WO2026003122A1

WO2026003122A1 - Method for producing isobutene from acetic acid

Info

Publication number: WO2026003122A1
Application number: PCT/EP2025/067984
Authority: WO
Inventors: Benoit VILLIERS; Guillaume JANNOT; Ronan BESSARD; Frédéric OLLIVIER
Original assignee: Global Bioenergies SA
Current assignee: Global Bioenergies SA
Priority date: 2024-06-26
Filing date: 2025-06-25
Publication date: 2026-01-02
Anticipated expiration: 2026-12-26

Abstract

The present invention relates to a method for the production of isobutene, a precursor, or a derivative thereof, from acetic acid, or a salt thereof, in a recombinant microorganism, the method comprising the steps of: (i) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA; and (ii) enzymatically converting said produced acetyl-CoA into isobutene, or a precursor thereof.

Description

METHOD FOR PRODUCING ISOBUTENE FROM ACETIC ACID

The present invention relates to a method for the production of isobutene, a precursor, or a derivative thereof, from acetic acid, or a salt thereof, in a recombinant microorganism, the method comprising the steps of: (i) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA; and (ii) enzymatically converting said produced acetyl-CoA into isobutene, a precursor, or a derivative thereof.

BACKGROUND OF THE INVENTION

A large number of chemical compounds are currently derived from petrochemicals. Alkenes (such as ethylene, propylene, the different butenes, or else the pentenes, for example) are used in the plastics industry, for example for producing polypropylene or polyethylene, and in other areas of the chemical industry and that of fuels. Butylene exists in four forms, one of which, isobutene (also referred to as isobutylene), enters into the composition of methyl-tert- butyl-ether (MTBE), an anti-knock additive for automobile fuel. Isobutene can also be used to produce oligomers such as isooctene, isododecane, and/or isohexadecane or mixtures thereof. Isooctene can be reduced to isooctane (2,2,4-trimethylpentane); the very high octane rating of isooctane makes it the best fuel for so-called "gasoline" engines. Isododecane is widly used as a cosmetic emollient in makeup compositions as well as in skin care and hair care compositions. A mixture of isododecane and isohexadecane (85/15) has also been recently approved as sustainable aviation fuel by the ASTM. Alkenes such as isobutene are currently produced by catalytic cracking of petroleum products (or by a derivative of the Fischer-Tropsch process in the case of hexene, from coal or gas). The production costs are therefore tightly linked to the price of oil. Moreover, catalytic cracking is sometimes associated with considerable technical difficulties which increase process complexity and production costs. The production by a biological pathway of alkenes such as isobutene is called for in the context of a sustainable industrial operation in harmony with geochemical cycles. The first generation of biofuels consisted in the fermentative production of ethanol, as fermentation and distillation processes already existed in the food processing industry. The production of second-generation biofuels is in an exploratory phase, encompassing in particular the production of long chain alcohols (butanol and pentanol), terpenes, linear alkanes and fatty acids. Two reviews provide a general overview of research in this field: Ladygina et al. (Process Biochemistry 41 (2006), 1001) and Wackett (Current Opinions in Chemical Biology 21 (2008), 187).

The conversion of isovalerate to isobutene by the yeast Rhodotorula minuta has been described (Fujii et al. (Appl. Environ. Microbiol. 54 (1988), 583)).

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 conversions wherein the last step of the proposed pathway is the conversion of 3-hydroxy-3-methyl butyric acid (also referred to as 3-hydroxyisovalerate (HIV)) by making use of a mevalonate diphosphate decarboxylase. This reaction for the production of isobutene from 3-hydroxy-3-methylbutyric acid is also described in W02010/001078 which, in general terms, describes methods for generating alkenes through a biological process, in particular methods for producing terminal alkenes (in particular propylene, ethylene, 1-butylene, isobutylene or isoamylene) from molecules of the 3- hydroxyalkanoate type.

WO2012/052427 also describes a method for generating alkenes through a biological process while, in particular, a method for producing alkenes (for example propylene, ethylene, 1- butylene, isobutylene or isoamylene) from molecules of the 3-hydroxyalkanoate type is described. In this context, the reaction for the production of isobutene from 3-hydroxy-3- methylbutyric acid is also described in WO2012/052427.

WO 2016/042012 describes methods for producing said 3-hydroxy-3-methylbutyric acid. In particular, WO 2016/042012 describes methods for producing 3-hydroxy-3-methyl butyric acid comprising the step of enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid and the step of enzymatically further converting the thus produced 3-methylcrotonic acid into 3-hydroxy-3-methylbutyric acid.

In Gogerty et al. (loc. cit.) and in van Leeuwen et al. (loc. cit.) the production of 3-hydroxy-3- methylbutyric acid is proposed to be achieved by the conversion of 3-methylcrotonyl-CoAvia 3-hydroxy-3-methylbutyryl-CoA. In order to further improve the efficiency and variability of methods for producing isobutene from renewable resources, alternative routes for the provision of isobutene and its precursors have been developed by providing methods for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid (also termed 3-methyl-2-butenoic acid, 3,3-dimethylacrylic acid or senecioic acid) into isobutene.

In particular, in WO 2017/085167, methods for the production of isobutene have been described comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene, wherein the enzymatic conversion of 3-methylcrotonic acid into isobutene is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) into a flavin-derived cofactor while these enzymes have artificially been implemented in a pathway which ultimately leads to the production of isobutene. Moreover, in WO 2017/085167, methods have been described, wherein such a method further comprises (a) providing the 3-methylcrotonic acid by the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid, or (b) providing the 3-methylcrotonic acid by the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3- methylcrotonic acid.

WO 2017/085167 also describes that this method which has been developed for the production of isobutene from 3-methylcrotonyl-CoA via 3-methylcrotonic acid or from 3- hydroxyisovalerate (HIV) via 3-methylcrotonic acid may be embedded in a pathway for the production of isobutene starting from acetyl-CoA which is a central component and an important key molecule in metabolism used in many biochemical reactions. The corresponding reactions are schematically shown in Figure 1. In WO 2018/206262 it is described that 3-methylcrotonic acid is enzymatically converted into isobutene by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase when dimethylallyl pyrophosphate (DMAPP) instead of DMAP is used.

WO 2018/206262, moreover, describes that the enzymatic conversion of 3-methylcrotonic acid into isobutene which is achieved by making use of an FMN-dependent decarboxylase associated with an FMN prenyl transferase, wherein said FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) into a flavin-derived cofactor is a key step of the above overall metabolic pathway from acetyl-CoA into isobutene. It has been found that in this key step, the availability of dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) as well as the availability of the flavin cofactor FMN are limiting factors while in WO 2018/206262 improved methods by increasing the pool/amount of dimethylallyl phosphate (DMAP) and/or dimethylallyl pyrophosphate (DMAPP) in order to ensure the efficient biosynthesis of the prenylated flavin cofactor (FMN or FAD) are described.

Moreover, WO 2020/188033 is based on the concept of increasing the yield of isobutene by providing and maintaining a high pool of acetyl-CoA in a cells used for isobutene production wherein the acetyl-CoA pool is kept high by ensuring an increased uptake of pantothenate by the cell and/or an increased conversion of pantothenate into CoA.

Further, WO 2014/086780 describes a fermentation method for producing a hydrocarbon compound, preferably isobutene, comprising the culturing of an organism in a liquid fermentation medium, wherein said organism produces a desired hydrocarbon compound by an enzymatic pathway, said enzymatic pathway comprising an intermediate which evaporates into the gaseous phase and wherein said intermediate is recovered from the gaseous phase and is reintroduced into the liquid fermentation medium. Moreover, WO 2014/086781 describes a process for the fermentative production of a hydrocarbon, preferably isobutene, wherein a microorganism producing the hydrocarbon is cultured in a liquid fermentation medium in a fermenter, wherein an inlet gas comprising oxygen is fed into the fermenter and the total pressure of the inlet gas before introduction into the fermenter is about 1.5 bar to about 15 bar (about 150 kPa to about 1500 kPa), wherein the hydrocarbon is obtained in a gaseous state in the fermentation off-gas, and wherein the concentration of oxygen in the fermentation off-gas is controlled to be below about 10 vol-%.

Although, as described above, various approaches have been described in the prior art for producing isobutene by enzymatic conversions in biological systems and in fermentation processes/fermenters, thereby allowing to use renewable resources as raw material, the acetyl-CoA that is used as starting point for the production of isobutene is always produced from sugars, such as glucose or sucrose. The use of sugars as carbon source not only contributes significantly to the overall cost of the production process, but also consumes resources that could be used for human nutrition.

Accordingly, there is still a need for improvements, in particular, regarding the sustainability of such (fermentation) methods. In particular, it would be desirable to produce isobutene from renewable resources and/or waste products that do not directly compete with human nutrition, and that allow reducing the CO2 emissions associated with isobutene production when compared to isobutene produced from fossil resources or from sugar fermentation.

SUMMARY OF THE INVENTION

The present invention meets this demand by providing, in a first aspect, a method for the production of isobutene, a precursor, or a derivative thereof, from acetic acid, or a salt thereof, in a recombinant microorganism, the method comprising the steps of: (i) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA; and (ii) enzymatically converting said produced acetyl-CoA into isobutene, or a precursor thereof.

Surprisingly, it has been shown that isobutene can be produced from acetic acid in a microbial fermentation process. The enzymatic conversion of acetyl-CoA to isobutene has previously been described in WO 2020/188033, where acetyl-CoA is typically generated from pyruvate, a central metabolite in carbon metabolism. Although it was known in the art that acetyl-CoA can be obtained from acetate and CoA by the enzyme acetyl-CoA synthase, the use of acetic acid as a starting material for isobutene biosynthesis had neither been considered nor demonstrated. This is primarily because acetic acid is known to inhibit cellular growth. Contrary to this prevailing prejudice, the inventors have unexpectedly found that acetic acid can indeed be used as a starting material for the production of isobutene, as well as its precursors and derivatives.

Acetic acid is one of the most common waste products from microbial fermentation and is therefore an inexpensive chemical that is available in large quantities. By using acetic acid as starting product for the production of isobutene, the need for sugars in the microbial fermentation process is obviated or at least significantly reduced. This is advantageous in that the acetic acid-based process does not rely on materials that could otherwise be used for human nutrition. In addition, acetic acid is significantly cheaper than second-generation sugars (i.e., sugars derived from non-food biomass) and could therefore contribute to the economic viability of the process. Last but not least, acetic acid can be obtained with a process that captures carbon dioxide, which further contributes to the sustainability of the process described here. Currently, in Europe, only second-generation sugars qualify as feedstock for producing sustainable aviation fuel, but they are not readily available at volumes and costs compatible with SAF.

In a particular embodiment, the invention relates to a recombinant microorganism which is capable of enzymatically converting acetic acid, or a salt thereof, into isobutene, or a precursor thereof, wherein said recombinant microorganism expresses a plurality of enzymes catalyzing: (i) the conversion of acetic acid, or a salt thereof, into acetyl-CoA; and (ii) the conversion of acetyl-CoA into isobutene, or a precursor thereof.

Conversion of acetic acid, or a salt thereof, into acetyl-CoA

In a first step of the process of the invention, acetic acid, or a salt thereof is converted into acetyl-CoA by a recombinant microorganism. In a second step, the resulting acetyl-CoA is then converted into isobutene, a precursor, or a derivative thereof as known in the art and as described in more detail herein below.

The first step of the method of the invention starts from acetic acid, or a salt thereof. Acetic acid, or the salt thereof, may be enzymatically converted by the recombinant microorganism of the invention into acetyl-CoA by any suitable metabolic pathway. Synthetic routes from acetate to acetyl-CoA are depicted in Figure 2.

For example, acetate may be enzymatically converted into acetyl-CoA in a single reaction step. Alternatively, or in parallel, acetate may be converted into acetyl-CoA in a two-step method, wherein acetate is first enzymatically converted into acetate phosphate and the resulting acetate phosphate is then enzymatically converted into acetyl-CoA.

Accordingly, in a particular embodiment, the invention relates to the method according to the invention, wherein the conversion of acetic acid, or a salt thereof, into acetyl-CoA comprises the step(s) of (i) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA with an acetyl-CoA synthase (EC 6.2.1.1); and/or (ii) enzymatically converting acetic acid, or a salt thereof, into acetyl-phosphate with an acetate kinase (EC 2.7.2.1) and enzymatically converting the resulting acetyl-phosphate into acetyl-CoA with a phosphate acetyltransferase (EC 2.3.1.8).

In another particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein said microorganism expresses: (i) an acetyl-CoA synthase (EC

6.2.1.1) to enzymatically convert acetate into acetyl-CoA by; and/or (ii) an acetate kinase (EC

2.7.2.1) to enzymatically convert acetate into acetyl-phosphate and a phosphate acetyltransferase (EC 2.3.1.8) to enzymatically convert the resulting acetyl-phosphate into acetyl-CoA.

In certain embodiments, acetate is enzymatically converted into acetyl-CoA by an acetyl-CoA synthase (EC 6.2.1.1). "Acetyl-CoA synthase", also referred to as acetyl-CoA synthetase, is an enzyme that catalyzes the formation of acetyl-CoA from acetate and coenzyme A (CoA) in the presence of ATP. The reaction typically involves the conversion of acetate (a two-carbon molecule) and CoA into acetyl-CoA, which is a crucial intermediate in various metabolic pathways, including the citric acid cycle (Krebs cycle), fatty acid synthesis, and amino acid metabolism.

The general reaction catalyzed by acetyl-CoA synthase can be summarized as follows: ATP + Acetate + CoA AMP + Pyrophosphate + Acetyl-CoA

Acetyl-CoA synthase plays a vital role in cellular metabolism by providing acetyl-CoA, which is essential for energy production and biosynthetic processes. This enzyme is found in various organisms, including bacteria, plants, and animals, and is crucial for maintaining metabolic homeostasis.

The acetyl-CoA synthase used in the method of the present invention may be an endogenous acetyl-CoA synthase or a recombinantly expressed acetyl-CoA synthase.

That is, in certain embodiments, the recombinant microorganism of the invention may be a microorganism that endogenously expresses an acetyl-CoA synthase to catalyze the conversion of acetate into acetyl-CoA.

However, it is preferred herein that the recombinant microorganism of the invention recombinantly overexpresses an acetyl-CoA synthase to increase the metabolic flux from acetate to acetyl-CoA and/or increase the intracellular pool of acetyl-CoA that can then serve as substrate for the production of isobutene.

Generally, "recombinant" in the context of the present invention denotes the artificial genetic modification of a microorganism, either by addition, removal, or modification of a chromosomal or extra-chromosomal gene or regulatory motif such as a promoter, or by fusion of organisms, or by addition of a vector of any type, for example plasmidic.

The term "recombinant expression" denotes the production of a protein involving a genetic modification, preferably in order to produce a protein of exogenous or heterologous origin with respect to its host, that is, which does not naturally occur in the production host, or in order to produce a modified or mutated endogenous protein.

The "recombinant expression" in the context of the present invention is preferably an "overexpression". "Overexpression" or "overexpressing" in this context denotes the recombinant expression of a protein in a host organism, preferably originating from an organism different from the one in which it is expressed, increased by at least 10% and preferably by 20%, 50%, 100%, 500% and possibly more as compared to the natural expression of said protein occurring in said host microorganism. This definition also encompasses the case where there is no natural expression of said protein.

Recombinant expression of an acetyl-CoA synthase may be achieved by placing an endogenous gene encoding an acetyl-CoA synthase under control of a recombinant promoter. Alternatively, recombinant expression of an acetyl-CoA synthase may be achieved by introducing a heterologous gene encoding an acetyl-CoA synthase into the recombinant microorganism of the invention, wherein the heterologous gene encoding an acetyl-CoA synthase may be under control of its own promoter or a recombinant promoter.

In a preferred embodiment, an endogenous or heterologous gene encoding an acetyl-CoA synthase is under control of a recombinant promoter. Thus, in a particular embodiment, the invention relates to the method of the invention, wherein the acetyl-CoA synthase is expressed from a recombinant promoter.

The term "recombinant promoter", as used herein, refers to a promoter which is not naturally occurring in the genome in the upstream region of the gene encoding the acetyl-Co synthetase within a host cell in order to control the transcription of said gene. The recombinant promoter can be a promoter derived from the same or another organism or a heterologous promoter being derived from any other source provided that the recombinant promoter is functional (i.e. is able to control the transcription of the gene operably linked thereto) in the host cell. The term "promoter" includes also fragments of a wild-type promoter, provided that said fragments are able to control the transcription rate of a gene to which said promoter fragment is operably linked.

The gene encoding the acetyl-CoA synthase may be under control of an inducible or a constitutive recombinant promoter. That is, the gene encoding the acetyl-CoA synthase may be under control of a constitutive recombinant promoter to ensure constant expression of acetyl-CoA synthase in the cell and thus an efficient conversion of acetate into acetyl-CoA. However, it is particularly preferred therein, that the promoter is an inducible promoter. Thus, in a particular embodiment, the invention relates to the method of the invention, wherein the acetyl-CoA synthase is expressed from an inducible promoter.

Using inducible promoters for the (over-)expression of acetyl-CoA synthase in the method of the present invention has the advantage that the conversion of acetate into acetyl-CoA can be switched on at a desired point in time. For example, in certain embodiments, the conversion of acetate into acetyl-CoA may be switched on once a sufficient amount of biomass has been obtained.

An "inducible promoter" is a segment of DNA that initiates transcription of a gene in response to specific external or internal stimuli. Unlike constitutive promoters, which are always active, inducible promoters are only activated under certain conditions, allowing for controlled expression of the associated gene. These stimuli can include various factors, including light, temperature, or chemicals (i.e., chemical inducers). Inducible promoters are widely used in genetic engineering and research to study gene function and regulation.

Various inducible promoters have been described in the art and are known to the skilled person. Chemically inducible promoters known in the art include, inter alia, lac promoter (Inducer: Isopropyl P-D-l-thiogalactopyranoside (IPTG) or lactose), araBAD promoter (Inducer: Arabinose), tet promoter (Inducer: Tetracycline or its analogs (e.g., anhydrotetracycline)), trp promoter (Inducer: Tryptophan depletion), rhaBAD promoter (Inducer: Rhamnose), pLac/ara-1 promoter (Inducer: IPTG and arabinose), or phage Lambda promoter (Inducer: Temperature shift).

In a particular embodiment, the invention relates to the method of the invention, wherein the acetyl-CoA synthase is expressed from a heat-inducible promoter.

That is, by placing the gene encoding the acetyl-CoA synthase under the control of a heatinducible promoter, the conversion of acetate to acetyl-CoA can be selectively induced by exposing the recombinant microorganisms to temperatures at which the promoter is activated.

A "heat-inducible promoter" is a type of inducible promoter that activates transcription of a gene in response to elevated temperatures. When the temperature reaches a certain threshold, the heat-inducible promoter triggers the expression of the downstream gene. Heatinducible promoters are commonly found in organisms that need to rapidly respond to changes in temperature, such as bacteria, plants, and some animal cells.

In certain embodiments, the gene encoding the acetyl-CoA synthase is under control of the heat-inducible promoter PL or PR.

The PL and PR promoters are regulatory DNA sequences derived from bacteriophage lambda (A) that control the transcription of adjacent genes. The PL promoter (leftward promoter) and PR promoter (rightward promoter) are integral to the phage's ability to switch between lysogenic and lytic cycles. These promoters are tightly regulated by the lambda repressor protein (cl), which binds to operator sites to repress transcription under lysogenic conditions. Upon inactivation of the repressor, such as through a temperature shift in temperaturesensitive mutants, the promoters are derepressed, initiating transcription of downstream genes. The PL and PR promoters are widely utilized in genetic engineering and synthetic biology for inducible and controlled gene expression systems, enabling precise temporal regulation of target genes in response to specific environmental cues.

In certain embodiments, the gene encoding the acetyl-CoA synthase is under control of the heat-inducible promoter PR.

In a particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein said acetyl-CoA synthase is encoded by a nucleic acid comprising a recombinant promoter, preferably wherein the recombinant promoter is an inducible promoter, more preferably a heat-inducible promoter. The gene encoding the acetyl-CoA synthase can originate from any organism, provided that an active acetyl-CoA synthase can be expressed from said gene in the host organism. The gene encoding the acetyl-CoA synthase may be of any origin, including prokaryotic, archaeal, or eukaryotic.

In certain embodiments where the host organism is a bacterial host organism, the gene encoding the acetyl-CoA synthase is preferably of bacterial origin. The gene encoding the acetyl-CoA synthase may be from the same species as the host organism or from a different (bacterial) host organism.

The acetyl-CoA synthase may be a wild-type acetyl-CoA synthase. However, it is preferred herein that the acetyl-CoA synthase is an engineered acetyl-CoA synthase.

Preferably, the acetyl-CoA synthase is engineered such that it is resistant to inactivation through acetylation. That is, in a particular embodiment, the invention relates to the method according to the invention, wherein the acetyl-CoA synthase comprises at least one mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase, thereby maintaining the acetyl-CoA synthase in its active state.

In another particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein the acetyl-CoA synthase comprises at least one mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase, thereby maintaining the acetyl-CoA synthase in its active state.

It has been demonstrated by Starai et al. (J Biol Chem. 2005 Jul 15;280(28):26200-5. doi: 10.1074/jbc.M504863200. Epub 2005 May 17) that one residue in acetyl-CoA synthase from Salmonella enterica, Leu-641, is critical for the acetylation of acetyl-CoA synthase by the protein acetyltransferase enzyme. In vivo and in vitro evidence shows that mutations at Leu- 641 prevent the acetylation of acetyl-CoA synthase by protein acetyltransferase, maintain the acetyl-CoA synthase enzyme in its active state, and bypass the need for sirtuin deacetylase activity during growth on acetate. The engineered acetyl-CoA synthase may thus be an acetyl-CoA synthase from Salmonella enterica comprising a mutation in position Leu-641. Such engineered acetyl-CoA synthetases are disclosed in WO 2003/104403, which is fully incorporated herein by reference.

However, the engineered acetyl-CoA synthase may be derived from a different organism and may comprise a mutation at a position that is homologous to position Leu-641 of S. enterica acetyl-CoA synthase. Preferably, the position that is homologous to position Leu-641 of S. enterica acetyl-CoA synthase is a leucine residue. More preferably, the position that is homologous to position Leu-641 of S. enterica acetyl-CoA synthase is replaced by a proline residue.

Suitable assays to identify whether an engineered acetyl-CoA synthase is resistant to inactivation through acetylation are known in the art and have been disclosed by Starai et al. (J Biol Chem. 2005 Jul 15;280(28):26200-5. doi: 10.1074/jbc.M504863200. Epub 2005 May 17) and in WO 2003/104403.

In certain embodiments, the engineered acetyl-CoA synthase is derived from E. coli and comprises a mutation at a position that is homologous to position Leu-641 of S. enterica acetyl-CoA synthase. That is, in a particular embodiment, the invention relates to the method of the invention, wherein the acetyl-CoA synthase comprises or consists of an amino acid sequence as set forth in SEQ ID NO:1 and wherein the mutation that prevents acetylation of the acetyl-CoA synthase is a mutation of leucine at position 641 of SEQ ID NO:1, preferably wherein the leucine at position 641 of SEQ ID NO:1 is replaced by a proline.

In another particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein the acetyl-CoA synthase comprises or consists of an amino acid sequence as set forth in SEQ ID NO:1, and wherein the mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase is a mutation of a leucine at position 641 of SEQ ID NO:1, preferably wherein the leucine (L) at position 641 of SEQ ID NO:1 is replaced by a proline.

E. coli acetyl-CoA synthase comprises the following amino acid sequence (SEQ ID NO :1): MSQIHKHTIP ANIADRCLIN PQQYEAMYQQ SINVPDTFWG EQGKILDWIK PYQKVKNTSF APGNVSIKWY EDGTLNLAAN CLDRHLQENG DRTAI IWEGD DASQSKHISY KELHRDVCRF ANTLLELGIK KGDWAIYMP MVPEAAVAML ACARIGAVHS VIFGGFSPEA VAGRIIDSNS RLVITSDEGV RAGRSIPLKK NVDDALKNPN VTSVEHVWL KRTGGKIDWQ EGRDLWWHDL VEQASDQHQA EEMNAEDPLF ILYTSGSTGK PKGVLHTTGG YLVYAALTFK YVFDYHPGDI YWCTADVGWV TGHSYLLYGP LACGATTLMF EGVPNWPTPA RMAQWDKHQ VNILYTAPTA IRALMAEGDK AIEGTDRSSL RILGSVGEPI NPEAWEWYWK KIGNEKCPW DTWWQTETGG FMITPLPGAT ELKAGSATRP FFGVQPALVD NEGNPLEGAT EGSLVITDSW PGQARTLFGD HERFEQTYFS TFKNMYFSGD GARRDEDGYY WITGRVDDVL NVSGHRLGTA EIESALVAHP KIAEAAWGI PHNIKGQAIY AYVTLNHGEE PSPELYAEVR NWVRKEIGPL ATPDVLHWTD SLPKTRSGKI MRRILRKIAA GDTSNLGDTS TLADPGWEK LLEEKQAIAM PS

Engineered E. coli acetyl-CoA synthase comprising the L641P mutation comprises the following amino acid sequence (SEQ ID NO :2):

MSQIHKHTIP ANIADRCLIN PQQYEAMYQQ SINVPDTFWG EQGKILDWIK PYQKVKNTSF APGNVSIKWY EDGTLNLAAN CLDRHLQENG DRTAI IWEGD DASQSKHISY KELHRDVCRF ANTLLELGIK KGDWAIYMP MVPEAAVAML ACARIGAVHS VIFGGFSPEA VAGRIIDSNS RLVITSDEGV RAGRSIPLKK NVDDALKNPN VTSVEHVWL KRTGGKIDWQ EGRDLWWHDL VEQASDQHQA EEMNAEDPLF ILYTSGSTGK PKGVLHTTGG YLVYAALTFK YVFDYHPGDI YWCTADVGWV TGHSYLLYGP LACGATTLMF EGVPNWPTPA RMAQWDKHQ VNILYTAPTA IRALMAEGDK AIEGTDRSSL RILGSVGEPI NPEAWEWYWK KIGNEKCPW DTWWQTETGG FMITPLPGAT ELKAGSATRP FFGVQPALVD NEGNPLEGAT EGSLVITDSW PGQARTLFGD HERFEQTYFS TFKNMYFSGD GARRDEDGYY WITGRVDDVL NVSGHRLGTA EIESALVAHP KIAEAAWGI PHNIKGQAIY AYVTLNHGEE PSPELYAEVR NWVRKEIGPL ATPDVLHWTD SLPKTRSGKI MRRILRKIAA GDTSNLGDTS TLADPGWEK PLEEKQAIAM PS

Accordingly, in a preferred embodiment, the recombinant microorganism of the invention encodes an engineered E. coli acetyl-CoA synthase having the amino acid sequence set forth in SEQ ID NO:2. More preferably, the gene encoding the engineered E. coli acetyl-CoA synthase having the amino acid sequence set forth in SEQ ID NO:2 is under control of an inducible promoter, even more preferably a heat-inducible promoter, such as the heat-inducible promoter PR.

Alternatively, or in addition, acetic acid, or a salt thereof, may be converted into acetyl-CoA via acetyl-phosphate. That is, in certain embodiments, acetic acid, or a salt thereof, may be first enzymatically converted into acetyl-phosphate by an acetate kinase (EC 2.7.2.1) and the resulting acetyl-phosphate may then be enzymatically converted into acetyl-CoA with a phosphate acetyltransferase (EC 2.3.1.8).

Acetate kinase (EC 2.7.2.1) is an enzyme that catalyzes the reversible phosphorylation of acetate to form acetyl-phosphate, using ATP as the phosphate donor. The reaction it facilitates can be summarized as follows:

Acetate + ATP acetyl-phosphate + ADP

This enzyme plays a crucial role in the metabolism of acetate in various microorganisms, including bacteria and archaea, where it is involved in pathways such as the acetyl-CoA pathway and the mixed acid fermentation pathway. Acetate kinase is essential for energy production and regulation, as it helps in the conversion of acetate, a key metabolic intermediate, into a high-energy compound, acetyl phosphate, which can then be used in various biosynthetic processes or further converted to acetyl-CoA.

Phosphate acetyltransferase (EC 2.3.1.8) is an enzyme that catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. The reaction it facilitates can be summarized as follows:

Acetyl-phosphate + CoA Acetyl-CoA + Phosphate

This enzyme plays a critical role in the metabolism of acetate and other short-chain fatty acids in various microorganisms, including bacteria and archaea. It is a key component of the acetate fermentation pathway, where it works in conjunction with acetate kinase to convert acetyl phosphate into acetyl-CoA, a central metabolite involved in numerous biosynthetic and energy-producing pathways, such as the citric acid cycle (Krebs cycle) and fatty acid synthesis. Phosphate acetyltransferase is essential for cellular energy production and the regulation of metabolic flux, enabling the efficient utilization of acetate as a carbon and energy source. The acetate kinase and the phosphate acetyltransferase may be of any origin, provided that they can catalyze the conversion of acetate into acetyl-CoA in a host organism. In certain embodiments, the acetate kinase and the phosphate acetyltransferase are from the same species as the host organism. In certain embodiments, at least one of the acetate kinase and/or the phosphate acetyltransferase are from a different species as the host organism.

The recombinant microorganism of the invention may use its endogenous acetate kinase and/or phosphate acetyltransferase to convert acetic acid, or a salt thereof, into acetyl-CoA.

However, it is preferred herein that the acetate kinase and/or the phosphate acetyltransferase are overexpressed in the recombinant microorganism of the invention. Accordingly, the invention relates to the method according to the invention, wherein the recombinant microorganism overexpresses an acetate kinase (EC 2.7.2.1) and/or a phosphate acetyltransferase (EC 2.3.1.8).

In another particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein said recombinant microorganism comprises a recombinant nucleic acid encoding an acetate kinase (EC 2.7.2.1) and/or a phosphate acetyltransferase (EC 2.3.1.8), preferably wherein the recombinant nucleic acid is suitable for overexpressing the acetate kinase (EC 2.7.2.1) and/or the phosphate acetyltransferase (EC 2.3.1.8).

The acetate kinase and/or the phosphate acetyltransferase may be overexpressed from a recombinant promoter, including inducible promoters and constitutive promoters. In certain embodiments, both the acetate kinase and the phosphate acetyltransferase are expressed from an inducible promoter, such as any of the inducible promoters disclosed above in the context of the acetyl-CoA synthetase. In certain embodiments, the acetate kinase and/or the phosphate acetyltransferase are (over)expressed from a heat-inducible promoter.

In certain embodiments, genes encoding the acetyl-CoA synthase, the acetate kinase and the phosphate acetyltransferase are (over)expressed from a recombinant promoter. In certain embodiments, genes encoding the acetyl-CoA synthase, the acetate kinase and the phosphate acetyltransferase are (over)expressed from an inducible promoter, preferably a heatinducible promoter as disclosed herein above.

Preferably, genes encoding the acetyl-CoA synthase, the acetate kinase and the phosphate acetyltransferase are (over)expressed from a plasmid comprising an inducible promoter. In certain embodiments, all three genes may be encoded in a single plasmid comprising an inducible promoter, preferably a heat-inducible promoter.

In certain embodiments, it may be advantageous to delete or inactivate the endogenous genes encoding the acetate kinase and/or the phosphate acetyltransferase. Since the reactions catalyzed by these enzymes are reversible, expression of these enzymes in the absence of acetate may result in the accumulation of acetate, which may be harmful to the cells. That is particularly relevant in processes in which biomass is generated in an initial stage of the process in the absence of acetate.

Thus, in certain embodiments, the endogenous gene(s) of the recombinant microorganism encoding the acetate kinase and/or the phosphate acetyltransferase is/are deleted or inactivated. In certain embodiments, the recombinant microorganism of the invention is an E. coli strain in which at least one of the endogenous genes ackA and/or pta is deleted or inactivated.

If the endogenous genes encoding the acetate kinase and/or the phosphate acetyltransferase are deleted or inactivated, it is preferred that the recombinant microorganism of the invention comprises recombinant variants of these genes, preferably under control of a (heat-)inducible promoter. The recombinant variants of these genes are preferably encoded on a plasmid.

Controlling the expression of the acetate kinase and/or the phosphate acetyltransferase with an inducible promoter may be advantageous, as it allows inducing the expression of these enzymes only when acetate is present in the culture medium.

In certain embodiments, the recombinant microorganism may further lack an endogenous gene encoding an acetyl-CoA synthase (acs in case of E. coli) and instead comprise a recombinant gene encoding an acetyl-CoA synthase, preferably under control of a (heat- )inducible promoter as described herein above.

In certain embodiments, the endogenous genes encoding an acetyl-CoA synthase, an acetate kinase and a phosphate acetyltransferase are deleted or inactivated in the recombinant microorganism of the invention. Preferably, the recombinant microorganism of the invention comprises recombinant variants of these genes under control of an inducible promoter, preferably a heat-inducible promoter.

However, conversion of acetate into isobutene may also be achieved in recombinant microorganisms comprising functional endogenous genes encoding an acetate kinase, a phosphate acetyltransferase and/or an acetyl-CoA synthase.

The recombinant microorganism of the invention may be further engineered to increase the amount of acetyl-CoAthat is available in the cells for the production of isobutene, a precursor, or a derivative thereof.

For example, in certain embodiments, the recombinant microorganism of the invention may lack a functional gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3. l._). Thus, in a particular embodiment, the invention relates to the method of the invention, wherein at least one endogenous gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3. l._) is inactivated in the recombinant microorganism.

In another particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein at least one endogenous gene encoding a peptidyl-lysine Inacetyltransferase (EC 2.3. l._) is inactivated in said recombinant microorganism.

Peptidyl-lysine N-acetyltransferase (EC 2.3. l._) is an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to the e-amino group of a lysine residue within a peptide or protein. This enzymatic activity results in the formation of Ne-acetyllysine, a post-translational modification that can affect the function, stability, localization, and interactions of the modified protein. The general reaction it facilitates can be summarized as follows: Peptidyl-lysine + Acetyl-CoA Ne-acetylpeptidyl-lysine + CoA

Inactivating an endogenous gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3. l._) in the recombinant host organism will eliminate a potential sink of acetyl-CoA and thus increase the amount of acetyl-CoA that will be available for the production of isobutene.

In certain embodiments, the recombinant microorganism of the invention is Escherichia coli and the peptidyl-lysine /V-acetyltransferase (EC 2.3. l._) is the peptidyl-lysine N- acetyltransferase PatZ (EC 2.3.1.286) encoded by the gene patZ.

The skilled person is aware of methods for inactivating an endogenous gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3. l._). For example, the endogenous gene encoding the peptidyl-lysine /V-acetyltransferase and/or its promoter may be fully or partially deleted. Alternatively, one or more mutations may be introduced into the promoter or another regulatory element to reduce or prevent expression of the gene encoding the peptidyl-lysine /V-acetyltransferase (EC 2.3. l._).

In certain embodiments, the recombinant microorganism of the invention overexpresses at least one acetate transporter. Overexpressing an acetate transporter will result in increased levels of acetate inside the cell which can then be converted into acetyl-CoA.

The acetate transporter that is overexpressed in the recombinant microorganism of the invention may be an endogenous or a heterologous acetate transporter. Overexpression may be driven by a recombinant promoter, such as an inducible or constitutive recombinant promoter.

The skilled person is capable of identifying acetate transporters that may result in increased uptake of acetate in the recombinant microorganism of the invention. In a preferred embodiment, the recombinant microorganism is E. coli, and the acetate transporter may be encoded by the genes yJcH_actP and/or satP. The source of acetic acid

The acetic acid, or the salt thereof, that is used as starting point for the production of isobutene, a precursor, or a derivative thereof may be of any origin.

In certain embodiments, acetic acid is obtained through carbonylation of methanol with carbon monoxide, as shown in the following equation:

Such a process is disclosed, for example, in WO 2020/092874.

In certain embodiments, the methanol and/or the carbon monoxide used in the equation above may be obtained from carbon dioxide (CO2).

For example, in certain embodiments, carbon monoxide may be obtained from CO2 in a reverse water-gas shift (RWGS) reaction following the equation:

CO₂+ H₂ ^ CO + H₂O

In certain embodiments, methanol may be obtained from H2 and CO2 as described, inter alia, by Liu et al. (J Phys Chem A. 2010 Mar 25;114(ll):3888-95. doi: 10.1021/jp906780a) following the equation:

CO₂ + 3 H2 - CH3OH + H₂O

The H2 used in the equations above may be obtained, without limitation, by the electrolysis of water.

Accordingly, acetic acid for the production of isobutene, a precursor thereof or a derivative thereof can be obtained in a sustainable way, mainly from CO2 and (green) energy. Alternatively, the acetic acid used in the method of the invention may be obtained from green or renewable methane. That is, methane may be converted into carbon monoxide and H2 using a steam methane reforming reaction:

CH4 + H2O -> CO + 3H₂

Methanol may be obtained from green or renewable methane in a reaction catalyzed by a methane monooxygenase (MMO):

CH₄ + O₂ + NAD(P)H + H⁺ -> CH3OH + NAD(P)⁺ + H₂O

Accordingly, the substrates for the carbonylation of methanol, i.e., methanol and carbon monoxide, may be exclusively derived from green or renewable methane, resulting in green acetic acid.

Green methane, also known as biomethane or renewable methane, is methane produced from renewable resources rather than fossil fuels. It is generated through processes such as anaerobic digestion of organic waste materials (e.g., agricultural residues, manure, food waste, and sewage sludge), power-to-gas (P2G) technology that utilizes renewable electricity to produce hydrogen and subsequently methane, and gasification of biomass. Green methane serves as a sustainable and environmentally friendly alternative to conventional natural gas, helping to reduce greenhouse gas emissions and reliance on fossil fuels while being compatible with existing natural gas infrastructure.

Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the acetic acid, or the salt thereof, used in the method is derived from carbon monoxide and methanol, preferably wherein the carbon monoxide and/or the methanol are obtained from carbon dioxide or methane, preferably green methane.

The term "acetic acid, or a salt thereof" refers to acetic acid (CH3COOH) and its corresponding salts. The salts of acetic acid are formed when the hydrogen atom in the carboxyl group (COOH) is replaced by a metal cation or another positively charged ion. Common examples of acetic acid salts include sodium acetate (CH₃COONa), potassium acetate (CH₃COOK), and calcium acetate (Ca(CH₃COO)₂).

The enzymatic conversion of acetyl-CoA into isobutene

The recombinant microorganism of the present invention is capable of producing isobutene, or a precursor thereof, from acetyl-CoA.

Isobutene, also known as isobutylene, is an organic compound with the chemical formula C₄H₈. It is a colorless gas at room temperature and is a type of olefin (alkene) with a double bond between two of its carbon atoms. Isobutene is an important building block in the petrochemical industry, used primarily in the production of various polymers, fuels, and other chemicals.

In the following, the major reactions of the individual enzymatic conversions as described in the prior art WO 2017/085167, WO 2018/206262, W02010/001078, WO2012/052427 and WO 2016/042012, and as schematically illustrated in Figure 1 are described in more detail.

However, the present invention is not limited to these major reactions but also relates to all other routes for the individual steps of the conversion of acetyl-CoA into isobutene as described in the prior art documents WO 2017/085167, WO 2018/206262, W02010/001078, WO2012/052427 and WO 2016/042012. The disclosure of these documents, in particular with respect to preferred embodiments of the enzymes for the individual conversions of the pathways described therein, is herewith incorporated by reference in its entirety. Accordingly, in preferred embodiments, it is preferable to use the enzymes selected from the preferred embodiments described in these prior art documents in connection with the respective enzymatic conversion. Thus, the same applies to the enzymatic conversions of the present invention described in the following as has been set forth in WO 2017/085167, WO 2018/206262, W02010/001078, WO2012/052427 and WO 2016/042012, respectively. In a particular embodiment, the invention relates to the method according to the invention, wherein the enzymatic conversion of acetyl-CoA into isobutene, or a precursor thereof, comprises one or more of the steps selected from (i) to (vi):

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA,

(ii) enzymatically converting said produced acetoacetyl-CoA into 3-hydroxy-3- methylglutaryl-CoA,

(iii) enzymatically converting said produced 3-hydroxy-3-methylglutaryl-CoA into 3- methylglutaconyl-CoA,

(iv) enzymatically converting said produced 3-methylglutaconyl-CoA into 3- methylcrotonyl-CoA,

(v) enzymatically converting said produced 3-methylcrotonyl-CoA into 3,3- dimethyl acrylic acid, and

(vi) enzymatically converting said produced 3,3-dimethylacrylic acid into isobutene.

The enzymatic conversion of acetyl-CoA into acetoacetyl-CoA

According to the present invention, the conversion of acetyl-CoA into acetoacetyl-CoA can be achieved by different routes. One possibility is to first convert acetyl-CoA into malonyl-CoA (step XIV as shown in Figure 1) and then to further condense said malonyl-CoA and acetyl- CoA into acetoacetyl-CoA (step XV as shown in Figure 1). Another possibility is to directly condense in a single enzymatic reaction two molecules of acetyl-CoA into acetoacetyl-CoA (step XIII as shown in Figure 1).

The enzymatic conversion of acetyl-CoA into malonyl-CoA preferably makes use of an acetyl- CoA carboxylase (EC 6.4.1.2) (step XIV as shown in Figure 1). This naturally occurring reaction fixes CO2 on acetyl-CoA utilizing ATP resulting in malonyl-CoA.

Moreover, the enzymatic condensation of malonyl-CoA and acetyl-CoA into said acetoacetyl-

CoA preferably makes use of an acetoacetyl-CoA synthase (EC 2.3.1.194) (step XV as shown in Figure 1). This is a natural occurring reaction and condenses malonyl-CoA and acetyl-CoA in a decarboxylation reaction.

Alternatively, the enzymatic conversion of acetyl-CoA into said acetoacetyl-CoA consists of a single enzymatic reaction in which acetyl-CoA is directly converted into acetoacetyl-CoA by the enzymatic condensation of two molecules of acetyl-CoA into acetoacetyl-CoA. Preferably, this enzymatic conversion is achieved by making use of an acetyl-CoA acetyltransferase (EC 2.3.1.9). This reaction is a naturally occurring reaction (step XIII as shown in Figure 1).

The enzymatic conversion of acetoacetyl-CoA into 3-hvdroxy-3-methylglutaryl-CoA

The enzymatic conversion of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA is an enzymatic condensation of acetoacetyl-CoA and acetyl-CoA into said 3-hydroxy-3- methylglutaryl-CoA (see step IX of Figure 1).

This condensation preferably makes use of a 3-hydroxy-3-methylglutaryl-CoA synthase (also referred to as HMG-CoA synthase). HMG-CoA synthases are classified in EC 2.3.3.10 (formerly, HMG-CoA synthase has been 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 which is able to catalyze the reaction where acetyl-CoA condenses with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). HMG-CoA synthase is part of the mevalonate pathway. Several pathways have been identified for the synthesis of isopentenyl pyrophosphate (IPP), i.e. the mevalonate pathway and the 2-C-methyl-D-erythritol 4-phosphate/l-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway. HMG-CoA synthase catalyzes the biological Claisen condensation of acetyl-CoA with acetoacetyl-CoA and is a member of a superfamily of acyl-condensing enzymes that includes beta-ketothiolases, fatty acid synthases (beta-ketoacyl carrier protein synthase) and polyketide synthases.

The enzymatic conversion of 3-hvdroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA

The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA is an enzymatic dehydration reaction which occurs naturally, and which is catalyzed, e.g., by enzymes classified as 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18). Accordingly, the enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA preferably makes use of a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18) (as shown in step VIII of Figure 1).

The conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA can also be achieved by making use of a 3-hydroxy-3-methylglutaryl-coenzyme A dehydratase activity which has been identified, e.g., in Myxococcus xanthus and which is encoded by the liuC gene (Li et al., Angew. Chem. Int. Ed. 52 (2013), 1304-1308). The 3-hydroxy-3-methylglutaryl- coenzyme A dehydratase derived from Myxococcus xanthus has the Uniprot accession number Q1D5Y4.

The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA can also be achieved by making use of a 3-hydroxyacyl-CoA dehydratase or an enoyl-CoA hydratase. 3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases catalyze the same reaction while the name of one of these enzymes denotes one direction of the corresponding reaction while the other name denotes the reverse reaction. As the reaction is reversible, both enzyme names can be used. 3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases belong to enzymes classified as EC 4.2.1.

The enzymatic conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA

The conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA may be catalyzed by different enzymes, e.g., by making use of (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) a geranoyl-CoA carboxylase (EC 6.4.1.5) (as shown in step VII of Figure 1).

In another preferred embodiment the conversion of 3-methylglutaconyl-CoA via decarboxylation into 3-methylcrotonyl-CoA is catalyzed by a 3-methylglutaconyl-CoA decarboxylase, e.g. a 3-methylglutaconyl-CoA decarboxylase of Myxococcus xanthus composed of the two subunits AibA and AibB (Li et al., Angew. Chem. Int. Ed. 52 (2013), 1304- 1308). The enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid (3,3- dimethylacrylic acid)

The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid (also referred to as 3,3- dimethylacrylic acid) can, e.g., be achieved in different ways, e.g., by three alternative enzymatic routes described in the following and as shown in Figure 1 (step Via, step Vlb or step Vic as shown in Figure 1).

Thus, the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid may be achieved by

(a) a single enzymatic reaction in which 3-methylcrotonyl-CoA is directly converted into 3- methylcrotonic acid, preferably by making use of a CoA transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA transferase (EC 2.8.3.1), an acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18) (step Via as shown in Figure 1);

(b) a single enzymatic reaction in which 3-methylcrotonyl-CoA is directly converted into 3- methylcrotonic acid, preferably by making use of a thioester hydrolase (EC 3.1.2), preferably an acetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20) (step Vlb as shown in Figure 1); or

(c) two enzymatic steps comprising

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

(ii) then enzymatically converting the thus obtained 3-methylcrotonyl phosphate into said 3-methylcrotonic acid (step Vic as shown in Figure 1).

As regards (c), the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by two enzymatic steps comprising (i) first enzymatically converting 3- methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and (ii) then enzymatically converting the thus obtained 3-methylcrotonyl phosphate into said 3-methylcrotonic acid. T1

The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate can, e.g., be achieved by the use of a phosphate butyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).

The conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase. Such enzymes use a carboxy group as acceptor. Thus, the conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-). In a preferred embodiment, the conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).

As mentioned above, the conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid can also be achieved by two alternative conversions wherein 3-methylcrotonyl-CoA is directly converted into 3-methylcrotonic acid.

Preferably, in one embodiment, 3-methylcrotonyl-CoA is directly converted into 3- methylcrotonic acid by hydrolyzing the thioester bond of 3-methylcrotonyl-CoA into 3- methylcrotonic acid by making use of an enzyme which belongs to the family of thioester hydrolases (in the following referred to as thioesterases (EC 3.1.2.-)); step Vlb as shown in Figure 1.

Thioesterases (TEs; also referred to as thioester hydrolases) are enzymes which are classified as EC 3.1.2. Presently thioesterases are classified as EC 3.1.2.1 through EC 3.1.2.30 while TEs which are not yet classified/unclassified are grouped as enzymes belonging to EC 3.1.2.-. Cantu et al. (Protein Science 19 (2010), 1281-1295) describe that there are 23 families of thioesterases which are unrelated to each other as regards the primary structure. However, it is assumed that all members of the same family have essentially the same tertiary structure. Thioesterases hydrolyze the thioester bond between a carbonyl group and a sulfur atom. In a preferred embodiment, a thioesterase employed according to the present invention for converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid is selected from the group consisting of: acetyl-CoA hydrolase (EC 3.1.2.1); palmitoyl-CoA hydrolase (EC 3.1.2.2);

3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4); oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);

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

ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19); l,4-dihydroxy-2-naphthoyl-CoA hydrolase (EC 3.1.2.28); and acyl-CoA hydrolase (EC 3.1.2.20).

In more preferred embodiments, a thioesterase/thioester hydrolase (EC 3.1.2.-) employed according to the present invention is an acetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18), a l,4-dihydroxy-2-naphthoyl-CoA hydrolase (EC 3.1.2.28), and an acyl-CoA hydrolase (EC 3.1.2.20).

In an alternative embodiment, 3-methylcrotonyl-CoA is directly converted into 3- methylcrotonic acid, preferably by making use of an enzyme which belongs to the family of CoA-transferases (EC 2.8.3.-) capable of transferring the CoA group of 3-methylcrotonyl-CoA to a carboxylic acid (step Via as shown in Figure 1).

CoA-transferases are found in organisms from all lines of descent. Most of the CoA- transferases belong to two well-known enzyme families (referred to in the following as families I and II) and there exists a third family which had been identified in anaerobic metabolic pathways of bacteria. A review describing the different families can be found in Heider (FEBS Letters 509 (2001), 345-349).

Preferably, the CoA-transferase employed according to the present invention for the direct conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid is selected from the group consisting of: propionate:acetate-CoA transferase (EC 2.8.3.1); acetate CoA-transferase (EC 2.8.3.8); and butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

In more preferred embodiments, CoA transferases (EC 2.8.3.-) are a propionate:acetate-CoA transferase (EC 2.8.3.1), an acetate CoA-transferase (EC 2.8.3.8) and a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18).

The enzymatic conversion of 3-methylcrotonic acid (3,3-dimethylacrylic acid) into isobutene

The enzymatic conversion of 3-methylcrotonic acid (also referred to as 3,3-dimethylacrylic acid) into isobutene is schematically shown in step I of Figure 1). This conversion can be achieved by a decarboxylation making use of a prenylated FMN-dependent decarboxylase associated with an FMN prenyl transferase. "Decarboxylation" is generally a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2).

The enzymatic conversion of 3-methylcrotonic acid into isobutene utilizing a prenylated FMN- dependent decarboxylase associated with an FMN prenyl transferase relies on a reaction of two consecutive steps catalyzed by the two enzymes, i.e., the prenylated FMN-dependent decarboxylase (catalyzing the actual decarboxylation of 3-methylcrotonic acid into isobutene) with an associated FMN prenyl transferase which provides the modified flavin cofactor.

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

Thus, in the conversion of 3-methylcrotonic acid into isobutene, in a first step, a flavin cofactor (FMN or FAD) is modified into a (modified) flavin-derived cofactor. This modification is catalyzed by said FMN prenyl transferase. FMN prenyl transferase prenylates the flavin ring of the flavin cofactor (FMN or FAD) into a (modified) prenylated flavin cofactor. More specifically, FMN prenyl transferase catalyzes the prenylation of a flavin cofactor (FMN or FAD) utilizing dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP) into a flavinderived cofactor.

In a second step, the actual conversion of 3-methylcrotonic acid into isobutene is catalyzed by said prenylated FMN-dependent decarboxylase via a 1,3-dipolar cycloaddition based mechanism wherein said prenylated FMN-dependent decarboxylase uses the prenylated flavin cofactor (FMN or FAD) provided by the associated FMN prenyl transferase.

In a preferred embodiment, said FMN prenyl transferase which modifies the flavin cofactor (FMN or FAD) into a (modified) flavin-derived cofactor (utilizing dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP)) is a phenylacrylic acid decarboxylase (PAD)- type protein, or the closely related prokaryotic enzyme UbiX, an enzyme which is involved in ubiquinone biosynthesis in prokaryotes.

In Escherichia coli, the protein UbiX (also termed 3-octaprenyl-4-hydroxybenzoate carboxylyase) has been shown to be involved in the third step of ubiquinone biosynthesis.

In certain embodiments, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by the FMN-containing protein phenylacrylic acid decarboxylase (PAD). The enzymes involved in the modification of the flavin cofactor (FMN or FAD) into the corresponding modified flavin-derived cofactor were initially annotated as decarboxylases (EC 4.1.1.-). Some phenylacrylic acid decarboxylases (PAD) are now annotated as flavin prenyl transferases as EC 2.5.1.-. Enzymes capable of catalyzing the enzymatic reaction described herein for flavin prenyl transferases have recently also been annotated as flavin prenyl transferases as EC 2.5.1.129.

In certain embodiments, the conversion of 3-methylcrotonic acid into isobutene makes use of a phenylacrylic acid decarboxylase (PAD)-type protein as the FMN prenyl transferase which modifies a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor wherein said phenylacrylic acid decarboxylase (PAD)-type protein is derived from Candida albicans (Uniprot accession number Q5A8L8), Aspergillus niger (Uniprot accession number A3F715), Saccharomyces cerevisiae (Uniprot accession number P33751) or Cryptococcus gattii (Uniprot accession number E6R9Z0).

In certain embodiments, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by the FMN-containing protein 3-octaprenyl-4-hydroxybenzoate carboxy-lyase also termed UbiX (initially annotated EC 4.1.1.-). As mentioned above, the enzymes involved in the modification of the flavin cofactor (FMN or FAD) into the corresponding modified flavin-derived cofactor were initially annotated as decarboxylases. Some phenylacrylic acid decarboxylases (PAD) are now annotated as flavin prenyl transferases as EC 2.5.1.-.

As mentioned above, enzymes capable of catalyzing the enzymatic reaction described herein for flavin prenyl transferases have recently also been annotated as flavin prenyl transferases as EC 2.5.1.129.

In certain embodiments, the conversion of 3-methylcrotonic acid into isobutene makes use of a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also termed UbiX) as the FMN prenyl transferase which modifies the flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor wherein said 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also termed UbiX) is derived from Escherichia coli (Uniprot accession number P0AG03), Bacillus subtilis (Uniprot accession, number A0A086WXG4), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1).

In certain embodiments, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by an UbiX-like flavin prenyl transferase derived from E. coli encoded by kpdB and ecdB, respectively (UniProt accession number A0A023LDW3 and UniProt accession number P69772, respectively), and an UbiX-like flavin prenyl transferase derived from Klebsiella pneumoniae encoded by kpdB (UniProt accession number Q462H4). In certain embodiments, the modification of a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor is catalyzed by a flavin prenyl transferase.

As mentioned above, the actual decarboxylation, i.e., the conversion of 3-methylcrotonic acid into isobutene is catalyzed by a prenylated FMN-dependent decarboxylase via a 1,3-dipolar cycloaddition based mechanism wherein said prenylated FMN-dependent decarboxylase uses the prenylated flavin cofactor (FMN or FAD) provided by any of the above described associated FMN prenyl transferases.

In certain embodiments, said decarboxylation of 3-methylcrotonic acid into isobutene is catalyzed by a ferulic acid decarboxylase (FDC). Ferulic acid decarboxylases (FDC) belong to the enzyme class EC 4.1.1.-.

In certain embodiments, the conversion of 3-methylcrotonic acid into isobutene makes use of a ferulic acid decarboxylases (FDC) which is derived from Saccharomyces cerevisiae (Uniprot accession number Q03034), Enterobacter sp. (Uniprot accession number V3P7U0), Bacillus pumilus (Uniprot accession number Q45361), Aspergillus niger (Uniprot accession number A2R0P7) or Candida dubliniensis (Uniprot accession number B9WJ66).

In certain embodiments, the conversion of 3-methylcrotonic acid into isobutene makes use of a protocatechuate decarboxylase (EC 4.1.1.63).

In certain embodiments of the present invention, the PCA decarboxylase employed in the method of the present invention is a PCA decarboxylase which is derived from Klebsiella pneumoniae (Uniprot accession number B9AM6), Leptolyngbya sp. (Uniprot accession number A0A0S3U6D8), or Phascolarctobacterium sp. (Uniprot accession number R6IIV6).

In certain embodiments, said prenylated FMN-dependent decarboxylase catalyzing the decarboxylation of 3-methylcrotonic acid into isobutene is an enzyme which is closely related to the above ferulic acid decarboxylase (FDC), namely a 3-polyprenyl-4-hydroxybenzoate decarboxylase (also termed UbiD). 3-polyprenyl-4-hydroxybenzoate decarboxylase belongs to the UbiD decarboxylase family classified as EC 4.1.1.-. In certain embodiments, the conversion of 3-methylcrotonic acid into isobutene makes use of a 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) which is derived from Hypocrea atroviridis (UniProt Accession number G9NLP8), Sphaerulina musiva (UniProt Accession number M3DF95), Penicillinum roqueforti (UniProt Accession number W6QKP7), Fusarium oxysporum f. sp. lycopersici (UniProt Accession number W9LTH3), Saccharomyces kudriavzevii (UniProt Accession number J8TRN5), Saccaromyces cerevisiae, Aspergillus parasiticus, Candida albicans, Grosmannia clavigera, Escherichia coli (Uniprot accession number P0AAB4), Bacillus megaterium (Uniprot accession number D5DTL4), Methanothermobacter sp. CaT2 (Uniprot accession number T2GKK5), Mycobacterium chelonae 1518 (Uniprot accession number X8EX86) or Enterobacter cloacae (Uniprot accessin number V3DX94).

In certain embodiments, the conversion of 3-methylcrotonic acid into isobutene makes use of an UbiD-like decarboxylase which is derived from Streptomyces sp (UniProt Accession number A0A0A8EV26). In certain embodiments, the UbiD-like decarboxylase may be any one of the variants disclosed in WO 2022/207684, which is fully incorporated herein by reference.

Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the enzymatic conversion of acetyl-CoA into isobutene, or a precursor thereof, comprises one or more of the steps selected from (i) to (vi):

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA,

(vi) enzymatically converting said produced 3,3-dimethylacrylic acid into isobutene. In another particular embodiment, the invention relates to the method according to the invention, wherein the enzymatic conversion of acetyl-CoA into isobutene, or a precursor thereof, comprises one or more of the steps selected from (i) to (vi):

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA with a thiolase, preferably an acetyl-CoA acetyltransferase (EC 2.3.1.9),

(ii) enzymatically converting said produced acetoacetyl-CoA into 3-hydroxy-3- methylglutaryl-CoA with a HMG-CoA synthase (EC 2.3.3.10),

(iii) enzymatically converting said produced 3-hydroxy-3-methylglutaryl-CoA into 3- methylglutaconyl-CoA with an enoyl-CoA hydratase (4.2.1.17),

(iv) enzymatically converting said produced 3-methylglutaconyl-CoA into 3- methylcrotonyl-CoA with a 3-methylglutaconyl-CoA decarboxylase (EC 4.1.1-),

(v) enzymatically converting said produced 3-methylcrotonyl-CoA into 3,3- dimethyl acrylic acid with a thioesterases (EC 3.1.2.-), and

(vi) enzymatically converting said produced 3,3-dimethylacrylic acid into isobutene with a ferulic acid decarboxylase (EC 4.1.1.-.) and a flavin prenyltransferase (2.5.1.129)

In yet another particular embodiment, the invention relates to the method according to the invention, wherein the enzymatic conversion of acetyl-CoA into isobutene, or a precursor thereof, comprises one or more of the steps selected from (i) to (vi):

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA with thl (thiolase) from Clostridium acetobutylicum (Uniprot Accession number P45359),

(ii) enzymatically converting said produced acetoacetyl-CoA into 3-hydroxy-3- methylglutaryl-CoA with mvaS (Hydroxymethylglutaryl-CoA synthase) from Enterococcus faecalis (Uniprot Accession number Q835L4),

(iii) enzymatically converting said produced 3-hydroxy-3-methylglutaryl-CoA into 3- methylglutaconyl-CoA with ech (enoyl-CoA hydratase) from Pseudomonas sp. (Uniprot Accession number K9NHK2),

(iv) enzymatically converting said produced 3-methylglutaconyl-CoA into 3- methylcrotonyl-CoA with aibA and aibB that code for the two subunits of 3- methylglutaconyl-CoA decarboxylase from Myxococcus hansupus (Uniprot Accession number A0A0H4WQB1 and A0A0H4WWJ4),

(v) enzymatically converting said produced 3-methylcrotonyl-CoA into 3,3- dimethyl acrylic acid with menl (ydil) (l,4-dihydroxy-2-naphthoyl-CoA hydrolase) from Escherichia coli (strain K12) (Uniprot Accession number P77781), and

(vi) enzymatically converting said produced 3,3-dimethylacrylic acid into isobutene with FDC1 (ferulic acid decarboxylase) from Streptomyces sp. 769 (Uniprot Accession number A0A0A8EV26) and UbiX (flavin prenyltransferase) from Escherichia coli (strain K12) (Uniprot Accession number P0AG03).

That is, the microorganism according to the invention may be used in an industrial process for the production of isobutene. In certain embodiments, all enzymatic steps for the synthesis of isobutene will be carried out in the microorganism of the invention. In such embodiments, the microorganism preferably comprises the enzyme ferulic acid decarboxylase, which catalyzes the conversion of 3-methylcrotonic acid into isobutene.

Precursors of isobutene

In certain embodiments, the method of the invention is used for the production of a precursor of isobutene from acetic acid or a salt thereof. A precursor of isobutene may be any molecule that can be converted into isobutene in a limited number of enzymatic or chemical reaction steps, preferable in not more than 3, not more than 2, or not more than one enzymatic or chemical reaction steps.

In certain embodiments, the precursor of isobutene is any one of the intermediates of the metabolic pathways described herein above. That is, in certain embodiments, the precursor of isobutene is 3-methylcrotonic acid, 3-methylcrotonyl-CoA, 3-methylglutaconyl-CoA or 3- hydroxy-3-methylglutaryl-CoA.

In a particular embodiment, the invention relates to the method of the invention, wherein the precursor of isobutene is 3-methylcrotonic acid. That is, the recombinant microorganism of the invention may be used for the production of 3-methylcrotonic acid from acetic acid, or a precursor thereof. The resulting 3-methylcrotonic acid may then be converted into isobutene in a subsequent reaction.

That is, isobutene may be produced in a two-step process in which the recombinant microorganism according to the invention is used for the production of the precursor molecule 3-methylcrotonic acid. The produced 3-methylcrotonic acid may then be converted into isobutene in vivo or in vitro in a biotransformation reaction.

That is, in a particular embodiment, the invention relates to a method comprising the steps of: a) producing 3-methylcrotonic acid by culturing a recombinant microorganism of the invention in a suitable culture medium under suitable conditions; and b) enzymatically converting said produced 3-methylcrotonic acid into isobutene in vivo or in vitro.

In a particular embodiment, the invention relates to the method of the invention, wherein the enzymatic conversion of acetyl-CoA into isobutene, or a precursor thereof, comprises one or more of the steps selected from (i) to (v):

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA,

(iv) enzymatically converting said produced 3-methylglutaconyl-CoA into 3- methylcrotonyl-CoA, and

(v) enzymatically converting said produced 3-methylcrotonyl-CoA into 3- methylcrotonic acid; preferably wherein said produced 3-methylcrotonic acid is enzymatically converted into isobutene, and wherein the conversion of 3-methylcrotonic acid into isobutene takes place within the recombinant microorganism or outside of the recombinant microorganism. 31

Processes for the production of isobutene, or a precursor thereof

The method of the present invention may be separated into two stages. The first step may be characterized by the production of biomass. During this first stage of the method, no or only little product is produced by the cells. This may be achieved by placing the genes that are required for the production of the product under control of an inducible promoter. Accordingly, the first stage of the method of the invention may be characterized by reduced expression of at least one gene that is responsible for the conversion of acetic acid, or a salt thereof, into isobutene, or a precursor thereof. Expression of the at least one gene that is responsible for the conversion of acetic acid, or a salt thereof, into isobutene, or a precursor thereof, may be reduced due to the absence of a suitable stimuli that induces the promoter that controls expression of said gene.

Once sufficient biomass has been generated, the second stage of the method may be initiated. The second stage of the method is mainly characterized by the production of the product, i.e., by the production of isobutene, or a precursor thereof. The second stage of the method may be initiated by addition of a compound or stimulus that induces expression of the genes that are responsible for the conversion of acetic acid, or a salt thereof, into isobutene, or a precursor thereof.

In certain embodiments, the genes that are responsible for the conversion of acetic acid, or a salt thereof, into isobutene, or a precursor thereof, may be under control of a heat-inducible promoter. In such embodiments, cells may be cultured at low temperatures, e.g., at 34°C, during the first stage of the method, to accumulate a sufficient amount of biomass. The production of isobutene, or a precursor thereof, may then be induced during the second stage of the process by elevating the temperature to 39°C.

In certain embodiments, cells may be cultured in the presence of a sugar (1^st or 2^nd generation sugar) or another suitable carbon source, such as, without limitation, glycerol, as the main carbon source during the first stage of the method to rapidly accumulate biomass. Thus, in a particular embodiment, the invention relates to the method of the invention, wherein during a first stage of production, biomass is produced from sugar or another suitable carbon source. In such embodiments, it is preferred that the recombinant microorganism lacks the endogenous acetate kinase and/or phosphate acetyltransferase genes to prevent accumulation of acetate in the cells and/or culture medium, as described in more detail above. Instead, it is preferred that the recombinant microorganism comprises recombinant variants of these genes that are under control of an inducible promoter and can be switched on when acetate is added to the culture medium. Even more preferably, the recombinant microorganism further lacks the endogenous acetyl-CoA synthase gene and instead comprises a recombinant variant of said gene under control of an inducible promoter.

During the second stage of the method, the carbon source may then be switched to acetic acid, such that isobutene, or the precursor thereof, is exclusively produced from acetic acid. Thus, in a particular embodiment, the invention relates to the method of the invention, wherein during a second stage of production, isobutene, or the precursor thereof, is exclusively or primarily produced from acetic acid, or a salt thereof.

In preferred embodiment, the invention relates to the method of the invention, wherein during a first stage of production, biomass is produced from sugar or any substrate allowing efficient growth of the biomass such as e.g.: glycerol) and during a second stage of production, isobutene, or a precursor thereof, is exclusively or primarily produced from acetic acid, or a salt thereof.

Within the present invention, isobutene, the precursor, or the derivative thereof, is said to be primarily produced from acetic acid, or a salt thereof, if at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the carbon atoms comprised in the produced isobutene, or the precursor thereof, are derived from acetic acid, or the salt thereof.

In certain embodiments, both the first stage and the second stage of the method may be conducted in the presence of a mixture of a sugar and acetic acid, or a salt thereof. Thus, in certain embodiments, the invention relates to the method of the invention, wherein during a first stage of production, biomass is produced from a sugar and acetic acid, or a salt thereof, and during a second stage of production, isobutene, or the precursor thereof, is produced from a sugar and acetic acid, or a salt thereof.

In certain embodiments, both the first stage and the second stage of the method may be conducted in the presence of a mixture of a substrate allowing accumulation of biomass, such as e.g.: glycerol, and acetic acid, or a salt thereof. Thus, in certain embodiments, the invention relates to the method of the invention, wherein during a first stage of production, biomass is produced from said substrate and acetic acid, or a salt thereof, and during a second stage of production, isobutene, or the precursor thereof, is produced from said substrate and acetic acid, or a salt thereof.

In certain embodiments, biomass is produced in the first stage of the method from a mixture of a sugar and acetic acid, or a salt thereof, and isobutene, or the precursor thereof, is produced during the second stage from acetic acid, or a salt thereof. Thus, in certain embodiments, the invention relates to the method of the invention, wherein during a first stage of production, biomass is produced from a sugar and acetic acid, or a salt thereof, and during a second stage of production, isobutene, or the precursor thereof, is exclusively or primarily produced from acetic acid, or a salt thereof.

In certain embodiments, biomass is produced in the first stage of the method from a mixture of acetic acid, or a salt thereof, with another carbon source and isobutene, or the precursor thereof, is produced during the second stage exclusively from acetic acid, or a salt thereof. Thus, in certain embodiments, the invention relates to the method of the invention, wherein during a first stage of production, biomass is produced from a substrate allowing accumulation of biomass, such as e.g.: glycerol, and acetic acid, or a salt thereof, and during a second stage of production, isobutene, or the precursor thereof, is exclusively or primarily produced from acetic acid, or a salt thereof.

Preferably, the sugar used for the production of biomass is glucose, xylose or sucrose.

In certain embodiments, both biomass and isobutene, or the precursor thereof, are produced from acetic acid, or a salt thereof. Thus, in certain embodiments, the invention relates to the method of the invention, wherein during a first stage of production, biomass is exclusively or primarily produced from acetic acid, or a salt thereof, and during a second stage of production, isobutene, or the precursor thereof, is exclusively or primarily produced from acetic acid, or a salt thereof.

Within the present invention, biomass is said to be primarily produced from acetic acid, or a salt thereof, if at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the carbon atoms comprised in the culture medium are comprised in the acetic acid, or the salt thereof. Preferably, when biomass and/or isobutene is said to be primarily produced from acetic acid, or a salt thereof, the culture medium is free of sugars or contains only trace amounts of sugars.

In another embodiment, the method of the invention comprises the step of providing the microorganism of the present invention in the form of a (cell) culture, preferably in the form of a liquid cell culture, a subsequent step of cultivating the microorganism in a fermenter (often also referred to a bioreactor) under suitable conditions and further comprising the step of effecting an enzymatic conversion of a method of the invention as described herein. Suitable fermenter or bioreactor devices and fermentation conditions are known to the person skilled in the art. A bioreactor or a fermenter refers to any manufactured or engineered device or system known in the art that supports a biologically active environment. Thus, a bioreactor or a fermenter may be a vessel in which a chemical/biochemical reaction like the method of the present invention is carried out which involves microorganisms and/or biochemically active substances. In a bioreactor or a fermenter, this process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, and may range in size from liters to cubic meters, and are often made of stainless steel. In this respect, without being bound by theory, the fermenter or bioreactor may be designed in a way that it is suitable to cultivate the microorganisms in, e.g., a batch-culture, fed-batch-culture, perfusion culture or chemostate-culture, all of which are generally known in the art.

The culture medium can be any culture medium suitable for cultivating the respective microorganism. When carried out by making use of an microorganism, the method according to the present invention may, e.g. be designed as a continuous fermentation culturing method or as a batch culture or any suitable culture method known to the person skilled in the art.

The term "culturing" as used in this context refers to the keeping the cells in a liquid medium, thereby keeping them vital and allowing the production of the required enzymes for the production of the desired product(s). Preferably, the term "culturing" also encompasses the growing of microorganism cells under conditions that allow their propagation, proliferation and cell division, thereby increasing the number of the cells in the liquid culture medium. Thus, the term "culturing" refers to maintaining the microorganism in culture conditions which allow for the survival of cells as well as for the occurrence of the metabolic processes which are required for the cells so as to convert the carbon source into isobutene. Such conditions generally comprise the provision of cells with a carbon source in the culture medium, agitation of the culture medium, maintaining the temperature of the culture medium at a value which allows for the required metabolic conversions to occur (and, if desired, of the growth of the microorganism) and supplying the cultured cells with air or a gas mixture which allows survival and metabolic activity of the cells.

Thus, the term "culturing" as used in this context is to be understood as a "fermentation", i.e. a metabolic process that produces chemical changes in organic substrates, preferably through the action of enzymes and, accordingly, refers to a process wherein products are synthesized from growth substrates via the microorganisms' native or genetically modified metabolism and are accomplished by metabolic intermediates. Therefore, the term culturing makes possible the occurrence of metabolism of the cultured cells, their growth and their survival. Moreover, the cultivation step, in order to allow this, requires carbon and energy sources to be present (e.g. in the form of glucose and oxygen).

In a preferred embodiment, the method according to the present invention also comprises the step of recovering the isobutene produced by the method. For example, when the method according to the present invention is carried out in vivo by fermenting a corresponding microorganism expressing the necessary enzymes/transporters, the isobutene can be recovered from the fermentation off-gas by methods known to the person skilled in the art. In a particular embodiment, the recovered isobutene is purified from other gases comprised in the off-gas, such as CO2, using methods known in the art.

As mentioned above, the recombinant organism and microorganism as well as the method according to the present invention is in particular useful for large scale production of isobutene in vivo, in particular for a commercial production. The present invention describes novel means and ways to commercially and sustainably produce large quantities of isobutene. The generated large quantities of isobutene can then be further converted, in a commercial setting, to produce large quantities of, e.g., drop-in gasoline (e.g. isooctane, ETBE, MTBE), jetfuel, cosmetics, chemicals, such as methacrylic acid, polyisobutene, or butyl rubber.

As used herein, "large scale production", "commercial production" and "bioprocessing" of isobutene in a fermentation reactor or in vitro is carried out at a capacity greater than at least 100 liters, and preferably greater than at least 400 liters, or more preferably production of 1,000 liters of scale or more, even more preferably production of 5,000 liters of scale or more. As used herein, "large quantities" specifically excludes trace amounts that may be produced inherently in an microorganism.

Derivatives of isobutene

The method of the invention may further be used for producing a derivative of isobutene. The term "derivative of isobutene" as used herein refers to any chemical compound that is structurally related to isobutene (C HS) and is formed by modifying its molecular structure through one or more chemical reactions. These modifications can include processes such as polymerization, oligomerization, hydrogenation, halogenation, or functional group substitutions. Common derivatives of isobutene include isobutylene oligomers, polyisobutylene, methyl tert-butyl ether (MTBE), and various alkylated compounds. These derivatives retain the core carbon skeleton of isobutene but exhibit different chemical and physical properties, making them useful in a wide range of industrial applications, including fuel additives, synthetic lubricants, adhesives, and intermediates in organic synthesis. In a preferred embodiment, the produced isobutene may be oligomerized into longer chain alkenes. Oligomerization of isobutene may take place in vivo, i.e., in the recombinant organism or organism of the invention. However, it is preferred that the isobutene that is produced by the recombinant microorganism of the invention is collected and/or purified and then subjected to oligomerization.

Isobutene may be oligomerized into alkenes through a catalytic process. The catalytic process may involve the use of a solid acid catalyst, such as a zeolite (e.g., H-ZSM-5, H-Beta, or H-Y), ion-exchange resin, or supported phosphoric acid. The isobutene, preferably purified to remove impurities, may be introduced into a reactor containing the catalyst. The reaction may be conducted under controlled conditions, typically at temperatures ranging from 50°C to 250°C and pressures from 1 bar to 50 bar. Under these conditions, isobutene molecules may undergo oligomerization, forming higher molecular weight alkenes. The reaction mixture may then be processed to separate the desired oligomers from unreacted isobutene, which can be recycled back into the reactor for further oligomerization.

The alkenes obtained from the oligomerization of isobutene may then be further processed through a hydrogenation reaction to convert them into alkanes. This hydrogenation process may involve contacting the alkenes with hydrogen gas in the presence of a suitable hydrogenation catalyst, such as palladium, platinum, or nickel, under elevated temperatures and pressures. The reaction conditions may range from 50°C to 300°C and pressures from 1 bar to 100 bar. During this process, the double bonds in the alkenes are saturated with hydrogen atoms, resulting in the formation of the corresponding alkanes. The hydrogenated products, being more stable and less reactive, are valuable as high-quality fuels, lubricants, and chemical intermediates.

Thus, in a particular embodiment, the invention relates to the method of the invention, wherein the method comprises a further step of (i) purifying the Isobutene, (ii) oligomerizing the isobutene into alkenes, preferably into C12 to C16 alkenes, and iii) hydrogenating the alkenes into alkanes. Preferably, the oligomerization of isobutene into alkenes and the subsequent hydrogenation of the alkenes into alkanes is carried out as described in US 2023/0181432, preferably in paragraphs [0082] to [0116] of US 2023/0181432, which is fully incorporated herein by reference.

Preferably, the resulting alkanes are a mixture of isododecane and isohexadecane, preferably in a ratio of about 85% isododecane and about 15% isohexadecane to meet the requirements for ASTM certified Sustainable Aviation Fuels (SAF).

ASTM certified Sustainable Aviation Fuel (SAF) refers to aviation fuel that has been produced from sustainable feedstocks and processes, and has met the rigorous specifications set by the ASTM International standards organization, specifically under ASTM D7566. This certification ensures that the SAF is chemically equivalent to conventional jet fuel (Jet-A or Jet-Al) and can be safely used in existing aircraft engines and fuel infrastructure without modifications. The certification process involves extensive testing to verify that the SAF meets all performance and safety criteria, including energy content, combustion characteristics, and compatibility with aircraft materials. By adhering to these standards, ASTM certified SAF provides a reliable and environmentally friendly alternative to traditional fossil-based aviation fuels, contributing to reduced greenhouse gas emissions and enhanced sustainability in the aviation industry.

The recombinant microorganism

Within the present invention, the recombinant microorganism may be any microorganism, preferably any microorganism that is suitable for the use in biotechnological processes at an industrial scale. Preferably, the microorganism is a microorganism that can be used for the biotechnological production of isobutene or its precursor molecule 3-methylcrotonic acid at an industrial scale.

In a particular embodiment, the invention relates to the method of the invention, wherein said microorganism is a bacterium, a yeast, a fungus, an algae or an archaeon. The term "microorganism" in the context of the present invention refers to bacteria, as well as to fungi, such as yeasts, and also to algae and archaea. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the present invention are bacteria of the genus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas, Acinetobacter or Escherichia. In a particularly preferred embodiment, the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli. In another preferred embodiment, the bacterium belongs to the species Pseudomonas putida or to the species Zymomonas mobilis or to the species Corynebacterium glutamicum or to the species Bacillus subtilis. It is also possible to employ an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae.

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

In another embodiment, the present invention makes use of a photosynthetic microorganism expressing at least one enzyme for the conversion according to the invention as described above. Preferably, the microorganism is a photosynthetic bacterium, or a microalgae. In a further embodiment the microorganism is an algae, more preferably an algae belonging to the diatomeae.

In a further embodiment, the microorganism is an archaeon. Archaea are single-celled microorganisms that form a distinct domain of life, differing from bacteria in their genetic and biochemical characteristics. Within the present invention, "archaeon" refers to any archaeal species suitable for use in industrial biotechnological processes, including, for example, methanogenic archaea such as Methanococcus or Methanosarcina, Halobacterium species, and thermophilic archaea such as Sulfolobus, Pyrococcus, or Thermococcus.

It is also conceivable to use in accordance with the present invention a combination of microorganisms, wherein different microorganisms express different enzymes as described above. In a preferred embodiment, the microorganism of the present invention is a microorganism which is capable of consuming acetic acid, or a salt thereof. In another preferred embodiment, the microorganism, preferably the microorganism, of the present invention is a microorganism which is capable of producing acetate from suitable precursors. In certain embodiments, the microorganism of the present invention is an microorganism which is capable of producing acetate from carbon monoxide and methanol.

In another preferred embodiment, the microorganism of the present invention is a microorganism consuming sucrose, wherein said microorganism has genetically been modified by the introduction of at least one gene of a non-Phosphotransferase Transport System (non-PTS). Without being bound to theory, such an microorganism has genetically been modified by introducing a gene selected from the group consisting of cscA, cscB, and cscK from Escherichia coli W (M. Bruschi etal., Biotechnology Advances 30 (2012) 1001-1010).

In a particular embodiment, the invention relates to the use of the recombinant microorganism of the invention for the production of isobutene.

List of embodiments

1. A method for the production of isobutene, a precursor, or a derivative thereof, from acetic acid, or a salt thereof, in a recombinant microorganism, the method comprising the steps of:

(i) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA; and

(ii) enzymatically converting said produced acetyl-CoA into isobutene, or a precursor thereof.

2. The method according to embodiment 1, wherein the conversion of acetic acid, or a salt thereof, into acetyl-CoA comprises the step(s) of: (i) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA with an acetyl-CoA synthase (EC 6.2.1.1); and/or

(ii) enzymatically converting acetic acid, or a salt thereof, into acetyl-phosphate with an acetate kinase (EC 2.7.2.1) and enzymatically converting the resulting acetyl-phosphate into acetyl-CoA with a phosphate acetyltransferase (EC 2.3.1.8). The method according to embodiment 2, wherein the acetyl-CoA synthase is expressed from a recombinant promoter, preferably wherein the recombinant promoter is an inducible promoter, more preferably a heat-inducible promoter. The method according to embodiment 2 or 3, wherein the acetyl-CoA synthase comprises at least one mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase, thereby maintaining the acetyl-CoA synthase in its active state. The method according to embodiment 4, wherein the acetyl-CoA synthase comprises or consists of an amino acid sequence as set forth in SEQ ID NO:1 and wherein the mutation that prevents acetylation of the acetyl-CoA synthase is a mutation of leucine at position 641 of SEQ ID NO:1, preferably wherein the leucine at position 641 of SEQ ID NO:1 is replaced by a proline. The method according to any one of embodiments 1 to 5, wherein at least one endogenous gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3. l._) is inactivated in the recombinant microorganism. The method according to any one of embodiments 1 to 6, wherein the recombinant microorganism overexpresses an acetate kinase (EC 2.7.2.1) and/or a phosphate acetyltransferase (EC 2.3.1.8), preferably wherein the acetate kinase (EC 2.7.2.1) and/or the phosphate acetyltransferase (EC 2.3.1.8) are expressed from an inducible promoter. The method according to any one of embodiments 1 to 7, wherein the acetic acid, or the salt thereof, used in the method is derived from carbon monoxide and methanol, preferably wherein the carbon monoxide and/or the methanol are obtained from carbon dioxide or methane. The method according to any one of embodiments 1 to 8, wherein the enzymatic conversion of acetyl-CoA into isobutene, or a precursor thereof, comprises one or more of the steps selected from (i) to (vi):

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA,

(vi) enzymatically converting said produced 3,3-dimethyl acrylic acid into isobutene. The method according to any one of embodiments 1 to 9, wherein during a first stage of production, biomass is produced from sugar and/or acetic acid, or a salt thereof. The method according to embodiment 10, wherein during a second stage of production, isobutene, or the precursor or derivative thereof, is exclusively or primarily produced from acetic acid, or a salt thereof. The method according to any one of embodiments 1 to 11, further comprising a step of recovering and/or purifying the produced isobutene. 13. The method according to embodiment 12, wherein the recovered and/or purified isobutene is further converted into alkanes through oligomerization and hydrogenation, preferably wherein the alkanes comprise a mixture of C12 (isododecane) and C16(isohexadecane) alkanes.

14. The method according to any one of embodiments 1 to 13, wherein said microorganism is a bacterium, a yeast, a fungus, an algae or an archaeon.

15. A recombinant microorganism which is capable of enzymatically converting acetic acid, or a salt thereof, into isobutene, a precursor, or a derivative thereof, wherein said recombinant microorganism expresses a plurality of enzymes catalyzing:

(i) the conversion of acetic acid, or a salt thereof, into acetyl-CoA; and

(ii) the conversion of acetyl-CoA into isobutene, or a precursor thereof.

16. The recombinant microorganism according to embodiment 15, wherein said microorganism expresses:

(i) an acetyl-CoA synthase (EC 6.2.1.1) to enzymatically convert acetate into acetyl-CoA by; and/or

(ii) an acetate kinase (EC 2.7.2.1) to enzymatically convert acetate into acetylphosphate and a phosphate acetyltransferase (EC 2.3.1.8) to enzymatically convert the resulting acetyl-phosphate into acetyl-CoA.

17. The recombinant microorganism according to embodiment 16, wherein said acetyl-CoA synthase is encoded by a nucleic acid comprising a recombinant promoter, preferably wherein the recombinant promoter is an inducible promoter, more preferably a heatinducible promoter.

18. The recombinant microorganism according to embodiment 16 or 17, wherein the acetyl-

CoA synthase comprises at least one mutation that prevents acetylation of the acetyl- CoA synthase by a protein acetyltransferase, thereby maintaining the acetyl-CoA synthase in its active state.

19. The recombinant microorganism according to embodiment 18, wherein the acetyl-CoA synthase comprises or consists of an amino acid sequence as set forth in SEQ ID NO:1, and wherein the mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase is a mutation of a leucine at position 641 of SEQ ID NO:1, preferably wherein the leucine at position 641 of SEQ ID NO:1 is replaced by a proline.

20. The recombinant microorganism according to any one of embodiments 15 to 19, wherein at least one endogenous gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3.1._) is inactivated in said recombinant microorganism.

21. The recombinant microorganism according to any one of embodiments 15 to 20, wherein said recombinant microorganism comprises a recombinant nucleic acid encoding an acetate kinase (EC 2.7.2.1) and/or a phosphate acetyltransferase (EC 2.3.1.8), preferably wherein the recombinant nucleic acid is suitable for overexpressing the acetate kinase (EC 2.7.2.1) and/or the phosphate acetyltransferase (EC 2.3.1.8).

22. The recombinant microorganism according to any one of embodiments 15 to 21, wherein said recombinant microorganism further recombinantly expresses one or more enzyme selected from (i) to (vi):

(i) an enzyme for catalyzing the conversion of acetyl-CoA into acetoacetyl-CoA,

(ii) an enzyme for catalyzing the conversion of the resulting acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA,

(iii) an enzyme for catalyzing the conversion of the resulting 3-hydroxy-3- methylglutaryl-CoA into 3-methylglutaconyl-CoA,

(iv) an enzyme for catalyzing the conversion of the resulting 3-methylglutaconyl- CoA into 3-methylcrotonyl-CoA,

(v) an enzyme for catalyzing the conversion of the resulting 3-methylcrotonyl-CoA into 3,3-dimethyl acrylic acid, and (vi) enzymatically converting said produced 3,3-dimethylacrylic acid into isobutene.

23. The recombinant microorganism according to any one of embodiments 15 to 22, wherein the recombinant microorganism is a bacterium, a yeast, a fungus, an algae or an archaeon.

24. Use of the recombinant microorganism as defined in any one of embodiments 15 to 23 for the production of isobutene, a precursor, or a derivative thereof.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: Overview of artificial pathways for isobutene production from acetyl-CoA via 3- methylcrotonic acid. Enzymatic recycling of metabolites which may occur during the pathway are shown in steps Xa, Xb, XI and XII.

Figure 2: Overview of isobutene pathway starting from acetate.

Figure 3: Isobutene production by fermentation from acetic acid as sole carbon source. From t = 21 h until t = 90 h the sole carbon source of the fermentation was acetic acid and 141 g/L was added (dotted line), 122 g/L was consumed and 5.4 g/L isobutene was produced (solid line).

EXAMPLES

Example 1: In vivo isobutene (IBN) production from acetic acid from a strain with an increased expression of acetyl-CoA synthetase

This Example shows the direct production of isobutene by a recombinant E. coli strain which expresses exogenous genes, thereby constituting the isobutene pathway. Like most microorganisms, E. coli converts glucose into acetyl-CoA. The enzymes used in this study to convert acetyl-CoA into isobutene are summarized in the following.

Expression of a thermoinducible isobutene biosynthetic pathway in E. coli The following genes were codon-optimized for the expression in E. coli and synthesized by GeneArt (Life Technologies):

- thl (thiolase) from Clostridium acetobutylicum (Uniprot Accession number P45359)

- mvaS (Hydroxymethylglutaryl-CoA synthase) from Enterococcus faecalis (Uniprot Accession number Q835L4)

- ech (enoyl-CoA hydratase) from Pseudomonas sp. (Uniprot Accession number K9NHK2)

- aibA and aibB that code for the two subunits of 3-methylglutaconyl-CoA decarboxylase from Myxococcus hansupus (Uniprot Accession number A0A0H4WQB1 and A0A0H4WWJ4)

- menl (ydil) (l,4-dihydroxy-2-naphthoyl-CoA hydrolase) from Escherichia coli (strain K12) (Uniprot Accession number P77781)

FDC1 (ferulic acid decarboxylase) from Streptomyces sp. 769 (Uniprot Accession number A0A0A8EV26)

UbiX (flavin prenyltransferase) from Escherichia coli (strain K12) (Uniprot Accession number P0AG03)

An expression vector containing the origin of replication of pSClOl (Bernadi and Bernadi, Nucleic Acids Res. 1984 Dec 21; 12(24): 9415-9426) was used for the expression of these genes. In the plasmid pGB5771 (described in WO2017/085167, Example 12, Table L), the DNA sequence between the restriction sites EcoRI and Ncol was replaced by the following DNA sequences cloned by NEBuilder HiFi DNA Assembly (New England Biolabs) in the following order:

- ci857(ts)-PRM promoter-PR promoter: the DNA sequence was amplified from the commercial plasmid pPL451 (NovoPro Bioscience Inc., #V004050) between the Pstl restriction site (2636) and the PR promoter (3669) after having introduced the mutation (TGC)A67T(TGT) in ci857, so that this repressor binding to the PR promoter was made thermosensitive.

- menl-ech-mvaS-aibA-aibB-UbiX-FDCl-thl: the ribosome binding site included in the thermoinducible PR promoter was used for menl whereas the ribosome binding site T7 (GAAGGAGATATA) was used for all the other enzymes. Genetic modifications to increase acetic acid conversion to acetyl-CoA

To increase the acetic acid conversion to acetyl-CoA, the acetyl-CoA synthetase acs gene was amplified from the genome of E. coli K12 MG1655 (Uniprot: P27550) and cloned by NEBuilder HiFi DNA Assembly (New England Biolabs) (with the ribosome binding site T7) after the thl gene in the plasmid described above, so under the control of the thermoinducible PR promoter. The mutation (CTG)L641P(CCG) was introduced in the acs gene to reduce its inactivation by acetylation.

To further reduce acs inactivation by acetylation, the peptidyl-lysine N-acetyltransferase patZ gene was deleted from the chromosome of the strain E. coli W (DSM 1116). The strain was also modified to increase its native capacity to use sucrose as carbon source by the integration of an additional sucrose operon cscAKB from E. coli W (DSM 1116) in the poxB locus and the deletion of its repressor cscR.

The resulting E. Coli W (DSM 1116) modified strain was made electro-competent and transformed with the thermoinducible expression vector. The transformed cells were then plated on LB plates with 100 mg/L spectinomycin, and the plates were incubated overnight at 30°C. An isolated colony was used to prepare a pre-culture as described in the following.

Production of isobutene from acetic acid in IL bioreactor

A I L vessel was filled with 0.5 L of a culture medium containing, 10 g/L L-glutamic acid monosodium monohydrate, 0.71 g/L sodium sulfate, 1.34 g/L ammonium sulfate, 3.4 g/L potassium phosphate monobasic, 4.45 g/L sodium phosphate dibasic, 80 pL/L antifoam (Struktol J673, Schill und Seilacher) and sterilized at 121°C for 20 minutes. After cooling, filter sterilized solutions were added to obtain the following final concentrations: 1 g/L magnesium sulfate heptahydrate, 100 pM iron III chloride, 40 pM calcium chloride, 20 pM manganese chloride, 20 pM zinc sulfate, 4 pM copper chloride, 100 mg/L spectinomycin, 30 g/L sugar beet molasses. A filter sterilized fed batch solution of sucrose was prepared at 850 g/L with 1000 pL/L antifoam. A 25 % acetic acid solution was also prepared to be used as fed batch solution. The culture medium was inoculated with 30 mL of a pre-culture at D0600 = 0.5 (previously grown at 30°C in the same medium as in the bioreactor but with 2 g/L sugar beet molasses and no antifoam).

All along the culture, the pH of the culture medium was regulated with an acid solution of 5 M phosphoric acid and a base solution of 25 % ammonium hydroxide.

For the first 11 hours of culture, temperature was kept at 34 °C, pH regulated at pH7.2, aeration was set at 1 L/min. The agitation was regulated to maintain dissolved oxygen at 45% of saturation until reaching the maximum agitation value of 1200 rpm, a value at which the dissolved oxygen starts decreasing to reach 0 % if sucrose concentration is sufficient.

At t = 11 h, the temperature was increased to 39°C and the pH was increased to pH7.6 in 15 min. The dissolved oxygen set point was decreased to 3%. The feed of the sucrose fed batch solution was started at 13 g/L.h to reach 20 g/L.h at t = 15 h. It was then decreased from 20 g/L.h to reach 14 g/L.h at t = 21 h.

At t = 21 h no residual sugar was present in the culture medium (sucrose, glucose or fructose) and the sucrose fed batch solution was replaced by a 25% acetic acid fed batch solution. The feed was decreased to 2 g/L.h and kept constant until t = 66 h. At t = 66h the feed was increased to 2.5 g/L.h and kept constant until the end of the fermentation at t = 90 h.

The production of isobutene in the gas phase was measured by mass spectrometry and the concentrations of sucrose, glucose, fructose and acetic acid in the culture medium were monitored by HPLC (Hiplex column at 40°C with a mobile phase of 5 mM sulfuric acid at 0.8 mL/min).

Claims

(iii) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA; and

(iv) enzymatically converting said produced acetyl-CoA into isobutene, or a precursor thereof.

2. The method according to claim 1, wherein the conversion of acetic acid, or a salt thereof, into acetyl-CoA comprises the step(s) of:

(i) enzymatically converting acetic acid, or a salt thereof, into acetyl-CoA with an acetyl-CoA synthase (EC 6.2.1.1); and/or

(ii) enzymatically converting acetic acid, or a salt thereof, into acetyl-phosphate with an acetate kinase (EC 2.7.2.1) and enzymatically converting the resulting acetyl-phosphate into acetyl-CoA with a phosphate acetyltransferase (EC 2.3.1.8).

3. The method according to claim 2, wherein the acetyl-CoA synthase is expressed from a recombinant promoter, preferably wherein the recombinant promoter is an inducible promoter, more preferably a heat-inducible promoter.

4. The method according to claim 2 or 3, wherein the acetyl-CoA synthase comprises at least one mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase, thereby maintaining the acetyl-CoA synthase in its active state.

5. The method according to claim 4, wherein the acetyl-CoA synthase comprises or consists of an amino acid sequence as set forth in SEQ ID NO:1 and wherein the mutation that prevents acetylation of the acetyl-CoA synthase is a mutation of leucine at position 641 of SEQ ID NO:1, preferably wherein the leucine at position 641 of SEQ ID NO:1 is replaced by a proline.

6. The method according to any one of claims 1 to 5, wherein at least one endogenous gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3. l._) is inactivated in the recombinant microorganism.

7. The method according to any one of claims 1 to 6, wherein the recombinant microorganism overexpresses an acetate kinase (EC 2.7.2.1) and/or a phosphate acetyltransferase (EC 2.3.1.8), preferably wherein the acetate kinase (EC 2.7.2.1) and/or the phosphate acetyltransferase (EC 2.3.1.8) are expressed from an inducible promoter.

8. The method according to any one of claims 1 to 7, wherein the acetic acid, or the salt thereof, used in the method is derived from carbon monoxide and methanol, preferably wherein the carbon monoxide and/or the methanol are obtained from carbon dioxide or methane.

9. The method according to any one of claims 1 to 8, wherein the enzymatic conversion of acetyl-CoA into isobutene, or a precursor thereof, comprises one or more of the steps selected from (i) to (vi):

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA,

(iv) enzymatically converting said produced 3-methylglutaconyl-CoA into 3- methylcrotonyl-CoA, (v) enzymatically converting said produced 3-methylcrotonyl-CoA into 3,3- dimethyl acrylic acid, and

(vi) enzymatically converting said produced 3,3-dimethyl acrylic acid into isobutene.

10. The method according to any one of claims 1 to 9, wherein during a first stage of production, biomass is produced from sugar and/or acetic acid, or a salt thereof.

11. The method according to claim 10, wherein during a second stage of production, isobutene, or the precursor or derivative thereof, is exclusively or primarily produced from acetic acid, or a salt thereof.

12. The method according to any one of claims 1 to 11, further comprising a step of recovering and/or purifying the produced isobutene.

13. The method according to claim 12, wherein the recovered and/or purified isobutene is further converted into alkanes through oligomerization and hydrogenation, preferably wherein the alkanes comprise a mixture of C12 (isododecane) and C16(isohexadecane) alkanes.

14. The method according to any one of claims 1 to 13, wherein said microorganism is a bacterium, a yeast, a fungus, an algae or an archaeon.

(iii) the conversion of acetic acid, or a salt thereof, into acetyl-CoA; and

(iv) the conversion of acetyl-CoA into isobutene, or a precursor thereof.

16. The recombinant microorganism according to claim 15, wherein said microorganism expresses: (i) an acetyl-CoA synthase (EC 6.2.1.1) to enzymatically convert acetate into acetyl-CoA by; and/or

17. The recombinant microorganism according to claim 16, wherein said acetyl-CoA synthase is encoded by a nucleic acid comprising a recombinant promoter, preferably wherein the recombinant promoter is an inducible promoter, more preferably a heatinducible promoter.

18. The recombinant microorganism according to claim 16 or 17, wherein the acetyl-CoA synthase comprises at least one mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase, thereby maintaining the acetyl-CoA synthase in its active state.

19. The recombinant microorganism according to claim 18, wherein the acetyl-CoA synthase comprises or consists of an amino acid sequence as set forth in SEQ ID NO:1, and wherein the mutation that prevents acetylation of the acetyl-CoA synthase by a protein acetyltransferase is a mutation of a leucine at position 641 of SEQ ID NO:1, preferably wherein the leucine at position 641 of SEQ ID NO:1 is replaced by a proline.

20. The recombinant microorganism according to any one of claims 15 to 19, wherein at least one endogenous gene encoding a peptidyl-lysine /V-acetyltransferase (EC 2.3. l._) is inactivated in said recombinant microorganism.

21. The recombinant microorganism according to any one of claims 15 to 20, wherein said recombinant microorganism comprises a recombinant nucleic acid encoding an acetate kinase (EC 2.7.2.1) and/or a phosphate acetyltransferase (EC 2.3.1.8), preferably wherein the recombinant nucleic acid is suitable for overexpressing the acetate kinase (EC 2.7.2.1) and/or the phosphate acetyltransferase (EC 2.3.1.8).

22. The recombinant microorganism according to any one of claims 15 to 21, wherein said recombinant microorganism further recombinantly expresses one or more enzyme selected from (i) to (vi):

(i) an enzyme for catalyzing the conversion of acetyl-CoA into acetoacetyl-CoA,

(v) an enzyme for catalyzing the conversion of the resulting 3-methylcrotonyl-CoA into 3,3-dimethyl acrylic acid, and

23. The recombinant microorganism according to any one of claims 15 to 22, wherein the recombinant microorganism is a bacterium, a yeast, a fungus, an algae or an archaeon.

24. Use of the recombinant microorganism as defined in any one of claims 15 to 23 for the production of isobutene, a precursor, or a derivative thereof.