Mg2+. Figure 12: Enzymatic activity as a function of temperature. Ratio: amount of IBN formed in the presence of enzyme in relation to background noise. Figure 13: IBN production as a function of HIV substrate concentration. Figure 14: Measurement of the optimized reaction and comparison to background noise. Measured by gas chromatography coupled to a flame ionization detector. Figure 15: improvement of the expression level by optimizing the nucleotide sequence encoding SEQ ID NO: 6. Lane M: molecular weight markers. Lanes 1,2,3: native nucleotide sequence (1) Cell lysate, soluble fraction deposited on the purification column (2) Lysate fraction not retained by the purification column (3) Purified fraction: 10 μg purified enzyme Lanes 4, 5 , 6: Optimized nucleotide sequence (4) Cell lysate, soluble fraction deposited on the purification column (5) Lysate fraction not retained by the purification column (6) Purified fraction: 10 μg purified enzyme

Example 1: cloning and expression of several MDP decarboxylases.

The gene coding for MDP decarboxylase from Saccharomyces cerevisiae is generated by synthesis from overlapping oligonucleotides, then inserted into a pET plasmid (Novagen) allowing bacterial expression. This plasmid is then transformed by electroporation into BL21 strain bacteria (Invitrogen). The bacteria are measured in a Petri dish containing ampicillin, and placed to incubate at 37 °C. The following day, a bacterial colony is selected at random and used to inseminate a 50 ml culture of LB medium containing ampicillin. The culture is incubated for 24 hours under shaking. At the end of 24 hours, the culture is centrifuged, the bacteria are lysed by sonication, and a total protein extract is performed. An aliquot of the extract is deposited on an electrophoresis gel, at the same time as a protein extract from bacteria of the same strain, but not transformed, and a molecular weight marker. A single band at approximately 30 kD is observed on the track corresponding to the transformed bacterial strain, corresponding to the expected size for the protein, this band does not exist on the track corresponding to non-transformed bacteria.

Example 2: measurement of the activity of protein extracts for 3-hydroxy-3-methyl-butyrate

3-hydroxy-3-methyl-butyrate (commanded in Sigma, reference 55453 as p-hydroxy-isovaleric acid) and suspended at a concentration of 10 g/L/mevalonate-diphosphate is synthesized from mevalonolactone and others reagents (Sigma), according to the classical technique, and is suspended again at a concentration of 10 g/L.

Prepare six chromatography bottles. In all flasks, 50 μl of buffer containing 50 mM Bistris/HCI, 1 mM dithiothreitol, 10 mM MgCI2, 5mM ATP are introduced.

In flasks 1 and 4, 5 μl of water is not added (no substrate).

In bottles 2 and 5, 5 μL of the di-phosphate mevalonate preparation (positive control) are added. In bottles 3 and 6, 5 μL of the 3-hydroxy-3-methyl-butyrate (HIV) preparation are added.

In flasks 1, 2 and 3, 5 μL of water (no enzyme) are then added

In bottles 4, 5 and 6, 5 μl of the enzyme preparation described in example 1 is added.

The vials are sealed using a stopper with septum and clamping forceps. All vials are incubated at 37°C for 4 hours to 3 days.

At the end of this incubation, a sample of the gas present in each bottle is taken, using a syringe with gas, and the concentration of CO2 in these samples is measured, using gas chromatography. It can be seen that the CO2 concentration is very significant in flask 5, and that a weaker CO2 concentration is also observed in flask 6, which reflects a significant reaction of the enzymatic preparation on 3-hydroxy- 15 3-methyl - butyrate.

The presence of iso-butylene is then measured in the gas sample corresponding to bottle 6, using a gas chromatography system coupled to an infrared or flame ionization detector.

Example 3: optimization of reaction conditions using a 20 cofactor

The same reaction is carried out as that described in bottle 6 of the previous example, but using, in one of the samples, ethyl diphosphate as a cofactor, synthesized in the fashion. In this example, three bottles are available. The first contains the buffers, ATP, and enzyme extract in the quantities described in the previous example. The second contains the same elements, such as, in addition, 3-hydroxy-3-methyl-butyrate in the quantities described in the preceding example. The third vial contains, in addition to 3-hydroxy-3-methyl-butyrate, 10 μL of ethyl diphosphate at 10 mg/L.

The formation of iso-butylene is measured as in the previous example, using gas chromatography equipment coupled to infrared detection or flame ionization. It can be seen that, when ethyl diphosphate is present, the amount of iso-butylene produced per unit of time is clearly higher.

Example 4: screening a collection of enzymes

A collection of sixty-three genes coding for enzymes of the MDP decarboxylase family was obtained and tested for their activity on the HIV substrate.

Cloning, bacterial cultures and protein expression

The genes corresponding to the mevalonate diphosphate (MDP) family decarboxylases EC 4.1.1.33 were cloned into the vector pET 25b (Novagen) for genes of eukaryotic origin and PET 22b (Novagen) for genes of prokaryotic origin with 6 His residues added in N- terminal, exactly after the initiator methionine codon. E.Coli BL21(DE3) competent cells were transformed with the vectors by heat shock. The cells were cultured in TB medium containing 0.5 M sorbitol, 5 mM betaine, 100 μg/ml ampicillin at 30 °C, under shaking (160 rpm) until an OD (600 nm) 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 hours). Cells were collected by centrifugation at 4 °C, 10000 rpm, 20 minutes and the residues were frozen at -80 °C.

Cell lysis

1.6 g of cells were thawed on ice; the cells were resuspended in 5 ml of 50 mM Na2HPO4, containing 300 mM NaCl, 5 mM MgCl2, 1 mM DTT ph 8. 20 μl of lysonase (Novagen) was added, then an incubation for 10 minutes at room temperature was done. , and followed by a 20-minute return on ice. Bacterial cell lysis was completed by sonication in a water bath with ultrasound 3 times for 5 minutes at 0°C with homogenization of the samples between pulses. The bacterial extracts were then clarified by centrifugation at 4 °C, at 10,000 rpm, for 20 minutes.

Purification and concentration of proteins (PROTINO kit)

Clarified bacterial lysates were deposited on the PROTINO-1000 Ni-IDA column (Macherey Nagel) allowing the absorption of proteins carrying a tag of 6 consecutive histidines (His tag). Columns were washed and enzymes of interest were purified with 4 ml of 50 mM Na2HPO4 containing 300 mM NaCl, 5 mM Mg CI2, 1 mM DTT, 250 mM imidazole pH8. The purifiers were then concentrated in Amicon Ultra-4 10 kDa cells (Millipore) to a final volume of 250 μl. The amount of enzymes was determined by the Bradford method.

Enzyme reactions

The enzymatic activity sought (conversion of 3-methyl 3-hydroxy butyrate or 3-hydroxy isovalerate or even HIV) was tested in two experimental conditions differing by the buffer and pH of the reaction. Experimental Conditions No. 1 100 mM citrate 10 mM ATP 20 mM KCI 200 mM HIV Final pH was adjusted to 5.5. Experimental conditions no. 2 100 mMTris-HCIpH 7.0 10mM MgCI2 10 mM ATP 20 MM KCI 200 mM HIV Final pH was adjusted to 7.0

The enzyme was then added to the reaction mixture. The protein production yield being variable, the amount of enzyme added varied from one sample to another between 0.01 and 1 mg/ml. Control reactions, without enzyme, were carried out in parallel.

The reactions (1 mL) were placed in 2 mL vials (Interchim), anchored with Teflon/silica/Teflon septa (Interchim). Reactions were incubated at 37°C without shaking for 72 hours.

Analysis of reactions

The gas present above the reactions was removed with a syringe equipped with an anti-retention system. The gas was then analyzed by gas chromatography (CPG) coupled to a mass spectrometer (SM). The device was previously calibrated using a range of iso-butylene concentrations. Column: BPX5 (SGE) CPG/SM: MSD 5973 (HP) For each chromatogram performed, three main peaks are obtained, the first corresponding to air, the second to water, and the third to isobutene. Of the sixty-three enzymes produced and tested, eleven potential candidates were identified in the first screening. Certain of these candidates were identified by an arrow in Figure 7. Their identity is presented in the list below, and their sequences are presented in SEQ NO 6 to 16 (his-tag not represented). Candidate 1: SEQ IP NO: 7 Genebank accession number: CAI97800.1 Swissprot/TrRMBL accession number: Q1GAB2 Microorganisms: Lactobacillus delbrueckii subsp. Bulgaricus (strain ATCC 11842/ DSM 20081) Candidate 2: SEQ ID NO: 8 Genebank accession number: CAJ51653 Swissprot/TrRMBL accession number: Q18K00 Microorganisms: Haloquadratum walsbyi PSM 16790 Candidate 3: SEQ IP NO: 9 Accession number Genebank: ABD99494.1 Swissprot/TrRMBL accession number: Q1WI41 Microorganisms: Lactobacillus salivarius subsp. Salivaryus (strain UCC 118) Candidate 4: SEQ IP NO: 10 Genebank accession number: ABJ57000.1 Swissprot/TrRMBL accession number: Q04EX2 Microorganisms: Oenococcus oeni (strain BAA-331 /PSU-1) Candidate 5: SEQ IP NO: 11 Genebank accession number: ABJ67984.1 Swissprot/TrRMBL accession number: Q03FN8 Microorganisms: Pediococcus pentosaceus ATCC 25745 Candidate 6: SEQ IP NO: 12 Genebank accession number: ABV09606.1 Swissprot/TrRMBL accession number : A8AUU9 Microorganisms: Streptococcus gordonii (strain Challis I ATCC 35105 / CH1 /DL1 /V288) Candidate 7: SEQ ID NO: 13 Genebank accession number: ABQ14154.1 Swissprot/TrRMBL accession number: A5EVP2 Microorganisms: Dichelonacter nodosus VCS1703A Candidate 8 : SEQ ID NO: 14 Genebank accession number: EDT95457.1 Swissprot/TrRMBL accession number: B2DRT0 Microorganisms: Streptococcus pneumoniae CDC0288-04 Candidate 9: SEQ ID NO: 15 Genebank accession number: AAT86835 Swissprot/accession number TrRMBL: Q5XCM8 Microorganisms: Streptococcus pyogenes serotype M6 (ATCC BAA-9461 MGAS 10394) Candidate 10: SEQ ID NO: 6 Genebank accession number: AAT43941 Swissprot/TrRMBL accession number: Q6KZB1 Microorganisms: Picrophilus torridus DSM 9790 Candidate 11: SEQ ID NO: 16 Genebank accession number: AAV43007.1 Swissprot/TrRMBL accession number: Q5FJW7 Microorganisms: Lactobacillus acidophillus NCFM

The highest production of IBN was observed with candidate 10, that is, with the purified decarboxylase enzyme of SEQ ID NO: 6 from Picrophilus torridus. This enzyme was retained to be characterized in detail.

Example 5: characterization of the enzyme of SEQ IP NO: 6

The recombinant enzyme was purified as described in example 4. The results, presented in figure 8, show that the enzyme purity in the final protein sample is estimated at 90%.

The activity of the isolated enzyme was confirmed. The reaction was carried out under the following conditions: 100 mM Tris-HCI pH 7.0 19 mM MgCI2 WmMATP 20 mM KCI 250 mM HIV Final pH was adjusted to 6.0 3 mg/ml enzyme.

After incubation at 30 °C for 72 hours, the signal is measured by CPG/SM. The results are presented in figure 9. In the presence of enzyme, IBN production is increased by a factor of approximately 2.3, compared to background noise. The background noise observed in this case is homogeneous with the organic chemistry literature, which mentions that 3-hydroxy-isovaleric acid in aqueous solution and at a temperature of the order of 100 °C slowly decarboxylates into tert-butanol, which partially converts by dehydration into iso-butylene, according to an equilibrium favorable to the formation of tert-butanol (Pressman et Luca, J. Am. Chem. Soc. 1940). Effect of ATP cosubstrate Test conditions 100 mM citrate 50 mMKCI 10 mM MgCI2 200 mM HIV (to be specified) 1 mg/ml purified enzyme pH 5.5 Incubation at 30 °C for 72 hours

The results are presented in figure 10 and show that enzymatic activity is only observed in the presence of ATP cosubstrate. Other molecules, and notably molecules containing a phospho-anhydride bond, are capable of constituting effective cosubstrates for the enzyme. Effect of cofactor Mg2* Test conditions 100 mM citrate pH5.5 50 mM KCI WmMATP 200 mM HIV (to be specified) pH 5.5 1 mg/ml purified enzyme

The results are presented in figure 10 and show that the enzyme activity is improved in the presence of Mg2+ ions. Other ions and notably other divalent ions are capable of being used as a cofactor in place of or in addition to Mg2+ ions. Enzymatic activity as a function of temperature Test conditions 100 mM buffer 50 mM KCI WmMATP 200 mM HIV (to be specified) 1 mg/ml purified enzyme Incubation for 72 hours at a variable temperature.

The results presented in figure 12 show that the enzyme is moderately thermoactive and has an optimum temperature of approximately 50 °C. Activity as a function of pH Test conditions 100 mM buffer 50 mM KCI WmMATP 200 mM HIV (to be specified) 1 mg/ml purified enzyme Incubation at 30 °C for 72 hours Optimal conditions were obtained at pH 5.5, in 100 mM citrate

Enzyme parameters

A substrate strip was performed under the conditions previously described, with an incubation at 50 °C. The km of the enzyme was estimated to be 30 mM HIV.

Optimization of reaction conditions

The reaction conditions were researched. The following conditions were kept: 100 mM citrate 50 mM KCI 10 mM ATP 200 mM HIV 1 mg/ml enzyme Incubation at 50 °C for 48 hours As shown in figure 14, the signal to background noise ratio is approximately 100.

Example 6: optimization of the expression level of MDP decarboxylase from P.torridus in E.coli

The initial expression level in E. coli BL21 was low, as the band was hardly visible in SDS-PASGE before purification. The Codon Optimization Index (CAI) of the native sequence for an expression in E.coli was measured using the "Optimizer" program available at http://qenomes.urv.es/OPTIMIZER/ and taking inspiration from Sharp's method et Li (1987). The value obtained was only 0.23, reflecting the limited expression level of the studied protein in E.coli.

A sequence coding for an identical protein, but having codons more adapted to expression in E.coli, was generated. The CAI of this sequence, 0.77, was closer to the optimum of 1.

The native sequence and the optimized sequence are presented in SEQ ID NO: 17 (optimized sequence of MDP decarboxylase from P. torridus (AAT43941), including the HIS Tag) and SEQ ID NO: 19 (native sequence of MDP decarboxylase from P. . torridus (AAT43941) including His Tag).

The optimized sequence was synthesized by concatenation of oligonucleotides, and cloned into a pET25 expression vector. After transformation of the vector into the E.coli BL21 (DE3) strain and induction, using the previously described protocol, the proteins were produced, purified and analyzed on gel in a similar way to that described previously. The same protocol was performed with the native sequence for 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 presented in figure 15 show that the protein corresponding to the optimized gene is clearly visible on gel in the unpurified cell lysate (lane 4), which translates into a very significant gain in expression. The purity of the protein after the purification step is also more important in the case of the optimized gene.

Activity was measured on crude lysate. Indeed, this activity was undetectable on the crude lysate obtained with the native nucleic sequence. The expression of the protein was improved in such a way that the crude lysate obtained from the improved nucleic sequence (optimized clone 224) then presents this activity. The reaction medium used in this test was as follows: Reaction Medium: Incubation 2 days at 50 °C Results Condition #1: lysate from optimized clone 224 Condition #2: lysate from clone GB6 (empty pET plasmid)

Example 7: process of synthesis of iso-butylene from 3-hydroxy-3-methyl-butyrate and conversion to iso-octane

A reaction identical to that in flask 3 of example 3 is carried out in a volume of 1 liter, arranged in a fermenter that has a gas extraction system. The presence of the recombinant enzyme induces the conversion of 3-hydroxy-3-methyl-butyrate into iso-butylene, which naturally degass 15 and is recovered by a gas extraction system located in the upper part of the fermenter. Iso-butylene is then used to generate iso-octene by addition catalyzed by Amberlysrt 35 wet or 36 ewet resin (Rohm and Haas). Iso-octene is finally reduced by catalytic hydrogenation to iso-octane.

Example 8: engineering the enzyme to improve its effectiveness for substrates

Random mutagenesis technology is applied to generate a bank containing thousands of mutants, based on the gene described in example 1. This bank of mutants is then integrated into the expression plasmid, and the bank is transformed into competent bacteria of strain BL21. One million bacteria are then isolated and placed in eppendorf tubes containing 500 μl of LB medium supplemented with ampicillin. These samples are incubated on a shaker for 15 hours. The following morning, the amount of iso-butylene produced is measured, using one or another of the experimental protocols presented in the preceding examples.

The clones showing a significantly increased amount of iso-butylene are then validated again, using the same experimental protocol. Once your improvement is validated, the plasmid is extracted from each improved strain and sequenced. The mutations responsible for improving activity are identified and combined on the same plasmid. The plasmid containing the different improvement mutations is, in turn, transformed into competent bacteria, and the same analysis system is performed.

The clone, which combines the different mutations, presenting a significantly higher activity than that which contains only a single improving mutation, is then used as the basis for a new round of mutation/screening, in search of mutants that present an even improved activity.

At the end of this protocol, the clone containing several mutations and having the best activity is selected.

Example 9: ethylene synthesis process from 3-hydroxypropionate.

The gene coding for the enzyme described in example 1 is inserted into a plasmid that allows the expression of recombinant proteins in a strain of E. coli. The plasmid is transformed into bacteria of that strain. The transformation bacteria are 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 induces the conversion of 3-hydroxy-propionate into ethylene, which spontaneously degasses and is recovered by a gas extraction system located in the upper part of the fermenter. The presence of ethylene is then measured in the corresponding gas sample, using a gas chromatography system coupled to an infrared detector, part of the spectrum in which ethylene presents a strong emission.

Example 10: Propylene synthesis process from 3-hydroxybutyrate.

The gene coding for the enzyme described in example 1 or for an enzyme described in example 4 is inserted into a plasmid that allows the expression of recombinant proteins in an E. coli strain. The plasmid is transformed into bacteria of that strain. The transformation bacteria are then incubated in a fermenter in the presence of ethyl diphosphate (10 mg/L) and 3-hydroxypropionate (1 g/L) (Sigma, reference 166898). The presence of the recombinant enzyme induces the conversion of 3-hydroxy-butyrate into propylene, which spontaneously degasses and is recovered by a gas extraction system located in the upper part of the fermenter. The presence of propylene in the corresponding gas sample is then measured using a gas chromatography system coupled to an infrared detector, part of the spectrum in which ethylene presents a strong emission.

Example 11: propylene synthesis process from glucose

The gene coding for the enzyme described in example 1 or for an enzyme described in example 4 is inserted into a plasmid that allows the expression of recombinant proteins in the bacterium Alcaligenes eutrophus. The plasmid is transformed into bacteria of that strain.

The bacteria are then incubated in a fermenter in the presence of ethyl diphosphate and under microaerophilic conditions, then subjected to thermal stress which results in the production of large quantities of 3-hydroxy-butyrate in propylene. The propylene naturally degasses and is recovered by a gas extraction system located in the upper part of the fermenter.

Example 12: propylene synthesis process from glucose.

The present example describes a process very close to that of example 11. The main difference consists in the use of a modified strain of E.coli, in order to produce 3-hydroxy-butyrate and not a natural strain, such as Alcaligenes eutrophus. This strain was obtained by engineering metabolic pathways in order to achieve the accumulation of 3-hydroxybutyrate. The addition of an MDP decarboxylase, as described in example 1 or example 4, allows 3-hydroxy-butyrate to be converted into propylene.

Example 13: iso-butylene synthesis process from glucose

The gene coding for the enzyme described in example 1 is inserted into a plasmid that allows the expression of recombinant proteins in a strain of E. coli, having, on the other hand, undergone metabolic changes, in order to synthesize endogenously 3- hydroxy-3-methyl-butyrate. The bacteria are then incubated in a fermenter in the presence of glucose and under microaerophilic conditions. The presence of the recombinant enzyme induces the simultaneous conversion of 3-hydroxy-3-methyl-butyrate into isobutylene, which naturally degasses and is recovered by a gas extraction system located in the upper part of the fermenter.

Claims (17)

1. Method for producing a terminal alkene characterized by comprising a step of converting a 3-hydroxyalkanoate by an enzyme having mevalonate diphosphate decarboxylase (MDP) activity (EC 4.1.1.33), in which the 3-hydroxyalkanoate is a compound of formula: HO-CO-CH2-C(R1)(R2)-OH or O--CO-CH2-C(R1)(R2)-OH in which R1 and R2 are selected, independently of each other, from a hydrogen atom, a methyl group and an ethyl group, and in which the conversion occurs in the presence of a cosubstrate, said cosubstrate being adenosine triphosphate (ATP), a ribonucleoside triphosphate (rNTP), a deoxyribonucleoside triphosphate (dNTP) or a mixture of some of these molecules, a polyphosphate or pyrophosphate. 2. Method according to claim 1, characterized in that the 3-hydroxyalkanoate is 3-hydroxybutyrate and the terminal alkene is propylene. 3. Method according to claim 1, characterized in that the 3-hydroxyalkanoate is 3-hydroxyvalerate and the terminal alkene is 1-butylene. 4. Method according to claim 1, characterized in that the 3-hydroxyalkanoate is 3-hydroxy-3-methylbutyrate and the terminal alkene is isobutylene. 5. Method according to claim 1, characterized in that the 3-hydroxyalkanoate is 3-hydroxy-3-methylvalerate and the terminal alkene is isoamylene. 6. Method according to any one of claims 1 to 5, characterized in that the enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6-16. 7. Method according to any one of claims 1 to 6, characterized in that the enzyme comprises the sequence SEQ ID NO: 6. 8. Method according to claim 1, characterized in that the 3-hydroxyalkanoate is 3-hydroxy-3-methylbutyrate, the enzyme comprises the sequence SEQ ID NO: 6, and the terminal alkene is isobutylene. 9. Method according to any one of claims 1 to 8, characterized in that a cofactor is added to the reaction, and said cofactor has the general formula R-O-PO2H-O-PO3H2 in which R is a hydrogen atom, a group methyl, ethyl or propyl, and may also be any linear, branched or cyclic alkyl group, or any other monovalent organic group. 10. Method according to any one of claims 1 to 8, characterized in that a cofactor is added to the reaction, and said cofactor has the general formula R-O-PO2H-CH2-PO3H2 in which R is a hydrogen atom, a group methyl, ethyl or propyl, and may also be any linear, branched or cyclic alkyl group, or any other monovalent organic group. 11. Method according to any one of claims 1 to 10, characterized in that the conversion step is carried out in vitro, in a cell-free system. 12. Method according to any one of claims 1 to 10, characterized in that the conversion step is carried out in the presence of a microorganism that produces said MDP decarboxylase, wherein the microorganism has the natural or artificial property of endogenously produce one or more 3-hydroxyalkanoates, and also express or overexpress said MDP decarboxylase, in order to produce terminal alkenes directly from a carbon source, and in which the microorganism is a bacterium of the strain Alcaligenes eutrophus or Bacillus megaterium, or a bacterium from an Escherichia coli strain that is recombinant in order to overproduce one or more 3-hydroxyalkanoates as defined in claim 1. 13. Method according to any one of claims 1 to 12, characterized in that it comprises a step of collecting gas from terminal alkenes that degas as a result of the reaction. 14. Method according to any one of claims 1 to 13, characterized in that the method is carried out under microaerophilic conditions. 15. Use of an MDP decarboxylase enzyme (EC 4.1.1.33), or a microorganism that produces an MDP decarboxylase enzyme (EC 4.1.1.33), according to claim 12, characterized in that it serves to produce compounds of terminal alkene of 3-hydroxyalkanoates according to claim 1. 16. Use of an MDP decarboxylase enzyme according to claim 15, characterized in that the enzyme comprises the sequence SEQ ID NO: 6. 17. Composition characterized by comprising a microorganism that produces an MDP decarboxylase enzyme (EC 4.1.1.33), according to claim 12, a suitable culture medium and a 3-hydroxyalkanoate compound, according to claim 1 .