PROCESS FOR THE ENZYMATIC PRODUCTION OF 1 -BUTENE FROM 2-PENTENOYL-COA

The present invention relates to a method for the production of 2-pentenoic acid comprising the enzymatic conversion of 2-pentenoyl-CoA into 2-pentenoic acid comprising two enzymatic steps comprising (i) first enzymatically converting 2- pentenoyl-CoA into 2-pentenoyl phosphate; and (ii) then enzymatically converting the thus obtained 2-pentenoyl phosphate into said 2-pentenoic acid; or a single enzymatic reaction in which 2-pentenoyl-CoA is directly converted into 2-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). The thus obtained 2-pentenoic acid can further be converted into pentanoic acid. The enzymatic conversion of 2-pentenoic acid into pentanoic acid can be achieved by making use of an (NADH) 2-enoate reductase (EC 1.3.1.31). Said pentanoic acid can be further converted into 1-butene. The enzymatic conversion of pentanoic acid into 1-butene can be achieved by making use of a cytochrome P450 fatty acid decarboxylase. The 2-pentenoyl-CoA can be obtained by the enzymatic conversion of 3-hydroxypentanoyl-CoA into 2- pentenoyl-CoA. 3-hydroxypentanoyl-CoA can be obtained by the enzymatic conversion of 3-oxopentanoyl-CoA into said 3-hydroxypentanoyl-CoA. 3- oxopentanoyl-CoA can be obtained by the enzymatic condensation of propionyl-CoA and acetyl-CoA. Said propionyl-CoA can be obtained by the enzymatic conversion of acrylyl-CoA into propionyl-CoA. Said acrylyl-CoA can be achieved 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 into said 3-hydroxypropionyl-CoA. Finally, 3- hydroxypropionaldehyde can be achieved by the enzymatic conversion of glycerol into said 3-hydroxypropionaldehyde.

1-butene, like many other alkenes and a large number of chemical compounds, is currently derived from petrochemicals and, accordingly, its large scale production relies on the availability of fossil resources. Recently, the microbial production of 1-butene from various carbon sources has been described by making use of the biotransformation of coronamic acid into 1-butene catalyzed by an ACC oxidase (US 2011/0177575 A1 ). This artificial metabolic pathway is not suitable for an application on an industrial scale because it involves several enzymatic steps which are "expensive" in carbon sources or require energy. Moreover, a product which is released during the reaction (i.e., cyanidric acid) is toxic which renders it difficult to reconstitute this pathway in microorganisms and to use it in an industrial fermentative process.

Other possibilities to produce 1-butene from renewable resources are known which involve the chemical dehydration of butanol while butanol is obtained by a fermentative process. In this context, a process for making butene from dry 1 -butanol has previously been described (US20080045754 A1 ). However, its production involves hybrid processes involving both, biological fermentation and chemical catalysis. Such hybrid processes are known to be cost intensive due to the chemical step of dehydration. Moreover, during the biological process, the accumulation of the produced alcohol is toxic to the cell, which is in particular disadvantageous if the process is carried out in a batch culture. Accordingly, there is a need for a direct fermentative route for the production of olefins, such as 1-butene.

Previously, compositions and methods for producing long chain olefins by making use of a fatty acid decarboxylase, i.e., a cytochrome P450 from Jeotgalicoccus ATCC 8456 have been described (US2011/0196180). The oxidative decarboxylation catalyzed by this CYP450 was demonstrated for the conversion of long chain carboxylic acids (C _n ) into their corresponding C _n- i alkene. However, no metabolic pathway for the biosynthesis of the corresponding metabolic precursor (i.e., pentanoic acid) for this enzymatic step has been proposed.

Thus, it is desirable to develop a process for the microbial production of pentanoic acid (also known as valeric acid) and, in particular, its precursor 2-pentenoic acid, from renewable resources because pentanoic acid not only serves as a precursor for the above-described production of 1-butene but may also be involved in the production of several industrial products such as perfumes, cosmetics or food additives. Furthermore, the C5 linear carboxylic acid pentanoic acid is suitable as a biofuel as it can deliver both, gasoline and diesel components that are fully compatible with transportation fuels. Recently, a method for the synthesis of valeric acid (i.e., pentanoic acid) from ligno-cellulosic materials has been described (Angew. Chem. Int. Ed. 49 (2010), 4479-4483). The bioproduction of valeric acid (i.e., pentanoic acid) has already been described previously (Microbiology 160 (2014), 1513-1522 and Appl. Environ. Microbiol. 80 (2014), 1042-1050). However, the yield of this process is too low to permit a viable industrial process.

Accordingly, in order to satisfy the increasing demand for 1-butene and its precursors pentanoic acid and 2-pentenoic acid it is desirable to provide for an alternative process for its production which is independent of fossil resources and which could be effected in living organisms, thereby being environmentally sound and inexpensive.

The present invention meets this demand for an alternative process for the enzymatic production of 1-butene and, in particular, its precursor pentanoic acid which is based on biological resources and which allows to produce 1-butene in vitro or in vivo in a microorganism and in other species.

Therefore, in the present invention, a direct route for the conversion of glycerol into propionyl-coenzyme A (propionyl-CoA also known as propanoyl-CoA) is described which is a precursor for the production of 2-pentenoic acid, pentanoic acid and, ultimately, 1-butene.

The biosynthesis of propionyl-CoA in microorganisms 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 propionyl-CoA from glucose or glycerol with a high efficiency (productivity up to 0.42 g. Γ ¹ . H ^"1 ). Engineered metabolic pathways producing propionyl-CoA have already been established in modified microorganisms. For example, the heterologous pathway to convert the D-lactic acid to propionyl-CoA from Clostridium propionicum was established in Escherichia coli (Appl. Microbiol. Biotechnol. 97 (2013), 1191-2000).

In the literature, there are mainly three pathways for the biosynthesis of propionyl- CoA (or propionic acid) from a carbon source such as glucose or glycerol described: 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).

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 propionyl-coenzyme A.

In the present invention, the most direct route for the conversion of glycerol to propionyl-CoA is described.

Moreover, the biosynthesis of pentanoyl-CoA from acetyl-CoA and propionyl-CoA via 3-oxopentanoyl-CoA, 3-hydroxypentanoyl-CoA and 2-pentenoyl-CoA has already been described. More specifically, the condensation of propionyl-CoA and acetyl-CoA to produce 3-oxopentanoyl-CoA and its subsequent conversion into pentanoyl-CoA has previously been described in WO2012/151489, Proc. Natl. Acad. Sci. 109 (2012), 17925-17930, Appl. Environ. Microbiol. 80 (2014), 1042-1050 and Microbiology 160 (2014), 1513-1522.

Further, the biosynthesis of pentanol from propionyl-CoA and acetyl-CoA has previously been described; WO2012/151489 and Proc. Natl. Acad. Sci. 109 (2012), 17925-17930. Also the biosynthesis of 3-hydroxypentanoyl-CoA from propionyl-CoA and acetyl-CoA has been described; Microbial Cell Factory 9 (2010), 96. Further, the condensation of propionyl-CoA and acetyl-CoA for the biosynthesis of 3- hydroxypentanoyl-CoA has been described in Nature Biotech. 17 (1999) 1011-1016. The conversion of pentanoyl-CoA into pentanoic acid has been described by making use of a CoA transferase, more specifically a butyryl-CoA:acetoacetate CoA- transferase (J. Biol. Chem. 253 (1978), 1219-1225). Moreover, the conversion of pentanoyl-CoA into pentanoic acid has been described by making use of thioester hydrolase utilizing TesB from E. coli; Appl. Environ. Microbiol. 80 (2014), 1042-1050. Further, the conversion of pentanoyl-CoA into pentanoic acid has been described by making use of phosphate transbutyryiase/butyrate kinase in vitro for both, the first part of the double biocatalytic step (i.e., the formation of pentanoyl phosphate from pentanoyl-CoA; Appl. Environ. Microbiol. 55 (1989); 317-322) and for the second part of this double biocatalytic step while for the latter the reverse reaction has been described (i.e., the formation of pentanoyl phosphate from pentanoic acid and ATP; J. Biol. Chem. 262 (1987), 617-612).

In the present invention, this direct route for the conversion of glycerol into propionyl- CoA is supplemented with the subsequent enzymatic condensation of propionyl-CoA and acetyl-CoA into 3-oxopentanoyl-CoA which is supplemented with a direct pathway of enzymatic conversions to 2-pentenoic acid and pentanoic acid.

This pathway is further supplemented with an oxidative decarboxylation of pentanoic acid into 1-butene.

These three catalytic segments allow the efficient, economic and direct biosynthesis of 1-butene from glycerol, e.g., in a metabolically engineered strain of Escherichia coli.

In a nutshell, the present invention, therefore, provides a process for converting 2- pentenoyl-CoA into 2-pentenoic acid and, subsequently, into pentanoic acid and 1- butene. The present invention also provides a process by which 2-pentenoyl-CoA can be produced enzymatically starting from glycerol via propionyl-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, 3-hydroxypropionyl-CoA can enzymatically be converted to propionyl-CoA via the intermediate acrylyl-CoA. Moreover, the thus produced propionyl-CoA can further be enzymatically converted into 3-oxopentanoyl- CoA by an enzymatic condensation with acetyl-CoA. Further, the thus produced 3- oxopentanoyl-CoA can further be enzymatically converted into 3-hydroxypentanoyl- CoA. The produced 3-hydroxypentanoyl-CoA can further be converted into 2- pentenoyl-CoA. Further, the thus produced 2-pentenoyl-CoA can be enzymatically converted into 2-pentenoic acid via alternative routes. In one alternative, the produced 2-pentenoyl-CoA can enzymatically be converted into 2-pentenoic acid while the thus produced 2-pentenoic acid can further be enzymatically converted into pentanoic acid. Finally, the thus produced pentanoic acid can further be enzymatically converted into 1-butene. Alternatively, the produced 2-pentenoyl-CoA can be enzymatically converted into pentanoyl-CoA. The thus produced pentanoyl- CoA can further be enzymatically converted into pentanoic acid. The corresponding reactions are schematically shown in Figure 3.

According to the present invention, the conversion of 2-pentenoyl-CoA into pentanoic acid can be achieved via different routes. One possibility, i.e., to first convert 2- pentenoyl-CoA into 2-pentenoic acid and then to further convert 2-pentenoic acid into pentanoic acid is described in the following. Another possible route by first enzymatically converting 2-pentenoyl-CoA into pentanoyl-CoA and then further by converting pentanoyl-CoA into pentanoic acid is described further below.

Route for the enzymatic conversion of 2-pentenoyl-CoA into pentanoic acid (steps IXd and Xd or step IXe or step IXf as well as step XI as shown in Figure 3)

The enzymatic conversion of 2-pentenoyl-CoA into 2-pentenoic acid (steps IXd and Xd or step IXf or step IXe as shown in Figure 3)

The present invention relates to a method for the production of 2-pentenoic acid comprising the enzymatic conversion of 2-pentenoyl-CoA into 2-pentenoic acid. According to the present invention, the enzymatic conversion of 2-pentenoyl-CoA into 2-pentenoic acid can be achieved via different routes. One possibility is a two-step conversion via 2-pentenoyl phosphate. Another option involves a direct conversion of 2-pentenoyl-CoA into 2-pentenoic acid. These options will be outlined in the following and these reactions are schematically illustrated in Figure 3.

Thus, the present invention relates to a method for the production of 2-pentenoic acid comprising the enzymatic conversion of 2-pentenoyl-CoA into 2-pentenoic acid comprising:

(a) two enzymatic steps comprising

(i) first enzymatically converting 2-pentenoyl-CoA into 2-pentenoyl phosphate (step IXd as shown in Figure 3); and

(ii) then enzymatically converting the thus obtained 2-pentenoyl phosphate into said 2-pentenoic acid (step Xd as shown in Figure 3); or (b) a single enzymatic reaction in which 2-pentenoyl-CoA is directly converted into 2-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) (step IXf as shown in Figure 3).

Thus, in one embodiment, the enzymatic conversion of 2-pentenoyl-CoA into 2- pentenoic acid can be achieved by a two-step conversion via 2-pentenoyl phosphate. Accordingly, in one embodiment, the enzymatic conversion of 2-pentenoyl-CoA into 2-pentenoic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting 2-pentenoyl-CoA into 2-pentenoyl phosphate; and (ii) then enzymatically converting the thus obtained 2-pentenoyl phosphate into said 2- pentenoic acid.

The corresponding reaction is schematically shown in Figure 4.

In a preferred embodiment, the present invention relates to a method for the production of 2-pentenoic acid comprising the enzymatic conversion of 2-pentenoyl- CoA into 2-pentenoic acid, wherein the enzymatic conversion of said 2-pentenoyl- CoA into said 2-pentenoylphosphate 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-pentenoylphosphate into said 2-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).

The conversion of 2-pentenoyl-CoA into 2-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).

Phosphate butyryltransferase (EC 2.3.1.19) naturally catalyzes the following reaction Butyryl-CoA + H ₃ P0 ₄ butyryl phosphate + CoA

It has been described by Wiesenborn et al. (Appl. Environ. Microbiol. 55 ( 989), 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 organisms, 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 P58255; 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 2-pentenoyl-CoA into 2-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: 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 2-pentenoyl-CoA into 2-pentenoyl 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 + H ₃ PO ₄ T*' 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/entrv/IPR012147

http://www.ebi.ac.uk/interpro/entry/IPR002505)

See also http://pfam.sanger.ac.uk/family/PF015 5

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.

The conversion of 2-pentenoyl phosphate into 2-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-pentenoyl phosphate into 2-pentenoic 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-pentenoyl phosphate into 2-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). 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).

Thus, in a preferred embodiment, the conversion of 2-pentenoyl phosphate into 2- 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: 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 2-pentenoyl phosphate into 2-pentenoic acid. As regards the determination of the sequence identity, the same applies as has been set forth above.

Branched-chain-fatty-acid kinases (EC 2.7.2.14) naturally catalyze the following reaction 2-methylpropanoate + ATP 2-methylpropanoyl phosphate + ADP

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 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 or Escherichia coli.

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, 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 2-pentenoyl phosphate into 2-pentenoic acid 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.pl7PS01 075

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 2-pentenoyl-CoA into 2-pentenoic acid can also be achieved by an alternative conversion wherein 2-pentenoyl-CoA into is directly converted into 2-pentenoic acid.

In a preferred embodiment, the direct conversion of 2-pentenoyl-CoA into 2- 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 2-pentenoyl- 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:(f?)-benzylsuccinate CoA-transferase, (E)- cinnamoyl-CoA:(f?)-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 CO 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-transferase 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 2-pentenoyl-CoA into 2-pentenoic acid is an acetate CoA-transferase (EC 2.8.3.8) (also termed butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8)). This reaction is schematically shown in Figure 4.

Thus, in one preferred embodiment the direct conversion of 2-pentenoyl-CoA into 2- pentenoic acid is achieved by making use of an acetate CoA-transferase (EC 2.8.3.8) which are also termed butyryl-CoA:acetate-CoA transferases (EC 2.8.3.8). Butyryl- CoA:acetate-CoA transferase (EC 2.8.3.8) 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 2-pentenoyl-CoA into 2- pentenoic 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 2-pentenoyl-CoA into 2- pentenoic 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.

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 2-pentenoyl-CoA into 2- pentenoic acid is achieved by making use of a succinyl-CoA:acetate CoA transferase (EC 2.8.3.18). Propionate:succinate-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.

Alternatively to the above, 2-pentenoyl-CoA can also be directly converted into 2- pentenoic acid by hydrolysing the thioester bond of 2-pentenoyl-CoA to 2-pentenoic acid (as schematically shown in step IXe of Figure 3) 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 4. The hydrolysis of trans-2-pentenoyl-CoA into 2-pentenoic acid catalyzed by a thioesterase (pr655) has previously been described (Appl. Environ. Microbiol. 80 (2014), 1042- 1050).

Thus, in one alternative, 2-pentenoyl-CoA is directly converted into 2-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).

Thus, in an alternative, the enzymatic conversion of 2-pentenoyl-CoA into 2- pentenoic acid (step IXe as shown in Figure 3) is achieved by a single enzymatic reaction in which 2-pentenoyl-CoA is directly converted into 2-pentenoic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably an acetyl-CoA hydrolase (EC 3.1.2.1 ) and an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).

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. (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 2-pentenoyl-CoA into 2-pentenoic 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).

Thus, in one preferred embodiment the direct conversion of 2-pentenoyl-CoA into 2- pentenoic 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 + H ₂ 0 ► 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 2-pentenoyl-CoA into 2- pentenoic 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 + H ₂ 0 ► 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 2-pentenoyl-CoA into 2- pentenoic 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 + H ₂ 0 ► 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 2-pentenoyl-CoA into 2- pentenoic 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] + H2O ► 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 2-pentenoyl-CoA into 2- pentenoic 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.

In yet another preferred embodiment the direct conversion of 2-pentenoyl-CoA into 2- pentenoic 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. In a further preferred embodiment the direct conversion of 2-pentenoyl-CoA into 2- pentenoic 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, 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). Moreover, this enzyme is described to hydrolyse pentanoyl-CoA and 2-pentenoyl-CoA (Applied and Environmental Microbiology 80 (2014), 1042-1050). In another preferred embodiment the acetyl- CoA hydrolase is an enzyme from Homo sapiens (Cao et al., Biochemistry 48 (2009), 1293-1304).

Particularly preferred are 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, Proc. Nat. Acad. Sci. USA 109 (2012), 17925-17930). 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: 3 to 5 or shows an amino acid sequence which is at least x% homologous to any one of SEQ ID NOs: 3 to 5 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 2-pentenoyl-CoA into 2-pentenoic acid.

Preferably, 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. The enzymatic conversion of 2-pentenoic acid into pentanoic acid (step XI as shown in Figure 3)

The 2-pentenoic acid which is produced according to any of the above described methods may further be converted into pentanoic acid by an enzymatic reaction, namely by the enzymatic conversion of 2-pentenoic acid into pentanoic acid. The conversion of 2-pentenoic acid into pentanoic acid is schematically illustrated in Figure 5.

Thus, the present invention also relates to a method for the production of pentanoic acid in which 2-pentenoyl-CoA is first converted into 2-pentenoic acid as described herein above wherein said 2-pentenoic acid is then further enzymatically converted into pentanoic acid.

According to the present invention, the enzymatic conversion of 2-pentenoic acid into pentanoic acid (step XI as shown in Figure 3), i.e., the reduction of 2-pentenoic acid into pentanoic 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. Biochem. 97 (1979), 103-112). 2-enoate reductases have also been described to accept substrates having an aliphatic chain which is longer than C4 as well as substrates having an aromatic substitution.

The enzymatic conversion of pentanoic acid into 1-butene (step XII as shown in Figure 3)

The pentanoic acid which is produced according to any of the above described methods may further be converted into 1-butene by an enzymatic reaction, namely by an oxidative decarboxylation of pentanoic acid into 1-butene. The conversion of pentanoic acid into 1-butene is schematically illustrated in Figures 3 and 6 and in the following:

Pentanoic acid + NADPH + O ₂ ► 1-butene + CO ₂ + NADP

Thus, the present invention also relates to a method for the production of 1-butene in which 2-pentenoyl-CoA is first converted into 2-pentenoic acid as described herein above wherein said 2-pentenoic acid is then further enzymatically converted into pentanoic acid as described herein above and wherein said pentanoic acid is then further enzymatically converted into 1-butene.

In one possibility, the enzymatic conversion of pentanoic acid into 1-butene 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 pentanoic acid into 1-butene is achieved by making use of a cytochrome P450, more preferably of the cytochrome P450 of R. minuta. The sequence of this enzyme is available under UniProt Accession number Q12668. In a preferred embodiment such an enzyme has an amino acid sequence as shown in SEQ ID NO: 6 or shows an amino acid sequence which is at least x% homologous to SEQ ID NO: 6 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 catalyze the conversion of pentanoic acid into 1-butene.

Preferably, the enzymatic conversion of pentanoic acid into 1-butene 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, pentanoic acid is enzymatically converted into 1-butene 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 decarboxyiate 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, 3 ^rd 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 hydroxylation of fatty acids in a- and/or β-position:

Fatty acid + H2O2 = 3- or 2-hydroxy fatty acid + H ₂ O

As reported by Rude et al. (Appl. Environ. Microbiol. 77 (2011 ), 1718-1727) some cyp 52 P450 enzymes have the ability to decarboxyiate 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 favour 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 olefin-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 cyp152 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 pentanoic acid as a substrate and convert it into 1-butene.

In a preferred embodiment the Ole T JE is an enzyme which

(a) comprises the sequence as shown in SEQ ID NO:7 or a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 7; and

(b) shows the activity of converting pentanoic acid via oxidative decarboxylation into 1-butene.

As mentioned above in (a), the enzyme comprises the sequence as shown in SEQ ID NO:7 or a sequence which is at least 60% identical to the amino acid sequence shown in SEQ ID NO: 7. SEQ ID NO: 7 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: 7. 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 pentanoic acid via oxidative decarboxylation into 1-butene. 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:7. 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:7.

Structure/function analyses of the Ole T JE protein have shown that the residue in position 85 of SEQ ID NO:7 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: 7 and in which the amino acid residue corresponding to position 85 of SEQ ID NO: 7 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: 7 and in which the amino acid residue corresponding to position 85 of SEQ ID NO: 7 is histidine.

As regards the determination of sequence identity, the same applies as has been set forth above.

Amino acid residues located at a position corresponding to a position as indicated herein-below in the amino acid sequence shown in SEQ ID NO:7 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 SEQ ID NO:7 and by identifying the positions which correspond to the above indicated positions of SEQ ID NO:7. 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.

As mentioned above in (b), the enzyme shows the activity of converting pentanoic acid via oxidative decarboxylation into 1-butene. This activity can be assayed as described in the appended Examples.

In another embodiment the conversion of pentanoic acid into 1-butene 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: 8 (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: 8. 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:8 and the enzyme has the enzymatic activity of converting pentanoic acid into 1-butene. As regards the determination of the sequence identity, the same applies as has been set forth above.

In a preferred embodiment the conversion of pentanoic acid into 1-butene 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. 84 (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 6. 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 pentanoic acid into 1 -butene 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:

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 pentanoic acid into 1- butene according to the present invention.

Accordingly, in a preferred embodiment, the conversion of pentanoic acid into 1- butene 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: 27 to 29, respectively.

It is, of course, not only possible to use an enzyme exactly showing any of these amino acid sequences of SEQ ID NOs:27 to 29. 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: 27 to 29. 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:27 to 29 and the enzyme has the enzymatic activity of converting pentanoic acid into 1-butene. 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 pentanoic acid into 1-butene.

Alternative route for the enzymatic conversion of 2-pentenoyl-CoA into pentanoic acid (step VIII as well as steps IXa and Xa or step IXb or step tXc as shown in Figure 3)

As mentioned above, the conversion of 2-pentenoyl-CoA into pentanoic acid can be achieved via different routes. One possibility, i.e., to first convert 2-pentenoyl-CoA into 2-pentenoic acid and then to further convert 2-pentenoic acid into pentanoic acid has been described above. In the alternative to the above, in another possible route, pentanoic acid can also be provided by the enzymatic conversion of 2-pentenoyl-CoA into pentanoyl-CoA which can then further be enzymatically converted into pentanoic acid. This alternative route for the production of pentanoic acid from 2-pentenoyl-CoA via pentanoyl-CoA is described in the following and is illustrated in Figure 3.

The enzymatic conversion of 2-pentenoyl-CoA into pentanoyl-CoA (step VIII as shown in Figure 3)

The pentanoyl-CoA which is further converted into pentanoic acid according the method as described further below may be provided by an organism or microorganism which produces pentanoyl-CoA. The biosynthesis of pentanoyl-CoA from acetyl-CoA and propionyl-CoA via 3-oxopentanoyl-CoA, 3-hydroxypentanoyl- CoA and 2-pentenoyl-CoA has already been described. More specifically, the condensation of propionyl-CoA and acetyl-CoA to produce 3-oxopentanoyl-CoA and its subsequent conversion into pentanoyl-CoA has previously been described in WO2012/151489, Proc. Natl. Acad. Sci. 109 (2012), 17925-17930, Appl. Environ. Microbiol. 80 (2014), 1042-1050 and Microbiology 160 (2014), 1513-1522.

Accordingly, organisms which produce pentanoyl-CoA or organisms which have been genetically modified so as to produce pentanoyl-CoA may be used as a host for expressing the enzymes as described below for the conversion of pentanoyl-CoA into pentanoic acid according to any of the below described methods (which may further be converted into 1-butene according to the method as described above).

The present invention also relates to a method for the production of pentanoyl-CoA comprising the enzymatic conversion of 2-pentenoyl-CoA into pentanoyl-CoA (step VIII as shown in Figure 3).

The enzymatic conversion of 2-pentenoyl-CoA into pentanoyl-CoA (step VIII as shown in Figure 3) can, for example, be achieved by making use of an enzyme, i.e., an oxido-reductase acting on the CH-CH group of a donor molecule which is classified as EC1.3.-.-. Preferably, the enzymatic conversion of 2-pentenoyl-CoA into pentanoyl-CoA can 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. 95 (1984), 1315-1321 ). Other enzymes which are able to catalyze the reduction of the double bond may use FADH as a reducing factor. The conversions using such enzymes are schematically shown in Figure 14. 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 2-pentenoyl-CoA into said pentanoyl-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 2-pentenoyl-CoA into said pentanoyl-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 2-pentenoyl-CoA into said pentanoyl-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 2-pentenoyl-CoA into said pentanoyl-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 2-pentenoyl-CoA into said pentanoyl-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 2-pentenoyl-CoA into said pentanoyl-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 2-pentenoyl-CoA into said pentanoyl-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. 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 2-pentenoyl-CoA into said pentanoyl-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 2-pentenoyl-CoA into said pentanoyl-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 preferred embodiment, the enzymatic conversion of 2-pentenoyl-CoA into pentanoyl-CoA can be achieved by making use of a trans-2-enoyl-CoA reductase (NAD ⁺ ) (EC 1.3.1.44) from Treponema denticola (Uniprot Q73Q47) as this enzyme is described to be capable of reducing 2-pentenoyl-CoA into pentanoyl-CoA (Applied and Environmental Microbiology 80 (2014), 1042-1050).

Thus, in a preferred embodiment, the conversion of 2-pentenoyl-CoA into pentanoyl- CoA is achieved by making use of an enoyl-CoA reductase from Treponema denticola (Uniprot Q73Q47). The amino acid sequence of said protein is shown in SEQ ID NO: 19.

It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO: 19. 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: 19. 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: 19 and the enzyme has the enzymatic activity of converting 2-pentenoyl-CoA into pentanoyl-CoA. As regards the determination of the sequence identity, the same applies as has been set forth above.

In a further preferred embodiment, the conversion of 2-pentenoyl-CoA into said pentanoyl-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 ⁺ _¾ » ^» acryloyl-CoA + NADH + H ⁺

This enzyme occurs in a variety of prokaryotic organisms and the enzyme has, e.g., been described in Clostridium kluyveri, Clostridium propionicum, Metallosphaera sedula and Sufolobus tokodaii.

In a preferred embodiment, the enzymatic conversion of 2-pentenoyl-CoA into said pentanoyl-CoA is achieved by making use of an NADPH dependent trans-2-enoyl- CoA reductase (EC 1.3.1.38), a crotonyl-CoA reductase (EC 1.3.1.86), an NADPH- dependent acrylyl-CoA reductase (EC 1.3.1.84) or an NAD dependent trans-2-enoyl- CoA reductase (EC 1.3.1.44).

The enzymatic conversion of pentanoyl-CoA into pentanoic acid (steps IXa and Xa or step IXb or step IXc as shown in Figure 3)

The pentanoyl-CoA which is produced according to any of the above described methods may further be converted into pentanoic acid by an enzyme which is known to produce pentanoic acid from pentanoyl-CoA. As mentioned above, the conversion of pentanoyl-CoA into pentanoic acid has been described by making use of a CoA transferase, more specifically a butyryl-CoA:acetoacetate CoA-transferase (J. Biol. Chem. 253 (1978), 1219-1225). Moreover, the conversion of pentanoyl-CoA into pentanoic acid has been described by making use of thioester hydrolase utilizing TesB from E. coli; Appl. Environ. Microbiol. 80 (2014), 1042-1050. Further, the conversion of pentanoyl-CoA into pentanoic acid has been described by making use of phosphate transbutyrylase/butyrate kinase in vitro for both, the first part of the double biocatalytic step (i.e., the formation of pentanoyl phosphate from pentanoyl- CoA; Appl. Environ. Microbiol. 55 (1989); 317-322) and for the second part of this double biocatalytic step while for the latter the reverse reaction has been described (i.e., the formation of pentanoyl phosphate from pentanoic acid and ATP; J. Biol. Chem. 262 (1987), 617-612). Accordingly, organisms which are capable of converting pentanoyl-CoA into pentanoic acid or organisms which have been genetically been modified so as to convert pentanoyl-CoA into pentanoic acid may be used as a host for expressing the enzymes as described above for the above conversions according to any of the above described methods and which may further be converted according to any of the methods described herein above.

In a preferred embodiment, the pentanoyl-CoA which is produced according to any of the above described methods may further be converted into pentanoic acid by an enzymatic reaction. Thus, the present invention relates to a method for the production of pentanoic acid comprising the enzymatic conversion of 2-pentenoyl- CoA into pentanoyl-CoA according to any of the methods as described herein above and the enzymatic conversion of the thus produced pentanoyl-CoA into pentanoic acid. According to the present invention, the enzymatic conversion of pentanoyl-CoA into pentanoic acid can be achieved via different routes. One possibility is a two-step conversion via pentanoyl phosphate (steps IXa and Xa as shown in Figure 3). Two other options involve a direct conversion of pentanoyl-CoA into pentanoic acid (steps IXb and IXc as shown in Figure 3). These options will be outlined in the following and these reactions are schematically illustrated in Figure 3.

Thus, the present invention relates to a method for the production of pentanoic acid comprising the enzymatic conversion of pentanoyl-CoA into pentanoic acid comprising:

(a) two enzymatic steps comprising

(i) first enzymatically converting pentanoyl-CoA into pentanoyl phosphate (step IXa as shown in Figure 3)); and (ii) then enzymatically converting the thus obtained pentanoyi phosphate into said pentanoic acid (step Xa as shown in Figure 3); or

(b) a single enzymatic reaction in which pentanoyl-CoA is directly converted into pentanoic acid by making use of a thioester hydrolase (EC 3.1.2.-), preferably an acyl-CoA hydrolase (EC 3.1.2.20) (step IXb as shown in Figure 3); or

(c) a single enzymatic reaction in which pentanoyl-CoA is directly converted into pentanoic acid by making use of a CoA transferase (EC 2.8.1.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8) (step IXc as shown in Figure 3).

Thus, in one embodiment, the enzymatic conversion of pentanoyl-CoA into pentanoic acid can be achieved by a two-step conversion via pentanoyi phosphate. Accordingly, in one embodiment, the enzymatic conversion of pentanoyl-CoA into pentanoic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting pentanoyl-CoA into pentanoyi phosphate; and (ii) then enzymatically converting the thus obtained pentanoyi phosphate into said pentanoic acid.

The corresponding reaction is schematically shown in Figure 15.

Accordingly, in a preferred embodiment, the present invention relates to a method for the production of pentanoic acid comprising the enzymatic conversion of pentanoyl- CoA into pentanoic acid, wherein the enzymatic conversion of said pentanoyl-CoA into said pentanoyi phosphate is achieved by making use of a phosphate butyry transferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the enzymatic conversion of said pentanoyi phosphate into said pentanoic 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 pentanoyl-CoA into pentanoyl-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) for the conversion of pentanoyl-CoA into pentanoyi phosphate have already been described above in the context of the conversion of 2- pentenoyl-CoA into 2-pentenoyl phosphate. As regards these enzymes, the same applies for the conversion of pentanoyl-CoA into pentanoyi phosphate as has been set forth above in the context of the conversion of 2-pentenoyl-CoA into 2-pentenoyl phosphate.

In a preferred embodiment, the conversion of pentanoyl-CoA into pentanoyl- 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: 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 pentanoyl-CoA into pentanoyl-phosphate. As regards the determination of the sequence identity, the same applies as has been set forth above.

The conversion of pentanoyi phosphate into pentanoic 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 pentanoyi phosphate into pentanoic 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 pentanoyi phosphate into pentanoic 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) for the conversion of pentanoyl phosphate into pentanoic acid have already been described above in the context of the conversion of 2-pentenoyl phosphate into 2-pentenoic acid. As regards these enzymes, the same applies for the conversion of pentanoyl phosphate into pentanoic acid as has been set forth above in the context of the conversion of 2- pentenoyl phosphate into 2-pentenoic acid.

In a preferred embodiment, the conversion of pentanoyl phosphate into pentanoic 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: 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 pentanoyl phosphate into pentanoic acid. As regards the determination of the sequence identity, the same applies as has been set forth above.

As mentioned above, the conversion of pentanoyl-CoA into pentanoic acid can also be achieved by an alternative conversion wherein pentanoyl-CoA into is directly converted into pentanoic acid.

In a preferred embodiment, the direct conversion of pentanoyl-CoA into pentanoic 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-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 16.

The enzymes which are classified as a CoA-transferase (EC 2.8.3.-) capable of transferring the CoA group of 2-pentenoyl-CoA to a carboxylic acid as well as the enzyme CoA transferase (EC 2.8.3.8) for the conversion of pentanoyl-CoA into pentanoic acid have already been described above in the context of the conversion of 2-pentenoyl-CoA into 2-pentenoic acid. As regards these enzymes, the same applies for the conversion of pentanoyl-CoA into pentanoic acid as has been set forth above in the context of the conversion of 2-pentenoyl-CoA into 2-pentenoic acid.

Alternatively to the above, pentanoyl-CoA can also be directly converted into pentanoic acid by hydrolysing the thioester bond of pentanoyl-CoA to pentanoic 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 17 and has previously been described (Appl. Environ. Microbiol. 80 (2014), 1042-1050).

Thus, in one alternative, pentanoyl-CoA can also be directly converted into pentanoic 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) for the conversion of pentanoyl-CoA into pentanoic acid have already been described above in the context of the conversion of 2-pentenoyl- CoA into 2-pentenoic acid. As regards these enzymes, the same applies for the conversion of pentanoyl-CoA into pentanoic acid as has been set forth above in the context of the conversion of 2-pentenoyl-CoA into 2-pentenoic acid.

The pentanoic acid which is produced according to any of the above described methods may further be converted into 1-butene by an enzymatic reaction, namely by an oxidative decarboxylation of pentanoic acid into 1-butene as described above. The enzymatic conversion of 3-hydroxypentanoyl-CoA into 2-pentenoyl-CoA

(step VII as shown in Figure 3)

The 2-pentenoyl-CoA which is converted according to the present invention into 2- pentenoic acid according to any of the above described methods (and further converted to pentanoic acid according to any of the above described methods which is further converted into 1-butene according to any of the above described methods) or the 2-pentenoyl-CoA which is converted according to the present invention into pentanoyl-CoA according to any of the above described methods (and further converted to pentanoic acid according to any of the above described methods which is further converted into 1-butene according to any of the above described methods) may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of 3-hydroxypentanoyl-CoA into said 2-pentenoyl-CoA (step VII as shown in Figure 3). The conversion of 3-hydroxypentanoyl-CoA into 2-pentenoyl-CoA is schematically illustrated in Figure 7.

Thus, the present invention also relates to a method for producing 1-butene from 3- hydroxypentanoyl-CoA in which 3-hydroxypentanoyl-CoA is first converted into 2- pentenoyl-CoA which is then converted into 2-pentenoic acid. Further, 2-pentenoic acid is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

Moreover, the present invention also relates to a method for producing 1-butene from 3-hydroxypentanoyl-CoA in which 3-hydroxypentanoyl-CoA is first converted into 2- pentenoyl-CoA, which is then converted into pentanoyl-CoA. Further, pentanoyl-CoA is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

According to the present invention, the conversion of 3-hydroxypentanoyl-CoA into said 2-pentenoyl-CoA preferably makes use of an enzyme catalyzing 3- hydroxypentanoyl-CoA dehydration. The term "dehydration" is generally referred to a reaction involving the removal of H ₂ 0. Enzymes catalyzing 3-hydroxypentanoyl-CoA dehydration are enzymes which catalyze the reaction as shown in Figure 7.

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 conversion of 3- hydroxypentanoyl-CoA into said 2-pentenoyl-CoA by making use of an enzyme catalyzing 3-hydroxypentanoyl-CoA dehydration, preferably of a 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-). Examples for enzymes catalyzing 3- hydroxypentanoyl-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-hydroxypentanoyl-CoA dehydration have in common that they use a natural substrate having the following minimal structural motif:

wherein

R is a hydrogen atom or an alkyl group or CH ₂ COO ^" ;

R ² is a hydrogen atom or a methyl group; and

R ³ is coenzyme A or acyl-carrier protein.

Thus, the above mentioned enzymes which can catalyze the dehydration of 3- hydroxypentanoyl-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] + H ₂ 0

The enzymes of this group share a common structural motif which is referenced in the InterPro as InterPro IPRQ13114

(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- hydroxypentanoyl-CoA into said 2-pentenoyl-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 ■< acryloyl-CoA + H ₂ O

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. _f 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-hydroxypentanoyl-CoA into said 2-pentenoyl-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- hydroxypentanoyl-CoA into said 2-pentenoyl-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-hydroxypentanoyl-CoA into said 2-pentenoyl-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 + H ₂ O

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-hydroxypentanoyl- CoA into said 2-pentenoyl-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-hydroxypentanoyl- CoA into said 2-pentenoyl-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 conversion of 3-hydroxypentanoyl-CoA into said 2-pentenoyl- CoA.

Thus, in a preferred embodiment, the conversion of 3-hydroxypentanoyl-CoA into 2- pentenoyl-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: 18.

It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO: 18. 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: 18. 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:18 and the enzyme has the enzymatic activity of converting 3-hydroxypentanoyl-CoA into 2-pentenoyl-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 conversion of 3- hydroxypentanoyl-CoA into said 2-pentenoyl-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 + H ₂ O 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- hydroxypentanoyl-CoA into said 2-pentenoyl-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- hydroxypentanoyl-CoA into said 2-pentenoyl-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] + 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 (3R)-3- hydroxyoctanoyl-facyl-carrier-protein] hydro-lyase (oct-2-enoy!-[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- hydroxypentanoyl-CoA into said 2-pentenoyl-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- hydroxypentanoyl-CoA into said 2-pentenoyl-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.

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-hydroxypentanoyl-CoA into said 2-pentenoyl-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- hydroxypentanoyl-CoA into said 2-pentenoyl-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] + H ₂ O (2) (3R)-3-hydroxydecanoyl-[acyl-carrier protein] a cis-dec-3-enoyl-[acyl- carrier protein] + H ₂ O

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-hydroxypentanoyl-CoA into said 2-pentenoyl-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- hydroxypentanoyl-CoA into said 2-pentenoyl-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] + H ₂ O

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-hydroxypentanoyl-CoA into said 2-pentenoyl-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- hydroxypentanoyl-CoA into said 2-pentenoyl-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. 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-hydroxypentanoyl-CoA into said 2-pentenoyl-CoA can be employed in the method of the invention.

In another embodiment of the method according to the invention the conversion of 3- hydroxypentanoyl-CoA into said 2-pentenoyl-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 + 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-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:15. 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:15 or an amino acid sequence which is at least x% homologous SEQ ID NO: 15 and which has the activity of a 3-methylglutaconyl- CoA hydratase/3-hydromethylglutaryl-CoA dehydratase and which shows the activity of converting 3-hydroxypentanoyl-CoA into said 2-pentenoyl-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.

Yet, in a more preferred embodiment, the enzymatic conversion of 3- hydroxypentanoyl-CoA into said 2-pentenoyl-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 3-oxopentanoyl-CoA into 3-hvdroxypentanoyl- CoA (step VI as shown in Figure 3)

The 3-hydroxypentanoyl-CoA which is converted according to the present invention into 2-pentenoyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may be provided by an organism or microorganism which produces 3-hydroxypentanoyl-CoA. As mentioned above, the biosynthesis of 3-hydroxypentanoyl-CoA from propionyl- CoA and acetyl-CoA has been described; Microbial Cell Factory 9 (2010), 96. Further, the condensation of propionyl-CoA and acetyl-CoA for the biosynthesis of 3- hydroxypentanoyl-CoA has been described in Nature Biotech. 17 (1999) 1011-1016. Accordingly, organisms which produce 3-hydroxypentanoyl-CoA or organisms which have been genetically modified so as to produce 3-hydroxypentanoyl-CoA may be used as a host for expressing the enzymes as described above for the conversion of 3-hydroxypentanoyl-CoA into 2-pentenoyl-CoA according to any of the above described methods and which may further be converted according to any of the methods described herein above.

Further, in a preferred embodiment, the 3-hydroxypentanoyl-CoA which is converted according to the present invention into 2-pentenoyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of 3-oxopentanoyl-CoA into said 3- hydroxypentanoyl-CoA (step VI as shown in Figure 3). The conversion of 3- oxopentanoyl-CoA into said 3-hydroxypentanoyl-CoA is schematically illustrated in Figure 7. The reaction involves the formation of a chiral carbon bearing hydroxyl group (indicated with an (*) in Figure 8) and the reaction may be stereoselective.

Thus, the present invention also relates to a method for producing 1-butene from 3- oxopentanoyl-CoA in which 3-oxopentanoyl-CoA is first converted into 3- hydroxypentanoyl-CoA which is then converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then further converted into 2-pentenoic acid as described herein above. Further, 2-pentenoic acid is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

Moreover, the present invention also relates to a method for producing 1-butene from 3-oxopentanoyl-CoA in which 3-oxopentanoyl-CoA is first converted into 3- hydroxypentanoyl-CoA which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then converted into pentanoyl- CoA. Further, pentanoyl-CoA is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

According to the present invention, the conversion of 3-oxopentanoyl-CoA into said 3-hydroxypentanoyl-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 8. 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 NADH 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 NADPH 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-oxopentanoyl-CoA into said 3-hydroxypentanoyl-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 conversion of 3-oxopentanoyl- CoA into said 3-hydroxypentanoyl-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 conversion of 3-oxopentanoyl-CoA into said 3- hydroxypentanoyl-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: 17.

It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO:17. 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: 17. 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:17 and the enzyme has the enzymatic activity of converting 3-oxopentanoyl-CoA into said 3-hydroxypentanoyl- 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 condensation of propionyl-CoA and acetyl-CoA into 3- oxopentanoyl-CoA (step V as shown in Figure 3)

The 3-oxopentanoyl-CoA which is converted according to the present invention into 3-hydroxypentanoyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may itself be provided by an enzymatic reaction, namely by the enzymatic condensation of propionyl-CoA and acetyl-CoA into 3-oxopentanoyl-CoA (step V as shown in Figure 3). The enzymatic condensation of propionyl-CoA and acetyl-CoA into 3-oxopentanoyl-CoA is schematically illustrated in Figure 9.

Thus, the present invention also relates to a method for producing 1-butene from propionyl-CoA and acetyl-CoA in which propionyl-CoA and acetyl-CoA are first condensed into 3-oxopentanoyl-CoA which is then converted into 3- hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then further converted into 2-pentenoic acid as described herein above. Further, 2- pentenoic acid is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

Moreover, the present invention also relates to a method for producing 1-butene from propionyl-CoA and acetyl-CoA in which propionyl-CoA and acetyl-CoA are first condensed into 3-oxopentanoyl-CoA which is then converted into 3- hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then converted into pentanoyl-CoA as described herein above. Further, pentanoyl-CoA is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

According to the present invention, the enzymatic condensation of propionyl-CoA and acetyl-CoA into 3-oxopentanoyl-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 propionyl-CoA and acetyl- CoA into 3-oxopentanoyl-CoA preferably makes use of the acetyl-CoA C- acyltransferase thiolase from Cupriavidus necator (Uniprot number:Q0KBP1 ). 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 propionyl-CoA and acetyl-CoA into 3-oxopentanoyl-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: 16.

It is, of course, not only possible to use an enzyme exactly showing this amino acid of SEQ ID NO: 16. 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: 6. 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:16 and the enzyme has the enzymatic activity of condensating propionyl-CoA and acetyl-CoA into 3- oxopentanoyl-CoA. As regards the determination of the sequence identity, the same applies as has been set forth above.

The enzymatic conversion of acrylyl-CoA into propionyl-CoA (step IV as shown in Figure 3)

The propionyl-CoA which is condensed with acetyl-CoA according to the present invention into 3-oxopentanoyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may be provided from an external source. Accordingly, propionyl-CoA may be added to the above enzyme(s) capable of converting propionyl-CoA into 1-butene or to a microorganism.

The propionyl-CoA which is converted according to the method of the present invention into 1-butene according to any of the above described methods may also be provided by an organism or microorganism which produces propionyl-CoA. The biosynthesis of propionyl-CoA in microorganisms has already been described and bacteria such as Propionibacterium acidipropionici or Propionibacterium freudenreichii ssp. shermanii are well known to naturally produce propionyl-CoA from glucose or glycerol. Moreover, engineered metabolic pathways producing propionyl- CoA have already been established in genetically modified microorganisms. For example, the heterologous pathway to convert D-lactic acid to propionyl-CoA from Clostridium propionicum was established in Escherichia coli (Appl. Microbiol. Biotechnol. 97 (2013), 1191-2000). Accordingly, organisms which naturally produce propionyl-CoA or organisms which have been genetically modified so as to produce propionyl-CoA may be used as a host for expressing the enzymes as described above for the conversion of propionyl-CoA into 1-butene according to any of the above described methods.

In addition, the literature describes mainly three pathways for the biosynthesis of propionyl-CoA (or propionic acid) 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).

Moreover, propionyl-CoA is biosynthetically produced in archaea. 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. Bacterid. 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.

Thus, 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.

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 propionyl-CoA into 1-butene is realized in the presence of an organism or microorganism capable of producing propionyl-CoA. Such an organism or microorganism has the capability 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. These organisms or microorganisms may be naturally occurring organisms or microorganisms which naturally have the capability to produce propionyl-CoA as described further below or may be an organism or microorganism which is derived from an organism or microorganism which naturally does not produce propionyl-CoA but which has been genetically modified so as to produce propionyl-CoA, i.e., by introducing the gene(s) necessary for allowing the production of propionyl-CoA in the organism or microorganism as described further below. Any such organism will also produce acetyl-CoA since this compound is a central metabolite.

The propionyl-CoA which is converted according to the method of the present invention into 1-butene according to any of the above described methods may also be provided by enzymatic reactions by which propionyl-CoA is produced, for example by enzymatic reactions by which propionyl-CoA is produced enzymatically starting from glycerol as schematically shown in Figure 3.

In one preferred embodiment, the propionyl-CoA which is condensed with acetyl-CoA according to the present invention into 3-oxopentanoyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of acrylyl-CoA into propionyl-CoA (step IV as shown in Figure 3). The enzymatic conversion of acrylyl-CoA into propionyl-CoA is schematically illustrated in Figure 10.

Thus, the present invention also relates to a method for producing 1-butene from acrylyl-CoA in which acrylyl-CoA is first converted into propionyl-CoA which is then further condensed with acetyl-CoA into 3-oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3-hydroxypentanoyl- CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then further converted into 2- pentenoic acid as described herein above. Further, 2-pentenoic acid is then further converted into pentanoic acid, which is then further enzymatically converted into 1- butene as described herein above.

Moreover, the present invention also relates to a method for producing 1-butene from acrylyl-CoA in which acrylyl-CoA is first converted into propionyl-CoA which is then further condensed with acetyl-CoA into 3-oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3-hydroxypentanoyl- CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then converted into pentanoyl-CoA as described herein above. Further, pentanoyl-CoA is then further converted into pentanoic acid, which is then further enzymatically converted into 1- butene as described herein above.

The enzymatic conversion of acrylyl-CoA into propionyl-CoA (step IV as shown in Figure 3) 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 10.

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 ⁺ < 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. 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, Burkholderia 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 enzyme 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 3-hydroxypropionyl-CoA into acrylyl-CoA (step 111 as shown in Figure 3)

The acrylyl-CoA which is converted into propionyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA (step III as shown in Figure 3). The enzymatic conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA is schematically illustrated in Figure 11. 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 ).

Thus, the present invention also relates to a method for producing 1-butene from 3- hydroxypropionyl-CoA in which 3-hydroxypropionyl-CoA is first converted into acrylyl- CoA which is then further converted into propionyl-CoA as described herein above. Further, said propionyl-CoA is then further condensed with acetyl-CoA into 3- oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3-hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2- pentenoyl-CoA is then further converted into 2-pentenoic acid as described herein above. Further, 2-pentenoic acid is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

Moreover, the present invention also relates to a method for producing 1-butene from 3-hydroxypropionyl-CoA in which 3-hydroxypropionyl-CoA is first converted into acrylyl-CoA which is then further converted into propionyl-CoA as described herein above. Further, said propionyl-CoA is then further condensed with acetyl-CoA into 3- oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3-hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2- pentenoyl-CoA is then converted into pentanoyl-CoA as described herein above. Further, pentanoyl-CoA is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene 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 H ₂ 0. Enzymes catalyzing 3-hydroxypropionyl-CoA dehydration are enzymes which catalyze the reaction as shown in Figure 11. 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. .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).

In a preferred embodiment, 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-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) or a enoyl-CoA hydratase (EC 4.2.1.17).

The enzymes classified as 3-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (EC 4.2.1.-), and the enzymes classified as 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59), crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58), 3-hydroxydecanoyl- [acyl-carrier-protein] dehydratase (EC 4.2.1.60), 3-hydroxypalmitoyl-[acyl-carrier- protein] dehydratase (EC 4.2.1.61 ), long-chain-enoyl-CoA hydratase (EC 4.2.1.74), and 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18) have already been described above in the context of the enzymatic conversion of 3-hydroxypentanoyl-CoA into said 2-pentenoyl-CoA. As regards these enzymes, the same applies for the enzymatic conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA as has been set forth above in the context of the enzymatic conversion of 3-hydroxypentanoyl- CoA into said 2-pentenoyl-CoA.

The enzymatic conversion of 3-hydroxypropionyldehvde into 3- hydroxypropionyl-CoA (step II as shown in Figure 3)

The 3-hydroxypropionyl-CoA which is converted into acrylyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of 3-hydroxypropionaldehyde into 3- hydroxypropionyl-CoA (step II as shown in Figure 3). The enzymatic conversion of 3- hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is schematically illustrated in Figure 12.

Thus, the present invention also relates to a method for producing 1-butene from 3- hydroxypropionaldehyde in which 3-hydroxypropionyldehyde is first converted into 3- hydroxypropionyl-CoA which is then further converted into acrylyl-CoA which is then further converted into propionyl-CoA as described herein above. Further, said propionyl-CoA is then further condensed with acetyl-CoA into 3-oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3- hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then further converted into 2-pentenoic acid as described herein above. Further, 2- pentenoic acid is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

Moreover, the present invention also relates to a method for producing 1-butene from 3-hydroxypropionaldehyde in which 3-hydroxypropionyldehyde is first converted into 3-hydroxypropionyl-CoA which is then further converted into acrylyl-CoA which is then further converted into propionyl-CoA as described herein above. Further, said propionyl-CoA is then further condensed with acetyl-CoA into 3-oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3- hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then converted into pentanoyl-CoA as described herein above. Further, pentanoyl-CoA is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

The enzymatic conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionyl-CoA preferably makes 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-coenzyme A 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 1 12. 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 12.

The enzymatic conversion of glycerol into said 3-hvdroxypropionyldehvde (step I as shown in Figure 3)

The 3-hydroxypropionaldehyde which is converted into 3-hydroxypropionyl-CoA according to any of the above described methods and which is further converted according to any of the methods described herein above may itself be provided by an enzymatic reaction, namely by the enzymatic conversion of glycerol into 3- hydroxypropionaldehyde (step I as shown in Figure 3). The enzymatic conversion of glycerol into 3-hydroxypropionaldehyde (also known as 3-hydroxypropanal) is schematically illustrated in Figure 13.

Thus, the present invention also relates to a method for producing 1-butene from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then further converted into 3-hydroxypropionyl-CoA as described herein above. Further, said 3-hydroxypropionyl-CoA is then further converted into acrylyl-CoA which is then further converted into propionyl-CoA as described herein above. Further, said propionyl-CoA is then further condensed with acetyl-CoA into 3-oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3- hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then further converted into 2-pentenoic acid as described herein above. Further, 2- pentenoic acid is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene as described herein above.

Moreover, the present invention also relates to a method for producing 1-butene from glycerol in which glycerol is first converted into 3-hydroxypropionaldehyde which is then further converted into 3-hydroxypropionyl-CoA as described herein above. Further, said 3-hydroxypropionyl-CoA is then further converted into acrylyl-CoA which is then further converted into propionyl-CoA as described herein above. Further, said propionyl-CoA is then further condensed with acetyl-CoA into 3-oxopentanoyl-CoA as described herein above. Further, the 3-oxopentanoyl-CoA is then converted into 3- hydroxypentanoyl-CoA as described herein above which is then further converted into 2-pentenoyl-CoA as described herein above. Further, 2-pentenoyl-CoA is then converted into pentanoyl-CoA as described herein above. Further, pentanoyl-CoA is then further converted into pentanoic acid, which is then further enzymatically converted into 1-butene 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 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-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.

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 dehytratase 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 dehytratase 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 dehytratase 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- hydroxypropionaldehvde, 3-hvdroxypropionyl-CoA and acrylyl-CoA (steps I, II, III and IV as shown in Figure 3)

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 3);

(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 3);

(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-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) or a enoyl-CoA hydratase (EC 4.2.1.17) (step III as shown in Figure 3); 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 IV as shown in Figure 3).

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-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), the 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) or the 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.

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 plasmid.

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 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. As mentioned above, propionyl-CoA is biosynthetically produced in microorganisms such as Propionibacterium acidipropionici, Propionibacterium freudenreichii ssp. shermanii or Clostridium propionicum. Moreover, engineered metabolic pathways producing propionyl-CoA have already been established in modified microorganisms. For example, the heterologous pathway to convert the D-lactic acid to propionyl-CoA 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 the enzymes as described above for the conversion of propionyl-CoA into -butene according to any of the above described methods.

In addition, the literature describes mainly three pathways for the biosynthesis of propionyl-CoA 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 propionyl-CoA may be used as a host for expressing the above enzyme(s) for the conversion of propionyl-CoA into pentanoic acid and a cytochrome P450 or a non-heme iron oxygenase as described above for the conversion of pentanoic acid into 1-butene 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 propionyl-CoA.

As mentioned above, propionyl-CoA is biosynthetically produced in archaea. 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. Thus, 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.

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 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., any of the enzymes catalyzing the enzymatic conversions of propionyl-CoA into pentanoic acid as described herein above and an enzyme catalyzing the subsequent enzymatic conversion into 1-butene as defined above, preferably a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase as described above for the production of 1-butene.

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 propionyl- CoA but which has been genetically modified so as to produce propionyl-CoA, i.e., by introducing the gene(s) necessary for allowing the production of propionyl-CoA in the organism/microorganism. In principle, any microorganism can be genetically modified in this way. The enzymes responsible for the synthesis of propionyl-CoA 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 propionyl- CoA, preferably from any of the precursors of propionyl-CoA (i.e., 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 1-butene 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 1-butene 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 propionyl-CoA (and preferably further into pentanoic acid and, even more preferably, into 1-butene), 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 ligated 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 pentanoic acid into 1-butene and the enzymes required for the enzymatic conversion of 2-pentenoyl-CoA into pentanoic acid via 2-pentenoic acid. Thus, the present invention relates to a recombinant organism or microorganism which expresses (i) an enzyme catalyzing the enzymatic conversion of 2-pentenoyl-CoA into 2- pentenoic acid as defined above and an enzyme catalyzing the enzymatic conversion of 2-pentenoic acid into pentanoic acid as defined above (step IXd and step Xd or step IXe or step IXf as well as step XI as shown in Figure 3); and

(ii) an enzyme catalyzing the enzymatic conversion of pentanoic acid into 1- butene as defined above (step XII as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (i) catalyzing the direct conversion of 2-pentenoyl-CoA into 2-pentenoic acid is a CoA transferase (EC 2.8.3.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8) as described herein above (step IXf as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (i) catalyzing the direct conversion of 2-pentenoyl-CoA into 2-pentenoic acid is 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) as described herein above (step IXe as shown in Figure 3).

In a further preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (i) are an enzyme catalyzing the enzymatic conversion of 2-pentenoyl-CoA into 2- pentenoyl phosphate as described herein above (step IXd as shown in Figure 3); and enzyme further catalyzing the enzymatic conversion of the thus obtained 2-pentenoyl phosphate into said 2-pentenoic acid as described herein above (step Xd as shown in Figure 3).

In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme catalyzing the enzymatic conversion of said 2-pentenoyl-CoA into said 2-pentenoyl-phosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) as described herein above and the enzyme catalyzing the enzymatic conversion of said 2-pentenoyl-phosphate into said 2-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) as described herein above. In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme in (i) catalyzing the enzymatic conversion of 2-pentenoic acid into pentanoic acid is an (NADH) 2-enoate reductase (EC 1.3.1.31 ) as described herein above (step XI as shown in Figure 3). In a further preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (ii) catalyzing the enzymatic conversion of pentanoic acid into 1-butene (step XII as shown in Figure 3) is a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase as described herein above.

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 3-hydroxypentanoyl-CoA into 2-pentenoyl- CoA as defined above (step VII as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-hydroxypentanoyl-CoA into 2-pentenoyl-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) as described herein above (step VII as shown in Figure 3).

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 3-oxopentanoyl-CoA into 3-hydroxypentanoyl- CoA as described herein above (step VI as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-oxopentanoyl-CoA into said 3-hydroxypentanoyl-CoA is a 3-hydroxyacyl-CoA dehydrogenase (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 described herein above (step VI as shown in Figure 3).

In a further aspect, the above recombinant organism or microorganism is a recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic condensation of propionyl-CoA and acetyl-CoA into said 3- oxopentanoyl-CoA as described herein above (step V as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic condensation of propionyl-CoA and acetyl-CoA into said 3-oxopentanoyl- CoA is an acetyl-CoA C-acyltransferase (EC 2.3.1.16) as described herein above (step V as shown in Figure 3).

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 said propionyl-CoA as described herein above (step IV as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of said acrylyl-CoA into said propionyl-CoA is an enoyl-CoA reductase (EC 1.3.1.-), preferably an acrylyl-CoA reductase (EC 1.3.1.95) as described herein above (step IV as shown in Figure 3).

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 3-hydroxypropionyl-CoA into said acrylyl-CoA as described herein above (step III as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA is a 3- hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (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 described herein above (step III as shown in Figure 3).

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 3-hydroxypropionaldehyde into said 3- hydroxypropionyl-CoA as described herein above (step II as shown in Figure 3). In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is a CoA-acylating aldehyde dehydrogenase (EC 1.2.1.-), preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10) as described herein above (step II as shown in Figure 3).

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 glycerol into said 3-hydroxypropionaldehyde as described herein above (step I as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of glycerol into said 3-hydroxypropionaldehyde is a cobalamine (B12 vitamin)-dependent or B12-indepentent/radical-S-adenosyl methionine- dependent) glycerol dehydratase (EC 4.2.1.30) as described herein above (step I as shown in Figure 3).

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 pentanoic acid into 1-butene and the enzymes required for the enzymatic conversion of 2-pentenoyl-CoA into pentanoic acid via pentanoyl-CoA. Thus, the present invention relates to a recombinant organism or microorganism which expresses

(i) an enzyme catalyzing the enzymatic conversion of 2-pentenoyl-CoA into pentanoyl-CoA as defined above and an enzyme catalyzing the enzymatic conversion of pentanoyl-CoA into pentanoic acid as defined above (step VIII as well as steps IXa and Xa or step IXb or step IXc as shown in Figure 3); and

(ii) an enzyme catalyzing the enzymatic conversion of pentanoic acid into 1- butene as defined above (step XII as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (i) catalyzing the conversion of pentanoyl-CoA into pentanoic acid is 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) and an acyl-CoA hydrolase (EC 3.1.2.20) as described herein above (step IXb as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism wherein the enzyme in (i) catalyzing the conversion of pentanoyl-CoA into pentanoic acid is a CoA transferase (EC 2.8.1.-), preferably a butyryl-CoA:acetate-CoA transferase (EC 2.8.3.8) as described herein above (step IXc as shown in Figure 3).

In a further preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (i) are an enzyme catalyzing the enzymatic conversion of pentanoyl-CoA into pentanoyl phosphate as described herein above (step IXa as shown in Figure 3) and an enzyme further catalyzing the enzymatic conversion of pentanoyl phosphate into pentanoic acid as described herein above (step Xa as shown in Figure 3).

In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the conversion of said pentanoyl-CoA into said pentanoyl phosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) as described herein above and the enzyme catalyzing the conversion of said pentanoyl phosphate into said pentanoic 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) as described herein above.

In a preferred embodiment, the above recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (i) catalyzing the conversion of 2-pentenoyl-CoA into said pentanoyl-CoA (step VIII as shown in Figure 3) is an oxidoreductase acting on the CH-CH group of donors with NAD(+) or NADP(+) as acceptors (EC 1.3.1.-), preferably an NADPH dependent trans-2-enoyl- CoA reductase (EC 1.3.1.38), a crotonyl-CoA reductase (EC 1.3.1.86), an NADPH- dependent acrylyl-CoA reductase (EC 1.3.1.84) or an NAD dependent trans-2-enoyl- CoA reductase (EC 1.3.1.44) as described herein above.

In a further preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme in (ii) catalyzing the enzymatic conversion of pentanoic acid into 1-butene (step XII as shown in Figure 3) is a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase as described herein above. 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 3-hydroxypentanoyl-CoA into 2-pentenoyl- CoA as defined above (step VII as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-hydroxypentanoyl-CoA into 2-pentenoyl-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) as described herein above (step VII as shown in Figure 3).

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 3-oxopentanoyl-CoA into 3-hydroxypentanoyl- CoA as described herein above (step VI as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-oxopentanoyl-CoA into said 3-hydroxypentanoyl-CoA is a 3-hydroxyacyl-CoA dehydrogenase (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 described herein above (step VI as shown in Figure 3).

In a further aspect, the above recombinant organism or microorganism is a recombinant organism or microorganism which further expresses an enzyme catalyzing the enzymatic condensation of propionyl-CoA and acetyl-CoA into said 3- oxopentanoyl-CoA as described herein above (step V as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic condensation of propionyl-CoA and acetyl-CoA into said 3-oxopentanoyl- CoA is an acetyl-CoA C-acyltransferase (EC 2.3.1.16) as described herein above (step V as shown in Figure 3).

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 said propionyl-CoA as described herein above (step IV as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of said acrylyl-CoA into said propionyl-CoA is an enoyl-CoA reductase (EC 1.3.1.-), preferably an acrylyl-CoA reductase (EC 1.3.1.95) as described herein above (step IV as shown in Figure 3).

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 3-hydroxypropionyl-CoA into said acrylyl-CoA as described herein above (step III as shown in Figure 3).

In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-hydroxypropionyl-CoA into said acrylyl-CoA is a 3- hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase (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 a enoyl-CoA hydratase (EC 4.2.1.17) as described herein above (step III as shown in Figure 3).

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 3-hydroxypropionaldehyde into said 3- hydroxypropionyl-CoA as described herein above (step II as shown in Figure 3). In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionyl-CoA is a CoA-acylating aldehyde dehydrogenase (EC 1.2.1.-), preferably a CoA-acylating propionaldehyde dehydrogenase (EC 1.2.1.87) or an acetaldehyde dehydrogenase (EC 1.2.1.10) as described herein above (step II as shown in Figure 3).

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 glycerol into said 3-hydroxypropionaldehyde as described herein above (step I as shown in Figure 3). In a preferred embodiment, the recombinant organism or microorganism is a recombinant organism or microorganism, wherein the enzyme catalyzing the enzymatic conversion of glycerol into said 3-hydroxypropionaldehyde is a cobalamine (B12 vitamin)-dependent or B12-indepentent/radical-S-adenosyl methionine- dependent) glycerol dehydratase (EC 4.2.1.30) as described herein above (step I as shown in Figure 3).

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 1-butene.

Moreover, the present invention also relates to the use of one or more enzymes as defined above for the production of 1-butene.

As regards the preferred embodiments of the enzymes, recombinant organisms and microorganisms applied in the uses for the production of 1-butene 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 a recombinant organism and microorganism which expresses enzymes catalyzing the enzymatic conversion of 2-pentenoyl-CoA into pentanoic acid via 2-pentenoic acid and enzymes catalyzing the enzymatic conversion of pentanoic acid into 1-butene as defined above, preferably a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase as described above for the production of 1-butene.

In another preferred embodiment, the present invention relates to the use of an enzyme catalyzing the enzymatic conversion of 2-pentenoyl-CoA into pentanoic acid via 2-pentenoic acid and enzymes catalyzing the enzymatic conversion of pentanoic acid into 1-butene as defined above, preferably a cytochrome P450 fatty acid decarboxylase or a non-heme iron oxygenase as described above for the production of 1-butene. 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 3);

(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 3);

(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-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) or a enoyl-CoA hydratase (EC 4.2.1.17) (step III as shown in Figure 3); 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 IV as shown in Figure 3).

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-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116), the 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) or the 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.

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 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. shows a GC chromatogram obtained for the enzyme-catalyzed oxidative decarboxylation of pentanoic acid with the cytochrome P450 from Jeotgalicoccus sp (Uniprot Accession number: E9NSU2) as outlined in Example 2. shows chromatograms of 1-butene produced by E.coli strain, expressed cytochrome P450 from Jeotgalicoccus sp, as outlined in Example 3.

Figure 3: shows an artificial metabolic pathway for 1-butene production from glycerol via propionyl-CoA, 2-pentenoyl-CoA, 2-pentenoic acid and pentanoic acid or an alternative route for 1-butene production from glycerol via propionyl-CoA, 2-pentenoyl-CoA, pentanoyl-CoA and pentanoic acid.

Figure 4: Schematic reactions for the alternative conversions of 2-pentenoyl-CoA into 2-pentenoic acid.

Figure 5: Schematic reaction for the enzymatic conversion of 2-pentenoic acid into pentanoic acid.

Figure 6: Schematic reaction for the enzymatic conversion of pentanoic acid into

1-butene. Figure 7: Schematic reaction for the enzymatic conversion of 3- hydroxypentanoyl-CoA into 2-pentenoyl-CoA.

Figure 8: Schematic reaction for the conversion of 3-oxopentanoyl-CoA into 3- hydroxypentanoyl-CoA. The reaction involves the formation of a chiral carbon bearing hydroxyl group (indicated with an (*)).

Figure 9: Schematic reaction for the enzymatic condensation of propionyl-CoA and acetyl-CoA into 3-oxopentanoyl-CoA.

Figure 10: Schematic reaction of the enzymatic conversion of acrylyl-CoA into propionyl-CoA.

Figure 11 : Schematic reaction of 3-hydroxypropionyl-CoA into acrylyl-CoA.

Figure 12: Schematic reaction of the conversion of 3-hydroxypropionaldehyde into

3-hydroxypropionyl-CoA using either NAD or NADPH as a reducing cofactor.

Figure 13: Schematic reaction of the conversion of glycerol into 3- hydroxypropionaldehyde.

Figure 14: Schematic reaction for the conversion of 2-pentenoyl-CoA into pentanoyl-CoA.

Figure 15: Schematic reaction for the conversion of pentanoyl-CoA into pentanoic acid via pentanoyl phosphate.

Figure 16: Schematic reaction for the conversion of pentanoyl-CoA into pentanoic acid.

Figure 17: Schematic reaction for the conversion of pentanoyl-CoA into pentanoic acid. Figure 18: shows a GC chromatogram obtained for the enzyme-catalyzed oxidative decarboxylation of pentanoic acid with the cytochrome P450 from Staphylococcus aureus C0673 (Uniprot Accession number: A0A033V973) as outlined in Example 5.

Figure 19: shows a metabolic pathway for the conversion of acetyl-CoA and propionyl-CoA into pentanoic acid via pentanoyl-CoA as exemplified in Example 6.

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 {aka flavodoxin reductase) from E.coli (Uniprot Accession number: P28861 ) was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection). The 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). Flavodoxin reductase 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 expression of recombinant cytochrome P450 fatty acid decarboxylases

The sequences of the studied enzymes inferred from the genome of prokaryotic organisms were 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 genes thus synthesized were 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, 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 pentanoic acid into 1-butene 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 pentanoic 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 pentanoic acid

0.2 mg/ml purified flavodoxin reductase from E.coli

Assays were started by adding 30μΙ of cell lysate containing the recombinant P450 fatty acid decarboxylase from Jeotgalicoccus sp. (total volume 300 μΙ)

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 1-butene 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 a 1-butene standard. Under these GC conditions, the retention time of 1-butene was 2.12 min. A significant production of 1-butene from pentanoic acid was observed in the assay, contained cytochrome P450 fatty acid decarboxylase and redox partners (Table 1, Figure 1).

Table 1

Assay 1-butene peak area, arbitrary units

Enzymatic assay 3320

Control assay without cytochrome P450 fatty acid 0.5

decarboxylase

Control assay without flavodoxine reductase 33

Control assay without cytochrome P450 fatty acid 4 decarboxylase and without flavodoxine reductase

Control assay without NADPH 14 Example 3: In vivo oxidative decarboxylation of pentanoic acid into 1-butene catalyzed by cytochrome P450 fatty acid decarboxylase from Jeotgalicoccus sp.

BL21(DE3) competent cells were transformed with pET-25b(+) expression vector, harboring the gene of cytochrome P450 fatty acid decarboxylase and plated out onto LB agar plates supplemented with ampicillin (100 pg/ml).

BL21(DE3) strain transformed with empty pET-25b(+) vector was used as a negative control in the subsequent assays (control strain). Plates were incubated overnight at 30°C. Single transformants 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 (2005), local citation), supplemented with 0.5 mM 5-aminolevulinic acid. The cultures were grown for 20 hours at 30°C and 160 rpm shaking.

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 pentanoic acid. These cultures were then incubated in 160 ml bottles, sealed with a screw cap, at 30°C with shaking for 22 h. The pH value of the cultures was adjusted to 8.5 after 8 hours of incubation by using 30 % NH ₄ OH.

After an incubation period, the 1-butene 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.

Figure 2 shows that 1-butene was produced with strain expressing P450 fatty acid decarboxylase. These results clearly indicate that a production of 1-butene from pentanoic acid can be achieved in vivo by E.coli strain expressing cytochrome P450 fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456. Example 4: Cloning and overexpression of recombinant cytochrome P450 fatty acid decarboxylase 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 pentanoic acid into -butene 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 pentanoic acid was prepared in water and adjusted to pH 7.0 with 0 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 pentanoic 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 2).

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 1-butene 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 a 1-butene standard. Under these GC conditions, the retention time of 1-butene was 2.22 min. A significant production of 1-butene from pentanoic acid was observed in the assay, contained cytochrome P450 and redox partners (Table 2 and Figure 18).

Table 2

Example 6: Microorganism for the production of pentanoic acid from acetyl- CoA and exogenous propionic acid

This example shows the production of pentanoic acid by a recombinant E. coli expressing several exogenous genes.

Like most organisms, E. coli converts glucose to acetyl-CoA. Furthermore, it is known that E. coli is able to biosynthesize propionyl-CoA from propionic acid by using endogenous acetate kinase (ackA) and phosphotransacetylase (pta) (Microbial cell factory 9 (2010), 96). The enzymes used in this study to convert acetyl-CoA and propionyl-CoA into pentanoic acid (also known as valeric acid), according to the metabolic pathway shown in Figure 19, are summarized in Table 3.

Table 3

Expression of pentanoic acid biosynthetic pathway in E. coli

The modified version of pUC18 (New England Biolabs), containing a modified Multiple Cloning Site (pUC18 MCS) (WO 2013/007786), was used as an expression vector. A terminator sequence was inserted into pUC18 MCS between the HindlW and Nar\ restriction sites and the resulting vector was termed pGBE 992. The corresponding genes were codon optimized for expression in E.coli and synthesized by GeneArt® (Life Technologies).

The master plasmid pGB2281 (commercially provided by GeneArt) contained the 3- hydroxybutyryl-CoA dehydrogenase gene (hbd) and the 3-hydroxybutyryl-CoA dehydratase gene (crt). pGB2281 was digested with the restriction enzymes Clal and Notl to create a .7 kbp product. The 1.7 kbp restriction fragment containing the hbd and crt gene was ligated into the modified pUC18 plasmid. The resulting recombinant plasmid pGB2368 was verified by sequencing. The master plasmid pGB3073 (commercially provided by GeneArt) contained the ptb and bukl genes. pGB3073 was digested with the restriction enzymes Kpnl and Xbal to create a 2 kbp product. The 2 kbp restriction fragment containing the ptb and bukl gene was ligated into the pGB2368 plasmid. The resulting recombinant plasmid pGBE3177 was verified by sequencing.

The master plasmid pGB3320 (commercially provided by GeneArt) contained the TDE_0597 gene coding for enoyl-CoA reductase from Treponema denticola. pGB3320 was digested with the restriction enzymes EcoRI and Kpnl to create a 1.2 kbp product. The 1.2 kbp restriction fragment containing the TDE-0597 gene was ligated into the pGB3177 plasmid. The resulting recombinant plasmid pGBE3361 was verified by sequencing.

The beta-ketothiolase (bktB) gene was PCR amplified from pGB3389 (master plasmid provided by GeneArt) using primers 7005 (SEQ ID NO:1 ) and 7006 (SEQ ID NO:2) shown in Table 4. A Pad restriction site at the 5' end of the PCR product was introduced. At the 3' end of the PCR product a C/al restriction site was introduced. The resulting 1.2 kbp PCR product and pGBE3361 were digested with the Pad and C/al restriction enzymes and then ligated together resulting in the pGBE3741 plasmid. The recombinant pGBE3741 plasmid was verified by sequencing.

Strain MG1655 E.coli was made electrocompetent. MG1655 electrocompetent cells were then transformed with the expression vector pGBE3741. An empty plasmid pUC18 was transformed as well 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 20h at 30°C and 160 rpm shaking.

A volume of cultures corresponding to OD ₆ oo 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, loc. cit.) containing glucose (45 g/L), and MgS0 ₄ (1 mM). Then, the culture medium was supplemented with 200 mM propionic acid.

The cultures were then incubated in 160 ml bottles and sealed with a screw cap, at 30°C with shaking for 48 hours. The pH value of the cultures was adjusted to 8.5 twice per day using 30 % NH ₄ OH.

At the end of incubation 2 ml of culture mediums was removed and centrifuged at 4°C, 10,000 rpm for 5 min. The supernatants were filtered through a 0.22 pm filter and diluted at 1/5 with H ₂ 0. The production of pentanoic acid was then analyzed.

Analysis of pentanoic acid production

The amount of pentanoic acid produced was measured using a HPLC-based procedure. HPLC analysis was performed using a 1260 Inifinity LC System (Agilent), equipped with column heating module, and refracto meter. 5 μΙ of samples were separated using a Hi-Plex column (100 x 7.7 mm, 8 μιτι particle size) (Agilent) heated at 30°C.The mobile phase consisted of aqueous sulfuric acid (8.4mM), mobile phase flow rate was 1.0 ml/min. Commercial pentanoic acid (Sigma-Aldrich) was used as reference. Retention time of pentanoic acid under these conditions was 3.96 min. Up to 32 mM pentanoic acid was produced in these shake-flask experiments by engineered E.coliexpressing the genes of pentanoic acid biosynthetic pathway. No pentanoic acid production was observed with the control strain, contained empty vector.