(19)
(11)EP 2 630 236 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
26.06.2019 Bulletin 2019/26

(21)Application number: 11784957.0

(22)Date of filing:  18.10.2011
(51)International Patent Classification (IPC): 
C12N 9/88(2006.01)
C12P 5/02(2006.01)
(86)International application number:
PCT/EP2011/068174
(87)International publication number:
WO 2012/052427 (26.04.2012 Gazette  2012/17)

(54)

PRODUCTION OF ALKENES BY COMBINED ENZYMATIC CONVERSION OF 3-HYDROXYALKANOIC ACIDS

HERSTELLUNG VON ALKENEN MITTELS KOMBINIERTER ENZYMATISCHER UMWANDLUNG VON 3-HYDROXYALKANSÄUREN

PRODUCTION D'ALCÈNES PAR CONVERSION ENZYMATIQUE COMBINÉE D'ACIDES 3-HYDROXYALCANOÏQUES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 19.10.2010 EP 10188001

(43)Date of publication of application:
28.08.2013 Bulletin 2013/35

(73)Proprietors:
  • Global Bioenergies
    91030 Evry Cedex (FR)
  • Scientist of Fortune S.A.
    1940 Luxembourg (LU)

(72)Inventors:
  • MARLIÈRE, Philippe
    2718 Luxembourg (LU)
  • DELCOURT, Marc
    F-75013 Paris (FR)
  • ANISSIMOVA, Maria
    F-91620 Nozay (FR)
  • TALLON, Richard
    F-91400 Orsay (FR)

(74)Representative: Vossius & Partner Patentanwälte Rechtsanwälte mbB 
Siebertstrasse 3
81675 München
81675 München (DE)


(56)References cited: : 
WO-A2-2010/001078
  
     
    Remarks:
    The file contains technical information submitted after the application was filed and not included in this specification
     
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description


    [0001] The present invention relates to a method for generating alkenes through a biological process. More specifically, the invention relates to a method for producing alkenes (for example propylene, ethylene, 1-butylene, isobutylene or isoamylene) from molecules of the 3-hydroxyalkanoate type.

    [0002] 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 production of 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 octane rating of isooctane makes it the best fuel for so-called "gasoline" engines.
    Amylene, hexene and heptene exist in many forms according to the position and configuration of the double bond. These products have real industrial applications but are less important than ethylene, propylene or butenes.
    All these 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.

    [0003] 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 MS et al., Biotech. Bioeng.99 (2008), 919-928). 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.

    [0004] The biological production of alkanes or other hydrocarbon 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.

    [0005] 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 pathways, 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 ST et al., Appl. Environ. Microbiol., 2007, 73:6277).

    [0006] The production of alkenes, in particular terminal alkenes, [ethylene mono- or disubstituted at position 2: H2C=C(R1)(R2)] has apparently been less extensively investigated. The conversion of isovalerate to isobutylene by the yeast Rhodotorula minuta has been described (Fujii T. et al., Appl. Environ. Microbiol., 1988, 54:583), but the efficiency of this reaction, characterized by a very low value of the turnover number (kcat is 1x10-5 sec-1), 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).
    The 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 the production of a two-carbon molecule of ethylene consumes a four- or five-carbon molecule precursor, these pathways appear materially and energetically unfavorable for their industrial application.
    Thus, there is a need for efficient methods for producing alkenes such as ethylene, propylene, 1-butylene, isobutylene, 1-amylene or isoamylene.
    WO2010/001078 describes a process for producing alkenes by enzymatic conversion of 3-hydroxyalkanoic acids with an enzyme having the activity of a decarboxylase. Such a method is advantageous because it helps to avoid the use of petroleum products, to lower the costs of producing plastics and fuels and can have a considerable global environmental impact by allowing carbon to be stored in solid form. Although the method described in WO 2010/001078 allows to produce alkenes by enzymatically converting 3-hydroxyalkanoates, there is still a need for improvements, in particular as regards efficiency of the process so as to make it suitable for industrial purposes. The present application addresses this need.

    [0007] The present invention describes a method for producing alkene compounds starting from a 3-hydroxyalkanoate through a biological process, in particular an enzymatic process, in which two types of enzymes are combined in order to increase the efficiency of the production rate. More specifically, the present invention relates to a method for producing an alkene, characterized in that it comprises the conversion of a 3-hydroxyalkanoate into said alkene by
    1. (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and
    2. (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene by decarboxylation and dephosphorylation.
    The present invention also relates to the use of at least two enzymes, wherein one enzyme is selected from (i) as specified above and the other enzyme is selected from (ii) as specified above or of a microorganism producing said combination of enzymes, for producing an alkene compound from a 3-hydroxyalkanoate.
    The present invention also relates to organisms, preferably microorganisms, which recombinantly produce at least two enzymes, wherein one enzyme is selected from (i) as specified above and the other enzyme is selected from (ii) as specified above.

    [0008] More specifically, the present invention relates to the embodiments as characterized in the claims. Thus, it relates to the following items:
    1. 1. A method for producing an alkene, characterized in that it comprises the conversion of a 3-hydroxyalkanoate into said alkene by
      1. (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and
      2. (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene by decarboxylation and dephosphorylation.
    2. 2. The method of item 1 wherein
      1. (i) the first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate is selected from the group consisting of
        1. (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 1 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 1;
        2. (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 2 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 2;
        3. (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 3 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 3 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 3; and
        4. (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 4 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 4.
    3. 3. The method of item 1 or 2, wherein
      (ii) the second enzyme having an activity of converting said 3-phosphonoxyalkanoate into said alkene selected from the group consisting of
      1. (a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 5 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 5 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 5;
      2. (b) a protein comprising the amino acid sequence as shown in SEQ ID NO: 6 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 6 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 6;
      3. (c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 7 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 7;
      4. (d) a protein comprising the amino acid sequence as shown in SEQ ID NO: 8 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 8 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 8;
      5. (e) a protein comprising the amino acid sequence as shown in SEQ ID NO: 9 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 9 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 9
      6. (f) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 10 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 10;
      7. (g) a protein comprising the amino acid sequence as shown in SEQ ID NO: 11 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 11 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 11;
      8. (h) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 12 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 12;
      9. (i) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 13 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 13;
      10. (j) a protein comprising the amino acid sequence as shown in SEQ ID NO: 14 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 14 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 14; and
      11. (k) a protein comprising the amino acid sequence as shown in SEQ ID NO: 15 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 15 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 15.
    4. 4. The method of any one of item 1 to 3, comprising the step of converting 3-hydroxyproprionate to ethylene.
    5. 5. The method of any one of item 1 to 3, comprising the step of converting 3-hydroxybutyrate to propylene.
    6. 6. The method of any one of item 1 to 3, comprising the step of converting 3-hydroxyvalerate to 1-butylene.
    7. 7. The method of any one of item 1 to 3, comprising the step of converting 3-hydroxy-3-methylbutyrate to isobutylene.
    8. 8. The method of any one of item 1 to 3, comprising the step of converting 3-hydroxy-3-methylvalerate to isoamylene.
    9. 9. The method of any one of the previous item, characterized in that the conversion step is carried out in vitro, in a cell-free system.
    10. 10. The method of any one of item 1 to 8 characterized in that the method is carried out in the presence of a microorganism recombinantly producing both said enzymes as defined in (i) and (ii) of item 1.
    11. 11. The method of any one of item 1 to 8, characterized by the use of a plant, animal cell, plant cell or microorganism recombinantly producing both said enzymes as defined in (i) and (ii) of item 1.
    12. 12. The method according to any one of the previous item, comprising a step of collecting gaseous alkenes degassing out of the reaction.
    13. 13. A plant, animal cell, plant cell or a microorganism which recombinantly produces at least two enzymes, wherein one enzyme is selected from (i) and the other enzyme is selected from (ii), wherein (i) and (ii) are as follows:
      1. (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and
      2. (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene.
    14. 14. A composition comprising the microorganism of item 13, a suitable culture medium and a 3-hydroxyalkanoate compound or a carbon source that can be converted by the microorganism to a 3-hydroxyalkanoate compound.
    15. 15. Use of a combination of at least two enzymes, wherein one enzyme is selected from the following (i) and the other enzyme is selected from the following (ii) or of a microorganism of item 13 or of a composition of item 14, for producing alkene compounds from 3-hydroxyalkanoates,
      wherein (i) and (ii) are as follows:
      1. (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and
      2. (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene.
    16. 16. The use of item 14, wherein
      1. (i) the first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate is selected from the group consisting of
        1. (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 1 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 1;
        2. (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 2 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 2;
        3. (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 3 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 3 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 3; and
        4. (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 4 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 4.
    17. 17. The use of item 15 or 16, wherein
      (ii) the second enzyme having an activity of converting said 3-phosphonoxyalkanoate into said alkene selected from the group consisting of
      1. (a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 5 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 5 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 5;
      2. (b) a protein comprising the amino acid sequence as shown in SEQ ID NO: 6 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 6 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 6;
      3. (c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 7 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 7;
      4. (d) a protein comprising the amino acid sequence as shown in SEQ ID NO: 8 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 8 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 8;
      5. (e) a protein comprising the amino acid sequence as shown in SEQ ID NO: 9 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 9 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 9;
      6. (f) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 10 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 10;
      7. (g) a protein comprising the amino acid sequence as shown in SEQ ID NO: 11 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 11 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 11;
      8. (h) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 12 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 12;
      9. (i) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 13 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 13;
      10. (j) a protein comprising the amino acid sequence as shown in SEQ ID NO: 14 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 14 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 14; and
      11. (k) a protein comprising the amino acid sequence as shown in SEQ ID NO: 15 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 15 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 15.
    18. 18. A method for producing an alkene comprising the step of enzymatically converting a 3-phosphonoxyalkanoate into the corresponding alkene by use of an enzyme which can catalyze the conversion via decarboxylation and dephosphorylation, wherein said 3-phosphonoxyalkanoate has been produced by a different enzyme.
    19. 19. The method of any one of item 1 to 12 or the method of item 18, characterized in that the method is carried out with ATP, dATP, ADP, AMP, an NTP other than ATP, a dNTP or pyrophosphate as co-substrate.
    "3-hydroxyalkanoate", as used herein, denotes a molecule responding to the following general formula:

            Cn+1H2n+2O3

    , with 1<n<7, and comprising 3-hydroxypropionate as a common motif (Figure 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 Figure 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 embodiment, 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.

    [0009] The term "3-phosphonoxyalkanoate" denotes a molecule which responds to the following general formula:

            Cn+1H2n+3O6P

    , with 1<n<7, and comprising 3-phosphonoxypropionate as a common motif, and optionally one or two alkyl substitutions on carbon 3.

    [0010] The term "alkene", as used herein, denotes molecules composed solely of carbon and hydrogen, containing one carbon-carbon double bond and having the chemical formula of a mono-unsaturated hydrocarbon, CnH2n, where n equals at least two. Preferably, n equals at least 3, 4, 5 or 6. Most preferably n is at most 6. Thus, generally, the term "alkene" refers to a molecule responding to the formula CnH2n, with 1<n<7,

    [0011] In a preferred embodiment alkenes are represented by the structural formula H2C=C(R1)(R2) wherein R1 and R2 are selected, independently, from the group consisting of a hydrogen atom and a linear or branched alkyl radical, so that the total number of carbon atoms in the alkene molecule is at most 6.

    [0012] Preferred examples of alkene compounds according to the invention are in particular ethylene, propylene, isobutylene, and isoamylene (Figure 4), or else 1-butylene and 1-amylene.

    [0013] "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.

    [0014] "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.

    [0015] "Overexpression" or "overexpressing", as used herein, denotes the recombinant expression of a protein in a host organism, 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 occurring in said host organism. This definition also encompasses the case where there is no natural expression of said protein.

    [0016] A "co-substrate" is a compound or molecule 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.

    [0017] 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".

    [0018] 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.

    [0019] "Homology", as used herein, denotes the existence of a similarity between two sequences as measured by the percent identity between said two sequences. In a preferred embodiment the term "homology" means sequence identity.

    [0020] 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.


    [0021] The present invention describes a method for producing alkene compounds starting from a 3-hydroxyalkanoate through a biological process, in particular an enzymatic process, in which two types of enzymes are combined in order to increase the efficiency of the production rate. More specifically, the present invention relates to a method for producing an alkene, characterized in that it comprises the conversion of a 3-hydroxyalkanoate into said alkene by
    1. (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and
    2. (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene -
      by decarboxylation and dephosphorylation.
    As mentioned above, WO 2010/001078 describes a process for producing alkenes by enzymatic conversion of 3-hydroxyalkanoic acids with an enzyme having the activity of a decarboxylase. It has been described in WO 2010/001078 that generally the conversion of a 3-hydroxyalkanoate into an alkene by an enzyme having a decarboxylase activity, e.g. a mevalonate diphosphate (MDP) decarboxylase (E.C. 4.1.1.33) takes place by the conversion of the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is then decarboxylated to lead to the corresponding alkene. The generic reaction carried out by MDP decarboxylase using various 3-hydroxyalkanoates is depicted in Figure 2B.
    It has now been found that different decarboxylases, in particular mevalonate diphosphate decarboxylases, catalyze the two above mentioned steps with different efficiencies, i.e. that some decarboxylases catalyze the first step with a higher efficiency than other decarboxylases and that some decarboxylases show a preference for the second step, i.e. the decarboxylation step, and that therefore the efficiency of the conversion of the 3-hydroxyalkanoate into the alkene as described in WO 2010/001078 can be significantly increased by combining corresponding enzymes. Thus, the present invention in particular relates to a method for achieving a higher efficiency in the enzymatic production of alkenes from 3-hydroxyalkanoates, i.e. a method for improving the efficiency of such an enzymatic production.
    The term "an enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate" means an enzyme which can phosphorylate a 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate. The phosphate group comes preferably from an ATP molecule.
    This activity can, e.g., be measured as described in the attached Examples, in particular Example 5. One possibility is thus to incubate the respective enzyme with the 3-hydroxyalkanoate and ATP and to measure the production of ADP (which reflects the production of the corresponding 3-phosphonoxyalkanoate). Assays for measuring the production of ADP are known to the person skilled in the art. One of these methods is the pyruvate kinase/lactate dehydrogenase assay described in Example 5. In this case the assay measures the rate of NADH absorbance decrease at 340 nm which is proportional to the ADP quantity. In a preferred embodiment the term "an enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate" means an enzyme which can convert 3-hydroxyisovalerate and ATP into 3-phosphonoxyisovalerate and ADP. Even more preferably such an enzyme can catalyze the reaction of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate, preferably the reaction of converting 3-hydroxyisovalerate and ATP into 3-phosphonoxyisovalerate and ADP, with a KM of 10 mM or lower, e.g. with a KM of 5 mM or lower, preferably of 1 mM or lower and even more preferably of 0.1 mM or lower. In a particularly preferred embodiment such an enzyme can catalyze the reaction of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate, preferably the reaction of converting 3-hydroxyisovalerate and ATP into 3-phosphonoxyisovalerate and ADP, with a kcat of at least 0.2 s-1, preferably with a kcat of at least 0.5 s-1, particularly preferred with a kcat of at least 1.0 s-1, more preferred of at least 2.0 s-1 and even more preferred with a kcat of at least 5.0 s-1.
    In a particularly preferred embodiment the capacity to convert 3-hydroxyisovalerate and ATP into 3-phosphonoxyisovalerate and ADP is measured in an assay as described in Example 5.

    [0022] The term "an enzyme having an activity of converting said 3-phosphonoxyalkanoate into said alkene" means an enzyme which can catalyze a reaction by which there is a decarboxylation and dephosporylation of the 3-phosphonoxyalkanoate thereby leading to the corresponding alkene.
    This activity can, e.g., be measured as described in the appended Examples, in particular in Example 8. One possibility is thus to incubate the respective enzyme with the corresponding phosphonoxyalkanoate under conditions which in principle allow the decarboxylation and the dephosphorylation and to detect the production of the corresponding alkene, e.g. by gas chromatography. In a preferred embodiment the term "an enzyme having an activity of converting said 3-phosphonoxyalkanoate into said alkene" means an enzyme which can convert 3-phosphonoxyisovalerate into isobutene, preferably under the conditions described in Example 8. Even more preferably such an enzyme can catalyze the reaction of converting the 3-phosphonoxyalkanoate into the corresponding alkene (via decarboxylation and dephosphorylation) with a KM of 100 mM or lower, e.g. with a KM of 75 mM or lower, or with a KM of 50 mM or lower, preferably of 10 mM or lower or 5 mM or lower or 1 mM or lower, and even more preferably of 0.1 mM or lower. In a particularly preferred embodiment such an enzyme can catalyze the reaction of converting the 3-phosphonoxyalkanoate into the corresponding alkene, preferably the reaction of converting 3-phosphonoxyisovalerate into isobutene, with a kcat of at least 10-6 s-1, preferably with a kcat of at least 10-4 s-1, e.g. with a kcat of at least 10-3 s-1 or with a kcat of at least 10-2 s-1, such as with a kcat of at least 10-1 s-1, for example with a kcat of at least 0.2 s-1, preferably with a kcat of at least 0.5 s-1, particularly preferred with a kcat of at least 1.0 s-1, more preferred of at least 2.0 s-1 and even more preferred with a kcat of at least 5.0 s-1.
    In a particularly preferred embodiment the capacity to convert 3-phosphonoxyisovalerate into isobutene is measured in an assay as described in Example 8.

    [0023] In one preferred embodiment an enzyme mentioned in (i) and (ii), above, is an enzyme which is considered by NCBI or an equivalent engine as having a COG3407 domain.

    [0024] In a preferred embodiment of the method according to the invention the first enzyme (i) having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate is selected from the group consisting of
    1. (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 1 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 1;
    2. (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 2 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 2;
    3. (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 3 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 3 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 3; and
    4. (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 4 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 4.

    SEQ ID NO: 1 shows the amino acid sequence of an enzyme from Picrophilus torridus DSM 9790 (GenBank accession number AAT43941; Swissprot/TrEMBL accession number Q6KZB1).

    SEQ ID NO: 2 shows the amino acid sequence of an enzyme from Thermoplasma acidophilum (GenBank accession number CAC12426; Swissprot/TrEMBL accession number Q9HIN1).

    SEQ ID NO: 3 shows the amino acid sequence of an enzyme from Thermoplasma volcanium (GenBank accession number BAB59465; Swissprot/TrEMBL accession number Q97BY2).

    SEQ ID NO: 4 shows the amino acid sequence of an enzyme from Ferroplasma acidarmanus fer1 (GenBank accession number ZP_05571615).



    [0025] In a further preferred embodiment of the method according to the invention the second enzyme (ii) having an activity of converting said 3-phosphonoxyalkanoate into said alkene is selected from the group consisting of
    1. (a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 5 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 5 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 5;
    2. (b) a protein comprising the amino acid sequence as shown in SEQ ID NO: 6 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 6 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 6;
    3. (c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 7 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 7;
    4. (d) a protein comprising the amino acid sequence as shown in SEQ ID NO: 8 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 8 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 8;
    5. (e) a protein comprising the amino acid sequence as shown in SEQ ID NO: 9 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 9 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 9;
    6. (f) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 10 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 10;
    7. (g) a protein comprising the amino acid sequence as shown in SEQ ID NO: 11 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 11 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 11;
    8. (h) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 12 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 12;
    9. (i) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 13 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 13;
    10. (j) a protein comprising the amino acid sequence as shown in SEQ ID NO: 14 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 14 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 14; and
    11. (k) a protein comprising the amino acid sequence as shown in SEQ ID NO: 15 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 15 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 15.


    [0026] SEQ ID NO: 5 shows the amino acid sequence of an enzyme cloned from Streptococcus gordonii. SEQ ID NO: 6 shows the amino acid sequence of an enzyme from Streptococcus gordonii str. Challis substr. CH1 (GenBank accession number AAT43941; Swissprot/TrEMBL accession number A8UU9). SEQ ID NO: 7 shows the amino acid sequence of an enzyme from Streptococcus infantarius subsp infantarius ATCC BAA-102 (GenBank accession number EDT48420.1; Swissprot/TrEMBL accession number B1SCG0). SEQ ID NO: 8 shows the amino acid sequence of an enzyme from Homo sapiens (GenBank accession number AAC50440.1; Swissprot/TrEMBL accession number P53602.1). SEQ ID NO: 9 shows the amino acid sequence of an enzyme from Lactobacillus delbrueckii (GenBank accession number CAl97800.1; Swissprot/TrEMBL accession number Q1GAB2). SEQ ID NO: 10 shows the amino acid sequence of an enzyme from Streptococcus mitis (strain B6) (GenBank accession number CBJ22986.1). SEQ ID NO: 11 shows the amino acid sequence of an enzyme from Streptococcus gallolyticus UCN34 (GenBank accession number CBI13757.1). SEQ ID NO: 12 shows the amino acid sequence of an enzyme from Streptococcus sanguinis SK36 (GenBank accession number ABN43791.1). SEQ ID NO: 13 shows the amino acid sequence of an enzyme from Streptococcus sp. M143 (GenBank accession number EFA24040.1), SEQ ID NO: 14 shows the amino acid sequence of an enzyme from Streptococcus suis 89/1591 (GenBank accession number EEF63672.1). SEQ ID NO: 15 shows the amino acid sequence of an enzyme from Streptococcus salivarius SK126 (GenBank accession number EEK09252).

    [0027] In a preferred embodiment of the method according to the invention the first enzyme (i) is as defined in (A) above and the second enzyme (ii) is as defined in (a) or (b) mentioned above, even more preferably the second enzyme is as defined in (f), (g), (h), (i), (j) or (k) mentioned above. As illustrated in the examples, the combination of these enzymes is particularly efficient at producing alkene compounds according to the present invention.

    [0028] In another preferred embodiment of the method according to the invention the second enzyme (ii) having an activity of converting said 3-phosphonoxyalkanoate into said alkene is selected from any one of the proteins listed in the following Table or from a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence of such a protein and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of said protein.
    Table 1
    OrganismRef sequence GenBank
    Methanosarcina mazei AAM31457.1
    Methanocaldococcus jannaschii AAB98390.1
    Staphylococcus saprophyticus BAE19266.1
    Streptococcus agalactiae EAO73731.1
    Enterococcus faecalis AAO80711.1
    Flavobacterium johnsoniae ABQ04421.1
    Bdellovibrio bacteriovorus CAE79505.1
    Chloroflexus aurantiacus A9WEU8.1
    Legionella pneumophila CAH13175.1
    Listeria monocytogenes EAL09343.1
    Metallosphaera sedula ABP95731.1
    Staphylococcus epidermidis AAO03959.1
    Streptococcus thermophilus AAV60266.1
    Bacillus coagulans EAY45229.1
    Chloroflexus aggregans EAV09355.1
    Lactobacillus brevis ABJ64001.1
    Lactobacillus fermentum BAG27529.1
    Lactobacillus plantarum CAD64155.1
    Lactobacillus salivarius ABD99494.1
    Lactococcus lactis sp. lactis AAK04503.1
    Dichelobacter nodosus ABQ14154.1
    Flavobacterium psychrophilum CAL42423.1
    Streptococcus pneumoniae EDT95457.1
    Streptococcus pyogenes AAT86835.1
    Streptococcus suis ABP91444.1
    Staphylococcus haemolyticus BAE05710.1
    Streptococcus equi ACG62435.1
    Arabidopsis thaliana AAC67348.1
    Borrelia afzelii ABH01961.1
    Encephalitozoon cuniculi CAD25409.1
    Streptomyces sp. BAB07791.1
    Streptococcus agalactiae EAO73731.1
    Streptococcus uberis CAR41735.1
    Gallus gallus XP_423130
    Salmo salmar ACI34234
    Natromonas pharaonis CAI48881.1
    Haloarcula marismortui AAV46412.1
    Haloquadratum walsbyi CAJ51653.1


    [0029] As mentioned above, not only the proteins having the specifically mentioned amino acid sequences listed in the respective SEQ ID NOs or in Table 1 can be used, but also proteins which are considered by NCBI or an equivalent engine as having a COG3407 domain and, more preferred, proteins the amino acid sequence of which shows a homology of at least 15% to the specifically mentioned amino acid sequence and which have a respective enzymatic activity at least as high as the activity of a protein having the specifically mentioned amino acid sequence. Preferred enzymes advantageously have at least x% homology, wherein x is selected from the group consisting of 20, 25, 20, 35, 40, 45, 50, 55 and 60. In a further preferred embodiment the enzyme has at least 65% sequence homology, preferably at least 70%, more preferably at least 75%, even more preferably, at least 80, 85, 90, 95, 96, 97, 98 or 99% homology to one of the sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or to one of the sequences shown in Table 1. 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).

    [0030] Such proteins showing the indicated degree of homology can, e.g., be other enzymes which occur naturally or which have been prepared synthetically. They include in particular enzymes which can be selected for their ability to produce 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 respective compound, i.e. the 3-phosphonoxyalkanoate or the alkene. Such tests are described in the experimental section. Such selection tests can also be used to screen for enzymes with an optimized enzymatic activity for the substrate to be converted into the 3-phosphonoxyalkanoate or the alkene, i.e. having an optimized activity with respect to one or more 3-hydroxyalkanoates or 3-phosphonoxyalkanoates.

    [0031] Such screening methods are well-known in the art and include, e.g. protein engineering techniques such as random mutagenesis, massive mutagenesis, site-directed mutagenesis, DNA shuffling, synthetic shuffling, in vivo evolution, or complete synthesis of genes and subsequent screening for the desired enzymatic activity.

    [0032] The enzymes 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.

    [0033] It has been found that enzymes which are able to catalyze the above described reactions for converting a 3-hydroxyalkanoate into an alkene via a 3-phospho-hydroxyalkanoate are often enzymes which can be classified in the phylogenetic superfamily of mevalonate diphosphate (MDP) decarboxylases (enzyme nomenclature EC 4.1.1.33). 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., Phytochemistry 24 (11), (1985), 2569-2571). 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 diphosphate (Figure 2).
    Accordingly, in a preferred embodiment, the enzyme defined in (i) or (ii) above, is a MDP decarboxylase. In the context of the present invention a MDP decarboxylase is defined as an enzyme which can at least catalyze the conversion of 5-diphospho-3-phosphomevalonate into isopentenyl-5-diphosphate and CO2 or which can at least catalyze the reaction of converting mevalonate diphosphate and ATP into 5-diphospho-3-phosphomevalonate and ADP. Preferably, such an enzyme can catalyze both reactions.
    In another preferred embodiment the enzyme defined in (i) above, is an enzyme as defined in (i) (B). The sequence shown in SEQ ID NO: 2 represents an enzyme identified in Thermoplasma acidophilum. In Genbank this enzyme is classified as a mevalonate diphosphate decarboxylase. However, it is known from Chen and Poulter (Biochemistry 49 (2010), 207-217) that in Th. acidophilum there exists an alternative mevalonate pathway which involves the action of a mevalonate-5-monophosphate decarboxylase. Thus, it is possible that the enzyme represented by SEQ ID NO: 2 actually represents a mevalonate-5-monophosphate decarboxylase. The same may hold true for other archae bacteria. Therefore, in another preferred embodiment the enzyme defined in (i) or (ii) above, is a mevalonate-5-monophosphate decarboxylase. Such an enzyme is capable of converting mevalonate-5-monophosphate into isopentenylpyrophosphate.

    [0034] In preferred embodiments of the invention:
    • 3-hydroxypropionate is converted via 3-phosphonoxypropionate into ethylene; or
    • 3-hydroxybutyrate is converted via 3-phosphonoxybutyrate into propylene; or
    • 3-hydroxyvalerate is converted via 3-phosphonoxyvalerate into 1-butylene; or
    • 3-hydroxy-3-methylbutyrate (or 3-hydroxyisovalerate) is converted via 3-phosphonoxy-3-methylbutyrate (3-phosphonoxyisovalerate) into isobutylene; or
    • 3-hydroxy-3-methylvalerate is converted via 3-phosphonoxy-3-methylvalerate into isoamylene.


    [0035] The method according to the invention can be carried out in vitro, in the presence of isolated enzymes (or enzyme systems additionally comprising one or more cofactors). In vitro preferably means in a cell-free system.

    [0036] In one embodiment, the enzymes employed in the method are used in purified form to convert 3-hydroxyalkanoates to alkenes. However, such a method may be costly, since enzyme and substrate production and purification costs are high.
    Thus, in another preferred embodiment, the enzymes employed in the method are 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 such a method may still be quite high due to the costs of producing and purifying the substrates.

    [0037] Accordingly, in one preferred embodiment, the enzymes, native or recombinant, purified or not, are used to convert a 3-hydroxyalkanoate to an alkene. To do this, the enzymes are incubated in the presence of the substrate in physicochemical conditions allowing the enzymes 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 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.

    [0038] In a preferred embodiment, cofactors are added so as to best mimic the natural reaction or so as to provide steric or electronic complementation in the catalytic cleft. For example, if one of the enzymes used in the method according to the invention is an enzyme which naturally uses mevalonate disphosphate (MDP) as a substrate, the structure of 3-hydroxyalkanoates leaves a large space in the catalytic cleft empty during enzyme-substrate binding since generally a 3-hydroxyalkanoate corresponds to a fragment of MDP. 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 Figure 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 global formula R-PO2H-O-PO3H2, in which R is in particular H, 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 analogous motifs corresponding to methylene diphosphonate monoesters, having the general formula R-O-PO2H-CH2-PO3H2 in which phosphyanhydride 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. The cofactor is advantageously selected from the group consisting of the pyrophosphate ion, methyl diphosphate, ethyl diphosphate, or propyl diphosphate.

    [0039] 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 from 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 Figure 2).
    Although the decarboxylation step, i.e. the reaction defined as (ii) herein-above, does not require ATP consumption, it could be shown that the presence of ATP in the reaction could be beneficial. This has been demonstrated in Example 7, using 3-phosphonoxyisovalerate as a substrate. It is assumed that ATP might have an effect on the folding of the protein by the binding of ATP to the ATP-binding site of the diphosphomevalonate decarboxylase. In fact, this can be observed by eye: the purified enzyme has a tendency to precipitate, and the addition of ATP prevents this effect. It is considered that not only ATP but also other similar compounds like dATP, ADP, AMP or other NTPs or dNTPs have this effect. Thus, in a preferred embodiment, the method according to the present invention is carried with ATP, dATP, ADP, AMP or an NTP other than ATP or a dNTP as co-substrate.

    [0040] In another preferred embodiment the method according to the invention is carried out in culture, in the presence of an organism, preferably a microorganism, recombinantly producing the enzymes. Thus, in such an embodiment of the invention, an organism, preferably a microorganism, that recombinantly produces the enzymes specified in (i) and (ii) above is used. The (micro)organism is recombinant in that the enzymes specified in (i) and (ii) produced by the host are heterologous 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 enzymes. In an especially advantageous manner, a (micro)organism is used having the natural or artificial property of endogenously producing one or more 3-hydroxyalkanoates, and also expressing or overexpressing the enzymes specified in (i) and (ii) above, natural or modified, so as to produce alkenes directly from a carbon source present in solution.
    For example, the method according to the invention can be carried out by using microorganisms which produce one or more 3-hydroxyalkanoates [for example Alcaligenes eutrophus or Bacillus megaterium, or else an E. coli strain genetically modified so as to produce said product(s)] and which have been genetically engineered such that they overexpress the enzymes as defined in (i) and (ii) above, said enzymes preferably originating from an organism different from the host microorganism. The genetic modification can consist, e.g. in integrating the corresponding genes encoding the enzymes into the chromosome, expressing the enzymes from 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 can be chosen. For instance, a yeast such as Saccharomyces cerevisiae, an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae, microalgae, or photosynthetic bacteria can be used.

    [0041] The organisms used in the invention can be prokaryotes or eukaryotes, preferably, they are microorganisms such as bacteria, yeasts, fungi or molds, or plant cells or animal cells. In a particular embodiment, the microorganisms are bacteria, preferably of the genus Escherichia, Alcaligenes or Bacillus and even more preferably of the species Escherichia coli, Alcaligenes eutrophus or Bacillus megaterium.
    In another preferred embodiment, the microorganisms are recombinant bacteria of the genus Escherichia, preferably of the species Escherichia coli, having been modified so as to endogenously produce one or more 3-hydroxyalkanoates, and converting them to alkenes.

    [0042] In a further preferred embodiment the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus or Trichoderma and even more preferably of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger or of the species Trichoderma reesei. In a particularly preferred embodiment the microorganism is a recombinant yeast producing 3-hydroxyalkanoates and converting them to alkenes due to the expression of the enzymes specified in (i) and (ii) above.

    [0043] In another preferred embodiment, the method according to the invention makes use of a photosynthetic microorganism expressing the enzymes as specified in (i) and (ii) above. Preferably, the microorganism is a photosynthetic bacterium, or a microalgae. Even more preferably such a microorganism has the natural or artificial property of endogenously producing one or more 3-hydroxyalkanoates. In this case the microorganism would be capable of producing alkenes directly from CO2 present in solution.

    [0044] It is also conceivable to use in the method according to the invention one microorganism that produces an enzyme as defined in (i) above and another microorganism which produces an enzyme as defined in (ii) above. Moreover, in a further embodiment at least one of the microorganisms is capable of producing one or more 3-hydroxyalkanoates or, in an alternative embodiment, a further microorganism is used in the method which is capable of producing one or more 3-hydroxyalkanoates.

    [0045] In another preferred embodiment the method according to the invention makes use of a multicellular organism expressing the enzymes as defined in (i) and (ii) above. Examples for such organisms are plants or animals.

    [0046] 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 thermophilic organisms, for example).

    [0047] In a further preferred embodiment the method of the invention is carried out in microaerophilic conditions. This means that the quantity of injected air is limiting so as to minimize residual oxygen concentrations in the gaseous effluents containing the alkene hydrocarbons.

    [0048] In another preferred embodiment the method according to the invention furthermore comprises the step of collecting gaseous alkenes degassing out of the reaction, i.e. recovering the products which degas, e.g., out of the culture. Thus in a preferred embodiment, the method is carried out in the presence of a system for collecting alkene under gaseous form during the reaction.
    As a matter of fact, short alkenes, and particularly ethylene, propylene and butene isomers, adopt the gaseous state at room temperature and atmospheric pressure. The method according to the invention therefore does not require extraction of the product from the liquid culture medium, a step which is always very costly when performed at 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.
    It is also disclosed that the method also comprises detecting the alkene (for example propylene, ethylene or isobutylene) which is present in the gaseous phase. The presence of the compound to be produced 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.

    [0049] In a particular embodiment, the alkenes produced by a method according to the invention 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. For example, isobutylene can be used to synthesize isooctane: the catalytic methods for successfully carrying out this reaction have already been fully described.

    [0050] In another embodiment, the method according to the invention is characterized by the conversion of a carbon source such as glucose, to 3-hydroxyalkanoate, followed by the conversion of said 3-hydroxyalkanoate into the corresponding alkene. The different steps of said method are outlined in Figure 6.
    In a particular embodiment, the method is characterized by the conversion of polyhydroxyalkanoates into 3-hydroxyalkanoate by using an enzyme or a suitable physicochemical method, followed by the conversion of said 3-hydroxyalkanoate into said alkene. Optionally, the polyhydroxyalkanoate has been produced by a microorganism or a plant whose metabolic pathways have been modified to as to produce high yields of polyhydroxyalkanoate.
    In another embodiment, the method according to the invention comprises the production of alkenes from atmospheric CO2 or from CO2 artificially added to the culture medium. In this case the method is implemented in an organism which is able to carry out photosynthesis, such as for example microalgae.

    [0051] The present invention also relates to a method for producing an alkene comprising the step of enzymatically converting a 3-phosphonoxyalkanoate into the corresponding alkene by use of an enzyme which can catalyze the conversion via decarboxylation and dephosphorylation, wherein said 3-phosphonoxyalkanoate, in a first step, is produced by a different enzyme.

    [0052] As regards the preferred enzyme to be used in such a method, the same applies as has been set forth above in connection with (ii) of the method according to the invention as described herein-above.

    [0053] Moreover, also with respect to the other preferred embodiments described above for the method according to the invention, the same applies to the method for producing an alkene from a 3-phosphonoxyalkanoate.

    [0054] The present invention also relates to organisms, preferably microorganisms, which recombinantly
    produce at least two enzymes, wherein one enzyme is selected from (i) as specified above and the other enzyme is selected from (ii) as specified above.

    [0055] Such an organism is a recombinant organism in the sense that it is genetically modified due to the introduction of at least one nucleic acid molecule encoding at least one of the above mentioned enzymes. Preferably such a nucleic acid molecule is heterologous with regard to the organism which means that it does not naturally occur in said organism.

    [0056] Thus, the present invention also relates to an organism, preferably a microorganism, comprising a nucleic acid molecule coding for an enzyme as defined in (i) above and comprising a nucleic acid molecule coding for an enzyme as defined in (ii) above. In a preferred embodiment at least one of the nucleic acid molecules is heterologous to the organism which means that it does not naturally occur in said organism. The microorganism is preferably a bacterium, a yeast or a fungus. In another preferred embodiment the organism is a plant or non-human animal. As regards other preferred embodiments, the same applies as has been set forth above in connection with the method according to the invention.

    [0057] Moreover, the present invention also relates to a composition comprising a microorganism according to the present invention, a suitable culture medium and a 3-hydroxyalkanoate compound or a carbon source that can be converted by the microorganism to a 3-hydroxyalkanoate compound.

    [0058] The present invention also relates to the use of a combination of at least two enzymes, wherein one enzyme is selected from the following (i) and the other enzyme is selected from the following (ii) or of an organism, preferably a microorganism, according to the invention or of a composition according to the invention, for producing alkene compounds from 3-hydroxyalkanoates, wherein (i) and (ii) are as follows:
    1. (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and
    2. (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene.


    [0059] As regards the preferred embodiments of the different components recited, the same applies as has been set forth above in connection with the method according to the invention.

    [0060] Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation.

    Figures legends



    [0061] 
    Figure 1:
    The 3-hydroxypropionate motif.
    Figure 2:
    Reaction catalyzed by mevalonate diphosphate decarboxylase.
    Figure 3:
    Examples of 3-hydroxyalkanoates.
    Figure 4:
    Production of alkenes from 3-hydroxyalkanoates by combining two enzymatic steps.
    Figure 5:
    Cofactors that can be used in the reaction for the purpose of structural complementation in the catalytic site of mevalonate diphosphate decarboxylase.
    Figure 6:
    Integrated method for producing an alkene from glucose.
    Figure 7:
    Screening of MDP decarboxylases in a complementation assay. The reaction catalyzed by the P. torridus enzyme alone (0.1 mg) without a second enzyme, was taken as reference.
    Figure 8:
    Combined effect of MDP decarboxylase enzymes from P. torridus and S. gordonii for converting 3-hydroxyisovalerate (HIV) into isobutene (IBN). IBN production was measured as a function of the concentration of S. gordonii MDP decarboxylase added to a pre-incubated reaction mixture of HIV with 100 µg of P. torridus MDP decarboxylase.
    Figure 9:
    Screening of enzyme homologs of S. gordonii MDP decarboxylase. The peak area of isobutene obtained for the reaction with Th. acidophilum (0.1 mg) enzyme alone (no second enzyme), was used as reference (ratio=1).
    MDP decarboxylases from the Streptococcus genus are particularly efficient when used in combination with an enzyme of the P. torridus phylum.
    Figure 10:
    Scheme of the ADP quantification assay, monitoring NADH consumption by the decrease of absorbance at 340 nm.
    Figure 11:
    Plot of the velocity as a function of substrate concentration for the phosphotransferase reaction catalyzed by P. torridus MDP decarboxylase. Initial rates were computed from the kinetics over the 30 first minutes of the reaction.
    Figure 12:
    Isobutene production from 3-hydroxyisovalerate in the following assays:
    Without enzyme
    In the presence of S. mitis MDP decarboxylase
    In the presence of Th. acidophilum MDP decarboxylase
    In the presence of both Th. acidophilum and S. mitis enzymes.
    Figure 13:
    Scheme for the chemical synthesis of 3-phosphonoxyisovalerate.
    Figure 14:
    GC analysis of assays for isobutene production from 3-phosphonoxyisovalerate in the absence and presence of ATP. Assays:
    1. 1. Without enzyme, 0 mM ATP
    2. 2. 2 mg/ml enzyme, 0 mM ATP
    3. 3. Without enzyme, 10 mM ATP
    4. 4. 2 mg/ml enzyme, 10 mM ATP


    [0062] The following Examples serve to illustrate the invention.

    Examples


    Example 1: Cloning, expression and purification of an MDP decarboxylase library.



    [0063] A library of 55 genes encoding representatives of the diphosphomevalonate decarboxylase (MDP decarboxylase) family across eukaryotic, prokaryotic and archaeal organisms was constructed and tested to identify the most active candidates for improving isobutene (IBN) production.

    [0064] Cloning, bacterial cultures and expression of proteins.

    [0065] The genes encoding mevalonate diphosphate (MDP) decarboxylase EC 4.1.1.33 were cloned in the pET 25b vector (Novagen) in the case of eukaryotic genes and in pET 22b (Novagen) in the case of prokaryotic genes. A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. Competent E. coli BL21(DE3) cells (Novagen) were transformed with these vectors according to the heat shock procedure. The transformed cells were grown with shaking (160 rpm) on ZYM-5052 auto-induction medium (Studier FW, Prot.Exp.Pur. 41, (2005), 207-234) for 6h at 37°C and protein expression was continued at 28°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.

    [0066] Protein purification and concentration.

    [0067] The pellets from 200 ml of culture cells were thawed on ice and resuspended in 5 ml of Na2HPO4 pH 8 containing 300 mM NaCl, 5 mM MgCl2 and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 3 x 15 seconds. The bacterial extracts were then clarified by centrifugation at 4°C, 10,000 rpm for 20 min. The clarified bacterial lysates were loaded on PROTINO-1000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged 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 and desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and resuspended in 0.25 ml 50 mM Tris-HCl pH 7.4 containing 0.5 mM DTT and 5 mM MgCl2. Protein concentrations were quantified according to the Bradford method. The purity of proteins thus purified varied from 40 % to 90 %.

    Example 2: Screening of the MDP decarboxylase library.



    [0068] MDP decarboxylases were evaluated using a complementation assay. P. torridus MDP decarboxylase was incubated together with each tested enzyme from the library.
    The enzymatic assay was carried out under the following conditions:

    50 mM Tris HCl pH 7,0

    10 mM MgCl2

    20 mM KCl

    40 mM ATP

    50 mM 3-hydroxyisovalerate (HIV)

    The pH was adjusted to 7.0

    [0069] 100 µg of the MDP decarboxylase from P. torridus and 1 mg of the MDP decarboxylase to be tested were added to 1 ml of reaction mixture. A reaction mixture containing only 100 µg of P. torridus MDP decarboxylase was used as reference. The mixture was then incubated without shaking at 45°C for 90h in a sealed vial (Interchim).
    One ml of the gaseous phase was collected and injected into a HP5890 gas chromatograph (HP) equipped with an FID detector and a CP SilicaPlot column (Varian). Commercial isobutene was used as reference.

    [0070] This screening procedure led to the identification of several MDP decarboxylase enzymes increasing the isobutene production rate. As shown in Figure 7, a higher production of isobutene was observed for the following MDP decarboxylases.

    Candidate 1:



    [0071] 

    Accession number Genbank : CAI97800

    Accession number SwissProt/TrEMBL : Q1GAB2

    Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842


    Candidate 2:



    [0072] 

    Accession number Genbank : AAC50440.1

    Accession number SwissProt/TrEMBL : P53602.1

    Organism: Homo sapiens


    Candidate 3:



    [0073] 

    Accession number Genbank : ABV09606

    Accession number SwissProt/TrEMBL : A8AUU9

    Organism: Streptococcus gordonii str. Challis substr. CH1



    [0074] The highest production of isobutene was observed with purified MDP decarboxylase from Streptococcus gordonii.
    This indicated that the two enzymes present in the assay (the one from P. torridus and the other from S. gordonii) were performing complementarily the two steps of reaction producing IBN from HIV: transfer of the terminal phosphoryl group from ATP to the C3-oxygen of 3-hydroxyisovalerate followed by combined dephosphorylation-decarboxylation of the intermediate 3-phosphonoxyisovalerate.

    Example 3: Effect of enzyme concentration on isobutene production yield.



    [0075] The effect of Streptococcus gordonii MDP decarboxylase concentration was assessed under the following conditions:

    50 mM Tris-HCl pH 7,0

    10 mM MgCl2

    20 mM KCl

    40 mM ATP

    50 mM 3-hydroxyisovalerate (HIV)

    The pH was adjusted to 7,0
    100 µg of MDP decarboxylase from P. torridus and a varying amount (from 0 to 1 mg) of purified MDP decarboxylase from Streptococcus gordonii were added to 1 ml of reaction mixture. The mixture was then incubated without shaking at 45°C for 90h in a sealed vial (Interchim).

    [0076] One ml of the headspace phase was collected and injected into a HP5890 gas chromatograph (HP) equipped with an FID detector and a CP SilicaPlot column (Varian). Commercial isobutene was used as reference.

    [0077] Increasing the S. gordonii enzyme concentration resulted in an increase of the amount of isobutene produced (Figure 8).

    Example 4: Screening of a library of Streptococcus gordonii MDP decarboxylase homologs.



    [0078] Using the BLAST online program hosted by NCBI, sequences were searched against non redundant protein sequence database to generate a list of enzymes with high sequence similarity (> 40% identity) to the Streptococcus gordonii enzyme. The resulting list included 18 candidates.
    Microorganisms% Identity with MDP decarboxylase from Streptococcus gordoniiAccession number Genbank
    Streptococcus oralis ATCC 35037 75 EFE56694.1
    Leptotrichia goodfellowii F0264 61 EEY36155.1
    Carnobacterium sp. AT7 40 EDP67928.1
    Enterococcus faecium TX1330 40 EEI60970.1
    Staphylococcus aureus JH1 40 ABR51487.1
    Streptococcus agalactiae NEM316 70 CAD47054.1
    Streptococcus mutans UA159 71 AAN58642.1
    Streptococcus uberis 0140J 71 CAR41735.1
    Streptococcus infantarius subsp infantarius ATCC BAA-102 71 EDT48420.1
    Streptococcus gallolyticus UCN34 71 CBI13757.1
    Streptococcus dysgalactiae subsp equisimilis GGS_124 71 BAH81333.1
    Streptococcus sp. M143 76 EFA24040.1
    Streptococcus salivarius SK126 74 EEK09252.1
    Streptococcus suis 89/1591 40 EEF63672.1
    Streptococcus parasanguinis ATCC 15912 73 EFH19018.1
    Streptococcus sanguinis SK36 98 ABN43791.1
    Streptococcus sp. 2_1_36FAA 98 EEY81027.1
    Streptococcus mitis B6 74 CBJ22986.1


    [0079] Sequences of MDP decarboxylase enzymes inferred from the genomes of the above species as well as from the genome of S. gordonii were generated by oligonucleotide concatenation to fit the codon usage of E. coli. A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The genes thus synthesized were cloned in a pET25b expression vector (the vectors were constructed by GENEART AG). After transformation of the E. coli strain BL21(DE3), the proteins were produced according to the protocol described in Example 1. The enzymes were then assayed using the method described in Example 2, using Th. acidophilum MDP decarboxylase instead of the P. torridus enzyme. This screening procedure led to the identification of enzymes more efficient for isobutene production than the S. gordonii enzyme (Figure 9), in particular MDP decarboxylases from S. infantarius, S. gallolyticus, S. sp. M143, S. salivarius, S. suis, S. sanguinis and S. mitis.

    Example 5: Characterisation of the phosphotransferase activity.



    [0080] The release of ADP that is associated with IBN production from HIV was quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (Figure 10). The MDP decarboxylases from P. torridus, Th. acidophilum, S. infantarius, S. mitis were evaluated for their ability to phoshorylate HIV, releasing ADP.
    The studied enzymatic reaction was carried out under the following conditions at 40°C:

    50 mM Tris-HCl pH 7,0

    10 mM MgCl2

    100 mM KCl

    5 mM ATP

    0,2 mM NADH

    0,5 mM Phosphoenolpyruvate

    3 U/ml Lactate dehydrogenase

    1,5 U/ml Pyruvate kinase

    0-50 mM 3-Hydroxyisovalerate (HIV)

    The pH was adjusted to 7,0
    Each assay was started by addition of particular enzyme (at a concentration from 0.05 to 1 mg/ml) and the disappearance of NADH was monitored by following the absorbance at 340 nM.
    Assays with MDP decarboxylases from the P. torridus phylum as well from the Streptococcus genus gave rise to a reproducible increase in ADP production in the presence of HIV. Figure 11 shows an example of a Michaelis-Menten plot corresponding to the data collected for P. torridus enzyme. The kinetic parameters are shown in the following Table.

    [0081] The enzymes from the P. torridus phylum displayed higher phosphotransferase
    OrganismKM, mMkcat, sec-1kcat/ KM x 10-3, mM-1 sec-1
    Thermoplasma acidophilum 4.02 0.26 60
    Picrophilus torridus 9.17 0.19 20
    Streptococcus mitis 12.1 0.04 3
    Streptococcus infantarius 13.4 0.03 2
    activities than those of the Streptococcus genus.

    Example 6: Isobutene production from 3-hydroxyisovalerate by combining two enzymes.



    [0082] The desired enzymatic reaction was carried out under the following conditions:

    50 mM Tris HCl pH 7,5

    10 mM MgCl2

    20 mM KCl

    40 mM ATP

    50 mM HIV



    [0083] The pH was adjusted to 7,5

    [0084] 100 µg of MDP decarboxylase from Th. acidophilum and 500 µg of MDP decarboxylase from S. mitis were added to 1 ml of reaction mixture. Control reactions with only one of the two enzymes were run in parallel. The assays were incubated without shaking at 37°C in a sealed vial (Interchim).

    [0085] The production of IBN was measured by analyzing aliquots sampled over a 142 hour incubation period.

    [0086] One ml of the gaseous phase was collected and injected into a HP5890 gas chromatograph (HP) equipped with an FID detector and a CP SilicaPlot column (Varian). Commercial isobutene was used as reference.

    [0087] The kinetics of isobutene production is shown in Figure 12. MDP decarboxylase from Th. acidophilum catalyzed the production of isobutene from HIV. The addition of MDP decarboxylase from S. mitis led to a 3-fold increase of isobutene production after 142 h of incubation.

    [0088] MDP decarboxylase from S. mitis alone produced only small amounts of isobutene after 6 days of incubation, indicating a low phosphotransferase activity.

    [0089] Isobutene production can thus be increased by combining two types of enzymes performing complementarily the two reaction steps.

    Example 7: Effect of ATP on isobutene production from 3-phosphonoxyisovalerate (PIV).



    [0090] The compound 3-phosphonoxyisovalerate (PIV) was chemically synthesized from 3-hydroxyisovalerate according to the scheme depicted in Figure 13 by SYNTHEVAL (France).

    [0091] The assays of isobutene production were carried out under the following conditions:

    50 mM Tris-HCl pH 7,5

    10 mM MgCl2

    20 mM KCl

    0 mM ATP (assay Nº1 and Nº2)

    10 mM ATP (assay Nº3 and Nº4)

    25 mM 3-phosphonoxyisovalerate



    [0092] The pH was adjusted to 7.5

    [0093] The reaction was initiated by addition of 2 mg of purified MDP decarboxylase from S. mitis to 0.5 ml of reaction mixture. Control reactions were run in the absence of enzyme (assays Nº1 and Nº3).

    [0094] The mixture was incubated without shaking at 37°C for 26 h in a sealed vial of 2 ml (Interchim).

    [0095] One ml of the gaseous phase was collected and injected into a Varian 430-GC gas chromatograph equipped with an FID detector and a CP SilicaPlot column (Varian). Commercial isobutene was used as reference.

    [0096] Addition of 10 mM ATP to the reaction mixture increased 120 fold isobutene production from 3-phosphonoxyisovalerate (PIV) (Figure 14).

    Example 8: Kinetic parameters of isobutene production from 3-phosphonoxyisovalerate (PIV).



    [0097] The kinetic parameters of isobutene production were measured under the following conditions:

    50 mM Tris-HCl pH 7,5

    10 mM MgCl2

    50 mM KCl

    40 mM ATP

    0-100 mM 3-phosphonoxyisovalerate



    [0098] The pH was adjusted to 7,5

    [0099] The reaction was initiated by addition of 1 mg of purified MDP decarboxylase from S. mitis to 0.5 ml of reaction mixture. The mixture was then incubated without shaking at 37°C for 44 h in a sealed vial of 2 ml (Interchim).

    [0100] One ml of the gaseous phase was collected and injected into a Varian 430-GC gas chromatograph equipped with an FID detector and a CP SilicaPlot column (Varian). Commercial isobutene was used as reference.

    [0101] The assays with MDP decarboxylase from S. mitis showed a 160-400 fold increase in IBN production over the background level (spontaneous decomposition of 3-phosphonoxyisovalerate) in the presence of ATP as co-factor (see the following Table).
    PIV concentration, mMPeak area, arbitrary units
    No enzyme2 mg/ml enzyme
    25 164,9 26945,2
    50 328,7 65720,4
    75 561,5 239249,2
    100 2078,7 339363,7


    [0102] MDP decarboxylase from S. mitis was found to have a KM higher than 60 mM and a kcat of at least 1.3 x 10-3 sec-1.

    SEQUENCE LISTING



    [0103] 

    <110> Global Bioenergies, Scientist of Fortune S.A.

    <120> Production of alkenes by combined enzymatic conversion of
    3-hydroxyalkanoic acids

    <130> S1768 EP S3

    <140> EP 11 78 4957.0
    <141> 2011-10-18

    <150> EP 10 18 8001.1
    <151> 2010-10-19

    <150> PCT/EP2011/068174

    <150> 2011-10-18

    <160> 15

    <170> PatentIn version 3.5

    <210> 1
    <211> 324
    <212> PRT
    <213> Picrophilus torridus

    <400> 1



    <210> 2
    <211> 318
    <212> PRT
    <213> Thermoplasma acidophilum

    <400> 2





    <210> 3
    <211> 320
    <212> PRT
    <213> Thermoplasma volcanium

    <400> 3



    <210> 4
    <211> 322
    <212> PRT
    <213> Ferroplasma acidarmanus fer1

    <400> 4



    <210> 5
    <211> 315
    <212> PRT
    <213> Streptococcus gordonii

    <400> 5



    <210> 6
    <211> 315
    <212> PRT
    <213> Streptococcus gordonii str. Challis substr. CH1

    <400> 6



    <210> 7
    <211> 311
    <212> PRT
    <213> Streptococcus infantarius subsp infantarius ATCC BAA-102

    <400> 7





    <210> 8
    <211> 400
    <212> PRT
    <213> Homo sapiens

    <400> 8



    <210> 9
    <211> 319
    <212> PRT
    <213> Lactobacillus delbrueckii

    <400> 9





    <210> 10
    <211> 317
    <212> PRT
    <213> Streptococcus mitis (strain B6)

    <400> 10



    <210> 11
    <211> 316
    <212> PRT
    <213> Streptococcus gallolyticus UCN34

    <400> 11



    <210> 12
    <211> 315
    <212> PRT
    <213> Streptococcus sanguinis SK36

    <400> 12



    <210> 13
    <211> 317
    <212> PRT
    <213> Streptococcus sp. M143

    <400> 13



    <210> 14
    <211> 341
    <212> PRT
    <213> Streptococcus suis 89/1591

    <400> 14





    <210> 15
    <211> 314
    <212> PRT
    <213> Streptococcus salivarius SK126

    <400> 15






    Claims

    1. A method for producing an alkene, characterized in that it comprises the conversion of a 3-hydroxyalkanoate into said alkene by

    (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and

    (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene by decarboxylation and dephosphorylation.


     
    2. The method of claim 1 wherein

    (i) the first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate is selected from the group consisting of

    (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 1 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 1;

    (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 2 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 2;

    (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 3 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 3 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 3; and

    (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 4 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 4.


     
    3. The method of claim 1 or 2, wherein
    (ii) the second enzyme having an activity of converting said 3-phosphonoxyalkanoate into said alkene selected from the group consisting of

    (a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 5 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 5 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 5;

    (b) a protein comprising the amino acid sequence as shown in SEQ ID NO: 6 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 6 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 6;

    (c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 7 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 7;

    (d) a protein comprising the amino acid sequence as shown in SEQ ID NO: 8 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 8 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 8;

    (e) a protein comprising the amino acid sequence as shown in SEQ ID NO: 9 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 9 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 9

    (f) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 10 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 10;

    (g) a protein comprising the amino acid sequence as shown in SEQ ID NO: 11 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 11 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 11;

    (h) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 12 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 12;

    (i) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 13 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 13;

    (j) a protein comprising the amino acid sequence as shown in SEQ ID NO: 14 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 14 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 14; and

    (k) a protein comprising the amino acid sequence as shown in SEQ ID NO: 15 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 15 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 15.


     
    4. The method of any one of claims 1 to 3, comprising the step of converting 3-hydroxyproprionate to ethylene.
     
    5. The method of any one of claims 1 to 3, comprising the step of converting 3-hydroxybutyrate to propylene.
     
    6. The method of any one of claims 1 to 3, comprising the step of converting 3-hydroxyvalerate to 1-butylene.
     
    7. The method of any one of claims 1 to 3, comprising the step of converting 3-hydroxy-3-methylbutyrate to isobutylene.
     
    8. The method of any one of claims 1 to 3, comprising the step of converting 3-hydroxy-3-methylvalerate to isoamylene.
     
    9. The method of any one of the previous claims, characterized in that the conversion step is carried out in vitro, in a cell-free system.
     
    10. The method of any one of claims 1 to 8 characterized in that the method is carried out in the presence of a microorganism recombinantly producing both said enzymes as defined in (i) and (ii) of claim 1.
     
    11. The method of any one of claims 1 to 8, characterized by the use of a plant, animal cell, plant cell or microorganism recombinantly producing both said enzymes as defined in (i) and (ii) of claim 1.
     
    12. The method according to any one of the previous claims, comprising a step of collecting gaseous alkenes degassing out of the reaction.
     
    13. A plant, animal cell, plant cell or a microorganism which recombinantly produces at least two enzymes, wherein one enzyme is selected from (i) and the other enzyme is selected from (ii), wherein (i) and (ii) are as follows:

    (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and

    (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene.


     
    14. A composition comprising the microorganism of claim 13, a suitable culture medium and a 3-hydroxyalkanoate compound or a carbon source that can be converted by the microorganism to a 3-hydroxyalkanoate compound.
     
    15. Use of a combination of at least two enzymes, wherein one enzyme is selected from the following (i) and the other enzyme is selected from the following (ii) or of a microorganism of claim 13 or of a composition of claim 14, for producing alkene compounds from 3-hydroxyalkanoates,
    wherein (i) and (ii) are as follows:

    (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and

    (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene.


     
    16. The use of claim 14, wherein

    (i) the first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate is selected from the group consisting of

    (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 1 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 1;

    (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 2 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 2;

    (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 3 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 3 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 3; and

    (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 4 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 4.


     
    17. The use of claim 15 or 16, wherein
    (ii) the second enzyme having an activity of converting said 3-phosphonoxyalkanoate into said alkene selected from the group consisting of

    (a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 5 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 5 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 5;

    (b) a protein comprising the amino acid sequence as shown in SEQ ID NO: 6 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 6 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 6;

    (c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 7 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 7;

    (d) a protein comprising the amino acid sequence as shown in SEQ ID NO: 8 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 8 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 8;

    (e) a protein comprising the amino acid sequence as shown in SEQ ID NO: 9 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 9 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 9;

    (f) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 10 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 10;

    (g) a protein comprising the amino acid sequence as shown in SEQ ID NO: 11 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 11 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 11;

    (h) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 12 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 12;

    (i) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 13 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 13;

    (j) a protein comprising the amino acid sequence as shown in SEQ ID NO: 14 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 14 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 14; and

    (k) a protein comprising the amino acid sequence as shown in SEQ ID NO: 15 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 15 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 15.


     
    18. A method for producing an alkene comprising the step of enzymatically converting a 3-phosphonoxyalkanoate into the corresponding alkene by use of an enzyme which can catalyze the conversion via decarboxylation and dephosphorylation, wherein said 3-phosphonoxyalkanoate, in a first step, is produced by a different enzyme.
     
    19. The method of any one of claims 1 to 12 or the method of claim 18, characterized in that the method is carried out with ATP, dATP, ADP, AMP, an NTP other than ATP, a dNTP or pyrophosphate as co-substrate.
     


    Ansprüche

    1. Verfahren zur Herstellung eines Alkens, dadurch gekennzeichnet, dass es die Umsetzung eines 3-Hydroxyalkanoats in das Alken umfasst durch

    (i) ein erstes Enzym, das eine Aktivität aufweist, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen; und

    (ii) ein zweites Enzym, das unterschiedlich zu dem ersten Enzym ist und eine Aktivität aufweist, das 3-Phosphonoxyalkanoat durch Decarboxylierung und Dephosphorylierung in das Alken umzusetzen.


     
    2. Verfahren nach Anspruch 1, wobei

    (i) das erste Enzym, das eine Aktivität aufweist, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, ausgewählt ist aus der Gruppe bestehend aus

    (A) einem Protein, das die in SEQ ID NO:1 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:1 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:1 dargestellten Aminosäuresequenz;

    (B) einem Protein, das die in SEQ ID NO:2 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:2 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:2 dargestellten Aminosäuresequenz;

    (C) einem Protein, das die in SEQ ID NO:3 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:3 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:3 dargestellten Aminosäuresequenz; und

    (D) einem Protein, das die in SEQ ID NO:4 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:4 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:4 dargestellten Aminosäuresequenz.


     
    3. Verfahren nach Anspruch 1 oder 2, wobei
    (ii) das zweite Enzym, das eine Aktivität aufweist, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, ausgewählt ist aus der Gruppe bestehend aus

    (a) einem Protein, das die in SEQ ID NO:5 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:5 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:5 dargestellten Aminosäuresequenz;

    (b) einem Protein, das die in SEQ ID NO:6 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:6 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:6 dargestellten Aminosäuresequenz;

    (c) einem Protein, das die in SEQ ID NO:7 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:7 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:7 dargestellten Aminosäuresequenz;

    (d) einem Protein, das die in SEQ ID NO:8 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:8 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:8 dargestellten Aminosäuresequenz;

    (e) einem Protein, das die in SEQ ID NO:9 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:9 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:9 dargestellten Aminosäuresequenz;

    (f) einem Protein, das die in SEQ ID NO:10 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:10 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:10 dargestellten Aminosäuresequenz;

    (g) einem Protein, das die in SEQ ID NO:11 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:11 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:11 dargestellten Aminosäuresequenz;

    (h) einem Protein, das die in SEQ ID NO:12 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:12 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:12 dargestellten Aminosäuresequenz;

    (i) einem Protein, das die in SEQ ID NO:13 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:13 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:13 dargestellten Aminosäuresequenz;

    (j) einem Protein, das die in SEQ ID NO:14 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:14 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:14 dargestellten Aminosäuresequenz; und

    (k) einem Protein, das die in SEQ ID NO:15 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die mindestens zu 15% mit der in SEQ ID NO:15 dargestellten Aminosäuresequenz identisch ist, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:15 dargestellten Aminosäuresequenz.


     
    4. Verfahren nach einem der Ansprüche 1 bis 3, das den Schritt des Umsetzens von 3-Hydroxyproprionat in Ethylen umfasst.
     
    5. Verfahren nach einem der Ansprüche 1 bis 3, das den Schritt des Umsetzens von 3-Hydroxybutyrat in Propylen umfasst.
     
    6. Verfahren nach einem der Ansprüche 1 bis 3, das den Schritt des Umsetzens von 3-Hydroxyvalerat in 1-Butylen umfasst.
     
    7. Verfahren nach einem der Ansprüche 1 bis 3, das den Schritt des Umsetzens von 3-Hydroxy-3-methylbutyrat in Isobutylen umfasst.
     
    8. Verfahren nach einem der Ansprüche 1 bis 3, das den Schritt des Umsetzens von 3-Hydroxy-3-methylvalerat in Isoamylen umfasst.
     
    9. Verfahren nach einem der vorangehenden Ansprüche, dadurch gekennzeichnet, dass der Umsetzungsschritt in vitro in einem Zell-freien System durchgeführt wird.
     
    10. Verfahren nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, dass das Verfahren in Anwesenheit eines Mikroorganismus durchgeführt wird, der die beiden wie unter (i) und (ii) von Anspruch 1 definierten Enzyme rekombinant herstellt.
     
    11. Verfahren nach einem der Ansprüche 1 bis 8, das durch die Verwendung einer Pflanze, einer Tierzelle, Pflanzenzelle oder eines Mikroorganismus gekennzeichnet ist, die/der die beiden wie unter (i) und (ii) von Anspruch 1 definierten Enzyme rekombinant herstellt.
     
    12. Verfahren nach einem der vorangehenden Ansprüche, das einen Schritt des Gewinnens von gasförmigen Alkenen umfasst, die aus der Reaktion entgasen.
     
    13. Pflanze, Tierzelle, Pflanzenzelle oder ein Mikroorganismus, die/der mindestens zwei Enzyme rekombinant herstellt, wobei ein Enzym aus (i) ausgewählt ist und das zweite Enzym aus (ii) ausgewählt ist, wobei (i) und (ii) wie folgt sind:

    (i) ein erstes Enzym, das eine Aktivität aufweist, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen; und

    (ii) ein zweites Enzym, das sich von dem ersten Enzym unterscheidet und das eine Aktivität aufweist, das 3-Phosphonoxyalkanoat in das Alken umzusetzen.


     
    14. Zusammensetzung, die den Mikroorganismus nach Anspruch 13, ein geeignetes Kulturmedium und eine 3-Hydroxyalkanoatverbindung oder eine Kohlenstoffquelle umfasst, die von dem Mikroorganismus in eine 3-Hydroxyalkanoatverbindung umgesetzt werden kann.
     
    15. Verwendung einer Kombination von mindestens zwei Enzymen, wobei ein Enzym ausgewählt ist aus dem folgenden (i) und das zweite Enzym ausgewählt ist aus dem folgenden (ii) oder von einem Mikroorganismus nach Anspruch 13 oder einer Zusammensetzung nach Anspruch 14, zur Herstellung von Alkenverbindungen aus 3-Hydroxyalkanoaten,
    wobei (i) und (ii) wie folgt sind:

    (i) ein erstes Enzym, das eine Aktivität aufweist, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen; und

    (ii) ein zweites Enzym, das sich von dem ersten Enzym unterscheidet und das eine Aktivität aufweist, das 3-Phosphonoxyalkanoat in das Alken umzusetzen.


     
    16. Verwendung nach Anspruch 14, wobei

    (i) das erste Enzym, das eine Aktivität aufweist, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, ausgewählt ist aus der Gruppe bestehend aus

    (A) einem Protein, das die in SEQ ID NO:1 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:1 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:1 dargestellten Aminosäuresequenz;

    (B) einem Protein, das die in SEQ ID NO:2 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:2 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:2 dargestellten Aminosäuresequenz;

    (C) einem Protein, das die in SEQ ID NO:3 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz zeigt, die zu mindestens 15% identisch ist mit der in SEQ ID NO:3 dargestellten Aminosäuresequenz, und das eine Aktivität aufweist, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:3 dargestellten Aminosäuresequenz;

    (D) einem Protein, das die in SEQ ID NO:4 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:4 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Hydroxyalkanoat in das entsprechende 3-Phosphonoxyalkanoat umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:4 dargestellten Aminosäuresequenz.


     
    17. Verwendung nach Anspruch 15 oder 16, wobei
    (ii) das zweite Enzym, das eine Aktivität aufweist, das 3 -Phosphonoxyalkanoat in das Alken umzusetzen, ausgewählt ist aus der Gruppe bestehend aus

    (a) einem Protein, das die in SEQ ID NO:5 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:5 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:5 dargestellten Aminosäuresequenz;

    (b) einem Protein, das die in SEQ ID NO:6 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:6 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:6 dargestellten Aminosäuresequenz;

    (c) einem Protein, das die in SEQ ID NO:7 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:7 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:7 dargestellten Aminosäuresequenz;

    (d) einem Protein, das die in SEQ ID NO:8 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:8 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:8 dargestellten Aminosäuresequenz;

    (e) einem Protein, das die in SEQ ID NO:9 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:9 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:9 dargestellten Aminosäuresequenz;

    (f) einem Protein, das die in SEQ ID NO:10 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:10 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:10 dargestellten Aminosäuresequenz;

    (g) einem Protein, das die in SEQ ID NO:11 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:11 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:11 dargestellten Aminosäuresequenz;

    (h) einem Protein, das die in SEQ ID NO:12 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:12 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:12 dargestellten Aminosäuresequenz;

    (i) einem Protein, das die in SEQ ID NO:13 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:13 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:13 dargestellten Aminosäuresequenz;

    (j) einem Protein, das die in SEQ ID NO:14 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:14 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:14 dargestellten Aminosäuresequenz; und

    (k) einem Protein, das die in SEQ ID NO:15 dargestellte Aminosäuresequenz umfasst, oder einem Protein, das eine Aminosäuresequenz umfasst, die zu mindestens 15% identisch ist mit der in SEQ ID NO:15 dargestellten Aminosäuresequenz, und das eine Aktivität zeigt, das 3-Phosphonoxyalkanoat in das Alken umzusetzen, die mindestens so hoch ist wie die entsprechende Aktivität des Proteins mit der in SEQ ID NO:15 dargestellten Aminosäuresequenz.


     
    18. Verfahren zur Herstellung eines Alkens, umfassend den Schritt des enzymatischen Umsetzens eines 3-Phosphonoxyalkanoats in das entsprechende Alken durch Verwendung eines Enzyms, das die Umsetzung über Decarboxylierung und Dephosphorylierung katalysieren kann, wobei das 3-Phosphonoxyalkanoat in einem ersten Schritt von einem anderen Enzym hergestellt wird.
     
    19. Verfahren nach einem der Ansprüche 1 bis 12 oder das Verfahren nach Anspruch 18, dadurch gekennzeichnet, dass das Verfahren mit ATP, dATP, ADP, AMP, einem NTP, das nicht ATP ist, einem dNTP oder Pyrophosphat als Co-Substrat durchgeführt wird.
     


    Revendications

    1. Méthode pour produire un alcène, caractérisée en ce qu'elle comprend la conversion d'un 3-hydroxyalcanoate en ledit alcène par

    (i) une première enzyme ayant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant ; et

    (ii) une seconde enzyme différente de la première enzyme et ayant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène par décarboxylation et déphosphorylation.


     
    2. Méthode selon la revendication 1 dans laquelle

    (i) la première enzyme ayant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant est sélectionnée dans le groupe consistant en

    (A) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 1 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 1 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 1 ;

    (B) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 2 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 2 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 2 ;

    (C) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 3 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 3 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 3 ; et

    (D) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 4 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 4 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 4.


     
    3. Méthode selon la revendication 1 ou 2, dans laquelle
    (ii) la seconde enzyme ayant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène est sélectionnée dans le groupe consistant en

    (a) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 5 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 5 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 5 ;

    (b) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 6 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 6 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 6 ;

    (c) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 7 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 7 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 7 ;

    (d) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 8 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 8 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 8 ;

    (e) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 9 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 9 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 9 ;

    (f) une protéine comprenant la séquence d'acides aminés, telle que représentée dans SEQ ID NO : 10 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 10 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 10 ;

    (g) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 11 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 11 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 11 ;

    (h) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 12 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 12 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 12 ;

    (i) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 13 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 13 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 13 ;

    (j) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 14 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 14 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 14 ; et

    (k) une protéine comprenant la séquence d'acides aminés, telle que représentée dans SEQ ID NO : 15 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 15 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 15.


     
    4. Méthode selon l'une quelconque des revendications 1 à 3, comprenant l'étape de conversion du 3-hydroxy-propionate en éthylène.
     
    5. Méthode selon l'une quelconque des revendications 1 à 3, comprenant l'étape de conversion du 3-hydroxy-butyrate en propylène.
     
    6. Méthode selon l'une quelconque des revendications 1 à 3, comprenant l'étape de conversion du 3-hydroxy-valérate en 1-butylène.
     
    7. Méthode selon l'une quelconque des revendications 1 à 3, comprenant l'étape de conversion du 3-hydroxy-3-méthylbutyrate en isobutylène.
     
    8. Méthode selon l'une quelconque des revendications 1 à 3, comprenant l'étape de conversion du 3-hydroxy-3-méthylvalérate en isoamylène.
     
    9. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que l'étape de conversion est réalisée in vitro, dans un système exempt de cellule.
     
    10. Méthode selon l'une quelconque des revendications 1 à 8 caractérisée en ce que la méthode est réalisée en présence d'un micro-organisme produisant de manière recombinante à la fois lesdites enzymes telles que définies en (i) et (ii) de la revendication 1.
     
    11. Méthode selon l'une quelconque des revendications 1 à 8, caractérisée par l'utilisation d'une plante, d'une cellule animale, d'une cellule végétale ou d'un micro-organisme produisant de manière recombinante à la fois lesdites enzymes telles que définies en (i) et (ii) de la revendication 1.
     
    12. Méthode selon l'une quelconque des revendications précédentes, comprenant une étape de collecte d'alcènes gazeux se dégageant de la réaction.
     
    13. Plante, cellule animale, cellule végétale ou micro-organisme qui produit de manière recombinante au moins deux enzymes, dans lequel une enzyme est sélectionnée parmi (i) et l'autre enzyme est sélectionnée parmi (ii), dans lequel (i) et (ii) sont les suivants :

    (i) une première enzyme ayant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant ; et

    (ii) une seconde enzyme différente de la première enzyme et ayant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène.


     
    14. Composition comprenant le micro-organisme selon la revendication 13, un milieu de culture approprié et un composé de 3-hydroxyalcanoate ou une source de carbone qui peut être convertie par le micro-organisme en un composé de 3-hydroxyalcanoate.
     
    15. Utilisation d'une combinaison d'au moins deux enzymes, dans laquelle une enzyme est sélectionnée parmi le (i) suivant et l'autre enzyme est sélectionnée parmi le (ii) suivant ou d'un micro-organisme selon la revendication 13 ou d'une composition selon la revendication 14, pour produire des composés d'alcène à partir de 3-hydroxyalcanoates,
    dans laquelle (i) et (ii) sont les suivants :

    (i) une première enzyme ayant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant ; et

    (ii) une seconde enzyme différente de la première enzyme et ayant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène.


     
    16. Utilisation selon la revendication 14, dans laquelle

    (i) la première enzyme ayant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant est sélectionnée dans le groupe consistant en

    (A) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 1 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 1 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 1 ;

    (B) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 2 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 2 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 2 ;

    (C) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 3 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 3 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 3 ; et

    (D) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 4 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 4 et présentant une activité de conversion du 3-hydroxyalcanoate en le 3-phosphonoxyalcanoate correspondant qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 4.


     
    17. Utilisation selon la revendication 15 ou 16, dans laquelle
    (ii) la seconde enzyme ayant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène est sélectionnée dans le groupe consistant en

    (a) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 5 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 5 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 5 ;

    (b) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 6 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 6 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 6 ;

    (c) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 7 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 7 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 7 ;

    (d) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 8 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 8 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 8 ;

    (e) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 9 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 9 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 9 ;

    (f) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 10 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 10 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 10 ;

    (g) une protéine comprenant la séquence d'acides aminés, telle que représentée dans SEQ ID NO : 11 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 11 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés "représentée dans SEQ ID NO : 11 ;

    (h) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 12 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 12 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 12 ;

    (i) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 13 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 13 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 13 ;

    (j) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 14 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 14 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 14 ; et

    (k) une protéine comprenant la séquence d'acides aminés telle que représentée dans SEQ ID NO : 15 ou une protéine comprenant une séquence d'acides aminés qui est identique à au moins 15 % à la séquence d'acides aminés représentée dans SEQ ID NO : 15 et présentant une activité de conversion dudit 3-phosphonoxyalcanoate en ledit alcène qui est au moins aussi élevée que l'activité correspondante de la protéine ayant la séquence d'acides aminés représentée dans SEQ ID NO : 15.


     
    18. Méthode pour produire un alcène comprenant l'étape de conversion enzymatique d'un 3-phosphonoxyalcanoate en l'alcène correspondant par l'utilisation d'une enzyme qui peut catalyseur la conversion via décarboxylation et déphosphorylation, dans laquelle ledit 3-phosphonoxyalcanoate, dans une première étape, est produit par une enzyme différente.
     
    19. Méthode selon l'une quelconque des revendications 1 à 12 ou méthode selon la revendication 18, caractérisée en ce que la méthode est réalisée avec de l'ATP, du dATP, de l'ADP, de l'AMP, un NTP autre que l'ATP, un dNTP ou un pyrophosphate comme co-substrat.
     




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