Improved means and methods for producing isobutylene from acetyl-CoA

Abstract

A recombinant organism or microorganism, (A) wherein in the organism or microorganism: (i) acetyl-CoA is enzymatically converted to acetoacetyl-CoA, (ii) acetoacetyl-CoA is enzymatically converted to 3-hydroxy-3-methylglutaryl-CoA, (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted to 3-methylglutaconyl-CoA, (iv) 3-methylglutaconyl-CoA is enzymatically converted to 3-methylcrotonyl-CoA, and (v) wherein the 3-methylcrotonyl-CoA is converted to isobutylene by: (a) enzymatic conversion of 3-methylcrotonyl-CoA To 3-methylcrotonic acid, which is then further enzymatically converted into the isobutylene; or (b) 3-methylcrotonyl-CoA is enzymatically converted into 3-hydroxy-3-methylbutyryl-CoA, which is then further enzymatically converted into 3-hydroxy-3-methylbutyric acid, which is then further enzymatically converted into 3-phosphino-3-methylbutyric acid, which is then further enzymatically converted into the isobutylene; (B) wherein the recombinant organism or microorganism has an increased coenzyme A (CoA) pool exceeding the organism or microorganism from which it is derived, due to: (i) increased pantothenic acid uptake; and / or (ii) increased conversion of pantothenic acid to CoA. In addition, the use of such recombinant organisms or microorganisms for the production of isobutylene is described. In addition, a method for producing isobutylene by culturing such a recombinant organism or microorganism under suitable conditions in a suitable culture medium is described.

Description

[0001] The present invention relates to a recombinant organism or microorganism capable of enzymatically converting acetyl-CoA into isobutylene, (A) wherein in said organism or microorganism: (i) acetyl-CoA is enzymatically converted into acetoacetyl-CoA, (ii) acetoacetyl-CoA is enzymatically converted into 3-hydroxy-3-methylglutaryl-CoA, (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted into 3-methylglutaconyl-CoA, (iv) 3-methylglutaconyl-CoA is enzymatically converted into 3-methylcrotonyl-CoA, and (v) wherein said 3-methylcrotonyl-CoA is converted into isobutylene by: (a) converting 3-methyl (B) wherein the recombinant organism or microorganism has an increased coenzyme A (CoA) pool over the organism or microorganism from which it is derived, due to: (i) increased pantothenate uptake; and / or (ii) increased conversion of pantothenate to CoA. In addition, the present invention relates to the use of such a recombinant organism or microorganism for the production of isobutylene. In addition, the present invention relates to a method for producing isobutylene by culturing such a recombinant organism or microorganism in a suitable culture medium under suitable conditions.

[0002] At present, a large number of compounds are derived from petrochemical products. Olefins (such as ethylene, propylene, different butenes or pentenes, for example) are used in the plastics industry, for example for the production of polypropylene or polyethylene, as well as in other areas of the chemical industry and in the fuel sector. Butenes exist in four forms, one of which is isobutene (also known as isobutylene), which enters the composition of methyl tert-butyl ether (MTBE), an antiknock additive for automobile fuels. Isobutene can also be used to produce isooctene, which in turn can be reduced to isooctane (2,2,4-trimethylpentane); the very high octane number of isooctane makes it an optimal fuel for so-called "gasoline" engines. Olefins (such as isobutene) are currently produced by catalytic cracking of petroleum products (or in the case of hexene, by derivatives of the Fischer-Tropsch process from coal or natural gas). Therefore, the production costs are closely related to the price of oil. In addition, catalytic cracking is sometimes associated with considerable technical difficulties, which increase the process complexity and production costs.

[0003] In the context of sustainable industrial operations that are coordinated with geochemical cycles, the production of olefins such as isobutylene by biological pathways is needed. The first generation of biofuels includes the fermentative production of ethanol, as fermentation and distillation processes already exist in the food processing industry. The production of second-generation biofuels is at an exploratory stage and includes in particular the production of long-chain alcohols (butanol and pentanol), terpenes, linear alkanes, and fatty acids. Two recent reviews provide a general overview of research in this area: Ladygina et al. (Process Biochemistry 41 (2006), 1001) and Wackett (Current Opinions in Chemical Biology 21 (2008), 187).

[0004] The conversion of isovaleric acid to isobutylene by the yeast Rhodotorula minuta has been described (Fujii et al. (Appl. Environ. Microbiol. 54 (1988), 583)), but the efficiency of the reaction is far from allowing industrial application. Fukuda et al. (BBRC 201 (1994), 516) elucidated the reaction mechanism and involved a cytochrome P450 enzyme, which oxidizes the iron-based Fe by reducing the oxidized iron. V =O decarboxylates isovaleric acid. Large-scale biosynthesis of isobutene by this pathway seems very disadvantageous because it requires the synthesis and degradation of one leucine molecule to form one isobutene molecule. In addition, the enzyme catalyzing the reaction uses heme as a cofactor and is not very suitable for recombinant expression in bacteria and improvement of enzyme parameters. For all these reasons, this pathway seems unlikely to serve as a basis for industrial development. Other microorganisms have been described as being barely able to produce isobutene naturally from isovaleric acid; the yields obtained are even lower than those obtained with red yeast (Fukuda et al. (Agric. Biol. Chem. 48 (1984), 1679)).

[0005] Gogerty et al. (Appl. Environm. Microbiol. 76 (2010), 8004-8010) and van Leeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387) describe the production of isobutene from acetoacetyl-CoA by enzymatic conversion, where the last step of the proposed pathway is the conversion of 3-hydroxy-3-methylbutyrate (also known as 3-hydroxyisovalerate (HIV)) by using mevalonate diphosphate decarboxylase.

[0006] This reaction for producing isobutene from 3-hydroxy-3-methylbutyric acid is also described in WO 2010 / 001078, which generally describes methods for producing olefins by biological processes, in particular the production of terminal olefins (in particular propylene, ethylene, 1-butene, isobutene or isopentene) from molecules of the 3-hydroxyalkanoate type.

[0007] WO2012 / 052427 also describes a method for producing olefins by a biological process, in particular a method for producing olefins (such as propylene, ethylene, 1-butene, isobutylene or isopentene) from molecules of the 3-hydroxyalkanoate type. In this context, WO2012 / 052427 also describes a reaction for producing isobutylene from 3-hydroxy-3-methylbutyric acid.

[0008] WO 2016 / 042012 describes a method for producing the 3-hydroxy-3-methylbutyric acid. In particular, WO 2016 / 042012 describes a method for producing 3-hydroxy-3-methylbutyric acid, comprising the step of enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid and further enzymatically converting the 3-methylcrotonic acid thus produced into 3-hydroxy-3-methylbutyric acid.

[0009] In Gogerty et al. (loc. cit.) and van Leeuwen et al. (loc. cit.), it is proposed to produce 3-hydroxy-3-methylbutyrate via the conversion of 3-methylcrotonyl-CoA to 3-hydroxy-3-methylbutyryl-CoA. In order to further improve the efficiency and variability of the process for producing isobutylene from renewable resources, alternative pathways for providing isobutylene and its precursors are developed by providing an isobutylene production process comprising enzymatic conversion of 3-methylcrotonate (also known as 3-methyl-2-butenoate, 3,3-dimethylacrylic acid or isopentenoate) to isobutylene.

[0010] In particular, in WO 2017 / 085167, a method for producing isobutene has been described, comprising enzymatically converting 3-methylcrotonate to isobutene, wherein the enzymatic conversion of 3-methylcrotonate to isobutene is achieved by using an FMN-dependent decarboxylase associated with an FMN prenyltransferase, wherein the FMN prenyltransferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) to a flavin-derived cofactor using dimethylallyl phosphate (DMAP), and these enzymes have been artificially used in pathways that ultimately lead to the production of isobutene. In addition, in WO 2017 / 085167, a method has been described, wherein such a method further comprises (a) providing 3-methylcrotonate by enzymatically converting 3-methylcrotonyl-CoA to 3-methylcrotonate, or (b) providing 3-methylcrotonate by enzymatically converting 3-hydroxyisovalerate (HIV) to 3-methylcrotonate.

[0011] WO 2017 / 085167 also describes that the method developed for producing isobutene from 3-methylcrotonyl-CoA via 3-methylcrotonic acid or from 3-hydroxyisovaleric acid (HIV) via 3-methylcrotonic acid can be embedded in the pathway for producing isobutene starting from acetyl-CoA, which is a central component and an important key molecule in metabolism used in many biochemical reactions. The corresponding reaction diagram is shown in Figure 1 shown.

[0012] In WO 2018 / 206262, the enzymatic conversion of 3-methylcrotonate to isobutylene is described by using an FMN-dependent decarboxylase associated with FMN prenyltransferase when dimethylallyl pyrophosphate (DMAPP) is used instead of DMAP.

[0013] In addition, WO 2018 / 206262 describes the enzymatic conversion of 3-methylcrotonate to isobutene by using an FMN-dependent decarboxylase associated with an FMN prenyltransferase, which catalyzes the prenylation of a flavin cofactor (FMN or FAD) using dimethylallyl phosphate (DMAP) and / or dimethylallyl pyrophosphate (DMAPP) to a flavin-derived cofactor, which is a key step in the above-mentioned entire metabolic pathway from acetyl-CoA to isobutene. It has been found that the availability of dimethylallyl phosphate (DMAP) and / or dimethylallyl pyrophosphate (DMAPP) and the availability of the flavin cofactor FMN are limiting factors in this critical step, and in WO 2018 / 206262, an improved method is described to ensure efficient biosynthesis of the isoprenylated flavin cofactor (FMN or FAD) by increasing the pool / amount of dimethylallyl phosphate (DMAP) and / or dimethylallyl pyrophosphate (DMAPP).

[0014] Although, as mentioned above, various methods for producing isobutene by enzymatic conversion in biological systems have been described in the prior art, allowing the use of renewable resources as raw materials, there is still a need to improve the efficiency and effectiveness of these methods in order to increase the yield and make them commercially attractive.

[0015] The present invention meets this need by providing a recombinant organism or microorganism capable of enzymatically converting acetyl-CoA to isobutylene.

[0016] (A) wherein in said organism or microorganism:

[0017] (i) enzymatic conversion of acetyl-CoA into acetoacetyl-CoA,

[0018] (ii) enzymatic conversion of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA,

[0019] (iii) enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA,

[0020] (iv) enzymatic conversion of 3-methylglutaconyl-CoA to 3-methylcrotonyl-CoA, and

[0021] (v) wherein the 3-methylcrotonyl-CoA is converted to isobutylene by:

[0022] (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid, which is then further enzymatically converted into said isobutylene; or

[0023] (b) Enzymatic conversion of 3-methylcrotonyl-CoA to 3-hydroxy-3-methylbutyryl-

[0024] CoA, which is then further enzymatically converted into 3-hydroxy-3-methylbutyrate, which is then further enzymatically converted into 3-phosphinoyl-3-methylbutyrate, which is then further enzymatically converted into said isobutylene;

[0025] (B) wherein the recombinant organism or microorganism has an increased coenzyme A (CoA) pool over the organism or microorganism from which it was derived due to:

[0026] (i) increased pantothenic acid uptake; and / or

[0027] (ii) Increased conversion of pantothenate to CoA.

[0028] Furthermore, the present invention provides the use of a recombinant organism or microorganism as defined above for producing isobutene.

[0029] Furthermore, the present invention provides a method for producing isobutene by culturing the recombinant organism or microorganism as defined above in a suitable culture medium under suitable conditions.

[0030] Finally, the present invention provides a method for producing isobutylene from acetyl-CoA in a recombinant organism or microorganism, comprising:

[0031] (A)(i) enzymatic conversion of acetyl-CoA into acetoacetyl-CoA,

[0032] (ii) enzymatically converting the produced acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA,

[0033] (iii) enzymatically converting the produced 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA,

[0034] (iv) enzymatically converting the produced 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA, and

[0035] (v) converting the produced 3-methylcrotonyl-CoA into isobutylene by:

[0036] (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid, which is then further enzymatically converted into said isobutylene; or

[0037] (b) Enzymatic conversion of 3-methylcrotonyl-CoA to 3-hydroxy-3-methylbutyryl-

[0038] CoA, which is then further enzymatically converted into 3-hydroxy-3-methylbutyrate, which is then further enzymatically converted into 3-phosphinoyl-3-methylbutyrate, which is then further enzymatically converted into said isobutylene;

[0039] (B) wherein the method further comprises:

[0040] From CoA, acetyl-CoA is provided enzymatically,

[0041] (C) wherein the method further comprises providing coenzyme A (CoA) by culturing the recombinant organism or microorganism in a suitable medium under suitable conditions, wherein the recombinant organism or microorganism has increased coenzyme A (CoA) over the organism or microorganism from which it is derived.

[0042] Pool, this is due to:

[0043] (i) increased pantothenic acid uptake; and / or

[0044] (ii) Increased conversion of pantothenate to CoA.

[0045] The present invention is based on the concept of increasing isobutene production by providing and maintaining a high acetyl-CoA pool in cells for isobutene production, wherein the high acetyl-CoA pool is maintained by ensuring increased pantothenate uptake by the cells and / or increased conversion of pantothenate to CoA.

[0046] Acetyl-CoA is the acetylated form of coenzyme A (CoASH or CoA). The chemical structure of CoA is as follows Figure 2 As shown in . CoA consists of a β-mercaptoethylamine group and 3'-phosphorylated ADP linked to the vitamin pantothenic acid via an amide bond. In acetyl-CoA, the acetyl group of acetyl-CoA is linked to the sulfhydryl substituent of the β-mercaptoethylamine group. This thioester bond is a "high energy" bond and is particularly reactive.

[0047] The recombinant organism or microorganism and the method according to the present invention are particularly useful for large-scale isobutene production in vitro or in vivo, in particular for commercial production. Therefore, the present invention relates to a method for large-scale production, in particular for commercial production of isobutene, wherein the method comprises the steps as described above.

[0048] Enzymatic conversion of acetyl-CoA to isobutylene

[0049] As mentioned above, methods for producing isobutylene from 3-methylcrotonyl-CoA via 3-methylcrotonic acid or from 3-hydroxyisovaleric acid (HIV) via 3-methylcrotonic acid have been described (see Figure 1 as well as Figure 3 Methods for utilizing these pathways and enzymatic transformations as well as recombinant organisms and microorganisms have been described in particular in WO 2017 / 085167 and WO

[0050] 2018 / 206262.

[0051] In addition, a process for producing isobutene from 3-methylcrotonyl-CoA via 3-hydroxy-3-methylbutyryl-CoA, which is then further enzymatically converted to 3-hydroxy-3-methylbutyrate, which is then further enzymatically converted to 3-phosphinoyl-3-methylbutyrate, which is then further enzymatically converted to the isobutene has been described (see Figure 3 In addition, for these pathways, it has been described that they can be inserted into the pathway for producing isobutene from acetyl-CoA. Methods and recombinant organisms and microorganisms using these pathways and enzymatic conversions have been described in particular in WO2010 / 001078, WO2012 / 052427 and WO 2016 / 042012.

[0052] Therefore, in the prior art, corresponding recombinant organisms or microorganisms are also described which are capable of enzymatically converting acetyl-CoA into isobutene, which utilize the above-mentioned pathway. Other possible pathways for the enzymatic conversion of acetyl-CoA into isobutene via different possible pathways are shown schematically in Figure 1 and Figure 3 middle. Figure 3 The main pathways relevant to the present invention are shown. However, as indicated above, other possible pathways exist and the present invention also relates to these pathways for the enzymatic conversion of acetyl-CoA to isobutene.

[0053] In the following, the prior art WO 2017 / 085167, WO

[0054] 2018 / 206262, WO2010 / 001078, WO2012 / 052427 and WO 2016 / 042012, respectively, and as described in Figure 1 and Figure 3 The main reactions of a single enzymatic transformation are shown schematically.

[0055] However, the present invention is not limited to these main reactions, but also relates to all other pathways for the individual steps of converting acetyl-CoA into isobutylene described in prior art documents WO 2017 / 085167, WO 2018 / 206262, WO 2010 / 001078, WO 2012 / 052427 and WO 2016 / 042012. The disclosures of these documents, in particular the preferred embodiments of the enzymes for the individual conversions of the pathways described therein, are hereby incorporated by reference in their entirety. Therefore, in a preferred embodiment, an enzyme selected from the preferred embodiments described in these prior art documents is preferably used, and combined with respective enzyme conversions. Therefore, this also applies to the enzymatic conversion of the present invention described below, as described in WO 2017 / 085167, WO 2018 / 206262, WO 2010 / 001078, WO 2012 / 052427 and WO 2016 / 042012, respectively.

[0056] Acetyl-CoA is enzymatically converted to acetoacetyl-CoA

[0057] According to the present invention, the conversion of acetyl-CoA into acetoacetyl-CoA can be achieved by different approaches. One possibility is to first convert acetyl-CoA into malonyl-CoA (e.g. Figure 1 Then, the malonyl-CoA and acetyl-CoA are further condensed to form acetoacetyl-CoA (eg, Figure 1 Another possibility is to directly condense two molecules of acetyl-CoA to acetoacetyl-CoA in a single enzymatic reaction (e.g. Figure 1 ), step XIII).

[0058] The acetyl-CoA is preferably converted to malonyl-CoA using an acetyl-CoA carboxylase (EC 6.4.1.2) (e.g. Figure 1 This naturally occurring reaction uses ATP to fix CO2 onto acetyl-CoA, thereby producing malonyl-CoA.

[0059] In addition, acetoacetyl-CoA and acetyl-CoA are preferably synthesized by condensation of acetoacetyl-CoA using acetoacetyl-CoA synthase (EC 2.3.1.194) (e.g. Figure 1 This is a spontaneous reaction and condenses malonyl-CoA and acetyl-CoA in a decarboxylation reaction.

[0060] Alternatively, the enzymatic conversion of acetyl-CoA to the acetoacetyl-CoA consists of a single enzymatic reaction in which acetyl-CoA is directly converted to acetoacetyl-CoA by enzymatic condensation of two molecules of acetyl-CoA. Preferably, this enzymatic conversion is achieved by using acetyl-CoA acetyltransferase (EC 2.3.1.9). This reaction is a naturally occurring reaction (e.g. Figure 1 ), step XIII).

[0061] Acetoacetyl-CoA is enzymatically converted to 3-hydroxy-3-methylglutaryl-CoA

[0062] The enzymatic conversion of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA is the enzymatic condensation of acetoacetyl-CoA and acetyl-CoA to synthesize the 3-hydroxy-3-methylglutaryl-CoA (see Figure 1 Step IX).

[0063] This condensation preferably uses 3-hydroxy-3-methylglutaryl-CoA synthase (also referred to as HMG-CoA synthase). HMG-CoA synthase is classified in EC 2.3.3.10 (previously, HMG-CoA synthase was classified as EC 4.1.3.5, but has been transferred to EC 2.3.3.10). The term "HMG-CoA synthase" refers to any enzyme that can catalyze the reaction in which acetyl-CoA and acetoacetyl-CoA condense to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). HMG-CoA is part of the mevalonate pathway. Several pathways for isopentenyl pyrophosphate (IPP) synthesis have been identified, i.e., the mevalonate pathway and the 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate (MEP / DOXP) pathway. HMG-CoA synthase catalyzes the bio-Claisen condensation of acetyl-CoA with acetoacetyl-CoA and is a member of the acyl-condensing enzyme superfamily, which includes β-ketothiolases, fatty acid synthases (β-ketoacyl carrier protein synthases), and polyketide synthases.

[0064] 3-Hydroxy-3-methylglutaryl-CoA is enzymatically converted to 3-methylglutaconyl-CoA

[0065] The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA to 3-methylglutaconyl-CoA is a naturally occurring enzymatic dehydration reaction and is catalyzed, for example, by an enzyme classified as 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18). Thus, the enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA to 3-methylglutaconyl-CoA preferably uses 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18) (e.g. Figure 1 ) as shown in step VIII.

[0066] The conversion of 3-hydroxy-3-methylglutaryl-CoA to 3-methylglutaconyl-CoA can also be achieved by utilizing 3-hydroxy-3-methylglutaryl-CoA dehydratase activity, which has been identified, for example, in Myxococcus xanthus and encoded by the liuC gene (Li et al., Angew. Chem. Int., ed., 52 (2013), 1304-1308). The 3-hydroxy-3-methylglutaryl-CoA dehydratase from Myxococcus xanthus has the Uniprot accession number Q1D5Y4.

[0067] The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA to 3-methylglutaconyl-CoA can also be achieved by utilizing 3-hydroxyacyl-CoA dehydratase or enol-CoA hydratase. 3-hydroxyacyl-CoA dehydratase and enol-CoA hydratase catalyze the same reaction, wherein the name of one enzyme indicates one direction of the corresponding reaction, while the other name indicates the reverse reaction. Since the reaction is reversible, both enzyme names can be used. 3-hydroxyacyl-CoA dehydratase and enoyl-CoA hydratase belong to enzymes classified as EC4.2.1.-.

[0068] 3-Methylglutaconyl-CoA is enzymatically converted to 3-methylcrotonyl-CoA

[0069] The conversion of 3-methylglutaconyl-CoA to 3-methylcrotonyl-CoA can be catalyzed by different enzymes, for example, using (i) methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) geranoyl-CoA carboxylase (EC 6.4.1.5) (e.g. Figure 1 as shown in step VII).

[0070] In another preferred embodiment, the conversion of 3-methylglutaconyl-CoA to 3-methylcrotonyl-CoA by decarboxylation is catalyzed by a 3-methylglutaconyl-CoA decarboxylase (e.g., 3-methylglutaconyl-CoA decarboxylase of Myxococcus xanthus encoded by the liuB gene). This gene encodes an enzyme with two subunits, AibA and AibB (Li et al., Angew. Chem. Int., ed., 52 (2013), 1304-1308).

[0071] Enzymatic conversion of 3-methylcrotonyl-CoA to isobutylene via 3-methylcrotonate

[0072] The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid can be achieved, for example, by different pathways, for example, by the following and Figure 1 As shown in Figure 1 The invention can be achieved by three alternative enzymatic pathways, step VIa, step VIb or step VIc), as shown in FIG.

[0073] Thus, 3-methylcrotonyl-CoA can be enzymatically converted to 3-methylcrotonate by

[0074] (a) A single enzymatic reaction in which 3-methylcrotonyl-CoA is directly converted to 3-methylcrotonate, preferably using a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1), an acetate CoA transferase (EC 2.8.3.8) or a succinyl-CoA:acetate-CoA transferase (EC 2.8.3.18) (e.g. Figure 1Step VIa) shown in FIG.

[0075] (b) a single enzymatic reaction in which 3-methylcrotonyl-CoA is directly converted to 3-methylcrotonate, preferably using a thioester hydrolase (EC 3.1.2.-), preferably an acetyl-CoA hydrolase (EC

[0076] 3.1.2.1), ADP-dependent short-chain acyl-CoA hydrolase (EC 3.1.2.18) or acyl-CoA hydrolase (EC 3.1.2.20) (e.g. Figure 1 or

[0077] (c) a double enzymatic step comprising

[0078] (i) first enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and

[0079] (ii) then enzymatically converting the 3-methylcrotonyl phosphate thus obtained into the 3-methylcrotonic acid (e.g. Figure 1 100 ] Step VIc) shown in FIG.

[0080] Regarding (c), the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by a double enzymatic step comprising (i) first enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and (ii) then enzymatically converting the 3-methylcrotonyl phosphate thus obtained into said 3-methylcrotonic acid.

[0081] For example, conversion of 3-methylcrotonyl-CoA to 3-methylcrotonyl phosphate can be achieved by using a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).

[0082] For example, the conversion of 3-methyl crotonyl phosphate to 3-methyl crotonic acid can be achieved by utilizing an enzyme classified as EC 2.7.2.- (i.e., a phosphotransferase). The enzyme utilizes a carboxyl group as an acceptor. Therefore, the conversion of 3-methyl crotonyl phosphate to 3-methyl phosphate can be achieved, for example, using an enzyme (EC 2.7.2.-) that uses a carboxyl group as an acceptor. In a preferred embodiment, the conversion of 3-methyl crotonyl phosphate to 3-methyl crotonic acid is achieved by using propionate kinase (EC 2.7.2.15), acetate kinase (EC 2.7.2.1), butyrate kinase (EC 2.7.2.7) or branched-chain fatty acid kinase (EC 2.7.2.14).

[0083] As described above, conversion of 3-methylcrotonyl-CoA to 3-methylcrotonic acid can be achieved by two alternative transformations, wherein 3-methylcrotonyl is directly converted to 3-methylcrotonic acid.

[0084] Preferably, in one embodiment, 3-methylcrotonyl-CoA is directly converted to 3-methylcrotonic acid by alkaline hydrolysis of the thioester of 3-methylcrotonyl-CoA to 3-methylcrotonic acid using an enzyme belonging to the thioester hydrolase family, hereinafter referred to as thioesterase (EC 3.1.2.-); e.g. Figure 1 Step VIb shown in FIG.

[0085] Thioesterases (TEs; also called thioester hydrolases) are enzymes classified as EC 3.1.2. Thioesterases are currently classified as EC 3.1.2.1 to EC 3.1.2.30, while unclassified / unclassified TEs are grouped as enzymes belonging to EC 3.1.2.-. Cantu et al. (Protein Science 19 (2010), 1281-1295) describe the existence of 23 thioesterase families, which are unrelated to each other with respect to primary structure. However, it is believed that all members of the same family have substantially the same tertiary structure. Thioesterases hydrolyze the thioester bond between the carbonyl group and the sulfur atom.

[0086] In a preferred embodiment, the thioesterase used according to the present invention for converting 3-methylcrotonyl-CoA to 3-methylcrotonate is selected from the group consisting of:

[0087] - Acetyl-CoA hydrolase (EC 3.1.2.1);

[0088] - Palmitoyl-CoA hydrolase (EC 3.1.2.2);

[0089] -3-Hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4);

[0090] -oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);

[0091] - ADP-dependent short-chain acyl-CoA hydrolase (EC 3.1.2.18);

[0092] - ADP-dependent medium-chain acyl-CoA hydrolase (EC 3.1.2.19);

[0093] -1,4-dihydro-2-naphthoyl-CoA hydrolase (EC 3.1.2.28); and

[0094] -Acyl-CoA hydrolase (EC 3.1.2.20).

[0095] In a more preferred embodiment, the thioesterase / thioester hydrolase (EC 3.1.2.-) used according to the present invention is acetyl-CoA hydrolase (EC 3.1.2.1), ADP-dependent short-chain acyl-CoA hydrolase (EC 3.1.2.18), 1,4-dihydro-2-naphthoyl-CoA hydrolase (EC 3.1.2.28) and acyl-CoA hydrolase (EC 3.1.2.20).

[0096] In an alternative embodiment, 3-methylcrotonyl-CoA is directly converted to 3-methylcrotonate, preferably by utilizing an enzyme belonging to the CoA transferase family (EC 2.8.3.-), which is capable of transferring the CoA group of 3-methylcrotonyl-CoA to a carboxylic acid (e.g. Figure 1 ) as shown in step VIa).

[0097] CoA-transferases are found in organisms from all lineages. Most CoA-transferases belong to two well-known enzyme families (hereinafter referred to as families I and II), and there is a third family that has been identified in the anaerobic metabolic pathways of bacteria. An overview describing the different families can be found in Heider (FEBS Letters 509 (2001), 345-349).

[0098] Preferably, the CoA-transferase used according to the invention for the direct conversion of 3-methylcrotonyl-CoA into 3-methylcrotonate is selected from the group consisting of:

[0099] - Propionate:acetate-CoA transferase (EC 2.8.3.1);

[0100] - acetate CoA-transferase (EC 2.8.3.8); and

[0101] -Butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

[0102] In a more preferred embodiment, the CoA transferase (EC 2.8.3-) is propionate:acetate-CoA transferase (EC 2.8.3.1), acetate CoA-transferase (EC 2.8.3.8) and succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18).

[0103] Alternative routes for providing 3-methylcrotonic acid

[0104] As shown above, acetyl-CoA can be enzymatically converted to acetoacetyl-CoA (e.g. Figure 1 acetoacetyl-CoA is enzymatically converted to 3-hydroxy-3-methylglutaryl-CoA ( Figure 1Step IX), 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted to 3-methylglutaconyl-CoA ( Figure 1 Step VIII), 3-methylglutaconyl-CoA is enzymatically converted to 3-methylcrotonyl-CoA ( Figure 1 3-methylcrotonate can be enzymatically provided from acetyl-CoA (which is then further enzymatically converted to isobutylene, as described in further detail below).

[0105] In an alternative route, according to the present invention, 3-methylcrotonate can be provided from acetyl-CoA by another possible pathway. In this pathway, acetyl-CoA is enzymatically converted to acetoacetyl-CoA as described above.

[0106] According to this alternative pathway, acetoacetyl-CoA is then enzymatically converted to acetoacetate ( Figure 1 Step Va or Vb), acetoacetate is further enzymatically converted into acetone ( Figure 1 Step IV), acetone is further enzymatically converted into 3-hydroxyisovaleric acid (HIV) ( Figure 1 Step III), which is then further enzymatically converted into the 3-methylcrotonic acid.

[0107] The individual enzymatic steps of this alternative pathway are described in more detail below.

[0108] Acetoacetyl-CoA is enzymatically converted to acetoacetate

[0109] The conversion of acetoacetyl-CoA to acetoacetate can be achieved by two different pathways. One possibility is to convert acetoacetyl-CoA to acetoacetate (e.g. Figure 1 In another more preferred aspect, the CoA group of acetoacetyl-CoA is transferred on the acetate, resulting in the formation of acetoacetate and acetyl-CoA (e.g. Figure 1 Step Vb) shown in FIG.

[0110] As described, in one aspect, the CoA thioester of acetoacetyl-CoA is hydrolyzed to obtain acetoacetate. According to this aspect of the invention, the enzymatic conversion of acetoacetyl-CoA to acetoacetate is achieved by preferably utilizing an acetoacetyl-CoA hydrolase (EC 3.1.2.11) that naturally catalyzes this reaction.

[0111] As described, in another more preferred possibility, the CoA group of acetoacetyl-CoA is transferred on acetate, resulting in the formation of acetoacetate and acetyl-CoA. According to this possibility of the invention, the enzymatic conversion of acetoacetyl-CoA into acetoacetate is achieved by preferably utilizing an enzyme that can transfer the CoA group of acetoacetyl-CoA on acetate.

[0112] Preferably, such an enzyme capable of transferring acetoacetyl-CoA on acetate belongs to the CoA transferase family (EC 2.8.3.-).

[0113] Thus, the present invention relates to a method for enzymatically converting acetoacetyl-CoA to acetoacetate by utilizing an enzyme capable of transferring the CoA group of acetoacetyl-CoA on acetate, preferably a CoA transferase (EC 2.8.3.-). Preferred examples of enzymes that catalyze the conversion of acetoacetyl-CoA to acetoacetate that can be used in the method of the present invention are enzymes classified as acetate CoA transferases (EC 2.8.3.8).

[0114] Enzymatic conversion of acetoacetate to acetone

[0115] The conversion of acetoacetate to acetone is shown in Figure 1 In step IV of . This reaction is a decarboxylation reaction and is a reaction that occurs naturally in organisms that are able to produce acetone, i.e. organisms of the genus Clostridia. According to the present invention, the conversion of acetoacetate into said acetone preferably utilizes acetoacetate decarboxylase (EC 4.1.1.4).

[0116] Enzymatic condensation of acetone and acetyl-CoA to 3-hydroxyisovalerate (HIV)

[0117] The condensation of acetone and acetyl-CoA to form 3-hydroxyisovaleric acid (HIV) is shown in FIG. Figure 1 This condensation preferably utilizes an enzyme that can catalyze the formation of a covalent bond between the carbon atom of the oxo (i.e., C=O) group of acetone and acetyl-CoA (particularly the methyl group of acetyl-CoA). According to this reaction scheme, the oxo group of acetone reacts as an electrophile, while the methyl group of acetyl-CoA reacts as a nucleophile.

[0118] Enzymes capable of enzymatically condensing acetone and acetyl-CoA to 3-hydroxyisovalerate (HIV) are known in the art and have for example been described in WO 2011 / 0322934.

[0119] Preferably, the enzyme used in the enzymatic condensation of acetone and acetyl-CoA to 3-hydroxyisovalerate (HIV) is an enzyme having HMG CoA synthase (EC 2.3.3.10) activity and / or a PksG protein and / or an enzyme having CC bond cleavage / condensation lyase (preferably classified as isopropylmalate synthase (EC 2.3.3.13), such as homocitrate synthase (EC 2.3.3.14) or 4-hydroxy-2-ketovalerate aldolase (EC 2.3.3.15).

[0120] 4.1.3.39)) activity, such as HMG CoA lyase (EC 4.1.3.4).

[0121] 3-Hydroxyisovalerate (HIV) is enzymatically converted to 3-methylcrotonate

[0122] The enzymatic conversion of 3-hydroxyisovalerate (HIV) to 3-methylcrotonate is shown in Figure 1 In step II of . This conversion preferably utilizes an enzyme that catalyzes the dehydration of a β-hydroxy acid (i.e., for example, 3-hydroxyisovaleric acid (HIV)) to an α,β-unsaturated acid (i.e., for example, 3-methylcrotonic acid). "Dehydration" generally refers to a reaction involving the removal of H2O. Preferably, such an enzyme belongs to the family of water-lyases (EC 4.2.-.-). Preferred examples of such enzymes classified as EC 4.2.-.- (i.e., water-lyases) are:

[0123] Aconitase (EC 4.2.1.3);

[0124] Fumarase (EC 4.2.1.2); and

[0125] Enoyl-CoA hydratase / dehydratase (EC 4.2.1.17).

[0126] Enzymatic conversion of 3-methylcrotonate to isobutylene

[0127] The enzymatic conversion of 3-methylcrotonate to isobutylene is shown in Figure 1 In step I of . This conversion can be achieved by decarboxylation using an FMN-dependent decarboxylase associated with FMN prenyltransferase. "Decarboxylation" is generally a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2).

[0128] The enzymatic conversion of 3-methylcrotonate to isobutene using an FMN-dependent decarboxylase associated with an FMN prenyltransferase relies on two sequential step reactions catalyzed by two enzymes, namely, an FMN-dependent decarboxylase (catalyzing the actual decarboxylation of 3-methylcrotonate to isobutene) associated with an FMN prenyltransferase (which provides the modified flavin cofactor).

[0129] The flavin cofactor is preferably FMN or FAD. FMN (flavin mononucleotide; also known as riboflavin-5'-phosphate) is a biomolecule produced from riboflavin (vitamin B2) by the enzyme riboflavin kinase and serves as a prosthetic group in various reactions. FAD (flavin adenine dinucleotide) is a redox cofactor, more specifically a prosthetic group, involved in several important reactions in metabolism.

[0130] Thus, in the conversion of 3-methylcrotonate to isobutylene, in a first step, a flavin cofactor (FMN or FAD) is modified to a (modified) flavin-derived cofactor. This modification is catalyzed by the FMN prenyltransferase. The FMN prenyltransferase prensers the flavin cycloprenylation of the flavin cofactor (FMN or FAD) to a (modified) prenylated flavin cofactor. More specifically, the FMN prenyltransferase catalyzes the prenylation of the flavin cofactor (FMN or FAD) to a flavin-derived cofactor using dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP).

[0131] In the second step, the actual conversion of 3-methylcrotonate to isobutene is catalyzed by the FMN-dependent decarboxylase via a 1,3-dipolar cycloaddition-based mechanism, which utilizes the prenylated flavin cofactor (FMN or FAD) provided by the associated FMN prenyltransferase.

[0132] In a preferred embodiment, the FMN prenyltransferase that modifies a flavin cofactor (FMN or FAD) to a (modified) flavin-derived cofactor (using dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP)) is a phenylacrylic acid decarboxylase (PAD)-type protein, or the closely related prokaryotic enzyme UbiX, which is an enzyme involved in the biosynthesis of ubiquinone in prokaryotes.

[0133] In Escherichia coli, the protein UbiX (also known as 3-octiprenyl-4-hydroxybenzoate carboxyl-lyase) has been shown to be involved in the third step of ubiquinone biosynthesis.

[0134] In a preferred embodiment, the modification of a flavin cofactor (FMN or FAD) to a corresponding (modified) flavin-derived cofactor is catalyzed by the FMN-containing protein phenylacrylic acid decarboxylase (PAD). Enzymes involved in the modification of a flavin cofactor (FMN or FAD) to a corresponding flavin-derived cofactor were initially annotated as decarboxylases (EC 4.1.1.-). Some phenylacrylic acid decarboxylases (PAD) are now annotated as flavin prenyltransferases of EC 2.5.1.-. For flavin prenyltransferases, enzymes capable of catalyzing the enzymatic reactions described herein have also recently been annotated as flavin prenyltransferases of EC 2.5.1.129.

[0135] In a more preferred embodiment, the conversion of 3-methylcrotonate to isobutylene utilizes a phenylacrylic acid decarboxylase (PAD)-type protein, such as an FMN prenyltransferase that modifies a flavin cofactor (FMN or FAD) to a corresponding (modified) flavin-derived cofactor, wherein the phenylacrylic acid decarboxylase (PAD)-type protein is derived from Candida albicans (Uniprot Accession No. Q5A8L8), Aspergillus niger (Uniprot Accession No. A3F715), Saccharomyces cerevisiae (Uniprot Accession No. P33751), or Cryptococcus gattii (Uniprot Accession No. E6R9Z0).

[0136] In another preferred embodiment, the cytokine is expressed by a protein also known as UbiX (originally annotated as EC

[0137] The FMN-containing protein 3-octaprenyl-4-hydroxybenzoate carboxyl-lyase of EC 2.4.1.1.- catalyzes the modification of a flavin cofactor (FMN or FAD) into a corresponding (modified) flavin-derived cofactor. As mentioned above, enzymes involved in the modification of a flavin cofactor (FMN or FAD) into a corresponding (modified) flavin-derived cofactor were initially annotated as decarboxylases. Some phenylacrylic acid decarboxylases (PADs) are now annotated as flavin isoprenyltransferases of EC 2.5.1.-.

[0138] As described above with respect to flavin prenyltransferase, enzymes capable of catalyzing the enzymatic reactions described herein have also recently been annotated as flavin prenyltransferases of EC 2.5.1.129.

[0139] In a more preferred embodiment, the conversion of 3-methylcrotonate into isobutene utilizes 3-octyl-4-hydroxybenzoate carboxyl-lyase (also known as UbiX) as FMN prenyltransferase, which modifies a flavin cofactor (FMN or FAD) into a corresponding (modified) flavin-derived cofactor, wherein the 3-octyl-4-hydroxybenzoate carboxyl-lyase (also known as UbiX) is derived from Escherichia coli (Uniprot Accession No. P0AG03), Bacillus subtilis (Uniprot Accession No. A0A086WXG4), Pseudomonas aeruginosa (Uniprot Accession No. A0A072ZCW8) or Enterobacter sp. DC4 (Uniprot Accession No. W7P6B1).

[0140] In another preferred embodiment, the modification of the flavin cofactor (FMN or FAD) to the corresponding (modified) flavin-derived cofactor is catalyzed by Ubx-like flavin prenyltransferases encoded by kpdB and ecdB, respectively, from Escherichia coli (Uniprot Accession No. A0A023LDW3 and Uniprot Accession No. P69772, respectively) and Ubx-oxyflavin prenyltransferase encoded by kpdB from Klebsiella pneumoniae.

[0141] In another preferred embodiment, the modification of a flavin cofactor (FMN or FAD) to the corresponding (modified) flavin-derived cofactor is catalyzed by a flavin isoprenyltransferase.

[0142] As described above, the actual decarboxylation, i.e., conversion of 3-methylcrotonate to isobutene, is catalyzed via a 1,3-dipolar cycloaddition based mechanism by an FMN-dependent decarboxylase utilizing a prenylated flavin cofactor (FMN or FAD) provided by any of the related FMN prenyltransferases described above.

[0143] In a preferred embodiment, the FMN-dependent decarboxylase catalyzes the decarboxylation of 3-methylcrotonate to isobutene by a ferulic acid decarboxylase (FDC).Ferulic acid decarboxylase (FDC) is an enzyme of the term class EC 4.1.1.-.

[0144] In an even more preferred embodiment, the conversion of 3-methylcrotonate to isobutene utilizes a ferulic acid decarboxylase (FDC) derived from Saccharomyces cerevisiae (Uniprot Accession No. Q03034), Enterobacter (Uniprot Accession No. V3P7U0), Bacillus pumilus (Uniprot Accession No. Q45361), Aspergillus niger (Uniprot Accession No. A2R0P7), or Candida dubliniensis (Uniprot Accession No. B9WJ66).

[0145] In another more preferred embodiment, the conversion of 3-methylcrotonate to isobutylene utilizes protocatechuate decarboxylase (EC 4.1.1.63).

[0146] In a preferred embodiment of the present invention, the PCA decarboxylase used in the method of the present invention is a PCA decarboxylase derived from Klebsiella pneumoniae (Uniprot Accession No. B9AM6), Leptolyngbya sp. (Uniprot Accession No. A0A0S3U6D8) or Phascolarctobacterium sp. (Uniprot Accession No. R6IIV6).

[0147] In another preferred embodiment, the FMN-dependent decarboxylase that decarboxylates 3-methylcrotonate to isobutylene is an enzyme closely related to the above ferulic acid decarboxylase (FDC), i.e., 3-polyisoprene-4-hydroxybenzoate decarboxylase (also known as UbiD). 3-polyisoprene-4-hydroxybenzoate decarboxylase belongs to the UbiD decarboxylase family classified as EC 4.1.1.-.

[0148] In a more preferred embodiment, the conversion of 3-methylcrotonate to isobutylene utilizes 3-polyisoprene-4-hydroxybenzoate decarboxylase (UbiD) from Hypocrea atroviridis (UniProt Accession No. G9NLP8), Sphaerulina musiva (UniProt Accession No. M3DF95), Penicillium requeforti (UniProt Accession No. W6QKP7), Fusarium oxysporum f. sp. lycopersici)

[0149] (UniProt accession number W9LTH3), Saccharomyces kudriavzevii (UniProt accession number J8TRN5), Saccharomyces cerevisiae, Aspergilus parasiticus, Candida albicans, Grosmannia clavigera, Escherichia coli (Uniprot accession number P0AAB4), Bacillus megaterium

[0150] (Uniprot accession number D5DTL4), Methanothermobacter CaT2 (Uniprot accession number T2GKK5), Mycobacterium chelonae 1518 (Uniprot accession number X8EX86), or Enterobacter cloacae

[0151] (Uniprot accession number V3DX94).

[0152] In another more preferred embodiment, the conversion of 3-methylcrotonate to isobutylene utilizes a UbiD-like decarboxylase derived from Streptomyces sp (UniProt Accession No. A0A0A8EV26).

[0153] In an even more preferred embodiment, the UbiD-like decarboxylase derived from Streptomyces is an enzyme comprising the amino acid sequence of SEQ ID NO:21, or a sequence that is at least n% identical to SEQ ID NO:21, n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has enzymatic activity for converting 3-methylcrotonic acid into isobutylene.

[0154] Regarding the determination of sequence identity, the following should apply: when the sequences being compared have different lengths, the degree of identity refers to the percentage of the amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence or the percentage of the amino acid residues in the longer sequence that are identical to the amino acid residues in the shorter sequence. Preferably, it refers to the percentage of the amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence. The degree of sequence identity can be determined using a preferred suitable computer algorithm (such as CLUSTAL) according to methods well known in the art.

[0155] For example, when using the Clustal analysis method to determine whether a particular sequence is at least 60% identical to a reference sequence, the default settings or settings preferably are as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extended gap penalty: 0.05; Delayed divergence: 40; Gap separation distance: 8, for comparison of amino acid sequences. For comparison of nucleotide sequences, it is best to set the extended gap penalty to 5.0.

[0156] In a preferred embodiment, ClustalW2 is used for comparing amino acid sequences. In the case of pairwise comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap opening: 10; gap extension: 0.1. In the case of multiple comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap opening: 10; gap extension: 0.2; gap distance: 5; no end gaps.

[0157] Preferably, the degree of identity is calculated over the entire length of the sequence.

[0158] For the conversion of 3-methylcrotonyl-CoA via 3-hydroxy-3-methylbutyryl-CoA, 3-hydroxy-3-methylbutyrate and 3- Alternative pathways for the enzymatic conversion of phosphinoyl-3-methylbutyrate to isobutylene

[0159] As described above, according to the present invention, in one possible pathway, 3-methylcrotonyl-CoA is enzymatically converted to isobutylene, and 3-methylcrotonyl-CoA is enzymatically converted to isobutylene via 3-methylcrotonate. This pathway is described above.

[0160] In an alternative pathway, isobutylene may be provided by another possible pathway in which 3-methylcrotonyl-CoA is converted to 3-hydroxy-3-methylbutyryl-CoA, which is then further enzymatically converted to 3-hydroxy-3-methylbutyrate, which is then further enzymatically converted to 3-phosphinoyl-3-methylbutyrate, which is then further enzymatically converted to the isobutylene. These individual steps are described below.

[0161] 3-Methylcrotonyl-CoA is enzymatically converted to 3-hydroxy-3-methylbutyrate via 3-hydroxy-3-methylbutyryl-CoA

[0162] According to the present invention, the enzymatic conversion of 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyrate comprises the steps of:

[0163] (a) enzymatically converting 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyryl-CoA; and

[0164] (b) the 3-hydroxy-3-methylbutyryl-CoA thus produced is further enzymatically converted into 3-hydroxy-3-methylbutyrate.

[0165] The enzymatic conversion of 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyryl-CoA according to step (a) of the above method can be achieved, for example, by using the following enzyme

[0166] (i) 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.1.116);

[0167] (ii) 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55);

[0168] (iii) Enol-CoA hydratase (EC 4.2.1.17);

[0169] (iv) 3-Hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59)

[0170] (v) Crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58)

[0171] (vi) 3-Hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60)

[0172] (vii) 3-Hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61)

[0173] (viii) Long-chain enol-CoA hydratase (EC 4.2.1.74)

[0174] (ix) 3-Methylglutaryl-CoA hydratase (EC 4.2.1.18).

[0175] The conversion of 3-hydroxy-3-methylbutyryl-CoA to 3-hydroxy-3-methylbutyrate (step (b)) can be achieved by direct conversion or alternatively by a two-step reaction via 3-hydroxy-3-methylbutyryl phosphate.

[0176] Direct conversion can be achieved, for example, using the following enzymes

[0177] (i) thioesterase (EC 3.1.2); or

[0178] (ii) CoA-transferase (EC 2.8.3)

[0179] The two-step transformation can be summarized as follows:

[0180] (i) 3-Hydroxy-3-methylbutyryl-CoA + H3PO4 → 3-Hydroxy-3-methylbutyryl phosphate + CoA

[0181] (ii) 3-Hydroxy-3-methylbutyryl phosphate + ADP → 3-Hydroxy-3-methylbutyrate + ATP

[0182] The conversion of 3-hydroxy-3-methylbutyryl-CoA to 3-hydroxy-3-methylbutyryl phosphate can be achieved, for example, by using a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).

[0183] The conversion of 3-hydroxy-3-methylbutyryl phosphate to 3-hydroxy-3-methylbutyrate can be achieved, for example, by using butyrate kinase (EC 2.7.2.7), branched chain fatty acid kinase (EC 2.7.2.14), propionate kinase (EC 2.7.2.15), or acetate kinase (EC 2.7.2.1).

[0184] A method for enzymatically converting 3-methylcrotonyl-CoA to 3-hydroxy-3-methylbutyrate via 3-methylcrotonate Replace Path

[0185] In the above-described alternative pathway for converting 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyrate via 3-hydroxy-3-methylbutyryl-CoA according to the invention, 3-methylcrotonyl-CoA can also be enzymatically converted into 3-hydroxy-3-methylbutyrate, still via 3-methylcrotonate.

[0186] Therefore, according to the present invention, the enzymatic conversion of 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyrate comprises the steps of:

[0187] (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid; and

[0188] (b) the 3-methylcrotonate thus produced is further enzymatically converted into 3-hydroxy-3-methylbutanoate.

[0189] The conversion of 3-methylcrotonyl-CoA to 3-methylcrotonate can be achieved by direct conversion, preferably using enzymes belonging to the thioester hydrolase family, referred to below as thioesterases (EC 3.1.2), or the CoA transferase family (EC 2.8.3).

[0190] Thioesterases have been described above. With regard to preferred embodiments, the same applies as described above and briefly summarized below. TEs (also known as thioester hydrolases) are enzymes classified as EC 3.1.2. Currently, thioesterases are classified as EC 3.1.2.1 to EC 3.1.2.27.

[0191] In a preferred embodiment, the thioesterase used to convert 3-methylcrotonyl-CoA to 3-methylcrotonate is selected from the group consisting of:

[0192] - Acetyl-CoA hydrolase (EC 3.1.2.1);

[0193] - Palmitoyl-CoA hydrolase (EC 3.1.2.2);

[0194] -3-Hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4);

[0195] -oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);

[0196] - ADP-dependent short-chain acyl-CoA hydrolase (EC 3.1.2.18);

[0197] - ADP-dependent medium-chain acyl-CoA hydrolase (EC 3.1.2.19); and

[0198] -Acyl-CoA hydrolase (EC 3.1.2.20)

[0199] Concerning CoA-transferases (EC 2.8.3) and preferred embodiments thereof, the same applies as stated above.

[0200] In an alternative embodiment, conversion of 3-methylcrotonyl-CoA to 3-methylcrotonic acid can be achieved by first converting 3-methylcrotonyl-CoA to 3-methylcrotonyl-phosphate, and then converting 3-methylcrotonyl-phosphate to 3-methylcrotonic acid.

[0201] The conversion of 3-methylcrotonyl-CoA to 3-methylcrotonyl-phosphate can be achieved, for example, by using a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.18).

[0202] The conversion of 3-methylcrotonyl-phosphate into 3-methylcrotonic acid can be achieved, for example, by using an enzyme classified as EC 2.7.2 (i.e., a phosphotransferase). Such an enzyme utilizes a carboxyl group as an acceptor. In a preferred embodiment, the conversion of 3-methylcrotonyl-phosphate into 3-methylcrotonic acid is achieved by using butyrate kinase (EC 2.7.2.7), branched-chain fatty acid kinase (EC 2.7.2.14), propionate kinase (EC 2.7.2.15) or acetate kinase (EC 2.7.2.1).

[0203] According to the present invention, the 3-methylcrotonic acid obtained in step (a) is further enzymatically converted into 3-hydroxy-3-methylbutyric acid. This conversion can be carried out, for example, by utilizing a water-lysing enzyme (EC

[0204] 4.2.1) enzymes, in particular aconitate hydratase (EC 4.2.1.3) or maleate hydratase (EC 4.2.1.31) or 2-methylcitrate dehydratase (EC 4.2.1.79). The reaction diagram is shown in Figure 3 middle.

[0205] Enzymatic conversion of 3-hydroxy-3-methylbutyrate to isobutylene via 3-phosphinoyl-3-methylbutyrate

[0206] According to the present invention, 3-hydroxy-3-methylbutyrate can be further converted into isobutylene. This conversion can be achieved, for example, by utilizing a decarboxylase, in particular a mevalonate diphosphate (MDP) decarboxylase. This conversion has been described in the prior art, for example, in WO 2010 / 001078, WO 2012 / 052427 and Gogerty et al. (Appl. Environm. Microbiol. 76 (2010), 8004-8010).

[0207] More specifically, according to the present invention, 3-hydroxy-3-methylbutyrate can be converted into isobutylene by a single enzyme having decarboxylase activity. Preferably, according to the present invention, the conversion of 3-hydroxy-3-methylbutyrate into isobutylene can be achieved by mevalonate diphosphate (MDP) decarboxylase (EC 4.1.1.33).

[0208] As mentioned above, WO 2010 / 001078 describes a method for producing olefins (such as, for example, isobutylene) by enzymatic conversion of 3-hydroxyalkanoic acid (such as, for example, 3-hydroxy-3-methylbutyric acid) with an enzyme having decarboxylase activity. WO 2010 / 001078 has described that the conversion of 3-hydroxyalkanoic acid to olefin is usually carried out by converting the 3-hydroxyalkanoic acid into the corresponding 3-phosphinoxyalkanoic acid, which is then decarboxylated to form the corresponding olefin.

[0209] In WO 2012 / 052427, it has been found that different decarboxylases, in particular mevalonate diphosphate (MDP) decarboxylases, catalyze the above-mentioned two steps with different efficiencies, i.e., some decarboxylases catalyze the first step with a higher efficiency than other decarboxylases, while some decarboxylases show a preference for the second step, i.e., the decarboxylation step, and thus the efficiency of the conversion of 3-hydroxyalkanoic acids into olefins as described in WO 2010 / 001078 can be significantly improved by combining the corresponding enzymes. Therefore, it is preferred to use two different MDP decarboxylases for this conversion, and in particular the MDP decarboxylases selected from the preferred embodiments described in WO 2010 / 001078.

[0210] Effects of increasing CoA levels on isobutylene-producing cells

[0211] As described above, the present invention is based on the concept of increasing isobutene production and / or improving the rate of isobutene production by providing and maintaining a high acetyl-CoA pool in the cell for isobutene production, wherein the acetyl-CoA pool is kept high by ensuring increased pantothenate uptake by the cell and / or increased conversion of pantothenate to CoA.

[0212] Increasing pantothenate uptake by cells and / or increasing the conversion of pantothenate to CoA not only has the beneficial effect of maintaining a high acetyl-CoA pool in the cell for isobutene production. In addition, increasing the CoA pool has a facilitating effect on overall cellular function, ultimately leading to a beneficial effect on isobutene production.

[0213] In fact, it is known that CoA acts as a cofactor for various oxidation and biosynthetic reactions in cellular metabolism. Coenzyme A and its thioesters (e.g., acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, succinyl-CoA, HMG-CoA, etc.) are essential for various metabolic pathways, regulation of gene expression, growth and cell proliferation. In addition, as mentioned above, the metabolic pathway leading to the production of isobutene includes a series of biochemical transformations of acyl-CoA intermediates.

[0214] Thus, the recombinant organism or microorganism of the present invention is also characterized in that it has an increased CoA pool, which has an overall positive effect on cell growth and / or isobutylene production and / or stability of the isobutylene producing strain. These positive effects are achieved through known biological mechanisms and processes that are regulated by CoA or depend on CoA content, such as the availability of CoA as a substrate or cofactor, regulation of proteins, optimized balance between free CoA and CoA thioesters. In a preferred embodiment, the recombinant organism or microorganism according to the present invention is characterized in that it has an increased pool of one or more acyl thioester intermediates of the isobutylene metabolic pathway, including acetyl-CoA, acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl-CoA (Hmg-CoA), 3-methylglutaconyl-CoA, 3-hydroxy-3-methylbutyryl-CoA, 3-methylcrotonyl-CoA.

[0215] Enzymatic provision of acetyl-CoA from CoA

[0216] In the recombinant organism or microorganism of the present invention, acetyl-CoA can be provided by different pathways, since it is a central component in metabolism used in many biochemical reactions.

[0217] In a preferred embodiment, acetyl-CoA is provided enzymatically by using CoA, preferably coenzyme A (CoA) from the increased CoA pool of the recombinant organism or microorganism of the present invention.

[0218] In a more preferred embodiment, acetyl-CoA is provided enzymatically by acetylation of CoA with acetate, preferably using an acetyl-CoA synthetase or an acyl-CoA synthetase.

[0219] In a preferred embodiment, the acetyl-CoA synthetase (also known as acetate-CoA ligase) is an enzyme classified as EC 6.2.1.1. Acetyl-CoA synthetase (EC 6.2.1.1) is an enzyme that catalyzes the following reaction:

[0220] ATP + acetate + CoA = AMP + diphosphate + acetyl-CoA

[0221] This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals, fungi and bacteria. It has been used in Acetobacter aceti, Aedes togoi, Aliivibriofischeri, Amaranthus sp., Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus nidulans, Aspergillus niger, Bacillus subtilis, Bos taurus, Bradyrhizobium japonicum, Cryptosporidium parvum, Escherichia coli, Euglena gracilis, Haloarcula marismortui, Homo sapiens, Hordeum vulgare, Ignicoccus hospitalis, Marmota monax, Methanosarcinasp.), Methanothermobacterthermautotrophicus, Methanothrix soehngenii, Methanothrix thermoacetophila (SwissProt accession number A0B8F1), Moorellathermoacetica, Mus musculus, Neurospora crassa, Oryctolagus cuniculus, Ovis aries, Penicillium chrysogenum, Phycomyces blakesleeanus, Pinus radiata, Pisum sativum, Pseudomonas putida (UniProt accession number Q6EMJ3), Pyrobaculum aerophilum, Pyrococcus furiosus, Rattus norvegicus, Rhodobacter sphaeroides, Roseovarius sp., Saccharomyces cerevisiae, Salmonella enterica, Spinacia oleracea, Taxus sp., and Zeamays.

[0222] In a preferred embodiment, acetyl-CoA synthetase (EC 6.2.1.1) is an enzyme derived from Escherichia coli MG1655 encoded by acs having UniProt accession number P27550 (SEQ ID NO: 22). Therefore, in a preferred embodiment of the present invention, acetyl-CoA synthetase (EC

[0223] 6.2.1.1) is an enzyme comprising the amino acid sequence of SEQ ID NO: 22 or a sequence at least n% identical to SEQ ID NO: 22, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity of converting CoA and acetate into acetyl-CoA, i.e., acetylation of CoA by using acetate. Regarding the determination of sequence identity, the same applies as described above.

[0224] As described above, in another preferred embodiment, acetyl-CoA is provided enzymatically by acetylation of CoA with acetate, using an acyl-CoA synthetase.

[0225] In a preferred embodiment, the acyl-CoA synthetase is an enzyme classified as EC 6.2.1.2. These short / medium chain acyl-CoA synthetases (EC 6.2.1.2) (also known as butyrate-CoA ligases, adK, IvaK, butyryl-CoA synthetase, fatty acid thiokinase (medium chain), acyl-activating enzyme, fatty acid elongase, fatty acid activating enzyme, fatty acyl-CoA synthetase, butyrate-CoA ligase, butyryl-CoA synthetase, L-(+)-3-hydroxybutyryl-CoA ligase or butyrate:CoA ligase (AMP forming)) are enzymes that catalyze the following reaction:

[0226]

[0227] This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, fungi and bacteria. This enzyme has been described, for example, in Bos taurus, Byssochlamys spectabilis, Caviaporcellus, Dictyostelium discoideum (UniProt Accession No. Q54CJ4), Homo sapiens, Methanosarcina acetivorans (UniProt Accession No. Q8TLW1), Mus musculus, Mycobacterium avium, Ovis aries, Penicillium chrysogenum (UniProt Accession No. O74725), Pseudomonas aeruginosa, Rattus norvegicus, Streptomyces coelicolor and Sus scrofa.

[0228] In a preferred embodiment, the short / medium chain acyl-CoA synthetase (EC 6.2.1.-) is an enzyme derived from E. coli MG1655 encoded by fadK or ydiD (SEQ ID NO: 23) with UniProt accession number P38135. Therefore, in a preferred embodiment of the present invention, the short / medium chain acyl-CoA synthetase (EC 6.2.1.-) is an enzyme comprising the amino acid sequence of SEQ ID NO: 23 or a sequence at least n% identical to SEQ ID NO: 23, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzyme activity of converting CoA and acetate into acetyl-CoA, i.e., by acetylation of CoA with acetate. With regard to the determination of sequence identity, the same applies as described above.

[0229] The physiological substrates for the E. coli derived short / medium chain acyl-CoA synthetase are the short chain acids with maximal activity towards dodecanoate, hexanoate and octanoate.

[0230] In a preferred embodiment, the acyl-CoA synthetase is an enzyme classified as EC 6.2.1.3. These long-chain acyl-CoA synthetases (EC 6.2.1.3) are enzymes that catalyze the following reaction:

[0231]

[0232] This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as animals, plants, fungi and bacteria. This enzyme has been described, for example, in Agrypnus binodulus, Arabidopsis thaliana, Aspergillus nidulans, Babesia bovis (TrEMBL accession number Q6QLU3), Brassica napus, Caenorhabditis elegans, Caulobacter vibrioides, Chaenocephalus aceratus, Drosophila melanogaster, Escherichia coli, Geobacillus thermodenitrificans, Homo sapiens, Komagataella pastoris, Luciola cruciata, Mortierella alpina (UniProt accession number C8KHM6), Mus musculus, Mycobacterium tuberculosis, Notothenia coriiceps), Oryctolagus cuniculus, Photinus pyralis, Pisum sativum, Plasmodium falciparum, Plasmodium knowlesi, Pseudomonas aeruginosa, Pseudomonas chlororaphis (UniProt accession number Q5CD72), Rattus norvegicus, Saccharomyces cerevisiae, Sus scrofa, Iberian Guadyerbas×Landrace hybrid, Thalassiosira pseudonana, Thermus thermophilus, Tribolium castaneum, Trypanosoma brucei, Ulmus sp., Yarrowia lipolytica lipolytica) and corn (Zea mays).

[0233] In a preferred embodiment, the long-chain acyl-CoA synthetase (EC 6.2.1.3) is produced by

[0234] An enzyme derived from E. coli MG1655 encoded by fadD (SEQ ID NO: 24) of UniProt accession number P69451. Therefore, in a preferred embodiment of the present invention, the short / medium chain acyl-CoA synthetase (EC 6.2.1.3) is an enzyme comprising the amino acid sequence of SEQ ID NO: 24 or a sequence at least n% identical to SEQ ID NO: 24, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity of converting CoA and acetate into acetyl-CoA, i.e., acetylation of CoA by using acetic acid. With regard to the determination of sequence identity, the same applies as described above.

[0235] Adding a CoA pool

[0236] The recombinant organism or microorganism according to the present invention is characterized in that it has an increased coenzyme A (CoA) pool due to the organism or microorganism from which it is derived, and this increased coenzyme A (CoA) pool is due to:

[0237] (i) increased pantothenic acid uptake; and / or

[0238] (ii) Increased conversion of pantothenate to CoA.

[0239] The term "increased coenzyme A (CoA) pool" as used in the present invention generally refers to a recombinant (genetically modified) organism or microorganism with a content and / or availability of CoA that is higher than a corresponding unmodified organism or microorganism. In a preferred embodiment, in the context of the present invention, "increased coenzyme A (CoA) pool" means that the content and / or availability of CoA in a genetically modified recombinant organism or microorganism is at least 1%, 2%, 5%, 7% or 10% higher than a corresponding unmodified organism or microorganism, preferably at least 20% higher, more preferably at least 30% or 50% higher, even more preferably at least 70% or 80% higher, and particularly preferably at least 90% or 100% higher. In an even more preferred embodiment, compared with a corresponding unmodified organism or microorganism, the content and / or availability of CoA increases by at least 150%, at least 200% or at least 500%. In a particularly preferred embodiment, the content and / or availability of CoA is at least 2 times higher than a corresponding unmodified organism or microorganism, and more preferably at least 5 times higher.

[0240] The term "increased pantothenic acid uptake" as used in the present invention generally represents uptake, i.e. pantothenic acid is transferred to the inside from the outside of a recombinant (genetically modified) organism or microorganism, higher than in a corresponding unmodified organism or microorganism. In a preferred embodiment, in the context of the present invention, "increased pantothenic acid uptake" means that compared with a corresponding unmodified organism or microorganism, pantothenic acid is transferred to the inside from the outside of a genetically modified recombinant organism or microorganism by at least 1%, 2%, 5%, 7% or 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80%, and particularly preferably at least 90% or 100%. In even more preferred embodiments, compared with a corresponding unmodified organism or microorganism, pantothenic acid is transferred to the inside from the outside of a genetically modified recombinant organism or microorganism by at least 150%, at least 200% or at least 500%. In a particularly preferred embodiment, pantothenic acid is transferred to the inside from the outside of a genetically modified recombinant organism or microorganism by at least 2 times, 5 times, 7 times and more preferably at least 10 times higher than a corresponding unmodified organism or microorganism.

[0241] The term "increased pantothenic acid to the conversion of CoA" as used in the present invention generally represents that the expression and / or activity of the corresponding enzyme described below in recombinant (genetically modified) organism or microorganism is higher than in the corresponding unmodified organism or microorganism. In a preferred embodiment, in the content of the present invention, "increased pantothenic acid to the conversion of CoA" represents that the expression and / or activity of the corresponding enzyme described below in the recombinant organism or microorganism of genetic modification is higher than in the corresponding unmodified organism or microorganism by at least 1%, 2%, 5%, 7% or 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80%, and particularly preferably at least 90% or 100%. In even more preferred embodiments, compared with the corresponding unmodified organism or microorganism, the expression and / or activity of the corresponding enzyme described below in the recombinant organism or microorganism of genetic modification is high by at least 150%, at least 200% or at least 500%. In a particularly preferred embodiment, the expression and / or activity of the corresponding enzymes described below in the genetically modified recombinant organism or microorganism is at least 2-fold, 5-fold, 7-fold and more preferably at least 10-fold higher than in the corresponding unmodified organism or microorganism.

[0242] Methods for measuring the (increased) pantothenate uptake compared to the organism or microorganism from which it is derived and for measuring the (increased) conversion of pantothenate to CoA are well known to those skilled in the art. Since increased pantothenate uptake and / or increased conversion of pantothenate to CoA ultimately results in an increased CoA pool over that of the organism or microorganism from which it is derived, it is also preferred that the (increased) pantothenate uptake and (increased) conversion of pantothenate to CoA over that of the organism or microorganism from which it is derived can be measured by indirectly measuring the (increased) CoA pool.

[0243] Means and methods for measuring the (increased) CoA pool and / or acetyl-CoA over the organism or microorganism from which it is derived are known to the person skilled in the art and described in the literature.

[0244] Assays for measuring (increased) CoA pools and / or acetyl-CoA have been described, for example, in Rock et al. (J. Bacteriol. 185(11), 3410-3415 (2003); see, in particular page 3412 therein); Lin et al. (Biotechnol. Prog. 20(5), 1599-1604 (2004); see, in particular page 1600 therein); and Vadali et al. (Metab. Eng. 6(2), 133-139 (2004); see, in particular page 135).

[0245] Without being bound by theory, an example of such an assay for increased CoA pools is given in the Examples section below and can be performed, for example, as follows:

[0246] One possibility to quantify CoA (and / or its thioester intermediates) is therefore first to extract the compounds and subsequently to analyze them by LC-MA or HPLC analysis.

[0247] Therefore, for extraction, one possibility is to directly sample 120 μL of bacterial culture from the fermenter and filter through a membrane filter under vacuum. Immediately after filtration, the filter is placed in aluminum foil and inserted into liquid nitrogen to stop all metabolic reactions. Then, the aluminum foil containing the filter is stored at -80 ° C until extraction. Intracellular metabolites are extracted in a Falcon tube at -80 ° C in 15 min with 2 mL of cold MeOH / H2O (80 / 20) mixture (from a membrane filter). Then, the supernatant is taken out and transferred to a new test tube. According to the same process just described (except for the two extraction times at -80 ° C and the centrifugation step is shortened to 5 min), the second extraction of the membrane filter is carried out with 1 mL of cold MeOH / H2O mixture. The supernatant (about 3 mL) merged is filtered through a 0.2 μm syringe filter and 2.4 mL is evaporated to dryness by a Speed-Vac concentrator. The dried extract was then reconstituted with 120 μL ACN / H 2 O (50 / 50, v / v), filtered again through a 0.2 μm syringe filter and transferred to an HPLC vial for LC-MS analysis.

[0248] LC-MS analysis was performed in negative ionization mode on a UHPLC system coupled to a Q-Exactive mass spectrometer (ThermoFisher Scientific, Massachusetts, USA). For the LC part, a BEH amide column (1.7 μm, 2.1×100 mm, Waters) conditioned at 25 °C was used. The flow rate was set at 0.5 mL / min and 2 μL of the sample was injected. The mobile phase consisted of a binary gradient (A: ammonium formate (10 mM) + 0.1% ammonium hydroxide and B: acetonitrile): starting from 95% B in 1.5 min, then decreasing to 55% B in 8.5 min and holding at 55% B for 2 min, then returning to the initial conditions. The total analysis time was 19 min. In the mass level, the analysis was performed in negative ion mode in full MS + ddMS2 mode. If needed, the ion group between 80 < m / z < 1200 Da was considered, and all ions with intensities greater than 1e5 were fragmented using a collision energy of 35 eV to obtain additional structural information. The calibration curve of the target compound was recorded under the same LC / MS conditions. Data analysis was carried out using Xcalibur v3.0.63 software (ThermoFisher Scientific).

[0249] Alternatively, another analytical possibility is HPLC analysis.

[0250] Two LC methods can be used to detect and quantify coenzyme A-derived compounds.

[0251] The first LC method was carried out on an HPLC 1260 system coupled to a Multiple wavelength detector (MWD) (Agilent, Santa Clara, USA). Separation was performed by Zorbax Eclipse coupled to a C18 column (3.5 μm, 4.6×100 mm, Agilent) conditioned at 30 °C. The mobile phase consisted of an isocratic elution using acetonitrile (5%) and phosphate buffer (100 mM) at pH 5 (95%) in 10 min. The flow rate was set at 1.5 mL / min and 5 μL of the sample was injected.

[0252] The second method was carried out on an HPLC 1260 system coupled to a Diode array detector (DAD) (Agilent, Santa Clara, USA). A ZorbaxsbAq column (5 μm, 4.6×250 mm, Agilent) conditioned at 30 °C was used to separate the metabolites. 5 μL of the sample was injected. The flow rate was set at 1.5 mL / min. The mobile phase consisted of a binary gradient (A: acetonitrile; and B: H2SO4

[0253] 8.4 mM), starting at 100% B, and then decreasing to 30% B until 8 min, and remaining at 30% B for 1 min. During this 1 min 30% B period, the flow rate was increased to 2 mL / min. After 9 min, the binary gradient returned to the initial conditions (i.e., 100% B and a flow rate of 1.5 mL / min) within 2 min and remained at 100% B for 3 min. The total run time was 14 min.

[0254] In both LC methods, detection of target metabolites was performed at λ 260 nm and calibration curves of pure compounds were recorded under the respective LC conditions used.

[0255] Generally, a recombinant organism or microorganism having an increased coenzyme A (CoA) pool over that of the organism or microorganism from which it was derived (although such increased coenzyme A (CoA) pool is due to (i) increased pantothenate uptake; and / or (ii) increased conversion of pantothenate to CoA) can be achieved through various recombinant modifications, which are described in more detail further below.

[0256] Typically, (i) increased pantothenate uptake and / or (ii) increased conversion of pantothenate to CoA is achieved through recombinant expression of specific proteins.

[0257] Generally, "recombination" in this content means the artificial genetic modification of an organism or microorganism, by adding, removing or modifying chromosome or extrachromosomal genes or regulatory motifs, such as promoters, or by the fusion of an organism, or by adding any type of vector, for example, plasmid. The term "recombinant expression" means the production of proteins related to genetic modification, preferably to produce proteins that are exogenous or heterologous relative to the host, that is, they are not naturally produced in the production host, or produce modified or mutated endogenous proteins. "Recombinant expression" in the content of the present invention is preferably "overexpression". "Overexpression" or "overexpression" in this content means the recombinant expression of the protein in the host organism, preferably derived from an organism different from the one expressed therein, compared with the natural expression of the protein occurring in the host organism or microorganism, increase by at least 10% and preferably increase by 20%, 50%, 100%, 500% and possibly more. This definition also includes the situation where there is no natural expression of the protein.

[0258] Thus, in short, the recombinant expression according to the present invention resulting in increased pantothenate uptake; and / or increased conversion of pantothenate to CoA may be due to (1) overexpression of the respective preferred endogenous gene, (2) introduction of the respective heterologous gene and / or (3) expression of a mutant protein with increased activity, e.g., increased activity catalyzing the corresponding reaction or increased transporter activity over the respective enzyme from which it is derived.

[0259] Without being bound by theory, methods and assays for measuring expressed proteins (levels) can be performed by measuring the content of the corresponding protein. Corresponding methods are well known to those skilled in the art and include Western blotting, ELISA, etc. In another embodiment, the measurement of expression (level) is performed by measuring the content of the corresponding RNA. Corresponding methods are well known to those skilled in the art and include, for example, Northern blotting.

[0260] Furthermore, methods and assays for measuring increased enzyme activity catalyzing the corresponding reaction or increased transporter activity over the enzyme and transporter, respectively, from which each was derived are known to the person skilled in the art.

[0261] Increase pantothenic acid intake

[0262] According to the present invention, the increased CoA pool can be achieved by increasing the uptake of pantothenate (i.e., the precursor of CoA) by a recombinant organism or microorganism. Preferably, the increased pantothenate uptake can be achieved by recombinant expression of a pantothenate uptake transporter.

[0263] Pantothenate uptake and pantothenate uptake transporters are known to the person skilled in the art and have been described in the prior art. In particular, pantothenate uptake and pantothenate uptake transporters in bacteria are well described (Leonardi et al. (Progress in Lipid Reasearch 44, 125-153 (2005)).

[0264] Pantothenate is known to be taken up by virtually all bacteria and is required by those that lack de novo pantothenate biosynthesis, such as Streptococcus pneumoniae, Lactobacillus lactis, and Hemophilus influenzae. In Escherichia coli, pantothenate uptake is mediated by a pantothenate permease, also known as the PanF protein, encoded by the panF gene. PanF concentrates pantothenate from the culture medium using a sodium-cotransport mechanism. PanF is predicted to contain 12 transmembrane hydrophobic domains connected by short hydrophilic chains, a characteristic topological motif of other cation-dependent permeases of the major facilitator superfamily of proteins. The transport system is highly specific for pantothenate, with a Kt of 0.4 μM and a Kt of 1.6 pmol / min / 10 8 The maximum rate of pantothenate uptake in cells. It has been found that overexpression of the PanF protein in E. coli leads to a 10-fold increase in the rate of pantothenate uptake and a concomitant increase in the steady-state intracellular concentration of pantothenate. Pantothenate effluxes from E. coli, which excretes large amounts of pantothenate that has been synthesized de novo. Pantothenate excretion is still active in psnF mutants that are unable to uptake the vitamin, indicating that uptake is unidirectional and pointing to the existence of a unique and uncharacterized efflux system.

[0265] Thus, pantothenate transporters are known from various organisms, including eukaryotic and prokaryotic organisms, such as yeast and bacteria. The enzyme has been described, for example, in Escherichia coli (where pantothenate transport is mediated by the pantothenate permease PanF (encoded by the gene panF; UniProt Accession No. P16256)), Lactobacillus lactis (Lactobacillus Lactococcus lactis subsp. Cremoris (MG1363 strain); the enzyme PanT is encoded by the gene panT; UniProt Accession No. A2RIQ0), Saccharomyces cerevisiae (strain ATCC 50106);

[0266] 204508 / S288c; enzyme Fen2 is encoded by gene fen2; UniProt accession number P25621) and Schizosaccharomyces pombe (strain 972 / ATCC 24843; enzyme Liz1 is encoded by gene liz1; UniProt accession number O43000).

[0267] Thus, in a more preferred embodiment, the pantothenate uptake transporter is an E. coli derived transporter called PanF encoded by the gene panF. Leonardi (Progress in Lipid Reasearch 44, 125-153 (2005)) describes that overexpression of PanF in E. coli results in a 10-fold increase in the rate of pantothenate uptake and is accompanied by an increase in the steady-state intracellular concentration of pantothenate. The amino acid sequence of the E. coli derived transporter is shown in SEQ ID NO:1. In other more preferred embodiments, the pantothenate uptake transporter is a Saccharomyces cerevisiae derived transporter called Fen2, a Schizosaccharomyces pombe derived transporter Liz1 or a Lactobacillus derived transporter PanT. The amino acid sequences of the Saccharomyces cerevisiae derived transporter, the Schizosaccharomyces pombe derived transporter or the Lactobacillus derived transporter are shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0268] Therefore, in a preferred embodiment of the present invention, the pantothenate uptake transporter is a transporter comprising an amino acid sequence selected from SEQ ID NOs: 1 to 4, or a sequence that is at least n% identical to any one of SEQ ID NOs: 1 to 4, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, wherein the transporter has the activity of transporting pantothenate into the cell.

[0269] With regard to the determination of sequence identity, the same applies as described above.

[0270] Increases conversion of pantothenate to CoA

[0271] According to the present invention, an increased CoA pool can be achieved by increasing the conversion of pantothenic acid to CoA. Pantothenic acid is the starting molecule for CoA biosynthesis. The biosynthesis of CoA from pantothenic acid is shown in Figure 4 In the figure, the various steps of the biosynthesis of coenzyme A (CoA) are shown. Figure 4 It can be concluded that CoA is enzymatically produced from pantothenate in the following 5 subsequent steps:

[0272] First, pantothenate is enzymatically converted to 4'-phosphopantothenate;

[0273] second, the 4'-phosphopantothenate and L-cysteine ​​are enzymatically converted into 4'-phosphopantothenate-L-cysteine;

[0274] Third, the 4'-phosphopanthenoyl-L-cysteine ​​is enzymatically converted to pantetheine-4-phosphate;

[0275] Fourth, the pantetheine-4-phosphate is enzymatically converted to desphospho-CoA; and

[0276] Fifth, the dephospho-CoA is enzymatically converted to CoA.

[0277] Figure 5 The five steps are also illustrated, while the diagram shows the key enzymes used to convert pantothenate to CoA and their respective names in bacteria, yeast and mammals. In the following, these individual steps are described in more detail.

[0278] Enzymatic conversion of pantothenate to 4'-phosphopantothenate

[0279] The enzymatic conversion of pantothenate to 4'-phosphopantothenate can be achieved by utilizing a pantothenate kinase. In a preferred embodiment, the pantothenate kinase is an enzyme classified as EC 2.7.1.33. Pantothenate kinase (EC 2.7.1.33) is an enzyme that catalyzes the following reaction:

[0280] ATP + (R)-pantothenate = ADP + (R)-4'-phosphopantothenate.

[0281] This enzyme is known from various organisms, including eukaryotic and prokaryotic organisms, such as plants, animals, fungi and bacteria. The enzyme has been described in Arabidopsis thaliana, Aspergillus nidulans, Bacillus anthracis, Bacillus subtilis, Brassicanapus, Corynebacterium ammoniagenes,

[0282] (Corynebacterium ammoniagenes), Drosophila melanogaster

[0283] melanogaster), Enterococcus faecalis (UniProt accession number Q839J7), Escherichia coli, Gorilla beringei, Helicobacter pylori, Homo sapiens, Klebsiella pneumoniae

[0284] (Klebsiella pneumoniae)(UniProt accession number B5XYG3), Lactobacillus plantarum

[0285] (Lactobacillus plantarum), Micrococcus luteus,

[0286] Morganella morganii, Mus musculus, Mycobacterium tuberculosis

[0287] (Mycobacterium tuberculosis),Orchesella cincta,Picrophilus torridus

[0288] (UniProt accession number Q6L2I5), Plasmodium falciparum, Pseudomonas aeruginosa (UniProt accession number Q9HWC1), Rattus norvegicus, Saccharomyces cerevisiae, Spinach

[0289] Pantothenate kinases are known as CoaA, Cab1, PanK, and CoaX in bacteria, yeast, and mammals, respectively; for a review, see Figure 5 .

[0290] Pantothenate kinase has been described in the literature as the rate-limiting enzyme for CoA synthesis (Liu et al., Biotechnol Biofuels 10(41)(2017)). The gene encoding pantothenate kinase is known to be reversely controlled by coenzyme A, thereby limiting its intracellular concentration. It is known that there is a mutation at position 106 of its amino acid sequence.

[0291] A mutant form of the pantothenate kinase-encoding gene CoaA derived from E. coli (i.e., R106A) is less sensitive to this feedback inhibition, thereby allowing the CoA pool to be increased in vivo, particularly by exogenous addition of a metabolic precursor, i.e., pantothenate (Rock et al., Journal of Bacteriology 18(11)

[0292] (2003); Vallari et al., Journal of Bacteriology 170(9)(1988)). In addition, overexpression of the pantothenate kinase expression gene PanK has recently been described (US 8,143,035 B2;

[0293] Vadali et al., Biotechnol. Prog. 20 (2004); Vadali et al., Metabolic Engineering 6 (2004)).

[0294] Thus, in a preferred embodiment, pantothenate kinase (EC 2.7.1.33) is an E. coli derived enzyme encoded by coaA (SEQ ID NO: 5). Thus, in a preferred embodiment of the present invention, pantothenate kinase (EC 2.7.1.33) is an amino acid sequence comprising SEQ ID NO: 5 or with

[0295] An enzyme having a sequence at least n% identical to SEQ ID NO:5, wherein n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,

[0296] 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzyme activity that converts pantothenate to 4'-phosphopantothenate.

[0297] In another preferred embodiment of the present invention, pantothenate kinase (EC 2.7.1.33) is

[0298] A variant of an enzyme derived from E. coli encoded by coaA, the enzyme comprising the amino acid sequence of SEQ ID NO:5 or a sequence at least n% identical to SEQ ID NO:5, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity of converting pantothenate into 4'-phosphopantothenate. The pantothenate kinase is characterized in that it shows a substitution, deletion and / or insertion compared to the corresponding sequence from which it is derived, preferably compared to SEQ ID NO:5, and wherein this substitution, deletion and / or insertion occurs at the position corresponding to position R106 in the amino acid sequence shown in SEQ ID NO:5.

[0299] Preferably, the pantothenate kinase variant is one in which the amino acid residue at position 106 or a position corresponding to the position in the amino acid sequence shown in SEQ ID NO:5 is deleted or substituted with alanine.

[0300] The corresponding amino acid sequence is shown in SEQ ID NO:6.

[0301] The amino acid residue at the position corresponding to the position shown in the amino acid shown in SEQ ID NO:5 can be identified by methods known in the art by those skilled in the art. For example, such amino acid residues can be identified by comparing the query sequence with the sequence shown in SEQ ID NO:5 and by identifying the position corresponding to the position of SEQ ID NO:5 shown above. The comparison can be performed in a manner and method known to those skilled in the art, for example, by using a known computer algorithm, such as the Lipman-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. Preferably, in such an comparison, the maximum homology is assigned to the conservative amino acid residues present in the amino acid sequence.

[0302] In a preferred embodiment, ClustalW2 is used for comparing amino acid sequences. In the case of a pairwise comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap opening: 10; gap extension: 0.1. In the case of a multiple comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap opening: 10; gap extension: 0.2; gap distance: 5; no end gaps.

[0303] Preferably, the degree of identity is calculated over the entire length of the sequence.

[0304] Therefore, in a preferred embodiment of the present invention, the pantothenate kinase is an enzyme comprising the amino acid sequence of SEQ ID NO:6 or a sequence that is at least n% identical to SEQ ID NO:6, n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has enzymatic activity for converting pantothenate into 4'-phosphopantothenate.

[0305] In addition, the pantothenate kinase CoaA from Staphylococcus aureus (Choudhry et al., Antimocrobial Agents and Chemotherapy 47 (6) (2003)) and the putative pantothenate kinase CoaA from Bacillus anthracis are moderately related to the eukaryotic panK protein and are not related to E. coli CoaA. In particular, the CoaA from Staphylococcus aureus is not inhibited by CoA, and this organism is known to have a considerable CoA pool. For CoaA from Staphylococcus aureus, see also

[0306] http: / / parts.igem.org / Part:BBa_K1692021 .

[0307] Furthermore, it is known that the gene CoaX from Helicobacter pylori and Bacillus subtilis is not affected by CoA and acetyl-CoA (Brand and Strauss, Journal of Biological Chemistry 21 (2005)).

[0308] Thus, in preferred embodiments, the pantothenate kinase (EC 2.7.1.33) is a Staphylococcus aureus derived enzyme encoded by coaA (SEQ ID NO:7), a Helicobacter pylori derived enzyme encoded by coaX (SEQ ID NO:8), or a Bacillus subtilis derived enzyme encoded by coaX (SEQ ID NO:9).

[0309] Therefore, in a preferred embodiment of the present invention, the pantothenate kinase (EC 2.7.1.33) is an enzyme comprising the amino acid sequence of SEQ ID NOs: 7 to 9, or a sequence that is at least n% identical to any one of SEQ ID NOs: 7 to 9, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has enzymatic activity for converting pantothenate into 4'-phosphopantothenate.

[0310] Enzymatic conversion of 4'-phosphopantothenate and L-cysteine ​​to 4'-phosphopantothenoyl-L-cysteine

[0311] According to the present invention, the increased conversion of pantothenate to CoA can be achieved by recombinant expression of an enzyme that enzymatically converts 4'-phosphopantothenate and L-cysteine ​​into 4'-phosphopantothenyl-L-cysteine, preferably by utilizing phosphopantothenylcysteine ​​synthetase. The phosphopantothenylcysteine ​​synthetase is preferably an enzyme classified as EC 6.3.2.5. Phosphopantothenylcysteine ​​synthetase (EC 6.3.2.5), also known as phosphopantothenate-cysteine ​​ligase (CTP), is an enzyme that catalyzes the following reaction:

[0312] CTP + (R)-4'-phosphopantothenic acid + L-cysteine ​​= CMP + diphosphate + N-[(R)-4'-phosphopantothenoyl]-L-cysteine

[0313] This enzyme is known from various organisms, including eukaryotic and prokaryotic organisms, such as animals and bacteria. The enzyme has been described, for example, in Anas platyrhynchos, Corynebacterium ammoniagenes, Drosophila melanogaster, Enterococcus faecalis, Escherichia coli, Homo sapiens, Methanocaldococcus jannaschii (UniProt accession number Q58323), Micrococcus luteus, Morganella morganii, Pseudomonas aeruginosa, Rattus norvegicus, and Saccharomyces cerevisiae. In yeast and mammals, phosphopantetheine synthetase is called Cab2 and PPCS, respectively; for a summary, see Figure 5 In E. coli and most bacteria, the synthesis of pantetheine-4-phosphate (corresponding to step 3 of CoA biosynthesis described further below) from 4'-phosphopantothenate and L-cysteine ​​is catalyzed by the bifunctional Dfp (also called CoaBC) flavoprotein in a multistep process (for a summary, see Figure 5 ). The two enzymatic activities, PPCS and PPCDS, are located within the C-terminal and N-terminal domains of the bifunctional (fusion) protein.

[0314] In contrast, bacteria of the genera Streptococcus and Enterococcus have two separate open reading frames (PRFs) predicted to encode CoaB and CoaC (Gerdes, Journal of Bacteriology 184(16) (2002)).

[0315] Phosphopantetheine synthetase (EC 6.3.2.5) has also been described, for example, in Saccharomyces cerevisiae.

[0316] Thus, in a preferred embodiment, the phosphopantetheine synthetase (EC 6.3.2.5) is an Enterococcus derived enzyme encoded by coaB (SEQ ID NO: 10) or a Saccharomyces cerevisiae derived enzyme encoded by cab2 (SEQ ID NO: 11). Therefore, in a preferred embodiment of the present invention, the phosphopantothenoylcysteine ​​synthetase (EC 6.3.2.5) is an enzyme comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or a sequence at least n% identical to SEQ ID NO: 10 or SEQ ID NO: 11, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity for converting 4'-phosphopantothenate and L-cysteine ​​into 4'-phosphopantothenoyl-L-cysteine. With regard to the determination of sequence identity, the same applies as described above.

[0317] As described above, in E. coli and most eubacteria, as well as, for example, S. pneumoniae, the synthesis of pantetheine-4-phosphate (corresponding to step 3 of CoA biosynthesis described further below) from 4'-phosphopantothenate and L-cysteine ​​is catalyzed by the bifunctional Dfp (also known as CoaBC) flavoprotein in a multistep process (for a summary, see Figure 5). Thus, in a preferred embodiment, the bifunctional enzyme having phosphopantotheine synthetase / 4'-phosphopantotheine decarboxylase activity is the E. coli derived enzyme CoaBC (SEQ ID NO: 12) or the Streptococcus pneumoniae derived enzyme CoaBC (SEQ ID NO: 13). Therefore, in a preferred embodiment of the present invention, the bifunctional enzyme having phosphopantotheine synthetase / 4'-phosphopantotheine decarboxylase activity is an enzyme comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or a sequence at least n% identical to SEQ ID NO: 12 or SEQ ID NO: 13, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity for converting 4'-phosphopantothenate and L-cysteine ​​into pantetheine-4-phosphate. With regard to the determination of sequence identity, the same applies as described above. Structural information and site-directed mutagenesis data are available for the E. coli derived bifunctional protein CoaBC (Dfp) (Kupke, J. Biol. Chem 276 (2001); Kupke, Eur. J. Biochem. 271 (2004)).

[0318] Enzymatic conversion of 4'-phosphopanthenoyl-L-cysteine ​​to pantetheine-4-phosphate

[0319] According to the present invention, the increased conversion of pantothenate to CoA can be achieved by recombinant expression of an enzyme that enzymatically converts 4'-phosphopantothenyl-L-cysteine ​​to pantetheine-4-phosphate, preferably utilizing 4'-phosphopantothenylcysteine ​​decarboxylase. In a preferred embodiment, the 4'-phosphopantothenylcysteine ​​decarboxylase is an enzyme classified as EC 4.1.1.36. 4'-phosphopantothenylcysteine ​​decarboxylase (EC 4.1.1.36), also known as phosphopantothenylcysteine ​​decarboxylase, is an enzyme that catalyzes the following reaction:

[0320] N-[(R)-4'-phosphopanthenoyl]-L-cysteine ​​= pantetheine 4'-phosphate + CO2

[0321] This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals and bacteria. This enzyme has been described, for example, in Anas platyrhynchos, Corynebacterium ammoniagenes, Bos taurus, Drosophila melanogaster, Enterococcus faecalis, Equus caballus, Escherichia coli, Homo sapiens, Methanocaldococcus jannaschii, Micrococcus luteus, Morganella morganii, Mycobacterium tuberculosis, Nicotiana tabacum, Oryctolagus cuniculus, Oryza sativa, Rattus norvegicus, Saccharomyces cerevisiae and Sus scrofa. In yeast and mammals, 4'-phosphopantetheine decarboxylase is called Cab3 and PPCDC, respectively; for a review, see Figure 5 .

[0322] As described above, in the context of step 2 of CoA biosynthesis, bacteria of the genera Streptococcus and Enterococcus have genes predicted to encode CoaB and CoaC separately (Genschel, Molecular Biology and Evolution 21(7) (2004)).

[0323] 4'-Phosphopantothenoylcysteine ​​decarboxylase (EC 4.1.1.36) has also been described in Saccharomyces cerevisiae.

[0324] Thus, in a preferred embodiment, 4'-phosphopantothenylcysteine ​​decarboxylase (EC

[0325] 4.1.1.36) is an Enterococcus derived enzyme encoded by coaC (SEQ ID NO: 14) or a Saccharomyces cerevisiae derived enzyme encoded by cab4 (SEQ ID NO: 15). Therefore, in a preferred embodiment of the present invention, 4'-phosphopantothenoylcysteine ​​decarboxylase (EC 4.1.1.36) is an enzyme comprising SEQ ID NO:

[0326] NO:14 or SEQ ID NO:15 or the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15

[0327] NO: 15 An enzyme having at least n% identical sequence, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity of converting 4'-phosphopanthenoyl-L-cysteine ​​into pantetheine-4-phosphate. Regarding the determination of sequence identity, the same applies as described above.

[0328] As described above, in E. coli and most eubacteria, the synthesis of pantetheine-4-phosphate is catalyzed by the bifunctional Dfp (also known as CoaBC) from 4'-phosphopantothenate and L-cysteine ​​in a multistep process (for a summary, see Figure 5 ). Therefore, with regard to the preferred embodiments of the bifunctional enzyme having phosphopantotheine synthetase / 4'-phosphopantotheine decarboxylase activity, the same applies as listed above in the context of step 3 of CoA biosynthesis.

[0329] Enzymatic conversion of pantetheine-4-phosphate to desphospho-CoA

[0330] According to the present invention, increased conversion of pantothenate to CoA is achieved by recombinant expression of an enzyme that catalyzes the enzymatic conversion of pantetheine-4-phosphate to dephospho-CoA, preferably utilizing pantetheine-phosphate adenylyltransferase. In a preferred embodiment, the pantetheine-phosphate adenylyltransferase is an enzyme classified as EC 2.7.7.3.

[0331] 2.7.7.3), also known as pantetheine-phosphate adenylyltransferase, is an enzyme that catalyzes the following reaction:

[0332] ATP + pantetheine 4'-phosphate = diphosphate + 3'-dephospho-CoA

[0333] This enzyme is known from a variety of organisms, including eukaryotic and prokaryotic organisms such as plants, animals and bacteria. The enzyme has been described, for example, in Anas sp., Arabidopsis thaliana, Burkholderia pseudomallei (UniProt Accession No. Q3JW91), Candida albicans (UniProt Accession No. B9WCR8), Columba sp., Corynebacterium ammoniagenes, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Haemophilus influenzae (UniProt Accession No. P44805), Helicobacter pylori (UniProt Accession No. O26010), Homo sapiens, Micrococcus luteus, Mycobacterium tuberculosis. tuberculosis) (UniProt accession number P9WPA5), Pseudomonas aeruginosa (UniProt accession number Q9I6D1), Pyrococcus abyssi, Rattus norvegicus, Staphylococcus aureus (UniProt accession number P63820), Streptococcus mutans (UniProt accession number Q8DVH2), Streptococcus pneumoniae (UniProt accession number Q8DNE6), Streptococcus pyogenes, Sus scrofa, and Thermus thermophilus. In bacteria and yeast, pantetheine-phosphate adenylyltransferase is called CoaD and Cab4, respectively. This enzyme is also called PPAT and KdtB; for a review, see. Figure 5 .

[0334] It is known that CoaD from E. coli is feedback inhibited by CoA, which competes with ATP, pantetheine-4-phosphate (its substrate), and dephospho-CoA (its product) (Leonardi et al., Progress in Lipid Research 44:125-153 (2005)).

[0335] Thus, in a preferred embodiment, the pantetheine-phosphate adenylyltransferase (EC 2.7.7.3) is the E. coli (Uniprot No. P0A616) derived enzyme encoded by coaD.

[0336] Therefore, in a preferred embodiment of the present invention, pantetheine-phosphate adenylyltransferase (EC 2.7.7.3) is an enzyme comprising the amino acid sequence of SEQ ID NO: 18 or a sequence at least n% identical to SEQ ID NO: 18, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzyme activity for converting pantetheine-4-phosphate into dephospho-CoA. With regard to the determination of sequence identity, the same applies as described above.

[0337] As mentioned above, S. aureus is known to have a considerable CoA pool. Therefore, in a preferred embodiment, the pantetheine-phosphate adenylyltransferase (EC 2.7.7.3) is an enzyme derived from S. aureus (strain MW2) (Uniprot No. P63820) encoded by coaD (SEQ ID NO: 16) or an enzyme derived from Burkholderia pseudomallei (strain 1710b) (Uniprot No. Q3JW91) encoded by coaD. Thus, in a preferred embodiment of the present invention, pantetheine-phosphate adenylyltransferase (EC 2.7.7.3) is an enzyme comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17 or a sequence at least n% identical to SEQ ID NO: 16 or SEQ ID NO: 17, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity of converting pantetheine-4-phosphate into dephospho-CoA. With regard to the determination of sequence identity, the same applies as described above. The amino acid sequence of CoaD from Staphylococcus aureus (strain MW2) (Uniprot No. P63820) and the amino acid sequence of CoaD from Burkholderia pseudomallei (strain 1710b) (Uniprot No. Q3JW91) have a sequence identity of about 47%.

[0338] Dephospho-CoA is enzymatically converted to CoA

[0339] According to the present invention, increased conversion of pantothenate to CoA is achieved by recombinant expression of an enzyme that enzymatically converts dephospho-CoA to CoA, preferably utilizing dephospho-CoA kinase.

[0340] In a preferred embodiment, the dephospho-CoA kinase is an enzyme classified as EC 2.7.1.24. Dephospho-CoA kinase (EC 2.7.1.24) is an enzyme that catalyzes the following reaction:

[0341] ATP + 3'-dephospho-CoA = ADP + CoA

[0342] This enzyme is known from various organisms, including eukaryotic and prokaryotic organisms, such as plants, animals and bacteria. The enzyme has been described, for example, in Anas platyrhynchos, Aquifex aeolicus, Columba sp., Corynebacterium ammoniagenes, Escherichia coli (UniProt Accession No. P0A619), Haemophilus influenzae (UniProt Accession No. P44920), Homo sapiens, Legionella pneumophila (UniProt Accession No. Q5ZVH3), Micrococcus luteus, Mycobacterium tuberculosis, Rattus norvegicus, Saccharomyces cerevisiae, Streptomyces peucetius

[0343] (UniProt accession number D5MBE3), Sus scrofa, and Thermus thermophilus. In bacteria and yeast, the dephospho-CoA kinase is called CoaE and Cab5, respectively. This enzyme is also called YacE; for a summary, see Figure 5 .

[0344] In a preferred embodiment, pantothenate kinase (EC 2.7.124) is an Escherichia coli derived enzyme encoded by coaE (SEQ ID NO: 19) or an enzyme derived from Saccharomyces cerevisiae encoded by cab5 (SEQ ID NO: 20). Therefore, in a preferred embodiment of the present invention, dephospho-CoA kinase (EC 2.7.1.24) is an enzyme comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20 or a sequence at least n% identical to SEQ ID NO: 19 or SEQ ID NO: 20, n being an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has an enzymatic activity of converting dephospho-CoA into CoA. With regard to the determination of sequence identity, the same applies as described above. The structure of the E. coli derived dephospho-CoA kinase (EC 2.7.1.24) has been described (O'Toole et al., Protein Science 12 (2003)).

[0345] Increased production and flux of acetyl-CoA

[0346] In a preferred embodiment, the recombinant organism or microorganism of the present invention is further characterized by:

[0347] a) having phosphoketolase activity;

[0348] b) (i) having a reduced or inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation of the gene encoding phosphofructokinase or by reducing the phosphofructokinase activity compared to an unmodified microorganism; or

[0349] (ii) No phosphofructokinase activity

[0350] and

[0351] c) (i) having a reduced or inactivated oxidative branch of the pentose phosphate pathway (PPP) by inactivating the enzyme encoding glucose-6-phosphate dehydrogenase or by reducing the activity of glucose-6-phosphate dehydrogenase compared to a non-modified microorganism; or

[0352] (ii) It has no glucose-6-phosphate dehydrogenase activity.

[0353] Corresponding recombinant organisms or microorganisms have recently been described in WO 2013 / 007786. The disclosure of this document, in particular with regard to the preferred embodiments of the enzymes described therein, is incorporated by reference in its entirety. Therefore, in a preferred embodiment, an enzyme selected from the preferred embodiments described in WO 2013 / 007786 is preferably used.

[0354] The term "phosphoketolase activity" as used in the present invention refers to an enzyme activity capable of converting D-xylulose-5-phosphate into D-glyceraldehyde-3-phosphate according to the following reaction:

[0355] D-xylulose-5-phosphate + phosphoric acid → D-glyceraldehyde-3-phosphate + acetyl-phosphate + water

[0356] Or an enzyme activity that is capable of catalyzing the reaction shown above and is also capable of converting D-fructose-6-phosphate into D-erythrose-4-phosphate according to the following reaction:

[0357] D-fructose-6-phosphate + phosphoric acid → acetyl phosphate + D-erythrose-4-phosphate + water

[0358] The former phosphoketolase is usually classified as EC 4.1.2.9, while the latter is classified as EC

[0359] 4.1.2.22. Both types of phosphoketolase can be used within the scope of the present invention. This enzyme activity can be measured by assays known in the art. Examples of such assays are given in WO 2013 / 007786.

[0360] In the context of the present invention, an organism or microorganism having phosphoketolase activity may be, for example, an organism or microorganism having phosphoketolase activity naturally or an organism or microorganism not having phosphoketolase activity naturally and having been genetically modified to express phosphoketolase or an organism or microorganism having phosphoketolase activity naturally and having been genetically modified (e.g., transformed with a nucleic acid encoding phosphoketolase (e.g., a vector)) to increase the phosphoketolase activity in the microorganism. Organisms or microorganisms having phosphoketolase activity intrinsically (i.e., naturally) are known in the art and any of them may be used in the context of the present invention. It is also possible in the context of the present invention that an organism or microorganism is an organism or microorganism that does not have phosphoketolase activity naturally but is genetically modified to contain a nucleotide sequence that allows phosphoketolase expression. Similarly, an organism or microorganism may also be an organism or microorganism that has phosphoketolase activity naturally but is genetically modified to enhance phosphoketolase activity (e.g., an exogenous nucleotide sequence encoding phosphoketolase by a primer). The genetic modification of an organism or microorganism to express a target enzyme will be described in detail below.

[0361] The phosphoketolase expressed in the organism or microorganism according to the present invention can be a naturally occurring phosphoketolase or can be a phosphoketolase derived from a naturally occurring phosphoketolase, for example, by introducing a mutation or other change, which for example changes or improves enzyme activity, stability, etc. Therefore, in a preferred embodiment, the recombinant organism or microorganism of the present invention is an organism or microorganism that has been genetically modified to have an improved phosphoketolase activity. Methods for changing and / or improving the desired enzymatic activity of a protein are well known to those skilled in the art and include for example random mutagenesis or site-directed mutagenesis and subsequent selection of an enzyme of the desired property or the so-called "directed evolution" method described in further detail below.

[0362] The organism or microorganism according to the invention is preferably further characterized by having a reduced or inactivated Embden-Meyerhof-Parnas pathway (EMPP), which is achieved by inactivating the gene encoding phosphofructokinase or by reducing the phosphofructokinase activity compared to an unmodified organism or microorganism or by having no phosphofructokinase activity. Thus, the organism or microorganism is an organism or microorganism that naturally has an EMPP including phosphofructokinase activity but has been modified (in particular genetically modified) such that the phosphofructokinase activity is completely eliminated or reduced compared to a corresponding non-modified organism or microorganism, or the organism or microorganism is an organism or microorganism that naturally has no phosphofructokinase activity.

[0363] Phosphofructokinase activity refers to the conversion of ATP and fructose-6-phosphate into ADP and fructose-

[0364] 1,6-diphosphodiesterase activity (EC 2.7.1.11). This enzyme activity can be measured by assays known in the art, for example, as described by Kotlarz et al. (Methods Enzymol. (1982) 90, 60-70).

[0365] The term "an organism or microorganism characterized by having a reduction or inactivation of the Embden-Meyerhof-Parnas pathway (EMPP), which is achieved by inactivating the gene encoding phosphofructokinase or by reducing the phosphofructokinase activity compared to an unmodified organism or microorganism" preferably refers to an organism or microorganism wherein the gene encoding phosphofructokinase is inactivated or the phosphofructokinase activity is reduced compared to a non-modified organism or microorganism, which is achieved by a genetic modification of the organism or microorganism resulting in said inactivation or reduction.

[0366] In a preferred embodiment, the recombinant organism or microorganism of the present invention is a recombinant organism or microorganism having an inactivated Embden-Meyerhof-Parnas pathway by inactivating the gene encoding phosphofructokinase. Inactivation of the gene encoding phosphofructokinase in the context of the present invention means that the gene encoding phosphofructokinase present in the organism or microorganism is inactivated so that they are no longer expressed and / or no longer lead to the synthesis of functional phosphofructokinase. Inactivation can be achieved by many different approaches known in the art. Inactivation can be achieved, for example, by destroying the gene encoding phosphofructokinase or completely deleting the gene by introducing a selection marker. Alternatively, the promoter of the gene encoding phosphofructokinase can be mutated in such a way that the gene is no longer transcribed into mRNA. Other ways of inactivating the gene encoding phosphofructokinase known in the art are: expressing a polynucleotide encoding an RNA having a nucleotide sequence complementary to the transcription product of the phosphofructokinase gene so that the mRNA is no longer translated into protein, to express a polynucleotide encoding an RNA that suppresses the expression of the gene through the RNAi effect; expressing a polynucleotide encoding an RNA having the activity of specifically cutting the transcription product of the gene; or expressing a polynucleotide encoding an RNA that suppresses the expression of the gene through a co-suppression effect. These polynucleotides can be incorporated into vectors that can be introduced into organisms or microorganisms by transformation to achieve inactivation of the gene encoding phosphofructokinase.

[0367] The term "inactivated" in the context of the present invention preferably means complete inactivation, ie the organism or microorganism exhibits no phosphofructokinase activity. This means in particular that independently of the growth conditions used, the organism or microorganism exhibits no phosphofructokinase activity.

[0368] Preferably, "inactivation" means that the gene encoding phosphofructokinase present in the organism or microorganism is genetically modified so that the expression of the enzyme is prevented. This can be achieved, for example, by deleting the gene or a part thereof, wherein the deletion of the part thereof prevents the expression of the enzyme, or by disrupting the gene in the coding region or the promoter region, wherein the disruption has the effect that the protein is not expressed or that a malfunctioning protein is expressed.

[0369] In a preferred embodiment, the recombinant organism or microorganism of the present invention is a recombinant organism or microorganism with a reduced Embden-Meyerhof-Parnas pathway (EMPP) by reducing the phosphofructokinase activity compared to an unmodified organism or microorganism. Preferably, this reduction is achieved by genetic modification of the organism or microorganism. This can be achieved, for example, by random mutagenesis or site-directed mutagenesis of promoters and / or enzymes and subsequent selection of promoters and / or enzymes with the desired properties, or by complementary nucleotide sequences as described above or RNAi effects.

[0370] In the context of the present invention, "reduced activity" means that the expression and / or activity of an enzyme (particularly phosphofructokinase) in a genetically modified organism or microorganism is at least 10% lower, preferably at least 20% lower, more preferably at least 30% or 50% lower, even more preferably at least 70% or 80% lower and particularly preferably at least 90% or 100% lower than in a corresponding unmodified organism or microorganism. Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. Assays for measuring reduced enzymatic activity of phosphofructokinase are known in the art.

[0371] In another embodiment, the organism or microorganism according to the present invention is an organism or microorganism that does not have phosphofructokinase activity. This preferably means that such an organism or microorganism does not naturally have phosphofructokinase activity. This means that such an organism or microorganism naturally does not contain a nucleotide sequence encoding an enzyme with phosphofructokinase activity in its genome. Examples of such microorganisms are Zymomonas mobilis (JS Suo et al., Nat. Biotechnol. 23: 63 (2005)) and Ralstonia eutropha (C. Fleige et al., Appl. Microb. Cell Physiol. 91: 769 (2011)).

[0372] Organism or microorganism according to the present invention is preferably further characterized by the oxidation branch of the pentose phosphate pathway (PPP) with reduction or inactivation, which is realized by the gene of inactivation coding glucose-6-phosphate dehydrogenase or by reducing glucose-6-phosphate dehydrogenase activity compared with unmodified organism or microorganism or by not having glucose-6-phosphate dehydrogenase activity. Therefore, organism or microorganism is a natural PPP that has a glucose-6-phosphate dehydrogenase but has been modified (particularly genetically modified) so that glucose-6-phosphate dehydrogenase activity is completely eliminated or reduced compared with the corresponding unmodified organism or microorganism, or organism or microorganism is a natural organism or microorganism that does not have glucose-6-phosphate dehydrogenase activity.

[0373] Glucose-6-phosphate dehydrogenase activity refers to the activity of glucose-6-phosphate dehydrogenase + Enzyme activity (EC 1.1.1.49) that converts into 6-phosphoglucono-δ-lactone and NADPH. This enzyme activity can be measured by assays known in the art, such as, for example, those described by Noltman et al. (J. Biol. Chem. (1961) 236, 1225-1230).

[0374] The term "an organism or microorganism characterized by having a reduced or inactivated oxidative branch of the pentose phosphate pathway (PPP), by inactivating the gene encoding glucose-6-phosphate dehydrogenase or by reducing the activity of glucose-6-phosphate dehydrogenase compared to an unmodified organism or microorganism" preferably refers to an organism or microorganism in which the gene encoding glucose-6-phosphate dehydrogenase is inactivated or the activity of glucose-6-phosphate dehydrogenase is reduced compared to an unmodified organism or microorganism, by genetic modification of the organism or microorganism resulting in said reduced inactivation.

[0375] In a preferred embodiment, recombinant organism or microorganism of the present invention is a recombinant organism or microorganism having an oxidative branch of the pentose phosphate pathway (PPP) of inactivation by the gene of inactivation coding glucose-6-phosphate dehydrogenase. In the context of the present invention, the inactivation of the gene of coding glucose-6-phosphate dehydrogenase means that the gene of glucose-6-phosphate dehydrogenase in the organism or microorganism is inactivated so that they are no longer expressed and / or no longer cause the synthesis of functional glucose-6-phosphate dehydrogenase. Inactivation can be achieved by many different approaches known in the art. Inactivation can be achieved by, for example, destroying the gene of coding glucose-6-phosphate dehydrogenase or thoroughly deleting the gene by introducing a selection marker. Alternatively, the promoter of the gene of the gene of glucose-6-phosphate dehydrogenase can be mutated in a way that the gene is no longer transcribed into mRNA. Other ways of inactivating genes encoding phosphofructokinase known in the art are: expressing a polynucleotide encoding an RNA having a nucleotide sequence complementary to the transcription product of the glucose-6-phosphate dehydrogenase gene, so that the mRNA is no longer translated into protein, to express a polynucleotide encoding an RNA that suppresses the expression of the gene through the RNAi effect; expressing a polynucleotide encoding an RNA having the activity of specifically cutting the transcription product of the gene; or expressing a polynucleotide encoding an RNA that suppresses the expression of the gene through a common suppression effect. These polynucleotides can be incorporated into a vector, and the vector can be introduced into an organism or microorganism by transformation to achieve the inactivation of the gene encoding glucose-6-phosphate dehydrogenase.

[0376] The term "inactivation" in the context of the present invention preferably means complete inactivation, i.e., the organism or microorganism does not show glucose-6-phosphate dehydrogenase activity. This particularly means that regardless of the growth conditions used, the organism or microorganism does not show glucose-6-phosphate dehydrogenase activity.

[0377] Preferably, "inactivation" means that the gene encoding glucose-6-phosphate dehydrogenase present in an organism or microorganism is genetically modified so as to prevent the expression of the enzyme. This can be achieved by, for example, deleting the gene or part thereof, wherein the deletion of its part prevents the expression of the enzyme, or by destroying the gene in the coding region or promoter region, wherein the destruction has the effect of not expressing the protein or expressing a malfunctioning protein.

[0378] In a preferred embodiment, recombinant organism of the present invention or microorganism is by reducing glucose-6-phosphate dehydrogenase activity compared with unmodified organism or microorganism and has the recombinant organism of the oxidation branch of pentose phosphate pathway (PPP) of reduction or microorganism.Preferably, realize this reduction by the genetic modification of organism or microorganism.This can realize by for example random mutagenesis or site-directed mutagenesis of promoter and / or enzyme and select promoter and / or enzyme with desired characteristic subsequently, or realize by complementary nucleotide sequence as above or RNAi effect.Below will further provide the detailed description of the genetic modification of organism or microorganism.

[0379] In the context of the present invention, "reduced activity" means that the expression and / or activity of the enzyme (particularly glucose-6-phosphate dehydrogenase) in the genetically modified organism or microorganism is at least 10% lower than that in the corresponding unmodified organism or microorganism, preferably at least 20% lower, more preferably at least 30% or 50% lower, even more preferably at least 70% or 80% lower and particularly preferably at least 90% or 100% lower. The method for measuring the expression level of a given protein in a cell is well known to those skilled in the art. The assay for measuring the enzymatic activity that glucose-6-phosphate dehydrogenase reduces is known in the art.

[0380] In another embodiment, the organism or microorganism according to the present invention is an organism or microorganism that does not have glucose-6-phosphate dehydrogenase activity. This preferably means that such an organism or microorganism does not naturally have glucose-6-phosphate dehydrogenase activity. This means that such an organism or microorganism naturally does not contain a nucleotide sequence encoding an enzyme with glucose-6-phosphate dehydrogenase activity in its genome. Examples of such microorganisms are Acinetobacter baylyi (Barbe et al., Nucl. Acids Res. 32 (2004), 5766-5779), hyperthermophilic archaea, such as Sulfolobus solfataricus (Nunn et al., J. Biol. Chem. 285 (2010), 33701-33709), Thermoproteus tenax, Thermoplasma acidophilum and Picrophilus torridus (Reher and FEBS Lett. 580(2006), 1198-1204).

[0381] In a further embodiment, the organism or microorganism according to the invention is further characterized by having fructose-1,6-bisphosphate phosphatase activity, preferably when grown on glucose. Fructose-1,6-bisphosphate phosphatase is an enzyme involved in the gluconeogenic hydrolysis of fructose-1,6-bisphosphate into fructose-6-phosphate and free phosphate. However, in essentially all organisms in the presence of glucose, fructose-1,6-bisphosphate phosphatase activity is inhibited and glucose is processed by EMPP (glycolysis). For an organism or microorganism of the invention having phosphoketolase activity but not having phosphofructokinase activity or wherein the phosphofructokinase activity is reduced or its gene encoding phosphofructokinase is inactivated, the yield of acetyl-CoA from fructose-6-phosphate converted with phosphoketolase (EC 4.1.2.9 or EC 4.1.2.22) can be enhanced by ensuring the presence of fructose-1,6-bisphosphate phosphatase activity, for example by removing the suppression of fructose-1,6-bisphosphate phosphatase activity in the presence of glucose. The presence of fructose-1,6-bisphosphate phosphatase results in the recycling of fructose-1,6-bisphosphate produced by fructose-1,6-bisphosphate aldolase to fructose-6-phosphate, which can then be converted again to acetyl-CoA by the phosphoketolase pathway. In fact, acetyl phosphate, the product of phosphoketolase, is interconverted into acetyl-CoA by the action of phosphate acetyltransferase EC 2.3.1.8. Thus, the recombinant organism or microorganism of the present invention is capable of producing acetyl-CoA from glucose at a stoichiometry close to 3:1.

[0382] The term "fructose-1,6-bisphosphate phosphatase activity" refers to an enzyme activity (EC 3.1.3.11) that converts fructose-1,6-bisphosphate and H2O into fructose-6-phosphate and phosphate. This enzyme activity can be measured by assays known in the art, for example, as described by Hines et al. (J. Biol. Chem. (2007) 282, 11696-11704). The terms "fructose-1,6-bisphosphate phosphatase activity" and "fructose-1,6-bisphosphate phosphatase" also include enzymes that are bifunctional in the sense that they exhibit fructose-1-6-bisphosphate aldolase / phosphatase activity. Such bifunctional enzymes are expressed in most archaea and deeply branched bacterial lineages and, in most cases, are thermostable. For example, such enzymes have been reported for Thermococcus kodakaraensis, Sulfolobus tokodaii, Ignicoccus hospitalis, Cenarchaeum symbiosum, Sulfolobus solfataricas, Thermus thermophilus, Thermoproteus neutrophilus, Moorella thermoacetica, and many others (see, e.g., Say and Fuchs (Nature 464 (2010), 1077); Fushinobu et al. (Nature 478 (2011), 538; Du et al. (Nature 478 (2011), 534).

[0383] The term "fructose-1,6-bisphosphate phosphatase activity when grown on glucose" means that the organism or microorganism expresses an enzyme having fructose-1,6-bisphosphate phosphatase activity when the organism or microorganism is grown on glucose. "Growing on glucose" means that the organism or microorganism is grown in a medium containing, among other things, glucose as a carbon source. Preferably, the term means that the organism or microorganism is grown in a medium containing glucose as the sole carbon source.

[0384] In the context of the present invention, an organism or microorganism having fructose-1,6-bisphosphate phosphatase activity, in particular when grown on glucose, may for example be an organism or microorganism that naturally has fructose-1,6-bisphosphate phosphatase activity, in particular when grown on glucose, or an organism or microorganism that does not naturally have fructose-1,6-bisphosphate phosphatase activity (in particular when grown on glucose) but has been genetically modified to express fructose-1,6-bisphosphate phosphatase (in particular when grown on glucose). It may also be an organism or microorganism that naturally has fructose-1,6-bisphosphate phosphatase activity (in particular when grown on glucose) and has been genetically modified (e.g., transformed with a nucleic acid (e.g., a vector) encoding fructose-1,6-bisphosphate phosphatase) to increase the phosphoketolase activity in the organism or microorganism. Organisms or microorganisms that intrinsically (i.e., naturally) have fructose-1,6-bisphosphate phosphatase activity, in particular when grown on glucose, are known in the art, and any of them may be used in the context of the present invention.

[0385] It is also possible in the context of the present invention that the organism or microorganism is an organism or microorganism that naturally does not have fructose-1,6-bisphosphate phosphatase activity (especially when grown on glucose) but which has been genetically modified to be able to express fructose-1,6-bisphosphate phosphatase (especially when grown on glucose). This can be achieved, for example, by mutating the promoter of the gene encoding fructose-1,6-bisphosphate phosphatase in such a way that the gene is no longer repressed when the organism or microorganism is grown on glucose or by replacing the promoter with another promoter (for example, a non-regulated constitutive promoter) when the organism or microorganism is grown on glucose.

[0386] Similarly, the organism or microorganism may also be an organism or microorganism that naturally has fructose-1,6-bisphosphate phosphatase activity (particularly when grown on glucose) but which has been genetically modified to enhance / increase fructose-1,6-bisphosphate phosphatase activity (particularly when grown on glucose), for example, by introducing an exogenous nucleotide sequence encoding fructose-1,6-bisphosphate phosphatase.

[0387] The genetic modification of an organism or microorganism to express an enzyme of interest will be described in detail below.

[0388] The fructose-1,6-bisphosphate phosphatase according to the present invention may be a naturally occurring fructose-1,6-bisphosphate phosphatase or may be a fructose-1,6-bisphosphate phosphatase derived from a naturally occurring fructose-1,6-bisphosphate phosphatase, for example, by introducing a mutation or other change that, for example, changes or improves enzyme activity, stability, etc. Methods for changing and / or improving the desired enzyme activity of a protein are well known to those skilled in the art and have been described above. The resulting fructose-1,6-bisphosphate phosphatase variant is then tested for properties, for example, enzyme activity or regulation. Assays for measuring the enzyme activity of fructose-1,6-bisphosphate phosphatase are known in the art. In one embodiment, the fructose-1,6-bisphosphate phosphatase is an enzyme that is not regulated by feedback inhibition.

[0389] In a preferred embodiment, the recombinant organism or microorganism has been genetically modified to have an increased fructose-1,6-bisphosphate phosphatase activity. This can be achieved, for example, by transforming the organism or microorganism with a nucleic acid encoding fructose-1,6-bisphosphate phosphatase. A detailed description of the genetic modification of the organism or microorganism will be further provided below.

[0390] In the context of the present invention, "increased activity" means that the expression and / or activity of an enzyme (particularly fructose-1,6-bisphosphate phosphatase) in a genetically modified organism or microorganism is at least 10% higher, preferably at least 20% higher, more preferably at least 30% or 50% higher, even more preferably at least 70% or 80% higher, and particularly preferably at least 90% or 100% higher when grown in glucose than a corresponding unmodified organism or microorganism. In even more preferred embodiments, the increase in expression and / or activity can be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments, expression is at least 10 times higher, more preferably at least 100 times higher and even more preferably at least 1000 times higher than a corresponding unmodified organism or microorganism (particularly when grown in glucose).

[0391] Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. In one embodiment, the expression level is measured by measuring the content of the corresponding protein. Corresponding methods are well known to those skilled in the art and include Western blotting, ELISA, etc. In another embodiment, the expression level is measured by measuring the content of the corresponding RNA. Corresponding methods are well known to those skilled in the art and include, for example, Northern blotting.

[0392] Methods for measuring the enzyme activity of fructose-1,6-bisphosphate are known in the art.

[0393] In another embodiment, a further feature of the organism or microorganism according to the invention is that EMPP is further reduced or inactivated by inactivating a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity compared to an unmodified organism or microorganism. Further reducing EMPP by reducing or inactivating glyceraldehyde 3-phosphate dehydrogenase in a further downstream step ensures that no or almost no glyceraldehyde-3-phosphate is produced in the organism or microorganism for processing to acetyl-CoA via glycolysis, thereby losing a carbon atom by the release of CO2 in the last step catalyzed by pyruvate dehydrogenase. Therefore, blocking EMPP by reducing or inactivating glyceraldehyde 3-phosphate dehydrogenase activity further ensures that the overall flux is directed to phosphoketolase.

[0394] "Glyceraldehyde 3-phosphate dehydrogenase activity" means the dehydrogenation of glyceraldehyde 3-phosphate, phosphate and NAD + Converted into 3-phospho-D-glycerophosphate and NADH+H + Enzyme activity (EC 1.2.1.12). This activity can be measured by assays known in the art, such as, for example, those described by D'Alessio et al. (J. Biol. Chem. (1971) 246, 4326-4333).

[0395] The term "an organism or microorganism characterized by an inactivation of a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by a reduction in the activity of glyceraldehyde 3-phosphate dehydrogenase compared to an unmodified organism or microorganism and having a further reduction or inactivation of the Embden-Meyerhof-Parnas pathway (EMPP)" preferably refers to an organism or microorganism in which the inactivation of a gene encoding glyceraldehyde 3-phosphate dehydrogenase or the reduction in the activity of glyceraldehyde 3-phosphate dehydrogenase compared to an unmodified organism or microorganism is achieved by a genetic modification of the organism or microorganism resulting in said inactivation or reduction.

[0396] In a preferred embodiment, the recombinant organism or microorganism of the present invention is a recombinant organism or microorganism in which EMPP is further reduced or inactivated by inactivating a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing the activity of glyceraldehyde 3-phosphate dehydrogenase compared to an unmodified organism or microorganism. In the context of the present invention, inactivation of a gene encoding glyceraldehyde 3-phosphate dehydrogenase means that the genes encoding glyceraldehyde 3-phosphate dehydrogenase present in the organism or microorganism are inactivated so that they are no longer expressed and / or no longer lead to the synthesis of functional glyceraldehyde 3-phosphate dehydrogenase. Inactivation can be achieved by many different approaches known in the art. Inactivation can be attempted, for example, by destroying a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by completely deleting the gene by introducing a selection marker. Alternatively, the promoter of the gene encoding glyceraldehyde 3-phosphate dehydrogenase is mutated in such a way that the gene is no longer transcribed into mRNA. Other ways of inactivating the gene of glyceraldehyde 3-phosphate dehydrogenase known in the art are: expressing a polynucleotide encoding an RNA having a nucleotide sequence complementary to the transcription product of the glyceraldehyde 3-phosphate dehydrogenase gene, so that the mRNA is no longer translated into protein, to express a polynucleotide encoding an RNA that suppresses the expression of the gene through the RNAi effect; expressing a polynucleotide encoding an RNA having the activity of specifically cutting the transcription product of the gene; or expressing a polynucleotide encoding an RNA that suppresses the expression of the gene through a common suppression effect. These polynucleotides can be incorporated into a vector, and the vector can be introduced into an organism or microorganism by transformation to achieve the inactivation of the gene encoding glyceraldehyde 3-phosphate dehydrogenase.

[0397] The term "inactivation" in the context of the present invention preferably means complete inactivation, i.e., the organism or microorganism does not show glyceraldehyde 3-phosphate dehydrogenase activity. This particularly means that regardless of the growth conditions used, the organism or microorganism does not show glyceraldehyde 3-phosphate dehydrogenase activity.

[0398] Preferably, "inactivation" means that the gene encoding glyceraldehyde 3-phosphate dehydrogenase present in an organism or microorganism is genetically modified so as to prevent the expression of the enzyme. This can be achieved, for example, by deleting the gene or a part thereof, wherein the deletion of a part thereof prevents the expression of the enzyme, or by destroying the gene in the coding region or the promoter region, wherein the destruction has the effect of not expressing the protein or of expressing a malfunctioning protein.

[0399] In a preferred embodiment, the recombinant organism or microorganism of the present invention is a recombinant organism or microorganism with reduced EMPP by reducing the activity of glyceraldehyde 3-phosphate dehydrogenase compared to an unmodified organism or microorganism. Preferably, this reduction is achieved by genetic modification of the organism or microorganism. This can be achieved, for example, by random mutagenesis or site-directed mutagenesis of promoters and / or enzymes and subsequent selection of promoters and / or enzymes with desired properties, or by complementary nucleotide sequences or RNAi effects as described above. A detailed description of the genetic modification of the organism or microorganism will be further provided below.

[0400] In the context of the present invention, "reduced activity" means that the expression and / or activity of an enzyme (particularly glyceraldehyde 3-phosphate dehydrogenase) in a genetically modified organism or microorganism is at least 10% lower than that in a corresponding unmodified organism or microorganism, preferably at least 20% lower, more preferably at least 30% or 50% lower, even more preferably at least 70% or 80% lower and particularly preferably at least 90% or 100% lower. Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. The assay for measuring the enzymatic activity that glyceraldehyde 3-phosphate dehydrogenase reduces is known in the art.

[0401] In a more preferred embodiment, in which the recombinant microorganism is a bacterium, the genes encoding the PEP-dependent PTS transporter have been inactivated. In the context of the present invention, inactivation means that the genes encoding the PEP-dependent PTS transporter present in the microorganism are inactivated so that they are no longer expressed and / or no longer lead to the synthesis of a functional PEP-dependent PTS transporter. The inactivation of the genes encoding the PEP-dependent PTS transporter should render the bacterium no longer able to transport glucose via the PEP-dependent PTS transporter.

[0402] PEP-dependent PTS transporters (eg, from E. coli, Bacillus subtilis) are known in the art. Examples for inactivation of PEP-dependent PTS transporters are shown in the Examples section below.

[0403] Inactivation can be achieved in many different ways known in the art. Inactivation can be achieved, for example, by destroying the gene encoding the PEP-dependent PTS transporter or completely deleting the gene by introducing a selection marker. Alternatively, the promoter of the gene encoding the PEP-dependent PTS transporter can be mutated in such a way that the gene is no longer transcribed into mRNA. Other ways of inactivating genes encoding PEP-dependent PTS transporters known in the art are: expressing a polynucleotide encoding an RNA having a nucleotide sequence complementary to the PEP-dependent PTS transporter gene transcript, so that the mRNA is no longer translated into protein, to express a polynucleotide encoding an RNA that suppresses the expression of the gene through the RNAi effect; expressing a polynucleotide encoding an RNA having the activity of specifically cutting the gene transcript; or expressing a polynucleotide encoding an RNA that suppresses the expression of the gene through a common suppression effect. These polynucleotides can be incorporated into a vector, which can be introduced into an organism or microorganism by transformation to achieve the inactivation of the gene encoding the PEP-dependent PTS transporter.

[0404] The present invention furthermore relates to the use of the recombinant organism or microorganism according to the invention and as defined above for the production of isobutene.

[0405] The present invention further relates to a method for producing isobutene by culturing a recombinant organism or microorganism according to the invention and as defined above in a suitable culture medium under suitable conditions. Suitable culture media and suitable culture conditions are known to the skilled person and are described in more detail further below.

[0406] The present invention further relates to a method for producing isobutylene from acetyl-CoA in a recombinant organism or microorganism, comprising:

[0407] (A)(i) enzymatic conversion of acetyl-CoA into acetoacetyl-CoA,

[0408] (ii) enzymatically converting the produced acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA,

[0409] (iii) enzymatically converting the produced 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA,

[0410] (iv) enzymatically converting the produced 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA, and

[0411] (v) converting the produced 3-methylcrotonyl-CoA into isobutylene by:

[0412] (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid, which is then further enzymatically converted into said isobutylene; or

[0413] (b) Enzymatic conversion of 3-methylcrotonyl-CoA to 3-hydroxy-3-methylbutyryl-

[0414] CoA, which is then further enzymatically converted into 3-hydroxy-3-methylbutyrate, which is then further enzymatically converted into 3-phosphinoyl-3-methylbutyrate, which is then further enzymatically converted into said isobutylene;

[0415] (B) wherein the method further comprises:

[0416] From CoA, acetyl-CoA is provided enzymatically,

[0417] (C) wherein the method further comprises providing coenzyme A (CoA) by culturing the recombinant organism or microorganism in a suitable medium under suitable conditions, wherein the recombinant organism or microorganism has an increased coenzyme A (CoA) pool over that of the organism or microorganism from which it was derived due to:

[0418] (i) increased pantothenic acid uptake; and / or

[0419] (ii) Increased conversion of pantothenate to CoA.

[0420] Preferably, the present invention relates to the above method, wherein the acetyl-CoA is provided enzymatically by acetyl-CoA synthetase acetylation of coenzyme A (CoA) to acetyl-CoA using acetic acid.

[0421] In a more preferred embodiment, the present invention relates to the above method, wherein said increased pantothenate uptake in (C)(i) is due to recombinant expression of a pantothenate uptake transporter; and / or

[0422] wherein the increased conversion of pantothenate to CoA is due to recombinant expression of at least one of the following (i) to (vi):

[0423] (i) an enzyme that catalyzes the enzymatic conversion of pantothenate into 4'-phosphopantothenate, preferably pantothenate kinase;

[0424] (ii) an enzyme that catalyzes the enzymatic conversion of 4'-phosphopantothenate and L-cysteine ​​into 4'-phosphopantothenate-L-cysteine, preferably phosphopantothenate cysteine ​​synthetase;

[0425] (iii) an enzyme that catalyzes the enzymatic conversion of 4'-phosphopanthenoyl-L-cysteine ​​into pantetheine-4-phosphate, preferably 4'-phosphopanthenoylcysteine ​​decarboxylase;

[0426] (iv) an enzyme that catalyzes the enzymatic conversion of pantetheine-4-phosphate to desphospho-CoA, preferably pantetheine-phosphate adenylyltransferase; and

[0427] (v) an enzyme that catalyzes the enzymatic conversion of dephospho-CoA to CoA, preferably dephospho-CoA kinase.

[0428] With regard to the above enzymatic conversion, the above recombinant organism or microorganism or the above enzyme, with regard to preferred embodiments, what has already been listed above for the recombinant organism or microorganism according to the invention also applies to the process for producing isobutene.

[0429] In a preferred embodiment, the present invention relates to any of the above-mentioned methods, wherein the method further comprises supplementing the culture medium, wherein the organism or microorganism is cultured with pantothenic acid.

[0430] Without being bound by theory, it is believed that supplementation / supplementation of the culture medium with pantothenate further increases the CoA pool.

[0431] Because as mentioned above, pantothenic acid is the starting molecule for CoA biosynthesis. Known pantothenic acid is actually taken up by all known cells, because there is a corresponding pantothenic acid uptake transporter (see, e.g., Progress in Lipid Research 44:125.153 (2005)). As mentioned above, the pantothenic acid uptake according to the present invention can be further increased by the expression of, for example, recombinant pantothenic acid uptake transporters.

[0432] Thus, increasing the extracellular concentration of pantothenate in the culture medium further drives the method to increase the intracellular pantothenate concentration in a recombinant organism or microorganism and thus results in increased CoA production.

[0433] Indeed, supplementation of the culture medium with pantothenic acid has recently been described ( US 8,143,035 B2 ; Vadai et al., Biotechnol. Prog. 20 (2004 ); Vadai et al., Metabolic Endineering 6 (2004 )).

[0434] In a preferred embodiment, the present invention relates to any of the above-mentioned microorganisms, wherein the microorganism is a C1-fixing microorganism, preferably a recombinant C1-fixing microorganism as described in more detail below.

[0435] Furthermore, in a preferred embodiment, the present invention relates to any of the uses and methods described more generally above, wherein the microorganism is a C1-fixing microorganism, preferably a C1-fixing microorganism as described in more detail below.

[0436] Use of C1-fixing bacteria for the production of isobutylene

[0437] Nowadays, technologies to limit greenhouse gas (GHG) emissions are urgently needed in order to slow down global warming of the Earth. The concepts of carbon capture and storage (CCS) and carbon capture and utilization (CCU) are suitable to overcome these needs. While CCS focuses only on the permanent storage of CO2 underground to avoid CO2 accumulation in the atmosphere, CCU has the potential to replace chemical processes based on fossil raw materials for the production of commodity chemicals such as isobutylene. Gaseous raw materials containing C1 carbon compounds can be obtained from different sources. For example they are emitted in large quantities by industrial facilities using combustion processes such as the steel, cement or petrochemical industries. Other possible sources are the gasification of biomass, such as agricultural waste or wood residues, but the gasification of municipal solid waste is also considered as a potential source. Recent literature (Chauvy et al., Applied Energy 236 (2019), 662); et al., Proc Natl Acad Sci US A 116 (2019), 11187) have extensively discussed this topic.

[0438] For CCU, either chemical or biological conversion processes are suitable. Chemical methods (such as the Fischer-Tropsch process) use industrial waste gases to produce a large number of fuels and chemicals. Biological solutions use C1-fixing bacteria or algae for gas fermentation. Among the bacteria capable of fixing C1, acetogens are of particular interest. Using the reductive acetyl-CoA pathway (the "Wood-Ljungdahl" pathway), they are able to convert CO and / or CO2 and H2 into acetyl-CoA, the central metabolic precursor used primarily for the production of acetate. Under certain conditions, alternative products such as ethanol or 2,3-butanediol are also produced. The most studied acetogens are probably Clostridium ljungdahlii and Clostridium autothanogenum. More information on the biotechnological applications of these and other acetogens can be found in recent reviews (Bengelsdorf et al., Adv Appl Microbiol 103 (2018), 143; Diender et al., Front Microbiol 6 (2015), 1275).

[0439] For example, the use of Clostridium ljungdahlii for the production of acetic acid and ethanol is described in US5173429 and US5593886, while the discovery of Clostridium autoethanogenum as a microorganism exhibiting an increased ethanol / acetic acid product ratio is described in WO2009 / 064200. The first reported genetic manipulation of acetogenic bacteria for the recombinant production of chemicals was the introduction of the butanol pathway from Clostridium acetobutylicum. et al., Proc Natl Acad Sci U SA 107 (2010), 13087). Since then, acetogenic bacteria have been engineered to improve the production of their natural fermentation products, such as ethanol or 2,3-butanediol (WO2009 / 151342 and WO2014 / 197746). In addition, non-natural pathways have been introduced to produce acetone / isopropanol, 3-hydroxypropionic acid, terpenes, 1- or 2-butanol or propanol (WO2012 / 115527; WO2013 / 180581;

[0440] WO2013 / 180584; WO5390103 / 185123 and WO2014 / 036152).

[0441] Methods for producing isobutylene in C1-fixing bacteria (i.e., Clostridium autoethanogenum and Clostridium ljungdahlii) using different metabolic pathways have been described (WO2016 / 034691 and WO2017 / 066498). WO2016 / 034691 discloses the use of a metabolic pathway utilizing a 3-methylcrotonate intermediate, the decarboxylation of which leads to the production of isobutylene, such as the pathway described in WO2017 / 085167.

[0442] The metabolic pathway disclosed in WO2017 / 064498 includes a 3-hydroxyisovalerate intermediate as described by Gogerty and Bobik, Appl Environ Microbiol, December 2010;

[0443] 76(24):8004-10, van Leeuwen et al., February 2012; 93(4):1377-87, WO 2010 / 001078, WO 2012 / 052427, WO 2012 / 0524027, WO 2012 / 052427 and WO 2017 / 085167.

[0444] However, although various methods have been described in the prior art for the production of C1-fixing bacteria, thereby allowing the use of non-organic renewable resources as feedstock, there is still a need to improve the efficiency and effectiveness of these methods in order to increase the yields and make them commercially attractive.

[0445] The present invention meets this need by providing, in further aspects, the following:

[0446] A recombinant C1-fixing microorganism capable of enzymatically converting acetyl-CoA to isobutylene, wherein in the C1-fixing microorganism:

[0447] (i) enzymatic conversion of acetyl-CoA into acetoacetyl-CoA,

[0448] (ii) enzymatic conversion of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA,

[0449] (iii) enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA,

[0450] (iv) enzymatic conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA,

[0451] (v) enzymatic conversion of 3-methylcrotonyl-CoA to 3-methylcrotonic acid;

[0452] (vi) enzymatic conversion of 3-methylcrotonate to isobutylene by:

[0453] (a) 3-polyisoprene-4-hydroxybenzoate decarboxylase (UbiD) having the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 60% sequence identity to SEQ ID NO:25 and having activity of converting 3-methylcrotonic acid into isobutylene; or

[0454] (b) A UbiD-like decarboxylase having the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 26 and having activity of converting 3-methylcrotonate into isobutylene.

[0455] Thus, in a recombinant C1-fixing microorganism capable of enzymatically converting acetyl-CoA to isobutylene, the conversion of 3-methylcrotonate to isobutylene in C1-fixing bacteria utilizes 3-polyisoprene-4-hydroxybenzoate dehydrogenase (UbiD), which is derived from Hypocrea atroviridis (Uniprot Accession No.

[0456] G9NLP8), which has the amino acid sequence shown in SEQ ID NO:25 (and amino acid sequences with at least 60% sequence identity to SEQ ID NO:25), or utilizing a UbiD-like decarboxylase derived from Streptomyces (UniProt Accession No. A0A0A8EV26), which has the amino acid sequence shown in SEQ ID NO:26 (and amino acid sequences with at least 60% sequence identity to SEQ ID NO:26).

[0457] In a preferred embodiment, the decarboxylase derived from Hypocrea atroviridis is an enzyme comprising the amino acid sequence of SEQ ID NO: 25, or a sequence that is at least n% identical to SEQ ID NO: 25, n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has enzymatic activity for converting 3-methylcrotonic acid into isobutylene.

[0458] In a preferred embodiment, the decarboxylase derived from Streptomyces is an enzyme comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least n% identical to SEQ ID NO: 26, n is an integer from 10 to 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 94, 94, 95, 96, 97, 98 or 99, and wherein the enzyme has enzymatic activity for converting 3-methylcrotonic acid into isobutylene.

[0459] In another preferred embodiment, the decarboxylase is a variant of the above decarboxylase derived from Hypocrea atroviridis having one or more amino acid modifications as disclosed in WO 2017 / 191239, wherein the enzyme has enzymatic activity for converting 3-methylcrotonate into isobutylene.

[0460] Regarding the determination of sequence identity, the following should apply: when the sequences being compared have different lengths, the degree of identity refers to the percentage of the amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence or the percentage of the amino acid residues in the longer sequence that are identical to the amino acid residues in the shorter sequence. Preferably, it refers to the percentage of the amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence. The degree of sequence identity can be determined using a preferred suitable computer algorithm (such as CLUSTAL) according to methods well known in the art.

[0461] For example, when using the Clustal analysis method to determine whether a particular sequence is at least 60% identical to a reference sequence, the default settings or settings preferably are as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extended gap penalty: 0.05; Delayed divergence: 40; Gap separation distance: 8, for comparison of amino acid sequences. For comparison of nucleotide sequences, it is best to set the extended gap penalty to 5.0.

[0462] In a preferred embodiment, ClustalW2 is used for comparing amino acid sequences. In the case of pairwise comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap opening: 10; gap extension: 0.1. In the case of multiple comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap opening: 10; gap extension: 0.2; gap distance: 5; no end gaps.

[0463] Preferably, the degree of identity is calculated over the entire length of the sequence.

[0464] The activity of an enzyme capable of converting 3-methylcrotonic acid into isobutylene can be determined by methods known to those skilled in the art. In one embodiment, the activity is determined as described in the accompanying examples. In a specific embodiment, the activity can be measured by in vitro incubation of the enzyme, preferably containing a cell lysate of an overexpressed recombinant protein. Alternatively, a purified enzyme can be used in an in vitro assay.

[0465] More specifically, the activity of the decarboxylase for converting 3-methylcrotonate to isobutylene can be assessed in an enzymatic in vitro assay based on purified protein and detection of isobutylene by gas chromatography. The turnover rate of the enzyme to be assessed can be examined as outlined below: The Michaelis-Menten k for the reaction of 3-methylcrotonate to isobutylene can be determined using the following experimental protocol: cat and K m Steady-state kinetic constant:

[0466] The enzyme assay for quantifying the conversion of 3-methylcrotonate to isobutylene was performed in 50 mM potassium phosphate pH 7.5 buffer; 20 mM NaCl, 3 mM MgCl2, 5 mM DTT, 0.5 mg / ml purified decarboxylase to be tested, 100 μl containing FMN prenyltransferase (i.e., flavin prenyltransferase UbiX protein expressed from E. coli and prepared as further outlined below) and varying concentrations of the substrate 3-methylcrotonate ranging from 0 to 128 mM, at 30°C in 2 ml glass vials. A control without decarboxylase was performed in parallel. After 60 minutes, the reaction was stopped by incubation at 80°C for 2 min. The rate of isobutylene production was quantified by gas chromatography as follows.

[0467] The isobutylene formed in the headspace of the reaction was analyzed by gas chromatography (GC) equipped with a flame ionization detector (FID). For GC headspace analysis, 1 ml of headspace gas was separated at 130°C using isothermal mode in a Bruker GC-450 system equipped with a GS-alumina column (30 m×0.53 mm) (Agilent). Nitrogen was used as a carrier gas with a flow rate of 6 ml / min. The products of the enzymatic reaction were identified by comparison with an isobutylene standard. Under these GC conditions, the retention time of isobutylene was 2.42 minutes. From the production rate of isobutylene, and using the Michaelis-Menten approximation, the enzyme catalytic efficiency can then be calculated. The production rate of isobutylene (PV moles / mole enzyme /

[0468] sec) were plotted as a function of 3-methylcrotonic acid concentration and the curve was fitted using the Michaelis Menten equation (V = (V max *(substrate)) / (K m + (substrate))) to find k cat (s -1 ) and K m Value (mM).

[0469] Regarding the enzymatic transformation of the above-mentioned recombinant C1-fixing microorganism, the following enzymatic transformation

[0470] (i) conversion of acetyl-CoA into acetoacetyl-CoA,

[0471] (ii) conversion of acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA,

[0472] (iii) 3-hydroxy-3-methylglutaryl-CoA is converted into 3-methylglutaconyl-CoA,

[0473] (iv) conversion of 3-methylglutaconyl-CoA to 3-methylcrotonyl-CoA, and

[0474] (v) 3-methylcrotonyl-CoA is converted into 3-methylcrotonic acid,

[0475] As well as in relation to the preferred embodiments of these transformations, the same applies as listed above.

[0476] In a preferred embodiment, with respect to (i) acetyl-CoA is enzymatically converted to acetoacetyl-CoA by an acetyl-CoA transferase from Clostridium kluyverii (encoded by gene thlA3; NCBI reference EDK35683.1; UniProt accession A5N317).

[0477] In a preferred embodiment, with respect to (ii), acetoacetyl-CoA is enzymatically converted to 3-hydroxy-3-methylglutaryl-CoA by hydroxymethylglutaryl-CoA synthetase from Enterococcus faecalis (encoded by gene mvaS; NCBI reference WP_002357756.1; UniProt accession Q9FD71).

[0478] In a preferred embodiment, with respect to (iii), 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted to 3-methylglutaconyl-CoA by an enoyl-CoA hydratase / isomerase from Pseudomonas sp. UW4 (ECH) (encoded by gene PputUW4_01474; NCBI reference number WP_015094072.1; UniProt accession number K9NHK2).

[0479] In a preferred embodiment, with respect to (iv), 3-methylglutaconyl-CoA is enzymatically converted to 3-methylcrotonyl-CoA by a glutaconyl-CoA-transferase (AibA / B) from Myxococcus xanthus (encoded by genes MXAN_4264 or MXAN_4265; NCBI reference number WP_011554268.1 or WP_011554267.1; UniProt accession number Q1D413 or Q1D414).

[0480] In a preferred embodiment, with respect to (v), 3-methylcrotonyl-CoA is enzymatically converted to 3-methylcrotonate by an acyl-CoA thioesterase (encoded by gene tesB; NCBI reference AAC73555.1; UniProt accession P0AGG2).

[0481] In a preferred embodiment, with respect to (vi), the flavin cofactor (FMN or FAD) is modified to the corresponding (modified) flavin-derived cofactor by flavin isoprenyltransferase from Escherichia coli (encoded by gene ubiX; NCBI reference WP_000825700.1; UniProt accession P0AG03).

[0482] The nature of the C1-fixing microorganism is not particularly limited, as long as it is a microorganism capable of utilizing carbon monoxide (CO) and a gaseous substrate comprising CO (such as, for example, syngas) as a carbon source and energy. Syngas or synthetic gas is a gas mixture consisting primarily of H2, CO and often some CO2. Corresponding naturally occurring (or genetically modified) microorganisms capable of utilizing CO and converting it to acetyl-CoA are known in the art. These organisms are often referred to as acetogenic microorganisms or acetogenic bacteria (sometimes also referred to as carbon monoxide nutrition, acetogenic microorganisms). These microorganisms utilize the Wood-Ljungdahl pathway to fix CO and / or CO2 and convert it to acetyl-CoA. Examples of such microorganisms belong to the family Clostridiae and are described, for example, in WO 2009 / 094485; WO 2012 / 05905; WO 2013 / 180584; US

[0483] 2011 / 0236941; PNAS107(29):13087-13092(2010); Current Opinion inBiotechnology 23:364-381(2012); Applied and Environmental Microbiology77(15):5467-5475(2011).

[0484] In certain embodiments, the C1-fixing microorganism is selected from the group consisting of carboxydothermus ferrireducens, Carboxydothermus hydrogenoformans, Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Delsufitobacterium hafniense, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorellathermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, Thermincola potens (for mesophilic strains, the optimal growth temperature is 30-37°C, and for thermophilic strains, the optimal growth temperature is 55-65°C) and strict anaerobes (Tanner et al., Int J Syst Bacteriol 1993, 43:

[0485] 232-236; Abrini et al., Arch Microbiol 1994,4:345-351; WO

[0486] 2008 / 028055). In addition, they all share the same major phylogenetic traits, such as the same pH range (pH 4-7.5, with an optimal initial pH of 5.5-6), strong autotrophic growth under CO2-containing gases at similar growth rates, and similar metabolic profiles, with ethanol and acetate as the main fermentation end products, and under certain conditions forming small amounts of 2,3-butanediol and lactic acid (Tanner et al., Int J Syst Bacteriol 1993, 43: 232-236; Abrini et al., Arch Microbiol 1994, 4: 345-351; WO 2008 / 028055).

[0487] In one embodiment, the C1-fixing, carboxydotrophic acetogenic microorganism is selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorellathermautotrophica, Oxobacter prausnitzii, and the like. pfennigii) and Thermoanaerobacterkiuvi.

[0488] In a particular embodiment, the C1-fixing microorganism is selected from the group consisting of carboxydotrophic Clostridia, including Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, and Clostridium magnum.

[0489] In one embodiment, the microorganism is selected from the carboxydotrophic Clostridium cluster, including species C. autoethanogenum, C. ljungdahlii and C. ragsdalei and related isolates. These include, but are not limited to, strain C. autoethanogenum JAI01 T (DSM10061) (Abrini et al., Arch Microbiol 1994, 4: 345-351), Clostridium autoethanogenum LBS1560 (DSM19630) (WO2009 / 064200), Clostridium autoethanogenum LBS1561 (DSM23693), Clostridium ljungdahlii PETC T (DSM13528 = ATCC 55383) (Tanner et al., Int J Syst Bacteriol 1993, 43: 232-236), Clostridium ljungdahlii 'EM-2 (ATCC 55380) (US Patent 5,593,886), Clostridium ljungdahlii C-01 (ATCC 55988) (US Patent 6,368,819), Clostridium ljungdahlii O-52 (ATCC 55989) (US Patent 6,368,819) or C. ragsdalei P11 T(ATCC BAA-622) (WO 2008 / 028055), and related isolates such as C. coskatii (US Patent 2011 / 0229947), Clostridium MT351 (Michael et al., J. Biotech Res. 4: 1-12), and mutants thereof, such as Clostridium ljungdahlii OTA-1 (Tirado-Acevedo O. Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii. PhD thesis, North Carolina State University, 2010).

[0490] These strains form a subcluster within Clostridium rRNA cluster I (Collins et al., 1994) and share at least 99% identity at the 16S rRNA gene level, although they are distinct species as determined by DNA-DNA reassociation and DNA fingerprinting experiments (WO 2008 / 028055, US Patent 2011 / 0229947).

[0491] The strains of this cluster are defined by common features, have similar genotypes and phenotypes, and they all share the same energy conservation and fermentative metabolic mode. The strains of this cluster lack cytochromes and conserve energy through the Rnf complex.

[0492] All strains of this cluster have a genome size of about 4.2 MBp and a GC composition of about 32% mol (Abrini et al., Arch Microbiol 1994, 4:345-351; Kopke et al. (2011) Curr. Opin. Biotechnol. 22:320-325 and Appl. Environ. Microbiol. 77:5467-75; Tanner et al., Int J Syst Bacteriol 1993, 43:232-236) (WO 2008 / 028055; US Patent No. 2011 / 0229947), as well as conserved essential key genes encoding enzymes of the Wood-Ljungdahl pathway (carbon monoxide dehydrogenase, formyltetrahydrofolate synthetase, methylenetetrahydrofolate dehydrogenase, formyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate reductase, 5-methyltetrahydrofolate:corrinoid / iron-sulfur protein comethyltransferase and carbon monoxide dehydrogenase / acetyl-CoA synthetase), hydrogenase, formate dehydrogenase, Rnf complex (rnfCDGEAB), pyruvate:ferredoxin oxidoreductase, aldehyde:ferredoxin oxidoreductase (Kopke et al., (2011) Curr. Opin. Biotechnol. 22:320-325 and Appl. Environ. Microbiol. 77:5467-75). The organization and number of Wood-Ljungdahl pathway genes responsible for gas absorption have been found to be identical in all species, despite differences in nucleic acid and amino acid sequences (Kopke et al., (2011) Curr. Opin. Biotechnol. 22:320-325 and Appl. Environ. Microbiol. 77:5467-75).

[0493] The strains all have similar morphology and size (logarithmically growing cells of 0.5-0.7×3-5 μm), are mesophilic (optimal growth temperature of 30-37° C.) or thermophilic, and are strictly anaerobic (Abrini et al., 1994, cited above; Tanner et al., 1993, cited above; WO 2008 / 028055). In addition, they all share the same major phylogenetic traits, such as the same pH range (pH 4-7.5, with an optimal initial pH of 5.5-6), strongly autotrophic growth under CO2-containing gases at similar growth rates, and similar metabolic profiles, with ethanol and acetic acid as the main fermentation end products, and under certain conditions, small amounts of 2,3-butanediol and lactic acid are formed (Abrini et al., 1994, cited above; Kopke et al., 2011, cited above; Tanner et al., 1993, cited above). However, species differ in substrate utilization of various sugars (e.g., rhamnose, arabinose), acids (e.g., gluconic acid, citric acid), amino acids (e.g., arginine, histidine), or other substrates (e.g., betaine, butanol). Some species have been found to be auxotrophic for certain vitamins (e.g., thiamine, biotin), while others are not. Reduction of carboxylic acids to their corresponding alcohols has been shown in a range of these organisms (Perez, Richter, Loftus, & Angenent, 2012).

[0494] The trait is therefore not specific to one organism (such as C. autoethanogenum or C. ljungdahlii), but is a general trait of carboxydotrophic, ethanol-synthesizing Clostridium. Therefore, although there may be differences in performance, it is expected that the present invention will work in these strains.

[0495] Recombinant C1-fixing microorganisms, preferably C1-fixing, carboxydotrophic acetogenic microorganisms of the present invention, can be prepared from a parent C1-fixing microorganism, preferably a carboxydotrophic acetogenic microorganism, and one or more nucleic acid molecules encoding enzymes / transporters as defined above, using any number of techniques known in the art for producing recombinant microorganisms. By way of example only, transformation (including transduction or transfection) can be achieved by electroporation, electrofusion, ultrasound, polyethylene glycol-mediated transformation, conjugation or chemical and innate ability. Suitable transformation techniques are described, for example, in Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989.

[0496] With regard to C1-fixing microorganisms, preferably C1-fixing, carboxydotrophic acetogenic microorganisms, electroporation has been described for several carboxydotrophic acetogenic bacteria, such as Clostridium ljungdahlii ( et al., 2011, cited above; Leang, Ueki, Nevin & Lovley, 2012)

[0497] (PCT / NZ201 1 / 000203; WO2012 / 053905), Clostridium autoethanogenum

[0498] (PCT / NZ2011 / 000203; WO2012 / 053905), Acetobacterium woodii (Stratz, Sauer, Kuhn & Diirre, 1994) or Moorella thermoacetica (Kita et al., 2012), and is a standard method used in many Clostridia, such as C. acetobutylicum (Mermelstein, Welker, Bennett & Papoutsakis, 1992), C. cellulolyticum (Jennert, Tardif, Young & Young, 2000) or C. thermocellum (C.

[0499] thermocellum) (MV Tyurin, Desai & Lynd, 2004). Electrofusion has been described for Clostridium acetogenicum MT351 (Tyurin and Kiriukhin, 2012).

[0500] Furthermore, the present invention provides the use of a C1-fixing microorganism as defined above for the production of isobutene.

[0501] Furthermore, the present invention provides a method for producing isobutene by culturing a C1-fixing microorganism as defined above in a suitable culture medium under suitable conditions.

[0502] The C1-fixing microorganisms and methods according to the invention are particularly useful for large-scale in vivo isobutene production, in particular for commercial production. Therefore, the present invention relates to a method for large-scale production, in particular for commercial production of isobutene, wherein the method comprises the steps as described above.

[0503] With regard to the above enzymatic conversion, the above recombinant organisms or microorganisms or the above enzymes, with regard to the preferred embodiments, the same applies to the use of C1-fixing microorganisms and to the method for producing isobutene by culturing C1-fixing microorganisms, as listed above for the recombinant organisms or microorganisms according to the invention.

[0504] In a preferred embodiment, the present invention relates to any of the above methods, wherein the method further comprises collecting the produced isobutylene. For example, when the organism or microorganism according to the present invention is cultivated in a suitable culture medium, preferably a C1-fixing microorganism expressing the desired enzyme as defined above, isobutylene can be collected from the fermentation off-gas by methods known to those skilled in the art.

[0505] The method according to the invention can be carried out in culture, using a recombinant organism or microorganism according to the invention, preferably a C1-fixed microorganism as defined above, producing the above-mentioned enzymes for the conversion according to the invention as described above, or producing a transporter as described above. Methods using an organism or microorganism, preferably a C1-fixed microorganism as defined above, for carrying out the method according to the invention are referred to as "in vivo" methods.

[0506] An organism or microorganism may be used, preferably a C1-fixing microorganism as defined above, which according to the invention may be an organism or microorganism that naturally produces the above-mentioned enzyme / transporter, or an organism or microorganism that has been genetically modified to express (including overexpression) one or more such enzymes / transporters, preferably a C1-fixing microorganism as defined above. Thus, the organism or microorganism may be an engineered organism or microorganism that expresses an enzyme for the above-mentioned conversion or a transporter as described above, i.e., it has in its genome a nucleotide sequence encoding such an enzyme / transporter and it has been modified to overexpress them. The expression may occur constitutively or in an induced or regulated manner.

[0507] In another embodiment, the organism or microorganism, preferably the C1-fixing microorganism as defined above, may be an organism or microorganism that has been genetically modified by the introduction of one or more nucleic acid molecules containing nucleotide sequences encoding one or more enzymes for the above-mentioned conversion or transporters as described above. The nucleic acid molecules may be stably integrated into the genome of the organism or microorganism or may be present in an extrachromosomal manner, for example, on a plasmid.

[0508] Such a genetically modified organism or microorganism can be, for example, an organism or microorganism that does not naturally express such an enzyme / transporter and has been genetically modified to express such an enzyme / transporter, or an organism or microorganism that naturally expresses such an enzyme / transporter and has been genetically modified, for example, by transformation with a nucleic acid (e.g., a vector) encoding the respective enzyme / transporter, and / or by inserting a promoter in front of the endogenous nucleotide sequence encoding the enzyme / transporter, in order to increase the respective activity in said organism or microorganism.

[0509] However, the present invention preferably excludes naturally occurring organisms or microorganisms found in nature that express the above enzymes / transporters at naturally occurring levels. Instead, the organisms or microorganisms of the present invention, preferably C1-fixing microorganisms as defined above, and preferably non-naturally occurring organisms or microorganisms used in the methods of the present invention, have been genetically modified to express (including overexpression) exogenous enzymes / transporters not normally present in their genome or have been engineered to overexpress endogenous enzymes / transporters.

[0510] Therefore, the protein (micro) organism used in conjunction with the present invention is preferably a non-natural protein or (micro) organism, that is, they are significantly different from naturally occurring protein or organism or microorganism and do not produce protein or (micro) organism in nature. About enzyme / transporter, they are preferably variants of naturally occurring enzyme / transporter, which can not produce as naturally occurring enzyme / transporter in nature. Such variants include for example mutants, particularly prepared by molecular biology methods, which demonstrate improved characteristics, such as higher enzymatic activity, higher substrate specificity, higher temperature tolerance, higher transport capacity etc. About (micro) organism, preferably C1-fixed microorganisms as defined above, they are preferably genetically modified organisms as described above, which are different from naturally occurring organisms due to genetic modification. Genetically modified organisms are not naturally occurring organisms, that is, can not be found in nature, and owing to introducing exogenous nucleic acid molecules it is substantially different from naturally occurring organisms.

[0511] By overexpressing the exogenous or endogenous enzymes / transporters described above, the concentration of the enzyme / transporter is substantially higher than that found in nature, which can then unexpectedly drive the reactions of the present invention, which utilize the non-natural respective enzyme / transporter. Preferably, the concentration of the overexpressed enzyme / transporter is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.

[0512] A "non-natural" substrate is understood to be a molecule that is not acted upon by the respective enzyme in nature, even if it actually coexists with the endogenous enzyme in the organism or microorganism. Such a "non-natural" substrate is not converted by the organism or microorganism in nature, because other substrates are preferred (e.g., a "natural substrate"). Therefore, the present invention relates to the use of non-natural substrates and the above-mentioned enzymes in an environment not found in nature.

[0513] Thus, in the context of the present invention, an organism or microorganism, preferably a C1-fixing microorganism as defined above, may also be an organism or microorganism that does not naturally have the corresponding enzyme / transporter activity but has been genetically modified to contain a nucleotide sequence allowing the expression of the corresponding enzyme / transporter. Similarly, an organism or microorganism, preferably a C1-fixing microorganism as defined above, may also be an organism or microorganism that naturally has the corresponding enzyme / transporter activity but has been genetically modified to enhance this activity, for example, by introducing an exogenous nucleotide sequence encoding the corresponding enzyme / transporter or by introducing a promoter for an endogenous gene encoding the enzyme / transporter to increase endogenous production to overexpression (non-natural) levels.

[0514] If an organism or microorganism that naturally expresses the corresponding enzyme / transporter is used, preferably a C1-fixing microorganism as defined above, it is possible to modify such an organism or microorganism so that the respective activity is overexpressed in the organism or microorganism. This can be achieved, for example, by achieving mutations in the promoter region of the corresponding gene or by introducing high-expression promoters to generate promoters that ensure higher expression of the gene. Alternatively, the gene itself can also be mutated to generate an enzyme / transporter that exhibits a higher activity.

[0515] In one embodiment, the organism or microorganism according to the present invention, preferably the C1-fixing microorganism as defined above, is an exogenous nucleic acid molecule that has been modified to contain at least one of the enzymes / transporters described. In this context, the term "exogenous" or "exogenous" means that the nucleic acid molecule does not naturally occur in the organism or microorganism. This means that it will not occur in the same structure or the same position of the organism or microorganism. In a preferred embodiment, the exogenous nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding the corresponding enzyme / transporter, wherein the promoter driving the expression of the coding sequence is heterologous relative to the coding sequence. "Heterologous" in this context means that the promoter is not a promoter that naturally drives the expression of the coding sequence, but a promoter that naturally drives the expression of a different coding sequence, that is, it is derived from another gene, or is a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter heterologous to the organism or microorganism, that is, a promoter that is not naturally present in the respective organism or microorganism. Even more preferably, the promoter is an inducible promoter. Promoters used to drive expression in different types of organisms, in particular in organisms or microorganisms, are well known to those skilled in the art.

[0516] In a further embodiment, nucleic acid molecules are exogenous to organisms or microorganisms, because the encoded enzyme / transporter is not endogenous to microorganisms, that is, when it is not genetically modified, organisms or microorganisms will not express naturally. In other words, the encoded enzyme / transporter is heterologous to organisms or microorganisms. Exogenous nucleic acid molecules can be present in organisms or microorganisms in an extrachromosomal form (for example, as a plasmid), or stably integrated into the chromosome. Preferably, stable integration. Therefore, genetic modification can, for example, include integrating the corresponding gene of the encoded enzyme / transporter into the chromosome, or expressing the enzyme / transporter from a plasmid containing an enzyme encoding or transporter encoding sequence upstream promoter, and promoters and encoding sequences are preferably derived from different organisms, or any other method known to those skilled in the art.

[0517] The term "microorganism" in the context of the present invention refers to bacteria, as well as fungi, such as yeast, algae and archaea. In a preferred embodiment, the microorganism is a bacterium. In principle, any bacterium can be used. The preferred bacterium used in the present invention is a bacterium of the genus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas or Escherichia. In a particularly preferred embodiment, the bacterium belongs to the genus Escherichia coli, and even more preferably belongs to the species of Escherichia coli. In another preferred embodiment, the bacterium belongs to the species of Pseudomonas putida or the species of Zymomonas mobilis or the species of Corynebacterium glutamicum or the species of Bacillus subtilis.

[0518] Extremely thermophilic bacteria, such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiaceae may also be used.

[0519] In another particularly preferred embodiment, as detailed above, the microorganism is a microorganism capable of using carbon monoxide (CO) and a gaseous substrate comprising CO (e.g., synthesis gas) as a carbon source and energy. Synthesis gas or synthesis gas is a mixture of CO and CO2 and H2. Therefore, in a particularly preferred embodiment, the microorganism is a C1-fixing microorganism as defined above. As mentioned above, corresponding naturally occurring (or genetically modified) microorganisms that can utilize CO and convert it into acetyl-CoA are known in the art. These organisms are generally referred to as acetogenic microorganisms (sometimes also referred to as carbon monoxide nutrient acetogenic microorganisms) and are referred to herein as "C1-fixed microorganisms". These microorganisms use the Wood-Ljungdahl pathway to fix CO and convert it into acetyl-CoA. Examples of such microorganisms belong to the family Clostridium and are described, for example, in WO 99 / 0566.

[0520] 2009 / 094485; WO 2012 / 05905; WO 2013 / 180584; US2011 / 0236941;

[0521] PNAS107(29):13087-13092(2010); Current Opinion in Biotechnology23:364-381(2012); Applied and Environmental Microbiology 77(15):5467-5475(2011).

[0522] In another preferred embodiment, the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus, Trichoderma, Kluyveromyces, Clostridium or Pichia pastoris, and even more preferably a species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger, Trichoderma reesei, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris, Pichia toro or Pichia utilis.

[0523] In another embodiment, the present invention utilizes photosynthetic microorganisms expressing at least one enzyme for conversion according to the present invention as described above. Preferably, the microorganism is a photosynthetic bacterium or a microalgae. In yet another embodiment, the microorganism is an algae, more preferably an algae belonging to the family Bacillariophyceae.

[0524] Combinations of microorganisms may also be used according to the invention, preferably combinations of C1-immobilized microorganisms as defined above, wherein different microorganisms express different enzymes as described above. The genetic modification of a microorganism to express an enzyme of interest will also be described in further detail below.

[0525] In a preferred embodiment, the organism, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism capable of consuming glucose.

[0526] In a preferred embodiment, the organism, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism capable of consuming fructose.

[0527] In a preferred embodiment, the organism, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism capable of consuming xylose.

[0528] In a preferred embodiment, the organism, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism capable of consuming mannose.

[0529] In a preferred embodiment, the organism of the present invention, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, is an organism capable of consuming more than one sugar. Preferably, the more than one sugar comprises fructose, glucose, mannose and / or xylose. In a more preferred embodiment, in a preferred embodiment, the organism of the present invention, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, is an organism capable of consuming two or more sugars selected from fructose, glucose, mannose and xylose. Organisms and / or microorganisms capable of consuming glucose, fructose, xylose and / or mannose are naturally occurring and known in the art.

[0530] In a preferred embodiment, in a preferred embodiment, the organism of the present invention, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, is an organism capable of consuming CO and / or syngas. In another preferred embodiment, in a preferred embodiment, the organism of the present invention, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, is an organism capable of consuming CO and / or a mixture of CO2 and H2.

[0531] In another embodiment, the organism and / or microorganism is genetically modified to be able to consume glucose, fructose, xylose, mannose and / or CO (or syngas), and / or genetically modified to increase the ability of the organism and / or microorganism to consume glucose, fructose, xylose, mannose and / or CO (or syngas). Corresponding genetic modifications are known in the art.

[0532] In another preferred embodiment, the organism, preferably microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism capable of consuming sugars via a phosphotransferase transport system (PTS).

[0533] In a preferred embodiment, the organism, preferably microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism capable of consuming sugars via a non-phosphotransferase transport system (non-PTS).

[0534] Organisms and / or microorganisms that are able to consume sugars by a phosphotransferase transport system (PTS) and / or by a non-phosphotransferase transport system (non-PTS) are known in the art.

[0535] In another embodiment, the organism and / or microorganism is genetically modified to be able to consume sugars by a phosphotransferase transport system (PTS) or by a non-phosphotransferase transport system (non-PTS). In another preferred embodiment, the organism and / or microorganism is genetically modified to improve the ability of the organism and / or microorganism to consume sugars by a phosphotransferase transport system (PTS) or by a non-phosphotransferase transport system (non-PTS). Corresponding genetic modifications are known in the art.

[0536] In another preferred embodiment, the organism, preferably microorganism, more preferably C1-fixing microorganism as defined above, of the present invention is an organism with a reduced or inactivated phosphotransferase transport system (PTS). Such an organism, preferably a microorganism, is preferably genetically modified by deleting or inactivating the phosphotransferase transport system (PTS) gene. Corresponding genetic modifications are known in the art.

[0537] In another preferred embodiment, the organism of the present invention, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, is an organism with an enhanced non-phosphotransferase transport system (non-PTS) for sugar uptake. Such an organism, preferably a microorganism, is preferably genetically modified by overexpressing the non-phosphotransferase transport system (PTS) gene for sugar uptake. Corresponding genetic modifications are known in the art.

[0538] In another preferred embodiment, the organism, preferably microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism with a reduced or inactivated phosphotransferase transport system (PTS) and an enhanced non-phosphotransferase transport system (non-PTS) for sugar uptake.

[0539] In another preferred embodiment, the organism, preferably microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism capable of consuming sucrose via a non-phosphotransferase transport system (non-PTS).

[0540] In another preferred embodiment, the organism, preferably microorganism, more preferably C1-fixing microorganism as defined above, of the present invention is an organism that consumes sucrose, wherein said organism, preferably said microorganism, has been genetically modified by introducing at least one gene of a non-phosphotransferase transport system (non-PTS). Without being bound by theory, such organisms and / or microorganisms are genetically modified by introducing a gene selected from cscA, cscB and cscK from Escherichia coli W (M. Bruschi et al., Biotechnology Advances 30 (2012) 1001-1010).

[0541] In another preferred embodiment, the organism, preferably microorganism, more preferably a C1-fixing microorganism as defined above, of the present invention is an organism that has been genetically modified to have a reduced or inactivated phosphotransferase transport system (PTS) and overexpression of at least one gene selected from galP, glk and glf.

[0542] In another preferred embodiment, the organism of the present invention, preferably a microorganism, more preferably a C1-fixing microorganism as defined above, is an organism that has been genetically modified to avoid acetyl-CoA seepage, thereby increasing the intracellular concentration of acetyl-CoA. The genetic modification that causes the intracellular concentration of acetyl-CoA to increase is known in the art. Such an organism, preferably a microorganism, can preferably be genetically modified by deleting or inactivating the following genes: ΔackA (acetate kinase), Δldh (lactate dehydrogenase), ΔadhE (alcohol dehydrogenase), ΔfrdB and / or ΔfrdC (fumarate reductase and fumarate dehydrogenase).

[0543] In another embodiment, the method of the present invention comprises the step of providing an organism of the present invention, preferably a microorganism, more preferably a C1-fixed microorganism as defined above, in the form of a (cell) culture, preferably in the form of a liquid cell culture, followed by a step of culturing the organism (preferably a microorganism) in a fermenter (often also referred to as a bioreactor) under suitable conditions that allow expression of the respective enzymes / transporters, and further comprising a step of implementing the enzymatic transformation of the method of the present invention as described above. Suitable fermenter or bioreactor equipment and fermentation conditions are known to those skilled in the art. A bioreactor or fermenter refers to any manufactured or engineered device or system known in the art that supports a biologically active environment. Thus, a bioreactor or fermenter can be a container in which a chemical / biochemical reaction as in the method of the present invention is carried out, the method involving an organism, preferably a microorganism and / or a biochemically active substance, i.e., the above-mentioned enzyme / transporter derived from such an organism or an organism with the above-mentioned enzyme / transporter. In a bioreactor or fermenter, this process can be aerobic or anaerobic. These bioreactors are typically cylindrical and can range in size from a few liters to a few cubic meters, and are often made of stainless steel. In this regard, without being bound by theory, fermentor tanks or bioreactors can be designed in a manner suitable for, for example, cultivating an organism (preferably a microorganism) in batch culture, fed-batch culture, perfusion culture or chemostat culture, all of which are generally known in the art. Substratum can be any substratum suitable for cultivating a respective organism or microorganism. When utilizing an organism or microorganism to carry out, the method according to the present invention can, for example, be designed as a continuous fermentation culture method or a distribution culture or any suitable culture method known to those skilled in the art.

[0544] In a preferred embodiment, the method according to the invention further comprises the step of collecting the isobutene produced by the method. For example, when the method according to the invention is carried out in vivo by fermentation of a corresponding organism or microorganism expressing the required enzyme / transporter (preferably a C1-fixing microorganism as defined above), isobutene is collected from the fermentation off-gas by methods known to those skilled in the art.

[0545] The enzymes and transporters used according to the invention may be naturally occurring enzymes or transporters or enzymes or transporters derived from naturally occurring enzymes, e.g. by introducing mutations or other changes which, e.g., alter or improve enzyme activity, stability, etc.

[0546] Methods for altering and / or improving the desired enzymatic activity of proteins and the activity / capacity of transporters are well known to the person skilled in the art and comprise, for example, random mutagenesis or site-directed mutagenesis and subsequent selection of enzymes / transporters with the desired properties or so-called "directed evolution" methods.

[0547] For example, for genetic modification in prokaryotic cells, the nucleic acid molecules encoding the corresponding enzyme or transporter can be introduced into a plasmid that allows mutagenesis or sequence changes by DNA sequence reorganization. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchange or the addition of natural or synthetic sequences. DNA fragments can be connected by using an adaptor and a joint complementary to the fragment. In addition, engineering measures that provide suitable restriction sites or remove unnecessary DNA or restriction sites can be used. In those cases, wherein insertion, deletion or displacement are possible, in vitro mutagenesis, "primer repair", restriction or connection can be used. Typically, sequence analysis, restriction analysis and biochemical and molecular biological alternatives are carried out according to analytical methods. Then for desired activity, use as described above assay, and particularly for the enzyme activity and transporter activity / ability that they improve respectively, for example, enzyme activity, test the resulting enzyme or transporter.

[0548] As mentioned above, recombinant organisms or microorganisms of the present invention, preferably C1-fixed microorganisms as defined above, can be organisms or microorganisms that have been genetically modified by introducing nucleic acid molecules encoding corresponding enzymes or transporters. Therefore, in a preferred embodiment, organisms or microorganisms are recombinant organisms or microorganisms that have been genetically modified to have the activity of at least one enzyme for conversion according to the present invention or to have the activity / ability of at least one above-mentioned transporter that is improved. This can be realized by, for example, transforming organisms or microorganisms with nucleic acid encoding corresponding enzymes / transporters. Preferably, the nucleic acid molecule introduced into the microorganism is a nucleic acid molecule heterologous to the organism or microorganism, that is, it is not naturally produced in the organism or microorganism.

[0549] In the context of the present invention, "increased activity" or "improved activity" means that the expression and / or activity of the enzyme / transporter in the genetically modified organism or microorganism is at least 10% higher than in the corresponding unmodified organism or microorganism, preferably at least 20% higher, more preferably at least 30% or 50% higher, even more preferably at least 70% or 80% higher, and particularly preferably at least 90% or 100% higher. In even more preferred embodiments, the increase in expression and / or activity can be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments, the expression is at least 100 times higher than in the corresponding unmodified organism or microorganism, and even more preferably, 1000 times higher.

[0550] The term "increased" / "improved" expression / activity also encompasses situations in which the corresponding unmodified organism or microorganism does not express the corresponding enzyme / transporter, such that the corresponding expression / activity in the non-modified organism or microorganism is zero. Preferably, the concentration of the overexpressed enzyme / transporter is at least 5%, 10%, 20%, 30% or 40% of the total host cell protein.

[0551] Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. Examples have been described above.

[0552] In the context of the present invention, the term "recombinant" means that an organism or microorganism, preferably a C1-fixing microorganism as defined above, is genetically modified so that it contains a nucleic acid molecule encoding an enzyme / transporter as defined above compared to a wild-type or unmodified organism or microorganism. The nucleic acid molecule encoding an enzyme / transporter as defined above can be used alone or as part of a vector.

[0553] The nucleic acid molecule may further comprise an expression control sequence operably linked to the polynucleotide contained in the nucleic acid molecule. The term "operably linked" or "operably linked" as used throughout the description of the present invention refers to the connection 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 sequences.

[0554] Expression includes transcription of the heterologous DNA sequence, preferably into translatable mRNA. Regulatory elements that ensure expression in fungi and bacteria are known to those skilled in the art. They include promoters, enhancers, termination signals, targeting sequences, etc. Examples are further given below in conjunction with the explanation of the vector.

[0555] The promoter used in conjunction with the nucleic acid molecule can be homologous or heterologous with respect to its source and / or with respect to the gene to be expressed. Suitable promoters are, for example, promoters that allow their own constitutive expression. Then, promoters that are activated only at a time point determined by external influences can also be used. In this context, artificial and / or chemically inducible promoters can be used.

[0556] The vector may further comprise an expression control sequence operably linked to the polynucleotide contained in the vector. These expression control sequences are suitable for ensuring transcription and synthesis of translatable RNA in bacteria or fungi.

[0557] In addition, different mutations can be inserted into polynucleotides by common methods in molecular biology (see, e.g., Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), resulting in the synthesis of polypeptides that may have altered biological properties. It is possible to introduce point mutations at modified positions of the amino acid sequence that, for example, affect biological activity or polypeptide regulation.

[0558] In addition, mutants with altered substrate or product specificity can be prepared. Preferably, such mutants show increased activity. Alternatively, mutants whose catalytic activity is eliminated but without losing substrate binding activity can be prepared.

[0559] Furthermore, the introduction of mutations into a polynucleotide encoding an enzyme / transporter as defined above allows the gene expression rate and / or the activity of the enzyme / transporter encoded by said polynucleotide to be reduced or increased.

[0560] For genetically modified bacteria or fungi, the polynucleotides encoding the enzyme / transporter as defined above or a part of these molecules can be introduced into a plasmid, which allows sudden change or sequence change by DNA sequence recombination. Standard methods (see, Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchange or the addition of natural or synthetic sequences. DNA fragments can be connected to each other by applying fitters and joints to fragments. In addition, engineering measures that provide suitable restriction sites or remove redundant DNA or restriction sites can be used. In those cases where insertion, deletion or displacement can be performed, in vitro mutagenesis, "introduction repair", restriction or connection can be used. Usually, sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out according to analytical methods.

[0561] Thus, according to the present invention, a recombinant organism or microorganism, preferably a C1-fixing microorganism as defined above, can be produced by genetically modifying a fungus or a bacterium, comprising introducing the above-mentioned polynucleotide, nucleic acid molecule or vector into a fungus or a bacterium.

[0562] The polynucleotide encoding the respective enzyme / transporter is expressed so that a polypeptide having any of the above-mentioned activities is produced. An overview of different expression systems is contained, for example, in Methods in Enzymology 153 (1987), 385-516, see Bitter et al. (Methods in Enzymology 153 (1987), 516-544), 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 given, for example, 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).

[0563] Expression vectors have been extensively described in the literature. Usually, they not only comprise a selectable marker gene and a replication origin that ensures replication in a selected host, but also comprise a bacterial or viral promoter, and a termination signal that is transcribed in most cases. There is usually at least one restriction site or polylinker between the promoter and the termination signal, which can insert the coding DNA sequence. The DNA sequence that naturally controls the transcription of the corresponding gene can be used as a promoter sequence if it is active in a selected host organism. However, this sequence can also be exchanged into other promoter sequences. Promoters that ensure that the genome is expressed and inducible promoters that allow intentional control of gene expression can be used. Bacteria and viral promoter sequences with these characteristics are described in detail in the literature. The regulatory sequences expressed in microorganisms (e.g., Escherichia coli, Saccharomyces cerevisiae) are fully described in the literature. Promoters which allow particularly high expression of downstream sequences are, for example, 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;

[0564] Praeger, New York; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), lp1, rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often produce higher polypeptide yields than constitutive promoters. In order to obtain the optimal amount of polypeptide, a two-step process is usually used. First, the host cells are cultured under optimal conditions to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, the tac promoter is particularly suitable, which can be induced by lactose or IPTG (=isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

[0565] Transformation of host cells with the above-mentioned polynucleotides or vectors can be carried out by standard methods, for example Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; 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. Host cells are cultured in a nutrient medium that meets the requirements of the specific host cells used, especially with regard to pH, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements, etc.

[0566] As described above, recombinant organisms and microorganisms, preferably C1-fixing microorganisms as defined above, and the methods according to the invention are particularly suitable for large-scale production of isobutylene in vivo, in particular for commercial production. The present invention describes new means and methods for commercially and cost-effectively producing large amounts of isobutylene that have not been obtained so far. The large amounts of isobutylene produced can then be further converted in a commercial environment to produce large amounts, such as plug-in gasoline (e.g., isooctane, ETBE, MTBE), jet fuel, cosmetics, chemicals, such as methacrylic acid, polyisobutylene or butyl rubber. As used herein, "large-scale production", "commercial production" and "bioprocessing" of isobutylene in a fermentation reactor or in vitro are carried out at a capacity of greater than at least 100 liters, preferably greater than at least 400 liters, or more preferably a scale of 1,000 liters or more, even more preferably a scale of 5,000 liters or more. As used herein, "large amounts" specifically exclude trace amounts that can be inherently produced in an organism or microorganism.

[0567] For example, in preferred embodiments, the yield of continuous cultures according to the methods described herein is at least about 0.2 grams of isobutylene per liter per day, at least about 0.3 grams of isobutylene per liter per day, at least about 0.4 grams of isobutylene per liter per day, at least about 0.5 grams of isobutylene per liter per day, at least about 0.6 grams of isobutylene per liter per day, at least about 0.7 grams of isobutylene per liter per day, at least about 0.8 grams of isobutylene per liter per day, or at least about 1.0 grams of isobutylene per liter per day. In further embodiments, the yield of continuous cultures according to the methods described herein is about 0.3 grams to about 1.0 grams of isobutylene per liter per day, about 0.4 grams to about 1.0 grams of isobutylene per liter per day, and about 0.5 grams to 1.0 grams of isobutylene per liter per day. In other specific embodiments, the yield of continuous cultures according to the methods described herein is about 0.5 grams to about 0.75 grams of isobutylene per liter per day. In other specific embodiments, continuous cultures according to the methods described herein produce between about 0.5 grams and about 1.5 grams of isobutylene per liter per day.

[0568] In further embodiments, the yield of a batch culture according to the methods described herein is at least about 2 grams per liter in a batch culture, at least about 5 grams per liter in a batch culture, at least about 10 grams per liter in a batch culture, and at least about 15 grams per liter in a batch culture. In some embodiments, the yield of a batch culture according to the methods described herein is about 2 grams to about 5 grams per liter in a batch culture, about 5 grams to about 10 grams per liter in a batch culture, and even more preferably about 10 grams to about 20 grams per liter in a batch culture. In other specific embodiments, the yield of a batch culture according to the methods described herein is about 2.4 grams to about 4.8 grams per liter, and preferably about 4.8 grams to about 9.4 grams per liter in a batch culture, and even more preferably about 9.4 grams to about 18.6 grams per liter in a batch culture.

[0569] In other embodiments, the concentration of CoA in an organism or microorganism used for commercial production of isobutylene is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% or 2.0% compared to all other molecules found in the microorganism or organism.

[0570] As used herein, the term "about" is used to refer to an amount that is approximately, nearly, nearly, or close to being equal to or equal to the stated amount, for example, the stated amount plus / minus about 5%, about 4%, about 3%, about 2%, or about 1%.

[0571] Figure 1 : An artificial pathway for the production of isobutene from acetyl-CoA via 3-methylcrotonate is shown. In addition, the enzymatic cycling of metabolites that may occur in this pathway is shown in steps Xa, Xb, XI and XII.

[0572] Figure 2: Chemical structure of coenzyme A (CoA).

[0573] Figure 3 : The main artificial pathway for the production of isobutene from acetyl-CoA via 3-methylcrotonyl-CoA and two possible main pathways from 3-methylcrotonyl-CoA to isobutene are shown.

[0574] Figure 4 : Shows the individual steps in the biosynthesis of coenzyme A (CoA) from pantothenate.

[0575] Figure 5 : The key enzymes for the conversion of pantothenate to CoA and their respective names in bacteria, yeast and mammals are shown. This figure is obtained from Martinez et al., Biochemical Society Transactions 42(4) (2014) 1112-1117 and supplements thereof.

[0576] Figure 6 : Metabolic pathway for the biosynthesis of isobutene from acetyl-CoA via 3-methylcrotonate in Escherichia coli.

[0577] Figure 7 : shows the growth of strains E (-CoaAR106A, dotted line) and F (+CoaAR106A, solid line). Both strains are described in Example 1.

[0578] Figure 8 : shows a comparison of the IBN volumetric productivity of strains E (-CoaAR106A, dashed line) and F (+CoaAR106A, solid line). Both strains are described in Example 1. The volumetric productivity is the mass of isobutene produced per unit volume of fermentation broth and per unit time.

[0579] Fig. 9 : Comparison of the IBN specific productivity of strains E (-CoaAR106A, dotted line) and F (+CoaAR106A, solid line) is shown. Both strains are described in Example 1. The specific productivity is the mass of isobutylene produced per unit of biomass and per unit of time. The volume was not measured during the entire growth period. Therefore, the specific productivity is not indicated before t = 25h for strain E and t = 39h for strain F.

[0580] Fig.10 : Shown is a strain containing 3-methylcrotonate decarboxylase and prenyltransferase (SGP244) compared to a strain containing the corresponding empty vector (SGP184).

[0581] Fig.11: Shown is a strain containing 3-methylcrotonate decarboxylase and prenyltransferase (SGP 339) compared to a strain containing the corresponding empty vector (SGP184).

[0582] Fig.12 : Shown is a strain containing an isobutene pathway (SGP353) compared to a strain containing the corresponding empty vector (SGP348).

[0583] Fig.13 : It shows that SGP353 produces a large amount of isobutylene, while only trace amounts can be found in SGP348.

[0584] In this specification, many documents, including patent applications, are cited. The disclosures of these documents, although considered irrelevant to the patentability of the present invention, are incorporated herein by reference in their entirety. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0585] The present invention will now be described with reference to the following examples, which are merely illustrative and should not be construed as limiting the scope of the present invention. Example

[0586] Example 1: Construction of a new E. coli chassis for isobutylene production

[0587] Like most organisms, E. coli converts glucose to acetyl-CoA. A modified E. coli base has been previously described in which the production and flux of acetyl-CoA has been increased to optimize the yield of acetyl-CoA production (WO2013 / 007786). Among them, a bacterial base with the following genotype was constructed, strain A:

[0588] MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB

[0589] Plasmid-based overexpression of the PKT gene of the phosphoketolase YP 003354041.1 from Lactococcus lactis in strain A resulted in strain B. This strain B is characterized by a "rewiring" of central carbon metabolism, wherein a new phosphoketolase-based carbon catabolic pathway replaces the inactivated Embden-Meyerhoff-Parnas pathway (EMPP), pentose phosphate pathway (PPP), and Entner Doudoroff pathway (EDP). After the acetone pathway was introduced into strain B, higher acetone production was observed compared to the wild-type MG1655 strain expressing the same acetone pathway.

[0590] To construct a strain that possesses the PKT pathway and can grow robustly on sucrose as a carbon source, strain A was further engineered as follows.

[0591] The PKT gene was introduced into the chromosome of strain A at the kdgk locus (kdgK::P1_RBST7_pkt). The resulting strain had the following genotype:

[0592] MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB kdgK::P1_RBST7_pkt

[0593] The strain was passaged for several months on minimal medium supplemented with glucose as a carbon source with continual selection of clones or populations with the highest growth rates until a doubling time of less than 5 hours was reached.

[0594] To allow efficient sucrose consumption, the CscA, CscB and CscK encoding genes were inserted into the chromosome within the zwf locus (Δzwf::P1_cscA_cscB_cscK_FRT). CscA (Uniprot O86076; NCBI reference sequence WP_000194515.1), CscB (Uniprot Q7WZY9; NCBI reference sequence WP_001197025.1) and CscK (Uniprot Q7WZY7; NCBI reference sequence WP_001274855.1), encoding sucrose hydrolase, non-PTS sucrose permease and fructokinase, respectively, allow sucrose uptake, hydrolysis to glucose and fructose, and phosphorylation of fructose to fructose-6-phosphate, thereby efficiently metabolizing fructose (reviewed in Biotechnology Advances 32 (2014) 905-919). These modifications resulted in a strain that possessed the PKT pathway and was able to grow efficiently on sucrose.

[0595] To further increase sugar consumption, the glk (glucokinase gene, UniprotP0A6V8; NCBI reference sequence: NP_416889.1) gene from E. coli was overexpressed by inserting additional copies under the control of the PN25 promoter at the pfkA locus.

[0596] The resulting strain is referred to as strain C hereinafter.

[0597] Example 2: Construction of E. coli strains for producing isobutylene from acetyl-CoA

[0598] This working example shows the production of isobutylene by a recombinant E. coli strain expressing the following genes: (i) constituting an isobutylene pathway and (ii) encoding pantothenate kinase, CoaA.

[0599] The enzyme used in this study to convert acetyl-CoA to isobutylene (IBN) via 3-methylcrotonate ( Figure 6 ) are listed in Table A. The source of pantothenate kinase is the coaA gene from Escherichia coli MG1655 strain (Uniprot accession number: P0A6I3).

[0600]

[0601]

[0602] Table A

[0603] Expression of the isobutene biosynthetic pathway in Escherichia coli

[0604] The strain C described in Example 1 was used as the host microorganism.

[0605] All genes listed were codon-optimized for expression in E. coli and were (Thermofisher) or Twist Bioscience synthesis, except for the genes YdiI, UbiX and CoaA. These last were directly amplified from the genomic DNA of E. coli MG1655. The mutant of CoaA (R106A) was then constructed by site-directed mutagenesis. The genes thlA and AibA / B were integrated into the ssrS and mgsA loci in the bacterial chromosome, respectively, to generate strain D.

[0606] An expression vector containing the replication origin pSC and a tetracycline resistance marker was used to express genes MvaS, ECH, YdiI and UbiD. The constructed vector was named pGB12762.

[0607] A modified version of pUC18 containing a modified multiple cloning site (pUC18 MCS) (WO 2013 / 007786) and an ampicillin resistance gene (New England Biolabs) was used for overexpression of the UbiX and YdiI genes (plasmid pGB6546) or for a combination of UbiX, YdiI and CoaA (R106A) genes (plasmid pGB13095). Different combinations of the above plasmids were transformed into strain D by electroporation. The strains generated in this way, strains E and F, are summarized in Table B.

[0608] Table B

[0609]

[0610]

[0611] Example 3: Growth of E. coli strains and production of isobutylene from acetyl-CoA

[0612] Pre-culture conditions

[0613] The transformed cells were then inoculated on LB plates supplemented with ampicillin (100 μg / ml) and tetracycline (10 μg / ml). The plates were incubated at 30°C for 2 days. The isolated colonies were used to inoculate LB medium supplemented with ampicillin, tetracycline and 50 mM glutamate. These pre-cultures were grown at 30°C to reach an optical density of 0.6.

[0614] Growing conditions

[0615] Fermentation was performed in a 1 L bioreactor (Multifors 2, Infors HT) with pH and temperature control. Preculture cells were used to inoculate 500 ml of fermentation medium (Table C) supplemented with ampicillin (100 μg / ml), tetracycline (10 μg / ml), thiamine (0.6 mM), glutamate (50 mM), pantothenic acid (5 mM), sucrose (1 g / l) and glycerol (5 g / l) to achieve an initial optical density (OD) of 0.05. 600 ). During the growth period, the temperature (T = 32°C), pH = 6.5 and pO2 = 5% were kept constant. The sucrose feed was kept at 0.1 g / g DCW / h. 5 g / L of yeast extract was added at 16, 24 and 30 hours.

[0616] At the end of the growth period (t = 40 h), the cell density of strain E was lower than that of strain F, with strain E being 13 g / l and strain F being 15.5 g / l ( Figure 7 ).

[0617] IBN production stage

[0618] During this phase, the temperature T=34°C, pH 6.5 and pO2=5% were kept constant. The sucrose feed was started at 0.30 g sucrose / g DCW / h and then adjusted according to strain consumption. The glycerol concentration was kept above 2 g / l.

[0619] Isobutylene (IBN) production of strains E and F was continuously analyzed using a gas chromatograph 7890A (Agilent Technology) equipped with a flame ionization detector (FID) to measure IBN. Volatile organic compounds were chromatographed on a PoraBond Q column (25 m×0.25 mm×0.35 mm) (Agilent), and standard gas (Sigma) was used for IBN quantification.

[0620] Figure 8 A comparison of the volumetric productivity of IBN (mass of IBN produced per unit volume of fermentation broth and per unit time) of strains E and F during the fermentation run is shown. Throughout the production phase, the volumetric productivity (mass of IBN produced per unit volume of fermentation broth and per unit time) of strain F was significantly higher than that of strain E, with the maximum volumetric productivity of strain E being within 66% of the maximum volumetric productivity achieved by strain F.

[0621] Furthermore, this higher volumetric productivity was not only due to the higher biomass concentration of strain F, but also due to the higher yield per unit biomass. Fig. 9 As shown, during most of the production phase (more precisely, from t=42 h to the end of the run), the specific productivity (mass of isobutene produced per unit of biomass and per unit of time) of strain F was higher compared to the reference strain E. The maximum IBN specific productivity of strain E was in the range of 82% of the maximum specific productivity achieved by strain F.

[0622] Table C: Fermentation medium composition (derived from ZYM-5052 medium (Studier FW, Prot. Exp. Pur. 41, (2005), 207-234)).

[0623]

[0624] Example 4: Quantification of Coenzyme A and its Thioester Intermediates

[0625] extract

[0626] 120 μL of bacterial culture was directly sampled from the fermenter and filtered through a membrane filter under vacuum. Immediately after filtration, the filter was placed in aluminum foil and immersed in liquid nitrogen to stop all metabolic reactions. Then, the aluminum foil containing the filter was stored at -80 °C until extraction. Intracellular metabolites (from the membrane filter) were extracted in a Falcon tube at -80 °C with 2 mL of cold MeOH / H2O (80 / 20) mixture for 15 minutes. After this extraction time, the Falcon tube was centrifuged at -9 °C for 20 minutes. Then, the supernatant was taken out and transferred to a new tube. The second extraction of the membrane filter was carried out using 1 mL of cold MeOH / H2O mixture following the same procedure as above (except that the extraction time and centrifugation step at -80 °C were both reduced to 5 min). The combined supernatant (about 3 mL) was filtered through a 0.2 μm syringe filter, and 2.4 mL was evaporated to dryness by a Speed-Vac concentrator. Then, the dry extract was redissolved with 120 μL of ACN / H2O (50 / 50, v / v), filtered again through a 0.2 μm syringe filter, and transferred to an HPLC vial for LC-MS analysis.

[0627] LC-MS analysis

[0628] Analysis was performed in negative ionization mode on a UHPLC system coupled to a Q-Exactive mass spectrometer (ThermoFisher Scientific, Massachusetts, USA). For the LC part, a BEH amide column (1.7 μm, 2.1×100 mm, Waters) adjusted at 25 °C was used. The flow rate was set at 0.5 mL / min, and 2 μL of the sample was injected. The mobile phase consisted of a binary gradient (A: ammonium formate (10 mM) + 0.1% ammonium hydroxide and B: acetonitrile), starting from 95% B in 1.5 min, then decreasing to 55% B until 8.5 min and remaining at 55% B for 2 min, and then returning to the initial conditions. The total analysis time was 19 min. At the mass level, the analysis was performed in Full MS+ddMS2 mode in negative ionization mode. If needed, the ion group between 80 < m / z < 1200 Da was considered, and all ions with an ion intensity greater than 1e5 were fragmented using a collision energy of 35 eV to obtain additional structural information. The calibration curve of the target compound was recorded under the same LC / MS conditions. Data analysis was carried out using Xcalibur v3.0.63 software (ThermoFisher Scientific). The LC-MS characteristics of the target compound are summarized in Table D.

[0629] Table D: LC-MS characteristics of coenzyme A and its thioesters

[0630] <![CDATA[Compound 1 > Molecular formula Molecular weight (g / mol) RT(min) Observed m / z CoA <![CDATA[C 21 H 36 N7O 16 P3S]]> 767.535 6.96 <![CDATA[766.1096[M-H] - ;382.5509[M-H] 2+ ]]> AcCoA <![CDATA[C 23 H 38 N7O 17 P3S]]> 809.570 6.83 <![CDATA[808.1204[M-H] - ;403.5561[M-H] 2+ ]]> AcAcCoA <![CDATA[C 25 H 40 N7O 18 P3S]]> 851.607 6.78 <![CDATA[850.1310[M-H] - ;424.5618[M-H] 2+ ]]> HMG-CoA <![CDATA[C 27 H 44 N7O 20 P3S]]> 911.659 7.27 <![CDATA[910.1526[M-H]-;454.5722[M-H] 2+ <!-- 53 -->]]> MC-CoA <![CDATA[C 26 H 42 N7O 17 P3S]]> 849.635 6.53 <![CDATA[848.1515[M-H] - ;423.5719[M-H] 2+ ]]>

[0631] 1 CoA: coenzyme A; AcCoA: acetyl-coenzyme A; AcAcCoA: acetoacetyl-coenzyme A; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; MC-CoA: 3-methylcrotonyl-coenzyme A

[0632] HPLC analysis

[0633] There are two LC methods that can be used to detect and quantify CoA-derived compounds.

[0634] The first LC method (“Method 1”) was performed by using a multi-wavelength detector (MWD)

[0635] (Agilent, Santa Clara, USA) coupled HPLC 1260 system. Separation was performed by coupling Zorbax Eclipse with C18 column (3.5 μm, 4.6×100 mm, Agilent) adjusted to 30°C. The mobile phase consisted of isocratic elution with acetonitrile (5%) and phosphate buffer (100 mM) at pH 5 (95%) in 10 min. The flow rate was set at 1.5 mL / min and the injection volume was 5 μL.

[0636] The second method (“Method 2”) uses a diode array detector (DAD)

[0637] (Agilent, Santa Clara, USA) coupled HPLC 1260 system. A Zorbax sbAq column (5 μm, 4.6×250 mm, Agilent) conditioned at 30°C was used to separate metabolites. 5 μL of sample was injected. The flow rate was set to 1.5 mL / min. The mobile phase consisted of a binary gradient (A: acetonitrile; and B: H2SO4 8.4 mM) starting at 100% B, then decreasing to 30% B until 8 min and held at 30% B for 1 min. During this 1 min at 30% B, the flow rate was increased to 2 mL / min. After 9 min, the binary gradient was returned to the initial conditions (i.e., 100% B and a flow rate of 1.5 mL / min) within 2 min and held at 100% B for 3 min. The total run time was 14 min.

[0638] In both methods, detection of target metabolites was performed at λ260 nm and calibration curves of pure compounds were recorded under the respective LC conditions used. ChemStation software (Agilent) was used for data analysis.

[0639] Table E: Retention times of CoA and its thioesters in HPLC-based quantification methods

[0640]

[0641] 1 CoA: coenzyme A; AcCoA: acetyl-coenzyme A; AcAcCoA: acetoacetyl-coenzyme A; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; MC-CoA: 3-methylcrotonyl-coenzyme A

[0642] Example 5: Conversion of 3-methylcrotonic acid into isobutylene by recombinant Clostridium ljungdahlii expressing ubiDHav6

[0643] This working example shows the production of isobutylene from 3-methylcrotonate by recombinant Clostridium ljungdahlii expressing genes encoding (i) 3-methylcrotonate decarboxylase ubiD HAv6 and (ii) isoprenyltransferase ubiX. 3-Methylcrotonate decarboxylase converts 3-methylcrotonate to isobutylene and is derived from Hypocrea atroviride (Trichoderma atroviride) and further engineered to achieve efficient IBN production. Isoprenyltransferase converts FMN to isoprene-FMN, providing a cofactor for isoprene decarboxylase. The source of this enzyme is Escherichia coli MG1655.

[0644] Expression of 3-methylcrotonate decarboxylase and prenyltransferase in Clostridium ljungdahlii DSM 13528

[0645] These genes were codon optimized for expression in Clostridium ljungdahlii and synthesized by Genscript. An expression vector containing the pCB102 replicon for maintenance in Clostridium ljungdahlii and the erythromycin resistance gene for selection was used for gene expression. Expression was driven by the th1 gene promoter of Clostridium acetobutylicum ATCC 824 (combined with the ribosome binding site from bacteriophage T7) and terminated by the rrnB terminator from Escherichia coli MG1655.

[0646] Culture conditions

[0647] 2YT medium supplemented with 20 g / L fructose and 5 μg / mL clarithromycin was inoculated from a frozen culture of transformed cells and pressurized with 2 bar of CO2. The cells were grown to mid-exponential phase. The pH of the culture was adjusted to maintain the pH between 5 and 6. The culture was carried out at 150 rpm and 37°C in serum bottles. The cells were collected by centrifugation and stored at -80°C or directly resuspended in PETC 1754 medium supplemented with 20 g / L fructose at an OD600 of 13. The cells were grown in glass vials at 150 rpm and 37°C for 72 h. The amount of IBN was determined by GC-MS analysis of the headspace.

[0648] The strain containing 3-methylcrotonate decarboxylase and prenyltransferase (SGP244) was compared with the strain containing the corresponding empty vector (SGP184) ( Fig.10 ). Compared with SGP184, the isobutylene accumulation of SGP244 increased by 201 times.

[0649] Example 6: Conversion of 3-methylcrotonic acid into isobutylene by recombinant Clostridium ljungdahlii expressing FDCSs5v2

[0650] This working example shows the production of isobutylene from 3-methylcrotonate by recombinant Clostridium ljungdahlii expressing genes encoding (i) 3-methylcrotonate decarboxylase FDCSs5v2 and (ii) isoprenyltransferase ubiX. 3-Methylcrotonate decarboxylase converts 3-methylcrotonate to isobutylene and is derived from Streptomyces and further engineered to achieve efficient IBN production. Isoprenyltransferase converts FMN to isoprene-FMN, thereby providing a cofactor for isoprene decarboxylase. The source of this enzyme is Escherichia coli MG1655.

[0651] Expression of 3-methylcrotonate decarboxylase and prenyltransferase in Clostridium ljungdahlii DSM 13528 These genes were codon optimized for expression in Clostridium ljungdahlii and synthesized by Genscript. An expression vector containing the pCB102 replicon for maintenance in Clostridium ljungdahlii and an erythromycin resistance gene for selection was used for expression of the genes. Expression was driven by the th1 gene promoter of Clostridium acetobutylicum ATCC 824 (combined with a ribosome binding site from bacteriophage T7) and terminated by the rrnB terminator from Escherichia coli MG1655.

[0652] Culture conditions

[0653] 2YT medium supplemented with 20 g / L fructose and 5 μg / mL clarithromycin was inoculated from a frozen culture of transformed cells and pressurized with 2 bar of CO2. The cells were grown to mid-exponential phase. The pH of the culture was adjusted to maintain the pH between 5 and 6. The culture was carried out at 150 rpm and 37°C in serum bottles. The cells were collected by centrifugation and stored at -80°C or directly resuspended in PETC 1754 medium supplemented with 20 g / L fructose at an OD600 of 13. The cells were grown in glass vials at 150 rpm and 37°C for 72 h. The amount of IBN was determined by GC-MS analysis of the headspace.

[0654] The strain containing 3-methylcrotonate decarboxylase and prenyltransferase (SGP339) was compared with the strain containing the corresponding empty vector (SGP184) ( Fig.11). Compared with SGP184, the isobutylene accumulation of SGP244 increased 2093 times.

[0655] Example 7: Construction of Clostridium ljungdahlii strain for producing isobutylene from CO / CO2 / H2 gas via acetyl-CoA

[0656] This working example shows the production of isobutylene from a C1 gas mixture by recombinant Clostridium ljungdahlii expressing genes constituting the isobutylene pathway. The enzymes used in this study to convert acetyl-CoA to isobutylene (IBN) via 3-methylcrotonate ( Figure 6 ) are listed in Table F.

[0657]

[0658] Table F

[0659] Expression of the isobutene biosynthetic pathway in Clostridium ljungdahlii DSM 13528

[0660] These genes were codon optimized for expression in Clostridium ljungdahlii and synthesized by Genscript or IDT. An expression vector containing the pCB102 replicon for maintenance in Clostridium ljungdahlii and an erythromycin resistance gene for selection was used to express the genes divided into two operons. The expression of both operons was driven by the promoter of the thl gene of Clostridium acetobutylicum ATCC 824 and terminated by the rrnB terminator of Escherichia coli MG1655.

[0661] Culture conditions

[0662] For pre-culture, the PETC1754 culture medium supplemented with 20g / L fructose and 5μg / mL clarithromycin was inoculated from the frozen culture of the transformed cells and pressurized with 2 bars of CO2. The cells were grown overnight at 150rpm and 37°C in serum bottles. For main culture, the PETC1754 culture medium supplemented with 5μg / mL clarithromycin was inoculated from the pre-culture and pressurized with a gas mixture of 55%CO, 25%H2 and 20%CO2 (both volume percentages) at 2 bars. The pH of the culture was adjusted to maintain the pH between 5 and 6, and the gas was refilled every day to maintain a pressure of 2 bars. Cultivation was carried out for 6 days at 150rpm and 37°C in serum bottles. The amount of IBN was determined by GC-MS analysis of the headspace.

[0663] The strain containing the isobutylene pathway (SGP353) was compared with the strain containing the corresponding empty vector (SGP348) ( Fig.12 ). While SGP353 produced a large amount of isobutylene, only trace amounts were found in SGP348 ( Fig.13). Thus, by introducing the isobutene pathway into C. ljungdahlii, the isobutene signal was increased 1971-fold compared to a strain without the isobutene pathway.

Claims

1. A recombinant microorganism capable of enzymatically converting acetyl-CoA into isobutylene, (A) wherein in the microorganism: (i) enzymatic conversion of acetyl-CoA into acetoacetyl-CoA, (ii) enzymatic conversion of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA, (iii) Enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA to 3-methylglutaconyl- CoA, (iv) enzymatically converting 3-methylglutaconyl-CoA to 3-methylcrotonyl-CoA, and (v) wherein the 3-methylcrotonyl-CoA is converted to isobutylene by: 3-Methylcrotonyl-CoA is enzymatically converted to 3-methylcrotonate, which is then further enzymatically converted into said isobutylene; (B) wherein the recombinant microorganism has an increased coenzyme A (CoA) pool over that of the microorganism from which it was derived due to: Increased pantothenate uptake, wherein the increased pantothenate uptake is due to recombinant expression of a pantothenate uptake transporter.

2. The recombinant microorganism of claim 1, wherein the microorganism is further characterized by: a) having phosphoketolase activity; b) (i) having a reduced or inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivating the gene encoding phosphofructokinase or by reducing the phosphofructokinase activity compared to an unmodified microorganism; or (ii) No phosphofructokinase activity and c) (i) having a reduced or inactivated oxidative branch of the pentose phosphate pathway (PPP) by inactivating the enzyme encoding glucose-6-phosphate dehydrogenase or by reducing the activity of glucose-6-phosphate dehydrogenase compared to a non-modified microorganism; or (ii) It has no glucose-6-phosphate dehydrogenase activity.

3. The recombinant microorganism of claim 2, further characterized by: d) possessing fructose-1,6-bisphosphate phosphatase.

4. The recombinant microorganism of claim 2 or 3, wherein EMPP is further reduced or inactivated by inactivating a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity compared to an unmodified microorganism.

5. The recombinant microorganism of claim 2 or 3, which has been genetically modified to have increased phosphoketolase activity.

6. The recombinant microorganism of claim 2 or 3, wherein the microorganism is a fungus.

7. The recombinant microorganism of claim 2 or 3, wherein the microorganism is a bacterium.

8. The recombinant microorganism of claim 7, wherein the gene encoding the PEP-dependent PTS transporter has been inactivated.

9. Use of a recombinant microorganism as defined in any one of claims 1 to 8 for the production of isobutene.

10. A method for producing isobutene by culturing a recombinant microorganism as defined in any one of claims 1 to 8.

11. A method for producing isobutylene from acetyl-CoA in a recombinant organism or microorganism, comprising: (A)(i) enzymatic conversion of acetyl-CoA into acetoacetyl-CoA, (ii) enzymatically converting the acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA, (iii) enzymatically converting the 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA, (iv) enzymatically converting the 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA, and (v) wherein the 3-methylcrotonyl-CoA is converted to isobutylene by: enzymatically converting 3-methylcrotonyl-CoA to 3-methylcrotonate, which is then further enzymatically converted to the isobutylene; (B) wherein the method further comprises: Enzymatically provides acetyl-CoA from CoA, (C) wherein the method further comprises providing coenzyme A (CoA) by culturing a recombinant organism or microorganism, wherein the recombinant organism or microorganism has an increased coenzyme A (CoA) pool over the organism or microorganism from which it was derived due to: Increased pantothenate uptake, wherein the increased pantothenate uptake is due to recombinant expression of a pantothenate uptake transporter.

12. The method of claim 10 or 11, further comprising supplementing the culture medium with pantothenic acid.

13. The process of claim 10 or 11, further comprising collecting the produced isobutylene.

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