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WO2016097289A1 - Enzymatic production of acrylyl-coa or ethylene from glycerol - Google Patents

Enzymatic production of acrylyl-coa or ethylene from glycerol Download PDF

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Publication number
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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PCT/EP2015/080482
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French (fr)
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Mathieu Allard
Philippe Marliere
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Global Bioenergies
Scientist Of Fortune S.A.
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Publication of WO2016097289A1 publication Critical patent/WO2016097289A1/en

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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

Described is 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. Further enzymatic conversion of acrylyl-CoA into other products are disclosed. Further described is a method for the production of ethylene comprising the enzymatic conversion of propionic acid into ethylene. It is described that 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. Further, it is described that said propionic acid can be obtained by the enzymatic conversion of acrylyl-CoA into said propionic acid.

Description

Enzymatic production of acrylyl-CoA or ethylene from glycerol
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. Moreover, said acrylyl-CoA can further be enzymatically converted into acrylic acid. Moreover, 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. Finally, 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. Finally, 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.
Moreover, 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.
Thus, there is an increasing demand for acrylyl-CoA as well as its derivatives mentioned above. Hence, in order to satisfy the increasing demand for acrylyl-CoA (and its derivatives acrylic acid, propionic acid, 3-oxo-4-pentenoyl-CoA, 3-hydroxy-4- pententoic acid and 2,4-pentadienoic acid) it is desirable to provide for an alternative process for the production of acrylyl-CoA which could be effected in living organisms, thereby being environmentally sound and inexpensive.
In a first aspect, 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 (C2H4). 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 C02 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.
Several companies develop a semi-biologic process to chemically convert bio- ethanol to ethylene. However, these processes are expensive in energy since the dehydration of ethanol at high temperature, (i.e., 300-400 °C) is required and involve a purification step of the culture medium because ethanol is toxic for yeast at a concentration of 15%. A direct route to the biosynthesis of ethylene in microorganism is suggested to be a better way to produce bio-ethylene.
Several plants are well known to produce ethylene as a plant growth regulator. The metabolic pathway involves in the last step a metalloenzyme, i.e., an ACC oxidase, which converts 1-aminocyclopropane-1 -carboxylic acid to ethylene, C02 and HCN by an oxidative decarboxylation mechanism (Annual Review of Plant Physiology and Plant Molecular Biology 1993, 44, p283-307).
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 C02 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) 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). But, 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. These organisms produce ethylene by using an enzyme (called Ethylene Forming Enzyme or EFE) catalyzing the conversion of 2-oxo-glutarate, arginine and molecular oxygen to the olefin, succinate and CO2 (Biotech. Biofuel. 7 (2014), 33). The stoichiometry of this reaction is complex, as three precursors of five carbon atoms is necessary for producing 2 molecules of ethylene. The EFE protein from Pseudomonas syringae was cloned in an industrial strain, i.e., in Saccharomyces cerevisae, and a metabolically engineered strain allowed to obtain the conversion of 1 mg ethylene from 1g glucose (Metab Eng. 10 (2008), 276-80). But the carbon yield of this pathway turned out to be too low to permit a viable industrial process.
Accordingly, in order to satisfy the increasing demand for ethylene it is desirable to provide for an alternative process for the production of ethylene which is efficient and independent of inorganic production steps and which could be effected in living organisms, thereby being environmentally sound and inexpensive.
Therefore, in a second aspect, 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 CO2 using a cytochrome P450.
In addition, although the biosynthesis of propionic acid in microorganisms has already been described, 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 ).
The biosynthesis of propionic acid in microorganism is already described (Appl. Microbiol. Biotechnol. 53 (2000), 435-440). For example, bacteria such as Propionibacterium acidipropionici or Propionibacterium freudenreichii ssp. shermanii are well known to naturally produce propionic acid from glucose or glycerol with a high efficiency (productivity up to 0.42 g. Γ1. H"1). Engineered metabolic pathways producing propionic acid have already been established in modified microorganisms. For example, the heterologous pathway to convert the D-lactic acid to propionic acid from Clostridium propionicum was established in Escherichia coli (Appl. Microbiol. Biotechnol. 97 (20 3), 1191-2000).
In the literature, there are mainly three pathways for the biosynthesis of propionic acid (or propionyl-coenzyme A) from a carbon source such as glucose or glycerol described:
1 . The Propionobacterium pathway involving the oxaloacetate/ succinate pathway (Curr. Microbiol. 62 (201 1 ), 152-158).
2. The threonine pathway (Proc. Natl. Acad. Sci. 109 (2012), 17925-17930).
3. The 3-hydroxypropionate bicycle pathway (Appl. Environ. Microbiol. 78 (2012), 8564-8570).
Moreover, the biosynthetic production of 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 ).
All these pathways involve several enzymatic steps with more or less complex organic intermediates to ultimately convert the carbon source to the precursor of propionic acid, i.e., propionyl-coenzyme A.
In the present invention, the most direct route for the conversion of glycerol to propionic acid via propionyl-coenzyme A is described. In the present invention, this direct pathway is supplemented with the above- mentioned oxidative decarboxylation of propionic acid to ethylene and C02 catalyzed by a cytochrome P450.
These two catalytic segments allow the efficient, economic and direct biosynthesis of ethylene from glycerol, e.g., in a metabolically engineered strain of Escherichia coli.
In a nutshell, in a first aspect, 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. The thus obtained 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.
Further, in a nutshell, the present invention, therefore, 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.
More specifically, glycerol can enzymatically be converted to 3- hydroxypropionaldehyde which can be further enzymatically converted to 3- hydroxypropionyl-CoA. Further, in one alternative, 3-hydroxypropionyl-CoA can enzymatically be converted to propionic acid via the intermediate propionyl-CoA. In another alternative, 3-hydroxypropionyl-CoA can enzymatically be converted to propionic acid via the intermediate acrylic acid. Finally, 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)
Thus, 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 (step I as shown in Figure 14);
(b) the enzymatic conversion of said 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA (step II as shown in Figure 14); and
(c) the enzymatic conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA (step III as shown in Figure 14).
The enzymatic conversion of glycerol into 3-hvdroxypropionaldehvde (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 (step I as shown in Figure 14) 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.-.
In a preferred embodiment, 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 .«¾ 3-hydroxypropanal + H20
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.
In a preferred embodiment, 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).
In a preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing any one of the amino acid sequences of SEQ ID NOs:22 to 24. It is also possible to use 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. Preferably, 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.
As regards the determination of sequence identity, the following should apply: When the sequences which are compared do not have the same length, the degree of identity 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. 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.
When using the Clustal analysis method to determine 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. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.
In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiple comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.
Preferably, 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. For example, 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.
In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiple comparisons/alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.
In another embodiment, 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. Such 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 enzymatic conversion of 3-hvdroxypropionaldehvde into 3-hvdroxypropionyl-CoA (step II as shown in Figure 14)
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 (step II as shown in Figure 14) 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.-.
In a preferred embodiment, 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+ ^¾>ropanoyl-CoA + NADH + H+
These enzymes naturally catalyze the conversion of propionaldehyde (or propanal) to propanoyl-coenzyme A by using a reducing cofactor NAD or NADP and coenzyme A. This reaction is reversible.
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.
In a preferred embodiment, 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). The conversion of 3- hydroxypropionaldehyde to 3-hydroxypropionyl-CoA catalyzed by this enzyme in E. co// has already been described (Enz. Microbiol. Tech. 53 (2013), 235-242). In a preferred embodiment, 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.
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. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
Acetaldehyde dehydrogenases (acetylating) (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 (acetylating) enzymes 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. As mentioned, the enzyme classified as Coenzyme-A-acylating aldehyde dehydrogenases (EC 1.2.1.-) use NADH or NADPH as a co-factor. In a particularly preferred embodiment, 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 enzymatic conversion of 3-hvdroxypropionyl-CoA into acrylyl-CoA (step III as shown in Figure 14)
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 (step III as shown in Figure 14) 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 H2O. Enzymes catalyzing 3-hydroxypropionyl-CoA dehydration are enzymes which catalyze the reaction as shown in Figure 10. Preferably, such an enzyme belongs to the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases (EC 4.2.1.-).
Thus, 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.-). 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):
(a) a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116),
(b) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55),
(c) an enoyl-CoA hydratase (EC 4.2.1.17),
(d) a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59), (e) a crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58),
(f) a 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60),
(g) a 3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61 ),
(h) a long-chain-enoyl-CoA hydratase (EC 4.2.1.74), and
(i) a 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18).
All these enzymes which are capable of catalyzing 3-hydroxypropionyl-CoA dehydration have in common that they use a natural substrate having the following minimal structural motif:
Figure imgf000015_0001
wherein
R1 is a hydrogen atom or an alkyl group or CH2COO";
R2 is a hydrogen atom or a methyl group; and
R3 is coenzyme A or acyl-carrier protein.
Thus, the above mentioned enzymes which can catalyze the dehydration of 3- hydroxypropionyl-CoA can be divided into two groups as follows:
I. 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:
3-hydroxyacyl-[acyl-carrier protein] 2-enoyl-[acyl-carrier protein] + H20
The enzymes of this group share a common structural motif which is referenced in the InterPro as InterPro IPR013114
(http://www.ebi.ac.uk/interpro/entry/IPR013114). The accession number for these enzymes in the Pfam database is PF 07977 (http://pfam.sanger.ac.uk/familv/PF07977). II. 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 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
(http://www.ebi.ac.uk/interpro/entry/IPR018376). The accession number for these enzymes in the Pfam database is PF00378 (http://pfam.sanger.ac.uk/family/PF00378).
In one embodiment of the method according to the invention 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:
3-hydroxypropionyl-CoA ^~ **acrvlovl-CoA + H2O
Figure imgf000016_0001
The enzyme is known from various bacteria and archae. Thus, in a preferred embodiment of the invention 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. 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.
In a preferred embodiment, 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.
In principle any 3-hydroxypropionyl-CoA dehydratase can be employed in the method according to the invention. However, it is not only possible to employ in the method of 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.
Thus, in another embodiment of the method according to the invention 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 crotonyl-CoA + H2O
Figure imgf000017_0001
This reaction corresponds to a Michael elimination. 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. In principle, 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. In a preferred embodiment of the 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.
In a preferred embodiment, 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.
In another embodiment of the method according to the invention 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:
(3S)-3-hydroxyacyl-CoA →- trans-2(or 3)-enoyl-CoA + H2O
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. In principle, 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.
In another embodiment of the method according to the 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:
(3R)-3-hydroxyoctanoyl-[acyl-carrier protein] - ""^" oct-2-enoyl-[acyl-carrier protein] + H2O
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). Other names in common use include D-3-hydroxyoctanoyl-[acyl carrier protein] dehydratase, D-3-hydroxyoctanoyl-acyl carrier protein dehydratase, beta- hydroxyoctanoyl-acyl carrier protein dehydrase, beta-hydroxyoctanoyl thioester dehydratase, beta-hydroxyoctanoyl-ACP-dehydrase, and (3R)-3-hydroxyoctanoyl- [acyl-carrier-protein] hydro-lyase. 3-hydroxyoctanoyl-[acyI-carrier-protein] dehydratases has been described to exist, e.g., in E. coli (Mizugaki et al., Biochem. Biophys. Res. Commun. 33 (1968), 520-527). In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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. .58) catalyze the following reaction:
(3R)-3-hydroxybutanoyl-[acyl-carrier-protein] but-2-enoyl-[acyl-carrier- protein] + H20
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
Other names in common use include (3R)-3-hydroxybutanoyl-[acyl-carrier-protein] hydro-lyase, beta-hydroxybutyryl acyl carrier protein dehydratase, beta- hydroxybutyryl acyl carrier protein (ACP) dehydratase, beta-hydroxybutyryl acyl carrier protein dehydratase, enoyl acyl carrier protein hydratase, crotonyl acyl carrier protein hydratase, 3-hydroxybutyryl acyl carrier protein dehydratase, beta- hydroxybutyryl acyl carrier, and protein dehydratase. This enzyme participates in fatty acid biosynthesis. Crotonoyl-[acyl-carrier-protein] hydratase has been described to exist, e.g., in E. coli and Arabidopsis thaliana. In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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 (EC 4.2.1.60) catalyze the following reactions:
(1 ) (3R)-3-hydroxydecanoyl-[acyl-carrier protein] ^~*~ a trans-dec-2-enoyl-[acyl- carrier protein] + H2O
(2) (3R)-3-hydroxydecanoyl-[acyl-carrier protein] ^ ^- a cis-dec-3-enoyl-[acyl- carrier protein] + H2O
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. In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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 (EC 4.2.1.61 ) catalyze the following reaction:
(3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] ^ hexadec-2-enoyl-[acyl-carrier- protein] + H20
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
Other names in common use include D-3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase, beta-hydroxypalmitoyl-acyl carrier protein dehydratase, beta- hydroxypalmitoyl thioester dehydratase, beta-hydroxypalmityl-ACP dehydratase, and (3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] hydro-lyase. 3-hydroxypalmitoyl-[acyl- carrier-protein] dehydratase has been described to exist, e.g., in Candida albicans, Yarrowia lipolytica, S. cerevisiae, S. pombe, Cochliobolus carbonum, Mus musculus, Rattus norvegicus, Bos taurus, Gallus gallus and Homo sapiens. In principle, 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.
In another embodiment of the method according to the 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 + H2O
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. 252 (1977), 542- 547; Schulz, Biol. Chem. 249 (1974), 2704-2709) and in principle any long-chain- enoyl-CoA hydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA can be employed in the method of the invention.
In another embodiment of the method according to the invention 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
Figure imgf000023_0001
+ H20
Figure imgf000023_0002
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. The term "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.
Yet, in a more preferred embodiment, 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 enzymatic conversion of acrylyl-CoA into propionic acid (steps IV and V as shown in Figure 14)
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.
Thus, 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.
Moreover, 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 enzymatic conversion of acrylyl-CoA into propionyl-CoA (step IV as shown in Figure 14)
According to the present invention, the conversion of acrylyl-CoA into propionyl-CoA (step IV as shown in Figure 14) 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. In one embodiment, the enzyme is an enzyme which is classified as EC 1.3.1.- and which uses NADH or NADPH as a co-factor. In a particularly preferred embodiment, 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.
Thus, in one particularly preferred embodiment, the enzyme is an enzyme which uses NADPH as a co-factor. In a preferred embodiment the enzyme is selected from the group consisting of.
- acyl-CoA dehydrogenase (NADP+) (EC 1.3.1.8);
- enoyl-[acyl-carrier-protein] reductase (NADPH, Si-specific) (EC 1.3.1.10);
- cis-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.37);
- trans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38);
- enoyl-[acyl-carrier-protein] reductase (NADPH, Re-specific) (EC 1.3.1.39); and
- crotonyl-CoA reductase (EC 1.3.1.86). Thus, in one preferred embodiment 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:
Acyl-CoA + NADP+ « *~ 2,3-dehydroacyl-CoA + NADPH + H+
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.
In a further preferred embodiment 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+ „ »* 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.
In a further preferred embodiment 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+ ί: cis-2,3-dehydroacyl-CoA + NADPH + H+
This enzyme has been described to occur in Escherichia coli.
In a further preferred embodiment 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). Trans-2-enoyl-CoA reductases (NADPH) are enzymes which catalyze the following reaction:
Acyl-CoA + NADP+ ¾ »» trans-2,3-dehydroacyl-CoA + NADPH + H+
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.
In a further preferred embodiment 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.
In a further preferred embodiment 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.
In a further preferred embodiment, 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+ „ » 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.
In another particularly preferred embodiment the enzyme is an enzyme which uses NADH as a co-factor. The conversion using such an enzyme is schematically shown in Figure 4. In a preferred embodiment the enzyme is selected from the group consisting of:
- enoyl-[acyl-carrier-protein] reductase (NADH) (EC 1.3.1.9); and
- trans-2-enoyl-CoA reductase (NAD+) (EC .3. .44).
Thus, in one preferred embodiment 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). Enoyl-[acyl-carrier-protein] reductases (NADH) are 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.
In a further preferred embodiment 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). Trans-2-enoyl-CoA reductases (NAD+) are enzymes which catalyze the following reaction:
Acyl-CoA + NAD+ „ » trans-2,3-dehydroacyl-CoA + NADH + H+
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.
In a further preferred embodiment, 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). 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. In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO.26. 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: 26. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
In one preferred embodiment, the propionyl-CoA produced according to the above described method from glycerol is not further converted. Thus, in one embodiment, 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. In a further preferred embodiment, the above described method for producing propionyl- CoA further comprises the step of recovering the thus produced propionyl-CoA.
In another preferred embodiment, the propionyl-CoA produced according to the above described method from glycerol is not further converted into 3-oxopentanoyl- CoA. Thus, in another embodiment, 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. In a further preferred embodiment, the propionyl-CoA produced according to the above described method from glycerol is further converted into another compound which is not 3-oxopentanoyl-CoA. Thus, in another embodiment, 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.
In another preferred embodiment, 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. These reactions are described further below.
The enzymatic conversion of propionyl-CoA into propionic acid (step V as shown in Figure 14)
According to the present invention, the conversion of propionyl-CoA into propionic acid (step V as shown in Figure 1) 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.
Thus, in one embodiment, the enzymatic conversion of propionyl-CoA into propionic acid can be achieved by a two-step conversion via propionyl phosphate. Accordingly, in one embodiment, 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.
The corresponding reaction is schematically shown in Figure 5. The 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 + H3PO4 ^**" butyryl phosphate + CoA
It has been described by Wiesenborn et al. (Appl. Environ. Microbiol. 55 (1989), 317- 322) and by Ward et al. (J. Bacteriol. 181 (1999), 5433-5442) that 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.
The enzyme has been described to occur in a number of organism, in particular in bacteria and in protozoae. In one embodiment the enzyme is from the protozoae Dasytricha ruminantium. In a preferred embodiment 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. Most preferably, 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)).
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:20. 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: 20. Preferably, 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. As regards the determination of the 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 + H3PO4 acetyl phosphate + CoA
It has been described by Veit et al. (J. Biotechnol.140 (2009), 75-83) that phosphate acetyltransferase can also use as a substrate butyryl-CoA or propionyl-CoA.
The accession numbers for this enzyme family in InterPro database are IPR012147 and IPR002505, "http://www.ebi.ac.uk/interpro/entry/IPR002505"
(http://www.ebi.ac.uk/interpro/entry/IPR012147
http://www.ebi.ac.uk/interpro/entrv/IPR002505)
See also http://pfam.sanger.ac.uk/family/PF01515
The enzyme has been described in a variety of organisms, in particular bacteria and fungi. Thus, in one preferred embodiment 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, Bradyrhizobium japonicum, Lactobacillus fermentum, Lactobacillus sanfranciscensis, Moorella thermoacetica, Rhodopseudomonas palustris, Sinorhizobium meliloti, Streptococcus pyogenes, Thermotoga maritima or Bacillus subtilis. In another preferred embodiment the enzyme is an enzyme from a fungus, preferably from the genus Saccharomyces, more preferably of the species Saccharomyces cerevisiae.
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:21. 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: 21. Preferably, 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. As regards the determination of the 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. Thus, 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.-). In a preferred embodiment, 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).
Butyrate kinases (EC 2.7.2.7) naturally catalyze the following reaction Butyrate + ATP ^"**"butvryl phosphate + ADP
It has been described, e.g. by Hartmanis (J. Biol. Chem. 262 (1987), 617-621 ) that 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. In one preferred embodiment the enzyme is from a bacterium, preferably from a bacterium of the genus Clostridium, Butyrivibrio, Thermotoga or Enterococcus. Preferred is Clostridium. More preferably 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. Preferred is 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 carboxylic acid + ATP acyl phosphate + ADP
wherein "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).
This enzyme has been described to occur in a number of bacteria. Thus, in one preferred embodiment 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
Propanoate + ATP propanoyl phosphate + ADP
Acetate + ATP ^r^* acetyl phosphate + ADP
This enzyme has been described to occur in a number of bacteria, in particular Enterobacteriacea. Thus, in one preferred embodiment 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. In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:27. 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: 27. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
In another preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:28. 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: 28. Preferably, 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. As regards the determination of 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
This enzyme has been described to occur in a number of organisms, in particular bacteria and eukaryotes. In one preferred embodiment 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 Veillonella, more preferably from a bacterium of the species Methanosarcina thermophila, Cryptococcus neoformans, Ethanoligenens harbinense, Propionibacterium acidipropionici, Streptococcus pneumoniae, Streptococcus enterica, Streptococcus pyogenes, Acholeplasma laidlawii, Acinetobacter calcoaceticus, Ajellomyces capsulatus, Bacillus subtilis, Borrelia burgdorferi, Chaetomium globosum, Clostridium acetobutylicum, Clostridium thermocellum, Coccidioides immitis, Coprinopsis cinerea, Cryptococcus neoformans, Cupriavidus necator, Desulfovibrio vulgaris, Enterococcus faecalis, Escherichia coli, Ethanoligenes harbinense, Geobacillus stearothermophilus, Helicobacter pylori, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus sanfranciscensis, Lactococcus lactis, Listeria monocytogenes, Mesoplasma florum, Methanosarcina acetivorans, Methanosarcina mazei, Moorella thermoacetica, Mycoplasma pneumoniae, Oceanobacillus iheyensis, Propionibacterium freudenreichii, Propionibacterium acidipropionici, Rhodospeudomonas palustris, Salmonella enteric, Staphylococcus aureus, Thermotoga maritime or Veillonella parvula.
In another preferred embodiment 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.
In a further preferred embodiment the enzyme is an enzyme from a plant or an algae, preferably from the genus Chlamydomonas, even more preferably from the species Chlamydomonas reinhardtii. In another embodiment the enzyme is from an organism of the genus Entamoeba, more preferably of the species Entamoeba histolytica.
The above mentioned enzyme families suitable for the conversion of propionyl-CoA into propionyl phosphate have been shown to be evolutionary related and contain common sequence signatures. Theses signatures are referenced and described in Prosite database:
http://prosite.expasy.org/cgi-bin/prosite/nicedoc.pl7PS01075
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. These E. coli cells have been shown to be able to produce D-(-)-3-hydroxybutyric acid (3HB).
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.
Preferably, in one embodiment, 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.-)). This reaction is schematically shown in Figure 6.
Thus, in one alternative, 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).
In the alternative embodiment, 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).
Thus, in another alternative, 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).
Thus, in one embodiment, the enzymatic conversion of propionyl-CoA into propionic acid (step V as shown in Figure 1 ) according to step (b) 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: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 while 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.
In a preferred embodiment, 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).
Thus, in one preferred embodiment 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:
Acetyl-CoA + H2O ► acetate + 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 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 dionisii, Trypanosoma vespertilionis, Crithidia fasciculate, Clostridium aminovalericum, Acidaminococcus fermaentans, Bradyrhizobium japonicum and Methanosarcina barken.
In another preferred embodiment 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:
Palmitoyl-CoA + H20 ► palmitate + 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 (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 auratus, Drosophila melanogaster, Rattus norvegicus, Oryctolagus cuniculus, Gallus gallus, Anas platyrhynchos, Mycobacterium tuberculosis, Mycobacterium phlei, Mycobacterium smegmatis, Acinetobacter colcaceticus, Haemophilus influenza, Helicobacter pylori, Bacillus subtilis, Pseudomonas aeruginosa, Rhodobacter shpaeroides, Streptomyces coelicolor, Streptomyces venezuelae and E. coli.
In a further preferred embodiment 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:
3-hydroxyisobutyryl-CoA + H2O ► 3-hydroxyisobutyrate + 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, Homo sapiens, Canus lupus familiaris, Rattus norvegicus, Bacillus cereus, Pseudomonas fluorescens and Pseudomonas aeruginosa.
In yet another preferred embodiment 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] + H20 ► 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.
In yet another preferred embodiment 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 + H20 ► 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.
In yet another preferred embodiment 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 + H20 ► a carboxylate + CoA
This enzyme occurs in a variety of animals and has, e.g., been described in Rattus norvegicus and Mesocricetus auratus.
In a further preferred embodiment 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 + H2O * 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, Pseudomonas putida, Amycolatopsis mediterranei, Acinetobacter calcoaceticus, Helicobacter pylori, Rhodobacter spaeroides and Mycobacterium phlei. In a preferred embodiment 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). In another preferred embodiment 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) and acyl-CoA thioesterase II from Pseudomonas putida (Uniprot Q88DR1 ; 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).
In a particularly preferred embodiment, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
As described 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.
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).
Family I contains, e.g., the following CoA-transferases:
For 3-oxo acids: enzymes classified in EC 2.8.3.5 or EC 2.8.3.6;
For short chain fatty acids: enzymes classified in EC 2.8.3.8 or EC 2.8.3.9;
For succinate: succinyl-CoA:acetate CoA-transferases, i.e. enzymes classified in EC
2.8.3.18 (see also Mullins et al., Biochemistry 51(2012), 8422-34; Mullins et al., J.
Bacteriol. 190 (2006), 4933-4940).
Most enzymes of family I naturally use succinyl-CoA or acetyl-CoA as CoA donors.
These enzymes contain two dissimilar subunits in different aggregation states. Two conserved amino acid sequence motives have been identified:
Prosites entry PS01273 (http://prosite.expasy.org/cgi-bin/prosite/prosite-search- ac?PDOC00980)
COA_TRANSF_1 , PS01273; Coenzyme A transferases signature 1 (PATTERN) Consensus pattern:
[DN]-tGN]-x(2)-[LIVMFA](3)-G-G-F-x(3)-G-x-P
and
Prosites entries PS01273 (http://prosite.expasy.org/cgi-bin/prosite/prosite-search- ac?PDOC00980) COA_TRANSF_2, PS01274; Coenzyme A transferases signature 2 (PATTERN)
Consensus pattern:
[LF]-[HQ]-S-E-N-G-[LIVF](2)-[GA]
E (glutamic 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. 80 (1977), 479-488) and acetyl-CoA and succinyl-CoA in the case of citramalate lyase (Dimroth et al., Eur. J. Biochem. 80 (1977), 469-477).
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 C02 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.
In a preferred embodiment 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.
Preferably, 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:
propionate:acetate-CoA transferase (EC 2.8.3.1 );
acetate CoA-transferase (EC 2.8.3.8);
butyrate-acetoacetate CoA-transferase (EC 2.8.3.9); and succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18).
Thus, in one preferred embodiment 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:
Acyl-CoA + acetate a fatty acid anion + acetyl-CoA
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.
In another preferred embodiment 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:
Butanoyl-CoA + acetoacetate butanoate + acetoacetyl-CoA
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.
In another preferred embodiment 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:
Acetyl-CoA + propanoate acetate + propanoyl-CoA
This enzyme catalyzes the reversible transfer of CoA group from propionyl-CoA and acetate. The reaction is also schematically shown in Figure 7 for R= H. 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.
In another preferred embodiment 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. The reaction is also schematically shown in Figure 7 for R = CH2-CO2H.
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 enzymatic conversion of acrylyl-CoA into acrylic acid (step VI as shown in Figure 14)
The acrylyl-CoA which is produced according to the above method from glycerol may further be converted into acrylic acid.
Thus, 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.
According to the present invention, 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.
Thus, in one embodiment, 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 enzymes butyryltransferase (EC 2.3.1.19) and phosphate acetyltransferase (EC 2.3.1.8) have already been described above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionyl phosphate. As regards these enzymes, the same applies for the conversion of acrylyl-CoA into acrylyl phosphate as has been set forth above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionyl phosphate.
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. Such enzymes use a carboxy group as acceptor. Thus, 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.-). In a preferred embodiment, 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).
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. As regards these enzymes, the same applies for the conversion of acrylyl phosphate into acrylic acid as has been set forth above in the context of the enzymatic conversion of propionyl phosphate into propionic acid.
As mentioned above, 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.
Preferably, in one embodiment, 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.-)). This reaction is schematically shown in Figure 8. Thus, in one alternative, 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).
In the alternative embodiment, 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.-).
This reaction is schematically shown in Figure 8 for preferred enzymes, i.e., 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).
Thus, in another alternative, 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).
Thus, in one embodiment, 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.
In a preferred embodiment, 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);
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. .2.20).
The enzymes 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. 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. As described above, 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 (EC 2.8.3.-), 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.
In one preferred embodiment, the acrylic acid produced according to the above described method from glycerol is not further converted. Thus, in one embodiment, 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. In a further preferred embodiment, the above described method for producing acrylic acid further comprises the step of recovering the thus produced acrylic acid.
In another preferred embodiment, the acrylic acid produced according to the above described method from glycerol is not further converted into propionic acid. Thus, in another embodiment, 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.
In a further preferred embodiment, the acrylic acid produced according to the above described method from glycerol is further converted into another compound which is not propionic acid. Thus, in another embodiment, 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.
In another preferred embodiment, 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. These reactions are described further below.
The enzymatic conversion of acrylyl-CoA into 3-oxo-4-pentenoyl-CoA, 3- hydroxy-4-pentenoic acid and/or 2,4-pentadienoic acid (steps VII, VIII, IX, X and X as shown in Figure 14)
The acrylyl-CoA which is produced according to the above method from glycerol may further be converted into 3-oxo-4-pentenoyl-CoA.
Moreover, 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. Alternatively, the thus produced 3-oxo-4-pentenoyl-CoA may further be converted into 3-hydroxy-4-pentenoic acid via 3-hydroxy-4-pentenoyl-CoA.
Thus, 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.
Moreover, 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.
Moreover, 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 enzymatic condensation of acrylyl-CoA and acetyl-CoA into 3-oxo-4-pentenoyl- CoA (step VII as shown in Figure 14)
As mentioned, in accordance with the methods of the present invention, the produced acrylyl-CoA can further be converted into 3-oxo-4-pentenoyl-CoA. Thus, 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.
According to the present invention, 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 littoralis (SwissProt Q66Q58), Cupriavidus necator (Uniprot Q0KBP1 ) and Thermus thermophilus.
In a preferred embodiment, 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).
Thus, in a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:32. 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: 32. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
The enzymatic reduction of 3-oxo-4-pentenoyl-CoA into 3-hvdroxy-4-pentenoyl-CoA (step VIII as shown in Figure 14)
As mentioned, in accordance with the methods of the present invention, the produced 3-oxo-4-pentenoyl-CoA can further be converted into 3-hydroxy-4- pentenoyl-CoA. Thus, 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.
According to the present invention, 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.
Preferably, 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.-). In a preferred embodiment, the enzyme is an enzyme which uses NAD(+) as a co-factor. Several enzymes of the general family of 3-hydroxyacyl-CoA dehydrogenases classified as oxidoreductases acting on CH-OH groups of donors (EC 1.1.1.-) are also described to be able to use NADP(+) as reducing cofactor.
In a preferred embodiment, 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:
(S)-3-hydroxybutanoyl-CoA + NADP+ 3-acetoacetyl-CoA + NADPH + H+
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.
In a preferred embodiment, 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 ).
Thus, in a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:33. 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: 33. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
3-hydroxyacyl-CoA dehydrogenases (EC 1.1.1.35) catalyze the following reaction:
(S)-3-hydroxyacyl-CoA + NAD+ 3-oxoacyl-CoA + NADH + H+
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 enzymatic dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA
(step IX as shown in Figure 14)
As mentioned, in accordance with the methods of the present invention, the produced 3-hydroxy-4-pentenoyl-CoA can further be dehydrated into 2,4- pentadienoyl-CoA. Thus, 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.
According to the present invention, 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 H2O. 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. Preferably, such an enzyme belongs to the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases (EC 4.2.1.-).
Thus, 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.-). 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):
(a) a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116),
(b) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55),
(c) an enoyl-CoA hydratase (EC 4.2.1.17),
(d) a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59),
(e) a crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58),
(f) a 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60),
(g) a 3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61 ), (h) a long-chain-enoyl-CoA hydratase (EC 4.2.1.74), and
(i) a 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18).
All these enzymes which are capable of catalyzing 3-hydroxy-4-pentenoyl-CoA dehydration have in common that they use a natural substrate having the following minimal structural motif:
Figure imgf000057_0001
wherein
R1 is a hydrogen atom or an alkyl group or CH2COO";
R2 is a hydrogen atom or a methyl group; and
R3 is coenzyme A or acyl-carrier protein.
Thus, the above mentioned enzymes which can catalyze the dehydration of 3- hydroxy-4-pentenoyl-CoA can be divided into two groups as follows:
I. 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:
3-hydroxyacyl-[acyl-carrier protein] 2-enoyl-[acyl-carrier protein] + H2O
The enzymes of this group share a common structural motif which is referenced in the InterPro as InterPro IPR013114
(http://www.ebi.ac.uk/interpro/entrv/IPR013114). The accession number for these enzymes in the Pfam database is PF 07977 (http://pfam.sanger.ac.uk family/PF07977).
II. 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 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 IPR0018376
(http://www.ebi.ac.uk/interpro/entry/IPR018376). The accession number for these enzymes in the Pfam database is PF00378 (http://pfam.sanger.ac.uk/family/PF00378).
In one embodiment of the method according to the invention 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:
3-hyd
Figure imgf000058_0001
The enzyme is known from various bacteria and archae. Thus, in a preferred embodiment of the invention 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. 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.
In a preferred embodiment, 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.
In principle any 3-hydroxypropionyl-CoA dehydratase can be employed in the method according to the invention. However, it is not only possible to employ in the method of 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.
Thus, in another embodiment of the method according to the invention 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 crotonyl-CoA + H2O
Figure imgf000059_0001
This reaction corresponds to a Michael elimination. 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. In principle, 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. In a preferred embodiment of the 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.
In a preferred embodiment, 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.
In another preferred embodiment, the 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.
Thus, in a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:40. 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: 40. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
In another embodiment of the method according to the invention the dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-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:
(3S)-3-hydroxyacyl-CoA → trans-2(or 3)-enoyl-CoA + H2O
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. In principle, 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.
In another embodiment of the method according to the 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:
(3R)-3-hydroxyoctanoyl-[acyl-carrier protein] ^"^* oct-2-enoyl-[acyl-carrier protein] + H2O
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). Other names in common use include D-3-hydroxyoctanoyl-[acyl carrier protein] dehydratase, D-3-hydroxyoctanoyl-acyl carrier protein dehydratase, beta- hydroxyoctanoyl-acyl carrier protein dehydrase, beta-hydroxyoctanoyl thioester dehydratase, beta-hydroxyoctanoyl-ACP-dehydrase, and (3R)-3-hydroxyoctanoyl- [acyl-carrier-protein] hydro-lyase. 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratases has been described to exist, e.g., in E. coli (Mizugaki et al., Biochem. Biophys. Res. Commun. 33 (1968), 520-527). In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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] + H2O
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
Other names in common use include (3R)-3-hydroxybutanoyl-[acyl-carrier-protein] hydro-lyase, beta-hydroxybutyryl acyl carrier protein dehydratase, beta- hydroxybutyryl acyl carrier protein (ACP) dehydratase, beta-hydroxybutyryl acyl carrier protein dehydratase, enoyl acyl carrier protein hydratase, crotonyl acyl carrier protein hydratase, 3-hydroxybutyryl acyl carrier protein dehydratase, beta- hydroxybutyryl acyl carrier, and protein dehydratase. This enzyme participates in fatty acid biosynthesis. Crotonoyl-[acyl-carrier-protein] hydratase has been described to exist, e.g., in E. coli and Arabidopsis thaliana. In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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 (EC 4.2.1.60) catalyze the following reactions:
(2) (3R)-3-hydroxydecanoyl-[acyl-carrier protein] ^ a trans-dec-2-enoyl- [acyl-carrier protein] + H2O
(2) (3R)-3-hydroxydecanoyl-[acyl-carrier protein] ■ a cis-dec-3-enoyl-
[acyl-carrier protein] + H2O
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. In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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 (EC 4.2.1.61 ) catalyze the following reaction:
(3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] ^"^" hexadec-2-enoyl-[acyl- carrier-protein] + H2O
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
Other names in common use include D-3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase, beta-hydroxypalmitoyl-acyl carrier protein dehydratase, beta- hydroxypalmitoyl thioester dehydratase, beta-hydroxypalmityl-ACP dehydratase, and (3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] hydro-lyase. 3-hydroxypalmitoyl-[acyl- carrier-protein] dehydratase has been described to exist, e.g., in Candida albicans, Yarrowia lipolytica, S. cerevisiae, S. pombe, Cochliobolus carbonum, Mus musculus, Rattus norvegicus, Bos taurus, Gallus gallus and Homo sapiens. In principle, 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.
In another embodiment of the method according to the 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:
(3S)-3-hydroxyacyl-CoA trans-2-enoyl-CoA + H2O 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. 252 (1977), 542- 547; Schulz, Biol. Chem. 249 (1974), 2704-2709) and in principle any long-chain- enoyl-CoA hydratase which can catalyze the dehydration of 3-hydroxy-4-pentenoyl- CoA into 2,4-pentadienoyl-CoA can be employed in the method of the invention.
In another embodiment of the method according to the invention 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:
(S)-3-hydroxymethylglutaryl-CoA trans-3-methylglutaconyl-CoA + H20
Figure imgf000065_0001
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. The term "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. Although this gene was annotated as a 3-hydroxybutyryl-CoA dehydratase, Li et al. (loc. cit.) showed that its natural substrate is 3- hydroxymethylglutaryl-CoA. In a particularly preferred embodiment 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
Yet, in a more preferred embodiment, 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.
The enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid (step XI as shown in Figure 14)
As mentioned, in accordance with the methods of the present invention, the produced 2,4-pentadienoyl-CoA can further be converted into 2,4-pentadienoic acid. Thus, 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.
Thus, according to the present invention, the enzymatic conversion of 2,4- pentadienoyl-CoA into 2,4-pentadienoic acid comprises:
(a) 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; or (b) a single enzymatic reaction in which 2,4-pentadienoyl-CoA is directly converted into 2,4-pentadienoic acid by making use of a thioester hydrolase (EC 3.1 .2.-), preferably an acyl-CoA hydrolase (EC 3.1 .2.20); or
(c) a single enzymatic reaction in which 2,4-pentadienoyl-CoA is directly converted into 2,4-pentadienoic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8).
Thus, in one embodiment, 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. Accordingly, in one embodiment, 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.
Accordingly, in a preferred embodiment, 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 2.7.2.1 ), a butyrate kinase (EC 2.7.2.7) or a branched-fatty-acid kinase (EC 2.7.2.14).
As mentioned above, 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 enzymes phosphate butyryltransferase (EC 2.3.1.19) and phosphate acetyltransferase (EC 2.3. .8) have already been described above in the context of the conversion of propionyl-CoA into propionyl phosphate. As regards these enzymes, the same applies for the conversion of 2,4-pentadienoyl-CoA into said 2,4- pentadienoyl phosphate as has been set forth above of the enzymatic conversion of propionyl-CoA into propionyl phosphate.
In a preferred embodiment, 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. The amino acid sequence of said protein is shown in SEQ ID NO: 42.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:42. 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: 42. Preferably, 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. As regards the determination of the 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. Thus, 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.-). In a preferred embodiment, 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).
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. As regards these enzymes, the same applies for the conversion of 2,4-pentadienoyl phosphate into 2,4-pentadienoic acid as has been set forth above in the context of the conversion of propionyl phosphate into propionic acid.
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:43. 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: 43. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
As mentioned 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.
In a preferred embodiment, 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. Preferably, 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. As regards these enzymes, the same applies for the conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid as has been set forth above in the context of the conversion of propionyl-CoA into propionic acid.
Alternatively to the above, 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.
Thus, in one alternative, 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).
The enzymes which belong to the family of thioester hydrolases (referred to as thioesterases (EC 3.1.2.-) as well as the enzymes acetyl-CoA hydrolase (EC 3.1.2.1 ), ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) and acyl-CoA hydrolase (EC 3.1.2.20) have already been described above in the context of the conversion of propionyl-CoA into propionic acid. As regards these enzymes, the same applies for the conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid as has been set forth above in the context of the conversion of propionyl-CoA into propionic acid.
The enzymatic conversion of 3-hvdroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid (step X as shown in Figure 14)
As mentioned, in accordance with the methods of the present invention, the produced 3-hydroxy-4-pentenoyl-CoA can further be converted into 3-hydroxy-4- pentenoic acid. Thus, 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.
Thus, according to the present invention, the enzymatic conversion of 3-hydroxy-4- pentenoyl-CoA into 3-hydroxy-4-pentenoic acid comprises:
(a) 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; or
(b) a single enzymatic reaction in which 3-hydroxy-4-pentenoyl-CoA is directly converted into 3-hydroxy-4-pentenoic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably an acyl-CoA hydrolase (EC 3.1.2.20); or
(c) a single enzymatic reaction in which 3-hydroxy-4-pentenoyl-CoA is directly converted into 3-hydroxy-4-pentenoic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8).
Thus, in one embodiment, 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.
Accordingly, in a preferred embodiment, 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 kinase (EC 2.7.2.7) or a branched-fatty-acid kinase (EC 2.7.2.14).
As mentioned above, 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 enzymes phosphate butyryltransferase (EC 2.3.1.19) and phosphate acetyltransferase (EC 2.3.1.8) have already been described above in the context of the conversion of propionyl-CoA into propionyl phosphate. As regards these enzymes, the same applies for the conversion of 3-hydroxy-4-pentenoyl-CoA into said 3-hydroxy-4-pentenoyl phosphate as has been set forth above of the enzymatic conversion of propionyl-CoA into propionyl phosphate.
In a preferred embodiment, 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. The amino acid sequence of said protein is shown in SEQ ID NO: 42.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:42. 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: 42. Preferably, 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. As regards the determination of the 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. Thus, 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.-). In a preferred embodiment, 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).
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. As regards these enzymes, the same applies for the conversion of 3- hydroxy-4-pentenoyl phosphate into said 3-hydroxy-4-pentenoic acid as has been set forth above in the context of the conversion of propionyl phosphate into propionic acid.
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:43. 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: 43. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above. As mentioned 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.
In a preferred embodiment, 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. Preferably, the CoA-transferase is a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8). This reaction is schematically shown in Figure 18.
The enzymes which are classified as a CoA-transferase (EC 2.8.3.-) 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. As regards these enzymes, the same applies for the conversion of 3-hydroxy-4-pentenoyl-CoA into 3- hydroxy-4-pentenoic acid as has been set forth above in the context of the conversion of propionyl-CoA into propionic acid.
Alternatively to the above, 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.
Thus, in one alternative, 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).
The enzymes which belong to the family of thioester hydrolases (referred to as thioesterases (EC 3. .2.-) as well as the enzymes acetyl-CoA hydrolase (EC 3.1.2.1), ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) and acyl-CoA hydrolase (EC 3.1.2.20) have already been described above in the context of the conversion of propionyl-CoA into propionic acid. As regards these enzymes, the same applies for the conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4- pentenoic acid as has been set forth above in the context of the conversion of propionyl-CoA into propionic acid.
The enzymatic conversion of propionic acid into ethylene as shown !n Figure 1
As mentioned above, the present invention also relates to a method for the production of ethylene comprising the enzymatic conversion of propionic acid into ethylene.
According to the present invention, 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:
Propionic acid + NADPH + 02 *" Ethylene + C02 + NADP
In one possibility, the enzymatic conversion of propionic acid into ethylene can preferably be achieved by an oxidative decarboxylation by making use of a cytochrome P450. One example of 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. Commun. 21 (1994), 516-522 and Fukuda et al., J. Biochem. 119 (1996), 314-318). 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". Thus, in one preferred embodiment 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. In a preferred embodiment 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.
Preferably, 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.
According to one particularly preferred embodiment of the present invention, propionic acid is enzymatically converted into ethylene by an enzymatically catalyzed oxidative decarboxylation catalyzed by a cytochrome P450 olefin forming fatty acid decarboxylase. The term "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. In general, 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. (ed.), 2005, Cytochrome P450: structure, mechanism and biochemistry, 3rd ed. Kluwer Academics, New York, NY). Belcher et al. (J. Biol. Chem. 289 (2014), 6535-6550; Figure 2) shows an overview of members of the cyp152 family and their phylogenetic relationship. The family members of the cypl 52 family are characterized in that they are cytosolic hemoproteins with sequence homology to P450 monooxygenases. Enzymes of this type are produced by bacteria (e.g. Sphingomonas paucimobilis, Bacillus subtilis and those mentioned herein). 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 β-position:
Fatty acid + H2O2 = 3- or 2-hydroxy fatty acid + H2O
As reported by Rude et al. (Appl. Environ. Microbiol. 77 (2011 ), 1718-1727) some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in a- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. More preferably the term "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. cit.), 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).
Although it has been described in the literature that Ole T JE uses as substrates long chain fatty acids, the inventors surprisingly found that Ole T JE can actually accept propionic acid as a substrate and convert it into ethylene.
In a preferred embodiment the Ole T JE is an enzyme which
(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 ; and
(b) shows the activity of converting propionic acid via oxidative decarboxylation into ethylene.
As mentioned above in (a), 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. In one preferred embodiment 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. In another preferred embodiment 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. The term "structurally related" means that the amino acid sequence of the enzyme shows at least 60% sequence identity to the amino acid sequence shown in SEQ ID NO:1. Preferably, 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:1.
Structure/function analyses of the Ole T JE protein have shown that the residue in position 85 of SEQ ID NO:1 may play a role in the decarboxylation activity of the Ole T JE protein. In one preferred embodiment 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. In another preferred embodiment 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.
As regards the determination of the sequence identity, the same applies as has been set forth above.As mentioned above in (b), 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.
In another embodiment 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. Preferably, 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:2 and the enzyme has the enzymatic activity of converting propionic acid into ethylene. As regards the determination of the sequence identity, the same applies as has been set forth above.
In another embodiment, 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). The inventors surprisingly found that the cytochrome P450 fatty acid decarboxylase from Staphylococcus aureus strain C0673 can accept propionic acid as a substrate and convert it into ethylene.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:15. 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: 15. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
In a preferred embodiment 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.
In one preferred embodiment the reaction uses NADPH as a reducing agent. In such an embodiment it is preferred that 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). When used in connection with a cytochrome P450 enzyme or fused to a cytochrome P450 enzyme the resultant catalytic activity of the fusion CYP450 enzyme can be driven by NADPH.
In a further preferred embodiment 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).
In another preferred embodiment the reaction employs ferredoxin/ferredoxin reductase as redox partner.
In another possibility, 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. Sci., published ahead of print on December 8, 2014; doi:10.1073/pnas.1419701112) which is found in several Pseudomonas species. The x-ray structure of these non-heme iron oxygenases show a typical non-heme iron oxygenase active site having a coordination triade of His/His/Glu. These non-heme iron oxygenases have been shown to catalyze the following reaction:
Figure imgf000080_0001
Laurie acid (C12) 1 -undecene (C11 )
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.
Accordingly, in a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing any of these amino acid sequences of SEQ ID NOs:29 to 31. It is also possible to use 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. Preferably, 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. As regards the determination of the 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.
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. As mentioned above, the biosynthesis of propionic acid in microorganisms has already been described and bacteria such as Propionibacterium acidipropionici or Propionibacterium freudenreichii ssp. shermanii are well known to naturally produce propionic acid from glucose or glycerol. Moreover, engineered metabolic pathways producing propionic acid have already been established in genetically modified microorganisms. For example, the heterologous pathway to convert D-lactic acid to propionic acid from Clostridium propionicum was established in Escherichia coli (Appl. Microbiol. Biotechnol. 97 (2013), 1191-2000). Accordingly, 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.
In addition, the literature describes mainly three pathways for the biosynthesis of propionic acid (or propionyl-coenzyme A) from a carbon source such as glucose or glycerol:
1. The Propionobacterium pathway involving the oxaloacetate/ succinate pathway (Curr. Microbiol. 62 (201 1 ), 152-158).
2. The threonine pathway (Proc. Natl. Acad. Sci. 109 (2012), 17925-17930).
3. The 3-hydroxypropionate bicycle pathway (Appl. Environ. Microbiol. 78 (2012), 8564-8570).
Accordingly, as described in more detail further below, in preferred embodiments, 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. Such 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. These 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.
The enzymatic conversion of acrylyl-CoA into propionic acid (steps IVa and Va or, alternatively, steps IVb and Vb as shown in Figure 1)
In one preferred embodiment, 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.
According to the present invention, 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.
These reactions are schematically shown in Figure 1.
Thus, 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.
Moreover, 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 enzymatic conversion of acrylyl-CoA into propionic acid via propionyl-CoA (steps IVa and Va as shown in Figure 1)
Thus, in one preferred embodiment, 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. Thus, 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:
(a) enzymatically converting acrylyl-CoA into propionyl-CoA (step IVa as shown in Figure 1 ); and
(b) further enzymatically converting the thus produced propionyl-CoA into propionic acid (step Va as shown in Figure 1 ).
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. In one embodiment, the enzyme is an enzyme which is classified as EC 1.3.1.- and which uses NADH or NADPH as a co-factor. In a particularly preferred embodiment, 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. Thus, in one particularly preferred embodiment, the enzyme is an enzyme which uses NADPH as a co-factor. In a preferred embodiment the enzyme is selected from the group consisting of:
- acyl-CoA dehydrogenase (NADP+) (EC 1.3.1.8);
- enoyl-[acyl-carrier-protein] reductase (NADPH, Si-specific) (EC 1.3.1.10);
- cis-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.37);
- trans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38);
- enoyl-[acyl-carrier-protein] reductase (NADPH, Re-specific) (EC 1.3.1.39); and
- crotonyl-CoA reductase (EC 1.3.1.86).
Thus, in one preferred embodiment 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:
Acyl-CoA + NADP+ „ » 2,3-dehydroacyl-CoA + NADPH + H+
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.
In a further preferred embodiment 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.
In a further preferred embodiment 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.
In a further preferred embodiment 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). Trans-2-enoyl-CoA reductases (NADPH) are enzymes which catalyze the following reaction:
Acyl-CoA + NADP+ «. »" trans-2,3-dehydroacyl-CoA + NADPH + H+
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.
In a further preferred embodiment 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.
In a further preferred embodiment 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.
In a further preferred embodiment, 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+ „ »» 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.
In another particularly preferred embodiment the enzyme is an enzyme which uses NADH as a co-factor. The conversion using such an enzyme is schematically shown in Figure 4. In a preferred embodiment the enzyme is selected from the group consisting of:
- enoyl-[acyl-carrier-protein] reductase (NADH) (EC 1.3.1.9); and
- trans-2-enoyl-CoA reductase (NAD+) (EC 1.3.1.44).
Thus, in one preferred embodiment 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). Enoyl-[acyl-carrier-protein] reductases (NADH) are 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.
In a further preferred embodiment 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). Trans-2-enoyl-CoA reductases (NAD+) are enzymes which catalyze the following reaction:
Acyl-CoA + NAD+ « » trans-2,3-dehydroacyl-CoA + NADH + H+
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. In a further preferred embodiment, 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). 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.
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:26. 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: 26. Preferably, 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. As regards the determination of the 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. Thus, in one embodiment, the enzymatic conversion of propionyl-CoA into propionic acid according to step (b) can be achieved by a two-step conversion via propionyl phosphate. Accordingly, in one embodiment, the enzymatic conversion of propionyl- CoA into propionic acid (step Va as shown in Figure 1 ) according to step (b) 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.
The corresponding reaction is schematically shown in Figure 5.
The 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 + H3PO4 .^ ~*~ butyryl phosphate + CoA
It has been described by Wiesenborn et al. (Appl. Environ. Microbiol. 55 (1989), 317- 322) and by Ward et al. (J. Bacteriol. 181 (1999), 5433-5442) that 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.
The enzyme has been described to occur in a number of organism, in particular in bacteria and in protozoae. In one embodiment the enzyme is from the protozoae Dasytricha ruminantium. In a preferred embodiment 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. Most preferably, 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)).
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:20. 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: 20. Preferably, 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. As regards the determination of the 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 + H3PO4 ^-»· acetyl phosphate + CoA
It has been described by Veit et al. (J. Biotechnol.140 (2009), 75-83) that phosphate acetyltransferase can also use as a substrate butyryl-CoA or propionyl-CoA.
The accession numbers for this enzyme family in InterPro database are IPR012147 and IPR002505, "http://www.ebi.ac.uk/interpro/entry/IPR002505"
(http://www.ebi.ac.uk/interpro/entry/IPR012147
http://www.ebi.ac.uk/interpro/entrv/IPR002505)
See also http://pfam.sanger.ac.uk/family/PF01515
The enzyme has been described in a variety of organisms, in particular bacteria and fungi. Thus, in one preferred embodiment 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, Bradyrhizobium japonicum, Lactobacillus fermentum, Lactobacillus sanfranciscensis, Moorella thermoacetica, Rhodopseudomonas palustris, Sinorhizobium meliloti, Streptococcus pyogenes, Thermotoga maritima or Bacillus subtilis. In another preferred embodiment the enzyme is an enzyme from a fungus, preferably from the genus Saccharomyces, more preferably of the species Saccharomyces cerevisiae.
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:21. 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: 20. Preferably, 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. As regards the determination of the 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. Thus, 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.-). In a preferred embodiment, 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).
Butyrate kinases (EC 2.7.2.7) naturally catalyze the following reaction Butyrate + ATP butyryl phosphate + ADP
It has been described, e.g. by Hartmanis (J. Biol. Chem. 262 (1987), 617-621 ) that 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. In one preferred embodiment the enzyme is from a bacterium, preferably from a bacterium of the genus Clostridium, Butyrivibrio, Thermotoga or Enterococcus. Preferred is Clostridium. More preferably 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. Preferred is 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 carboxylic acid + ATP acyl phosphate + ADP
wherein "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).
This enzyme has been described to occur in a number of bacteria. Thus, in one preferred embodiment 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
Propanoate + propanoyl phosphate + ADP
Acetate + ATP
Figure imgf000094_0001
tyl phosphate + ADP
This enzyme has been described to occur in a number of bacteria, in particular Enterobacteriacea. Thus, in one preferred embodiment 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.
In a preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:27. 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: 27. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
In another preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:28. 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: 28. Preferably, 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. As regards the determination of 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 ^*" acetyl phosphate + ADP
This enzyme has been described to occur in a number of organisms, in particular bacteria and eukaryotes. In one preferred embodiment 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, Thermotoga or Veillonella, more preferably from a bacterium of the species Methanosarcina thermophila, Cryptococcus neoformans, Ethanoligenens harbinense, Propionibacterium acidipropionici, Streptococcus pneumoniae, Streptococcus enterica, Streptococcus pyogenes, Acholeplasma laidlawii, Acinetobacter calcoaceticus, Ajellomyces capsulatus, Bacillus subtilis, Borrelia burgdorferi, Chaetomium globosum, Clostridium acetobutylicum, Clostridium thermocellum, Coccidioides immitis, Coprinopsis cinerea, Cryptococcus neoformans, Cupriavidus necator, Desulfovibrio vulgaris, Enterococcus faecalis, Escherichia coli, Ethanoligenes harbinense, Geobacillus stearothermophilus, Helicobacter pylori, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus sanfranciscensis, Lactococcus lactis, Listeria monocytogenes, Mesoplasma florum, Methanosarcina acetivorans, Methanosarcina mazei, Moorella thermoacetica, Mycoplasma pneumoniae, Oceanobacillus iheyensis, Propionibacterium freudenreichii, Propionibacterium acidipropionici, Rhodospeudomonas palustris, Salmonella enteric, Staphylococcus aureus, Thermotoga maritime or Veillonella parvula. In another preferred embodiment 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.
In a further preferred embodiment the enzyme is an enzyme from a plant or an algae, preferably from the genus Chlamydomonas, even more preferably from the species Chlamydomonas reinhardtii.
In another embodiment the enzyme is from an organism of the genus Entamoeba, more preferably of the species Entamoeba histolytica.
The above mentioned enzyme families suitable for the conversion of propionyl-CoA into propionyl phosphate have been shown to be evolutionary related and contain common sequence signatures. Theses signatures are referenced and described in Prosite database:
http://prosite.expasy.org/cgi-bin/prosite/nicedoc.pl7PS01075
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. These E. coli cells have been shown to be able to produce D-(-)-3-hydroxybutyric acid (3HB).
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.
Preferably, in one embodiment, 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.-)). This reaction is schematically shown in Figure 6.
Thus, in one alternative, 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).
In the alternative embodiment, 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).
Thus, in another alternative, 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).
Thus, in one embodiment, the enzymatic conversion of propionyl-CoA into propionic acid (step Va as shown in Figure 1 ) according to step (b) 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: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 while 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.
In a preferred embodiment, 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 3.1.2.19); and acyl-CoA hydrolase (EC 3. .2.20).
Thus, in one preferred embodiment 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:
Acetyl-CoA + H2O ► acetate + 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 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 dionisii, Trypanosoma vespertilionis, Crithidia fasciculate, Clostridium aminovalericum, Acidaminococcus fermaentans, Bradyrhizobium japonicum and Methanosarcina barkeri. In another preferred embodiment 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:
Palmitoyl-CoA + H20 ► palmitate + 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 (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 auratus, Drosophila melanogaster, Rattus norvegicus, Oryctolagus cuniculus, Gallus gallus, Anas platyrhynchos, Mycobacterium tuberculosis, Mycobacterium phlei, Mycobacterium smegmatis, Acinetobacter colcaceticus, Haemophilus influenza, Helicobacter pylori, Bacillus subtilis, Pseudomonas aeruginosa, Rhodobacter shpaeroides, Streptomyces coelicolor, Streptomyces venezuelae and E. coli.
In a further preferred embodiment 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:
3-hydroxyisobutyryl-CoA + H2O ► 3-hydroxyisobutyrate + 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, Homo sapiens, Canus lupus familiaris, Rattus norvegicus, Bacillus cereus, Pseudomonas fluorescens and Pseudomonas aeruginosa. In yet another preferred embodiment 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] + H20 ► 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.
In yet another preferred embodiment 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 + H2O ► 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.
In yet another preferred embodiment 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 + H2O *► a carboxylate + CoA This enzyme occurs in a variety of animals and has, e.g., been described in Rattus norvegicus and Mesocricetus auratus.
In a further preferred embodiment 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 + H20 ► 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, Pseudomonas putida, Amycolatopsis mediterranei, Acinetobacter calcoaceticus, Helicobacter pylori, Rhodobacter spaeroides and Mycobacterium phlei. In a preferred embodiment 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). In another preferred embodiment 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) and acyl-CoA thioesterase II from Pseudomonas putida (Uniprot Q88DR1 ; 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).
In a particularly preferred embodiment, 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.
Preferably, the degree of identity is determined by comparing the respective sequence with the amino acid sequence of any one of the above-mentioned SEQ ID NOs. When the sequences which are compared do not have the same length, the degree of identity preferably either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. The degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.
When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0. Preferably, the degree of identity is calculated over the complete length of the sequence.
As described 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.
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).
Family I contains, e.g., the following CoA-transferases:
For 3-oxo acids: enzymes classified in EC 2.8.3.5 or EC 2.8.3.6;
For short chain fatty acids: enzymes classified in EC 2.8.3.8 or EC 2.8.3.9;
For succinate: succinyl-CoA:acetate CoA-transferases, i.e. enzymes classified in EC
2.8.3.18 (see also Mullins et al., Biochemistry 51(2012), 8422-34; Mullins et al., J.
Bacteriol. 190 (2006), 4933-4940).
Most enzymes of family I naturally use succinyl-CoA or acetyl-CoA as CoA donors.
These enzymes contain two dissimilar subunits in different aggregation states. Two conserved amino acid sequence motives have been identified:
Prosites entry PS01273 (http://prosite.expasy.org/cgi-bin/prosite/prosite-search- ac?PDOC00980)
COA_TRANSF_1 , PS01273; Coenzyme A transferases signature 1 (PATTERN) Consensus pattern:
[DN]-[GN]-x(2)-[LIVMFA](3)-G-G-F-x(3)-G-x-P
and
Prosites entries PS01273 (http://prosite.expasy.org/cgi-bin/prosite/prosite-search- ac?PDOC00980)
COA_TRANSF_2, PS01274; Coenzyme A transferases signature 2 (PATTERN)
Consensus pattern:
[LF]-[HQ]-S-E-N-G-[LIVF](2)-[GA]
E (glutamic 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. 80 (1977), 479-488) and acetyl-CoA and succinyl-CoA in the case of citramalate lyase (Dimroth et al., Eur. J. Biochem. 80 (1977), 469-477).
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.
In a preferred embodiment 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.
Preferably, 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:
propionate:acetate-CoA transferase (EC 2.8.3.1 );
acetate CoA-transferase (EC 2.8.3.8); and
butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).
Thus, in one preferred embodiment 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:
Acyl-CoA + acetate j^"*" a fatty acid anion + acetyl-CoA 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.
In another preferred embodiment 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:
Butanoyl-CoA + acetoacetate butanoate + acetoacetyl-CoA
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.
In another preferred embodiment 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:
Acetyl-CoA + propanoate acetate + propanoyl-CoA
This enzyme catalyzes the reversible transfer of CoA group from propionyl-CoA and acetate. The reaction is also schematically shown in Figure 7 for R= H.
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.
In another preferred embodiment 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. The reaction is also schematically shown in Figure 7 for R = CH2-C02H.
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 enzymatic conversion of acrylyl-CoA into propionic acid via acrylic acid (steps IVb and Vb as shown in Figure 1)
As mentioned above, according to the present invention, 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. In the following, 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.
Thus, in one preferred embodiment, 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 ).
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. Thus, in one embodiment, the enzymatic conversion of acrylyl-CoA into acrylic acid according to step (a) can be achieved by a two-step conversion via acrylyl phosphate. Accordingly, in one embodiment, the enzymatic conversion of acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1) according to step (a) 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 enzymes butyryltransferase (EC 2.3.1.19) and phosphate acetyltransferase (EC 2.3.1.8) for the conversion of acrylyl-CoA into acrylyl phosphate have already been described above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionyl phosphate. As regards these enzymes, the same applies for the conversion of acrylyl-CoA into acrylyl phosphate as has been set forth above in the context of the conversion of the enzymatic conversion of propionyl-CoA into propionyl phosphate.
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. Such enzymes use a carboxy group as acceptor. Thus, 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.-). In a preferred embodiment, 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).
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. As regards these enzymes, the same applies for the conversion of acrylyl phosphate into acrylic acid as has been set forth above in the context of the enzymatic conversion of propionyl phosphate into propionic acid.
As mentioned above, 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.
Preferably, in one embodiment, 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.-)). This reaction is schematically shown in Figure 8. Thus, in one alternative, 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).
In the alternative embodiment, 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.-).
This reaction is schematically shown in Figure 8 for preferred enzymes, i.e., 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. 8).
Thus, in another alternative, 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).
Thus, in one embodiment, 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
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.
In a preferred embodiment, 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);
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).
The enzymes 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. 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. As described above, 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 (EC 2.8.3.-), 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.
As mentioned above, 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 ).
The enzymatic conversion of acrylyl-CoA into acrylic acid has been described above while the enzymatic conversion of the thus produced acrylic acid into propionic acid according to step (b) (step IVb as shown in Figure 1 ) is schematically illustrated in Figure 9 and is described in the following.
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 ).
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., Burkholderia sp., Gluconobacter oxydans, Lactobacillus casei, Pseudomonas putida, Shewanella sp., Yersinia bercovieri, Bacillus subtilis, Moorella thermoacetica and Peptostreptococcus anaerobius. The enoate reductase of Clostridiae has been described, e.g., in Tischler et al. (Eur. J. Bioche. 97 (1979), 103-112).
The enzymatic conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA (step III as shown in Figure 1)
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 C02 fixation in various thermoacidophilic archaea (J. Bacteriol. 191 (2009), 4572-4581 ).
Thus, 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.
Moreover, 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.
According to the present invention, the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA preferably makes use of an enzyme catalyzing 3-hydroxypropionyl- CoA dehydration. The term "dehydration" is generally referred to a reaction involving the removal of H20. Enzymes catalyzing 3-hydroxypropionyl-CoA dehydration are enzymes which catalyze the reaction as shown in Figure 10. Preferably, such an enzyme belongs to the family of 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratases (EC 4.2.1.-).
Thus, 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.-). 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):
(a) a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116),
(b) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55),
(c) an enoyl-CoA hydratase (EC 4.2.1.17),
(d) a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59),
(e) a crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58),
(f) a 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60),
(g) a 3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61 ),
(h) a long-chain-enoyl-CoA hydratase (EC 4.2.1.74), and
(i) a 3-methylglutaconyl-CoA hydratase (EC 4.2. .18).
All these enzymes which are capable of catalyzing 3-hydroxypropionyl-CoA dehydration have in common that they use a natural substrate having the following minimal structural motif:
Figure imgf000113_0001
wherein
R1 is a hydrogen atom or an alkyl group or CH2COO";
R2 is a hydrogen atom or a methyl group; and
R3 is coenzyme A or acyl-carrier protein.
Thus, the above mentioned enzymes which can catalyze the dehydration of 3- hydroxypropionyl-CoA can be divided into two groups as follows:
I. 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:
3-hydroxyacyl-[acyl-carrier protein] 2-enoyl-[acyl-carrier protein] + H20
The enzymes of this group share a common structural motif which is referenced in the InterPro as InterPro IPR013114
(http://www.ebi.ac.uk/interpro/entry/IPR013114). The accession number for these enzymes in the Pfam database is PF 07977 (http://pfam.sanger.ac.uk/family/PF07977).
II. 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 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 IPR0018376
(http://www.ebi.ac.uk/interpro/entry/IPR018376). The accession number for these enzymes in the Pfam database is PF00378 (http://pfam.sanger.ac.uk/family/PF00378). In one embodiment of the method according to the invention 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:
3-hydroxypropionyl-CoA
Figure imgf000114_0001
acryloyl-CoA + H20
Figure imgf000114_0002
The enzyme is known from various bacteria and archae. Thus, in a preferred embodiment of the invention 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. 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.
In a preferred embodiment, 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.
In principle any 3-hydroxypropionyl-CoA dehydratase can be employed in the method according to the invention. However, it is not only possible to employ in the method of 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.
Thus, in another embodiment of the method according to the invention 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 crotonyl-CoA + H2O
Figure imgf000115_0001
This reaction corresponds to a Michael elimination. 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. In principle, 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. In a preferred embodiment of the 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.
In a preferred embodiment, 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.
In another embodiment of the method according to the invention 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:
(3S)-3-hydroxyacyl-CoA →- trans-2(or 3)-enoyl-CoA + H20
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. In principle, 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.
In another embodiment of the method according to the 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:
(3R)-3-hydroxyoctanoyl-[acyl-carrier protein] oct-2-enoyl-[acyl-carrier protein] + H2O
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). Other names in common use include D-3-hydroxyoctanoyl-[acyl carrier protein] dehydratase, D-3-hydroxyoctanoyl-acyl carrier protein dehydratase, beta- hydroxyoctanoyl-acyl carrier protein dehydrase, beta-hydroxyoctanoyl thioester dehydratase, beta-hydroxyoctanoyl-ACP-dehydrase, and (3R)-3-hydroxyoctanoyl- [acyl-carrier-protein] hydro-lyase. 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratases has been described to exist, e.g., in E. coli (Mizugaki et al., Biochem. Biophys. Res. Commun. 33 (1968), 520-527). In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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] + H20
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
Other names in common use include (3R)-3-hydroxybutanoyl-[acyl-carrier-protein] hydro-lyase, beta-hydroxybutyryl acyl carrier protein dehydratase, beta- hydroxybutyryl acyl carrier protein (ACP) dehydratase, beta-hydroxybutyryl acyl carrier protein dehydratase, enoyl acyl carrier protein hydratase, crotonyl acyl carrier protein hydratase, 3-hydroxybutyryl acyl carrier protein dehydratase, beta- hydroxybutyryl acyl carrier, and protein dehydratase. This enzyme participates in fatty acid biosynthesis. Crotonoyl-[acyl-carrier-protein] hydratase has been described to exist, e.g., in E. coli and Arabidopsis thaliana. In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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 (EC 4.2.1.60) catalyze the following reactions:
(1 ) (3R)-3-hydroxydecanoyl-[acyl-carrier protein] a trans-dec-2-enoyl-[acyl- carrier protein] + H2O
(2) (3R)-3-hydroxydecanoyl-[acyl-carrier protein] a cis-dec-3-enoyl-[acyl- carrier protein] + H2O
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. In principle, 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. In a preferred embodiment the enzyme from E. coli is used in a method according to the present invention.
In another embodiment of the method according to the 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 (EC 4.2.1.61 ) catalyze the following reaction:
(3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] ^"^" hexadec-2-enoyl-[acyl-carrier- protein] + H2O
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
Other names in common use include D-3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase, beta-hydroxypalmitoyl-acyl carrier protein dehydratase, beta- hydroxypalmitoyl thioester dehydratase, beta-hydroxypalmityl-ACP dehydratase, and (3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] hydro-lyase. 3-hydroxypalmitoyl-[acyl- carrier-protein] dehydratase has been described to exist, e.g., in Candida albicans, Yarrowia lipolytica, S. cerevisiae, S. pombe, Cochliobolus carbonum, Mus musculus, Rattus norvegicus, Bos taurus, Gallus gallus and Homo sapiens. In principle, 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.
In another embodiment of the method according to the 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
Figure imgf000120_0001
trans-2-enoyl-CoA + H2O
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. 252 (1977), 542- 547; Schulz, Biol. Chem. 249 (1974), 2704-2709) and in principle any long-chain- enoyl-CoA hydratase which can catalyze the conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA can be employed in the method of the invention.
In another embodiment of the method according to the invention 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 trans-3-methylglutaconyl-CoA + H20
Figure imgf000120_0002
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. The term "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. Yet, in a more preferred embodiment, 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 enzymatic conversion of 3-hydroxypropionaldehvde into 3- hydroxypropionyl-CoA (step II as shown in Figure 1)
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.
Thus, 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.
Moreover, 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 .-.
In a preferred embodiment, 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+
These enzymes naturally catalyze the conversion of propionaldehyde (or propanal) to propanoyl-coenzyme A by using a reducing cofactor NAD or NADP and coenzyme A. This reaction is reversible.
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.
In a preferred embodiment, 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). The conversion of 3- hydroxypropionaldehyde to 3-hydroxypropionyl-CoA catalyzed by this enzyme in E. coli has already been described (Enz. Microbiol. Tech. 53 (2013), 235-242).
In a preferred embodiment, 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. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
Acetaldehyde dehydrogenases (acetylating) (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 (acetylating) enzymes 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.
As mentioned, the enzyme classified as Coenzyme-A-acylating aldehyde dehydrogenases (EC 1.2.1.-) use NADH or NADPH as a co-factor. In a particularly preferred embodiment, 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 enzymatic conversion of glycerol into 3-hydroxypropionaldehvde (step I as shown in Figure 1)
The 3-hydroxypropionaldehyde which is converted into 3-hydroxypropionyl-CoA according to the method of the present (and further converted to ethylene via acrylyl- CoA and propionic acid according to any of the above described methods) 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.
Thus, 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.
Moreover, 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.-.
In a preferred embodiment, 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 + H2O
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.
In a preferred embodiment, 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).
In a preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
It is, of course, not only possible to use an enzyme exactly showing any one of the amino acid sequences of SEQ ID NOs:22 to 24. It is also possible to use 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. Preferably, 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. As regards the determination of the sequence identity, the same applies as has been set forth above.
In another embodiment, 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. Such 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 enzymatic conversion of glycerol into propionyl-CoA via 3- hvdroxypropionaldehvde. 3-hydroxypropionyl-CoA and acrylyl-CoA (steps I, II,
III, and IVa as shown in Figure 1)
The present invention also relates to a method for the production of propionyl-CoA from glycerol. In the 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. Further, 3-hydroxypropionyl-CoA is enzymatically converted to acrylyl-CoA which is further enzymatically converted to propionyl-CoA as described herein above.
In a preferred embodiment, the method for the production of propionyl-CoA from glycerol comprises the following steps:
(a) 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 );
(b) 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 );
(c) 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
(d) 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 ).
As regards the afore-mentioned embodiment, for the (cobalamine (B12 vitamin)- dependent or B12-indepentent/radical-S-adenosyl methionine-dependent) glycerol dehydratase (EC 4.2.1.30), the CoA-acylating aldehyde dehydrogenase, the CoA- acylating propionaldehyde dehydrogenase (EC 1.2.1.87), the acetaldehyde dehydrogenase (EC 1.2.1.10), the 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), the 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), the 3- hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) or a enoyl-CoA hydratase (EC 4.2.1.17), the enoyl-CoA reductase (EC 1.3.1.-), and the acrylyl-CoA reductase (EC 1.3.1.95), the same applies as has been set forth above in connection with the other methods of the present invention.
In a preferred embodiment, in the above method for the production of propionyl-CoA from glycerol the thus produced propionyl-CoA can enzymatically be converted into propionic acid by any of the enzymatic conversions of propionyl-CoA to propionic acid as described above. Moreover, in another preferred embodiment, in the above method for the production of propionyl-CoA from glycerol 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 enzymatic conversion of glycerol into propionic acid via 3- hvdroxypropionaldehyde, 3-hvdroxypropionyl-CoA, acrylyl-CoA and acrylic acid (steps I, II, III, IVb and Vb as shown in Figure 1)
The present invention also relates to a method for the production of propionic acid from glycerol. In the 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. Further, 3- hydroxypropionyl-CoA is enzymatically converted to acrylyl-CoA which is further converted to acrylic acid as described above. Further, the acrylic acid is enzymatically converted to propionic acid as described above.
In a preferred embodiment, the method for the production of propionic acid from glycerol comprises the following steps:
(a) 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 );
(b) 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 );
(c) 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
(d) the enzymatic conversion of said acrylyl-CoA into propionic acid by two enzymatic steps comprising
(i) first enzymatically converting acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ); and
(ii) 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 ).
In a preferred embodiment, 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
(a') 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; or
(b') 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
(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).
In a preferred embodiment, in the method for the production of propionic acid from glycerol, 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).
As regards the afore-mentioned embodiments of the method for the production of propionic acid from glycerol, for the (cobalamine (B12 vitamin)-dependent or B12- indepentent/radical-S-adenosyl methionine-dependent) glycerol dehydratase (EC 4.2.1.30), the CoA-acylating aldehyde dehydrogenase, the CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87), the acetaldehyde dehydrogenase (EC 1.2.1.10), the 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), the 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), the 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), the enoyl-CoA hydratase (EC 4.2.1.17), the (NADH) 2- enoate reductase (EC 1.3.1.31 ) the thioester hydrolase (EC 3.1.2.-), the ADP- dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18), the acetyl-CoA hydrolase (EC 3.1.2.1 ), the acyl-CoA hydrolase (EC 3.1.2.20), the CoA-transferase (EC 2.8.3.-), the propionate:acetate-CoA transferase (EC 2.8.3.1 ), the acetate CoA-transferase (EC 2.8.3.8), the succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18), the phosphate butyryltransferase (EC 2.3.1.19), the phosphate acetyltransferase (EC 2.3.1.8), the phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-), the propionate kinase (EC 2.7.2.15), the acetate kinase (EC 2.7.2.1 ) and the butyrate kinase (EC 2.7.2.7), the same applies as has been set forth above in connection with the other methods of the present invention.
In a preferred embodiment, in the above method for the production of propionic acid from glycerol 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. W
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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. Thus, 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). In one embodiment, the enzymes employed in the method are used in purified form.
For carrying out the method in vitro 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.
In another embodiment 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. Thus, 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. In another embodiment 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.
However, the invention preferably excludes naturally occurring microorganisms as found in nature expressing an enzyme as described above at levels as they exist in nature. Instead, 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. Thus, 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. As regards the enzymes, they 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. As regards 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.
By overexpressing an exogenous or endogenous enzyme as described herein above, 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. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.
A "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"). Thus, the present invention contemplates utilizing a non- natural substrate with the enzymes described above in an environment not found in nature.
Thus, it is also possible in the context of the present invention that 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. Similarly, 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.
If a microorganism is used which naturally expresses a corresponding enzyme, it is possible to 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. Alternatively, it is also possible to mutate the gene as such so as to lead to an enzyme showing a higher activity. By using microorganisms which express enzymes described above for the conversions of the methods according to the present invention, it is possible to carry out the methods according to the invention directly in the culture medium, without the need to separate or purify the enzymes.
In one embodiment 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. In one preferred embodiment, 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. Preferably, 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.
In a further embodiment 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. In other words, 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. Thus, the genetic modification can consist, e.g. 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.
The term "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. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas or Escherichia. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli. In another preferred embodiment 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.
It is also possible to employ an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae.
In another preferred embodiment 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.
In another embodiment, 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. Preferably, the microorganism is a photosynthetic bacterium, or a microalgae. In a further embodiment 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.
In a preferred embodiment, 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.
As mentioned above, propionic acid is biosynthetically produced in microorganisms such as Propionibacterium acidipropionici, Propionibacterium freudenreichii ssp. shermanii or Clostridium propionicum. Moreover, engineered metabolic pathways producing propionic acid have already been established in modified microorganisms. For example, 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.
In addition, the literature describes mainly three pathways for the biosynthesis of propionic acid (or propionyl-coenzyme A) from a carbon source such as glucose or glycerol:
1. The Propionobacterium pathway involving the oxaloacetate/ succinate pathway (Curr. Microbiol. 62 (2011 ), 152-158).
2. The threonine pathway (Proc. Natl. Acad. Sci. 109 (2012), 17925-17930).
3. The 3-hydroxypropionate bicycle pathway (Appl. Environ. Microbiol. 78 (2012), 8564-8570).
Accordingly, 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.
Thus, in one preferred embodiment, 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.
In a preferred embodiment, the method of the present invention makes use of 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.
In a further preferred embodiment, 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. In a preferred embodiment, 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.
In another preferred embodiment, 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. In principle, 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).
In another embodiment, 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. Suitable 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. Thus, 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). In a bioreactor or a fermenter, this process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, and may range in size from litres to cubic metres, and are often made of stainless steel. In this respect, without being bound by theory, 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.
In a preferred embodiment the method according to the present invention also comprises the step of recovering the ethylene produced by the method. For example, if the method according to the present invention is carried out in vivo by fermenting a corresponding microorganism expressing the necessary enzymes, the ethylene can be recovered from the fermentation off-gas by methods known to the person skilled in the art. In a preferred embodiment, 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".
For example, for genetic modification in prokaryotic cells, 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. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods. The resulting 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.
As described above, 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. Thus, in a preferred embodiment, 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. A detailed description of genetic modification of microorganisms will be given further below. Preferably, 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.
In the context of the present invention, 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. In even more preferred embodiments the increase in expression and/or activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments 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. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30%, or 40% of the total host cell protein.
Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot. In the context of the present invention 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. The term "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.
In addition, it is possible to insert different mutations into the polynucleotides by methods usual in molecular biology (see for instance Sambrook and Russell (2001 ), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), leading to the synthesis of polypeptides possibly having modified biological properties. The introduction of point mutations is conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide.
Moreover, 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.
Furthermore, 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.
For genetically modifying bacteria or fungi, 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. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.
Thus, in accordance with the present invention 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). An overview of yeast expression systems is 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. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481 ; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21 -25), Ip1 , rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters. In order to obtain an optimum amount of polypeptide, 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. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl^-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.
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. Thus, the present invention relates to a recombinant organism or microorganism which expresses
(a) an enzyme catalyzing the enzymatic conversion of glycerol into 3- hydroxypropionaldehyde;
(b) an enzyme catalyzing the enzymatic conversion of said 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA; and
(c) an enzyme catalyzing the enzymatic conversion of said 3-hydroxypropionyl- CoA into acrylyl-CoA (steps I to III as shown in Figure 14).
In a preferred embodiment, 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).
In a further preferred embodiment, 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).
In a further preferred embodiment, 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-
CoA dehydratase (EC 4.2.1.116) or an enoyl-CoA hydratase (EC 4.2.1.17).
As regards the preferred embodiments of these enzymes, 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 use of a recombinant organism or microorganism as defined above for the production of acrylyl-CoA from glycerol.
Moreover, the present invention also relates to the use of one or more enzymes as defined above for the production of acrylyl-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 acrylyl-CoA from glycerol, the same applies as has been set forth above in connection with the methods according to the present invention.
In a further aspect, 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).
In a preferred embodiment, 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
(EC 1.3.1.-), preferably an acrylyl-CoA reductase (EC 1.3.1.95).
As regards the preferred embodiments of these enzymes, 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 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.
In a further aspect, 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).
In a preferred embodiment, 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. In a more preferred embodiment, 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).
In another preferred embodiment, 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).
In another preferred embodiment, 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).
As regards the preferred embodiments of these enzymes, 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 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.
As regards the preferred embodiments of these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of propionic 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 and which further expresses an enzyme catalyzing the enzymatic conversion of acrylyl-CoA into acrylic acid (step VI as shown in Figure 14).
In a preferred embodiment, 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. In a more preferred embodiment, 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). In another preferred embodiment, 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).
In another preferred embodiment, 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).
As regards the preferred embodiments of these enzymes, 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 use of a recombinant organism or microorganism as defined above for the production of acrylic acid from glycerol.
Moreover, the present invention also relates to the use of one or more enzymes as defined above for the production of acrylic acid from glycerol.
As regards the preferred embodiments of these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of acrylic 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 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).
In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism which expresses an acetyl-CoA C- acyltransferase (EC 2.3.1.16). As regards the preferred embodiments of these enzymes, 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 use of a recombinant organism or microorganism as defined above for the production of 3-oxo-4-pentenoyl-CoA from glycerol.
Moreover, 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.
As regards the preferred embodiments of these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of 3-oxo-4- pentenoyl-CoA from glycerol, the same applies as has been set forth above in connection with the methods according to the present invention.
In a further aspect, 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).
In a preferred embodiment, 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).
As regards the preferred embodiments of these enzymes, 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 use of a recombinant organism or microorganism as defined above for the production of 3-hydroxy-4-pentenoyl-CoA from glycerol.
Moreover, 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. As regards the preferred embodiments of 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.
In a further aspect, 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).
In a preferred embodiment, 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).
As regards the preferred embodiments of these enzymes, 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 use of a recombinant organism or microorganism as defined above for the production of 2,4-pentadienoyl-CoA from glycerol.
Moreover, 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.
As regards the preferred embodiments of these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of 2,4- pentadienoyl-CoA from glycerol, the same applies as has been set forth above in connection with the methods according to the present invention.
In a further aspect, 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). In a preferred embodiment, 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. In a more preferred embodiment, 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 kinase (EC 2.7.2.14).
In another preferred embodiment, 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).
In another preferred embodiment, 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).
As regards the preferred embodiments of these enzymes, 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 use of a recombinant organism or microorganism as defined above for the production of 2,4-pentadienoic acid from glycerol.
Moreover, 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. As regards the 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).
In a preferred embodiment, 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. In a more preferred embodiment, 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 2.7.2.14).
In another preferred embodiment, 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).
In another preferred embodiment, 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).
As regards the preferred embodiments of these enzymes, 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 use of a recombinant organism or microorganism as defined above for the production of 3-hydroxy-4-pentenoic acid from glycerol.
Moreover, 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.
As regards the preferred embodiments of these enzymes and the recombinant organisms or microorganisms applied in the uses for the production of 3-hydroxy-4- pentenoic 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 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. Thus, the present invention relates to a bacterium which expresses
(I) an enzyme catalyzing the enzymatic conversion of propionic acid into ethylene as defined above; and
(II) an enzyme or enzymes catalyzing the enzymatic conversion of acrylyl-CoA into said propionic acid as described above.
In a preferred embodiment, 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. In another preferred embodiment, such a bacterium is a bacterium which is capable of converting glycerol into propionic acid, e.g., by the pathways as described herein above.
In a further preferred embodiment, 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 ).
In a preferred embodiment, 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).
In a preferred embodiment, 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) (i) an enzyme catalyzing the conversion of propionyl-CoA into propionyl phosphate; and (ii) an enzyme further catalyzing the thus obtained propionyl phosphate into said propionic acid; or
(b) an enzyme catalyzing the conversion of propionyl-CoA 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
(c) 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).
In a preferred embodiment, the bacterium is a bacterium, 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 ) or a butyrate kinase (EC 2.7.2.7).
In a further preferred embodiment, the above bacterium is a bacterium wherein the enzymes in (II) (steps IVb and Vb as shown in Figure 1 ) is an enzyme (a) catalyzing the conversion of acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ); and (b) and and enzyme catalyzing the conversion of the thus produced acrylic acid into propionic acid (step Vb as shown in Figure ).
In a preferred embodiment, the bacterium is a bacterium, wherein the enzyme(s) catalyzing the conversion of acrylyl-CoA into acrylic acid is
(a') (i) an enzyme catalyzing the conversion of acrylyl-CoA into acrylyl phosphate; and (ii) an enzyme catalyzing the conversion of the thus obtained acrylyl phosphate into said acrylic acid; or
(b') 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
(c') 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).
In a preferred embodiment, the bacterium is a bacterium wherein in (a'), the enzyme catalyzing the conversion of acrylyl-CoA into acrylyl phosphate is achieved a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme catalyzing the conversion of acrylyl phosphate into 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 ) or a butyrate kinase (EC 2.7.2.7).
In a preferred embodiment, the bacterium is a bacterium wherein in (b), the enzyme catalyzing the conversion of acrylic acid into propionic acid is an (NADH) 2-enoate reductase (EC 1.3.1.31 ) (step Vb as shown in Figure 1 ).
In a further aspect, the above bacterium is a bacterium which further expresses an enzyme catalyzing the enzymatic conversion of 3-hydroxypropionyl-CoA into acrylyl- CoA (step III as shown in Figure 1 ). In a preferred embodiment, the bacterium is a bacterium, wherein the enzyme catalyzing the enzymatic conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA is 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 ).
In a further embodiment, the present invention relates to a recombinant organism or microorganism, 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 enzymatic conversion of acrylyl-CoA into said propionic acid and the enzymes required for the enzymatic conversion of 3- hydroxypropionyl-CoA into acrylyl-CoA (step IVb as shown in Figure 1 ). Thus, the present invention relates to a recombinant organism or microorganism which expresses
(I) an enzyme catalyzing the enzymatic conversion of propionic acid into ethylene as defined above; and
(II) an enzyme catalyzing the enzymatic conversion of acrylyl-CoA into said propionic acid; and
(III) an enzyme catalyzing the enzymatic conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA.
In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme in (I) is a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase.
In another preferred embodiment, such a recombinant organism or microorganism is a recombinant organism or microorganism which is capable of converting glycerol into propionic acid, e.g., by the pathways as described herein above.
In a further preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism 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 (step 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).
In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism, 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).
In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme(s) catalyzing the enzymatic conversion of propionyl-CoA into propionic acid (step Va as shown in Figure 1) is/are
(a) (i) an enzyme catalyzing the conversion of propionyl-CoA into propionyl phosphate; and (ii) an enzyme further catalyzing the thus obtained propionyl phosphate into said propionic acid; or
(b) an enzyme catalyzing the conversion of propionyl-CoA 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
(c) 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).
In a preferred embodiment, the 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) or a butyrate kinase (EC 2.7.2.7).
In a further preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzymes in (II) (steps IVb and Vb as shown in Figure 1 ) are an enzyme (a) catalyzing the conversion of acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ); and (b) an enzyme catalyzing the conversion of the thus produced acrylic acid into propionic acid (step Vb as shown in Figure 1 ).
In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme(s) catalyzing the conversion of acrylyl-CoA into acrylic acid is
(a') (i) an enzyme catalyzing the conversion of acrylyl-CoA into acrylyl phosphate; and (ii) an enzyme catalyzing the conversion of the thus obtained acrylyl phosphate into said acrylic acid; or
(b') 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
(c') 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).
In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism wherein in (a'), the enzyme catalyzing the conversion of acrylyl-CoA into acrylyl phosphate is achieved a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme catalyzing the conversion of acrylyl phosphate into 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 ) or a butyrate kinase (EC 2.7.2.7).
In a preferred embodiment, the recombinant organism or microorganism is a bacterium wherein in (b), the enzyme catalyzing the conversion of acrylic acid into propionic acid (step IVb as shown in Figure 1 ) is an (NADH) 2-enoate reductase (EC 1.3.1.31 ) (step Vb as shown in Figure 1 ).
In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (III) catalyzing the enzymatic conversion of 3-hydroxypropionyl-CoA into acrylyl-CoA is 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 ).
In another aspect, the bacterium, recombinant organism or microorganism is a bacterium, recombinant organism or microorganism which further expresses an enzyme catalyzing the conversion of 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA (step II as shown in Figure 1 ).
In a preferred embodiment, the above bacterium, recombinant organism or microorganism is a bacterium, recombinant organism or microorganism wherein the enzyme catalyzing the conversion of 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA is 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 ).
In another aspect, the bacterium, recombinant organism or microorganism is a bacterium, recombinant organism or microorganism which further expresses an enzyme catalyzing the conversion of glycerol into 3-hydroxypropionaldehyde (step I as shown in Figure 1 ).
In a preferred embodiment, the above bacterium, recombinant organism or microorganism is a bacterium, recombinant organism or microorganism wherein the enzyme catalyzing the conversion of glycerol into 3-hydroxypropionaldehyde is 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 ).
The microorganism is preferably a bacterium, a yeast or a fungus. In another preferred embodiment, the organism is a plant or a non-human animal. As regards other preferred embodiments of the bacterium, recombinant organism or microorganism, 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 use of a bacterium as defined above or an organism or microorganism as defined above for the production of ethylene. Moreover, the present invention also relates to the use of one or more enzymes as defined above for the production of ethylene.
As regards the preferred embodiments of the enzymes, bacteria, organisms or microorganisms applied in the uses for the production of ethylene the same applies as has been set forth above in connection with the method according to the present invention.
In a preferred embodiment, the present invention relates to the use of an organism or microorganism which expresses 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.
In another preferred embodiment, the present invention relates to the use of 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.
In another aspect, the present invention relates to a recombinant organism or microorganism which expresses the enzymes catalyzing the conversion of glycerol to propionyl-CoA. In a preferred embodiment, the recombinant organism or microorganism which expresses enzymes catalyzing the conversion of glycerol into propionyl-CoA expresses
(a) an enzyme catalyzing the conversion of glycerol into 3- hydroxypropionaldehyde, preferably 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 );
(b) an enzyme catalyzing the conversion of 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA, preferably 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 );
(c) an enzyme catalyzing the conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA, preferably 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
(d) an enzyme catalyzing the conversion of said acrylyl-CoA into said propionyl- CoA, preferably 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 ).
As regards the afore-mentioned embodiment of the recombinant organism or microorganism, for the (cobalamine (B12 vitamin)-dependent or B12- indepentent/radical-S-adenosyl methionine-dependent) glycerol dehydratase (EC 4.2.1.30), the CoA-acylating aldehyde dehydrogenase, the CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87), the acetaldehyde dehydrogenase (EC 1.2.1.10), the 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), the 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), the 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) or a enoyl-CoA hydratase (EC 4.2.1.17), the enoyl-CoA reductase (EC 1.3.1.-), and the acrylyl-CoA reductase (EC 1.3.1.95), the same applies as has been set forth above in connection with the other methods of the present invention.
In a preferred embodiment, the above recombinant organism or microorganism which expresses the enzymes catalyzing the conversion of glycerol to propionyl-CoA, optionally expresses an enzyme catalyzing the conversion of propionyl-CoA into propionic acid (step Va as shown in Figure 1 ) wherein the enzyme(s) catalyzing the enzymatic conversion of propionyl-CoA into propionic acid (step Va as shown in Figure 1 ) is
(a) (i) an enzyme catalyzing the conversion of propionyl-CoA into propionyl phosphate; and (ii) an enzyme further catalyzing the thus obtained propionyl phosphate into said propionic acid; or
(b) an enzyme catalyzing the conversion of propionyl-CoA 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
(c) 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). In a preferred embodiment, the 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 ) or a butyrate kinase (EC 2.7.2.7).
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 the enzymes, 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 method according to the present invention.
In another aspect, the present invention relates to a recombinant organism or microorganism which expresses the enzymes catalyzing the conversion of glycerol to propionic acid. In a preferred embodiment, the recombinant organism or microorganism which expresses enzymes catalyzing the conversion of catalyzing the conversion of glycerol to propionic acid expresses
(a) an enzyme catalyzing the conversion of glycerol into 3- hydroxypropionaldehyde, preferably a (cobalamine (B12 vitamin)-dependent or B 2-indepentent/radical-S-adenosyl methionine-dependent) glycerol dehydratase (EC 4.2.1.30) (step I as shown in Figure 1 );
(b) an enzyme catalyzing the conversion of 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA, preferably 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);
(c) an enzyme catalyzing the conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA, preferably 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
(d) an enzyme catalyzing the conversion of acrylyl-CoA into acrylic acid (step IVb as shown in Figure 1 ); and (b) and and enzyme catalyzing the conversion of the thus produced acrylic acid into propionic acid (step Vb as shown in Figure
1 )·
In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme(s) catalyzing the conversion of acrylyl-CoA into acrylic acid is
(a') (i) an enzyme catalyzing the conversion of acrylyl-CoA into acrylyl phosphate; and (ii) an enzyme catalyzing the conversion of the thus obtained acrylyl phosphate into said acrylic acid; or
(b') 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
(c') 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).
In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism wherein in (a'), the enzyme catalyzing the conversion of acrylyl-CoA into acrylyl phosphate is achieved a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzyme catalyzing the conversion of acrylyl phosphate into 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 ) or a butyrate kinase (EC 2.7.2.7).
In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism wherein in (b), the enzyme catalyzing the conversion of acrylic acid into propionic acid (step Vb as shown in Figure 1) is an (NADH) 2-enoate reductase (EC 1.3.1.31 ) (step Vb as shown in Figure 1 ). 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.
As regards the preferred embodiments of the enzymes, the recombinant organisms or microorganisms applied in the uses for the production of propionic acid from glycerol the same applies as has been set forth above in connection with the method according to the present invention.
In another aspect, the present invention also relates to a composition comprising glycerol and a bacterium, organism or microorganism as defined above. In another aspect, the present invention also relates to a composition comprising or glycerol and an enzyme as defined above.
Figure 1 : shows an artificial metabolic pathway for ethylene production from glycerol via propionic acid.
Figure 2: shows a GC chromatogram obtained for the enzyme-catalyzed oxidative decarboxylation of propionic acid with the cytochrome P450 olefin-forming fatty acid decarboxylase from Jeotgalicoccus sp (Uniprot Accession number: E9NSU2) as outlined in Example 2.
Figure 3: shows a GC chromatogram of ethylene produced by E.coli strain, engineered for cytochrome P450 fatty acid decarboxylase and ferredoxin reductase overexpression (Figure 3b). No ethylene was observed in the E.coli control strain (Figure 3a).
Figure 4: Schematic reaction of the enzymatic conversion of acrylyl-CoA into propionyl-CoA.
Figure 5: Schematic reaction of the enzymatic conversion of propionyl-CoA into propionic acid by a two-step conversion via propionyl phosphate. Figure 6: Schematic reaction for the enzymatic conversion of propionyl-CoA into propionic acid by hydrolyzing the thioester bond of propionyl-CoA to propionic acid.
Figure 7: Schematic reaction for the enzymatic conversion of propionyl-CoA into propionic acid by making use of an enzyme which belongs to the family of CoA-transferases; R = H for propionate:acetate-CoA transferase; R = CH2-CO2H for succinyl-CoA:acetate CoA-transferase.
Figure 8: Schematic reactions for the three alternative enzymatic conversions of acrylyl-CoA into acrylic acid.
Figure 9: Schematic reaction for the reduction of acrylic acid to propionic acid.
Figure 10: Schematic reaction of 3-hydroxypropionyl-CoA into acrylyl-CoA.
Figure 11: Schematic reaction of the conversion of 3-hydroxypropionaldehyde into
3-hydroxypropionyl-CoA using either NAD or NADP as a cofactor.
Figure 12: Schematic reaction of the conversion of glycerol
hydroxypropionaldehyde.
Figure 13: shows a GC chromatogram obtained for the enzyme-catalyzed oxidative decarboxylation of propionic acid with the cytochrome P450 from Staphylococcus aureus C0673 (Uniprot Accession number: A0A033V973) as outlined in Example 4.
Figure 14: shows an artificial metabolic pathway for the production of acrylyl-CoA and derivatives thereof (i.e., acrylic acid, propionyl-CoA, propionic acid, 3-oxo-4-pentenoyl-CoA, 3-hyd roxy-4-pentenoyl-CoA, 2,4-pentad ienoyl- CoA, 3-hyd roxy-4-pentenoic acid and 2,4-pentadienoic acid) from glycerol. Figure 15: Schematic reaction of the condensation of acetyl-CoA and acrylyl-CoA into 3-oxo-4-pentenoyl-CoA.
Figure 16: Schematic reaction of the conversion of 3-oxo-4-pentenoyl-CoA into 3- hydroxy-4-pentenoyl-CoA.
Figure 17: Schematic reaction of the conversion of 3-hydroxy-4-pentenoyl-CoA into
2,4-pentadienoyl-CoA.
Figure 18: Schematic reactions for the three alternative enzymatic conversions of
3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid.
Figure 19: Schematic reactions for the three alternative enzymatic conversions of
2,4-pentadienoyl-CoA into 2,4-pentadienoic acid.
In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLES
GENERAL METHODS AND MATERIALS
All reagents and materials used in the experiences were obtained from Sigma-Aldrich Company (St. Luis, MO) unless otherwise specified. Materials and methods suitable for growth of bacterial cultures, genes cloning and protein expression are well known in the art.
Vector pCAN contained gene encoding ferredoxin-NADP reductase (a/ a flavodoxin reductase) from Escherichia coli (Uniprot Accession number: P28861 ) was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection). Provided vector contained a stretch of 6 histidine codons after the methionine initiation codon. Ferredoxin reductase thus cloned was overexpressed in E.coli BL21(DE3) strain and purified on PROTINO-2000 Ni-TED column (Macherey- Nagel) allowing adsorption of 6-His tagged proteins. Fractions contained the enzyme of interest were pooled and concentrated on Amicon Ultra-4 10 kDa filter unit (Millipore). Enzyme was then resuspended in 100 mM phosphate buffer pH 7.0, containing 100 mM NaCI to be used in subsequent enzymatic assays. Protein concentration was determined by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific).
Example 1 : Cloning and overexpression of recombinant cytochrome P450 terminal olefin-forming fatty acid decarboxylases
The sequence of the terminal olefin-forming fatty acid decarboxylase Jeotgalicoccus sp. ATCC 8456 gene (Uniprot Accession number: E9NSU2) was generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt®). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The gene thus synthesized was cloned in a pET-25b(+) expression vector (vectors were constructed by GeneArt®).
BL21(DE3) competent cells (Novagen) were transformed with these vectors using the heat shock method and plated out onto LB agar plates supplemented with the appropriate antibiotic. Single transformants were used to inoculate 200 ml of ZYM- 5052 auto-induction medium (Studier FW, Prot. Exp. Pur. 41 , (2005), 207-234). The cultures were incubated for 6h at 30°C in a shaker incubator. 0.5 mM 5- aminolevulinic acid was then added in the medium and protein expression was continued at 18°C overnight (approximately 16 h).
The cells were collected by centrifugation at 4°C, 10,000 rpm for 20 min and the pellets were stored at -80°C.
Example 2: In vitro oxidative decarboxylation of propionic acid into ethylene catalyzed by cytochrome P450 fatty acid decarboxylase from Jeotgalicoccus sp.
The pellet from 200 ml of cultured cells was resuspended in 50 ml of lysis buffer (100 mM potassium phosphate pH 7, 100 mM NaCI) supplemented with 20 μΙ of lysonase (Merck-Novagen). Cell suspensions were then incubated for 10 minutes at room temperature followed by 20 minutes on ice. The amount of cytochrome P450 fatty acid decarboxylase in the total cell lysate was estimated on SDS-PAGE using gel densitometry.
0.5 M stock solution of propionic acid was prepared in water and adjusted to pH 7.0 with 10 M solution of NaOH.
Enzymatic assays were set up in 2 ml glass vials (Interchim) in the following conditions:
00 mM potassium phosphate buffer pH 7.0
100 mM NaCI
1 mM NADPH
50 mM propionic acid
0.2 mg/ml purified ferredoxin reductase from E.coli
Assays were started by adding 30 μΙ of cell lysate containing the recombinant P450 fatty acid decarboxylase from Jeotgalicoccus sp. (total volume 300 pi)
A series of control assays were performed in parallel (Table 1 ). The vials were sealed and incubated for 30 minutes at 30°C. The assays were stopped by incubating for 1 minute at 80°C and ethylene present in the reaction headspace was analysed by Gas Chromatography (GC) equipped with Flame Ionization Detector (FID).
For the GC headspace analysis, one ml of the headspace gas was separated in a Bruker GC-450 system equipped with a GS-alumina column (30 m x 0.53 mm) (Agilent) using isothermal mode at 130°C. Nitrogen was used as carrier gas with a flow rate of 6 ml/min.
The enzymatic reaction product was identified by comparison with an ethylene standard. Under these GC conditions, the retention time of ethylene was .28 min. A significant production of ethylene from propionic acid was observed in the assay, contained cytochrome P450 fatty acid decarboxylase and redox partners (Table 1, Figure 2).
Table 1.
Figure imgf000169_0001
Example 3: In vivo transformation of propionic acid into ethylene catalyzed by E.coli strain, containing a piasmid harbouring the genes of cytochrome P450 fatty acid decarboxylase and ferredoxin reductase
Vector construction for genes overexpression
Gene encoding the terminal olefin-forming fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456 was synthesized according to the procedure described in Example 1. Master vector pMK-RQ + OleT_Js was commercially provided by GeneArt (Life Technologies).
Vector pCAN contained gene encoding a ferredoxin-NADP reductase from E. coli was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection).
The construction of a plasmid carrying both cytochrome P450 fatty acid decarboxylase and ferredoxin reductase was conducted using a Gibson Assembly approach (Gibson D., Nature Methods, 6, (2009), 343-347).
The fragments, containing the genes of interest, were amplified by PCR from original vectors to add the sequences designed for the appropriate overlaps. Primer pairs oRH1/oRH2 and oRH3/oRH4 were used to amplify the sequence of ferredoxin reductase and the sequence of cytochrome P450 fatty acid decarboxylase, respectively (Table 2).
Table 2.
Primer Primer Sequence 5' to 3'
name direction oRH1 Forward TAATTTTGTTTAACTTTAAG AAGG AG ATATACATATGG CTG ATTG G G
TAACAGGC oRH2 Reverse CATCTCAGTACCTCCTCATTTTGTTGAATTCCTATTACCAGTAATGC
TCCGC oRH3 Forward ATAGGAATTCAACAAAATGAGGAGGTACTGAGATGGCAACCCTGAA
ACGTGAA oRH4 Reverse GGCTAGCCCGTTTGATCTCGAGTGCGGCCGCTTATTAGGTGCGAT
CCACAACTTC Gibson Assembly Master Mix (NEB #E261 ) procedure was then used to assemble and clone the amplified fragments into Ndel - Notl digested pET-25b(+) vector. The resulting plasmid pGB 3354 was verified by sequencing.
Culture medium and flask fermentation conditions
Competent BL21(DE3) E.coli cells were transformed with pGB 3354 plasmid by heat- shock procedure. An empty plasmid pET-25b(+) was transformed in parallel to create a strain used as a negative control in the assay.
The transformed cells were then plated on LB plates, supplied with ampicillin (100 pg/ml). Plates were incubated overnight at 30°C. Isolated colonies were used to inoculate LB medium, supplemented with ampicillin, followed by incubation at 30°C overnight. 1 ml of this overnight culture was used to inoculate 300 ml of ZYM-5052 auto-inducing media (Studier FW, loc. cit.). This culture was grown for 7 hours at 30°C and 160 rpm shaking. 0.5 aminolevulinic acid was then added in the medium followed by further incubation at 18°C overnight.
A volume of cultures corresponding to OD600 of 30 was removed and centrifuged. The pellet was resuspended in 30 ml of MS medium (Richaud C, Mengin-Leucreulx D., Pochet S., Johnson EJ., Cohen GN. and Marliere P, The Journal of Biological Chemistry, 268, (1993), 26827-26835) containing glucose (45 g/L), and MgS04 (1 mM) and supplemented with 50 mM propionic acid.
These cultures were then incubated in 160 ml bottles, sealed with a screw cap, at 30°C with shaking for 8 h.
After an incubation period, the ethylene produced in the headspace was analysed by Gas Chromatography (GC) equipped Flame Ionization Detector (FID). One ml of the headspace gas phase was separated and analysed according to the method described in Example 2, in which the temperature was decreased to 90°C. In these conditions, the retention time of ethylene was 1.46 min. The specific ethylene formation from propionic acid was observed only with E.coli strain containing a plasmid harboring the genes of interest (Figure 3). Example 4: Cloning and overexpression of recombinant cytochrome P450 from Staphylococcus aureus C0673
The sequence of the cytochrome P450 from Staphylococcus aureus C0673 (Uniprot Accession number: A0A033V973) was generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt®). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The gene thus synthesized was cloned in a pET-25b(+) expression vector (vectors were constructed by GeneArt®).
BL21(DE3) competent cells (Novagen) were transformed with these vectors according to standard heat shock procedure and plated out onto LB agar plates supplemented with the appropriate antibiotic. Single transformants were used to inoculate 200 ml of ZYM-5052 auto-induction medium (Studier FW, loc. cit.), supplemented with 0.5 mM aminolevulinic acid for cytochrome P450 expression. The cultures were incubated for 6h at 30°C in a shaker incubator and protein expression was continued at 18°C overnight (approximately 16 h).
The cells were collected by centrifugation at 4°C, 10,000 rpm for 20 min and the pellets were stored at -80°C.
Example 5: In vitro oxidative decarboxylation of propionic acid into ethylene catalyzed by cytochrome P450 from Staphylococcus aureus C0673.
The pellet from 200 ml of cultured cells was resuspended in 50 ml of lysis buffer (100 mM potassium phosphate pH 7, 100 mM NaCI) supplemented with 20 μΙ of lysonase (Merck-Novagen). Cell suspensions were then incubated for 10 minutes at room temperature followed by 20 minutes on ice. The amount of cytochrome P450 in the total cell lysate was estimated on SDS-PAGE using gel densitometry.
0.5 M stock solution of propionic acid was prepared in water and adjusted to pH 7.0 with 10 M solution of NaOH.
Enzymatic assays were set up in 2 ml glass vials (Interchim) in the following conditions:
100 mM potassium phosphate buffer pH 7.0
100 mM NaCI
1 mM NADPH
50 mM propionic acid 0.2 mg/ml purified ferredoxin reductase from E.coli
Assays were started by adding 30 μΙ of cell lysate containing the recombinant P450 from Staphylococcus aureus C0673. (total volume 300 μΙ)
A series of control assays were performed in parallel (Table 3).
The vials were sealed and incubated for 30 minutes at 30°C. The assays were stopped by incubating for 1 minute at 80°C and the ethylene present in the reaction headspace was analysed by Gas Chromatography (GC) according to the procedure described in Example 2.
The enzymatic reaction product was identified by comparison with an ethylene standard. Under these GC conditions, the retention time of ethylene was .28 min. A significant production of ethylene from propionic acid was observed in the assay, contained cytochrome P450 and redox partners (Table 3; Figure 13).
Table 3
Assay Ethylene peak area, arbitrary units
Enzymatic assay 44
Control assay without cytochrome P450 0
Control assay without flavodoxine reductase 1.2
Control assay without cytochrome P450 fatty acid 0 decarboxylase and without flavodoxine reductase
Control assay without NADPH 0

Claims

1. 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.
2. The method according to claim 1 , wherein the enzymatic conversion of glycerol into 3-hydroxypropionaldehyde is achieved by making use of a (cobalamine (B12 vitamin)-dependent or B12-indepentent/radical-S-adenosyl methionine-dependent) glycerol dehydratase (EC 4.2.1.30).
3. The method of claim 1 or 2, wherein the enzymatic conversion of said 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is achieved by making use of a CoA-acylating aldehyde dehydrogenase.
4. The method of claim 3, wherein said CoA-acylating aldehyde dehydrogenase is a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10).
5. The method of any one of claims 1 to 4, wherein the enzymatic conversion of said 3-hydroxypropionyl-CoA into acrylyl-CoA is achieved by making use of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-).
6. The method of claim 5, wherein said 3-hydroxyacyl-CoA dehydratase/enoyl- CoA hydratase (EC 4.2.1.-) is 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).
7. The method of any one of claims 1 to 6, further comprising the enzymatic conversion of the thus produced acrylyl-CoA into propionyl-CoA.
8. The method of claim 7, wherein the enzymatic conversion of said acrylyl-CoA into said propionyl-CoA is achieved by making use of an enoyl-CoA reductase (EC 1.3.1.-), preferably an acrylyl-CoA reductase (EC 1.3.1.95).
9. The method of claim 7 or 8, further comprising the enzymatic conversion of the thus produced propionyl-CoA into propionic acid.
10. The method of claim 9, wherein the enzymatic conversion of propionyl-CoA into propionic acid is achieved by
(a) 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; or
(b) 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
(c) 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:acetate CoA- transferase (EC 2.8.3.18).
11. The method of claim 10(a), wherein the enzymatic conversion of said propionyl-CoA into said propionyl 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 propionyl phosphate into said propionic 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 kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
12. The method of any one of claims 1 to 6, further comprising the enzymatic conversion of the thus produced acrylyl-CoA into acrylic acid.
13. The method of claim 12, wherein the enzymatic conversion of acrylyl-CoA into acrylic acid is achieved by
(a') 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; or
(b') 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
(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).
14. The method of claim 13(a'), wherein the enzymatic conversion of said acrylyl- CoA into said acrylyl phosphate is achieved by making use of 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 2.7.2.15), 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).
15. The method of any one of claims 1 to 6, further comprising the enzymatic condensation of the thus produced acrylyl-CoA with acetyl-CoA into 3-oxo-4- pentenoyl-CoA.
16. The method of claim 5, wherein the enzymatic condensation of acrylyl-CoA and acetyl-CoA into 3-oxo-4-pentenoyl-CoA is achieved by making use of an acetyl-CoA C-acyltransferase (EC 2.3.1.16).
17. The method of claim 15 or 16, further comprising the enzymatic reduction of the thus produced 3-oxo-4-pentenoyl-CoA into 3-hydroxy-4-pentenoyl-CoA.
18. The method of claim 17, wherein the enzymatic reduction of 3-oxo-4- pentenoyl-CoA into 3-hydroxy-4-pentenoyl-CoA is achieved by making use of 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).
19. The method of claim 17 or 18, further comprising the enzymatic dehydration of 3-hydroxy-4-pentenoyl-CoA into 2,4-pentadienoyl-CoA.
20. The method of claim 19, wherein the enzymatic dehydration of 3-hydroxy-4- pentenoyl-CoA into 2,4-pentadienoyl-CoA is achieved by making use of 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).
21. The method of claims 19 or 20, further comprising the enzymatic conversion of 2,4-pentadienoyl-CoA into 2,4-pentadienoic acid.
22. The method of claim 21 , wherein the enzymatic conversion of 2,4- pentadienoyl-CoA into 2,4-pentadienoic acid comprises:
(a) 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; or
(b) a single enzymatic reaction in which 2,4-pentadienoyl-CoA is directly converted into 2,4-pentadienoic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably an acyl-CoA hydrolase (EC 3.1.2.20); or
(c) a single enzymatic reaction in which 2,4-pentadienoyl-CoA is directly converted into 2,4-pentadienoic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8).
23. The method of claim 22(a), 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 2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a branched-fatty-acid kinase (EC 2.7.2.14).
24. The method of any one of claims 15 to 18, further comprising the enzymatic conversion of 3-hydroxy-4-pentenoyl-CoA into 3-hydroxy-4-pentenoic acid.
25. The method of claim 24, wherein the enzymatic conversion of 3-hydroxy-4- pentenoyl-CoA into 3-hydroxy-4-pentenoic acid comprises: (a) 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; or
(b) a single enzymatic reaction in which 3-hydroxy-4-pentenoyl-CoA is directly converted into 3-hydroxy-4-pentenoic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably an acyl-CoA hydrolase (EC 3.1.2.20); or
(c) a single enzymatic reaction in which 3-hydroxy-4-pentenoyl-CoA is directly converted into 3-hydroxy-4-pentenoic acid by making use of a CoA transferase (EC 2.8.3.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8).
26. The method of claim 25(a), wherein the enzymatic conversion of said 3- hydroxy-4-pentenoyl-CoA into 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 kinase (EC 2.7.2.7) or a branched-fatty-acid kinase (EC 2.7.2.14).
27. A recombinant organism or microorganism which expresses an enzyme as defined in any one of claims 1 to 6.
28. The recombinant organism or microorganism of claim 27, further expressing an enzyme as defined in claim 7 or 8.
29. The recombinant organism or microorganism of claim 28, further expressing an enzyme as defined in any one of claims 9 to 11.
The recombinant organism or microorganism of claim 27, further expressing an enzyme as defined in any one of claims 12 to 14.
The recombinant organism or microorganism of claim 27, further expressing an enzyme as defined in claims 15 or 16.
The recombinant organism or microorganism of claim 31 , further expressing an enzyme as defined in claims 17 or 18.
The recombinant organism or microorganism of claim 32, further expressing an enzyme as defined in claims 19 or 20.
The recombinant organism or microorganism of claim 33, further expressing an enzyme as defined in any one of claims 21 to 23.
The recombinant organism or microorganism of claim 32, further expressing an enzyme as defined in any one of claims 24 to 26.
Use of a recombinant organism or microorganism as defined in claim 27 for the production of acrylyl-CoA.
Use of a recombinant organism or microorganism as defined in claim 27 or 28 for the production of propionic acid.
Use of a recombinant organism or microorganism as defined in claim 30 for the production of acrylic acid.
Use of a recombinant organism or microorganism as defined in any one of claims 31 to 34 for the production of 2,4-pentadienoic acid.
Use of a recombinant organism or microorganism as defined in any one of claims 31 , 32 and 35 for the production of 3-hydroxy-4-pentenoic acid.
41. A method for the production of ethylene comprising the enzymatic conversion of propionic acid into ethylene.
42. The method of claim 41 , wherein the enzymatic conversion of propionic acid into ethylene is achieved by making use of a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase.
43. The method of claim 41 or 42, further comprising the enzymatic conversion of acrylyl-CoA into said propionic acid.
44. The method of claim 43, wherein the enzymatic conversion of acrylyl-CoA into propionic acid comprises the steps of:
(a) enzymatically converting acrylyl-CoA into propionyl-CoA; and
(b) further enzymatically converting the thus produced propionyl-CoA into propionic acid.
45. The method of claim 44, wherein the enzymatic conversion of acrylyl-CoA into propionyl-CoA according to step (a) is achieved by making use of 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).
46. The method of claim 44, wherein the enzymatic conversion of propionyl-CoA into propionic acid according to step (b) is achieved by
(a) 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; or
(b) 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 (c) 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:acetate CoA- transferase (EC 2.8.3.18).
47. The method of claim 46(a), wherein the enzymatic conversion of said propionyl-CoA into said propionyl 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 propionyl phosphate into said propionic 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 kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).
48. The method of claim 43, wherein the enzymatic conversion of acrylyl-CoA into propionic acid comprises the steps of:
(a) enzymatically converting acrylyl-CoA into acrylic acid; and
(b) further enzymatically converting the thus produced acrylic acid into propionic acid.
49. The method of claim 48, wherein the enzymatic conversion of acrylyl-CoA into acrylic acid as defined in step (a) is achieved by
(a') 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; or
(b') 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 (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).
50. The method of claim 49(a'), wherein the enzymatic conversion of said acrylyl- CoA into said acrylyl phosphate is achieved by making use of 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 2.7.2.15), 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).
51. The method of claim 48, wherein the enzymatic conversion of acrylic acid into propionic acid according to step (b) is achieved by making use of an (NADH)
2- enoate reductase (EC 1.3.1.31 ).
52. The method of any one of claims 41 to 50 further comprising the enzymatic conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA.
53. The method of claim 52, wherein the enzymatic conversion of 3- hydroxypropionyl-CoA into said acrylyl-CoA is achieved 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).
54. The method of any one of claims 41 to 53, further comprising the enzymatic conversion of 3-hydroxypropionaldehyde into said 3-hydroxypropionyl-CoA.
55. The method of claim 54, wherein the enzymatic conversion of 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is achieved 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).
56. The method of any one of claims 41 to 55, further comprising the enzymatic conversion of glycerol into said 3-hydroxypropionaldehyde.
57. The method of claim 56, wherein the enzymatic conversion of glycerol into said 3-hydroxypropionaldehyde is achieved by making use of a cobalamine (B12 vitamin)-dependent or B12-indepentent radical-S-adenosyl methionine- dependent) glycerol dehydratase (EC 4.2.1.30).
58. A bacterium which expresses
(i) an enzyme as defined in claim 41 or 42; and
(ii) an enzyme as defined in claim 43.
59. The bacterium according to claim 58, wherein the enzyme in (ii) is an enzyme as defined in any one of claims 44 to 47 or an enzyme as defined in any one of claims 8 to 11.
60. The bacterium of claim 58 or 59, or a recombinant organism or microorganism expressing an enzyme as defined in (i) an enzyme as defined in claim 41 or 42; and (ii) an enzyme as defined in claim 43, preferably an enzyme as defined in any one of claims 44 to 47 or an enzyme as defined in any one of claims 48 to 51 , further expressing an enzyme as defined in claim 52 or 53.
61. The bacterium, organism or microorganism of claim 60, further expressing an enzyme as defined in claim 14 or 15.
62. The bacterium, organism or microorganism of claim 61 , further expressing an enzyme as defined in claim 56 or 57.
63. Use of a bacterium as defined in any one of claims 58 to 62, an organism or microorganism as defined in any one of claims 60 to 62 for the production of ethylene.
64. The use of a bacterium, organism, or microorganism of claim 63, wherein said bacterium, organism or microorganism expresses an enzyme catalyzing the enzymatic conversion of propionic acid into ethylene, preferably a cytochrome P450 fatty acid decarboxylase.
65. Use of an enzyme catalyzing the enzymatic conversion of propionic acid into ethylene, preferably a cytochrome P450 fatty acid decarboxylase, for the production of ethylene.
66. A composition comprising
glycerol and an organism or microorganism as defined in any one of claims 58 to 62; or glycerol and an enzyme as defined in any one of claims 41 to 57.
PCT/EP2015/080482 2014-12-19 2015-12-18 Enzymatic production of acrylyl-coa or ethylene from glycerol WO2016097289A1 (en)

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WO2018005770A3 (en) * 2016-06-30 2018-04-05 Invista North America S.á.r.l. Synthetic carbon fixation pathways
EP3816271A1 (en) * 2019-10-31 2021-05-05 The Procter & Gamble Company Detergent composition

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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

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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

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018005770A3 (en) * 2016-06-30 2018-04-05 Invista North America S.á.r.l. Synthetic carbon fixation pathways
EP3816271A1 (en) * 2019-10-31 2021-05-05 The Procter & Gamble Company Detergent composition
WO2021087510A1 (en) * 2019-10-31 2021-05-06 The Procter & Gamble Company Detergent composition

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