WO2016097289A1 - Production enzymatique d'acrylyl-coa ou d'éthylène à partir de glycérol - Google Patents

Production enzymatique d'acrylyl-coa ou d'éthylène à partir de glycérol Download PDF

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WO2016097289A1
WO2016097289A1 PCT/EP2015/080482 EP2015080482W WO2016097289A1 WO 2016097289 A1 WO2016097289 A1 WO 2016097289A1 EP 2015080482 W EP2015080482 W EP 2015080482W WO 2016097289 A1 WO2016097289 A1 WO 2016097289A1
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coa
enzyme
acrylyl
propionyl
phosphate
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Mathieu Allard
Philippe Marliere
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Global Bioenergies
Scientist Of Fortune S.A.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to a method for the production of acrylyl-CoA from glycerol comprising the following steps: (a) the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde; (b) the enzymatic conversion of said 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA; and (c) the enzymatic conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA.
  • Said acrylyl-CoA can further be enzymatically converted into propionic acid via propionyl-CoA.
  • said acrylyl-CoA can further be enzymatically converted into acrylic acid.
  • said acrylyl-CoA can further be enzymatically converted into 2,4-pentadienoic acid via 3-oxo-4-pentenoyl-CoA, 3-hydroxy-4-pentenoyl-CoA and 2,4-pentadienoyl-CoA.
  • said acrylyl-CoA can further be enzymatically converted into 3-hydroxy-4- pentenoic acid via 3-oxo-4-pentenoyl-CoA and 3-hydroxy-4-pentenoyl-CoA.
  • the present invention furthermore relates to a method for the production of ethylene comprising the enzymatic conversion of propionic acid into ethylene.
  • the enzymatic conversion of propionic acid into ethylene can be achieved by making use of a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase.
  • Said propionic acid can be obtained by the enzymatic conversion of acrylyl-CoA into said propionic acid.
  • Said acrylyl-CoA can be obtained by the enzymatic conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA while said 3-hydroxypropionyl-CoA can be obtained by the enzymatic conversion of 3-hydroxypropionaldehyde (also known as 3-hydroxypropanal) into said 3-hydroxypropionyl-CoA.
  • 3- hydroxypropionaldehyde can be achieved by the enzymatic conversion of glycerol into said 3-hydroxypropionaldehyde.
  • Acrylyl-CoA is a central metabolite and also its derivatives (like, e.g., acrylic acid, propionic acid, 3-oxo-4-pentenoyl-CoA, 3-hydroxy-4-pententoic acid and 2,4- pentadienoic acid) are useful compounds.
  • Acrylic acid is an important compound since, e.g., acrylic acid and its esters readily combine with themselves (to form polyacrylic acid) or other monomers (e.g., acrylamides, acrylonitrile, vinyl, styrene, and butadiene) by reacting at their double bond, forming homopolymers or copolymers. These products may, e.g., be used in the manufacture of various plastics, coatings, adhesives, elastomers, as well as floor polishes, and paints.
  • 2,4-pentadienoic acid is a precursor for the production of butadiene while 3-hydroxy-4-pententoic acid is an organic acid which is a frequently used precursor for the production of poly-hydroxyalkanoate.
  • Propionic acid is known to inhibit the growth of mold and some bacteria and, accordingly, is useful as a preservative for both animal feed and food for human consumption. Propionic acid is also useful as an intermediate in the production of other chemicals, especially polymers. Cellulose-acetate-propionate is a useful thermoplastic. In more specialized applications, it is also used to make pesticides and pharmaceuticals. The esters of propionic acid have fruit-like odors and are sometimes used as solvents or artificial flavorings. Moreover, in accordance with the present invention, propionic acid is a precursor for the enzymatic production of ethylene in vitro or in vivo in a microorganism as described in more detail further below.
  • the present invention therefore, meets this demand for an alternative process for the enzymatic production of acrylyl-CoA which is based on biological resources and which allows to produce acrylyl-CoA (as well as its derivatives acrylic acid, propionic acid, 3-oxo-4-pentenoyl-CoA, 3-hydroxy-4- pententoic acid and 2,4-pentadienoic acid) in vitro or in vivo in a microorganism and in other species by utilizing (a) the enzymatic conversion of glycerol into 3- hydroxypropionaldehyde; (b) the enzymatic conversion of said 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA; and (c) the enzymatic conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA (step III as shown in Figure 14).
  • the present invention also relates to ethylene and its production.
  • Ethylene is the lightest olefin (C 2 H 4 ). This compound is the essential component of the plastic industry for the synthesis of polyethylene (or polyethene), the most used plastic in the world. This polymer is obtained by the action of the Ziegler-Natta catalyst on ethylene monomer. Ethylene is produced by steam cracking or dehydrogenation of ethane, representing the largest C0 2 emitting process in the chemical industry. This process is based on several heating (700-900°C)/ compression and/or separation steps and are, therefore, complex and expensive in energy and high in pollutants, as all the petrochemical process.
  • ACC oxidase converts 1-aminocyclopropane-1 -carboxylic acid to ethylene, C0 2 and HCN by an oxidative decarboxylation mechanism
  • ACC oxidase was already cloned in Escherichia coli and purified as a recombinant protein (Biochem J. 1995, 307, p77-85).
  • the natural metabolic pathway to produce ethylene was already set up in recombinant microorganisms such as Synechocystis PCC6803, utilizing the photosynthetic conversion of C0 2 to ethylene (Energy Environ. Sci. 5 (2012), 8998-9006).
  • the nature of the substrate 1- aminocyclopropane-1-carboxylic acid does not allow a possible industrial application in genetically modified organisms since its biosynthesis from methionine involves several enzymatic steps which are "expensive" in carbon or energy (Proc. Natl. Acad. Sci. 76 (1979), 170-174) and, moreover, the released product during the course of the reaction (i.e., cyanidric acid) is toxic.
  • Some bacteria for example, Escherichia coli or Cryptococcus albidus
  • Escherichia coli or Cryptococcus albidus are known to produce traces of ethylene via oxidation of 2-keto-4-methylthiobutyric acid, a transaminated derivate of methionine (Phytopathology 91 (2001 ), 511-518).
  • this pathway involves a series of complicated steps which renders it difficult to reconstitute it in microorganisms and to use it in an industrial fermentative process.
  • Another way to biosynthesize ethylene is described in bacteria or fungi, such as Pseudomonas syringae or Penicillium cyclopium for example.
  • EFE Ethylene Forming Enzyme
  • the present invention meets this demand for an alternative process for the enzymatic production of ethylene acid which is based on biological resources and which allows to produce ethylene in vitro or in vivo in a microorganism and in other species by utilizing the oxidative decarboxylation of propionic acid which is a relatively non-toxic compound to the olefin ethylene and CO 2 using a cytochrome P450.
  • the present invention provides for a direct way to enzymatically provide propionic acid via propionyl-CoA from glycerol.
  • Glycerol (1 ,2,3- propanetriol or glycerin) is an interesting substrate, as this compound is a waste product from the production of bio-diesel and it is expected to be converted in value- added production by microbial conversion (Energies 6 (2013), 4739-4768), or, for example, by the production of bio-resourced propionic acid via classical chemical catalysis (US 20 3/0231504 A1 ).
  • propionyl-CoA has been described in archaea such as Sulfolobus tokodaii, Metallosphaera sedula and Chloroflexus aurantiacus as a metabolite of the 3-hydroxypropionate cycle (J. Bacteriol. 191 (2009), 4572-4581 ).
  • the present invention provides a method for the production of acrylyl-CoA from glycerol comprising (a) the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde; (b) the enzymatic conversion of said 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA; and (c) the enzymatic conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA.
  • acrylyl-CoA can further be enzymatically converted into its derivatives acrylic acid, propionic acid, 3-oxo-4-pentenoyl-CoA, 3-hydroxy-4-pententoic acid and 2,4- pentadienoic acid in different enzymatic pathways of the present invention.
  • the corresponding reactions are schematically shown in Figure 14.
  • the present invention also provides in a second aspect a process for converting propionic acid into ethylene.
  • the present invention also provides a process by which propionic acid can be produced enzymatically starting from glycerol via propionyl-CoA (also known as propanoyl-CoA) by employing certain enzymes.
  • glycerol can enzymatically be converted to 3- hydroxypropionaldehyde which can be further enzymatically converted to 3- hydroxypropionyl-CoA.
  • 3-hydroxypropionyl-CoA can enzymatically be converted to propionic acid via the intermediate propionyl-CoA.
  • 3-hydroxypropionyl-CoA can enzymatically be converted to propionic acid via the intermediate acrylic acid.
  • the thus produced propionic acid can further be enzymatically converted into ethylene.
  • the corresponding reactions are schematically shown in Figure 1.
  • the enzymatic conversion of glycerol into acrylyl-CoA steps I to III as shown in Figure 14)
  • the present invention relates to a method for the production of acrylyl-CoA from glycerol comprising the following steps:
  • step I (a) the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde (step I as shown in Figure 14);
  • the first step (a) in the method of the production of acrylyl-CoA from glycerol, i.e., the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde is schematically illustrated in Figure 12.
  • the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde preferably makes use of an enzyme which belongs to the family of glycerol dehydratases which naturally catalyze the conversion of glycerol into 3-hydroxypropionaldehyde.
  • Glycerol dehydratases are enzymes using cobalamine (B12 vitamin) as a prosthetic group. These enzymes belong to the family of hydro-lyases which are classified as EC 4.2.1.-.
  • the hydro-lyase (EC 4.2.1.-) employed in a method according to the invention for the conversion of glycerol into 3- hydroxypropionaldehyde is a glycerol dehydratase (EC 4.2.1.30), preferably a cobalamine (B12 vitamin)-dependent or, alternatively, a B12-indepentent/radical-S- adenosyl methionine-dependent glycerol dehydratase (EC 4.2.1.30).
  • Glycerol dehydratases (EC 4.2.1.30) catalyze the following reaction: glycerol . « 3 ⁇ 4 3-hydroxypropanal + H 2 0
  • Glycerol dehydratases occur in a variety of organism, including prokaryotic organisms, such as bacteria.
  • the enzyme has, e.g., been described in Citrobacter freundii, Citrobacter intermedicus, Clostridium butyricum, Clostridium pasteurianum, E. blattae, E. coli, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus brevis, Lactobacillus buchneri and Pantoea agglomerans.
  • the step of the enzymatic conversion of glycerol into 3- hydroxypropionaldehyde is catalyzed by a cobalamine (B12 vitamin)-dependent glycerol dehydratase from Klebsiella pneumoniae or Lactobacillus reuteri as their heterologous expression was already described in E. coli (Biotechnol. J. 6 (2007), 736-742 and Microbial Cell Factories 13 (2014), 76-86).
  • the conversion of glycerol into 3-hydroxypropionaldehyde is achieved by making use of a glycerol dehydratase from Klebsiella pneumoniae, preferably by the glycerol dehydratase alpha subunit from Klebsiella pneumoniae.
  • the amino acid sequence of said protein is shown in SEQ ID NO:22.
  • the conversion of glycerol into 3- hydroxypropionaldehyde is achieved by making use of a glycerol dehydratase from Klebsiella pneumoniae, preferably by the glycerol dehydratase medium subunit from Klebsiella pneumoniae.
  • the amino acid sequence of said protein is shown in SEQ ID NO:23.
  • the conversion of glycerol into 3- hydroxypropionaldehyde is achieved by making use of a glycerol dehydratase from Klebsiella pneumoniae, preferably by the glycerol dehydratase gamma subunit from Klebsiella pneumoniae.
  • the amino acid sequence of said protein is shown in SEQ ID NO:24.
  • an enzyme which comprises a sequence which is at least 60% identical to any one of the amino acid sequences shown in SEQ ID NOs: 22 to 24.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to any one of SEQ ID NOs:22 to 24 and the enzyme has the enzymatic activity of converting glycerol into 3-hydroxypropionaldehyde.
  • the degree of identity refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. Preferably, it refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence.
  • the degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.
  • the Clustal analysis method determines whether a particular sequence is, for instance, at least 60% identical to a reference sequence
  • default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences.
  • the Extend gap penalty is preferably set to 5.0.
  • ClustalW2 is used for the comparison of amino acid sequences.
  • the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1.
  • the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.
  • the degree of identity is calculated over the complete length of the sequence.
  • Amino acid residues located at a position corresponding to a position as indicated herein-below in the amino acid sequence shown in any one of SEQ ID NOs:22 to 24 can be identified by the skilled person by methods known in the art.
  • such amino acid residues can be identified by aligning the sequence in question with the sequence shown in any one of SEQ ID NOs:22 to 24 and by identifying the positions which correspond to the above indicated positions of any one of SEQ ID NOs:22 to 24.
  • the alignment can be done with means and methods known to the skilled person, e.g. by using a known computer algorithm such as the Lipman- Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. It is preferred that in such an alignment maximum homology is assigned to conserved amino acid residues present in the amino acid sequences.
  • ClustalW2 is used for the comparison of amino acid sequences.
  • the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1.
  • the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.
  • the step of the enzymatic conversion of glycerol into 3- hydroxypropionaldehyde is catalyzed by a B12-indepentent glycerol dehydratase which is radical-S-adenosyl methionine-dependent.
  • B12-indepentent glycerol dehydratase which is radical-S-adenosyl methionine-dependent have been described in Clostridium.
  • the family members of this type of glycerol dehydratases use a radical-SAM (S-Adenosyl methionine) instead of coenzyme B 12 based mechanism as it is described in Biochemistry. 43 (2004), 4635-4645. While these enzymes catalyze the above conversion, they operate strictly under anaerobic conditions. Accordingly, they are preferably employed in embodiments in which a method according to the present invention is carried out under anaerobic conditions.
  • the second step (b) in the method of the production of acrylyl-CoA from glycerol, i.e., the enzymatic conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is schematically illustrated in Figure 11.
  • the enzymatic conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionyl-CoA preferably makes preferably use of an enzyme which belongs to the family of Coenzyme-A-acylating aldehyde dehydrogenases. These dehydrogenases are oxidoreducates which act on the aldehyde or oxo group of donors and use either NAD(+) or NADP(+) as acceptor.
  • the family of Coenzyme-A-acylating aldehyde dehydrogenases is classified as EC 1.2.1.-.
  • the Coenzyme-A-acylating aldehyde dehydrogenase employed in a method according to the invention for the conversion of 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1 .10).
  • CoA-acylating propionaldehyde dehydrogenases (EC 1.2.1.87) (also termed propanal dehydrogenase (CoA-propanoylating)) catalyze the following reaction: propanal + CoA + NAD + ⁇ 3 ⁇ 4>ropanoyl-CoA + NADH + H +
  • This enzyme occurs in a number of organisms in particular in bacteria, and the enzyme has been described, e.g., for Burkholderia xenovorans and Thermus thermophilus.
  • the step of the enzymatic conversion of 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is catalyzed by the coenzyme A-acylating propionaldehyde dehydrogenase (gene: PduP, EC 1.2.1.87) from Lactobacillus reuteri (Uniprot accession number: B2G9K7).
  • PduP propionaldehyde dehydrogenase
  • Lactobacillus reuteri Uniprot accession number: B2G9K7
  • the conversion of 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA is achieved by making use of a CoA-acylating propionaldehyde dehydrogenase from Lactobacillus reuteri, preferably the CoA- dependent propionaldehyde dehydrogenase from Lactobacillus reuteri strain JCM 1112.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 25.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 25.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:25 and the enzyme has the enzymatic activity of converting 3-hydroxypropionaldehyde into 3-hydroxypropionyl- CoA.
  • sequence identity the same applies as has been set forth above.
  • Acetaldehyde dehydrogenases (EC 1.2.1.10) catalyze the following reaction: acetaldehyde + CoA + NAD + acetyl-CoA + NADH + H +
  • This reaction is the key step of the first segment of the metabolic pathway connecting the known formation of 3-hydroxypropionaldehyde from glycerol to the 3- hydroxypropionate bicycle pathway as already outlined above in the introductory section (Appl. Environ. Microbiol. 78 (2012), 8564-8570).
  • Acetaldehyde dehydrogenases occur in a variety of organism, including prokaryotic organisms, such as bacteria.
  • the enzyme has, e.g., been described in Acinobacter sp., Burkholderia xenovorans, Clostridium beijerinckii, Clostridium klyveri, E. coli, Giardia intestinalis, Leuconostoc mesenteroides, Propionibacterium freudenreichii, Pseudomonas sp., and Thermoanaerobacter ethanolicus.
  • the enzyme classified as Coenzyme-A-acylating aldehyde dehydrogenases use NADH or NADPH as a co-factor.
  • the enzyme is an enzyme which uses NADH as a co-factor.
  • Several enzymes of the general family of Coenzyme-A-acylating aldehyde dehydrogenases are also described to be able to use NADPH as reducing cofactor (Appl. Env. Microbiol. 56 (1990), 2591-2599). These conversions using either NADH or NADPH as a reducing cofactor are schematically shown in Figure 11.
  • the third step (c) in the method of the production of acrylyl-CoA from glycerol, i.e., the enzymatic conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA is schematically illustrated in Figure 10.
  • the enzymatic conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA preferably makes use of an enzyme catalyzing 3-hydroxypropionyl-CoA dehydration.
  • the term "dehydration" is generally referred to as a reaction involving the removal of H 2 O.
  • Enzymes catalyzing 3-hydroxypropionyl-CoA dehydration are enzymes which catalyze the reaction as shown in Figure 10.
  • such an enzyme belongs to the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases (EC 4.2.1.-).
  • the present invention relates to a method for the enzymatic conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA by making use of an enzyme catalyzing 3-hydroxypropionyl-CoA dehydration, preferably of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-).
  • an enzyme catalyzing 3-hydroxypropionyl-CoA dehydration preferably of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-).
  • Examples for enzymes catalyzing 3- hydroxypropionyl-CoA dehydration which can be employed in the method of the present invention are the following enzymes which are all classified as E.C. 4.2.1._ (i.e., hydro-lyases):
  • R 1 is a hydrogen atom or an alkyl group or CH 2 COO " ;
  • R 2 is a hydrogen atom or a methyl group
  • R 3 is coenzyme A or acyl-carrier protein.
  • R3 in the above shown formula is acyl-carrier protein
  • This group includes EC 4.2.1.58, EC 4.2.1.59, EC 4.2.1.60 and EC 4.2.1.61.
  • the enzymes of this group have in common that they catalyze a reaction of the following type:
  • This group includes EC 4.2.1.116, EC 4.2.1.55, EC 4.2.1.17, EC 4.2.1.74 and EC 4.2.1.18
  • the enzymes of this group share a common structural motif which is referenced in the InterPRO database as InterPro IPR001753 (http://www.ebi.ac.uk/interpro/entry/IPR001753) and IPR00 8376
  • the conversion of 3- hydroxypropionyl-CoA into acrylyl-CoA is achieved by the use of a 3- hydroxypropionyl-CoA dehydratase (EC 4.2.1.116).
  • 3-hydroxypropionyl-CoA dehydratases (EC 4.2.1.116) catalyze the following reaction:
  • a bacterial 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) is used, preferably a 3-hydroxypropionyl-CoA dehydratase from a bacterium or an archaebacterium of a genus selected from the group consisting of Metallosphaera, Sulfolobus and Brevibacillus and most preferably from a species selected from the group consisting of Metallosphaera cuprina, Metallosphaera sedula, Sulfolobus tokodaii and Brevibacillus laterosporus.
  • Examples for such bacterial 3-hydroxypropionyl-CoA dehydratases are the enzymes from Metallosphaera cuprina (Uniprot F4FZ85), Metallosphaera sedula (Uniprot A4YI89, Teufel et al., J. Bacteriol. 191 (2009), 4572-4581 ), Sulfolobus tokodaii (Uniprot F9VNG3) and Brevibacillus laterosporus (Uniprot F7TTZ1 ). Amino acid and nucleotide sequences for these enzymes are available.
  • SEQ ID NOs: 9 to 12 Examples for corresponding amino acid sequences are provided in SEQ ID NOs: 9 to 12 wherein SEQ ID NO:9 is the amino acid sequence of 3-hydroxypropionyl-CoA dehydratase of M. cuprina, SEQ ID NO: 10 is the amino acid sequence of 3-hydroxypropionyl-CoA dehydratase of M. sedula, SEQ ID NO:11 is the amino acid sequence of a 3-hydroxypropionyl-CoA dehydratase of S. tokodaii and SEQ ID NO: 2 is the amino acid sequence of a 3- hydroxypropionyl-CoA dehydratase of Brevibacillus laterosporus.
  • the 3-hydroxypropionyl-CoA dehydratase employed in the method of the invention has an amino acid sequence as shown in any one of SEQ ID NOs: 9 to 12 or shows an amino acid sequence which is at least x% homologous to any of SEQ ID NOs: 9 to 12 and has the activity of catalyzing the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA, with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99.
  • any 3-hydroxypropionyl-CoA dehydratase can be employed in the method according to the invention.
  • a 3-hydroxypropionyl-CoA dehydratase for converting 3- hydroxypropionyl-CoA into said acrylyl-CoA but also enzymes which show the structural and functional similarities as described above, i.e. enzymes as listed in items (b) to (f), above.
  • the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55).
  • 3-hydroxybutyryl-CoA dehydratases (EC 4.2.1.55) catalyze the following reaction:
  • 3-hydroxybutyryl-CoA dehydratase belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is (3R)-3- hydroxybutanoyl-CoA hydro-lyase (crotonoyl-CoA-forming).
  • Other names in common use include D-3-hydroxybutyryl coenzyme A dehydratase, D-3-hydroxybutyryl-CoA dehydratase, enoyl coenzyme A hydratase, and (3R)-3-hydroxybutanoyl-CoA hydro- lyase. This enzyme participates in the butanoate metabolism.
  • Enzymes belonging to this class and catalyzing the above shown conversion of 3-hydroxybutyryl-Coenzyme A into crotonyl-Coenzyme A have been described to occur, e.g. in rat (Rattus norvegicus), in Rhodospirillum rubrum, in Sulfolobus acidocaldarius and in Acidianus hospitalis. Nucleotide and/or amino acid sequences for such enzymes have been determined, e.g. for Aeropyrum pernix.
  • any 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) which can catalyze the conversion of 3-hydroxypropionyl- CoA into said acrylyl-CoA can be used in the context of the present invention.
  • a 3-hydroxybutyryl-CoA dehydratase from an archaebacterium is used, preferably a 3-hydroxybutyryl-CoA dehydratase from an archaebacterium of a genus selected from the group consisting of Sulfolobus and Acidianus and most preferably from a species selected from the group consisting of S. acidocaldarius and Acidianus hospitalis.
  • Examples for such bacterial 3- hydroxybutyryl-CoA dehydratases are the enzymes from Sulfolobus acidocaldarius (Uniprot Q4J8D5) and from Acidianus hospitalis ((Uniprot F4B9R3).
  • Examples for corresponding amino acid sequences are provided in SEQ ID NOs: 13 and 14 wherein SEQ ID NO:13 is the amino acid sequence of 3-hydroxybutyryl-CoA dehydratase of Sulfolobus acidocaldarius and SEQ ID NO: 14 is the amino acid sequence of 3-hydroxybutyryl-CoA dehydratase of Acidianus hospitalis.
  • the 3-hydroxybutyryl-CoA dehydratase employed in the method of the invention has an amino acid sequence as shown in SEQ ID NO: 13 or 14 or shows an amino acid sequence which is at least x% homologous to SEQ ID NO: 13 or 14 and has the activity of catalyzing the conversion of 3-hydroxypropionyl- CoA into acrylyl-CoA, with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of an enoyl-CoA hydratase (EC 4.2.1.17).
  • Enoyl-CoA hydratases (EC 4.2.1.17) catalyze the following reaction:
  • Enoyl-CoA hydratase is an enzyme that normally hydrates the double bond between the second and third carbons on acyl-CoA. However, it can also be employed to catalyze the reaction in the reverse direction. This enzyme, also known as crotonase, is naturally involved in metabolizing fatty acids to produce both acetyl-CoA and energy. Enzymes belonging to this class have been described to occur, e.g. in rat (Rattus norvegicus), humans (Homo sapiens), mouse (Mus musculus), wild boar (Sus scrofa), Bos taurus, E.coli, Clostridium acetobutylicum and Clostridium aminobutyricum.
  • Nucleotide and/or amino acid sequences for such enzymes have been determined, e.g. for rat, humans and Bacillus subtilis.
  • any enoyl- CoA hydratase (EC 4.2.1.17) which can catalyze the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59).
  • 3-hydroxyoctanoyl- [acyl-carrier-protein] dehydratases (EC 4.2.1.59) catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is (3R)-3- hydroxyoctanoyl-[acyl-carrier-protein] hydro-lyase (oct-2-enoyl-[acyl-carrier protein]- forming).
  • any 3-hydroxyoctanoyl- [acyl-carrier-protein] dehydratase which can catalyze the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a crotonoyl- [acyl-carrier-protein] hydratase (EC 4.2.1.58).
  • Crotonoyl-[acyl-carrier-protein] hydratases catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • Crotonoyl-[acyl-carrier-protein] hydratase has been described to exist, e.g., in E. coli and Arabidopsis thaliana.
  • any crotonoyl-[acyl-carrier- protein] hydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60).
  • 3- hydroxydecanoyl-[acyl-carrier-protein] dehydratases catalyze the following reactions:
  • the enzyme has been described to exist, e.g., in Pseudomonas aeruginosa, Pseudomonas fluorescens, Toxoplasma gondii, Plasmodium falciparum, Helicobacter pylori, Corynebacterium ammoniagenes, Enterobacter aerogenes, E. coli, Proteus vulgaris and Salmonella enterica.
  • any 3-hydroxydecanoyl-[acyl-carrier- protein] dehydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61 ).
  • 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratases catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • any 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a long-chain- enoyl-CoA hydratase (EC 4.2.1.74).
  • Long-chain-enoyl-CoA hydratases (EC 4.2.1.74) catalyze the following reaction :
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is long- chain-(3S)-3-hydroxyacyl-CoA hydro-lyase.
  • This enzyme is also called long-chain enoyl coenzyme A hydratase and it participates in fatty acid elongation in mitochondria and fatty acid metabolism.
  • This enzyme occurs in a number of organisms, e.g., in Rattus norvegicus (Wu et al., Org. Lett. 10 (2008), 2235-2238), Sus scrofa and Cavia porcellus (Fong and Schulz, J. Biol. Chem.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- methylglutaconyl-CoA hydratase (EC 4.2.1.18).
  • 3-methylglutaconyl-CoA hydratases (EC 4.2.1.18) catalyze the following reaction: (S)-3-hydroxymethylglutaryl -CoA + H 2 0
  • This enzyme occurs in a number of organisms in particular in bacteria, plants and animals.
  • the enzyme has been described, e.g., for Pseudomonas putida, Acinetobacter sp. (SwissProt accession number Q3HW12), Catharanthus roseus, Homo sapiens (SwissProt accession number Q13825), Bos taurus and Ovis aries and in principle any 3-methylglutaconyl-CoA hydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA can be employed in the method of the invention.
  • 3-methylglutaconyl-CoA hydratase also covers the enzyme encoded by the gene LiuC (Li et al., Angew. Chem. Int. Ed. 52 (2013), p. 1304-1308; Uniprot number Q1 D5Y4) from Myxococcus xanthus, preferably from strain DK 1622.
  • the enzymatic conversion of 3- hydroxypropionyl-CoA into acrylyl-CoA is achieved by making use of a 3- hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) or an enoyl-CoA hydratase (EC 4.2.1.17) as described above.
  • the acrylyl-CoA which is produced according to the above method from glycerol may further be converted into propionyl-CoA. Moreover, the thus produced propionyl-CoA may further be converted into propionic acid.
  • the present invention also relates to a method for producing propionyl-CoA from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into into acrylyl-CoA which is then further converted into propionyl- CoA.
  • the present invention also relates to a method for producing propionic acid from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into into acrylyl-CoA which is then further converted into propionyl- CoA which is then further converted into propionic acid.
  • the conversion of acrylyl-CoA into propionyl-CoA can, for example, be achieved by making use of an enzyme classified as EC 1.3.1.-.
  • Enzymes classified as EC 1.3.1.- are enoyl-CoA reductases.
  • the enzyme is an enzyme which is classified as EC 1.3.1.- and which uses NADH or NADPH as a co-factor.
  • the enzyme is an enzyme which uses NADH as a co-factor.
  • Several enzymes of the general family of enoyl-CoA reductase are also described to be able to use NADPH as reducing cofactor (J. Biochem. 1984, 95, p1315-1321). The conversion using such an enzyme is schematically shown in Figure 4.
  • the enzyme is an enzyme which uses NADPH as a co-factor.
  • the enzyme is selected from the group consisting of.
  • NADP+ - acyl-CoA dehydrogenase
  • NADPH - cis-2-enoyl-CoA reductase
  • NADPH - trans-2-enoyl-CoA reductase
  • acrylyl-CoA into propionyl-CoA is achieved by making use of an acyl-CoA dehydrogenase (NADP+) (EC 1.3.1.8).
  • Acyl-CoA dehydrogenases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Bos, taurus, Rattus novegicus, Mus musculus, Columba sp., Arabidopsis thaliana, Nicotiana benthamiana, Allium ampeloprasum, Euglena gracilis, Candida albicans, Streptococcus collinus, Rhodobacter sphaeroides and Mycobacterium smegmatis.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an enoyl-[acyl-carrier-protein] reductase (NADPH, Si- specific) (EC 1.3.1.10).
  • Enoyl-[acyl-carrier-protein] reductases (NADPH, Si-specific) are enzymes which catalyze the following reaction: acyl-[acyl-carrier-protein] + NADP + endeavour » * trans-2,3-dehydroacyl-[acyl- carrier-protein] + NADPH + H +
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, fungi and bacteria.
  • the enzyme has, e.g., been described in Carthamus tinctorius, Candida tropicalis, Saccharomyces cerevisiae, Streptococcus collinus, Streptococcus pneumoniae, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Porphyromonas gingivalis, Escherichia coli and Salmonella enterica.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a cis-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.37).
  • Cis- 2-enoyl-CoA reductases (NADPH) are enzymes which catalyze the following reaction: Acyl-CoA + NADP + ⁇ : cis-2,3-dehydroacyl-CoA + NADPH + H +
  • This enzyme has been described to occur in Escherichia coli.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a trans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38).
  • NADPH trans-2-enoyl-CoA reductases
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals and bacteria.
  • the enzyme has, e.g., been described in Homo sapiens, Rattus norvegicus, Mus musculus, Cavia porcellus, Caenorhabditis elegans, Phalaenopsis amabilis, Gossypium hirsutum, Mycobacterium tuberculosis, Streptococcus collinu and Escherichia coli.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an enoyl-[acyl-carrier-protein] reductase (NADPH, Re- specific) (EC 1.3.1.39).
  • Enoyl-[acyl-carrier-protein] reductases (NADPH, Re-specific) are enzymes which catalyze the following reaction: acyl-[acyl-carrier-protein] + NADP + ⁇ * " trans-2,3-dehydroacyl-[acyl- carrier-protein] + NADPH + H +
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as animals and bacteria.
  • the enzyme has, e.g., been described in Gallus gallus, Pigeon, Rattus norvegicus, Cavia porcellus, Staphylococcus aureus, Bacillus subtilis and Porphyromonas gingivalis.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a crotonyl-CoA reductase (EC 1.3.1.86).
  • Crotonyl-CoA reductases are enzymes which catalyze the following reaction: butanoyl-CoA + NADP + disturb > (E)-but-2-enoyl-CoA + NADPH + H +
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Bos taurus, Salinospora tropica, Clostridium difficile, Streptomyces collinus, Streptomyces cinnamonensis and Streptomyces hygroscopicus.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an NADPH-dependent acrylyl-CoA reductase (EC 1.3.1.84).
  • NADPH-dependent acrylyl-CoA reductases are enzymes which catalyze the following reaction: propanoyl-CoA + NADP +schreib » acryloyl-CoA + NADPH + H +
  • This enzyme occurs in a variety of organism, including prokaryotic organisms and the enzyme has, e.g., been described in Metallosphaera sedula and Sulfolobus tokodaii.
  • the enzyme is an enzyme which uses NADH as a co-factor.
  • the conversion using such an enzyme is schematically shown in Figure 4.
  • the enzyme is selected from the group consisting of:
  • NADH acyl-carrier-protein] reductase
  • NAD + trans-2-enoyl-CoA reductase
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an enoyl-[acyl-carrier-protein] reductase (NADH) (EC 1.3.1.9).
  • NADH enoyl-[acyl-carrier-protein] reductases
  • NADH enzymes which catalyze the following reaction: acyl-[acyl-carrier-protein] + NAD + « « * trans-2,3-dehydroacyl-[acyl- carrier-protein] + NADH + H + This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Plasmodium falciparum, Eimeria tenella, Toxoplasma gondii, Mycobacterium tuberculosis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Bacillus anthracis, Birkholderia mallei, Pseudomonas aeruginosa, Helicobacter pylori, Yersinia pestis and many others.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a trans-2-enoyl-CoA reductase (NAD + ) (EC 1.3.1.44).
  • NAD + trans-2-enoyl-CoA reductases
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals and bacteria.
  • the enzyme has, e.g., been described in Ratus norvegicus, Euglena gracilis, Mycobacterium smegmatis, Pseudomonas fluorescens, Clostridium acetobutylicum, Butyrivibrio fibrisolvens, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Treponema denticola.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an acrylyl-CoA reductase (aka acryloyl-CoA reductase) (EC 1.3.1.95).
  • acrylyl-CoA reductase aka acryloyl-CoA reductase
  • These enzymes are electron transferring flavoproteins (J. Bacteriol. 191 (2009), 4572-4581 ; Eur. J. Biochem. 270 (2003), 902-910).
  • An acrylyl-CoA reductase was already cloned in E. coli for a pathway to propionic acid biosynthesis (Appl. Microbiol. Biotechnol. 97 (2013), 1191-2000).
  • Acrylyl-CoA reductases are enzymes which catalyze the following reaction propanoyl-CoA + NAD + ⁇ * acryloyl-CoA + NADH + H +
  • This enzyme occurs in a variety of prokaryotic organisms and the enyzme has, e.g., been described in Clostridium kluyveri, Clostridium propionicum, Metallosphaera sedula and Sufolobus tokodaii.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an acryloyl-CoA reductase from Metallosphaera sedula, preferably from Metallosphaera sedula strain ATCC 51363.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 26.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 26.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:26 and the enzyme has the enzymatic activity of converting acrylyl-CoA into propionyl-CoA.
  • sequence identity the same applies as has been set forth above.
  • the propionyl-CoA produced according to the above described method from glycerol is not further converted.
  • the propionyl-CoA is the end product of the method of the present invention and, accordingly, the present invention relates to a method for producing propionyl-CoA from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into propionyl-CoA wherein said propionyl-CoA is not further converted into another compound.
  • the above described method for producing propionyl- CoA further comprises the step of recovering the thus produced propionyl-CoA.
  • the propionyl-CoA produced according to the above described method from glycerol is not further converted into 3-oxopentanoyl- CoA.
  • the present invention relates to a method for producing propionyl-CoA from glycerol in which glycerol is first converted into 3- hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into propionyl-CoA wherein said propionyl-CoA is not further converted into 3-oxopentanoyl-CoA.
  • the propionyl-CoA produced according to the above described method from glycerol is further converted into another compound which is not 3-oxopentanoyl-CoA.
  • the present invention relates to a method for producing another compound which is not 3- oxopentanoyl-CoA from glycerol in which glycerol is first converted into 3- hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into propionyl-CoA wherein said propionyl-CoA is converted into another compound which is not 3-oxopentanoyl-CoA.
  • the propionyl-CoA produced according to the above described method from glycerol may be further converted into propionic acid wherein said propionic acid may then further be converted into ethylene.
  • the conversion of propionyl-CoA into propionic acid can be achieved by three alternative enzymatic conversions.
  • One possibility is a two-step conversion via propionyl phosphate.
  • Two other options involve a direct conversion of propionyl-CoA into propionic acid. These three options will be discussed in the following.
  • the enzymatic conversion of propionyl-CoA into propionic acid can be achieved by a two-step conversion via propionyl phosphate.
  • the enzymatic conversion of propionyl-CoA into propionic acid (step V as shown in Figure 1 ) is achieved by two enzymatic steps comprising (i) first enzymatically converting propionyl-CoA into propionyl phosphate; and (ii) then enzymatically converting the thus obtained propionyl phosphate into said propionic acid.
  • propionyl-CoA into propionyl phosphate
  • conversion of propionyl-CoA into propionyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).
  • Phosphate butyryltransferase (EC 2.3.1.19) naturally catalyzes the following reaction Butyryl-CoA + H 3 PO 4 ⁇ * * " butyryl phosphate + CoA
  • phosphate butyryltransferases (EC 2.3.1.19) can use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA), in particular acetyl-CoA, propionyl-CoA, isobutyryl- CoA, valeryl-CoA and isovaleryl-CoA.
  • butyryl-CoA butyryl coenzyme A
  • the enzyme has been described to occur in a number of organism, in particular in bacteria and in protozoae.
  • the enzyme is from the protozoae Dasytricha ruminantium.
  • the phosphate butyryltransferase is a phosphate butyryltransferase from a bacterium, preferably from a bacterium of the genus Bacillus, Butyrivibrio, Enterococcus or Clostridium, more preferably Enterococcus or Clostridium, and even more preferably from Bacillus megaterium, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium butyricum, Clostridium kluyveri, Clostridium saccharoacetobutylicum, Clostridium sprorogenes or Enterococcus faecalis.
  • the enzyme is from Clostridium acetobutylicum, in particular the enzyme encoded by the ptb gene (Uniprot Accession number F0K6W0; Wiesenborn et al. (Appl. Environ. Microbiol. 55 (1989), 317-322)) or from Enterococcus faecalis (Untprot Accession number K4YRE8; Ward et al. (J. Bacteriol. 181 (1999), 5433-5442)).
  • ptb gene Uniprot Accession number F0K6W0; Wiesenborn et al. (Appl. Environ. Microbiol. 55 (1989), 317-322)
  • Enterococcus faecalis Untprot Accession number K4YRE8; Ward et al. (J. Bacteriol. 181 (1999), 5433-5442)
  • the conversion of propionyl-CoA into propionyl phosphate is achieved by making use of a phosphate butyryltransferase from Clostridium acetobutylicum, preferably from Clostridium acetobutylicum strain ATCC 824.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 20.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 20.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:20 and the enzyme has the enzymatic activity of converting propionyl-CoA into propionyl phosphate.
  • sequence identity the same applies as has been set forth above.
  • Phosphate acetyltransferase (EC 2.3.1.8) naturally catalyzes the following reaction Acetyl-CoA + H 3 PO 4 acetyl phosphate + CoA
  • accession numbers for this enzyme family in InterPro database are IPR012147 and IPR002505, "http://www.ebi.ac.uk/interpro/entry/IPR002505"
  • the enzyme has been described in a variety of organisms, in particular bacteria and fungi.
  • the enzyme is an enzyme from a bacterium, preferably of the genus Escherichia, Chlorogonium, Clostridium, Veillonella, Methanosarcina, Corynebacterium, Ruegeria, Salmonella, Azotobacter, Bradorhizobium, Lactobacillus, Moorella, Rhodopseudomonas, Sinorhizobium, Streptococcus, Thermotoga or Bacillus, more preferably of the species Escherichia coli, Chlorogonium elongatum, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium acidurici, Veillonella parvula, Methanosarcina thermophila, Corynebacterium glutamicum, Ruegeria pomeroyi, Salmonella enterica, Azotobacter vinelandii, Bradyr
  • the conversion of propionyl-CoA into propionyl phosphate is achieved by making use a phosphate acetyltransferase from Corynebacterium glutamicum, preferably from Corynebacterium glutamicum strain ATCC 13032.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 21.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 21.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:21 and the enzyme has the enzymatic activity of converting propionyl-CoA into propionyl phosphate.
  • sequence identity the same applies as has been set forth above.
  • the conversion of propionyl phosphate into propionic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2 -, i.e., a phosphotransferase. Such enzymes use a carboxy group as acceptor.
  • the conversion of propionyl phosphate into propionic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-).
  • the conversion of propionyl phosphate into propionic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
  • a propionate kinase EC 2.7.2.15
  • an acetate kinase EC 2.7.2.1
  • a butyrate kinase EC 2.7.2.7
  • a branched-chain-fatty-acid kinase EC 2.7.2.14
  • Butyrate kinases (EC 2.7.2.7) naturally catalyze the following reaction Butyrate + ATP ⁇ " * * "butvryl phosphate + ADP
  • butyrate kinase can use a number of substrates in addition to butyrate, e.g. valerate, isobutyrate, isovalerate and vinyl acetate.
  • the enzyme has been described in a variety of organisms, in particular bacteria.
  • the enzyme is from a bacterium, preferably from a bacterium of the genus Clostridium, Butyrivibrio, Thermotoga or Enterococcus. Preferred is Clostridium.
  • the enzyme is from a bacterium of the species Clostridium acetobutylicum, Clostridium proteoclasticum, Clostridium tyrobutyricum, Clostridium butyricum, Clostridium pasteurianum, Clostridium tetanomorphum, Butyrivibrio firbrosolvens, Butyrivibrio hungatei, Thermotoga maritime or Enterococcus durans.
  • Clostridium acetobutylicum For this organism two butyrate kinases have been described: butyrate kinase 1 (Uniprot Accession number: Q45829) and butyrate kinase II (Uniprot Accession number: Q97II19).
  • Branched-chain-fatty-acid kinases (EC 2.7.2.14) naturally catalyze the following reaction
  • alkyl may be 2-methylbutanoate, butanoate, isobutanoate, pentanoate or propionate.
  • the latter reaction with propionate has been described for a branched- chain fatty acid kinase from a spirochaete (J. Bacteriol. 152 (1982), 246-54).
  • the enzyme has been described to occur in a number of bacteria.
  • the enzyme is an enzyme from a bacterium, preferably of the genus Spirochaeta or Thermotoga, more preferably Thermotoga maritime.
  • the enzyme is an enzyme from a bacterium, preferably of the genus Salmonella or Escherichia, more preferably of the species Salmonella enterica, Salmonella typhimurium or Escherichia coli.
  • the conversion of propionyl phosphate into propionic acid is achieved by making use of a propionate kinase from Salmonella typhimurium, preferably from Salmonella typhimurium strain ATCC 700720.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 27.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 27.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:27 and the enzyme has the enzymatic activity of converting propionyl phosphate into propionic acid.
  • sequence identity the same applies as has been set forth above.
  • the conversion of propionyl phosphate into propionic acid is achieved by making use of a propionate kinase from Escherichia coli, preferably from Escherichia coli strain K12.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 28.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 28.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:28 and the enzyme has the enzymatic activity of converting propionyl phosphate into propionic acid.
  • the sequence identity the same applies as has been set forth above.
  • Acetate kinases (EC 2.7.2.1 ) naturally catalyze the following reaction Acetate + ATP ⁇ r ⁇ acetvl phosphate + ADP
  • the enzyme has been described to occur in a number of organisms, in particular bacteria and eukaryotes.
  • the enzyme is from a bacterium, preferably from a bacterium of the genus Methanosarcina, Cryptococcus, Ethanoligenens, Propionibacterium, Roseovarius, Streptococcus, Salmonella, Acholeplasma, Acinetobacter, Ajellomyces, Bacillus, Borrelia, Chaetomium, Clostridium, Coccidioides, Coprinopsis, Cryptococcus, Cupriavidus, Desulfovibrio, Enterococcus, Escherichia, Ethanoligenes, Geobacillus, Helicobacter, Lactobacillus, Lactococcus, Listeria, Mesoplasma, Moorella, Mycoplasma, Oceanobacillus, Propionibacterium, Rhodospeudomonas, Roseovarius, Salmonella, Staphylococcus, Thermotoga or
  • the enzyme is an enzyme from a fungus, preferably from a fungus of the genus Aspergillus, Gibberella, Hypocrea, Magnaporthe, Phaeosphaeria, Phanerochaete, Phytophthora, Sclerotinia, Uncinocarpus, Ustilago or Neurospora even more preferably from a fungus of the species Aspergillus fumigates, Aspergillus nidulans, Gibberella zeae, Hypocrea jecorina, Magnaporthe grisea, Phaeosphaeria nodorum, Phanerochaete chrysosporium, Phytophthora ramorum, Phytophthora sojae, Sclerotinia sclerotiorum, Uncinocarpus reesii, Ustilago maydis or Neurospora crassa.
  • the enzyme is an enzyme from a plant or an algae, preferably from the genus Chlamydomonas, even more preferably from the species Chlamydomonas reinhardtii.
  • the enzyme is from an organism of the genus Entamoeba, more preferably of the species Entamoeba histolytica.
  • Gao et al. FEMS Microbiol. Lett. 213 (2002), 59-65 already described genetically modified E. coli cells which have been transformed, inter alia, with the ptb gene and the buk gene from Clostridium acetobutylicum encoding a phosphate butyryltransferase (EC 2.3.1.19) and a butyrate kinase (EC 2.7.2.7), respectively.
  • EC coli cells have been shown to be able to produce D-(-)-3-hydroxybutyric acid (3HB).
  • propionyl-CoA As mentioned above, the conversion of propionyl-CoA into propionic acid can also be achieved by two alternative conversions wherein propionyl-CoA is directly converted into propionic acid.
  • propionyl-CoA is directly converted into propionic acid by hydrolyzing the thioester bond of propionyl-CoA to propionic acid by making use of an enzyme which belongs to the family of thioester hydrolases (in the following referred to as thioesterases (EC 3.1.2.-)).
  • thioesterases EC 3.1.2.-
  • propionyl-CoA is directly converted into propionic acid by a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • acetyl-CoA hydrolase EC 3.1.2.1
  • an ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.18
  • an acyl-CoA hydrolase EC 3.1.2.20
  • propionyl-CoA is directly converted into propionic acid by making use of an enzyme which belongs to the family of CoA-transferases (EC 2.8.3.-).
  • This reaction is schematically shown in Figure 7 for preferred enzymes, i.e., a propionate:acetate-CoA transferase (EC 2.8.3.1 ) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18).
  • propionyl-CoA is directly converted into propionic acid by a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18).
  • a CoA transferase EC 2.8.3.-
  • a propionate:acetate-CoA transferase EC 2.8.3.1
  • an acetate CoA-transferase EC 2.8.3.8
  • succinyl-CoA:acetate CoA- transferase EC 2.8.3.18
  • the enzymatic conversion of propionyl-CoA into propionic acid is achieved by a single enzymatic reaction in which propionyl-CoA is directly converted into propionic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20); or, in another embodiment, by a single enzymatic reaction in which propionyl-CoA is directly converted into propionic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl- CoA
  • a thioester hydrolase EC 3.1.2.-
  • Thioesterases are enzymes which are classified as EC 3.1.2.
  • TEs also referred to as thioester hydrolases
  • thioesterases are enzymes which are classified as EC 3.1.2.
  • TEs which are not yet classified/unclassified are grouped as enzymes belonging to EC 3.1.2.-.
  • Cantu et al. (Protein Science 19 (2010), 1281- 1295) describe that there are 23 families of thioesterases which are unrelated to each other as regards the primary structure. However, it is assumed that all members of the same family have essentially the same tertiary structure.
  • Thioesterases hydrolyze the thioester bond between a carbonyl group and a sulfur atom.
  • a thioesterase employed in a method according to the present invention for converting propionyl-CoA into propionic acid is selected from the group consisting of:
  • acetyl-CoA hydrolase (EC 3.1.2.1 );
  • palmitoyl-CoA hydrolase (EC 3.1.2.2);
  • 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4); oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);
  • ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);
  • ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3. .2.19); and acyl-CoA hydrolase (EC 3.1.2.20).
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an acetyl-CoA hydrolase (EC 3.1.2.1).
  • Acetyl-CoA hydrolases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Rattus norvegicus (Uniprot Accession number: Q99NB7), Mus musculus, Sus scrofa, Bos taurus, Gallus gallus, Platyrrhini, Ovis aries, Mesocricetus auratus, Ascaris suum, Homo sapiens (Uniprot Accession number: Q8WYK0), Pisum sativum, Cucumis sativus, Panicus sp., Ricinus communis, Solanum tuberosum, Spinacia oleracea, Zea mays, Glycine max, Saccharomyces cerevisiae, Neurospora crassa, Candida albicans, Trypanosoma brucei brucei, Trypanosoma cruzi, Trypanosoma di
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a palmitoyl-CoA hydrolase (EC 3.1.2.2).
  • Palmitoyl-CoA hydrolases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana (Uniprot Accession number: Q8GYW7), Pisum sativum, Spinacia oleracea, Bumilleriopsis filiformis, Eremosphaera viridis, Mougeotia scalaris, Euglena gracilis, Rhodotorula aurantiaca, Saccharaomyces cerevisiae, Candida rugosa, Caenorhabditis elegans, Mus musculus (Uniprot Accession number: P58137), Homo sapiens, Platyrrhini, Bos taurus, Canis lupus familiaris, Sus scrofa, Cavia procellus, Columba sp., Cricetulus griseus, Mesocricetus
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4).
  • 3-hydroxyisobutyryl-CoA hydrolases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Homo sapiens, Canus lupus familiaris, Rattus norvegicus, Bacillus cereus, Pseudomonas fluorescens and Pseudomonas aeruginosa.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14).
  • Oleoyl-[acyl-carrier-protein] hydrolases are enzymes which catalyze the following reaction: oleoyl-[acyl-carrier-protein] + H 2 0 ⁇ oleate + [acyl-carrier-protein]
  • This enzyme occurs in a variety of plants and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Allium ampeloprasum, Curcurbita moschata, Cuphea calophylla, Cuphea hookeriana, Cuphea lanceolata, Cuphea wrightii, Umbellularia californica, Coriandrum sativum, Spinacia oleracea, Elaeis sp., Elaeis guineensis, Glycine max, Persea americana, Pisum sativum, Sinapis alba, Ulmus americana, Zea mays, Brassica juncea, Brassica napus, Brassica rapa subsp.
  • campestris Jatropha curcas, Ricinus communis, Cinnamomum camphorum, Macadamia tetraphylla, Magnifera indica, Madhuca longifolia, Populus tomentosa, Chimonanthus praecox, Gossypium hirsutum, Diploknema butyracea, Helianthus annuus and Streptococcus pyogenes.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18).
  • ADP-dependent short-chain-acyl-CoA hydrolases are enzymes which catalyze the following reaction: an acyl-CoA + H 2 0 ⁇ a carboxylate + CoA
  • This enzyme occurs in a variety of animals and has, e.g., been described in Mus musculus, Rattus norvegicus and Mesocricetus auratus.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an ADP-dependent medium-chain-acyl- CoA hydrolase (EC 3.1.2.19).
  • ADP-dependent medium-chain-acyl-CoA hydrolases are enzymes which catalyze the following reaction: an acyl-CoA + H 2 0 ⁇ a carboxylate + CoA
  • This enzyme occurs in a variety of animals and has, e.g., been described in Rattus norvegicus and Mesocricetus auratus.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an acyl-CoA hydrolase (EC 3.1.2.20).
  • Acyl-CoA hydrolases are enzymes which catalyze the following reaction: an acyl-CoA + H 2 O * a carboxylate + CoA This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Rhodotorula aurantiaca, Bumilleriopsis filiformis, Eremosphaera viridis, Euglena gracilis, Mus musculus, Rattus norvegicus, Homo sapiens, Sus, scrofa, Bos taurus, Cais lupus familiaris, Cavia porcellus, Cricetus griseus, Drosophila melanogaster, Anas platyrhynchos, Gallus gallus, Caenorhabditis elegans, Saccharomyces cerevisia, Candida rugosa, Escherichia coli, Haemophilus influenzae, Xanthomonas campestris, Streptomyces sp., Streptomyces coelicolor, Alcaligenes faecalis, Pseudomonas aeruginosa, Pseudomon
  • the acyl-CoA hydrolase is an enzyme from Escherichia coli, from Pseudomonas putida or from Haemophilus influenza, more preferably the YciA enzyme from E. coli or its closely related homolog HI0827 from Haemophilus influenza (Zhuang et al., Biochemistry 47 (2008), 2789-2796).
  • the YciA enzyme from Haemophilus influenza is described to catalyze the hydrolysis of propionyl-CoA into propionic acid (Zhuang et al, Biochemistry 47 (2008), 2789-2796).
  • the acetyl-CoA hydrolase is an enzyme from Homo sapiens (UniProt: Q9NPJ3) which is described to hydrolyze propionyl-CoA (Cao et al., Biochemistry 48 (2009), 1293- 1304).
  • Particularly preferred enzymes are the above-described acyl-CoA hydrolase YciA enzyme from Haemophilus influenza strain R2866 (SEQ ID NO: 7) and the acetyl- CoA hydrolase enzyme from Homo sapiens (UniProt: Q9NPJ3; SEQ ID NO:8).Particularly preferred are also the enzymes acyl-CoA thioester hydrolase from E. coli (Uniprot P0A8Z0; SEQ ID NO: 4), acyl-CoA thioesterase 2 from E.
  • coli Uniprot P0AGG2; SEQ ID NO: 5
  • acyl-CoA thioesterase II from Pseudomonas putida
  • SEQ ID NO: 6 Particularly preferred is the thioesterase TesB from E.coli K12 (uniprot :P0AGG2), as this enzyme is already described to efficiently catalyze this reaction in E. Coli for the biosynthesis of propionic acid (Tseng and Prather, P.N.A.S. 2012, 109(44),p17925-17930).
  • the acyl-CoA hydrolase employed in the method of the invention has an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 8 or shows an amino acid sequence which is at least x% homologous to any one of SEQ ID NOs: 4 to 8 and has the activity of an acyl-CoA hydrolase with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 wherein such an enzyme is capable of catalyzing the conversion of propionyl-CoA into propionic acid.
  • sequence identity the same applies as has been set forth above.
  • the direct conversion of propionyl-CoA into propionic acid can also be achieved by making use of an enzyme which is classified as a CoA- transferase (EC 2.8.3.-) capable of transferring the CoA group of propionyl-CoA to a carboxylic acid.
  • an enzyme which is classified as a CoA- transferase (EC 2.8.3.-) capable of transferring the CoA group of propionyl-CoA to a carboxylic acid.
  • CoA-transferases are found in organisms from all lines of descent. Most of the CoA- transferase belong to two well-known enzyme families (referred to in the following as families I and II) and there exists a third family which had been identified in anaerobic metabolic pathways of bacteria. A review describing the different families can be found in Heider (FEBS Letters 509 (2001 ), 345-349).
  • succinate succinyl-CoA:acetate CoA-transferases, i.e. enzymes classified in EC
  • COA_TRANSF_1 COA_TRANSF_1 , PS01273; Coenzyme A transferases signature 1 (PATTERN) Consensus pattern:
  • E glutmic acid is an active site residue.
  • the family II of CoA-transferases consists of the homodimeric a-subunits of citrate lyase (EC 2.8.3.10) and citramalate lyase (EC 2.8.3.11 ). These enzymes catalyse the transfer of acyl carrier protein (ACP) which contains a covalently bound CoA- derivative. It was shown that such enzymes also accept free CoA-thioester in vitro, such as acetyl-CoA, propionyl-CoA, butyryl-CoA in the case of citrate lyase (Dimroth et al., Eur. J. Biochem.
  • ACP acyl carrier protein
  • CoA-transferases According to Heider (loc. cit.) family III of CoA-transferases consists of formyl-CoA: oxalate CoA-transferase, succinyl-CoA:(f?)-benzylsuccinate CoA-transferase, (E)- cinnamoyl-CoA:(R)-phenyllactate CoA-transferase and butyrobetainyl-CoA:(R)- carnitine CoA-transferase.
  • a further member of family III is succinyl-CoA: L-malate CoA-transferase whose function in autrophic C0 2 fixation of Chloroflexus aurantiacus is to activate L-malate to its CoA thioester with succinyl-CoA as the CoA donor (Friedman S. et al. J. Bacteriol. 188 (2006), 2646-2655).
  • the amino acid sequences of the CoA-tranferase of this family show only a low degree of sequence identity to those of families I and II. These CoA-transferases occur in prokaryotes and eukaryotes.
  • the CoA-transferase employed in a method according to the present invention is a CoA-transferase which belongs to family I or II as described herein-above.
  • the CoA-transferase employed in a method according to the present invention for the direct conversion of propionyl-CoA into propionic acid is selected from the group consisting of:
  • butyrate-acetoacetate CoA-transferase (EC 2.8.3.9); and succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18).
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an acetate CoA-transferase (EC 2.8.3.8).
  • Acetate CoA-transferases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of bacteria and has, e.g., been described in Anaerostipes caccae, Eubacterium hallii, Faecalibacterium prausnitzii, Roseburia hominis, Roseburia intestinalis, Coprococcus sp. and Escherichia coli.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).
  • Butyrate-acetoacetate CoA-transferase are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as animals and bacteria.
  • the enzyme has, e.g., been described in Bos taurus, Clostridium sp. and Escherichia coli.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a propionate:acetate-CoA transferase (EC 2.8.3.1 ).
  • Propionate:acetate-CoA transferases are enzymes which catalyze the following reaction:
  • This enzyme catalyzes the reversible transfer of CoA group from propionyl-CoA and acetate.
  • This enzyme occurs in a variety of organism including prokaryotic organisms and the enzyme has, e.g., been described in Clostridium kluyveri, Clostridium propionicum, Clostridium propionicum JCM1430, Cupriavidus necator and Emericella nidulans.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a succinyl-CoA.acetate -CoA transferase (EC 2.8.3.18).
  • Succinyl-CoA:acetate CoA-transferases are enzymes which catalyze the following reaction:
  • This enzyme catalyzes the reversible transfer of CoA group from propionyl-CoA and succinate.
  • This enzyme occurs in a variety of organism, including prokaryotic organisms, and the enzyme has, e.g., been described in Acetobacter aceti, Trichomonas vaginalis, Tritrichomonas foetus, Tritrichomonas foetus ATCC 30924 and Trypanosoma brucei.
  • the acrylyl-CoA which is produced according to the above method from glycerol may further be converted into acrylic acid.
  • the present invention also relates to a method for producing acrylic acid from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into acrylic acid.
  • the conversion of acrylyl-CoA into acrylic acid can be achieved by three alternative enzymatic conversions.
  • One possibility is a two-step conversion via acrylyl phosphate.
  • Two other options involve a direct conversion of acrylyl-CoA into acrylic acid. These three options will be discussed in the following and are schematically illustrated in Figure 8.
  • the enzymatic conversion of acrylyl-CoA into acrylic acid can be achieved by a two-step conversion via acrylyl phosphate. Accordingly, in one embodiment, the enzymatic conversion of acrylyl-CoA into acrylic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting acrylyl-CoA into acrylyl phosphate; and (ii) then enzymatically converting the thus obtained acrylyl phosphate into said acrylic acid.
  • the conversion of acrylyl-CoA into acrylyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).
  • the conversion of acrylyl phosphate into acrylic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase.
  • an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase.
  • Such enzymes use a carboxy group as acceptor.
  • the conversion of acrylyl phosphate into acrylic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-).
  • the conversion of acrylyl phosphate into acrylic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
  • a propionate kinase EC 2.7.2.15
  • an acetate kinase EC 2.7.2.1
  • a butyrate kinase EC 2.7.2.7
  • a branched-chain-fatty-acid kinase EC 2.7.2.14
  • the enzymes propionate kinase (EC 2.7.2.15), acetate kinase (EC 2.7.2.1 ), butyrate kinase (EC 2.7.2.7) and branched-chain-fatty-acid kinase (EC 2.7.2.14) have already been described above in the context of the enzymatic conversion of propionyl phosphate into propionic acid.
  • the conversion of acrylyl-CoA into acrylic acid can also be achieved by two alternative conversions wherein acrylyl-CoA is directly converted into acrylic acid.
  • acrylyl-CoA is directly converted into acrylic acid by hydrolyzing the thioester bond of acrylyl-CoA to acrylic acid by making use of an enzyme which belongs to the family of thioester hydrolases (in the following referred to as thioesterases (EC 3.1.2.-)).
  • thioesterases EC 3.1.2.-
  • acrylyl-CoA is directly converted into acrylic acid by a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • acrylyl-CoA is directly converted into acrylic acid by making use of an enzyme which belongs to the family of CoA-transferases (EC 2.8.3.-).
  • acrylyl-CoA is directly converted into acrylic acid by a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18).
  • a CoA transferase EC 2.8.3.-
  • a propionate:acetate-CoA transferase EC 2.8.3.1
  • an acetate CoA-transferase EC 2.8.3.8
  • succinyl-CoA:acetate CoA- transferase EC 2.8.3.18
  • the enzymatic conversion of acrylyl-CoA into acrylic acid is achieved by a single enzymatic reaction in which acrylyl-CoA is directly converted into acrylic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20); or, in another embodiment, by a single enzymatic reaction in which acrylyl-CoA is directly converted into acrylic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA- transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18).
  • Thioesterases (TEs; also referred to as thioester hydrolases) are enzymes which are classified as EC 3.1.2. Presently thioesterases are classified as EC 3.1.2.1 through EC 3.1.2.30 and EC 3.1.2.- for unclassified TEs. Cantu et al. (loc. cit.) describe that there are 23 families of thioesterases which are unrelated to each other as regards the primary structure. However, it is assumed that all members of the same family have essentially the same tertiary structure. Thioesterases hydrolyze the thioester bond between a carbonyl group and a sulfur atom.
  • a thioesterase employed in a method according to the present invention for converting acrylyl-CoA into acrylic acid is selected from the group consisting of:
  • acetyi-CoA hydrolase (EC 3.1.2.1 );
  • palmitoyl-CoA hydrolase (EC 3.1.2.2);
  • oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);
  • ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);
  • ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19); and acyl-CoA hydrolase (EC 3. .2.20).
  • acetyl-CoA hydrolase (EC 3.1.2.1 ), palmitoyl-CoA hydrolase (EC 3.1.2.2), 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4), oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14), ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18), ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19) and acyl- CoA hydrolase (EC 3.1.2.20) for the conversion of acrylyl-CoA into acrylic acid have already been described above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionic acid.
  • the direct conversion of acrylyl-CoA into acrylic acid can also be achieved by making use of an enzyme which is classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group of acrylyl-CoA to a carboxylic acid.
  • the enzymes classified as a CoA-transferase preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), a acetate CoA-transferase (EC 2.8.3.8), a butyrate-acetoacetate CoA-transferase (EC 2.8.3.9), a citrate lyase (EC 2.8.3.10) and citramalate lyase (EC 2.8.3.11 ) and a succinyl-CoA:acetate CoA- transferase (EC 2.8.3. 8) have already been described above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionic acid. As regards these enzymes, the same applies for the conversion of acrylyl-CoA into acrylic acid as has been set forth above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionic acid.
  • the acrylic acid produced according to the above described method from glycerol is not further converted.
  • the acrylic acid is the end product of the method of the present invention and, accordingly, the present invention relates to a method for producing acrylic acid from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into acrylic acid wherein said acrylic acid is not further converted into another compound.
  • the above described method for producing acrylic acid further comprises the step of recovering the thus produced acrylic acid.
  • the acrylic acid produced according to the above described method from glycerol is not further converted into propionic acid.
  • the present invention relates to a method for producing acrylic acid from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl- CoA is then converted into acrylyl-CoA which is then further converted into acrylic acid wherein said acrylic acid is not further converted into propionic acid.
  • the acrylic acid produced according to the above described method from glycerol is further converted into another compound which is not propionic acid.
  • the present invention relates to a method for producing another compound which is not propionic acid from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into acrylic acid wherein said acrylic acid is converted into another compound which is not propionic acid.
  • the acrylic acid produced according to the above described method from glycerol may be further converted into propionic acid wherein said propionic acid may then further be converted into ethylene.
  • the acrylyl-CoA which is produced according to the above method from glycerol may further be converted into 3-oxo-4-pentenoyl-CoA.
  • the thus produced 3-oxo-4-pentenoyl-CoA may further be converted into 2,4-pentadienoic acid via 3-hydroxy-4-pentenoyl-CoA and 2,4-pentadienoyl-CoA.
  • the thus produced 3-oxo-4-pentenoyl-CoA may further be converted into 3-hydroxy-4-pentenoic acid via 3-hydroxy-4-pentenoyl-CoA.
  • the present invention also relates to a method for producing 3-oxo-4-pentenoyl- CoA from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl- CoA is then converted into into acrylyl-CoA which is then further converted into 3- oxo-4-pentenoyl-CoA.
  • the present invention also relates to a method for producing 2,4- pentadienoic acid from glycerol in which glycerol is first converted into 3- hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into 3-oxo-4-pentenoyl-CoA. The thus produced 3-oxo-4-pentenoyl- CoA is then further converted into 3-hydroxy-4-pentenoyl-CoA which is then further converted into 2,4-pentadienoyl-CoA which is then further converted into 2,4- pentadienoic acid.
  • the present invention also relates to a method for producing 3-hydroxy-4- pentenoic acid from glycerol in which glycerol is first converted into 3- hydroxypropionaldehyde which is then converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into into acrylyl-CoA which is then further converted into 3-oxo-4-pentenoyl-CoA. The thus produced 3-oxo-4-pentenoyl- CoA is then further converted into 3-hydroxy-4-pentenoyl-CoA which is then further converted into 3-hydroxy-4-pentenoic acid.
  • the produced acrylyl-CoA can further be converted into 3-oxo-4-pentenoyl-CoA.
  • the methods of the present invention may further comprise the enzymatic condensation of acrylyl-CoA and acetyl-CoA into 3-oxo-4-pentenoyl-CoA.
  • the reaction of the enzymatic condensation of acrylyl-CoA and acetyl-CoA into 3-oxo-4- pentenoyl-CoA is schematically shown in Figure 15.
  • the enzymatic condensation of acrylyl-CoA and acetyl-CoA into 3-oxo-4-pentenoyl-CoA preferably makes use of an acetyl-CoA C- acyltransferase (EC 2.3.1.16).
  • Acetyl-CoA C-acyltransferase (EC 2.3.1.16) (also termed acyl-CoA:acetyl-CoA C- acetyltransferase or 3-ketoacyl CoA thiolase) catalyze the following reaction: acyl-CoA + acetyl-CoA ⁇ ⁇ " CoA + 3-oxoacyl-CoA
  • This enzyme occurs in a number of organisms in particular in bacteria, plants and animals, and the enzyme has been described, e.g., for Arabidopsis thaliana, (SwissProt Q56WD9), Bos taurus, Brassica napus, Caenorhabditis elegans (UniProt Q22100), Candida tropicalis, Escherichia coli, Glycine max (SwissProt Q6TXD0), Helianthus annuus (UniProt Q6W6X6), Homo sapiens (UniProt Q9H5J4), Mus musculus (Q921 H8, Q8VCH0), Parietochloris incisa (UniProt B8YJJ0), Pseudomonas fragi (UniProt P28790), Rattus norvegicus (SwissProt Q64428), Saccharomyces cerevisiae (UniProt P27796), Spodoptera
  • the enzymatic condensation of acrylyl-CoA and acetyl- CoA into 3-oxo-4-pentenoyl-CoA preferably makes use of the acetyl-CoA C- acyltransferase thiolase from Cupriavidus necator (Uniprot number.QOKBPI ).
  • This enzyme is already described to perform the catalysis of the enzymatic condensation of propionyl-CoA and acetyl-CoA into 3-oxopentanoyl-CoA (J. Bacteriol. 180 (1998), 1979-1987).
  • the condensation of acrylyl-CoA and acetyl-CoA into 3-oxo-4-pentenoyl-CoA is achieved by making use of a beta-ketothiolase from Cupriavidus necator (Uniprot Q0KBP1 ).
  • the amino acid sequence of said protein is shown in SEQ ID NO: 32.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 32.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:32 and the enzyme has the enzymatic activity of condensating acrylyl-CoA and acetyl-CoA into 3-oxo-4- pentenoyl-CoA.
  • the sequence identity the same applies as has been set forth above.
  • step VIII The enzymatic reduction of 3-oxo-4-pentenoyl-CoA into 3-hvdroxy-4-pentenoyl-CoA (step VIII as shown in Figure 14)
  • the produced 3-oxo-4-pentenoyl-CoA can further be converted into 3-hydroxy-4- pentenoyl-CoA.
  • the methods of the present invention further may comprise the enzymatic reduction of 3-oxo-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl-CoA.
  • the reduction of 3-oxo-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl-CoA is schematically illustrated in Figure 16.
  • the reaction involves the formation of a chiral carbon bearing a hydroxyl group (indicated with an ( * ) in Figure 16) and the reaction may be stereoselective.
  • the reduction of 3-oxo-4-pentenoyl-CoA into 3- hydroxy-4-pentenoyl-CoA preferably makes use of an enzyme which acts on a CH- OH group of a donor. Enzymes catalyzing this reaction are enzymes which catalyze the reaction as shown in Figure 16. These enzymes use either NAD(+) or NADP(+) as acceptor.
  • such an enzyme belongs to the family of 3-hydroxyacyl-CoA dehydrogenases classified as oxidoreductases acting on CH-OH groups of donors (EC 1.1.1.-).
  • the enzyme is an enzyme which uses NAD(+) as a co-factor.
  • the 3-hydroxyacyl-CoA dehydrogenase employed in a method according to the invention for the conversion of 3-oxo-4-pentenoyl-CoA into said 3-hydroxy-4-pentenoyl-CoA is a 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157) or a 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35).
  • 3-hydroxybutyryl-CoA dehydrogenases (EC 1.1.1.157) (also termed (S)-3- hydroxybutanoyl-CoA:NADP+ oxidoreductase or beta-hydroxybutyryl-CoA dehydrogenase) catalyze the following reaction:
  • This enzyme occurs in a number of organisms in particular in bacteria and animals, and the enzyme has been described, e.g., for Butyrivibrio fibrisolvens (Uniprot Q65Y06, Q65Y11), Clostridium acetobutylicum, Clostridium saccharobutylicum, Clostridium kluyveri, Mycobacterium tuberculosis (Uniprot 053753), Leishmania donovani, Leishmania major, Mycobacterium smegmatis, Trypanosoma brucei, Mus musculus and Rattus norvegicus.
  • the step of the enzymatic reduction of 3-oxo-4-pentenoyl- CoA into 3-hydroxy-4-pentenoyl-CoA is catalyzed by the 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157) from Clostridium acetobutylicum (Uniprot accession number: P52041 ).
  • the reduction of 3-oxo-4-pentenoyl-CoA into 3- hydroxy-4-pentenoyl-CoA is achieved by making use of a 3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum (Uniprot accession number: P52041).
  • the amino acid sequence of said protein is shown in SEQ ID NO: 33.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 33.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:33 and the enzyme has the enzymatic activity of reducing 3-oxo-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl- CoA.
  • sequence identity the same applies as has been set forth above.
  • 3-hydroxyacyl-CoA dehydrogenase enzymes occur in a variety of organism, including prokaryotic and eukaryotic organisms, such as bacteria, plants and animals.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Bos taurus, Brassica napus (SwissProt Q84X96, Q84X95), Clostridium kluyveri, Escherichia coli, Euglena gracilis, Giberella moniliformis, Homo sapiens (Uniprot Q99714, Q16836), Mus musculus, Mycobacterium smegmatis, Neurospora crassa, Pseudomonas putida, Rattus norvegicus and Sus scrofa.
  • the produced 3-hydroxy-4-pentenoyl-CoA can further be dehydrated into 2,4- pentadienoyl-CoA.
  • the methods of the present invention further comprise the enzymatic dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is schematically illustrated in Figure 17.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA preferably makes use of an enzyme catalyzing 3-hydroxy- 4-pentenoyl-CoA dehydration.
  • the term "dehydration” is generally referred to as a reaction involving the removal of H 2 O.
  • Enzymes catalyzing 3-hydroxy-4-pentenoyl- CoA dehydration are enzymes which catalyze the reaction as shown in Figure 17. Given the asymmetric hydroxyl group, the shown reaction may be stereo-specific.
  • such an enzyme belongs to the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases (EC 4.2.1.-).
  • the present invention relates to a method for the enzymatic dehydration of 3- hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA by making use of an enzyme catalyzing 3-hydroxy-4-pentenoyl-CoA dehydration, preferably of a 3-hydroxyacyl- CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-).
  • an enzyme catalyzing 3-hydroxy-4-pentenoyl-CoA dehydration preferably of a 3-hydroxyacyl- CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-).
  • Examples for enzymes catalyzing the dehydration of 3-hydroxy-4-pentenoyl-CoA which can be employed in the method of the present invention are the following enzymes which are all classified as E.C. 4.2.1.- (i.e., hydro-lyases):
  • R 1 is a hydrogen atom or an alkyl group or CH 2 COO " ;
  • R 2 is a hydrogen atom or a methyl group
  • R 3 is coenzyme A or acyl-carrier protein.
  • R3 in the above shown formula is acyl-carrier protein
  • This group includes EC 4.2.1.58, EC 4.2.1.59, EC 4.2.1.60 and EC 4.2.1.61.
  • the enzymes of this group have in common that they catalyze a reaction of the following type:
  • R3 in the above shown formula is coenzyme A This group includes EC 4.2.1.116, EC 4.2.1.55, EC 4.2.1.17, EC 4.2.1.74 and EC 4.2.1.18
  • the dehydration of 3- hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a 3- hydroxypropionyl-CoA dehydratase (EC 4.2.1.116).
  • 3-hydroxypropionyl-CoA dehydratases (EC 4.2.1.116) catalyze the following reaction:
  • a bacterial 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) is used, preferably a 3-hydroxypropionyl-CoA dehydratase from a bacterium or an archaebacterium of a genus selected from the group consisting of Metallosphaera, Sulfolobus and Brevibacillus and most preferably from a species selected from the group consisting of Metallosphaera cuprina, Metallosphaera sedula, Sulfolobus tokodaii and Brevibacillus laterosporus.
  • Examples for such bacterial 3-hydroxypropionyl-CoA dehydratases are the enzymes from Metallosphaera cuprina (Uniprot F4FZ85), Metallosphaera sedula (Uniprot A4YI89, Teufel et al., J. Bacteriol. 191 (2009), 4572-4581 ), Sulfolobus tokodaii (Uniprot F9VNG3) and Brevibacillus laterosporus (Uniprot F7TTZ1 ). Amino acid and nucleotide sequences for these enzymes are available.
  • SEQ ID NOs: 34 to 37 Examples for corresponding amino acid sequences are provided in SEQ ID NOs: 34 to 37 wherein SEQ ID NO:34 is the amino acid sequence of 3-hydroxypropionyl-CoA dehydratase of M. cuprina, SEQ ID NO:35 is the amino acid sequence of 3-hydroxypropionyl-CoA dehydratase of M. sedula, SEQ ID NO:36 is the amino acid sequence of a 3-hydroxypropionyl- CoA dehydratase of S. tokodaii and SEQ ID NO:37 is the amino acid sequence of a 3-hydroxypropionyl-CoA dehydratase of Brevibacillus laterosporus.
  • the 3-hydroxypropionyl-CoA dehydratase employed in the method of the invention has an amino acid sequence as shown in any one of SEQ ID NOs: 34 to 37 or shows an amino acid sequence which is at least x% homologous to any of SEQ ID NOs: 34 to 37 and has the activity of catalyzing the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA, with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99.
  • any 3-hydroxypropionyl-CoA dehydratase can be employed in the method according to the invention.
  • a 3-hydroxypropionyl-CoA dehydratase for the dehydration of 3- hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA but also enzymes which show the structural and functional similarities as described above, i.e. enzymes as listed in items (b) to (f), above.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55).
  • 3-hydroxybutyryl-CoA dehydratases (EC 4.2.1.55) catalyze the following reaction:
  • 3-hydroxybutyryl-CoA dehydratase belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is (3R)-3- hydroxybutanoyl-CoA hydro-lyase (crotonoyl-CoA-forming).
  • Other names in common use include D-3-hydroxybutyryl coenzyme A dehydratase, D-3-hydroxybutyryl-CoA dehydratase, enoyl coenzyme A hydratase, and (3R)-3-hydroxybutanoyl-CoA hydro- lyase. This enzyme participates in the butanoate metabolism.
  • Enzymes belonging to this class and catalyzing the above shown conversion of 3-hydroxybutyryl-Coenzyme A into crotonyl-Coenzyme A have been described to occur, e.g. in rat (Rattus norvegicus), in Rhodospirillum rubrum, in Sulfolobus acidocaldarius and in Acidianus hospitalis. Nucleotide and/or amino acid sequences for such enzymes have been determined, e.g. for Aeropyrum pernix.
  • any 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) which can catalyze the dehydration of 3-hydroxy-4- pentenoyl-CoA into 2,4-pentadienoyl-CoA can be used in the context of the present invention.
  • a 3-hydroxybutyryl-CoA dehydratase from an archaebacterium is used, preferably a 3-hydroxybutyryl-CoA dehydratase from an archaebacterium of a genus selected from the group consisting of Sulfolobus and Acidianus and most preferably from a species selected from the group consisting of S. acidocaldarius and Acidianus hospitalis.
  • Examples for such bacterial 3-hydroxybutyryl-CoA dehydratases are the enzymes from Sulfolobus acidocaldarius (Uniprot Q4J8D5) and from Acidianus hospitalis ((Uniprot F4B9R3).
  • Examples for corresponding amino acid sequences are provided in SEQ ID NOs: 38 and 39 wherein SEQ ID NO:38 is the amino acid sequence of 3-hydroxybutyryl-CoA dehydratase of Sulfolobus acidocaldarius and SEQ ID NO:39 is the amino acid sequence of 3-hydroxybutyryl-CoA dehydratase of Acidianus hospitalis.
  • the 3-hydroxybutyryl-CoA dehydratase employed in the method of the invention has an amino acid sequence as shown in SEQ ID NO: 38 or 39 or shows an amino acid sequence which is at least x% homologous to SEQ ID NO: 38 or 39 and has the activity of catalyzing the dehydration of 3-hydroxy-4- pentenoyl-CoA into 2,4-pentadienoyl-CoA, with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99.
  • 3-hydroxybutyryl-CoA dehydratase from Clostridium acetobutylicum (Uniprot P52046) can be used as 3-hydroxybutyryl-CoA dehydratase for the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyI- CoA.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by making use of a 3-hydroxybutyryl-CoA dehydratase from Clostridium acetobutylicum (Uniprot P52046).
  • the amino acid sequence of said protein is shown in SEQ ID NO: 40.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 40.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO.40 and the enzyme has the enzymatic activity of dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4- pentadienoyl-CoA.
  • sequence identity the same applies as has been set forth above.
  • Enoyl-CoA hydratase is an enzyme that normally hydrates the double bond between the second and third carbons on acyl-CoA. However, it can also be employed to catalyze the reaction in the reverse direction. This enzyme, also known as crotonase, is naturally involved in metabolizing fatty acids to produce both acetyl-CoA and energy. Enzymes belonging to this class have been described to occur, e.g. in rat (Rattus norvegicus), humans (Homo sapiens), mouse (Mus musculus), wild boar (Sus scrofa), Bos taurus, E.coli, Clostridium acetobutylicum and Clostridium aminobutyricum.
  • Nucleotide and/or amino acid sequences for such enzymes have been determined, e.g. for rat, humans and Bacillus subtilis.
  • any enoyl- CoA hydratase (EC 4.2.1.17) which can catalyze the dehydration of 3-hydroxy-4- pentenoyl-CoA into 2,4-pentadienoyl-CoA can be used in the context of the present invention.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a 3- hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59).
  • 3-hydroxyoctanoyl- [acyl-carrier-protein] dehydratases (EC 4.2.1.59) catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is (3R)-3- hydroxyoctanoyl-[acyl-carrier-protein] hydro-lyase (oct-2-enoyl-[acyl-carrier protein]- forming).
  • any 3-hydroxyoctanoyl- [acyl-carrier-protein] dehydratase which can catalyze the dehydration of 3-hydroxy-4- pentenoyl-CoA into 2,4-pentadienoyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a crotonoyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58).
  • Crotonoyl-[acyl-carrier- protein] hydratases (EC 4.2.1.58) catalyze the following reaction: (3R)-3-hydroxybutanoyl-[acyl-carrier-protein] — but-2-enoyl-[acyl-carrier-protein] + H 2 O
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • Crotonoyl-[acyl-carrier-protein] hydratase has been described to exist, e.g., in E. coli and Arabidopsis thaliana.
  • any crotonoyl-[acyl-carrier- protein] hydratase which can catalyze the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a 3- hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60).
  • 3- hydroxydecanoyl-[acyl-carrier-protein] dehydratases catalyze the following reactions:
  • the enzyme has been described to exist, e.g., in Pseudomonas aeruginosa, Pseudomonas fluorescens, Toxoplasma gondii, Plasmodium falciparum, Helicobacter pylori, Corynebacterium ammoniagenes, Enterobacter aerogenes, E. coli, Proteus vulgaris and Salmonella enterica.
  • any 3-hydroxydecanoyl-[acyl-carrier- protein] dehydratase which can catalyze the dehydration of 3-hydroxy-4-pentenoyl- CoA into 2,4-pentadienoyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61 ).
  • 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratases catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • any 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratase which can catalyze the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA can be used in the context of the present invention.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a long-chain-enoyl-CoA hydratase (EC 4.2.1.74).
  • Long-chain-enoyl-CoA hydratases (EC 4.2.1.74) catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is long- chain-(3S)-3-hydroxyacyl-CoA hydro-lyase.
  • This enzyme is also called long-chain enoyl coenzyme A hydratase and it participates in fatty acid elongation in mitochondria and fatty acid metabolism. This enzyme occurs in a number of organisms, e.g., in Rattus norvegicus (Wu et al., Org. Lett.
  • the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by the use of a 3- methylglutaconyl-CoA hydratase (EC 4.2.1.18).
  • 3-methylglutaconyl-CoA hydratases (EC 4.2.1.18) catalyze the following reaction:
  • This enzyme occurs in a number of organisms in particular in bacteria, plants and animals.
  • the enzyme has been described, e.g., for Pseudomonas putida, Acinetobacter sp. (SwissProt accession number Q3HW12), Catharanthus roseus, Homo sapiens (SwissProt accession number Q13825), Bos taurus and Ovis aries and in principle any 3-methylglutaconyl-CoA hydratase which can catalyze the conversion of 3-hydroxypentanoyl-CoA into said 2-pentenoyl-CoA can be employed in the method of the invention.
  • 3-methylglutaconyl-CoA hydratase also covers the enzyme encoded by the gene LiuC (Li et al., Angew. Chem. Int. Ed. 52 (2013), p. 1304-1308; Uniprot number Q1 D5Y4) from Myxococcus xanthus, preferably from strain DK 1622.
  • the amino acid sequence of this enzyme is shown in SEQ ID NO:41.
  • any protein can be employed in a method according to the present invention which comprises an amino acid as shown in SEQ ID NO:41 or an amino acid sequence which is at least x% homologous SEQ ID NO: 41 and which has the activity of a 3-methylglutaconyl- CoA hydratase/3-hydromethylglutaryl-CoA dehydratase and which shows the activity of dehydrating 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA, with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99.
  • sequence identity the same applies as has been set forth above.
  • the enzymatic dehydration of 3-hydroxy-4- pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by making use of a 3- hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) or an enoyl-CoA hydratase (EC 4.2.1.17) as described above.
  • step XI The enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid (step XI as shown in Figure 14)
  • the produced 2,4-pentadienoyl-CoA can further be converted into 2,4-pentadienoic acid.
  • the methods of the present invention may further comprise the enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid.
  • the enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid can by achieved by three alternative conversions which are schematically illustrated in Figure 19.
  • the enzymatic conversion of 2,4- pentadienoyl-CoA into 2,4-pentadienoic acid comprises:
  • the enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid can be achieved by a two-step conversion via 2,4-pentadienoyl phosphate.
  • the enzymatic conversion of 2,4- pentadienoyl-CoA into 2,4-pentadienoic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting 2,4-pentadienoyl-CoA into 2,4- pentadienoyl phosphate; and (ii) then enzymatically converting the thus obtained 2,4- pentadienoyl phosphate into said 2,4-pentadienoic acid.
  • the corresponding reaction is schematically shown in Figure 19.
  • the present invention relates to a method for the production of 2,4-pentadienoic acid comprising the enzymatic conversion of 2,4- pentadienoyl-CoA into 2,4-pentadienoic acid, wherein the enzymatic conversion of said 2,4-pentadienoyl-CoA into said 2,4-pentadienoyl phosphate is achieved by making use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzymatic conversion of said 2,4-pentadienoyl phosphate into said 2,4-pentadienoic acid is achieved by making use of a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.
  • the conversion of 2,4-pentadienoyl-CoA into said 2,4- pentadienoyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1 .19) or a phosphate acetyltransferase (EC 2.3.1.8).
  • the conversion of 2,4-pentadienoyl-CoA into said 2,4- pentadienoyl phosphate is achieved by making use of a phosphate butyryltransferase from Clostridium acetobutylicum (Uniprot: P58255), preferably from Clostridium acetobutylicum strain ATCC 824.
  • a phosphate butyryltransferase from Clostridium acetobutylicum (Uniprot: P58255), preferably from Clostridium acetobutylicum strain ATCC 824.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 42.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 42.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:42 and the enzyme has the enzymatic activity of converting 2,4-pentadienoyl-CoA into said 2,4-pentadienoyl phosphate.
  • sequence identity the same applies as has been set forth above.
  • the conversion of 2,4-pentadienoyl phosphate into said 2,4-pentadienoic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase. Such enzymes use a carboxy group as acceptor.
  • an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase.
  • Such enzymes use a carboxy group as acceptor.
  • the conversion of 2,4-pentadienoyl phosphate into said 2,4-pentadienoic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-).
  • the conversion of 2,4-pentadienoyl phosphate into said 2,4-pentadienoic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
  • a propionate kinase EC 2.7.2.15
  • an acetate kinase EC 2.7.2.1
  • a butyrate kinase EC 2.7.2.7
  • a branched-chain-fatty-acid kinase branched-chain-fatty-acid kinase
  • the enzymes with a carboxy group as acceptor (EC 2.7.2.-) as well as the enzymes propionate kinase (EC 2.7.2.15), acetate kinase (EC 2.7.2.1 ), butyrate kinase (EC 2.7.2.7) and branched-chain-fatty-acid kinase (EC 2.7.2.14) have already been described above in the context of the conversion of propionyl phosphate into propionic acid.
  • the conversion of 2,4-pentadienoyl phosphate into said 2,4-pentadienoic acid is achieved by making use of a butyrate kinase from Clostridium acetobutylicum (Uniprot Accession number: Q45829), preferably from Clostridium acetobutylicum strain ATCC 824.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 43.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 43.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:43 and the enzyme has the enzymatic activity of converting 2,4-pentadienoyl phosphate into said 2,4- pentadienoic acid.
  • sequence identity the same applies as has been set forth above.
  • the conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid can also be achieved by an alternative conversion wherein 2,4-pentadienoyl- CoA is directly converted into 2,4-pentadienoic acid.
  • the direct conversion of 2,4-pentadienoyl-CoA into 2,4- pentadienoic acid can be achieved by making use of an enzyme which is classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group of 2,4- pentadienoyl-CoA to a carboxylic acid.
  • a CoA-transferase EC 2.8.3.-
  • the CoA-transferase is a butyryl- CoA:acetate-CoA transferase (EC 2.8.3.8). This reaction is schematically shown in Figure 19.
  • the enzymes which are classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group to a carboxylic acid as well as the enzyme CoA transferase (EC 2.8.3.8) have already been described above in the context of the conversion of propionyl-CoA into propionic acid.
  • 2,4-pentadienoyl-CoA can also be directly converted into 2,4-pentadienoic acid by hydrolysing the thioester bond of 2,4-pentadienoyl-CoA to 2,4-pentadienoic acid by making use of an enzyme which belongs to the family of thioester hydrolases (referred to as thioesterases (EC 3.1.2.-)). This reaction is schematically shown in Figure 19.
  • 2,4-pentadienoyl-CoA can also be directly converted into 2,4-pentadienoic acid by a thioester hydrolase (EC 3.1.2.-), preferably an acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • acetyl-CoA hydrolase EC 3.1.2.1
  • an ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.1
  • an acyl-CoA hydrolase EC 3.1.2.20
  • thioesterases EC 3.1.2.-
  • acetyl-CoA hydrolase EC 3.1.2.1
  • ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.1
  • acyl-CoA hydrolase EC 3.1.2.20
  • step X The enzymatic conversion of 3-hvdroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid (step X as shown in Figure 14)
  • the produced 3-hydroxy-4-pentenoyl-CoA can further be converted into 3-hydroxy-4- pentenoic acid.
  • the methods of the present invention further comprise the enzymatic conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid.
  • the enzymatic conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid can by achieved by three alternative conversions which are schematically illustrated in Figure 18.
  • the enzymatic conversion of 3-hydroxy-4- pentenoyl-CoA into 3-hydroxy-4-pentenoic acid comprises:
  • the enzymatic conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid can be achieved by a two-step conversion via 3- hydroxy-4-pentenoyl phosphate. Accordingly, in one embodiment, the enzymatic conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting 3- hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl phosphate; and (ii) then enzymatically converting the thus obtained 3-hydroxy-4-pentenoyl phosphate into said 3-hydroxy-4-pentenoic acid.
  • the corresponding reaction is schematically shown in Figure 18.
  • the present invention relates to a method for the production of 3-hydroxy-4-pentenoic acid comprising the enzymatic conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid, wherein the enzymatic conversion of said 3-hydroxy-4-pentenoyl-CoA into said 3-hydroxy-4-pentenoyl phosphate is achieved by making use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzymatic conversion of said 3-hydroxy-4-pentenoyl phosphate into said 3-hydroxy-4-pentenoic acid is achieved by making use of a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kina
  • the conversion of 3-hydroxy-4-pentenoyl-CoA into said 3- hydroxy-4-pentenoyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).
  • the conversion of 3-hydroxy-4-pentenoyl-CoA into said 3- hydroxy-4-pentenoyl phosphate is achieved by making use of a phosphate butyryltransferase from Clostridium acetobutylicum (Uniprot: P58255), preferably from Clostridium acetobutylicum strain ATCC 824.
  • a phosphate butyryltransferase from Clostridium acetobutylicum (Uniprot: P58255), preferably from Clostridium acetobutylicum strain ATCC 824.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 42.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 42.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:42 and the enzyme has the enzymatic activity of 3-hydroxy-4-pentenoyl-CoA into said 3-hydroxy-4-pentenoyl phosphate.
  • sequence identity the same applies as has been set forth above.
  • the conversion of 3-hydroxy-4-pentenoyl phosphate into said 3-hydroxy-4-pentenoic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase.
  • Such enzymes use a carboxy group as acceptor.
  • the conversion of 2,4-pentadienoyl phosphate into said 2,4-pentadienoic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-).
  • the conversion of 3-hydroxy-4-pentenoyl phosphate into said 3-hydroxy-4-pentenoic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
  • a propionate kinase EC 2.7.2.15
  • an acetate kinase EC 2.7.2.1
  • a butyrate kinase EC 2.7.2.7
  • a branched-chain-fatty-acid kinase branched-chain-fatty-acid kinase
  • the enzymes with a carboxy group as acceptor (EC 2.7.2.-) as well as the enzymes propionate kinase (EC 2.7.2.15), acetate kinase (EC 2.7.2.1 ), butyrate kinase (EC 2.7.2.7) and branched-chain-fatty-acid kinase (EC 2.7.2.14) have already been described above in the context of the conversion of propionyl phosphate into propionic acid.
  • the conversion of 3-hydroxy-4-pentenoyl phosphate into said 3-hydroxy-4-pentenoic acid is achieved by making use of a butyrate kinase from Clostridium acetobutylicum (Uniprot Accession number: Q45829), preferably from Clostridium acetobutylicum strain ATCC 824.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 43.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 43.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:43 and the enzyme has the enzymatic activity of converting 3-hydroxy-4-pentenoyl phosphate into said 3- hydroxy-4-pentenoic acid.
  • sequence identity the same applies as has been set forth above.
  • the conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4- pentenoic acid can also be achieved by an alternative conversion wherein 3-hydroxy- 4-pentenoyl-CoA is directly converted into 3-hydroxy-4-pentenoic acid.
  • the direct conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid can be achieved by making use of an enzyme which is classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group of 3-hydroxy-4-pentenoyl-CoA to a carboxylic acid.
  • a CoA-transferase EC 2.8.3.-
  • the CoA-transferase is a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8). This reaction is schematically shown in Figure 18.
  • 3-hydroxy-4-pentenoyl-CoA can also be directly converted into 3-hydroxy-4-pentenoic acid by hydrolysing the thioester bond of 3-hydroxy-4- pentenoyl-CoA to 3-hydroxy-4-pentenoic acid by making use of an enzyme which belongs to the family of thioester hydrolases (referred to as thioesterases (EC 3.1.2.- )). This reaction is schematically shown in Figure 18.
  • 3-hydroxy-4-pentenoyl-CoA can also be directly converted into 3-hydroxy-4-pentenoic acid by a thioester hydrolase (EC 3.1.2.-), preferably an acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • acetyl-CoA hydrolase EC 3.1.2.1
  • an ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.1
  • an acyl-CoA hydrolase EC 3.1.2.20
  • thioesterases EC 3. .2.-
  • acetyl-CoA hydrolase EC 3.1.2.1
  • ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.1
  • acyl-CoA hydrolase EC 3.1.2.20
  • the present invention also relates to a method for the production of ethylene comprising the enzymatic conversion of propionic acid into ethylene.
  • the enzymatic conversion of propionic acid into ethylene can be achieved by an oxidative decarboxylation.
  • the reaction is schematically shown in Figure 1 and in the following:
  • the enzymatic conversion of propionic acid into ethylene can preferably be achieved by an oxidative decarboxylation by making use of a cytochrome P450.
  • a cytochrome P450 which can be employed in a method according to the present invention is an enzyme as described in van Leeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387). This enzyme, i.e., a cytochrome P450 from Rhodotorula minuta has been reported to be able to catalyze the conversion of 3-methylbutyric acid (isovalerate) into isobutene (Fukuda et al., Biochem. Biophys. Res.
  • This cytochrome P450 is referred to as P450rm. It is a membrane protein, in particular a microsomal protein and has been annotated as an "isobutene-forming enzyme and benzoate 4-hydroxylase".
  • the conversion of propionic acid into ethylene is achieved by making use of a cytochrome P450, more preferably of the cytochrome P450 of R. minuta.
  • the sequence of this enzyme is avaiblabe under UniProt Accession number Q12668.
  • such an enzyme has an amino acid sequence as shown in SEQ ID NO: 3 or shows an amino acid sequence which is at least x% homologous to SEQ ID NO: 3 and has the activity of an cytochrome P450 with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 wherein such an enzyme is capable to catalyze the conversion of propionic acid into ethylene.
  • the enzymatic conversion of propionic acid into ethylene can be achieved by an oxidative decarboxylation by making use of a cytochrome P450 fatty acid decarboxylase.
  • propionic acid is enzymatically converted into ethylene by an enzymatically catalyzed oxidative decarboxylation catalyzed by a cytochrome P450 olefin forming fatty acid decarboxylase.
  • cytochrome P450 olefin forming fatty acid decarboxylase refers to a cytochrome P450 which belongs to the cyp152 family and which has the ability to decarboxylate fatty acids to the terminal olefins.
  • P450s form a large superfamily of multifunctional proteins and are divided into different CYP families according to their sequence similarity (Ortiz de Montellano, P. R.
  • Catalytic turnover rates are high compared with those of monooxygenation reactions as well as peroxide shunt reactions catalyzed by the common P450s.
  • the catalyzed reaction is hydroxyiation of fatty acids in a- and/or ⁇ -position:
  • cytochrome P450 olefin forming fatty acid decarboxylase refers to the CYP450 olefin forming fatty acid decarboxylase of the bacterium Jeotgalicoccus sp. ATCC 8456 or a highly related enzyme which has the ability to decarboxylate fatty acids.
  • the CYP450 olefin forming fatty acid decarboxylase of the bacterium Jeotgalicoccus sp. 8456 is in the following referred to as "Ole T JE". In the literature this enzyme is also referred to as "CYP152L1" (Belcher et al., J. Biol. Chem. 289 (2014), 6535-6550).
  • Ole T JE had been identified in the bacterium Jeotgalicoccus sp. ATCC 8456 as a terminal olef in-forming fatty acid decarboxylase (Rude et al., Appl. Environm. Microbiol. 77 (2011 ), 1718-1727).
  • the nucleotide sequence of the gene encoding the protein has been deposited in GenBank under accession number HQ709266 and due to sequence homologies it has been assigned to the cyp 52 enzyme family of P450 peroxygenases (Rude et al., loc. cit).
  • the protein sequence is available at Uniprot accession number: E9NSU2. As reported in Rude et al. (loc.
  • Jeotgalicoccus sp. ATCC 8456 was able to produce terminal olefins with 18 to 20 C atoms. It has been reported by Belcher et al. (loc. cit.) that Ole T JE binds avidly to a range of long chain fatty acids and produces terminal alkenes form a range of saturated fatty acids (C12 - C20).
  • Ole T JE uses as substrates long chain fatty acids
  • (a) comprises the sequence as shown in SEQ ID NO:1 or a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 1 ;
  • (b) shows the activity of converting propionic acid via oxidative decarboxylation into ethylene.
  • the enzyme comprises the sequence as shown in SEQ ID NO:1 or a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 1.
  • SEQ ID NO: 1 represents the amino acid sequence of the Ole T JE protein.
  • the Ole T JE enzyme employed in a method according to the present invention is the Ole T JE protein comprising the amino acid sequence as shown in SEQ ID NO: 1.
  • the Ole T JE enzyme employed in the method according to the present invention is an enzyme which is structurally related to the Ole T JE protein and which also shows the property of being able to convert propionic acid via oxidative decarboxylation into ethylene.
  • sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:1.
  • the enzyme employed in the method according to the present invention is an enzyme which has an amino acid sequence which is at least 60% identical to the amino acid sequence as shown in SEQ ID NO: 1 and in which the amino acid residue corresponding to position 85 of SEQ ID NO: 1 is not glutamine.
  • the enzyme employed in the method according to the present invention is an enzyme which has an amino acid sequence which is at least 60% identical to the amino acid sequence as shown in SEQ ID NO: 1 and in which the amino acid residue corresponding to position 85 of SEQ ID NO: 1 is histidine.
  • the enzyme shows the activity of converting propionic acid via oxidative decarboxylation into ethylene. This activity can be assayed as described in the appended Examples.
  • the conversion of propionic acid into ethylene is achieved by making use a cytochrome P450 fatty acid decarboxylase from Macrococcus caseolyticus, preferably from strain JCSC5402.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 2 (Uniprot Accession number: B9EBA0). It is of course not only possible to use an enzyme exactly showing this amino acid. It is also possible to use an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 2.
  • sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:2 and the enzyme has the enzymatic activity of converting propionic acid into ethylene.
  • sequence identity the same applies as has been set forth above.
  • the conversion of propionic acid into ethylene is achieved by making use a cytochrome P450 fatty acid decarboxylase from Staphylococcus aureus, preferably from Staphylococcus aureus strain C0673.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 15 (Uniprot Accession number: A0A033V973).
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 15.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO: 5 and the enzyme has the enzymatic activity of converting propionic acid into ethylene.
  • the sequence identity the same applies as has been set forth above.
  • the conversion of propionic acid into ethylene is achieved by making use of a cytochrome P450 in combination with a cytochrome P450 reductase.
  • the reductase can be directly fused to the cytochrome P450 or it can be present as a separate enzyme.
  • the reaction uses NADPH as a reducing agent.
  • the P450 reductase is NADPH dependent.
  • An example and preferred embodiment is the Rhodococcus fusion reductase (RhFRED) domain from Rhodococcus, e.g. Rhodococcus sp. NCIMB 9784 (Roberts et al., J. Bacteriol. 184 (2002), 3898-3908).
  • RhFRED Rhodococcus fusion reductase
  • the resultant catalytic activity of the fusion CYP450 enzyme can be driven by NADPH.
  • the reaction employs a flavoprotein/flavodoxin reductase as redox mediator protein.
  • the corresponding reaction scheme is shown in Figure 1. Examples are the flavodoxin (Fid) and flavodoxin reductase (FdR) proteins from E. coli (Liu et al., Biotechnology for Biofuels 7 (2014), 28).
  • reaction employs ferredoxin/ferredoxin reductase as redox partner.
  • the enzymatic conversion of propionic acid into ethylene can preferably be achieved by making use a non-heme iron oxygenase.
  • Non-heme iron oxygenases have been reported to be able to catalyze the oxidative decarboxylation of a Cn carboxylic acid into the respective Cn-1 terminal alkene.
  • Rui et al. describes the biosynthesis of medium-chain 1-alkenes by a non-heme iron oxygenase (Rui et al., "Microbial biosynthesis of medium-chain 1-alkenes by non-heme oxidase"; Proc. Natl. Acad.
  • Rui et al. describe that the described non-heme iron oxygenases have a specificity for a chain length of the Cn carboxylic acid from C10 to C14. Yet, in the context of the present invention, these non-heme iron oxygenases which are capable of catalyzing the oxidative decarboxylation of a Cn carboxylic acid into the respective Cn-1 terminal alkene can be used to catalyze the conversion of propionic acid into ethylene according to the present invention.
  • the conversion of propionic acid into ethylene is achieved by making use a non-heme iron oxygenases capable of catalyzing the oxidative decarboxylation of a Cn carboxylic acid into the respective Cn-1 terminal alkene from Pseudomonas sp., preferably from Pseudomonas aeruginosa, more preferably Pseudomonas aeruginosa strain UCBPP-PA14, Pseudomonas syringae pv. Tomato, more preferably Pseudomonas syringae pv. Tomato strain DC3000, and Pseudomonas putida, more preferably Pseudomonas putida strain F1.
  • the amino acid sequences of said proteins are shown in SEQ ID NOs: 29 to 31 , respectively.
  • an enzyme which comprises a sequence which is at least 60% identical to any one of the amino acid sequences shown in SEQ ID NOs: 29 to 31.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to any one of SEQ ID NOs:29 to 31 and the enzyme has the enzymatic activity of converting propionic acid into ethylene.
  • sequence identity the same applies as has been set forth above.
  • the present invention also relates to the use of a cytochrome P450 or a non-heme iron oxygenase as described herein above or of a microorganism, preferably a recombinant microorganism, expressing such a cytochrome P450 or a non-heme iron oxygenase for the conversion of propionic acid into ethylene.
  • steps IVa and Va The enzymatic conversion of acrylyl-CoA into propionic acid (steps IVa and Va or, alternatively, steps IVb and Vb as shown in Figure 1)
  • the propionic acid which is converted according to the method of the present invention into ethylene according to any of the above described methods may be provided from an external source. Accordingly, propionic acid may be added to the above enzyme(s) capable of converting propionic acid into ethylene or to a microorganism, preferably a recombinant microorganism, expressing such a cytochrome P450 or a non-heme iron oxygenase for the conversion of propionic acid into ethylene.
  • the propionic acid which is converted according to the method of the present invention into ethylene according to any of the above described methods may also be provided by an organism or microorganism which produces propionic acid.
  • organisms which naturally produce propionic acid or organisms which have been genetically modified so as to produce propionic acid may be used as a host for expressing a cytochrome P450 as described above for the conversion of propionic acid into ethylene according to any of the above described methods.
  • the method according to the present invention is characterized in that the conversion of propionic acid into ethylene is realized in the presence of an organism or microorganism capable of producing propionic acid.
  • an organism or microorganism has the capability to produce propionic acid within the cell due to the presence of enzymes providing enzymatic activities allowing the production of propionic acid from metabolic precursors.
  • organisms or microorganisms may be naturally occurring organisms or microorganisms which naturally have the capability to produce propionic acid as described further below or may be an organism or microorganism which is derived from an organism or microorganism which naturally does not produce propionic acid but which has been genetically modified so as to produce propionic acid, i.e., by introducing the gene(s) necessary for allowing the production of propionic acid in the organism or microorganism as described further below.
  • Organisms or microorganisms naturally harbouring any of the above pathways for the biosynthesis of propionic acid may preferably be used as a host for expressing a cytochrome P450 as described above for the conversion of propionic acid into ethylene according to any of the above described methods.
  • the propionic acid which is converted according to the method of the present invention into ethylene according to any of the above described methods may also be provided by enzymatic reactions by which propionic acid is produced enzymatically starting from glycerol and propionyl-CoA as schematically shown in Figure 1.
  • steps IVa and Va The enzymatic conversion of acrylyl-CoA into propionic acid (steps IVa and Va or, alternatively, steps IVb and Vb as shown in Figure 1)
  • the propionic acid which is converted according to the method of the present invention into ethylene according to any of the above described methods may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of acrylyl-CoA (also known as acryloyl-CoA) into said propionic acid.
  • acrylyl-CoA also known as acryloyl-CoA
  • the conversion of acrylyl-CoA into said propionic acid can be achieved via different routes.
  • One possibility is to first convert acrylyl- CoA into propionyl-CoA and then to further convert propionyl-CoA into propionic acid.
  • Another possibility is to first convert acrylyl-CoA into acrylic acid and then to further convert acrylic acid into propionic acid.
  • the present invention also relates to a method for producing ethylene from acrylyl-CoA in which acrylyl-CoA is first converted into propionyl-CoA which is then converted into propionic acid and which is then further enzymatically converted into ethylene as described herein above.
  • the present invention also relates to a method for producing ethylene from propionyl- CoA in which propionyl-CoA is first converted into propionic acid which is then further enzymatically converted into ethylene as described herein above.
  • the present invention also relates to a method for producing ethylene from acrylyl-CoA in which acrylyl-CoA is first converted into acrylic acid which is then converted into propionic acid, and which is then further enzymatically converted into ethylene as described herein above.
  • the present invention also relates to a method for producing ethylene from acrylic acid in which acrylic acid is first converted into propionic acid and which is then further enzymatically converted into ethylene as described herein above.
  • the conversion of acrylyl-CoA into propionic acid is achieved by first converting acrylyl-CoA into propionyl-CoA and then by further converting propionyl-CoA into propionic acid.
  • the present invention relates to a method for the production of propionic acid by the enzymatic conversion of acrylyl- CoA into propionic acid comprises the steps of:
  • step IVa (a) enzymatically converting acrylyl-CoA into propionyl-CoA (step IVa as shown in Figure 1 );
  • step Va further enzymatically converting the thus produced propionyl-CoA into propionic acid
  • the enzymatic conversion of acrylyl-CoA into propionyl-CoA (step IVa as shown in Figure 1 ) according to step (a) can, for example, be achieved by making use of an enzyme classified as EC 1.3.1.-.
  • Enzymes classified as EC 1.3.1.- are enoyl-CoA reductases.
  • the enzyme is an enzyme which is classified as EC 1.3.1.- and which uses NADH or NADPH as a co-factor.
  • the enzyme is an enzyme which uses NADH as a co-factor.
  • Several enzymes of the general family of enoyl-CoA reductase are also described to be able to use NADPH as reducing cofactor (J. Biochem.
  • the enzyme is an enzyme which uses NADPH as a co-factor.
  • the enzyme is selected from the group consisting of:
  • NADP+ - acyl-CoA dehydrogenase
  • NADPH - cis-2-enoyl-CoA reductase
  • NADPH - trans-2-enoyl-CoA reductase
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an acyl-CoA dehydrogenase (NADP+) (EC 1.3.1.8).
  • Acyl-CoA dehydrogenases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Bos, taurus, Rattus novegicus, Mus musculus, Columba sp., Arabidopsis thaliana, Nicotiana benthamiana, Allium ampeloprasum, Euglena gracilis, Candida albicans, Streptococcus collinus, Rhodobacter sphaeroides and Mycobacterium smegmatis.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an enoyl-[acyl-carrier-protein] reductase (NADPH, Si- specific) (EC 1.3.1.10).
  • Enoyl-[acyl-carrier-protein] reductases (NADPH, Si-specific) are enzymes which catalyze the following reaction:
  • acyl-carrier-protein + NADP + ⁇ * trans-2,3-dehydroacyl-[acyl-carrier- protein] + NADPH + H +
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, fungi and bacteria.
  • the enzyme has, e.g., been described in Carthamus tinctorius, Candida tropicalis, Saccharomyces cerevisiae, Streptococcus collinus, Streptococcus pneumoniae, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Porphyromonas gingivalis, Escherichia coli and Salmonella enterica.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a cis-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.37).
  • NADPH cis-2-enoyl-CoA reductase
  • Cis- 2-enoyl-CoA reductases (NADPH) are enzymes which catalyze the following reaction:
  • This enzyme has been described to occur in Escherichia coli.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a trans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38).
  • NADPH trans-2-enoyl-CoA reductases
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals and bacteria.
  • the enzyme has, e.g., been described in Homo sapiens, Rattus norvegicus, Mus musculus, Cavia porcellus, Caenorhabditis elegans, Phalaenopsis amabilis, Gossypium hirsutum, Mycobacterium tuberculosis, Streptococcus collinu and Escherichia coli.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an enoyl-[acyl-carrier-protein] reductase (NADPH, Re- specific) (EC 1.3.1.39).
  • Enoyl-[acyl-carrier-protein] reductases (NADPH, Re-specific) are enzymes which catalyze the following reaction: acyl-[acyl-carrier-protein] + NADP + « « « " trans-2,3-dehydroacyl-[acyl-carrier- protein] + NADPH + H +
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as animals and bacteria.
  • the enzyme has, e.g., been described in Gallus gallus, Pigeon, Rattus norvegicus, Cavia porcellus, Staphylococcus aureus, Bacillus subtilis and Porphyromonas gingivalis.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a crotonyl-CoA reductase (EC 1.3.1.86).
  • Crotonyl-CoA reductases are enzymes which catalyze the following reaction: butanoyl-CoA + NADP + « *" (E)-but-2-enoyl-CoA + NADPH + H +
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Bos taurus, Salinospora tropica, Clostridium difficile, Streptomyces collinus, Streptomyces cinnamonensis and Streptomyces hygroscopicus.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an NADPH-dependent acrylyl-CoA reductase (EC 1.3.1.84).
  • NADPH-dependent acrylyl-CoA reductases are enzymes which catalyze the following reaction: propanoyl-CoA + NADP + think » » acryloyl-CoA + NADPH + H +
  • This enzyme occurs in a variety of organism, including prokaryotic organisms and the enzyme has, e.g., been described in Metallosphaera sedula and Sulfolobus tokodaii.
  • the enzyme is an enzyme which uses NADH as a co-factor.
  • the conversion using such an enzyme is schematically shown in Figure 4.
  • the enzyme is selected from the group consisting of:
  • NADH acyl-carrier-protein] reductase
  • NAD + trans-2-enoyl-CoA reductase
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an enoyl-[acyl-carrier-protein] reductase (NADH) (EC 1.3.1.9).
  • NADH enoyl-[acyl-carrier-protein] reductases
  • NADH enzymes which catalyze the following reaction: acyl-[acyl-carrier-protein] + NAD + participate *" trans-2,3-dehydroacyl-[acyl-carrier- protein] + NADH + H +
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Plasmodium falciparum, Eimeria tenella, Toxoplasma gondii, Mycobacterium tuberculosis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Bacillus anthracis, Birkholderia mallei, Pseudomonas aeruginosa, Helicobacter pylori, Yersinia pestis and many others.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of a trans-2-enoyl-CoA reductase (NAD + ) (EC 1.3.1.44).
  • NAD + trans-2-enoyl-CoA reductases
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals and bacteria.
  • the enzyme has, e.g., been described in Ratus norvegicus, Euglena gracilis, Mycobacterium smegmatis, Pseudomonas fluorescens, Clostridium acetobutylicum, Butyrivibrio fibrisolvens, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Treponema denticola.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an acrylyl-CoA reductase (aka acryloyl-CoA reductase) (EC 1.3.1.95).
  • acrylyl-CoA reductase aka acryloyl-CoA reductase
  • These enzymes are electron transferring flavoproteins (J. Bacteriol. 191 (2009), 4572-4581 ; Eur. J. Biochem. 270 (2003), 902-910).
  • An acrylyl-CoA reductase was already cloned in E. coli for a pathway to propionic acid biosynthesis (Appl. Microbiol. Biotechnol. 97 (2013), 1191-2000).
  • Acrylyl-CoA reductases are enzymes which catalyze the following reaction propanoyl-CoA + NAD + ⁇ » acryloyl-CoA + NADH + H +
  • This enzyme occurs in a variety of prokaryotic organisms and the enyzme has, e.g., been described in Clostridium kluyveri, Clostridium propionicum, Metallosphaera sedula and Sufolobus tokodaii.
  • the conversion of acrylyl-CoA into propionyl-CoA is achieved by making use of an acryloyl-CoA reductase from Metallosphaera sedula, preferably from Metallosphaera sedula strain ATCC 51363.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 26.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 26.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:26 and the enzyme has the enzymatic activity of converting acrylyl-CoA into propionyl-CoA.
  • sequence identity the same applies as has been set forth above.
  • the enzymatic conversion of propionyl-CoA into propionic acid (step Va as shown in Figure 1 ) according to step (b) can be achieved by three alternative enzymatic conversions.
  • One possibility is a two-step conversion via propionyl phosphate.
  • Two other options involve a direct conversion of propionyl-CoA into propionic acid. These three options will be discussed in the following.
  • the enzymatic conversion of propionyl-CoA into propionic acid according to step (b) can be achieved by a two-step conversion via propionyl phosphate.
  • the enzymatic conversion of propionyl- CoA into propionic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting propionyl-CoA into propionyl phosphate; and (ii) then enzymatically converting the thus obtained propionyl phosphate into said propionic acid.
  • propionyl-CoA into propionyl phosphate
  • conversion of propionyl-CoA into propionyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1 .19) or a phosphate acetyltransferase (EC 2.3.1.8).
  • Phosphate butyryltransferase (EC 2.3.1.19) naturally catalyzes the following reaction Butyryl-CoA + H 3 PO 4 . ⁇ ⁇ * ⁇ butyryl phosphate + CoA
  • phosphate butyryltransferases (EC 2.3.1.19) can use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA), in particular acetyl-CoA, propionyl-CoA, isobutyryl- CoA, valeryl-CoA and isovaleryl-CoA.
  • butyryl-CoA butyryl coenzyme A
  • the enzyme has been described to occur in a number of organism, in particular in bacteria and in protozoae.
  • the enzyme is from the protozoae Dasytricha ruminantium.
  • the phosphate butyryltransferase is a phosphate butyryltransferase from a bacterium, preferably from a bacterium of the genus Bacillus, Butyrivibrio, Enterococcus or Clostridium, more preferably Enterococcus or Clostridium, and even more preferably from Bacillus megaterium, Butyrivibrio fibrisolvens, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium butyricum, Clostridium kluyveri, Clostridium saccharoacetobutylicum, Clostridium sprorogenes or Enterococcus faecalis.
  • the enzyme is from Clostridium acetobutylicum, in particular the enzyme encoded by the ptb gene (Uniprot Accession number F0K6W0; Wiesenborn et al. (Appl. Environ. Microbiol. 55 (1989), 317-322)) or from Enterococcus faecalis (Uniprot Accession number K4YRE8; Ward et al. (J. Bacteriol. 181 (1999), 5433-5442)).
  • ptb gene Uniprot Accession number F0K6W0; Wiesenborn et al. (Appl. Environ. Microbiol. 55 (1989), 317-322)
  • Enterococcus faecalis Uniprot Accession number K4YRE8; Ward et al. (J. Bacteriol. 181 (1999), 5433-5442)
  • the conversion of propionyl-CoA into propionyl phosphate is achieved by making use of a phosphate butyryltransferase from Clostridium acetobutylicum, preferably from Clostridium acetobutylicum strain ATCC 824.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 20.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 20.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:20 and the enzyme has the enzymatic activity of converting propionyl-CoA into propionyl phosphate.
  • sequence identity the same applies as has been set forth above.
  • Phosphate acetyltransferase (EC 2.3.1.8) naturally catalyzes the following reaction Acetyl-CoA + H 3 PO 4 ⁇ -» ⁇ acetyl phosphate + CoA
  • accession numbers for this enzyme family in InterPro database are IPR012147 and IPR002505, "http://www.ebi.ac.uk/interpro/entry/IPR002505"
  • the enzyme has been described in a variety of organisms, in particular bacteria and fungi.
  • the enzyme is an enzyme from a bacterium, preferably of the genus Escherichia, Chlorogonium, Clostridium, Veillonella, Methanosarcina, Corynebacterium, Ruegeria, Salmonella, Azotobacter, Bradorhizobium, Lactobacillus, Moorella, Rhodopseudomonas, Sinorhizobium, Streptococcus, Thermotoga or Bacillus, more preferably of the species Escherichia coli, Chlorogonium elongatum, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium acidurici, Veillonella parvula, Methanosarcina thermophila, Corynebacterium glutamicum, Ruegeria pomeroyi, Salmonella enterica, Azotobacter vinelandii, Bradyr
  • the conversion of propionyl-CoA into propionyl phosphate is achieved by making use a phosphate acetyltransferase from Corynebacterium glutamicum, preferably from Corynebacterium glutamicum strain ATCC 13032.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 21.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 20.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:21 and the enzyme has the enzymatic activity of converting propionyl-CoA into propionyl phosphate.
  • sequence identity the same applies as has been set forth above.
  • the conversion of propionyl phosphate into propionic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase. Such enzymes use a carboxy group as acceptor.
  • the conversion of propionyl phosphate into propionic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-).
  • the conversion of propionyl phosphate into propionic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
  • a propionate kinase EC 2.7.2.15
  • an acetate kinase EC 2.7.2.1
  • a butyrate kinase EC 2.7.2.7
  • a branched-chain-fatty-acid kinase EC 2.7.2.14
  • Butyrate kinases (EC 2.7.2.7) naturally catalyze the following reaction Butyrate + ATP butyryl phosphate + ADP
  • butyrate kinase can use a number of substrates in addition to butyrate, e.g. valerate, isobutyrate, isovalerate and vinyl acetate.
  • the enzyme has been described in a variety of organisms, in particular bacteria.
  • the enzyme is from a bacterium, preferably from a bacterium of the genus Clostridium, Butyrivibrio, Thermotoga or Enterococcus. Preferred is Clostridium.
  • the enzyme is from a bacterium of the species Clostridium acetobutylicum, Clostridium proteoclasticum, Clostridium tyrobutyricum, Clostridium butyricum, Clostridium pasteurianum, Clostridium tetanomorphum, Butyrivibrio firbrosolvens, Butyrivibrio hungatei, Thermotoga maritime or Enterococcus durans.
  • Clostridium acetobutylicum For this organism two butyrate kinases have been described: butyrate kinase 1 (Uniprot Accession number: Q45829) and butyrate kinase II (Uniprot Accession number: Q97II19).
  • Branched-chain-fatty-acid kinases (EC 2.7.2.14) naturally catalyze the following reaction
  • alkyl may be 2-methylbutanoate, butanoate, isobutanoate, pentanoate or propionate.
  • the latter reaction with propionate has been described for a branched- chain fatty acid kinase from a spirochaete (J. Bacteriol. 152 (1982), 246-54).
  • the enzyme has been described to occur in a number of bacteria.
  • the enzyme is an enzyme from a bacterium, preferably of the genus Spirochaeta or Thermotoga, more preferably Thermotoga maritime.
  • Propionate kinases (EC 2.7.2.15) naturally catalyze the following reactions
  • the enzyme has been described to occur in a number of bacteria, in particular Enterobacteriacea.
  • the enzyme is an enzyme from a bacterium, preferably of the genus Salmonella or Escherichia, more preferably of the species Salmonella enterica, Salmonella typhimurium or Escherichia coli.
  • the conversion of propionyl phosphate into propionic acid is achieved by making use of a propionate kinase from Salmonella typhimurium, preferably from Salmonella typhimurium strain ATCC 700720.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 27.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 27.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:27 and the enzyme has the enzymatic activity of converting propionyl phosphate into propionic acid.
  • the sequence identity the same applies as has been set forth above.
  • the conversion of propionyl phosphate into propionic acid is achieved by making use of a propionate kinase from Escherichia coli, preferably from Escherichia coli strain K12.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 28.
  • an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 28.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:28 and the enzyme has the enzymatic activity of converting propionyl phosphate into propionic acid.
  • sequence identity the same applies as has been set forth above.
  • Acetate kinases (EC 2.7.2.1 ) naturally catalyze the following reaction Acetate + ATP ⁇ *" acetyl phosphate + ADP
  • the enzyme has been described to occur in a number of organisms, in particular bacteria and eukaryotes.
  • the enzyme is from a bacterium, preferably from a bacterium of the genus Methanosarcina, Cryptococcus, Ethanoligenens, Propionibacterium, Roseovarius, Streptococcus, Salmonella, Acholeplasma, Acinetobacter, Ajellomyces, Bacillus, Borrelia, Chaetomium, Clostridium, Coccidioides, Coprinopsis, Cryptococcus, Cupriavidus, Desulfovibrio, Enterococcus, Escherichia, Ethanoligenes, Geobacillus, Helicobacter, Lactobacillus, Lactococcus, Listeria, esoplasma, Moorella, Mycoplasma, Oceanobacillus, Propionibacterium, Rhodospeudomonas, Roseovarius, Salmonella, Staphylococcus, Thermotog
  • the enzyme is an enzyme from a fungus, preferably from a fungus of the genus Aspergillus, Gibberella, Hypocrea, Magnaporthe, Phaeosphaeria, Phanerochaete, Phytophthora, Sclerotinia, Uncinocarpus, Ustilago or Neurospora even more preferably from a fungus of the species Aspergillus fumigates, Aspergillus nidulans, Gibberella zeae, Hypocrea jecorina, Magnaporthe grisea, Phaeosphaeria nodorum, Phanerochaete chrysosporium, Phytophthora ramorum, Phytophthora sojae, Sclerotinia sclerotiorum, Uncinocarpus reesii, Ustilago maydis or Neurospora crassa.
  • the enzyme is an enzyme from a plant or an algae, preferably from the genus Chlamydomonas, even more preferably from the species Chlamydomonas reinhardtii.
  • the enzyme is from an organism of the genus Entamoeba, more preferably of the species Entamoeba histolytica.
  • Gao et al. FEMS Microbiol. Lett. 213 (2002), 59-65 already described genetically modified E. coli cells which have been transformed, inter alia, with the ptb gene and the buk gene from Clostridium acetobutylicum encoding a phosphate butyryltransferase (EC 2.3.1.19) and a butyrate kinase (EC 2.7.2.7), respectively.
  • EC coli cells have been shown to be able to produce D-(-)-3-hydroxybutyric acid (3HB).
  • propionyl-CoA As mentioned above, the conversion of propionyl-CoA into propionic acid can also be achieved by two alternative conversions wherein propionyl-CoA is directly converted into propionic acid.
  • propionyl-CoA is directly converted into propionic acid by hydrolyzing the thioester bond of propionyl-CoA to propionic acid by making use of an enzyme which belongs to the family of thioester hydrolases (in the following referred to as thioesterases (EC 3.1.2.-)).
  • thioesterases EC 3.1.2.-
  • propionyl-CoA is directly converted into propionic acid by a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • acetyl-CoA hydrolase EC 3.1.2.1
  • an ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.18
  • an acyl-CoA hydrolase EC 3.1.2.20
  • propionyl-CoA is directly converted into propionic acid by making use of an enzyme which belongs to the family of CoA-transferases (EC 2.8.3.-).
  • propionyl-CoA is directly converted into propionic acid by a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18).
  • a CoA transferase EC 2.8.3.-
  • a propionate:acetate-CoA transferase EC 2.8.3.1
  • an acetate CoA-transferase EC 2.8.3.8
  • succinyl-CoA:acetate CoA- transferase EC 2.8.3.18
  • the enzymatic conversion of propionyl-CoA into propionic acid is achieved by a single enzymatic reaction in which propionyl-CoA is directly converted into propionic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20); or, in another embodiment, by a single enzymatic reaction in which propionyl-CoA is directly converted into propionic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl- CoA
  • a thioester hydrolase EC 3.1.2.-
  • Thioesterases are enzymes which are classified as EC 3.1.2.
  • TEs also referred to as thioester hydrolases
  • thioesterases are enzymes which are classified as EC 3.1.2.
  • TEs which are not yet classified/unclassified are grouped as enzymes belonging to EC 3.1.2.-.
  • Cantu et al. (Protein Science 19 (2010), 1281- 1295) describe that there are 23 families of thioesterases which are unrelated to each other as regards the primary structure. However, it is assumed that all members of the same family have essentially the same tertiary structure.
  • Thioesterases hydrolyze the thioester bond between a carbonyl group and a sulfur atom.
  • a thioesterase employed in a method according to the present invention for converting propionyl-CoA into propionic acid is selected from the group consisting of:
  • acetyl-CoA hydrolase (EC 3.1.2.1);
  • palmitoyl-CoA hydrolase (EC 3.1.2.2);
  • oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);
  • ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);
  • ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19); and acyl-CoA hydrolase (EC 3. .2.20).
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an acetyl-CoA hydrolase (EC 3.1.2.1 ).
  • Acetyl-CoA hydrolases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Rattus norvegicus (Uniprot Accession number: Q99NB7), Mus musculus, Sus scrofa, Bos taurus, Gallus gallus, Platyrrhini, Ovis aries, Mesocricetus auratus, Ascaris suum, Homo sapiens (Uniprot Accession number: Q8WYK0), Pisum sativum, Cucumis sativus, Panicus sp., Ricinus communis, Solanum tuberosum, Spinacia oleracea, Zea mays, Glycine max, Saccharomyces cerevisiae, Neurospora crassa, Candida albicans, Trypanosoma brucei brucei, Trypanosoma cruzi, Trypanosoma di
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a palmitoyl-CoA hydrolase (EC 3.1.2.2).
  • Palmitoyl-CoA hydrolases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana (Uniprot Accession number: Q8GYW7), Pisum sativum, Spinacia oleracea, Bumilleriopsis filiformis, Eremosphaera viridis, Mougeotia scalaris, Euglena gracilis, Rhodotorula aurantiaca, Saccharaomyces cerevisiae, Candida rugosa, Caenorhabditis elegans, Mus musculus (Uniprot Accession number: P58137), Homo sapiens, Platyrrhini, Bos taurus, Canis lupus familiaris, Sus scrofa, Cavia procellus, Columba sp., Cricetulus griseus, Mesocricetus
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4).
  • 3-hydroxyisobutyryl-CoA hydrolases are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Homo sapiens, Canus lupus familiaris, Rattus norvegicus, Bacillus cereus, Pseudomonas fluorescens and Pseudomonas aeruginosa.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14).
  • Oleoyl-[acyl-carrier-protein] hydrolases are enzymes which catalyze the following reaction: oleoyl-[acyl-carrier-protein] + H 2 0 ⁇ oleate + [acyl-carrier-protein]
  • This enzyme occurs in a variety of plants and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Allium ampeloprasum, Curcurbita moschata, Cuphea calophylla, Cuphea hookeriana, Cuphea lanceolata, Cuphea wrightii, Umbellularia californica, Coriandrum sativum, Spinacia oleracea, Elaeis sp., Elaeis guineensis, Glycine max, Persea americana, Pisum sativum, Sinapis alba, Ulmus americana, Zea mays, Brassica juncea, Brassica napus, Brassica rapa subsp.
  • campestris Jatropha curcas, Ricinus communis, Cinnamomum camphorum, Macadamia tetraphylla, Magnifera indica, Madhuca longifolia, Populus tomentosa, Chimonanthus praecox, Gossypium hirsutum, Diploknema butyracea, Helianthus annuus and Streptococcus pyogenes.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18).
  • ADP-dependent short-chain-acyl-CoA hydrolases are enzymes which catalyze the following reaction: an acyl-CoA + H 2 O ⁇ a carboxylate + CoA
  • This enzyme occurs in a variety of animals and has, e.g., been described in Mus musculus, Rattus norvegicus and Mesocricetus auratus.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an ADP-dependent medium-chain-acyl- CoA hydrolase (EC 3.1.2.19).
  • ADP-dependent medium-chain-acyl-CoA hydrolases are enzymes which catalyze the following reaction: an acyl-CoA + H 2 O * ⁇ a carboxylate + CoA This enzyme occurs in a variety of animals and has, e.g., been described in Rattus norvegicus and Mesocricetus auratus.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an acyl-CoA hydrolase (EC 3.1.2.20).
  • Acyl-CoA hydrolases are enzymes which catalyze the following reaction: an acyl-CoA + H 2 0 ⁇ a carboxylate + CoA
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Arabidopsis thaliana, Rhodotorula aurantiaca, Bumilleriopsis filiformis, Eremosphaera viridis, Euglena gracilis, Mus musculus, Rattus norvegicus, Homo sapiens, Sus, scrofa, Bos taurus, Cais lupus familiaris, Cavia porcellus, Cricetus griseus, Drosophila melanogaster, Anas platyrhynchos, Gallus gallus, Caenorhabditis elegans, Saccharomyces cerevisia, Candida rugosa, Escherichia coli, Haemophilus influenzae, Xanthomonas campestris, Streptomyces sp., Streptomyces co
  • the acyl-CoA hydrolase is an enzyme from Escherichia coli, from Pseudomonas putida or from Haemophilus influenza, more preferably the YciA enzyme from E. coli or its closely related homolog HI0827 from Haemophilus influenza (Zhuang et al., Biochemistry 47 (2008), 2789-2796).
  • the YciA enzyme from Haemophilus influenza is described to catalyze the hydrolysis of propionyl-CoA into propionic acid (Zhuang et al., Biochemistry 47 (2008), 2789-2796).
  • the acetyl-CoA hydrolase is an enzyme from Homo sapiens (UniProt: Q9NPJ3) which is described to hydrolyze propionyl-CoA (Cao et al., Biochemistry 48 (2009), 1293- 1304).
  • Particularly preferred enzymes are the above-described acyl-CoA hydrolase YciA enzyme from Haemophilus influenza strain R2866 (SEQ ID NO: 7) and the acetyl- CoA hydrolase enzyme from Homo sapiens (UniProt: Q9NPJ3; SEQ ID NO:8). Particularly preferred are also the enzymes acyl-CoA thioester hydrolase from E. coli (Uniprot P0A8Z0; SEQ ID NO: 4), acyl-CoA thioesterase 2 from E.
  • coli Uniprot P0AGG2; SEQ ID NO: 5
  • acyl-CoA thioesterase II from Pseudomonas putida
  • SEQ ID NO: 6 Particularly preferred is the thioesterase TesB from E.coli K12 (uniprot :P0AGG2), as this enzyme is already described to efficiently catalyze this reaction in E. Coli for the biosynthesis of propionic acid (Tseng and Prather, P.N.A.S. 2012, 109(44),p17925-17930).
  • the acyl-CoA hydrolase employed in the method of the invention has an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 8 or shows an amino acid sequence which is at least x% homologous to any one of SEQ ID NOs: 4 to 8 and has the activity of an acyl-CoA hydrolase with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 wherein such an enzyme is capable of catalyzing the conversion of propionyl-CoA into propionic acid.
  • the degree of identity is determined by comparing the respective sequence with the amino acid sequence of any one of the above-mentioned SEQ ID NOs.
  • the degree of identity preferably either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence.
  • the degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.
  • the Clustal analysis method determines whether a particular sequence is, for instance, 80% identical to a reference sequence
  • default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences.
  • the Extend gap penalty is preferably set to 5.0.
  • the degree of identity is calculated over the complete length of the sequence.
  • the direct conversion of propionyl-CoA into propionic acid can also be achieved by making use of an enzyme which is classified as a CoA- transferase (EC 2.8.3.-) capable of transferring the CoA group of propionyl-CoA to a carboxylic acid.
  • an enzyme which is classified as a CoA- transferase (EC 2.8.3.-) capable of transferring the CoA group of propionyl-CoA to a carboxylic acid.
  • CoA-transferases are found in organisms from all lines of descent. Most of the CoA- transferase belong to two well-known enzyme families (referred to in the following as families I and II) and there exists a third family which had been identified in anaerobic metabolic pathways of bacteria. A review describing the different families can be found in Heider (FEBS Letters 509 (2001 ), 345-349).
  • succinate succinyl-CoA:acetate CoA-transferases, i.e. enzymes classified in EC
  • COA_TRANSF_1 COA_TRANSF_1 , PS01273; Coenzyme A transferases signature 1 (PATTERN) Consensus pattern:
  • E glutmic acid is an active site residue.
  • the family II of CoA-transferases consists of the homodimeric a-subunits of citrate lyase (EC 2.8.3.10) and citramalate lyase (EC 2.8.3.11 ). These enzymes catalyse the transfer of acyl carrier protein (ACP) which contains a covalently bound CoA- derivative. It was shown that such enzymes also accept free CoA-thioester in vitro, such as acetyl-CoA, propionyl-CoA, butyryl-CoA in the case of citrate lyase (Dimroth et al., Eur. J. Biochem.
  • ACP acyl carrier protein
  • CoA-transferases According to Heider (loc. cit.) family III of CoA-transferases consists of formyl-CoA: oxalate CoA-transferase, succinyl-CoA:(R)-benzylsuccinate CoA-transferase, (E)- cinnamoyl-CoA:(R)-phenyllactate CoA-transferase and butyrobetainyl-CoA:(R)- carnitine CoA-transferase.
  • a further member of family III is succinyl-CoA:L-malate CoA-transferase whose function in autrophic CO2 fixation of Chloroflexus aurantiacus is to activate L-malate to its CoA thioester with succinyl-CoA as the CoA donor (Friedman S. et al. J. Bacteriol. 188 (2006), 2646-2655).
  • the amino acid sequences of the CoA-tranferase of this family show only a low degree of sequence identity to those of families I and II. These CoA-transferases occur in prokaryotes and eukaryotes.
  • the CoA-transferase employed in a method according to the present invention is a CoA-transferase which belongs to family I or II as described herein-above.
  • the CoA-transferase employed in a method according to the present invention for the direct conversion of propionyl-CoA into propionic acid is selected from the group consisting of:
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of an acetate CoA-transferase (EC 2.8.3.8).
  • Acetate CoA-transferases are enzymes which catalyze the following reaction:
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).
  • Butyrate-acetoacetate CoA-transferase are enzymes which catalyze the following reaction:
  • This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as animals and bacteria.
  • the enzyme has, e.g., been described in Bos taurus, Clostridium sp. and Escherichia coli.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a propionate:acetate-CoA transferase (EC 2.8.3.1).
  • Propionate:acetate-CoA transferases are enzymes which catalyze the following reaction:
  • This enzyme catalyzes the reversible transfer of CoA group from propionyl-CoA and acetate.
  • This enzyme occurs in a variety of organism including prokaryotic organisms and the enzyme has, e.g., been described in Clostridium kluyveri, Clostridium propionicum, Clostridium propionicum JCM1430, Cupriavidus necator and Emericella nidulans.
  • the direct conversion of propionyl-CoA into propionic acid is achieved by making use of a succinyl-CoA:acetate -CoA transferase (EC 2.8.3.18).
  • Succinyl-CoA:acetate CoA-transferases are enzymes which catalyze the following reaction: Succinyl-CoA + acetate acetyl-CoA + succinate
  • This enzyme catalyzes the reversible transfer of CoA group from propionyl-CoA and succinate.
  • This enzyme occurs in a variety of organism, including prokaryotic organisms, and the enzyme has, e.g., been described in Acetobacter aceti, Trichomonas vaginalis, Tritrichomonas foetus, Tritrichomonas foetus ATCC 30924 and Trypanosoma brucei.
  • the conversion of acrylyl- CoA into propionic acid can be achieved via different routes.
  • One possibility i.e., to first convert acrylyl-CoA into propionyl-CoA and then to further convert propionyl-CoA into propionic acid has already described above.
  • the other possible route by first converting acrylyl-CoA into acrylic acid and then by further converting acrylic acid into propionic acid is described in the following.
  • the enzymatic conversion of acrylyl-CoA into propionic acid comprises the steps of: (a) enzymatically converting acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ); and (b) further enzymatically converting the thus produced acrylic acid into propionic acid (step Vb as shown in Figure 1 ).
  • step IVb as shown in Figure 1
  • Figure 8 The enzymatic conversion of acrylyl-CoA into acrylic acid according to step (a) (step IVb as shown in Figure 1 ) is schematically illustrated in Figure 8.
  • the enzymatic conversion of acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ) according to step (a) can be achieved by three alternative enzymatic conversions.
  • One possibility is a two-step conversion via acrylyl phosphate.
  • Two other options involve a direct conversion of acrylyl-CoA into acrylic acid. These three options will be discussed in the following and are schematically illustrated in Figure 8.
  • the enzymatic conversion of acrylyl-CoA into acrylic acid according to step (a) can be achieved by a two-step conversion via acrylyl phosphate.
  • the enzymatic conversion of acrylyl-CoA into acrylic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting acrylyl-CoA into acrylyl phosphate; and (ii) then enzymatically converting the thus obtained acrylyl phosphate into said acrylic acid.
  • the conversion of acrylyl-CoA into acrylyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).
  • the conversion of acrylyl phosphate into acrylic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase.
  • an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase.
  • Such enzymes use a carboxy group as acceptor.
  • the conversion of acrylyl phosphate into acrylic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-).
  • the conversion of acrylyl phosphate into acrylic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
  • a propionate kinase EC 2.7.2.15
  • an acetate kinase EC 2.7.2.1
  • a butyrate kinase EC 2.7.2.7
  • a branched-chain-fatty-acid kinase EC 2.7.2.14
  • the enzymes propionate kinase (EC 2.7.2.15), acetate kinase (EC 2.7.2.1 ), butyrate kinase (EC 2.7.2.7) and branched-chain-fatty-acid kinase (EC 2.7.2.14) for the conversion of acrylyl phosphate into acrylic acid have already been described above in the context of the enzymatic conversion of propionyl phosphate into propionic acid.
  • the conversion of acrylyl-CoA into acrylic acid can also be achieved by two alternative conversions wherein acrylyl-CoA is directly converted into acrylic acid.
  • acrylyl-CoA is directly converted into acrylic acid by hydrolyzing the thioester bond of acrylyl-CoA to acrylic acid by making use of an enzyme which belongs to the family of thioester hydrolases (in the following referred to as thioesterases (EC 3.1.2.-)).
  • thioesterases EC 3.1.2.-
  • acrylyl-CoA is directly converted into acrylic acid by a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • acrylyl-CoA is directly converted into acrylic acid by making use of an enzyme which belongs to the family of CoA-transferases (EC 2.8.3.-).
  • acrylyl-CoA is directly converted into acrylic acid by a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18).
  • a CoA transferase EC 2.8.3.-
  • a propionate:acetate-CoA transferase EC 2.8.3.1
  • an acetate CoA-transferase EC 2.8.3.8
  • succinyl-CoA:acetate CoA- transferase EC 2.8.3.18
  • the enzymatic conversion of acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ) according to step (a) is achieved by a single enzymatic reaction in which acrylyl-CoA is directly converted into acrylic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20); or, in another embodiment, by a single enzymatic reaction in which acrylyl-CoA is directly converted into acrylic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC
  • Thioesterases (TEs; also referred to as thioester hydrolases) are enzymes which are classified as EC 3.1.2. Presently thioesterases are classified as EC 3.1.2.1 through EC 3.1.2.30 and EC 3.1.2.- for unclassified TEs. Cantu et al. (loc. cit.) describe that there are 23 families of thioesterases which are unrelated to each other as regards the primary structure. However, it is assumed that all members of the same family have essentially the same tertiary structure. Thioesterases hydrolyze the thioester bond between a carbonyl group and a sulfur atom.
  • a thioesterase employed in a method according to the present invention for converting acrylyl-CoA into acrylic acid is selected from the group consisting of:
  • acetyl-CoA hydrolase (EC 3.1.2.1 );
  • palmitoyl-CoA hydrolase (EC 3.1.2.2);
  • oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);
  • ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);
  • ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19); and acyl-CoA hydrolase (EC 3.1.2.20).
  • the enzymes acetyl-CoA hydrolase (EC 3.1.2.1), palmitoyl-CoA hydrolase (EC
  • the direct conversion of acrylyl-CoA into acrylic acid can also be achieved by making use of an enzyme which is classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group of acrylyl-CoA to a carboxylic acid.
  • the enzymes classified as a CoA-transferase preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), a acetate CoA-transferase (EC 2.8.3.8), a butyrate-acetoacetate CoA-transferase (EC 2.8.3.9), a citrate lyase (EC 2.8.3.10) and citramalate lyase (EC 2.8.3.11 ) and a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18) for the conversion of acrylyl-CoA into acrylic acid have already been described above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionic acid. As regards these enzymes, the same applies for the conversion of acrylyl-CoA into acrylic acid as has been set forth above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionic acid
  • the enzymatic conversion of acrylyl-CoA into propionic acid comprises the steps of: (a) enzymatically converting acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ); and (b) further enzymatically converting the thus produced acrylic acid into propionic acid (step Vb as shown in Figure 1 ).
  • step (b) step IVb as shown in Figure 1
  • step IVb step IVb as shown in Figure 1
  • step IVb The enzymatic conversion of acrylic acid into propionic acid (step IVb as shown in Figure 1 ) according to step (b), i.e., the reduction of acrylic acid into propionic acid, can, for example, be achieved by making use of an (NADH) 2-enoate reductase (EC 1.3.1.31 ).
  • NADH NADH 2-enoate reductase
  • 2-enoate reductases are enzymes which naturally catalyze the following reaction:
  • Butanoate + NAD + but-2-enoate + NADH + H + This enzyme occurs in a variety of organism, including eukaryotic and prokaryotic organisms, such as animals, fungi and bacteria.
  • the enzyme has, e.g., been described in Cichorium intybus, Marchantia polymorpha, Solanum lycopersicum, Absidia glauca, Kluyveromyces lactis, Penicillium citrinum; Rhodosporidium, Saccharomyces cerevisiae, Clostridium kluyveri, Clostridium bifermentans, Clostridium botulinum, Clostridium difficile, Clostridium ghonii, Clostridium mangenotii, Clostridium oceanicum, Clostridium sordellii, Clostridium sporogenes, Clostridium sticklandii, Clostridium tyrobutyricum, Achromobacter sp.,
  • the acrylyl-CoA which is converted according to the method of the present invention into propionic acid according to any of the above described methods (and further converted to ethylene according to any of the above described methods) may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA.
  • the conversion of 3-hydroxypropionyl- CoA into said acrylyl-CoA (step III as shown in Figure 1 ) is schematically illustrated in Figure 10.
  • This reaction is a natural step of the 3-hydroxypropionate/4- hydroxybutyrate cycle in autotrophic C0 2 fixation in various thermoacidophilic archaea (J. Bacteriol. 191 (2009), 4572-4581 ).
  • the present invention also relates to a method for producing ethylene from 3- hydroxypropionyl-CoA in which 3-hydroxypropionyl-CoA is first converted into acrylyl- CoA which is then converted into propionyl-CoA. Further, propionyl-CoA is then further converted into propionic acid, which is then further enzymatically converted into ethylene as described herein above.
  • the present invention also relates to a method for producing ethylene from 3-hydroxypropionyl-CoA in which 3-hydroxypropionyl-CoA is first converted into acrylyl-CoA, which is then converted into acrylic acid. Further, acrylic acid is then further converted into propionic acid, which is then further enzymatically converted into ethylene as described herein above.
  • the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA preferably makes use of an enzyme catalyzing 3-hydroxypropionyl- CoA dehydration.
  • dehydration is generally referred to a reaction involving the removal of H 2 0.
  • Enzymes catalyzing 3-hydroxypropionyl-CoA dehydration are enzymes which catalyze the reaction as shown in Figure 10.
  • such an enzyme belongs to the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases (EC 4.2.1.-).
  • the present invention relates to a method for the enzymatic conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA by making use of an enzyme catalyzing 3-hydroxypropionyl-CoA dehydration, preferably of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-).
  • an enzyme catalyzing 3-hydroxypropionyl-CoA dehydration preferably of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-).
  • Examples for enzymes catalyzing 3- hydroxypropionyl-CoA dehydration which can be employed in the method of the present invention are the following enzymes which are all classified as E.C. 4.2.1._ (i.e., hydro-lyases):
  • R 1 is a hydrogen atom or an alkyl group or CH 2 COO " ;
  • R 2 is a hydrogen atom or a methyl group
  • R 3 is coenzyme A or acyl-carrier protein.
  • R3 in the above shown formula is acyl-carrier protein
  • This group includes EC 4.2.1.58, EC 4.2.1.59, EC 4.2.1.60 and EC 4.2.1.61.
  • the enzymes of this group have in common that they catalyze a reaction of the following type:
  • R 3 in the above shown formula is coenzyme A
  • This group includes EC 4.2.1.116, EC 4.2.1.55, EC 4.2.1.17, EC 4.2.1.74 and EC 4.2.1.18
  • a bacterial 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) is used, preferably a 3-hydroxypropionyl-CoA dehydratase from a bacterium or an archaebacterium of a genus selected from the group consisting of Metallosphaera, Sulfolobus and Brevibacillus and most preferably from a species selected from the group consisting of Metallosphaera cuprina, Metallosphaera sedula, Sulfolobus tokodaii and Brevibacillus laterosporus.
  • Examples for such bacterial 3-hydroxypropionyl-CoA dehydratases are the enzymes from Metallosphaera cuprina (Uniprot F4FZ85), Metallosphaera sedula (Uniprot A4YI89, Teufel et al., J. Bacteriol. 191 (2009), 4572-4581 ), Sulfolobus tokodaii (Uniprot F9VNG3) and Brevibacillus laterosporus (Uniprot F7TTZ1 ). Amino acid and nucleotide sequences for these enzymes are available.
  • SEQ ID NOs: 9 to 12 Examples for corresponding amino acid sequences are provided in SEQ ID NOs: 9 to 12 wherein SEQ ID NO:9 is the amino acid sequence of 3-hydroxypropionyl-CoA dehydratase of M. cuprina, SEQ ID NO:10 is the amino acid sequence of 3-hydroxypropionyl-CoA dehydratase of M. sedula, SEQ ID NO:11 is the amino acid sequence of a 3-hydroxypropionyl-CoA dehydratase of S. tokodaii and SEQ ID NO: 12 is the amino acid sequence of a 3- hydroxypropionyl-CoA dehydratase of Brevibacillus laterosporus.
  • the 3-hydroxypropionyl-CoA dehydratase employed in the method of the invention has an amino acid sequence as shown in any one of SEQ ID NOs: 9 to 12 or shows an amino acid sequence which is at least x% homologous to any of SEQ ID NOs: 9 to 12 and has the activity of catalyzing the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA, with x being an integer between 30 and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99.
  • any 3-hydroxypropionyl-CoA dehydratase can be employed in the method according to the invention.
  • a 3-hydroxypropionyl-CoA dehydratase for converting 3- hydroxypropionyl-CoA into said acrylyl-CoA but also enzymes which show the structural and functional similarities as described above, i.e. enzymes as listed in items (b) to (f), above.
  • the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55).
  • 3-hydroxybutyryl-CoA dehydratases (EC 4.2.1.55) catalyze the following reaction:
  • 3-hydroxybutyryl-CoA dehydratase belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is (3R)-3- hydroxybutanoyl-CoA hydro-lyase (crotonoyl-CoA-forming).
  • Other names in common use include D-3-hydroxybutyryl coenzyme A dehydratase, D-3-hydroxybutyryl-CoA dehydratase, enoyl coenzyme A hydratase, and (3R)-3-hydroxybutanoyl-CoA hydro- lyase. This enzyme participates in the butanoate metabolism.
  • Enzymes belonging to this class and catalyzing the above shown conversion of 3-hydroxybutyryl-Coenzyme A into crotonyl-Coenzyme A have been described to occur, e.g. in rat (Rattus norvegicus), in Rhodospirillum rubrum, in Sulfolobus acidocaldarius and in Acidianus hospitalis. Nucleotide and/or amino acid sequences for such enzymes have been determined, e.g. for Aeropyrum pernix.
  • any 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) which can catalyze the conversion of 3-hydroxypropionyl- CoA into said acrylyl-CoA can be used in the context of the present invention.
  • a 3-hydroxybutyryl-CoA dehydratase from an archaebacterium is used, preferably a 3-hydroxybutyryl-CoA dehydratase from an archaebacterium of a genus selected from the group consisting of Sulfolobus and Acidianus and most preferably from a species selected from the group consisting of S. acidocaldarius and Acidianus hospitalis.
  • Examples for such bacterial 3- hydroxybutyryl-CoA dehydratases are the enzymes from Sulfolobus acidocaldarius (Uniprot Q4J8D5) and from Acidianus hospitalis ((Uniprot F4B9R3).
  • Examples for corresponding amino acid sequences are provided in SEQ ID NOs: 13 and 14 wherein SEQ ID NO:13 is the amino acid sequence of 3-hydroxybutyryl-CoA dehydratase of Sulfolobus acidocaldarius and SEQ ID NO: 14 is the amino acid sequence of 3-hydroxybutyryl-CoA dehydratase of Acidianus hospitalis.
  • the 3-hydroxybutyryl-CoA dehydratase employed in the method of the invention has an amino acid sequence as shown in SEQ ID NO: 13 or 14 or shows an amino acid sequence which is at least x% homologous to SEQ ID NO: 13 or 14 and has the activity of catalyzing the conversion of 3-hydroxypropionyl- CoA into acrylyl-CoA, with x being an integer between 30 and 00, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of an enoyl-CoA hydratase (EC 4.2.1.17).
  • Enoyl-CoA hydratases (EC 4.2.1.17) catalyze the following reaction:
  • Enoyl-CoA hydratase is an enzyme that normally hydrates the double bond between the second and third carbons on acyl-CoA. However, it can also be employed to catalyze the reaction in the reverse direction. This enzyme, also known as crotonase, is naturally involved in metabolizing fatty acids to produce both acetyl-CoA and energy. Enzymes belonging to this class have been described to occur, e.g. in rat (Rattus norvegicus), humans (Homo sapiens), mouse (Mus musculus), wild boar (Sus scrofa), Bos taurus, E.coli, Clostridium acetobutylicum and Clostridium aminobutyricum.
  • Nucleotide and/or amino acid sequences for such enzymes have been determined, e.g. for rat, humans and Bacillus subtilis.
  • any enoyl- CoA hydratase (EC 4.2.1.17) which can catalyze the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59).
  • 3-hydroxyoctanoyl- [acyl-carrier-protein] dehydratases (EC 4.2.1.59) catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is (3R)-3- hydroxyoctanoyl-[acyl-carrier-protein] hydro-lyase (oct-2-enoyl-[acyl-carrier protein]- forming).
  • any 3-hydroxyoctanoyl- [acyl-carrier-protein] dehydratase which can catalyze the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a crotonoyl- [acyl-carrier-protein] hydratase (EC 4.2.1.58).
  • Crotonoyl-[acyl-carrier-protein] hydratases (EC 4.2.1.58) catalyze the following reaction: (3R)-3-hydroxybutanoyl-[acyl-carrier-protein] but-2-enoyl-[acyl-carrier-protein] + H 2 0
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • Crotonoyl-[acyl-carrier-protein] hydratase has been described to exist, e.g., in E. coli and Arabidopsis thaliana.
  • any crotonoyl-[acyl-carrier- protein] hydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60).
  • 3- hydroxydecanoyl-[acyl-carrier-protein] dehydratases catalyze the following reactions:
  • the enzyme has been described to exist, e.g., in Pseudomonas aeruginosa, Pseudomonas fluorescens, Toxoplasma gondii, Plasmodium falciparum, Helicobacter pylori, Corynebacterium ammoniagenes, Enterobacter aerogenes, E. coli, Proteus vulgaris and Salmonella enterica.
  • any 3-hydroxydecanoyl-[acyl-carrier- protein] dehydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA can be used in the context of the present invention.
  • the enzyme from E. coli is used in a method according to the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61 ).
  • 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratases catalyze the following reaction:
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • any 3- hydroxypalmitoyl-[acyl-carrier-protein] dehydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA can be used in the context of the present invention.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a long-chain- enoyl-CoA hydratase (EC 4.2.1.74).
  • Long-chain-enoyl-CoA hydratases (EC 4.2.1.74) catalyze the following reaction : (3S)-3-hydroxyacyl-CoA trans-2-enoyl-CoA + H 2 O
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is long- chain-(3S)-3-hydroxyacyl-CoA hydro-lyase.
  • This enzyme is also called long-chain enoyl coenzyme A hydratase and it participates in fatty acid elongation in mitochondria and fatty acid metabolism.
  • This enzyme occurs in a number of organisms, e.g., in Rattus norvegicus (Wu et al., Org. Lett. 10 (2008), 2235-2238), Sus scrofa and Cavia porcellus (Fong and Schulz, J. Biol. Chem.
  • the conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved by the use of a 3- methylglutaconyl-CoA hydratase (EC 4.2.1.18).
  • 3-methylglutaconyl-CoA hydratases (EC 4.2.1.18) catalyze the following reaction:
  • This enzyme occurs in a number of organisms in particular in bacteria, plants and animals.
  • the enzyme has been described, e.g., for Pseudomonas putida, Acinetobacter sp. (SwissProt accession number Q3HW12), Catharanthus roseus, Homo sapiens (SwissProt accession number Q13825), Bos taurus and Ovis aries and in principle any 3-methylglutaconyl-CoA hydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA can be employed in the method of the invention.
  • 3-methylglutaconyl-CoA hydratase also covers the enzyme encoded by the gene LiuC (Li et al., Angew. Chem. Int. Ed. 52 (2013), p. 1304-1308; Uniprot number Q1 D5Y4) from Myxococcus xanthus, preferably from strain DK 1622.
  • the enzymatic conversion of 3- hydroxypropionyl-CoA into acrylyl-CoA is achieved by making use of a 3- hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) or an enoyl-CoA hydratase (EC 4.2.1.17) as described above.
  • the 3-hydroxypropionyl-CoA which is converted into acrylyl-CoA according to the method of the present (and further converted to ethylene via propionic acid according to any of the above described methods) may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of 3-hydroxypropionaldehyde into said 3-hydroxypropionyl-CoA (step II as shown in Figure 1).
  • the conversion of 3- hydroxypropionaldehyde into said 3-hydroxypropionyl-CoA is schematically illustrated in Figure 11.
  • the present invention also relates to a method for producing ethylene from 3- hydroxypropionaldehyde in which 3-hydroxypropionaldehyde is first converted into 3- hydroxypropionyl-CoA, which is then converted into acrylyl-CoA. Acrylyl-CoA is then further converted into propionyl-CoA, which is then further converted into propionic acid, and which is then further enzymatically converted into ethylene as described herein above.
  • the present invention also relates to a method for producing ethylene from 3-hydroxypropionaldehyde in which 3-hydroxypropionaldehyde is first converted into 3-hydroxypropionyl-CoA, which is then converted into acrylyl-CoA. Acrylyl-CoA is then converted into acrylic acid, which is then further converted into propionic acid, and which is then further enzymatically converted into ethylene as described herein above.
  • the enzymatic conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionyl-CoA preferably makes preferably use of an enzyme which belongs to the family of Coenzyme-A-acylating aldehyde dehydrogenases.
  • These dehydrogenases are oxidoreducates which act on the aldehyde or oxo group of donors and use either NAD(+) or NADP(+) as acceptor.
  • the family of Coenzyme-A-acylating aldehyde dehydrogenases is classified as EC 1.2.1 .-.
  • the Coenzyme-A-acylating aldehyde dehydrogenase employed in a method according to the invention for the conversion of 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1 .10).
  • CoA-acylating propionaldehyde dehydrogenases (EC 1 .2.1.87) (also termed propanal dehydrogenase (CoA-propanoylating)) catalyze the following reaction: propanal + CoA + NAD + propanoyl-CoA + NADH + H +
  • This enzyme occurs in a number of organisms in particular in bacteria, and the enzyme has been described, e.g., for Burkholderia xenovorans and Thermus thermophilus.
  • the step of the enzymatic conversion of 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is catalyzed by the coenzyme A-acylating propionaldehyde dehydrogenase (gene: PduP, EC 1.2.1.87) from Lactobacillus reuteri (Uniprot accession number: B2G9K7).
  • PduP propionaldehyde dehydrogenase
  • Lactobacillus reuteri Uniprot accession number: B2G9K7
  • the conversion of 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA is achieved by making use of a CoA-acylating propionaldehyde dehydrogenase from Lactobacillus reuteri, preferably the CoA- dependent propionaldehyde dehydrogenase from Lactobacillus reuteri strain JCM 1 112.
  • the amino acid sequence of said protein is shown in SEQ ID NO: 25. It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:25. It is also possible to use an enzyme which comprises a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 25.
  • sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to SEQ ID NO:25 and the enzyme has the enzymatic activity of converting 3-hydroxypropionaldehyde into 3-hydroxypropionyl- CoA.
  • sequence identity the same applies as has been set forth above.
  • Acetaldehyde dehydrogenases (EC 1.2.1.10) catalyze the following reaction: acetaldehyde + CoA + NAD + acetyl-CoA + NADH + H +
  • This reaction is the key step of the first segment of the metabolic pathway connecting the known formation of 3-hydroxypropionaldehyde from glycerol to the 3- hydroxypropionate bicycle pathway as already outlined above in the introductory section (Appl. Environ. Microbiol. 78 (2012), 8564-8570).
  • Acetaldehyde dehydrogenases occur in a variety of organism, including prokaryotic organisms, such as bacteria.
  • the enzyme has, e.g., been described in Acinobacter sp., Burkholderia xenovorans, Clostridium beijerinckii, Clostridium klyveri, E. coli, Giardia intestinalis, Leuconostoc mesenteroides, Propionibacterium freudenreichii, Pseudomonas sp., and Thermoanaerobacter ethanolicus.
  • the enzyme classified as Coenzyme-A-acylating aldehyde dehydrogenases use NADH or NADPH as a co-factor.
  • the enzyme is an enzyme which uses NADH as a co-factor.
  • Several enzymes of the general family of Coenzyme-A-acylating aldehyde dehydrogenases are also described to be able to use NADPH as reducing cofactor (Appl. Env. Microbiol. 56 (1990), 2591-2599). These conversions using either NADH or NADPH as a reducing cofactor are schematically shown in Figure 11.
  • the 3-hydroxypropionaldehyde which is converted into 3-hydroxypropionyl-CoA according to the method of the present may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of glycerol into said 3-hydroxypropionaldehyde (step I as shown in Figure 1 ).
  • the conversion of glycerol into 3-hydroxypropionaldehyde is schematically illustrated in Figure 12.
  • the present invention also relates to a method for producing ethylene from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde, in which 3-hydroxypropionaldehyde is then further converted into 3-hydroxypropionyl-CoA. Further, 3-hydroxypropionyl-CoA is then converted into acrylyl-CoA which is then further converted into propionyl-CoA. Propionyl-CoA is then further converted into propionic acid which is then further enzymatically converted into ethylene as described herein above.
  • the present invention also relates to a method for producing ethylene from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde, in which 3-hydroxypropionaldehyde is then further converted into 3-hydroxypropionyl-CoA. 3- hydroxypropionyl-CoA is then converted into acrylyl-CoA, which is then converted into acrylic acid. Further, acrylic acid is then further converted into propionic acid which is then further enzymatically converted into ethylene as described herein above.
  • the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde preferably makes use of an enzyme which belongs to the family of glycerol dehydratases which naturally catalyze the conversion of glycerol into 3-hydroxypropionaldehyde.
  • Glycerol dehydratases are enzymes using cobalamine (B12 vitamin) as a prosthetic group. These enzymes belong to the family of hydro-lyases which are classified as EC 4.2.1.-.
  • the hydro-lyase (EC 4.2.1.-) employed in a method according to the invention for the conversion of glycerol into 3- hydroxypropionaldehyde is a glycerol dehydratase (EC 4.2.1.30), preferably a cobalamine (B12 vitamin)-dependent or, alternatively, a B 2-indepentent/radical-S- adenosyl methionine-dependent glycerol dehydratase (EC 4.2.1.30).
  • Glycerol dehydratases (EC 4.2.1.30) catalyze the following reaction: glycerol 3-hydroxypropanal + H 2 O
  • Glycerol dehydratases occur in a variety of organism, including prokaryotic organisms, such as bacteria.
  • the enzyme has, e.g., been described in Citrobacter freundii, Citrobacter intermedicus, Clostridium butyricum, Clostridium pasteurianum, E. blattae, E. coli, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus brevis, Lactobacillus buchneri and Pantoea agglomerans.
  • the step of the enzymatic conversion of glycerol into 3- hydroxypropionaldehyde is catalyzed by a cobalamine (B12 vitamin)-dependent glycerol dehydratase from Klebsiella pneumoniae or Lactobacillus reuteri as their heterologous expression was already described in E. coli (Biotechnol. J. 6 (2007), 736-742 and Microbial Cell Factories 3 (2014), 76-86).
  • the conversion of glycerol into 3-hydroxypropionaldehyde is achieved by making use of a glycerol dehydratase from Klebsiella pneumoniae, preferably by the glycerol dehydratase alpha subunit from Klebsiella pneumoniae.
  • the amino acid sequence of said protein is shown in SEQ ID NO:22.
  • the conversion of glycerol into 3- hydroxypropionaldehyde is achieved by making use of a glycerol dehydratase from Klebsiella pneumoniae, preferably by the glycerol dehydratase medium subunit from Klebsiella pneumoniae.
  • the amino acid sequence of said protein is shown in SEQ ID NO:23.
  • the conversion of glycerol into 3- hydroxypropionaldehyde is achieved by making use of a glycerol dehydratase from Klebsiella pneumoniae, preferably by the glycerol dehydratase gamma subunit from Klebsiella pneumoniae.
  • the amino acid sequence of said protein is shown in SEQ ID NO:24.
  • an enzyme which comprises a sequence which is at least 60% identical to any one of the amino acid sequences shown in SEQ ID NOs: 22 to 24.
  • the sequence identity is at least 70%, more preferably at least 80%, 85% or 90%, even more preferably 91 %, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularly preferred at least 99% to any one of SEQ ID NOs:22 to 24 and the enzyme has the enzymatic activity of converting glycerol into 3-hydroxypropionaldehyde.
  • sequence identity the same applies as has been set forth above.
  • the step of the enzymatic conversion of glycerol into 3- hydroxypropionaldehyde is catalyzed by a B12-indepentent glycerol dehydratase which is radical-S-adenosyl methionine-dependent.
  • B12-indepentent glycerol dehydratase which is radical-S-adenosyl methionine-dependent have been described in Clostridium.
  • the family members of this type of glycerol dehydratases use a radical-SAM (S-Adenosyl methionine) instead of coenzyme B 12 based mechanism as it is described in Biochemistry. 43 (2004), 4635-4645.
  • the present invention also relates to a method for the production of propionyl-CoA from glycerol.
  • the glycerol is enzymatically converted to 3-hydroxypropionaldehyde which is further enzymatically converted to 3-hydroxypropionyl-CoA as described herein above.
  • 3-hydroxypropionyl-CoA is enzymatically converted to acrylyl-CoA which is further enzymatically converted to propionyl-CoA as described herein above.
  • the method for the production of propionyl-CoA from glycerol comprises the following steps:
  • step I the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde, preferably by making use of a (cobalamine (B12 vitamin)-dependent or B12- indepentent/radical-S-adenosyl methionine-dependent) glycerol dehydratase (EC 4.2.1.30) (step I as shown in Figure 1 );
  • step II the enzymatic conversion of said 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA, preferably by making use of a CoA-acylating aldehyde dehydrogenase, preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10) (step II as shown in Figure 1 );
  • a CoA-acylating aldehyde dehydrogenase preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10)
  • step III the enzymatic conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA, preferably by making use of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), preferably a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) or a enoyl- CoA hydratase (EC 4.2.1.17) (step III as shown in Figure 1 ); and
  • step IVa the enzymatic conversion of said acrylyl-CoA into said propionyl-CoA, preferably by making use of an enoyl-CoA reductase (EC 1.3.1.-), preferably an acrylyl-CoA reductase (EC 1.3.1.95) (step IVa as shown in Figure 1 ).
  • an enoyl-CoA reductase EC 1.3.1.-
  • an acrylyl-CoA reductase EC 1.3.1.95
  • the thus produced propionyl-CoA in the above method for the production of propionyl-CoA from glycerol can enzymatically be converted into propionic acid by any of the enzymatic conversions of propionyl-CoA to propionic acid as described above.
  • the thus produced propionic acid produced via the intermediate propionyl-CoA can enzymatically be converted to ethylene by any of the enzymatic conversions of propionic acid to ethylene as described above.
  • the present invention also relates to a method for the production of propionic acid from glycerol.
  • the glycerol is enzymatically converted to 3-hydroxypropionaldehyde which is further enzymatically converted to 3-hydroxypropionyl-CoA as described above.
  • 3- hydroxypropionyl-CoA is enzymatically converted to acrylyl-CoA which is further converted to acrylic acid as described above.
  • the acrylic acid is enzymatically converted to propionic acid as described above.
  • the method for the production of propionic acid from glycerol comprises the following steps:
  • step I the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde, preferably by making use of a (cobalamine (B12 vitamin)-dependent or B12- indepentent/radical-S-adenosyl methionine-dependent) glycerol dehydratase (EC 4.2.1.30) (step I as shown in Figure 1 );
  • step II the enzymatic conversion of said 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA, preferably by making use of a CoA-acylating aldehyde dehydrogenase, preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10) (step II as shown in Figure 1 );
  • a CoA-acylating aldehyde dehydrogenase preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10)
  • step III the enzymatic conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA, preferably by making use of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), preferably a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) or a enoyl- CoA hydratase (EC 4.2.1.17) (step III as shown in Figure 1); and
  • step IVb (i) first enzymatically converting acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 );
  • step Vb then enzymatically converting the thus obtained acrylic acid into said propionic acid, preferably by making use of a (NADH) 2-enoate reductase (EC 1.3.1.31 ) (step Vb as shown in Figure 1 ).
  • step(d)(i) in the method for the production of propionic acid from glycerol, the enzymatic conversion of acrylyl-CoA into acrylic acid as defined in step(d)(i) is achieved by
  • (c') a single enzymatic reaction in which acrylyl-CoA is directly converted into acrylic acid by making use of a CoA-transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18).
  • a CoA-transferase EC 2.8.3.-
  • a propionate:acetate-CoA transferase EC 2.8.3.1
  • an acetate CoA-transferase EC 2.8.3.8
  • succinyl-CoA:acetate CoA-transferase EC 2.8.3.18
  • the enzymatic conversion of said acrylyl-CoA into said acrylyl phosphate of (a') is achieved by making use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzymatic conversion of said acrylyl phosphate into said acrylic acid is achieved by making use of a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2. 5), an acetate kinase (EC 2.7.2.1 ) or a butyrate kinase (EC 2.7.2.7).
  • a phosphotransferase with a carboxy group as acceptor EC 2.7.2.-
  • a propionate kinase EC 2.7.2. 5
  • an acetate kinase EC 2.7.2.1
  • the thus produced propionic acid can enzymatically be converted to ethylene by any of the enzymatic conversions of propionic acid to ethylene as described above.
  • a method according to the present invention may be carried out in vitro or in vivo.
  • An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction.
  • in vitro preferably means in a cell-free system.
  • the term "in vitro" in one embodiment means in the presence of isolated enzymes (or enzyme systems optionally comprising possibly required cofactors).
  • the enzymes employed in the method are used in purified form.
  • the substrates for the reaction and the enzymes are incubated under conditions (buffer, temperature, cosubstrates, cofactors etc.) allowing the enzymes to be active and the enzymatic conversion to occur.
  • the reaction is allowed to proceed for a time sufficient to produce the respective product.
  • the production of the respective products can be measured by methods known in the art, such as gas chromatography possibly linked to mass spectrometry detection.
  • the enzymes may be in any suitable form allowing the enzymatic reaction to take place. They may be purified or partially purified or in the form of crude cellular extracts or partially purified extracts. It is also possible that the enzymes are immobilized on a suitable carrier.
  • the method according to the invention is carried out in culture, in the presence of an organism, preferably a microorganism, producing the enzymes described above for the conversions of the method according to the present invention as described herein above.
  • a method which employs a microorganism for carrying out a method according to the invention is referred to as an "in vivo" method. It is possible to use a microorganism which naturally produces the enzymes described above for the conversions of the method according to the present invention or a microorganism which had been genetically modified so that it expresses (including overexpresses) one or more of such enzymes.
  • the microorganism can be an engineered microorganism which expresses enzymes described above for the conversions of the method according to the present invention, i.e. which has in its genome a nucleotide sequence encoding such enzymes and which has been modified to overexpress them. The expression may occur constitutively or in an induced or regulated manner.
  • the microorganism can be a microorganism which has been genetically modified by the introduction of one or more nucleic acid molecules containing nucleotide sequences encoding one or more enzymes described above for the conversions of the methods according to the present invention.
  • the nucleic acid molecule can be stably integrated into the genome of the microorganism or may be present in an extrachromosomal manner, e.g. on a piasmid.
  • Such a genetically modified microorganism can, e.g., be a microorganism that does not naturally express enzymes described above for the conversions of the method according to the present invention and which has been genetically modified to express such enzymes or a microorganism which naturally expresses such enzymes and which has been genetically modified, e.g. transformed with a nucleic acid, e.g. a vector, encoding the respective enzyme(s), and/or insertion of a promoter in front of the endogenous nucleotide sequence encoding the enzyme in order to increase the respective activity in said microorganism.
  • a nucleic acid e.g. a vector, encoding the respective enzyme(s)
  • a promoter in front of the endogenous nucleotide sequence encoding the enzyme in order to increase the respective activity in said microorganism.
  • the invention preferably excludes naturally occurring microorganisms as found in nature expressing an enzyme as described above at levels as they exist in nature.
  • the microorganism of the present invention and employed in a method of the present invention is preferably a non-naturally occurring microorganism, whether it has been genetically modified to express (including overexpression) an exogenous enzyme of the invention not normally existing in its genome or whether it has been engineered to overexpress an exogenous enzyme.
  • the enzymes and (micro)organisms employed in connection with the present invention are preferably non-naturally occurring enzymes or (micro)organisms, i.e. they are enzymes or (micro)organisms which differ significantly from naturally occurring enzymes or microorganism and which do not occur in nature.
  • the enzymes are preferably variants of naturally occurring enzymes which do not as such occur in nature. Such variants include, for example, mutants, in particular prepared by molecular biological methods, which show improved properties, such as a higher enzyme activity, higher substrate specificity, higher temperature resistance and the like.
  • the (micro)organisms they are preferably genetically modified organisms as described herein above which differ from naturally occurring organisms due to a genetic modification. Genetically modified organisms are organisms which do not naturally occur, i.e., which cannot be found in nature, and which differ substantially from naturally occurring organisms due to the introduction of a foreign nucleic acid molecule.
  • the concentration of the enzyme is substantially higher than what is found in nature, which can then unexpectedly force the reaction of the present invention which uses a non-natural for the respective enzyme.
  • the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.
  • non-natural substrate is understood to be a molecule that is not acted upon by the respective enzyme in nature, even though it may actually coexist in the microorganism along with the endogenous enzyme.
  • This "non-natural” substrate is not converted by the microorganism in nature as other substrates are preferred (e.g. the "natural substrate”).
  • the present invention contemplates utilizing a non- natural substrate with the enzymes described above in an environment not found in nature.
  • the microorganism is a microorganism which naturally does not have the respective enzyme activity but which is genetically modified so as to comprise a nucleotide sequence allowing the expression of a corresponding enzyme.
  • the microorganism may also be a microorganism which naturally has the respective enzyme activity but which is genetically modified so as to enhance such an activity, e.g. by the introduction of an exogenous nucleotide sequence encoding a corresponding enzyme or by the introduction of a promoter for the endogenous gene encoding the enzyme to increase endogenous production to overexpressed (non-natural) levels.
  • a microorganism which naturally expresses a corresponding enzyme
  • modify such a microorganism so that the respective activity is overexpressed in the microorganism. This can, e.g., be achieved by effecting mutations in the promoter region of the corresponding gene or introduction of a high expressing promoter so as to lead to a promoter which ensures a higher expression of the gene.
  • the organism employed in a method according to the invention is a microorganism which has been genetically modified to contain a foreign nucleic acid molecule encoding at least one enzyme described above for the conversions of the methods according to the present invention.
  • the term "foreign" or “exogenous” in this context means that the nucleic acid molecule does not naturally occur in said microorganism. This means that it does not occur in the same structure or at the same location in the microorganism.
  • the foreign nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding the respective enzyme in which the promoter driving expression of the coding sequence is heterologous with respect to the coding sequence.
  • Heterologous in this context means that the promoter is not the promoter naturally driving the expression of said coding sequence but is a promoter naturally driving expression of a different coding sequence, i.e., it is derived from another gene, or is a synthetic promoter or a chimeric promoter.
  • the promoter is a promoter heterologous to the microorganism, i.e. a promoter which does naturally not occur in the respective microorganism. Even more preferably, the promoter is an inducible promoter. Promoters for driving expression in different types of organisms, in particular in microorganisms, are well known to the person skilled in the art.
  • the nucleic acid molecule is foreign to the microorganism in that the encoded enzyme is not endogenous to the microorganism, i.e. is naturally not expressed by the microorganism when it is not genetically modified.
  • the encoded enzyme is heterologous with respect to the microorganism.
  • the foreign nucleic acid molecule may be present in the microorganism in extrachromosomal form, e.g. as a plasmid, or stably integrated in the chromosome. A stable integration is preferred.
  • the genetic modification can consist, e.g.
  • the promoter and coding sequence in integrating the corresponding gene(s) encoding the enzyme(s) into the chromosome, or in expressing the enzyme(s) 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.
  • microorganism in the context of the present invention refers to bacteria, as well as to fungi, such as yeasts, and also to algae and archaea.
  • the microorganism is a bacterium.
  • any bacterium can be used.
  • Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas or Escherichia.
  • the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli.
  • the bacterium belongs to the species Pseudomonas putida or to the species Zymomonas mobilis or to the species Corynebacterium glutamicum or to the species Bacillus subtilis.
  • the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus, Trichoderma, Kluyveromyces or Pichia and even more preferably of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger, Trichoderma reesei, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris, Pichia torula or Pichia utilis.
  • the method according to the invention makes use of a photosynthetic microorganism expressing at least one enzyme for the conversion according to the invention as described above.
  • the microorganism is a photosynthetic bacterium, or a microalgae.
  • the microorganism is an algae, more preferably an algae belonging to the diatomeae. It is also conceivable to use in the method according to the invention a combination of microorganisms wherein different microorganisms express different enzymes as described above. The genetic modification of microorganisms to express an enzyme of interest will also be further described in detail below.
  • the method of the present invention makes use of an organism, preferably a microorganism which is capable of producing propionic acid.
  • the term "which is capable of producing propionic acid" in the context of the present invention means that the organism/microorganism has the capacity to produce propionic acid within the cell due to the presence of enzymes providing enzymatic activities allowing the production of propionic acid from metabolic precursors.
  • propionic acid is biosynthetically produced in microorganisms such as Propionibacterium acidipropionici, Propionibacterium freudenreichii ssp. shermanii or Clostridium propionicum.
  • engineered metabolic pathways producing propionic acid have already been established in modified microorganisms.
  • the heterologous pathway to convert the D-lactic acid to propionic from Clostridium propionicum was established in Escherichia coli (Appl. Microbiol. Biotechnol. 97 (2013), 1191-200). Accordingly, these organisms or microorganisms may be used as a host for expressing a cytochrome P450 as described above for the conversion of propionic acid into ethylene according to any of the above described methods.
  • organisms or microorganisms harbouring any of the above pathways for the biosynthesis of propionic acid may be used as a host for expressing a cytochrome P450 as described above for the conversion of propionic acid into ethylene according to any of the above described methods.
  • the organism employed in the method according to the invention is an organism, preferably a microorganism, which naturally has the capacity to produce propionic acid.
  • the method of the present invention makes use of an organism, preferably a microorganism which is capable of producing propionyl-CoA.
  • an organism preferably a microorganism which is capable of producing propionyl-CoA.
  • the term "which is capable of producing propionyl-CoA" in the context of the present invention means that the organism/microorganism has the capacity to produce propionyl-CoA within the cell due to the presence of enzymes providing enzymatic activities allowing the production of propionyl-CoA from metabolic precursors.
  • Propionyl-CoA is biosynthetically produced in microorganisms. Its production has been described in archaea such as Sulfolobus tokodaii, Metallosphaera sedula and Chloroflexus aurantiacus as a metabolite of the 3-hydroxypropionate cycle (J. Bacteriol. 191 (2009), 4572-4581). Accordingly, organisms or microorganisms harboring this pathway for the biosynthesis of propionyl-CoA may be used as a host for expressing any of the enzymes as defined above to be employed in the methods according to the present invention as described above.
  • the organism employed in the method according to the invention is an organism, preferably a microorganism, which naturally has the capability to produce propionic acid (or propionyl-CoA) and which is recombinant in the sense that it has further been genetically modified so as to express an enzyme as defined above.
  • the organism has been genetically modified so as to contain a foreign nucleic acid molecule encoding an enzyme as defined above, e.g., an enzyme catalyzing the enzymatic conversion of propionic acid into ethylene as defined above, preferably a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase as described above for the production of ethylene.
  • the organism employed in the method according to the present invention is a genetically modified organism, preferably a microorganism, derived from an organism/microorganism which naturally does not produce propionic acid but which has been genetically modified so as to produce propionic acid or propionyl-CoA, i.e., by introducing the gene(s) necessary for allowing the production of propionic acid in the organism/microorganism.
  • any microorganism can be genetically modified in this way.
  • the enzymes responsible for the synthesis of propionic acid have been described above.
  • Genes encoding corresponding enzymes are known in the art and can be used to genetically modify a given microorganism so as to produce propionic acid, preferably from any of the precursors of propionic acid (i.e., propionyl-CoA, acrylic acid, acrylyl-CoA, 3-hydroxypropionyl-CoA, 3- hydroxypropionaldehyde and/or glycerol).
  • propionic acid i.e., propionyl-CoA, acrylic acid, acrylyl-CoA, 3-hydroxypropionyl-CoA, 3- hydroxypropionaldehyde and/or glycerol.
  • the method of the invention comprises the step of providing the organism, preferably the microorganism carrying the respective enzyme activity or activities in the form of a (cell) culture, preferably in the form of a liquid cell culture, a subsequent step of cultivating the organism, preferably the microorganism in a fermenter (often also referred to a bioreactor) under suitable conditions allowing the expression of the respective enzyme and further comprising the step of effecting an enzymatic conversion of a method of the invention as described herein above.
  • a fermenter or bioreactor devices and fermentation conditions are known to the person skilled in the art.
  • a bioreactor or a fermenter refers to any manufactured or engineered device or system known in the art that supports a biologically active environment.
  • a bioreactor or a fermenter may be a vessel in which a chemical/biochemical like the method of the present invention is carried out which involves organisms, preferably microorganisms and/or biochemically active substances, i.e., the enzyme(s) described above derived from such organisms or organisms harbouring the above described enzyme(s).
  • this process can either be aerobic or anaerobic.
  • bioreactors are commonly cylindrical, and may range in size from litres to cubic metres, and are often made of stainless steel.
  • the fermenter or bioreactor may be designed in a way that it is suitable to cultivate the organisms, preferably microorganisms, in, e.g., a batch-culture, feed-batch-culture, perfusion culture or chemostate-culture, all of which are generally known in the art.
  • the culture medium can be any culture medium suitable for cultivating the respective organism or microorganism.
  • the method according to the present invention also comprises the step of recovering the ethylene produced by the method.
  • the ethylene can be recovered from the fermentation off-gas by methods known to the person skilled in the art.
  • the present invention relates to a method as described herein above in which a microorganism as described herein above is employed, wherein the microorganism is capable of enzymatically converting glycerol into propionic acid (and preferably further into ethylene), wherein said method comprises culturing the microorganism in a culture medium which contains glycerol and/or which comprises the step of adding glycerol to the culture medium.
  • the enzymes used in the method according to the invention can be a naturally occurring enzymes or enzymes which are derived from a naturally occurring enzymes, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.
  • Methods for modifying and/or improving the desired enzymatic activities of proteins are well-known to the person skilled in the art and include, e.g., random mutagenesis or site-directed mutagenesis and subsequent selection of enzymes having the desired properties or approaches of the so-called "directed evolution".
  • a nucleic acid molecule encoding a corresponding enzyme can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences.
  • Standard methods see Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added.
  • DNA fragments can be Iigated by using adapters and linkers complementary to the fragments.
  • engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used.
  • a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.
  • the resulting enzyme variants are then tested for the desired activity, e.g., enzymatic activity, with an assay as described above and in particular for their increased enzyme activity.
  • the microorganism employed in a method of the invention or contained in the composition of the invention may be a microorganism which has been genetically modified by the introduction of a nucleic acid molecule encoding a corresponding enzyme.
  • the microorganism is a recombinant microorganism which has been genetically modified to have an increased activity of at least one enzyme described above for the conversions of the method according to the present invention. This can be achieved e.g. by transforming the microorganism with a nucleic acid encoding a corresponding enzyme.
  • the nucleic acid molecule introduced into the microorganism is a nucleic acid molecule which is heterologous with respect to the microorganism, i.e. it does not naturally occur in said microorganism.
  • an "increased activity” means that the expression and/or the activity of an enzyme in the genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-modified microorganism.
  • the increase in expression and/or activity may be at least 150%, at least 200% or at least 500%.
  • the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-modified microorganism.
  • the term "increased" expression/activity also covers the situation in which the corresponding non-modified microorganism does not express a corresponding enzyme so that the corresponding expression/activity in the non-modified microorganism is zero.
  • the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30%, or 40% of the total host cell protein.
  • the measurement of the level of expression is done by measuring the amount of the corresponding protein.
  • Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc.
  • the measurement of the level of expression is done by measuring the amount of the corresponding RNA.
  • Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot.
  • the term "recombinant" means that the microorganism is genetically modified so as to contain a nucleic acid molecule encoding an enzyme as defined above as compared to a wild-type or non-modified microorganism.
  • a nucleic acid molecule encoding an enzyme as defined above can be used alone or as part of a vector.
  • the nucleic acid molecules can further comprise expression control sequences operably linked to the polynucleotide comprised in the nucleic acid molecule.
  • operatively linked or “operably linked”, as used throughout the present description, refers to a linkage between one or more expression control sequences and the coding region in the polynucleotide to be expressed in such a way that expression is achieved under conditions compatible with the expression control sequence.
  • Expression comprises transcription of the heterologous DNA sequence, preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in fungi as well as in bacteria, are well known to those skilled in the art. They encompass promoters, enhancers, termination signals, targeting signals and the like. Examples are given further below in connection with explanations concerning vectors.
  • Promoters for use in connection with the nucleic acid molecule may be homologous or heterologous with regard to its origin and/or with regard to the gene to be expressed. Suitable promoters are for instance promoters which lend themselves to constitutive expression. However, promoters which are only activated at a point in time determined by external influences can also be used. Artificial and/or chemically inducible promoters may be used in this context.
  • the vectors can further comprise expression control sequences operably linked to said polynucleotides contained in the vectors. These expression control sequences may be suited to ensure transcription and synthesis of a translatable RNA in bacteria or fungi.
  • mutants possessing a modified substrate or product specificity can be prepared. Preferably, such mutants show an increased activity. Alternatively, mutants can be prepared the catalytic activity of which is abolished without losing substrate binding activity.
  • the introduction of mutations into the polynucleotides encoding an enzyme as defined above allows the gene expression rate and/or the activity of the enzymes encoded by said polynucleotides to be reduced or increased.
  • the polynucleotides encoding an enzyme as defined above or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences.
  • Standard methods see Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added.
  • DNA fragments can be connected to each other by applying adapters and linkers to the fragments.
  • engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used.
  • a recombinant microorganism can be produced by genetically modifying fungi or bacteria comprising introducing the above- described polynucleotides, nucleic acid molecules or vectors into a fungus or bacterium.
  • the polynucleotide encoding the respective enzyme is expressed so as to lead to the production of a polypeptide having any of the activities described above.
  • An overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440).
  • yeast expression systems are for instance given by Hensing et al. (Antonie van Leuwenhoek 67 (1995), 261 -279), Bussineau et al. (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991 ), 742-745) and Buckholz (Bio/Technology 9 (1991 ), 1067-1072).
  • Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence.
  • the DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene.
  • Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters.
  • a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used.
  • the transformation of the host cell with a polynucleotide or vector as described above can be carried out by standard methods, as for instance described in Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
  • the host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.
  • the present invention also relates to a recombinant organism or microorganism which is able to express the above described enzymes required for the enzymatic conversion of glycerol into acrylyl-CoA.
  • the present invention relates to a recombinant organism or microorganism which expresses
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme in (a) is a (cobalamine (B12 vitamin)-dependent or B12-indepentent/radical-S-adenosyl methionine- dependent) glycerol dehydratase (EC 4.2.1.30).
  • the enzyme in (a) is a (cobalamine (B12 vitamin)-dependent or B12-indepentent/radical-S-adenosyl methionine- dependent) glycerol dehydratase (EC 4.2.1.30).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme in (b) is a CoA-acylating aldehyde dehydrogenase, more preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10).
  • the enzyme in (b) is a CoA-acylating aldehyde dehydrogenase, more preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme in (c) is a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), more preferably a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), a 3-hydroxypropionyl-
  • the enzyme in (c) is a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), more preferably a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), a 3-hydroxypropionyl-
  • CoA dehydratase (EC 4.2.1.116) or an enoyl-CoA hydratase (EC 4.2.1.17).
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of acrylyl-CoA from glycerol.
  • the present invention also relates to the use of one or more enzymes as defined above for the production of acrylyl-CoA from glycerol.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of acrylyl-CoA into propionyl-CoA (step IV as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme catalyzing the enzymatic conversion of acrylyl-CoA into propionyl-CoA is an enoyl-CoA reductase
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of propionyl-CoA from glycerol. Moreover, the present invention also relates to the use of one or more enzymes as defined above for the production of propionyl-CoA from glycerol. As regards the preferred embodiments of these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of propionyl-CoA from glycerol, the same applies as has been set forth above in connection with the methods according to the present invention.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of propionyl-CoA into propionic acid (step V as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses and enzyme catalyzing the enzymatic conversion of propionyl-CoA into propionyl phosphate and which expresses an enzyme catalyzing the enzymatic conversion of propionyl phosphate into said propionic acid.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme catalyzing the enzymatic conversion of said propionyl-CoA into said propionyl phosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme catalyzing the conversion of said propionyl phosphate into said propionic acid is a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty- acid kinase (EC 2.7.2.14).
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of propionyl- CoA into propionic acid is a recombinant organism or microorganism which expresses a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1 .2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • acetyl-CoA hydrolase EC 3.1 .2.1
  • an ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.18
  • an acyl-CoA hydrolase EC 3.1.2.20
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of propionyl- CoA into propionic acid is a recombinant organism or microorganism which expresses a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl- CoA:acetate CoA-transferase (EC 2.8.3.18).
  • a CoA transferase EC 2.8.3.-
  • a propionate:acetate-CoA transferase EC 2.8.3.1
  • an acetate CoA-transferase EC 2.8.3.8
  • succinyl- CoA:acetate CoA-transferase EC 2.8.3.18.
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of propionic acid from glycerol. Moreover, the present invention also relates to the use of one or more enzymes as defined above for the production of propionic acid from glycerol.
  • the present invention also relates to the above recombinant organism or microorganism which is able to express the above described enzymes required for the enzymatic conversion of glycerol into acrylyl-CoA and which further expresses an enzyme catalyzing the enzymatic conversion of acrylyl-CoA into acrylic acid (step VI as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses an enzyme catalyzing the enzymatic conversion of acrylyl-CoA into acrylyl phosphate and which expresses an enzyme catalyzing the enzymatic conversion of acrylyl phosphate into acrylic acid.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme catalyzing the enzymatic conversion of said acrylyl-CoA into said acrylyl phosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme catalyzing the conversion of said acrylyl phosphate into said acrylic acid is a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of acrylyl- CoA into acrylic acid is a recombinant organism or microorganism which expresses a thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC 3.1.2.1 ), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • acetyl-CoA hydrolase EC 3.1.2.1
  • an ADP-dependent short-chain-acyl-CoA hydrolase EC 3.1.2.1
  • an acyl-CoA hydrolase EC 3.1.2.20
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of acrylyl- CoA into acrylic acid is is a recombinant organism or microorganism which expresses a CoA-transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA- transferase (EC 2.8.3.18).
  • a CoA-transferase EC 2.8.3.-
  • a propionate:acetate-CoA transferase EC 2.8.3.1
  • an acetate CoA-transferase EC 2.8.3.8
  • succinyl-CoA:acetate CoA- transferase EC 2.8.3.18
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of acrylic acid from glycerol.
  • the present invention also relates to the use of one or more enzymes as defined above for the production of acrylic acid from glycerol.
  • the present invention also relates to the above recombinant organism or microorganism which is able to express the above described enzymes required for the enzymatic conversion of glycerol into acrylyl-CoA and which further expresses an enzyme catalyzing the enzymatic condensation of the thus produced acrylyl-CoA with acetyl-CoA into 3-oxo-4-pentenoyl-CoA (step VII as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses an acetyl-CoA C- acyltransferase (EC 2.3.1.16).
  • an acetyl-CoA C- acyltransferase EC 2.3.1.16
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of 3-oxo-4-pentenoyl-CoA from glycerol.
  • the present invention also relates to the use of one or more enzymes as defined above for the production of 3-oxo-4-pentenoyl-CoA from glycerol.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which further expresses an enzyme catalyzing the the enzymatic reduction of 3-oxo-4-pentenoyl-CoA into 3-hydroxy-4- pentenoyl-CoA (step VIII as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses an enzyme of the family of 3-hydroxyacyl-CoA dehydrogenases (EC 1.1.1.-), preferably a 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157) or a 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35).
  • EC 1.1.1.- 3-hydroxyacyl-CoA dehydrogenases
  • EC 1.1.1.157 3-hydroxybutyryl-CoA dehydrogenase
  • a 3-hydroxyacyl-CoA dehydrogenase EC 1.1.1.35
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of 3-hydroxy-4-pentenoyl-CoA from glycerol.
  • the present invention also relates to the use of one or more enzymes as defined above for the production of 3-hydroxy-4-pentenoyl-CoA from glycerol.
  • one or more enzymes as defined above for the production of 3-hydroxy-4-pentenoyl-CoA from glycerol.
  • these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of 3-hydroxy-4- pentenoyl-CoA from glycerol the same applies as has been set forth above in connection with the methods according to the present invention.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4- pentadienoyl-CoA (step IX as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses an enzyme of the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases (EC 4.2.1.-), preferably, a 3- hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) or an enoyl-CoA hydratase (EC 4.2.1.17).
  • an enzyme of the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases EC 4.2.1.-
  • a 3-hydroxypropionyl-CoA dehydratase EC 4.2.1.116
  • a 3-hydroxybutyryl-CoA dehydratase EC 4.2.1.55
  • an enoyl-CoA hydratase EC 4.2.1.17
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of 2,4-pentadienoyl-CoA from glycerol.
  • the present invention also relates to the use of one or more enzymes as defined above for the production of 2,4-pentadienoyl-CoA from glycerol.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid (step XI as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses an enzyme catalyzing the enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoyl phosphate and which expresses an enzyme catalyzing the enzymatic conversion of 2,4-pentadienoyl phosphate into 2,4-pentadienoic acid.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme catalyzing the enzymatic conversion of said 2,4- pentadienoyl-CoA into said 2,4-pentadienoyl phosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme catalyzing the enzymatic conversion of said 2,4-pentadienoyl phosphate into said 2,4-pentadienoic acid is a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a branched-fatty-acid
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of 2,4- pentadienoyl-CoA into 2,4-pentadienoic acid is a recombinant organism or microorganism which expresses a thioester hydrolase (EC 3.1.2.-), preferably an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • an acyl-CoA hydrolase EC 3.1.2.20
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of 2,4- pentadienoyl-CoA into 2,4-pentadienoic acid is a recombinant organism or microorganism which expresses a CoA transferase (EC 2.8.3.-), preferably a butyryl- CoA:acetate-CoA transferase (EC 2.8.3.8).
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of 2,4-pentadienoic acid from glycerol.
  • the present invention also relates to the use of one or more enzymes as defined above for the production of 2,4-pentadienoic acid from glycerol.
  • enzymes as defined above for the production of 2,4-pentadienoic acid from glycerol.
  • preferred embodiments of these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of 2,4- pentadienoic acid from glycerol the same applies as has been set forth above in connection with the methods according to the present invention.
  • the present invention also relates to the above recombinant organism or microorganism which is able to express the above described enzymes required for the enzymatic conversion of glycerol into acrylyl-CoA, which is able to express the above described enzymes required for the enzymatic conversion of an enzyme catalyzing the enzymatic condensation of acrylyl-CoA with acetyl-CoA into 3-oxo-4- pentenoyl-CoA, which is able to express the above described enzymes required for the enzymatic conversion of 3-oxo-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl-CoA and which further expresses an enzyme catalyzing the enzymatic conversion of 3- hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid (step X as shown in Figure 14).
  • the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses an enzyme catalyzing the enzymatic conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl phosphate and which expresses an enzyme catalyzing the enzymatic conversion of 3-hydroxy-4-pentenoyl phosphate into 3-hydroxy-4-pentenoic acid.
  • the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme catalyzing the enzymatic conversion of said 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl phosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyitransferase (EC 2.3.1.8) and enzyme catalyzing the enzymatic conversion of said 3-hydroxy-4-pentenoyl phosphate into said 3-hydroxy-4-pentenoic acid is a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-fatty-acid kinase (EC 2.7.2
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of 3- hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid is a recombinant organism or microorganism which further expresses a thioester hydrolase (EC 3.1.2.-), preferably an acyl-CoA hydrolase (EC 3.1.2.20).
  • a thioester hydrolase EC 3.1.2.-
  • an acyl-CoA hydrolase EC 3.1.2.20
  • the above recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic conversion of 3- hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid is a recombinant organism or microorganism which further expresses a CoA transferase (EC 2.8.3.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8).
  • the present invention also relates to the use of a recombinant organism or microorganism as defined above for the production of 3-hydroxy-4-pentenoic acid from glycerol.
  • the present invention also relates to the use of one or more enzymes as defined above for the production of 3-hydroxy-4-pentenoic acid from glycerol.
  • the present invention also relates to a bacterium which is able to express the above described enzymes required for the enzymatic conversion of propionic acid into ethylene and the enzymes required for the enzymatic conversion of acrylyl-CoA into said propionic acid.
  • the present invention relates to a bacterium which expresses
  • the above bacterium is a bacterium wherein the enzyme in (I) is a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase as described above.
  • such a bacterium is a bacterium which is capable of converting glycerol into propionic acid, e.g., by the pathways as described herein above.
  • the above bacterium is a bacterium wherein the enzymes in (II) (steps IVa and Va as shown in Figure 1 ) are an enzyme catalyzing the conversion of acrylyl-CoA into propionyl-CoA (steps IVa as shown in Figure 1 ); and an enzyme further catalyzing the conversion of the thus produced propionyl-CoA into propionic acid (step Va as shown in Figure 1 ).
  • the above bacterium is a bacterium, wherein the enzyme catalyzing the conversion of acrylyl-CoA into propionyl-CoA (step IVa as shown in Figure 1 ) is an enoyl-CoA reductase (EC 1.3.1.-), preferably an acrylyl-CoA reductase (EC 1.3.1.95) or an NADPH-dependent acrylyl-CoA reductase (EC 1.3.1.84).
  • the enzyme catalyzing the conversion of acrylyl-CoA into propionyl-CoA is an enoyl-CoA reductase (EC 1.3.1.-), preferably an acrylyl-CoA reductase (EC 1.3.1.95) or an NADPH-dependent acrylyl-CoA reductase (EC 1.3.1.84).
  • the above bacterium is a bacterium, wherein the enzyme(s) catalyzing the enzymatic conversion of propionyl-CoA into propionic acid (step Va as shown in Figure 1 ) is/are
  • a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1 ), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18).

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Abstract

L'invention concerne un procédé pour la production d'acrylyl-CoA à partir de glycérol, comprenant les étapes suivantes : (a) la conversion enzymatique de glycérol en 3-hydroxypropionaldéhyde; (b) la conversion enzymatique dudit 3-hydroxypropionaldéhyde en 3-hydroxypropionyl-CoA; et (c) la conversion enzymatique dudit 3-hydroxypropionyl-CoA en acrylyl-CoA. L'invention concerne en outre la conversion enzymatique d'acrylyl-CoA en d'autres produits. L'invention concerne par ailleurs un procédé de production d'éthylène comprenant la conversion enzymatique d'acide propionique en éthylène. Selon l'invention, la conversion enzymatique d'acide propionique en éthylène peut être obtenue par utilisation d'une décarboxylase d'acide gras P450 cytochrome ou d'une oxygénase de fer non hémique. En outre, selon l'invention, ledit acide propionique peut être obtenu par la conversion enzymatique d'acrylyl-CoA en ledit acide propionique.
PCT/EP2015/080482 2014-12-19 2015-12-18 Production enzymatique d'acrylyl-coa ou d'éthylène à partir de glycérol WO2016097289A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018005770A3 (fr) * 2016-06-30 2018-04-05 Invista North America S.á.r.l. Voies de fixation de carbone synthétique
EP3816271A1 (fr) * 2019-10-31 2021-05-05 The Procter & Gamble Company Composition de détergent

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090325248A1 (en) * 2005-10-10 2009-12-31 Evonik Degussa Gmbh Microbiological Production of 3-Hydroxypropionic Acid
US20100291644A1 (en) * 2006-06-02 2010-11-18 Evonik Roehm Gmbh Process for preparing methacrylic acid or methacrylic esters
US20110105791A1 (en) * 2006-08-22 2011-05-05 Franz-Felix Kuppinger Process for preparing acrylic acid purified by crystallization from hydroxypropionic acid and apparatus therefore

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090325248A1 (en) * 2005-10-10 2009-12-31 Evonik Degussa Gmbh Microbiological Production of 3-Hydroxypropionic Acid
US20100291644A1 (en) * 2006-06-02 2010-11-18 Evonik Roehm Gmbh Process for preparing methacrylic acid or methacrylic esters
US20110105791A1 (en) * 2006-08-22 2011-05-05 Franz-Felix Kuppinger Process for preparing acrylic acid purified by crystallization from hydroxypropionic acid and apparatus therefore

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018005770A3 (fr) * 2016-06-30 2018-04-05 Invista North America S.á.r.l. Voies de fixation de carbone synthétique
EP3816271A1 (fr) * 2019-10-31 2021-05-05 The Procter & Gamble Company Composition de détergent
WO2021087510A1 (fr) * 2019-10-31 2021-05-06 The Procter & Gamble Company Composition détergente

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