Methods and means for production using prfmn-dependent enzymes under anaerobic conditions

Abstract

The present invention relates to a method for producing a product using an enzyme comprising a prenylated flavin mononucleotide (prFMN) cofactor in a recombinant microorganism, wherein the recombinant microorganism is grown in a culture medium in the absence of air, and wherein a prFMNH2 precursor is oxidised with molecular oxygen (O2) derived from decomposition of hydrogen peroxide (H2O2) to produce the active prFMN cofactor. Further provided are recombinant microorganism for use in such methods.

Description

[0001] New PCT-Patent Application

[0002] Global Bioenergies

[0003] Vossius Ref.: AJ4094 PCT BS

[0004] METHODSAND MEANS FOR PRODUCTION USING prFMN-DEPENDENT ENZYMES UNDER ANAEROBIC CONDITIONS

[0005] The present invention is in the field of microbial biotechnology and relates to a method for producing a product using an enzyme comprising a prenylated flavin mononucleotide (prFMN) cofactor in a recombinant microorganism, wherein the recombinant microorganism is grown in a culture medium in the absence of air, and wherein a prFMNH2 precursor is oxidised with molecular oxygen (O2) derived from decomposition of hydrogen peroxide (H2O2) to produce the active prFMN cofactor. Further provided are recombinant microorganism for use in such methods.

[0006] BACKGROUND TO THE INVENTION

[0007] prFMN-dependent enzymes are a versatile class of enzymes that utilize prenylated flavin mononucleotide (prFMN) as a cofactor to catalyze a variety of biochemical reactions, including decarboxylation, carboxylation, and other redox processes. These enzymes perform unique reactions by utilizing a prFMN as a cofactor, which is synthesized in a reduced form (prFMNH2) from FMNH2 and DMAP or DMAPP by a flavin prenyltransferase (UbiX-like enzyme) (see Figure 1A).

[0008] Among the prFMN-dependent enzymes, prFMN-dependent decarboxylases are particularly notable for their ability to catalyze the non-oxidative, reversible decarboxylation of a wide range of carboxylic acid substrates. UbiD and FDC are the most common and well-studied prFMN-dependent decarboxylases. UbiD enzymes are typically involved in ubiquinone biosynthesis and have been identified in a variety of organisms, including bacteria and fungi. FDC, or ferulic acid decarboxylase, is another prominent member of this enzyme class, knownfor its role in the decarboxylation of ferulic acid to produce vinyl guaiacol, a compound with applications in the flavour and fragrance industries. Both UbiD and FDC enzymes have been subjects of research for their potential applications in biocatalysis, where they are engineered to improve their efficiency and substrate range for industrial processes. Due to their substrate promiscuity, these enzymes can be utilized in various applications, making them versatile tools for the synthesis of biofuels, pharmaceuticals, and specialty chemicals.

[0009] The UbiX / UbiD system is widespread in microbes and responsible for the reversible decarboxylation of a,P-unsaturated carboxylic acids. UbiD and the associated flavin prenyltransferase UbiX were originally characterized and named as part of the bacterial ubiquinone biosynthesis pathway, with both UbiD and UbiX found to be essential for decarboxylation of the proposed 3-octaprenyl-4-hydroxybenzoic acid intermediate. A wide range of UbiD enzymes have since been implicated in (de)carboxylation of (hetero)aromatic and a,|3-unsaturated carboxylic acids, made feasible through reversible covalent adduct formation between prFMN and substrate (Bloor et al., The FEBS Journal, 2023, 290, 2232-2245).

[0010] FDCs are fungal homologues of the E. coli UbiD enzyme, placing FDCs within the broader UbiD enzyme family and representing a distinct class within this family. The presence of the fdcl gene and the associated padl gene (with PAD1 being homologous to UbiX in E. coli) has been shown to be essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae.

[0011] The oxidative maturation of the prFMN cofactor is essential for the catalytic activity of UbiD-type enzymes. The reduced form, prFMNFU, produced by a flavin prenyltransferase (UbiX / PADl), can undergo oxidation to form various species, including a stable prFMN radical and a prFMN hydroperoxide adduct (prFMN C4a-OOH). However, these species do not support decarboxylase activity. Instead, the active form, prFMNiminium, is generated through oxidation within the decarboxylase-prFMNH2 complex. This process mimics flavin reactivity with oxygen and involves a multistep pathway where prFMNH2 is oxidized to a transient prFMN C4a-OO_adduct, stabilized by a conserved arginine in the enzyme. This adduct eliminates H2O2, leading to the formation of the catalytically active prFMNiminium, a zwitterionicspecies resembling an azomethine ylide. This species can participate in reversible reactions, enabling the enzymes to perform non-oxidative reversible decarboxylation of various acids. Accordingly, the correct oxidative maturation of the prFMN cofactor is essential for the function of prFMN-dependent decarboxylases (Bloor et al., The FEBS Journal, 2023, 290, 2232-2245).

[0012] In 2024, the enzyme PhdC was described to function as a maturase to catalyse the oxidation of reduced prFMN (prFMNFh) to the catalytically active form. PhdC was shown to use molecular oxygen to oxidize the prFMN semiquinone radical, formed by spontaneous air oxidation, to the active cofactor (DiRocco et al., ACS Catalysis, 2024, 14 (13), 10223-10233).

[0013] One commercially relevant product of prFMN-dependent decarboxylation is isobutene (also referred to as isobutylene). Isobutene is a key component in the production of methyl-tert-butyl-ether (MTBE), an anti-knock additive for automobile fuel. Additionally, isobutene can be used to produce isooctene, which can subsequently be reduced to isooctane (2,2,4-trimethylpentane). The high octane rating of isooctane makes it an ideal fuel for gasoline engines. Furthermore, isobutene serves as a precursor for the synthesis of oligomers such as isododecene (C12), isohexadecene (C16), and mixtures thereof. These oligomers can be reduced to form isododecane, isohexadecane, and mixtures thereof, which are the main components of sustainable aviation fuels (SAF). Notably, a specific mixture of C12 / C16 at an 85 / 15 ratio has been certified as a sustainable aviation fuel by ASTM.

[0014] Traditionally, alkenes such as isobutene have been produced through catalytic cracking of petroleum products or via a derivative of the Fischer-Tropsch process in the case of hexene, derived from coal or gas. Consequently, production costs are closely tied to the price of oil. Moreover, catalytic cracking often involves significant technical challenges, increasing both process complexity and production costs. Thus, alternative processes for the production of isobutene have been explored in the past.

[0015] Different routes for the enzymatic production of isobutene have been described; see, e.g., 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) and W02010 / 001078.

[0016] However, the preferred route for the enzymatic production of isobutene utilizes the enzymatic conversion of 3-methylcrotonic acid into isobutene using a prFMN-dependent decarboxylase associated with a flavin prenyltransferase, as described, for example, in WO 2022 / 136207. The conversion of 3-methylcrotonic acid into isobutene catalysed by a prFMN-dependent decarboxylase is shown in Figure IB.

[0017] Given the dependency of the prFMN cofactor on molecular oxygen, biotechnological processes involving UbiD-type decarboxylases typically have to be carried out under aerobic conditions. However, aerobic processes face certain challenges that may be undesirable, particularly when producing gaseous products such as isobutene. The presence of oxygen in combination with flammable gases like isobutene poses a significant risk of explosions, necessitating stringent safety measures and specialized equipment to prevent ignition. Additionally, the large amounts of nitrogen introduced by air can dilute the gaseous product, leading to more laborious and costly purification processes. Moreover, aerobic fermentation processes require high agitation of the fermentation medium, which inevitably results in higher production costs compared to anaerobic processes.

[0018] Thus, despite recent advancements in the biotechnological production of alkenes and other products, there remains a need for improved biotechnological processes that utilize prFMN-dependent enzymes. Specifically, there is a need for processes that provide enhanced safety and efficiency over current aerobic fermentation processes.

[0019] SUMMARY OF THE INVENTION

[0020] The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

[0021] 1. A method for producing a product using an enzyme comprising a prenylated flavin mononucleotide (prFMN) cofactor in a recombinant microorganism, wherein therecombinant microorganism is grown in a culture medium in the absence of air, and wherein a prFMNH2 precursor is oxidised with molecular oxygen (O2) derived from decomposition of hydrogen peroxide (H2O2) to produce the active prFMN cofactor.

[0022] 2. The method of embodiment 1, wherein the decomposition of H2O2 is catalysed by a catalase.

[0023] 3. The method of embodiment 2, wherein the catalase (i) is added to the culture medium exogenously or (ii) is expressed by the recombinant microorganism or by a further recombinant microorganism present in the culture medium.

[0024] 4. The method of embodiment 2 or 3, wherein the catalase is a heme-containing catalase or a manganese-containing catalase.

[0025] 5. The method of embodiment 4, wherein the heme is added to the culture medium exogenously or produced in the recombinant microorganism.

[0026] 6. The method of any one of embodiments 1 to 5, wherein the H2O2 is added to the culture medium in the form of H2O2 or in the form of an adduct or precursor of H2O2, in particular wherein the adduct or precursor of H2O2 is urea hydrogen peroxide or a percarbonate salt, preferably sodium percarbonate.

[0027] 7. The method of any one of embodiments 1 to 6, wherein the H2O2 or the adduct or precursor of H2O2 is added to the culture medium at a concentration ranging from 50 to 1000 pM, preferably 300 to 500 pM.

[0028] 8. The method of any one of embodiments 1 to 7, wherein the recombinant microorganism expresses a flavin prenyltransferase.

[0029] 9. The method of any one of embodiments 1 to 8, wherein the product is formed by a decarboxylation reaction catalysed by the prFMN-dependent enzyme, preferably wherein the product is selected from the group consisting of: isobutene, styrene,butadiene, 1,3-pentadiene, hepta-l,3,5-triene and 4-vinylguiacol; or wherein the product is formed by a carboxylation reaction catalysed by the prFMN-dependent enzyme, preferably wherein the product is selected from the group consisting of: 2,5- furandicarboxylic acid (FDCA) and 2-naphthoic acid.

[0030] 10. The method of any one of embodiments 1 to 9, wherein the product is isobutene and, preferably, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity.

[0031] 11. The method of embodiment 10, wherein the prFMN-dependent enzyme having 3- methylcrotonic acid decarboxylase activity comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 1.

[0032] 12. The method of embodiment 10 or 11, wherein the isobutene is produced from 3- methylcrotonic acid.

[0033] 13. The method of any one of embodiments 1 to 12, wherein the recombinant microorganism is a bacterium, in particular Escherichia coli, or a Clostridium species, or a fungus, in particular wherein the fungus is a yeast, such as Saccharomyces cerevisiae.

[0034] 14. A recombinant microorganism comprising one or more nucleic acids encoding:

[0035] a) a prFMN-dependent enzyme;

[0036] b) a flavin prenyltransferase; and

[0037] c) a catalase.

[0038] 15. The recombinant microorganism of embodiment 14, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity and, preferably, comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 1, wherein the enzyme retains 3- methylcrotonic acid decarboxylase activity.16. The recombinant microorganism of embodiment 14 or 15, wherein the flavin prenyltransferase comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:15 or (ii) an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 15, wherein the amino acid sequence retains the enzymatic activity of modifying FMNH2into prFMNH2.

[0039] 17. The recombinant microorganism of any one of embodiments 14 to 16, wherein the catalase is a heme-containing catalase or a manganese-containing catalase.

[0040] 18. The recombinant microorganism of any one of embodiments 14 to 17, wherein the recombinant microorganism is a bacterium, in particular Escherichia coli, or a Clostridium species, or a fungus, in particular wherein the fungus is a yeast, such as Saccharomyces cerevisiae.

[0041] 19. Use of the recombinant microorganism of any one of embodiments 14 to 18 in the production of a product, wherein the product is produced by the prFMN-dependent enzyme.

[0042] Accordingly, in a particular embodiment, the invention relates to a method for producing a product using an enzyme comprising a prenylated flavin mononucleotide (prFMN) cofactor in a recombinant microorganism, wherein the recombinant microorganism is grown in a culture medium in the absence of air, and wherein a prFMNH2precursor is oxidised with molecular oxygen (O2) derived from decomposition of hydrogen peroxide (H2O2) to produce the active prFMN cofactor.

[0043] The present invention is based on the surprising finding that the prFMN cofactor of prFMN-dependent enzymes can be activated with molecular oxygen derived from the decomposition of hydrogen peroxide (H2O2), and thus in the absence of air. This allows for the efficient production of products catalysed by the prFMN-dependent enzyme, and without the need for maintaining aerobic conditions during the process. This innovative approach mitigates the risks associated with air and flammable gases, reduces energy consumption, and simplifies purification processes, thereby enhancing the overall safety and efficiency of biotechnologicalprocesses involving prFMN-dependent enzymes.

[0044] The molecular oxygen required for the oxidation of the inactive prFMNH2 precursor into the active prFMN cofactor of the enzyme is generated through the decomposition of hydrogen peroxide (H2O2). Using H2O2 for the oxidation of the prFMNFh precursor not only provides a reliable source of oxygen but also allows for precise control over the oxygen levels in the system, further enhancing the safety and efficiency of the process.

[0045] For example, it has been demonstrated in Example 4 that under anaerobic conditions, i.e., in the absence of air, the addition of H2O2 can increase isobutene productivity by a prFMN-dependent decarboxylase, as shown in Table 1. Notably, this is higher than the productivity measured under aerobic conditions in Example 3. Thus, in addition to addressing important safety concerns and cost aspects, employing prFMN-dependent enzymes under anaerobic conditions in the presence of H2O2 may even increase the productivity of these enzymes.

[0046] Decomposition of hydrogen peroxide (H2O2)

[0047] The decomposition of hydrogen peroxide (H2O2) is a chemical reaction that results in the formation of water (H2O) and oxygen (O2). This process can occur spontaneously, but it is significantly accelerated in the presence of catalysts such as catalase (EC 1.11.1.6) or catalaselike metal complexes or salts like MnCL Catalase is an enzyme commonly found in living organisms and catalyses the decomposition of hydrogen peroxide into water and oxygen, effectively reducing the potential oxidative damage that H2O2 may cause to cells. The reaction is exothermic, releasing energy in the form of heat.

[0048] The overall reaction of H2O2 decomposition can be represented as: 2 H2O2 -> 2 H2O + O2.

[0049] In certain embodiments, H2O2 spontaneously decomposes in the culture medium and the resulting oxygen can be used for the maturation of the prFMN cofactor. However, it is preferred herein that the decomposition of H2O2 is catalysed by a catalase. In a particular embodiment, the invention relates to the method according to the invention, wherein the decomposition of H2O2 is catalysed by a catalase.The term "catalase" as used herein refers to an enzyme that catalyses the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2). This enzyme is found in nearly all living organisms exposed to oxygen and serves to protect cells from oxidative damage by rapidly breaking down hydrogen peroxide, a potentially harmful by-product of metabolic processes. Catalase is highly efficient, capable of converting millions of hydrogen peroxide molecules per second, and is a key component of cellular antioxidant defence mechanisms. However, in anaerobic conditions, catalase is often poorly expressed.

[0050] Therefore, the catalase may be added to the culture medium comprising the recombinant microorganism or may be expressed by the recombinant microorganism itself. It has been demonstrated in the appended Examples that both the external addition of catalase to the culture medium (Example 4) and the recombinant expression of catalase within the microorganism (Example 6) significantly enhance the productivity of a prFMN-dependent enzyme in the presence of hydrogen peroxide, particularly under anaerobic conditions.

[0051] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the catalase (i) is added to the culture medium exogenously or (ii) is expressed by the recombinant microorganism or by a further recombinant microorganism present in the culture medium.

[0052] That is, in certain embodiments, a catalase is added to the culture medium exogenously. Preferably, the catalase is added to the culture medium in the form of a powder or in the form of a solution.

[0053] The catalase may be added to the culture medium at any suitable concentration to achieve sufficient release of molecular oxygen to oxidize the prFMNH2 cofactor. In certain embodiments, the catalase may be added to the culture medium at a concentration ranging from 100 to 100,000 units / L, preferably 500 to 50,000 units / L, more preferably 1,000 to 10,000 units / L, most preferably 1,500 to 3,000 units / L.Alternatively, the catalase may be expressed by the recombinant microorganism. In certain embodiments, the recombinant microorganism may express the catalase endogenously. However, due to the typically low expression of endogenous catalase under anaerobic conditions, it is preferred that the recombinant microorganism is engineered to express the catalase recombinantly. That is, the recombinant microorganism may comprise a recombinant nucleic acid encoding a catalase. In certain embodiments, the recombinant nucleic acid encoding the catalase may be expressed from an extrachromosomal nucleic acid, such as a vector or plasmid. In certain embodiments, the recombinant nucleic acid encoding the catalase may be integrated in the chromosomal DNA of the recombinant microorganism.

[0054] Alternatively, the catalase may be expressed by an additional recombinant microorganism present in the culture medium. In such embodiments, the culture medium may comprise two distinct recombinant microorganisms: the first recombinant microorganism expresses a prFMN-dependent enzyme for product production, while the second recombinant microorganism expresses the catalase to facilitate the decomposition of H2O2 and to generate sufficient oxygen for the oxidation of the prFMNH2 cofactor.

[0055] When the catalase is expressed by a recombinant microorganism, the skilled person is capable of identifying appropriate expression systems to achieve suitable levels of catalase to facilitate the decomposition of H2O2.

[0056] The catalase that is added to the culture medium or expressed by a recombinant microorganism may be any suitable catalase, in particular any catalase that remains active in an aqueous culture medium at temperatures suitable for growing recombinant microorganisms, e.g., temperatures ranging from 30 to 37°C.

[0057] In a particular embodiment, the invention relates to the method according to the invention, wherein the catalase is a heme-containing catalase or a manganese-containing catalase.

[0058] The term "heme-containing catalase", as used herein, refers to an enzyme that catalyses the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2) and contains a heme group as its prosthetic group. The heme group, which includes an iron ion coordinatedwithin a porphyrin ring, is essential for the enzyme's catalytic activity. Heme-containing catalases are found in nearly all aerobic organisms and are readily available from commercial sources.

[0059] The heme for the catalase may be added to the culture medium exogenously or may be produced by the recombinant microorganism. Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the heme is added to the culture medium exogenously or produced in the recombinant microorganism.

[0060] In certain embodiments, the catalase may be expressed by a recombinant organism, preferably the recombinant organism expressing the prFMN-dependent enzyme, and the culture medium may be exogenously supplemented with heme or a precursor thereof.

[0061] The term "manganese-containing catalase," as used herein, refers to an enzyme that catalyses the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2) and contains manganese ions as its essential cofactors. Unlike heme-containing catalases, which utilize a heme group, manganese-containing catalases rely on manganese ions to facilitate their catalytic activity. These enzymes are found in certain bacteria and archaea and play a crucial role in protecting these cells from oxidative damage by efficiently breaking down hydrogen peroxide. When manganese-containing catalases are used in the method of the invention, the culture medium is preferably supplemented with one or more manganese salt.

[0062] In certain embodiments, the catalase is derived from Saccharomyces cerevisiae (Uniprot accession numbers P15202 (SEQ ID NO: 26) and P06115 (SEQ ID NO:27)), from Bacillus subtilis (Uniprot accession number A0A7R7ZDZ6 (SEQ ID NO:28)), from Nostoc sp. (Uniprot accession number Q8YSJ5 (SEQ ID NO:29)), from Thermoascus aurantiacus (UniParc accession number UPI000DC0CC59 (SEQ ID NQ:30)) or from Mycothermus thermophilus (Uniprot accession number M4GGR5 (SEQ ID NO:31)). In certain embodiments, the catalase may be any one of the catalases disclosed in WO 2019 / 231944, which is fully incorporated by reference herein.

[0063] In a particular embodiment, the invention relates to the method according to the invention, wherein the H2O2 is added to the culture medium in the form of H2O2 or in the form of anadduct or precursor of H2O2, in particular wherein the adduct or precursor of H2O2 is urea hydrogen peroxide or a percarbonate salt, preferably sodium percarbonate.

[0064] That is, in certain embodiment, H2O2 is added to the culture medium comprising the recombinant microorganism. H2O2 is a clear, colourless liquid at room temperature that may be used as an aqueous solution at any suitable concentration. Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the H2O2 is added to the recombinant microorganism in the form of H2O2.

[0065] Alternatively, the H2O2 may be added to the culture medium comprising the recombinant microorganism in the form of an adduct or precursor of H2O2. As used herein, the term "adduct or precursor of H2O2" refers to any chemical compound or molecular entity that can either form H2O2 through a chemical reaction or is capable of releasing or generating H2O2 under certain conditions. An "adduct" typically involves a compound in which hydrogen peroxide is chemically bonded or associated with another molecule, while a "precursor" is a substance that undergoes a transformation to produce hydrogen peroxide.

[0066] The adduct or precursor of H2O2 may be any suitable compound that can be converted into or release H2O2in an aqueous culture medium.

[0067] In certain embodiments, the adduct or precursor of H2O2 is urea hydrogen peroxide. Urea hydrogen peroxide, also known as carbamide peroxide, is a solid, crystalline compound formed by the combination of urea and hydrogen peroxide. The compound releases hydrogen peroxide when dissolved in water, providing a controlled source of this reactive oxygen species. Urea hydrogen peroxide is often utilized for its stability and ease of handling compared to liquid hydrogen peroxide solutions.

[0068] In certain embodiments, the adduct or precursor of H2O2 is a percarbonate salt. The term "percarbonate salt" refers to a crystalline compound that consists of a carbonate salt, such as sodium carbonate, combined with hydrogen peroxide. When dissolved in water, percarbonate salts release hydrogen peroxide. Sodium percarbonate is a common example of a percarbonate salt and is widely used in laundry detergents and household cleaning productsdue to its stability, ease of handling, and ability to provide a controlled release of hydrogen peroxide.

[0069] The H2O2 or the precursor or adduct thereof may be added to the culture medium at any concentration that is suitable for oxidizing the prFMNH2cofactor without causing oxidative damage to the cofactor or other components of the recombinant microorganism.

[0070] In a particular embodiment, the invention relates to the method according to the invention, wherein the H2O2 or the adduct or precursor of H2O2 is added to the culture medium at a concentration ranging from about 50 to about 1000 pM, preferably about 300 to about 500 pM.

[0071] In certain embodiments, H2O2is added to the culture medium at a concentration of about 50 pM, about 100 pM, about 150 pM, about 200 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about 600 pM, about 650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, or about 1000 pM. The precise concentration of hydrogen peroxide is selected based on the specific requirements of the enzymatic process, the composition of the culture medium, the concentration of biomass, and the tolerance of the recombinant microorganism to oxidative stress.

[0072] It has been demonstrated in Example 4 that adding between 100 and 1000 pM of FhChto a culture medium comprising an exogenously added catalase can increase the activity of a prFMN-dependent UbiD decarboxylase. Particularly high activities of the prFMN-dependent UbiD decarboxylase were observed when between 300 and 500 pM of FhChwere added to the culture medium. However, the skilled person is aware that the optimal H2O2 concentrations may vary depending on the availability of the prFMN-dependent enzyme and the catalase, and the composition of the culture medium and biomass concentration. However, the skilled person is able to follow the teachings of Example 4 and identify the optimal concentration of H2O2 for a specific setup.In certain embodiments, H2O2 may be added continuously to the culture medium, for example as part of a feed solution. In certain embodiments, multiple doses of H2O2 may be added to the culture medium during the method of the invention. For example, two or more doses of about 300 to about 500 pM may be added to the culture medium during the method of the invention.

[0073] Of note, the optimal concentration of H2O2 in the culture medium may be lower when the catalase is expressed by the recombinant organism, as the released oxygen may be more efficiently utilized in situ for the activation of the prFMN cofactor. This may enhance the overall efficiency of the process and reduce the amount of H2O2 required, minimizing potential oxidative damage and improving the safety and cost-effectiveness of the biotechnological application.

[0074] Thus, in embodiments where the recombinant microorganism expresses a catalase, H2O2 may be added to the culture medium at a concentration ranging from about 50 to about 1000 pM, preferably from about 100 to about 500 pM.

[0075] The catalase, if added exogenously, and / or the hydrogen peroxide, or the precursor or adduct thereof, may be added to the culture medium at anytime during the method of the invention. However, it is preferred that the catalase, if added exogenously, and / or the hydrogen peroxide, or the precursor or adduct thereof, are added to the culture medium when the cells of the recombinant microorganism are in exponential growth phase. The skilled person is aware that the optimal time point for the addition of the catalase and / or the hydrogen peroxide, or the precursor or adduct thereof, depends on the choice of host cell and the culture conditions.

[0076] The

[0077]

[0078] The molecular oxygen generated by the decomposition of hydrogen peroxide is used to oxidize the prFMN cofactor of an enzyme, thereby activating it for catalytic functions. Accordingly, the present invention is based on the finding that the prFMNH2 precursor can be oxidized into the active prFMN cofactor in the absence of atmospheric air. This innovativeapproach enables the activation of prFMN-dependent enzymes under anaerobic conditions, broadening their applicability and enhancing the safety and efficiency of biotechnological processes.

[0079] The term "prFMN-dependent enzyme", as used herein, refers to an enzyme that requires prenylated flavin mononucleotide (prFMN) as a cofactor to carry out its catalytic activity. prFMN-dependent enzymes utilize the unique chemical properties of prFMN, which is derived from the modification of flavin mononucleotide (FMN) through prenylation, to facilitate various biochemical reactions.

[0080] In a particular embodiment, the prFMN-dependent enzyme is a prFMN-dependent decarboxylase.

[0081] The term "prFMN-dependent decarboxylase," as used herein, refers to an enzyme that catalyses the reversible decarboxylation and carboxylation of substrates, utilizing a prenylated flavin mononucleotide (prFMN) cofactor provided by an associated flavin prenyltransferase for their catalytic activity. prFMN-dependent decarboxylases facilitate non-oxidative decarboxylation reactions, converting carboxylic acids to alkenes or other products and vice versa, through a 1,3-dipolar cycloaddition-based mechanism. This mechanism allows the enzyme to remove a carboxyl group (decarboxylation) or add a carboxyl group (carboxylation), depending on the reaction conditions and specific substrate involved. Found in various microorganisms, these enzymes play a crucial role in metabolic pathways that transform aromatic and unsaturated carboxylic acids. Consequently, the product produced by the recombinant microorganism in the method according to the invention may involve a carboxylation or decarboxylation step catalysed by a prFMN-dependent decarboxylase. The versatility of this enzymatic system enables the efficient production of various products through either carboxylation or decarboxylation reactions.

[0082] The terms "prFMN" or "prFMN cofactor," as used herein, refer to the catalytically active form of prenylated flavin mononucleotide, specifically prFMNiminium. This form is crucial for the catalytic activity of prFMN-dependent enzymes, such as prFMN-dependent decarboxylases, enabling them to facilitate reversible decarboxylation and carboxylation reactions. In contrast,the terms "prFMNFh" or "prFMNH2 precursor" relate to the reduced form of the cofactor, which is the direct product of flavin prenyltransferases, as described herein below. The oxidation of the prFMNFh precursor into the catalytically active prFMNiminium cofactor is described in detail by Bloor et al. in The FEBS Journal, 2023, 290, 2232-2245.

[0083] Non-limiting examples of prFMN-dependent carboxylases for use in the method of the present invention are provided herein below:

[0084] In certain embodiments, the prFMN-dependent decarboxylase catalysing the carboxylation or decarboxylation of a substrate is a member of the UbiD decarboxylyase family. This family of proteins is found in prokaryotes, archaea and fungi. They are related to UbiD, a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also known as polyprenyl p-hydroxybenzoate decarboxylase) from Escherichia coli that is involved in ubiquinone biosynthesis. UbiD decarboxylases are classified under the InterPro protein family IPR002830.

[0085] Therefore, in certain embodiments, the prFMN-dependent decarboxylase used in the method according to the invention is a UbiD decarboxylase belonging to InterPro protein family IPR002830.

[0086] Currently, InterPro protein family IPR002830 includes enzymes classified under the enzyme class EC 4.1.1.-, including ferulic acid decarboxylase 1 (Fdcl; EC 4.1.1.102), anhydromevalonate phosphate decarboxylase (EC 4.1.1.126), gallate decarboxylase (EC.4.1.1.59), 4-hydroxybenzoate decarboxylase (EC 4.1.1.61), protocatechuate decarboxylase (EC 4.1.1.63), pyrrole-2-carboxylic acid decarboxylase (HudA; EC 4.1.1.93), and 4-hydroxy-3-polyprenylbenzoate decarboxylase (EC 4.1.1.98).

[0087] Thus, in a particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a UbiD decarboxylase belonging to enzyme class EC 4.1.1.-. In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a ferulic acid decarboxylase 1 (Fdcl) belonging to enzyme class EC 4.1.1.102.In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is an anhydromevalonate phosphate decarboxylase belonging to enzyme class EC 4.1.1.126.

[0088] In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a gallate decarboxylase belonging to enzyme class EC 4.1.1.59. In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a 4-hydroxybenzoate decarboxylase belonging to enzyme class EC 4.1.1.61.

[0089] In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a protocatechuate decarboxylase belonging to enzyme class EC 4.1.1.63.

[0090] In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a pyrrole-2-carboxylic acid decarboxylase HudA belonging to enzyme class EC 4.1.1.93.

[0091] In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a 4-hydroxy-3-polyprenylbenzoate decarboxylase belonging to enzyme class EC 4.1.1.98.

[0092] In certain embodiments, said prFMN-dependent decarboxylase catalysing the carboxylation or decarboxylation of a substrate is a ferulic acid decarboxylase (FDC)-like enzyme. Ferulic acid decarboxylases (FDC)-like enzymes are part of the UbiD decarboxylase family and are classified under the InterPro protein family IPR032903.

[0093] Therefore, in certain embodiments, the prFMN-dependent decarboxylase used in the method according to the invention is an FDC-like enzyme belonging to InterPro protein family IPR032903.

[0094] Currently, InterPro protein family IPR032903 includes enzymes classified under the enzyme class EC 4.1.1.-, including ferulic acid decarboxylase 1 (Fdcl; EC 4.1.1.102) from fungi and bacterial pyrrole-2-carboxylic acid decarboxylase HudA (EC 4.1.1.93).

[0095] Thus, in a particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is an FDC-like enzyme belonging to enzyme class EC 4.1.1.-.In another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a ferulic acid decarboxylase 1 (Fdcl) belonging to enzyme class EC 4.1.1.102.

[0096] In yet another particular embodiment, the prFMN-dependent decarboxylase used in the method according to the invention is a pyrrole-2-carboxylic acid decarboxylase (HudA) belonging to enzyme class EC 4.1.1.93.

[0097] In certain embodiments, the FDC-like enzyme is a fungal Fdcl (EC 4.1.1.102), such as an Fdcl derived from Saccharomyces cerevisiae (Uniprot accession number Q03034), Aspergillus niger (Uniprot accession number A2R0P7) or Candida dubliniensis (Uniprot accession number B9WJ66).

[0098] In certain embodiments, the FDC-like enzyme employed in the method of the present invention is an FDC-like enzyme derived from Saccharomyces cerevisiae (Uniprot accession number Q03034; SEQ ID NO:2), Aspergillus niger (Uniprot accession number A2R0P7; SEQ ID NO:3) or Candida dubliniensis (Uniprot accession number B9WJ66; SEQ ID NO:4) having the amino acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively. In certain embodiments, the FDC-like enzyme is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4 or a sequence which is at least n % identical to any of SEQ ID NOs: 2, 3 and 4 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains decarboxylase activity.

[0099] In certain embodiments, the FDC-like enzyme is a fungal Fdcl (EC 4.1.1.102) from Hypocrea atroviridis (Uniprot accession number G9NLP8).

[0100] In certain embodiments, the FDC-like enzyme employed in the method of the present invention is Fdcl from Hypocrea atroviridis (Uniprot accession number G9NLP8; SEQ ID NO:5) having the amino acid sequence as shown in SEQ ID NO:5. In certain embodiments, the FDC-like enzyme is an enzyme comprising an amino acid sequence as set forth in SEQ ID NO:5 or a sequence which is at least n % identical to SEQ ID NO:5 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains decarboxylase activity.In certain embodiments, the FDC-like enzyme employed in the method of the present invention is an engineered variant of Hypocrea atroviridis Fdcl (UniProt accession number G9NLP8; SEQ ID NO:5). Engineered variants of Hypocrea atroviridis Fdcl having increased catalytic activity have been disclosed in WO 2017 / 191239, which is fully incorporated herein by reference.

[0101] In certain embodiments, the FDC-like enzyme is a pyrrole-2-carboxylic acid decarboxylase (EC 4.1.1.93) from Streptomyces sp. 769 (Uniprot accession number A0A0A8EV26).

[0102] In certain embodiments, the FDC-like enzyme employed in the method of the present invention is a pyrrole-2-carboxylic acid decarboxylase (EC 4.1.1.93) from Streptomyces sp. 769 (Uniprot accession number A0A0A8EV26; SEQ ID NO:6) having the amino acid sequence as shown in SEQ ID NO:6. In certain embodiments, the FDC-like enzyme is an enzyme comprising an amino acid sequence as set forth in SEQ ID NO:6 or a sequence which is at least n % identical to SEQ ID NO:6 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains decarboxylase activity.

[0103] In certain embodiments, the FDC-like enzyme employed in the method of the present invention is an engineered variant of a pyrrole-2-carboxylic acid decarboxylase (EC 4.1.1.93) from Streptomyces sp. 769 (Uniprot accession number A0A0A8EV26; SEQ ID NO:6). Engineered variants of a pyrrole-2-carboxylic acid decarboxylase (EC 4.1.1.93) from Streptomyces sp. 769 having increased catalytic activity have been disclosed in WQ2020007886, which is fully incorporated herein by reference.

[0104] In a preferred embodiment, the FDC-like enzyme employed in the method of the present invention is an FDC-like enzyme derived from Yersinia frederiksenii (UniParc accession number UPI0005DC25B2, RefSeq WP_050108772.1; SEQ ID NO:1). In certain embodiments, the FDC-like enzyme employed in the method of the present invention is an enzyme comprising an amino acid sequence as set forth in SEQ ID NO:1 or a sequence which is at least n % identical to SEQ ID NO:1 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains decarboxylase activity.In certain embodiments, the FDC-like enzyme employed in the method of the present invention is an engineered variant of Yersinia frederiksenii FDC (UniParc accession number UPI0005DC25B2, RefSeq WP_050108772.1; SEQ ID NO:1). Engineered variants of Y. frederiksenii FDC having increased catalytic activity have been disclosed in WO 2022 / 207684, which is fully incorporated herein by reference.

[0105] In certain embodiments, the prFMN-dependent decarboxylase is a 4-hydroxy-3- polyprenylbenzoate decarboxylase (UbiD; EC 4.1.1.98).

[0106] In certain embodiments, the 4-hydroxy-3-polyprenylbenzoate decarboxylase (UbiD) employed in the method of the present invention is a 4-hydroxy-3-polyprenylbenzoate decarboxylase (UbiD) derived from Escherichia coli (Uniprot accession number P0AAB4; SEQ ID NO: 7).

[0107] In certain embodiments, the 4-hydroxy-3-polyprenylbenzoate decarboxylase (UbiD) is an enzyme comprising an amino acid sequence as set forth in SEQ ID NO:7 or a sequence which is at least n % identical to SEQ ID NO:7 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has retains decarboxylase activity.

[0108] In certain embodiments, the prFMN-dependent decarboxylase is a protocatechuate decarboxylase (EC 4.1.1.63).

[0109] Thus, in certain embodiments, the carboxylation or decarboxylation of a substrate is catalysed by a protocatechuate (PCA) decarboxylase (EC 4.1.1.63).

[0110] PCA decarboxylases (also termed AroY) have been described to catalyse the enzymatic conversion of protocatechuate (PCA) into catechol (Johnson et al., Metabolic Engineering Communications, 2016, 3, p.111-119). PCA decarboxylases occur in a variety of organisms and have, e.g., been described in Enterobacter aerogenes, Enterobacter cloacae, Rhodopseudomonas sp. and Sedimentibacter hydroxybenzoicus.

[0111] In certain embodiments, the PCA decarboxylase employed in the method of the present invention is a PCA decarboxylase which is derived from Klebsiella pneumoniae (Uniprot accession number B9A9M6), Leptolyngbya sp. (Uniprot accession number A0A0S3U6D8), or Phascolarctobacterium faecium (Uniprot accession number R6IIV6). In certain embodiments, the PCA decarboxylase employed in the method of the present invention is an enzyme derivedfrom Klebsiella pneumonia (SEQ ID NO:8), Leptolyngbya sp. (SEQ ID NO:9), or Phascolarctobacterium faecium (SEQ ID NO:10). In certain embodiments, the PCA decarboxylase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:8 to 10 or a sequence which is at least n % identical to any of SEQ ID NOs:8 to 10 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains decarboxylase activity.

[0112] In certain embodiments, the prFMN-dependent decarboxylase is a 2,5-furandicarboxylic acid (FDCA) decarboxylase.

[0113] FDCA decarboxylases have been described to catalyse the carboxylation of 2-furoic acid (Payne et al., ACS Catal., 2019, 9(4):2854-2865).

[0114] In certain embodiments, the FDCA decarboxylase employed in the method of the present invention is HmfF derived from Pelotomaculum thermopropionicum (UniProt accession number A5D4Z9; SEQ ID NO:11), or Geobacillus kaustophilus (UniProt accession number Q5QL39; SEQ ID NO:12) or any FDCA decarboxylase cited in WO 2019 / 094740; which is fully incorporated herein by reference.

[0115] In certain embodiments, the FDCA decarboxylase is an enzyme comprising an amino acid sequence selected from SEQ ID NOs:ll or 12 or a sequence which is at least n % identical to SEQ ID NOs:ll or 12 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains decarboxylase activity.

[0116] The flavin

[0117]

[0118] The method according to the invention requires the presence of a prFMN cofactor that has been oxidised with molecular oxygen obtained from the decomposition of H2O2. The precursor of the prFMN cofactor, i.e., the reduced form prFMNFh, is preferably produced by the recombinant microorganism from a suitable precursor, such as FMNH2. To achieve this, it is preferred that the recombinant microorganism used in the method of the invention expresses a flavin prenyltransferase.Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the recombinant microorganism expresses a flavin prenyltransferase.

[0119] The term "flavin prenyltransferase," as used herein, refers to an enzyme that catalyses the addition of a prenyl group to a flavin cofactor, such as reduced flavin mononucleotide (FMNH2). FMN, also known as riboflavin-5'-phosphate, is derived from riboflavin (vitamin B2) by the action of riboflavin kinase and serves as a prosthetic group in various biochemical reactions. FMNH2 is the reduced form of FMN. The prenylation of FMNH2 by flavin prenyltransferase uses DMAP or DMAPP as substrate (depending on the substrate specificity of the flavin prenyltransferase) (Figure 1A) and results in the formation of prFMNFh, the precursor to the catalytically active oxidized form prFMNiminium. This active form is essential for the function of certain enzymes, including decarboxylases of the UbiD-type.

[0120] Flavin prenyltransferases prenylate the flavin ring of FMNH2 into a (modified) prenylated flavin (prFMNFh). In a preferred embodiment, said flavin prenyltransferase is a fungal PAD1 enzyme, or the closely related prokaryotic enzyme UbiX, an enzyme which is involved in ubiquinone biosynthesis in prokaryotes and associated with UbiD decarboxylase, or the closely related prokaryotic enzyme LpdB associated with the gallate decarboxylase LpdC involved in tannin degradation.

[0121] In Escherichia coli, the protein UbiX has been shown to be involved in the third step of ubiquinone biosynthesis in association with UbiD. UbiD catalyses the reaction:

[0122] 3-octaprenyl-4-hydroxybenzoate = 2-octaprenylphenol + CO2.

[0123] Moreover, knockout of the homologous protein in yeast (PAD1) has been shown to confer sensitivity to phenylacrylic acid, showing that this enzyme is involved in phenylacrylic acid decarboxylation in yeast.

[0124] In Lactiplantibacillus plantarum, the protein LpdB has been shown to be associated to the gallate decarboxylase LpdC that catalyses the reactions:

[0125] 3,4,5-trihydroxybenzoate + H+ = 1,2,3-trihydroxybenzene + CO2; and3,4-dihydroxybenzoate + H+ = catechol + CO2

[0126] Non-limiting examples of flavin prenyltransferases of the PAD1, LpdB or UbiX-type will be provided herein below:

[0127] In certain embodiments, the modification of FMNH2 into prFMNH2 is catalysed by the FMN-containing protein phenylacrylic acid decarboxylase (PAD1). The enzymes involved in the modification of FMNH2 into prFMNFh were initially annotated as decarboxylases (EC 4.1.1.-). However, some phenylacrylic acid decarboxylases (PAD1) are now annotated as flavin prenyltransferases as EC 2.5.1.129.

[0128] Accordingly, in certain embodiments, the flavin prenyltransferase used in the method according to the invention is a flavin prenyltransferase belonging to enzyme class EC 2.5.1.129.

[0129] In certain embodiments, the flavin prenyltransferase is a phenylacrylic acid decarboxylase (PADl)-type protein derived from Candida albicans (Uniprot accession number A0A8H6C0A9), Aspergillus niger (Uniprot accession number A3F715), Saccharomyces cerevisiae (Uniprot accession number P33751) or Cryptococcus gattii (Uniprot accession number E6R9Z0) or Hypocrea atroviridis (also termed Trichoderma atroviride; Uniprot accession number G9NTN1).

[0130] In certain embodiments, the phenylacrylic acid decarboxylase (PADl)-type protein employed in the method of the present invention is a phenylacrylic acid decarboxylase (PADl)-type protein derived from Candida albicans (Uniprot accession number A0A8H6C0A9; SEQ ID NO:13), Aspergillus niger (Uniprot accession number A3F715; SEQ ID NO:14), Saccharomyces cerevisiae (Uniprot accession number P33751; SEQ ID NO:15), Cryptococcus gattii (Uniprot accession number E6R9Z0; SEQ ID NO:16) or Hypocrea atroviridis (also termed Trichoderma atroviride; Uniprot accession number G9NTN1; SEQ ID NO:17).

[0131] In a preferred embodiment of the present invention, the phenylacrylic acid decarboxylase (PADl)-type protein is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17 or a sequence which is at least n % identical to any of SEQ ID NOs: 13 to 17 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains the enzymatic activity of modifying FMNH2 into prFMNH2.In certain embodiments, the modification of FMNH2 into prFMNH2 is catalysed by the FMN-containing protein flavin prenyltransferase also termed UbiX (initially annotated EC 4.1.1.-). As mentioned above, enzymes capable of catalysing the enzymatic reaction described herein for flavin prenyltransferases have recently also been annotated as EC 2.5.1.129.

[0132] In certain embodiments, the flavin prenyltransferase which modifies FMNH2 into prFMNH2 is a flavin prenyltransferase (also termed UbiX) derived from Escherichia coli (Uniprot accession number P0AG03), Bacillus subtilis (Uniprot accession, number A0A7U5CMY8), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1).

[0133] In certain embodiments, the flavin prenyltransferase (also termed UbiX) employed in the method of the present invention is derived from Escherichia coli (Uniprot accession number P0AG03; SEQ ID NO:18), Bacillus subtilis (Uniprot accession, number A0A7U5CMY8; SEQ ID NO:19), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8; SEQ ID NO:20) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1; SEQ ID NO:21).

[0134] In certain embodiments, the flavin prenyltransferase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:18 to 21 or a sequence which is at least n % identical to any of SEQ ID NOs:18 to 21 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains the enzymatic activity of modifying FMNH2 into prFMNH2.

[0135] In certain embodiments, the modification of FMNH2 into prFMNH2 is catalysed by an UbiX-like flavin prenyltransferase derived from E. coli encoded by the genes kpdB and ecdB (UniProt accession numbers A0A023LDW3 and P69772; SEQ ID NOs: 22 and 23, respectively), or an UbiX-like flavin prenyl transferase derived from Klebsiella pneumoniae encoded by kpdB (UniProt accession number Q462H4; SEQ ID NO:24).

[0136] In certain embodiments, the UbiX-like flavin prenyltransferase is an enzyme comprising an amino acid sequence of selected from the group consisting of SEQ ID NOs:22 to 24 or a sequence which is at least n % identical to SEQ ID NOs:22 to 24 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains the enzymatic activity of modifying FMNH2 into prFMNH2.

[0137] In certain embodiments, the modification of FMNH2 into prFMNFh is catalysed by an UbiX-like flavin prenyltransferase derived from Lactiplantibacillus plantarum encoded by LpdB (Uniprot accession number F9UT67; SEQ ID NO:25).

[0138] In certain embodiments, the UbiX-like flavin prenyltransferase is an enzyme comprising an amino acid sequence of SEQ ID NO:25 or a sequence which is at least n % identical to SEQ ID NO:25 with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme retains the enzymatic activity of modifying FMNH2 into prFMNH2.

[0139] As regards the determination of sequence identity, the following should apply: When the sequences which are compared do not have the same length, the degree of identity either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. Preferably, it refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence. The degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.

[0140] When using the Clustal analysis method to determine whether a particular sequence is, for instance, at least 60% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

[0141] In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons / alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiplecomparisons / alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.

[0142] Preferably, the degree of identity is calculated over the complete length of the sequence.

[0143] Amino acid residues located at a position corresponding to a position as indicated hereinbelow in the amino acid sequence shown in any one of SEQ ID NOs:l to 31 can be identified by the skilled person by methods known in the art. For example, such amino acid residues can be identified by aligning the sequence in question with the sequence shown in any one of SEQ ID NOs:l to 31 and by identifying the positions which correspond to the above indicated positions of any one of SEQ ID NOs:l to 31. The alignment can be done with means and methods known to the skilled person, e.g. by using a known computer algorithm such as the Lipman-Pearson method (Science, 227 (1985), 1435) or the CLUSTAL algorithm. It is preferred that in such an alignment maximum homology is assigned to conserved amino acid residues present in the amino acid sequences.

[0144] In a preferred embodiment ClustalW2 is used for the comparison of amino acid sequences. In the case of pairwise comparisons / alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. In the case of multiple comparisons / alignments, the following settings are preferably chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.

[0145] Preferably, the degree of identity is calculated over the complete length of the sequence.

[0146] Culture conditions

[0147] Production of a desired product through the catalytic activity of a prFMN-dependent enzyme is achieved by growing a recombinant microorganism expressing the prFMN-dependent enzyme in a culture medium. The term "culture medium" refers to a nutrient-rich solution designed to support the growth and metabolic activity of microorganisms. It typically contains a balanced mixture of essential nutrients, including carbon and nitrogen sources, vitamins, and minerals.The culture medium may be a defined medium, which contains precise amounts of pure chemicals, or a complete medium, which includes complex ingredients such as yeast extract or peptone, or mixtures thereof. The composition of the culture medium can be tailored to meet the specific requirements of the recombinant microorganism, ensuring optimal expression of the decarboxylase and efficient production of the desired product. The skilled person is aware that the composition of the culture medium depends on the type of microorganism.

[0148] The term "grown," as used herein, refers to the process of cultivating the recombinant microorganism in a controlled environment, typically within a culture medium, to promote cell division, metabolic activity, and the expression of gene products. This process involves maintaining appropriate conditions such as temperature, pH, and nutrient availability to ensure optimal growth and productivity of the microorganism. The growth phase typically continues until the recombinant microorganism reaches a sufficient cell density or until the desired product is produced in adequate quantities.

[0149] The culture medium may be supplemented with various compounds that are essential or beneficial for the production of the target product.

[0150] Specifically, it is required to add a suitable precursor to the culture medium that can be converted into the desired product involving the catalytic activity of the prFMN-dependent enzyme. This precursor may be a commonly available substrate, such as glucose, which can be transformed into the target product via a series of enzymatic reactions. Alternatively, the culture medium may be supplemented with more specialized precursor molecules that can be converted into the desired product through one or only a few enzymatic reactions, with at least one of these reactions being catalysed by a prFMN-dependent enzyme.

[0151] In certain embodiments, the culture medium is further supplemented with FMN or its precursors (i.e. : riboflavin) at an appropriate concentration to provide sufficient substrate for the flavin prenyltransferase, which converts FMNH2 into the prFMNH2 precursor. Specifically, the culture medium may be supplemented with FMN in a range of about 0.1 to about 50 mg / L, with a preferred concentration of about 1 to about 10 mg / L. This supplementation ensuresthat there is an adequate supply of FMN to support the efficient production of the prFMNH2 precursor, thereby facilitating the optimal activity of the prFMN-dependent enzyme.

[0152] The recombinant organism is grown in the culture medium in the absence of air, meaning that the cultivation is conducted without introducing atmospheric air into the culture vessel. Instead, the molecular oxygen (O2) required for the oxidation of the prFMN cofactor is derived from the controlled decomposition of hydrogen peroxide (H2O2). This method ensures that the necessary oxygen is provided without the introduction of atmospheric gases, which may dilute and / or contaminate gaseous products.

[0153] To displace any residual air in the culture medium, the culture vessel may be initially purged with an inert gas, such as nitrogen, carbon dioxide or argon. This inert gas may also be continuously or intermittently provided during cultivation to ensure that the environment remains free of atmospheric gases. This approach allows for precise control over oxygen levels, supporting the optimal activity of the recombinant organism and its metabolic pathways, thereby ensuring efficient production of the desired product.

[0154] In another embodiment, the method of the invention includes cultivating the recombinant microorganism in a fermenter, also known as a bioreactor, under conditions that allow for the expression of the respective proteins. This method further comprises the step of effecting an enzymatic conversion as described herein. Suitable fermenters or bioreactor devices and fermentation conditions are well-known to those skilled in the art. A bioreactor or fermenter refers to any manufactured or engineered device or system that supports a biologically active environment, and it serves as the vessel in which the method of the present invention is carried out. The process within a bioreactor or fermenter can be either aerobic or anaerobic; however, it is preferred herein that the process is anaerobic, meaning it occurs in the absence of atmospheric air, with molecular oxygen required for the oxidation of the prFMN cofactor being supplied in the form of hydrogen peroxide. These bioreactors are typically cylindrical, ranging in size from litres to thousands of cubic meters, and are often constructed from stainless steel. The fermenter or bioreactor can be designed to cultivate organisms, preferably microorganisms, in various culture methods such as batch culture, fed-batch culture, perfusion culture, or chemostat culture, all of which are well-established in the art.The recombinant microorganism

[0155] The method of the invention requires the expression of a prFMN-dependent enzyme, which is involved in the production of a desired product, in a recombinant microorganism.

[0156] The recombinant microorganism may be derived from any microorganism that can be cultured in a culture medium, preferably in the absence of air. The term "recombinant microorganism," as used herein, refers to a microorganism that has been genetically engineered to contain and express foreign or endogenous DNA. This foreign or endogenous DNA can be introduced through various molecular biology techniques, such as transformation, transduction, or conjugation. The recombinant microorganism is designed to produce specific proteins, enzymes, or other molecules that are not naturally present in the organism in the desired growth conditions.

[0157] In certain embodiments, the recombinant microorganism is derived from a bacterium. Bacteria are commonly used in biotechnological applications for the production of target compounds due to their rapid growth rates, ease of genetic manipulation, and ability to express a wide range of proteins and enzymes. These microorganisms can be cultured in large-scale bioreactors, making them ideal for industrial processes that require the production of significant quantities of biochemicals, pharmaceuticals, or enzymes. Bacteria such as Escherichia coli and Bacillus subtilis are frequently employed as host organisms in recombinant DNA technology because they are well-characterized, have established genetic tools, and can be engineered to produce a variety of products. The genetic engineering of these bacteria allows for the introduction of foreign genes that encode for specific proteins or enzymes, enabling the synthesis of complex molecules that may be difficult or costly to produce through traditional chemical synthesis. Moreover, recombinant bacteria can be tailored to operate under specific conditions, such as anaerobic environments, which can be advantageous for certain bioprocesses.

[0158] In certain embodiments, the recombinant microorganism is a recombinant Escherichia coli (E. coli). E. coli is a well-studied and widely used bacterium in biotechnology due to its well-documented genetic makeup and the availability of various genetic manipulation tools. E. coli's rapid growth and ability to reach high cell densities make it ideal for large-scale production processes. It can be engineered to express high levels of recombinant proteins. Additionally, E. coli can utilize various carbon sources, including inexpensive and renewable feedstocks, reducing production costs and enhancing sustainability. Its versatility allows for tailored bioprocesses to meet specific production requirements, such as high-yield production of single or multiple target compounds. Notably, E. coli can also grow under anaerobic conditions, which is advantageous for the method of the present invention.

[0159] In certain embodiments, the recombinant microorganism is a recombinant Clostridium species, a group of bacteria known for their ability to thrive in anaerobic environments, making them particularly suitable for processes requiring the absence of air. Clostridium species are gaining attention in biotechnology due to their unique metabolic pathways and ability to produce a variety of valuable compounds, including solvents, biofuels, and enzymes. These bacteria can be genetically engineered to enhance their natural capabilities or to introduce new functionalities, allowing for the efficient production of target compounds. Clostridium species can utilize a wide range of substrates, including lignocellulosic biomass and other renewable feedstocks, which contributes to cost-effective and sustainable bioprocesses. Their ability to perform complex fermentations and produce high yields of desired products makes them an attractive choice for industrial applications. Among the most important Clostridium species in biotechnology are C. acetobutylicum for solvent production, C. thermocellum for cellulose degradation and biofuel production, and C. Ijungdahlii and C. autoethanogenum for converting syngas into ethanol and chemicals.

[0160] In certain embodiments, the recombinant microorganism is derived from yeast. Yeasts are eukaryotic microorganisms that are extensively used in biotechnological applications due to their ability to perform complex post-translational modifications, which are essential for the production of functional eukaryotic proteins. They are favoured for their ease of cultivation, robustness in large-scale fermentation processes, and the availability of advanced genetic tools for manipulation. Yeasts can be engineered to produce a wide range of biochemicals, pharmaceuticals, and enzymes, making them suitable for industrial applications that require the synthesis of complex molecules. Their ability to grow in both aerobic and anaerobicconditions adds to their versatility, allowing for tailored bioprocesses that can optimize production yields and reduce costs.

[0161] In certain embodiments, the recombinant microorganism is a recombinant Saccharomyces cerevisiae (5. cerevisiae). S. cerevisiae, commonly known as baker's yeast, is one of the most well-characterized and widely used yeast species in biotechnology. Its genetic makeup is thoroughly documented, and a variety of genetic engineering tools are available to manipulate its genome with precision. 5. cerevisiae is renowned for its ability to produce high levels of recombinant proteins with complex post-translational modifications, making it an ideal host for the production of therapeutic proteins and other biologically active compounds. Additionally, 5. cerevisiae can utilize a variety of carbon sources, including renewable feedstocks, which enhances the sustainability and cost-effectiveness of bioprocesses. Its robustness and adaptability to different fermentation conditions, including both aerobic and anaerobic environments, make it a versatile organism for diverse industrial applications.

[0162] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the recombinant microorganism is a bacterium, in particular Escherichia coli, or a Clostridium species, or a fungus, in particular wherein the fungus is a yeast, such as Saccharomyces cerevisiae.

[0163] While it is preferred that the recombinant microorganism used in the method of the invention is derived from a bacterium or yeast, it may also be derived from an archaeal species, a nonyeast fungus, or another microbial cell.

[0164] The recombinant microorganism for use in the method of the invention preferably has been engineered to express a prFMN-dependent enzyme, as described in more detail elsewhere herein. The genetic engineering process typically involves the insertion of a gene encoding the prFMN-dependent enzyme into the host microorganism's genome or the use of a plasmid or vector to express the gene in the host microorganism. This process is often accompanied by the necessary regulatory elements to ensure proper expression and activity of the enzyme, including promoters, terminators, and other genetic elements that optimize transcription and translation within the host organism.Additionally, the recombinant microorganism may be further modified to express cofactors or partner enzymes, such as flavin prenyltransferases, which provide the prenylated flavin mononucleotide (prFMN) cofactor essential for enzymatic activity.

[0165] In certain embodiments, the recombinant microorganism employed in the method of the invention may be further genetically engineered to express a catalase enzyme to facilitate the decomposition of hydrogen peroxide, such as any of the catalases disclosed herein.

[0166] In the context of the present invention the term "recombinant" means that the microorganism is genetically modified so as to contain a nucleic acid molecule encoding an enzyme as defined above as compared to a wild-type or non-modified microorganism. A nucleic acid molecule encoding an enzyme / transporter as defined above can be used alone or as part of a vector.

[0167] The nucleic acid molecules can further comprise expression control sequences operably linked to the polynucleotide comprised in the nucleic acid molecule. The term "operatively linked" or "operably linked", as used throughout the present description, refers to a linkage between one or more expression control sequences and the coding region in the polynucleotide to be expressed in such a way that expression is achieved under conditions compatible with the expression control sequence.

[0168] Expression comprises transcription of the heterologous DNA sequence, preferably into a translatable mRNA. Regulatory elements ensuring expression in fungi as well as in bacteria, are well known to those skilled in the art. They encompass promoters, enhancers, termination signals, targeting signals and the like. Examples are given further below in connection with explanations concerning vectors.

[0169] Promoters for use in connection with the nucleic acid molecule may be homologous or heterologous with regard to its origin and / or with regard to the gene to be expressed. Suitable promoters are for instance promoters which lend themselves to constitutive expression. However, promoters which are only activated at a point in time determined by externalinfluences can also be used. Artificial and / or chemically inducible promoters may be used in this context.

[0170] The vectors can further comprise expression control sequences operably linked to said polynucleotides contained in the vectors. These expression control sequences may be suited to ensure transcription and synthesis of a translatable RNA in bacteria or fungi.

[0171] In addition, it is possible to insert different mutations into the polynucleotides by methods usual in molecular biology (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), leading to the synthesis of polypeptides possibly having modified biological properties. The introduction of point mutations is conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide.

[0172] Moreover, mutants possessing a modified substrate or product specificity can be prepared. Preferably, such mutants show an increased activity. Alternatively, mutants can be prepared the catalytic activity of which is abolished without losing substrate binding activity.

[0173] Furthermore, the introduction of mutations into the polynucleotides encoding an enzyme as defined above allows the gene expression rate and / or the activity of the enzymes encoded by said polynucleotides to be reduced or increased.

[0174] For genetically modifying bacteria or fungi, the polynucleotides encoding an enzyme as defined above or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primerrepair", restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.

[0175] Thus, in accordance with the present invention a recombinant microorganism can be produced by genetically modifying fungi or bacteria comprising introducing the abovedescribed polynucleotides, nucleic acid molecules or vectors into a fungus or bacterium.

[0176] The nucleic acid encoding the respective enzyme is expressed so as to lead to the production of a polypeptide having any of the activities described above. An overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440). An overview of yeast expression systems is for instance given by Hensing et al. (Antonie van Leuwenhoek 67 (1995), 261-279), Bussineau et al. (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991), 742-745) and Buckholz (Bio / Technology 9 (1991), 1067-1072).

[0177] Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter or sometimes a fungal promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence. The DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) aresufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), Ipl, rac (Boros et al., Gene 42 (1986), 97-100). Suitable fungal promoters, including constitutive and inducible promoters, as well as terminators and other parts have been disclosed, without limitation, by Lee et al., (ACS Synth Biol, 2015, 18;4(9):975-86).

[0178] The expression vector may comprise an inducible promotor or a constitutive promoter. The term "inducible promoter" refers to a regulatory DNA sequence that initiates gene transcription in response to specific environmental or chemical stimuli, allowing controlled expression of the gene. In contrast, the term "constitutive promoter" refers to a regulatory DNA sequence that continuously drives gene transcription at a constant rate, regardless of external conditions.

[0179] Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters. In order to obtain an optimum amount of polypeptide, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-R-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

[0180] The transformation of the recombinant microorganism with a polynucleotide or vector as described above can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular recombinant microorganism used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.Products of a

[0181]

[0182] reaction

[0183]

[0184] prFMN-dependent decarboxylases may be used in the production of a wide range of products. In certain embodiments, the product may be the result of a decarboxylation reaction catalysed by the prFMN-dependent decarboxylase. Products that may be obtained in a prFMN-dependent decarboxylase catalysed decarboxylation reaction include, without limitation, isobutene, styrene, butadiene, 1,3-pentadiene, hepta-l,3,5-triene and 4-vinylguiacol.

[0185] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the product is formed by a decarboxylation reaction catalysed by the prFMN-dependent decarboxylase, preferably wherein the product is selected from the group consisting of: isobutene, styrene, butadiene, 1,3-pentadiene, hepta-l,3,5-triene and 4-vinylguiacol.

[0186] Isobutene

[0187] In a particular embodiment, the invention relates to the method according to the invention, wherein the product is isobutene.

[0188] The production of isobutene using a prFMN-dependent decarboxylase has been described in the art in great detail, for example in WO 2022 / 136207, WO 2022 / 207684, WO 2020 / 188033, WO 2020 / 007886, WO 2018 / 206262 and WO 2017 / 085167, which are all incorporated herein by reference.

[0189] Isobutene, also known as isobutylene, is a valuable hydrocarbon used as a building block in the production of various chemicals and materials, including synthetic rubber, plastics, fuel additives like methyl tert-butyl ether (MTBE), road fuel like isooctane, and aviation fuels like isododecane and isohexadecane. It can be produced biologically through the decarboxylation of 3-methylcrotonic acid, a process catalysed by prFMN-dependent decarboxylases. This enzymatic reaction converts 3-methylcrotonic acid into isobutene, offering a sustainable alternative to traditional petrochemical methods of isobutene production.Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein isobutene is produced from 3-methylcrotonic acid.

[0190] Conversion of 3-methylcrotonic acid into isobutene may be catalysed by any suitable prFMN-dependent decarboxylases disclosed herein. A prFMN-dependent decarboxylase having affinity for the substrate 3-methylcrotonic acid may also be referred to as a prFMN-dependent decarboxylase having 3-methylcrotonic acid decarboxylase activity. Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the prFMN-dependent decarboxylase has 3-methylcrotonic acid decarboxylase activity.

[0191] The term "3-methylcrotonic acid decarboxylase (MDC)" refers to an enzyme which can catalyze the decarboxylation of 3-methylcrotonic acid into isobutene. A decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide. In a preferred embodiment, the MDC is a Ferulic Acid Decarboxylase (FDC) or is derived from such an enzyme. Any enzyme capable of converting 3-methylcrotonic acid into isobutene is considered to possess 3-methylcrotonic acid decarboxylase activity. FDC-like proteins comprising 3-methylcrotonic acid decarboxylase activity have been disclosed, inter alia, in WO 2017 / 191239, WO 2020 / 007886 and WO 2022 / 207684, which are incorporated herein by reference.

[0192] Whenever reference is made to 3-methylcrotonic acid, both, its deprotonated carboxylate ion form (i.e., the COO-form, namely, 3-methylcrotonate) as well as its protonated acidic form (i.e., the COOH form, namely 3-methylcrotonic acid) are meant. In fact, its form depends on the pH of the solution and, accordingly, the definition of 3-methylcrotonic acid interchangeably denotes either form, i.e., 3-methylcrotonate as well as 3-methylcrotonic acid. When present as salt form, namely as 3-methylcrotonate, the preferred cation is Na+, Ca2+, Mg2+, K+or NH4+.

[0193] It is preferred herein that the conversion of 3-methylcrotonic acid into isobutene is catalysed by a ferulic acid decarboxylases (FDC) derived from Yersinia frederiksenii (UniParc accessionnumber UPI0005DC25B2, RefSeq WP_050108772.1; SEQ ID NO:1) or an engineered variant thereof, as disclosed elsewhere herein.

[0194] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the prFMN-dependent decarboxylase having 3-methylcrotonic acid decarboxylase activity comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0195] To produce isobutene from 3-methylcrotonic acid using a prFMN-dependent decarboxylase, the substrate 3-methylcrotonic acid may be added to the culture medium at a suitable concentration. In certain embodiments, 3-methylcrotonic acid may be added to the culture medium at a concentration ranging from about 0.1 to about 20 g / L, preferably about 0.5 to about 5 g / L. In certain embodiments, 3-methylcrotonic acid may be added to the culture medium when the cells in the culture medium have reached exponential growth phase.

[0196] Alternatively, the 3-methylcrotonic acid used as a substrate for the production of isobutene by a prFMN-dependent decarboxylase may be produced by the recombinant microorganism itself. Metabolic routes towards 3-methylcrotonic acid have been described in the art. For example, 3-methylcrotonic acid (also referred to as 3,3-dimethyl acrylic acid) may be produced from acetyl-CoA using the following enzymatic route:

[0197] (i) conversion of acetyl-CoA into acetoacetyl-CoA,

[0198] (ii) conversion of the resulting acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl- CoA,

[0199] (iii) conversion of the resulting 3-hydroxy-3-methylglutaryl-CoA into 3- methylglutaconyl-CoA,

[0200] (iv) conversion of the resulting 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA, and

[0201] (v) conversion of the resulting 3-methylcrotonyl-CoA into 3-methylcrotonic acid.The conversion of acetyl-CoA into 3-methylcrotonic acid is detailed more comprehensively in WO 2022 / 136207 and WO 2020 / 188033, both of which are fully incorporated herein by reference.

[0202] The enzymatic conversion of acetyl-CoA into acetoacetyl-CoA

[0203] 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 2) and then to further condense said malonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV as shown in Figure 2). 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 2).

[0204] 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 2). This naturally occurring reaction fixes CO2 on acetyl-CoA utilizing ATP resulting in malonyl-CoA.

[0205] 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 2). This is a natural occurring reaction and condenses malonyl-CoA and acetyl-CoA in a decarboxylation reaction.

[0206] 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 2).

[0207] The enzymatic conversion of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoAThe 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 2).

[0208] 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 catalyse 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 catalyses 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.

[0209] The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA

[0210] The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA is an enzymatic dehydration reaction which occurs naturally, and which is catalysed, 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 2).

[0211] 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.-.

[0212] The enzymatic conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA

[0213] The conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA may be catalysed 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 2).

[0214] In another preferred embodiment the conversion of 3-methylglutaconyl-CoA via decarboxylation into 3-methylcrotonyl-CoA is catalysed by a 3-methylglutaconyl-CoA decarboxylase, e.g. a 3-methylglutaconyl-CoA decarboxylase of Myxococcus xan th us encoded by the genes MXAN_4264 and MXAN_4265. These genes code for an enzyme having the two subunits AibA and AibB (Li et al., Angew. Chem. Int. Ed. 52 (2013), 1304-1308).

[0215] The enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid

[0216] The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic 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 2 (step Via, step Vlb or step Vic as shown in Figure 2).

[0217] Thus, the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid may be achieved by

[0218] (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 2);

[0219] (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 2); or

[0220] (c) two enzymatic steps comprising

[0221] (i) first enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and

[0222] (ii) then enzymatically converting the thus obtained 3-methylcrotonyl phosphate into said 3-methylcrotonic acid (step Vic as shown in Figure 2).

[0223] 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.

[0224] 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).

[0225] 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) ora 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.

[0226] Preferably, in one embodiment, 3-methylcrotonyl-CoA is directly converted into 3-methylcrotonic acid by hydrolysing 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 2.

[0227] 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 hydrolyse the thioester bond between a carbonyl group and a sulphur atom.

[0228] 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:

[0229] - acetyl-CoA hydrolase (EC 3.1.2.1);

[0230] - palmitoyl-CoA hydrolase (EC 3.1.2.2);

[0231] - 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4);

[0232] - oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);

[0233] - ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);

[0234] - ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19);

[0235] - l,4-dihydroxy-2-naphthoyl-CoA hydrolase (EC 3.1.2.28); and

[0236] - acyl-CoA hydrolase (EC 3.1.2.20).

[0237] 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-dependentshort-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).

[0238] 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 2).

[0239] 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).

[0240] 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:

[0241] - propionate:acetate-CoA transferase (EC 2.8.3.1);

[0242] - acetate CoA-transferase (EC 2.8.3.8); and

[0243] - butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

[0244] 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).

[0245] In certain embodiments, methods may be utilized wherein the yield, pool and / orflux of acetyl-CoA in the recombinant microorganism is increased. Corresponding methods as well as recombinant organisms and microorganisms having an increased pool of acetyl-CoA are described in the prior art, e.g., in W02013 / 007786, W02020 / 021051 and W02020 / 188033, the content of which is hereby incorporated by reference.In preferred embodiments, the yield, pool and / or flux of acetyl-CoA in the recombinant microorganism is increased by utilizing a recombinant organism or microorganism having a phosphoketolase (PKT) activity as described in, e.g., W02013 / 007786, W02020 / 021051 and W02020 / 188033, the content of which is hereby incorporated by reference.

[0246] Styrene

[0247] Another product that may be obtained in a decarboxylation reaction catalysed by a prFMN-dependent decarboxylase is styrene. Styrene is an important organic compound used extensively in the production of polystyrene plastics and resins. It serves as a precursor for various synthetic materials, including rubber, insulation, fiberglass, pipes, automobile and boat parts, and food containers. The ability to produce styrene through a biological pathway involving a prFMN-dependent decarboxylase offers a sustainable and environmentally friendly alternative to traditional petrochemical methods.

[0248] It was described previously that styrene can be produced with a prFMN-dependent decarboxylase using trans-cinnamic acid as the substrate. In particular, a ferulic acid decarboxylase (FDC) from Aspergillus niger (Uniprot accession number A2R0P7; SEQ ID NO:3) has been previously used for the enzymatic production of styrene from trans-cinnamic acid (see Messiha et al., ChemCatChem, 2023, 15, e202201102). Another prFMN-dependent decarboxylase that has been disclosed for the conversion of trans-cinnamic acid to styrene is the FDC from Capronia coronata (CcFDC; Uniprot accession number W9YNA8) (see Benfeldt et al., D0l:10.26434 / chemrxiv-2024-0npph). Moreover, Garcia-Franco et al. have reported the in silica design of a functional prFMN-dependent trans-cinnamic acid decarboxylase in Pseudomonas (Microb Cell Fact, 2024, 23, 69).

[0249] The production of styrene may be initiated by adding the substrate trans-cinnamic acid to the culture medium. However, trans-cinnamic acid may also be produced by the recombinant microorganism from the precursor L-phenylalanine using the enzyme phenylalanine ammonia-lyase (PAL) (see, e.g., Messiha et al., ChemCatChem, 2023, 15, e202201102; Noda et al., BioTech, 2024, 13, 2; and WO 2023 / 168315).Butadiene

[0250] Another product that may be obtained in a decarboxylation reaction catalysed by a prFMN-dependent decarboxylase is butadiene (1,3-butadiene). Butadiene is a valuable industrial raw material used primarily in the production of synthetic rubber, such as styrene-butadiene rubber (SBR) and polybutadiene rubber, as well as engineering plastics like acrylonitrile-butadiene-styrene (ABS) resin.

[0251] The synthesis of 1,3-butadiene using prFMN-dependent decarboxylases involves two key decarboxylation steps catalysed by prFMN-dependent enzymes. First, cis,cis-muconic acid (ccMA) is decarboxylated to form pentadienoic acid (PA). Subsequently, PA undergoes a second decarboxylation, also catalysed by the prFMN-dependent decarboxylase, to produce 1,3-butadiene.

[0252] These decarboxylation reactions are facilitated by the prFMN cofactor, which is essential for the catalytic activity of the decarboxylase. Mori et al. (Nat Commun, 2021;12(l):2195) disclose optimized FDC variants from Aspergillus niger (AnFDC) and Saccharomyces cerevisiae (ScFDC) that can be used in the formation of 1,3-butadiene.

[0253] The precursor cis,cis-muconic acid (ccMA) may be produced enzymatically by the recombinant microorganism through a series of metabolic engineering strategies. By introducing and optimizing specific biosynthetic pathways, ccMA may be synthesized from renewable carbon sources such as glucose. This process typically involves the expression of key enzymes that convert glucose into intermediate compounds, which are subsequently transformed into ccMA. For instance, enzymes such as 3-dehydroshikimic acid dehydratase, protocatechuic acid decarboxylase, and catechol dioxygenase may be employed to facilitate the conversion of glucose-derived intermediates into ccMA (see Mori et al., Nat Commun, 2021;12(l):2195).

[0254] Hepta-l,3,5-triene

[0255] Another product that may be obtained in a decarboxylation reaction catalysed by a prFMN-dependent decarboxylase is hepta-l,3,5-triene. Hepta-l,3,5-triene has valuable applicationsin the pharmaceutical and polymer industries, serving as a building block for various chemical syntheses.

[0256] Hepta-l,3,5-triene can be produced using a prFMN-dependent decarboxylase through the decarboxylation of 2,4,6-octatrienoic acid. This reaction may be catalysed using a fungal ferulic acid decarboxylase (Fdcl) derived from Aspergillus niger, with the assistance of the E. coli UbiX enzyme, which provides the essential prenylated flavin mononucleotide (prFMN) cofactor (see Messiha et al., ACS Synth Biol, 2021, 10(2):228-235). The decarboxylation reaction facilitated by Fdcl removes a carboxyl group from 2,4,6-octatrienoic acid, converting it into hepta-l,3,5-triene.

[0257] The precursor 2,4,6-octatrienoic acid can be obtained through a bioengineered metabolic pathway involving polyketide synthase (PKS) systems. This process begins with the deconstruction and repurposing of the andrimid biosynthesis pathway, typically found in certain bacteria such as Pantoea agglomerans. By employing a type II PKS, the pathway is engineered to produce 2,4,6-octatrienoic acid as a metabolic intermediate. The PKS system involves a series of enzymatic steps, including chain initiation and elongation, facilitated by ketosynthase-like condensing enzymes and an acyl carrier protein (ACP) that covalently tethers the growing chain. Extender units are recruited by a malonyl-CoA:ACP transferase, which can be shared between fatty acid synthases and PKS (see Messiha et al., ACS Synth Biol, 2021, 10(2):228-235).

[0258]

[0259] Another product that may be obtained in a decarboxylation reaction catalysed by a prFMN- dependent decarboxylase is 4-vinylguiacol. 4-vinylguaiacol (VG) is a known flavour and fragrance compound which is generally regarded as safe. VG and other aroma compounds (guaiacol, vanillin) of natural origin are of great interest in the fragrance industry.

[0260] 4-Vinylguaiacol can be produced using a prFMN-dependent decarboxylase through the decarboxylation of ferulic acid. The reaction may be catalysed by a ferulic acid decarboxylase(FDC), such as the variants from Schizophyllum commune (ScoFDC; Detering et al., PLoS One, 2020, 15(12):e0244290) or Bacillus pumilis (US 2007 / 0224668).

[0261] 1,3-pentadiene

[0262] Another product that may be obtained in a decarboxylation reaction catalysed by a prFMN-dependent decarboxylase is 1,3-pentadiene. 1,3-pentadiene is a volatile hydrocarbon with two conjugated double bonds, making it a valuable intermediate in the synthesis of polymers and other industrial chemicals.

[0263] 1,3-pentadiene can be produced using a prFMN-dependent decarboxylase through the decarboxylation of sorbic acid. Aspergillus niger ferulic acid decarboxylase (AnFDC) and Saccharomyces cerevisiae ferulic acid decarboxylase (ScFDC) have been demonstrated to effectively catalyse this reaction (Plumridge et al., Applied and Environmental Microbiology, 2008, 74(2):550-2; and Aleku et al.; ChemCatChem, 2018, 10(17):3736-3745)

[0264] Products of a ca

[0265]

[0266] reaction

[0267]

[0268] In certain embodiments, the product may be the result of a carboxylation reaction catalysed by a prFMN-dependent decarboxylase. Products that may be obtained in a prFMN-dependent decarboxylase catalysed carboxylation reaction include, without limitation, 2,5-furandicarboxylic acid (FDCA) and 2-naphthoic acid.

[0269] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the product is formed by a carboxylation reaction catalysed by the prFMN-dependent decarboxylase, preferably wherein the product is selected from the group consisting of: 2,5-furandicarboxylic acid (FDCA) and 2-naphthoic acid.

[0270] 2,5-Furandica

[0271]

[0272] ic acid

[0273]

[0274] 2,5-Furandicarboxylic acid (FDCA) is a significant bio-based chemical that has garnered substantial industrial interest as a renewable alternative to terephthalic acid (TPA) in theproduction of bioplastics. FDCA's importance stems from its ability to serve as a building block for the synthesis of polyethylene furanoate (PEF), a polyester that exhibits superior mechanical, thermal, and barrier properties compared to traditional polyethylene terephthalate (PET). The rigid furan ring structure of FDCA, adorned with two carboxyl groups, enables the formation of strong and durable polymers through polycondensation reactions.

[0275] FDCA can be produced using a prFMN-dependent decarboxylase through the enzymatic carboxylation of 2-furoic acid. In this process, a prFMN-dependent decarboxylase enzyme catalyses the addition of a carboxyl group to 2-furoic acid, converting it into 2,5-furandicarboxylic acid (FDCA). This reaction is facilitated by the presence of a prenylated flavin mononucleotide (prFMN) cofactor, which is essential for the enzyme's catalytic activity. The prFMN cofactor is synthesized by the UbiX enzyme, which works in conjunction with the decarboxylase to enable the carboxylation process. By expressing these enzymes in a microbial host, such as E. coli, and providing a source of carbon dioxide, the prFMN-dependent decarboxylase can efficiently convert 2-furoic acid into FDCA.

[0276] In previous approaches, a prFMN-dependent decarboxylase derived from P. thermopropionicum (PtHmfF) has been utilized for the production of FDCA (see Lopez-Lorenzo et al., ChemCatChem, 2023, 15, e202201483).

[0277] The precursor 2-furoic acid may be obtained enzymatically through the conversion of 2-butyl furoate, a compound derived from the biorefinery process of hemicellulose in the xylonic acid pathway (see Lopez-Lorenzo et al., ChemCatChem, 2023, 15, e202201483).

[0278] 2-naphtoic acid

[0279] Another product that may be obtained in a carboxylation reaction catalysed by a prFMN-dependent decarboxylase is 2-naphtoic acid. 2-naphthoic acid is an important chemical compound with a variety of industrial and research applications. It serves as a key intermediate in the synthesis of dyes, pigments, and pharmaceuticals. In the dye industry, 2-naphthoic acid is used to produce azo dyes, which are widely employed in textiles, printing, and other applications due to their vibrant colours and stability. In pharmaceuticals, it is aprecursor for the synthesis of various therapeutic agents, including anti-inflammatory and antimicrobial drugs. Additionally, 2-naphthoic acid is utilized in organic synthesis as a building block for more complex molecules, making it a valuable compound in chemical research and development. Its significance extends to environmental science, where it is studied as a model compound for understanding the degradation of polycyclic aromatic hydrocarbons (PAHs), which are persistent environmental pollutants.

[0280] 2-naphthoic acid can be produced through the carboxylation of naphthalene, facilitated by prFMN-dependent decarboxylases. The carboxylation mechanism involves the prFMN cofactor, which enables the reaction through a 1,3-dipolar cycloaddition process.

[0281] Aleku et al. (Nat Chem Biol. 2020, 16(ll):1255-1260) have demonstrated the suitability of an Aspergillus niger FDC (AnFDC) to catalyse the formation of 2-naphthoic acid from naphthalene. As in the case of FDCA, the synthesis of 2-naphthoic acid using a prFMN-dependent decarboxylase is performed in the presence of carbon dioxide, or a precursor thereof, such as bicarbonates.

[0282] It is to be understood that the products mentioned above are merely non-limiting examples of the various products that can be produced using the method of the invention. Furthermore, the product generated by the method of the invention does not necessarily have to be the direct product of the prFMN-dependent decarboxylase. Instead, the prFMN-dependent decarboxylase may produce an intermediate, which is subsequently converted into the final product through additional steps.

[0283] The method according to the present invention may further comprise a step of recovering the product produced by the method. When the product is a gaseous product, such as isobutene, it may be recovered from the fermentation off-gas by methods known to those skilled in the art. When the product is a liquid product, it may be recovered from the fermentation broth using techniques such as centrifugation, filtration, solvent extraction, or distillation. These recovery methods ensure the efficient separation and purification of the desired products from the fermentation medium, facilitating their subsequent use or commercialization.Further of the invention

[0284] In another aspect, the invention relates to a method of oxidizing prFMNFh into a prenylated flavin mononucleotide cofactor of a decarboxylase, wherein the molecular oxygen (O2) for said oxidation reaction is derived from decomposition of hydrogen peroxide (H2O2), preferably wherein the decomposition of H2O2 is catalysed by a catalase.

[0285] In another aspect, the invention relates to a recombinant microorganism comprising one or more nucleic acids encoding:

[0286] a) a prFMN-dependent enzyme;

[0287] b) a flavin prenyltransferase; and

[0288] c) a catalase.

[0289] That is, the present invention further relates to a recombinant microorganism that can be used in the method according to the invention.

[0290] The recombinant microorganism of the invention comprises a nucleic acid encoding a prFMN-dependent enzyme. Preferably, this prFMN-dependent enzyme is a prFMN-dependent decarboxylase that catalyses at least one carboxylation or decarboxylation step involved in the production of a desired product when expressed in the recombinant microorganism.

[0291] Additionally, the recombinant microorganism comprises a nucleic acid encoding a flavin prenyltransferase. The expression of this flavin prenyltransferase in the recombinant microorganism ensures the production of sufficient levels of the prFMNFh precursor, which is essential for the formation of the prFMN cofactor. This, in turn, is necessary for the optimal functionality of the prFMN-dependent enzyme.

[0292] In a particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein the flavin prenyltransferase comprises an amino acid sequence as set forth in SEQ ID NO:15 or an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity to SEQ ID NO:15, wherein the enzyme retains the enzymatic activity of modifying FMNH2 into prFMNH2.Furthermore, the recombinant microorganism comprises a nucleic acid encoding a catalase. The expression of catalase in the recombinant microorganism enables it to generate molecular oxygen from hydrogen peroxide, which can be used for the oxidation of the prFMN cofactor in the absence of air. In particular, the catalase, may be a heme-containing catalase or a manganese-containing catalase, as described herein above.

[0293] As regards the above-mentioned enzymes as well as preferred embodiments of said enzymes, the same applies as has been set forth above for the methods according to the present invention.

[0294] Moreover, the recombinant microorganism may comprise additional genetic modifications required for the production of a specific product. These modifications may include the introduction of genes encoding enzymes that are directly involved in the biosynthetic pathway of the target product, thereby enhancing the overall efficiency and yield of the production process. In addition to enzymes, other genetic modifications may be implemented to improve the microorganism's metabolic capabilities, stress tolerance, and overall robustness. For example, genes that enhance the uptake and utilization of specific substrates can be introduced, allowing the microorganism to efficiently convert available resources into the target product. Similarly, genes that confer resistance to toxic by-products or environmental stresses can be incorporated to ensure stable and high-level production under industrial conditions. Furthermore, regulatory elements such as promoters, terminators, and ribosome binding sites can be optimized to achieve precise control over the expression levels of the introduced genes, balancing the metabolic load and preventing bottlenecks. Overall, these additional genetic modifications enable the recombinant microorganism to be highly specialized and efficient in producing the specific target product, making it a valuable tool for various biotechnological applications.

[0295] The recombinant microorganism of the invention is preferably a microorganism that is suitable for the production of isobutene. That is, the recombinant microorganism of the invention preferably encodes a prFMN-dependent decarboxylase having 3-methylcrotonic acid decarboxylase activity, as defined elsewhere herein.Thus, in a particular embodiment, the invention relates to the recombinant microorganism according to the invention, wherein the prFMN-dependent decarboxylase has 3- methylcrotonic acid decarboxylase activity.

[0296] Accordingly, in a preferred embodiment, the recombinant microorganism expresses a prFMN-dependent decarboxylase having high 3-methylcrotonic acid decarboxylase activity. Exemplary non-limiting examples include the UbiD enzyme from Hypocrea atroviridis (UniProt Accession Number G9NLP8; SEQ ID NO:5), as disclosed in WO 2017 / 191239, the pyrrole-2-carboxylic acid decarboxylase (EC 4.1.1.93) from Streptomyces sp. 769 (Uniprot accession number A0A0A8EV26; SEQ ID NO:6), as disclosed in WO 2020 / 007886, and the FDC enzyme from Yersinia frederiksenii (UniParc Accession Number UPI0005DC25B2; SEQ ID NO:1), as disclosed in WO 2022 / 207684. WO 2017 / 191239, WO 2020 / 007886 and WO 2022 / 207684, which are fully incorporated herein by reference, also describe engineered variants of these enzymes, which may be encoded within the recombinant microorganism of the invention.

[0297] It is particularly preferred that the recombinant microorganism of the invention encodes an FDC enzyme from Yersinia frederiksenii (UniParc Accession Number UPI0005DC25B2; SEQ ID NO:1), or an engineered variant thereof. In particular, the engineered variant of the Yersinia frederiksenii FDC may be any one of the variants disclosed in WO 2022 / 207684. In a particular embodiment, the invention relates to the recombinant microorganism according to the invention, wherein the UbiD decarboxylase has 3-methylcrotonic acid decarboxylase activity and comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0298] The recombinant microorganism is preferably a bacterium or a fungus, including yeasts, as described in more detail elsewhere herein. Thus, in a particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein the recombinant microorganism is a bacterium, in particular Escherichia coli, or a Clostridium species, or a fungus, in particular wherein the fungus is a yeast, such as Saccharomyces cerevisiae.As regards other preferred embodiments of the recombinant microorganism, the same applies as has been set forth above in connection with the methods according to the present invention.

[0299] In a further aspect, the present invention also relates to the use of any of the above-described recombinant microorganisms for the production of a desired product by a prFMN-dependent enzyme. In a preferred embodiment, the recombinant microorganisms of the invention is used for the production of isobutene, wherein the isobutene is produced by a prFMN-dependent decarboxylase having 3-methylcrotonic acid decarboxylase activity.

[0300] As used herein, "about," "approximately" and "substantially" are understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. All ranges are inclusive of the endpoints of the range. For example, an amount between 1 and 10 includes both 1 and 10.

[0301] As used in this disclosure and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0302] The words "comprise," "comprises" and "comprising" are to be interpreted inclusively rather than exclusively. Likewise, the terms "include," "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Nevertheless, the compositions and methods disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term "comprising" includes a disclosure of embodiments "consisting essentially of" and "consisting of" the components or steps identified.The terms "at least one of" and "and / or" used respectively in the context of "at least one of X and Y" and "X and / or Y" should be interpreted as "X without Y," or "Y without X," or "both X and Y." Where used herein, the terms "example" and "such as," particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive.

[0303] In the foregoing description of the invention, a number of individual elements, characterizing features, techniques and / or steps are disclosed. It is readily recognized that each of these has benefit not only individually when considered or used alone, but also when considered and used in combination with one another. Accordingly, to avoid exceedingly repetitious and redundant passages, this description has refrained from reiterating every possible combination and permutation. Nevertheless, whether expressly recited or not, it is understood that such combinations are entirely within the scope of the presently disclosed subject matter.

[0304] All technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

[0305] In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0306] BRIEF DESCRIPTION OF THE DRAWINGS

[0307] Figure 1A shows a schematic reaction of the enzymatic prenylation of a flavin mononucleotide (FMNH2) into the corresponding modified (prenylated) flavin cofactor (prFMNH2) with DMAP(P) (R=ribityl-5-phosphate).Figure IB shows a schematic reaction of the enzymatic conversion of 3-methylcrotonic acid into isobutene.

[0308] Figure 2 shows artificial pathways for isobutene production from acetyl-CoA via 3-methylcrotonic acid. Moreover, enzymatic recycling of metabolites which may occur during the pathway are shown in steps Xa, Xb, XI and XII.

[0309] EXAMPLES

[0310] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0311] Example 1. Construction of a yeast strain converting 3-methylcrotonic acid into isobutene (SB2800)

[0312] Saccharomyces cerevisiae YPH499 (Sikorski et al. 1989. Genetics 122: 19-27) has the following genotype MATa ura3-52 lys2-801(amber mutation) ade2-101(ochre mutation) trpl-delta63 his3-delta200 Ieu2-deltal. It is a good starting material for genetic constructions and is available from ATCC (ATCC 204679).

[0313] Saccharomyces cerevisiae strain SB2800 was constructed by integrating in strain YPH499 two genes required for the conversion of 3-methylcrotonic acid into isobutene. The first gene coding for a mutant of prenylated FMN-dependent ferulic acid decarboxylase (FDC) from Yersinia frederiksenii (UniParc accession number UPI0005DC25B2, RefSeq WP_050108772.1; SEQ ID NO:1, WO 2022 / 207684) was integrated at the ura3-52 locus with a pRS306 yeast integrative vector with a URA3 marker (ATCC #77141), the yeast ADH1 promoter and the yeastCYC1 terminator (plasmid linearized with the Stul restriction enzyme). The second gene coding for an UbiX-like flavin prenyl transferase derived from Saccharomyces cerevisiae (PAD1; UniProt accession number P33751) was integrated at the Ieu2-deltal locus with a pRS305 yeast integrative vector with a LEU2 marker (ATCC #77140), a GPD promoter (p415 GPD, ATCC #87358) and the yeast CYC1 terminator (plasmid linearized with the EcoRV restriction enzyme).

[0314] The FDC and PAD1 gene sequences were codon-optimized for expression in Saccharomyces cerevisiae and synthesized by Twist Bioscience.

[0315] The integrations of the FDC and PAD1 genes in the genome of Saccharomyces cerevisiae YPH499 were done successively, with FDC first and then PAD1.

[0316] For each integration, the yeast strain was made chemically competent and transformed (100 pl of cells) with the linearized plasmid (2pg) by heat shock using the lithium acetate (LiAc) and single-stranded carrier DNA / polyethylene glycol (PEG) method. The transformed cells were plated on appropriate amino acid dropout plates (SD dropout plates) with 2% glucose as the carbon source, then incubated at 30°C for three days.

[0317] The integrations in the genome were controlled by PCR.

[0318] Example 2. Anaerobic isobutene production by Saccharomyces cerevisiae strain SB2800

[0319] A 1.4 L vessel was filled with 0.5 L of a culture medium containing 2.5 g / L yeast extract, 3 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulphate heptahydrate, 0.2 g / L sodium chloride, 0.26 g / L calcium chloride dihydrate, 5 g / L ammonium sulphate, 0.3 g / L adenine, 0.32 g / L L-tryptophan, 1 g / L L-lysine hydrochloride and 0.25 g / L L-histidine hydrochloride monohydrate and sterilized at 121°C for 20 minutes. After cooling, filter sterilized trace metals were added at a final concentration of 1.5 pM iron III chloride, 4.7 pM manganese sulphate, 2.8 pM zinc sulphate, 0.5 pM copper sulphate, 1.2 pM potassium iodide, 1.6 pM sodium molybdate and 16 pM boric acid. Several sterilized growth factors were also added at a final concentration of 60 mg / L ergosterol, 5 mg / L flavin mononucleotide (FMN), 500 mg / L Tween 80 and 15 ml / L corn oil (Merck). Then filter sterilized glucose was added at a final concentration of 40 g / L from a concentrated glucose solution at 600 g / L. This concentrated solution was used also as a fed batch solution and when the glucose concentration in theculture decreased below 15 g / L it was added continuously in order to maintain glucose concentration between 10 and 20 g / L.

[0320] The culture medium was inoculated with 30 mL of a pre-culture of strain SB2800 previously grown at 30°C in YP medium (10 g / L yeast extract and 20 g / L casein peptone) containing 1.7 g / L YNB medium without ammonium sulphate and without amino acids (Merck), 0.1 g / L adenine and 0.16 g / L L-tryptophan. The 600 nm optical density of the preculture was about 0.3. pH was set and regulated at pH 5.0 with ammonia 25% and phosphoric acid 5M. Temperature was set at 32 °C for 40 hours and then increased up to 34°C. Agitation was set at 250 RPM and a flow of 0.15 L / min of nitrogen was introduced in the vessel through a sparger to maintain the culture in anaerobic conditions. Exhaust gas was analysed using a Prima PRO process mass spectrometer (Thermo Onix).

[0321] After 40 hours of culture when the biomass level had reached about 5 to 6 g / L temperature was increased up to 34°C and potassium 3-methylcrotonate was added at a concentration of 1 g / L to start the production of isobutene. A small production of isobutene was detected and isobutene concentration in the exhaust gas was about 0.0075 %mol. The resulting specific productivity of biomass was about 0.45 mg isobutene per gram biomass per hour.

[0322] Example 3. Activation of isobutene production by air

[0323] The same experiment as described in Example 2 was performed but after 3 hours of production a small amount of air (10 ml) was added through the sparger using a syringe while increasing the agitation speed to 500 RPM for about 20 minutes at the same time. Then the agitation speed was decreased again to 250 RPM. As a result, the isobutene concentration in the exhaust gas increased from 0.0075 %mol to 0.055 %mol indicating that the specific activity of the biomass now reached 3 mg isobutene per g biomass per hour compared to 0.45 mg g-xh1in previous fully anaerobic conditions.

[0324] Interestingly, if the agitation speed was let at 250 RPM while introducing the same amount of air, no increase of isobutene productivity was observed and isobutene concentration in the exhaust gas remained close to 0.0075 %mol. In addition, no increase of dissolved oxygen inthe broth was observed at 250 RPM while at 500 RPM a small peak of about 2% oxygen saturation was detected. This shows that oxygen transfer is key for rising dissolved oxygen concentration to a level required for an efficient oxidation of prFMNH2 into the active cofactor of FDC enzyme.

[0325] Example 4. Activation of isobutene production by combination of the addition of hydrogen peroxide and exogenous catalase

[0326] The same experiment as described in Example 2 was performed again in six bioreactors but after 3 hours of production in anaerobic conditions 0.5 mg of catalase from bovine liver (Merck, 2000 to 5000 units / mg) was added to each broth and then increasing amounts (100 pM to 1 mM) of hydrogen peroxide were added to the six bioreactors. Few minutes later isobutene productivity increased as summarized in Table 1 below.

[0327] Table 1: Increase of specific isobutene productivity upon addition of hydrogen peroxide in the presence of catalase.

[0328]

[0329] The optimum concentration of hydrogen peroxide is about 400 to 500 pM. If less hydrogen peroxide is added, for example 200 pM, only a portion of the active FDC cofactor is formed, but the full activity (about 6 mg g-1h-1) could be reached again if a second addition of hydrogen peroxide (400 pM) was made. At the opposite adding too much hydrogen peroxide (1000 pM) led to lower specific activity by some irreversible oxidative damage to the FDC enzyme cofactor.Again, the same experiment as described in Example 2 was performed in five bioreactors but after 3 hours of production in anaerobic conditions increasing amounts (0 mg; 0.05 mg; 0.15 mg or 1.5 mg) of catalase from bovine liver (Merck, 2000 to 5000 units / mg) was added to each broth and then 450 pM of hydrogen peroxide were added to four bioreactors. A bioreactor without catalase nor hydrogen peroxide was the reference bioreactor. Few minutes later isobutene productivity increased as summarized in Table 2 below.

[0330] Table 2: Increase of specific isobutene productivity upon addition of hydrogen peroxide in the presence of increasing amounts of catalase.

[0331]

[0332] Once again, the same experiment as described in Example 2 was performed in six bioreactors but after 3 hours of production in anaerobic conditions increasing amounts (0 mg; 0.15 mg; 0.30 mg; 0.45 mg; 0.60 mg or 0.75 mg) of catalase from bovine liver (Merck, 2000 to 5000 units / mg) was added to each broth and then 450 pM of hydrogen peroxide were added to the six bioreactors. Few minutes later isobutene productivity increased as summarized in Table 3 below.

[0333] Table 3: Increase of specific isobutene productivity upon addition of hydrogen peroxide in the presence of increasing amounts of catalase.

[0334]

[0335]

[0336] Example 5. Construction of yeast strains converting 3-methylcrotonic acid into isobutene and expressing an additional catalase gene (SB2913, SB2918)

[0337] As described in Table 2 and 3, the addition of exogenous catalase increased IBN production significantly. To have the same effect without the addition of exogenous catalase, an additional catalase gene was integrated in the genome of the strain SB2800 (described in Example 1).

[0338] Construction ofSB2913: integration ofCTTl in SB2800 at the bud9 locus

[0339] The catalase gene CTT1, amplified by PCR from the genome of Saccharomyces cerevisiae (Uniprot accession number P06115), was cloned in the MCS of the pRS3O3 yeast integrative vector (ATCC #77138) (containing a HIS3 marker) with a GPD promoter (p415 GPD, ATCC #87358) and the yeast CYC1 terminator. The transcription unit "pGPD-CTTl-tCYCl" was then amplified by PCR from the resulting plasmid along with the HIS3 marker with 200 mer-oligonucleotides having 170 bp homology with the DNA sequences at the 5' and 3' ends of the bud9 gene (170 bp homology before the bud9 start codon and 170 bp homology after the bud9 stop codon).

[0340] This PCR fragment (5 pg) was used to transform SB2800 (100 pl of cells) by heat shock using the lithium acetate (LiAc) and single-stranded carrier DNA / polyethylene glycol (PEG) method. The transformed cells were plated on appropriate amino acid dropout plates (SD dropout plates) with 2% glucose as the carbon source, then incubated at 30°C for three days.

[0341] The integration in the genome was controlled by PCR.

[0342] Construction ofSB2918: integration ofCTAl in SB2800 at the rhr2 locus

[0343] The same strategy as the one described for the construction of SB2913 was used for the construction of SB2918, except that the catalase gene CTA1 derived from Saccharomyces cerevisiae (Uniprot accession number P15202) was integrated in SB2800 at the rhr2 locus. The integration in the genome was controlled by PCR.Example 6. Activation of isobutene production by combination of the addition of hydrogen peroxide and expression of an additional catalase gene (SB2913, SB2918)

[0344] The same experiment as described in Example 2 was performed again but with the strains SB2913 and SB2918 expressing an additional catalase gene (described in Example 5) and the control strain SB2800. After 3 hours of production in anaerobic conditions, 450 pM of hydrogen peroxide was added to the three bioreactors. Few minutes later isobutene productivity increased as summarized in Table 4 below.

[0345] Table 4: Increase of specific isobutene productivity upon addition of hydrogen peroxide in the strains expressing an additional catalase gene (SB2913, SB2918) and the control strain SB2800

[0346]

[0347] In the strains expressing an additional catalase gene (SB2913 and SB2918), the specific isobutene productivity upon addition of hydrogen peroxide was similar to the one obtained with the addition of exogenous catalase with SB2800 (see Table 1, Table 2, Table 3 and Table 4).

Claims

New PCT-Patent ApplicationGlobal BioenergiesVossius Ref.: AJ4094 PCT BSCLAIMS1. A method for producing a product using an enzyme comprising a prenylated flavin mononucleotide (prFMN) cofactor in a recombinant microorganism, wherein the recombinant microorganism is grown in a culture medium in the absence of air, and wherein a prFMNH2 precursor is oxidised with molecular oxygen (O2) derived from decomposition of hydrogen peroxide (H2O2) to produce the active prFMN cofactor.

2. The method of claim 1, wherein the decomposition of H2O2 is catalysed by a catalase.

3. The method of claim 2, wherein the catalase (i) is added to the culture medium exogenously or (ii) is expressed by the recombinant microorganism or by a further recombinant microorganism present in the culture medium.

4. The method of claim 2 or 3, wherein the catalase is a heme-containing catalase or a manganese-containing catalase.

5. The method of claim 4, wherein the heme is added to the culture medium exogenously or produced in the recombinant microorganism.

6. The method of any one of claims 1 to 5, wherein the H2O2 is added to the culture medium in the form of FhChor in the form of an adductor precursor of H2O2, in particular wherein the adduct or precursor of H2O2 is urea hydrogen peroxide or a percarbonate salt, preferably sodium percarbonate.

7. The method of any one of claims 1 to 6, wherein the H2O2 or the adduct or precursor of H2O2 is added to the culture medium at a concentration ranging from 50 to 1000 pM, preferably 300 to 500 pM.

8. The method of any one of claims 1 to 7, wherein the recombinant microorganism expresses a flavin prenyltransferase.

9. The method of any one of claims 1 to 8, wherein the product is formed by a decarboxylation reaction catalysed by the prFMN-dependent enzyme, preferably wherein the product is selected from the group consisting of: isobutene, styrene, butadiene, 1,3-pentadiene, hepta-l,3,5-triene and 4-vinylguiacol; or wherein the product is formed by a carboxylation reaction catalysed by the prFMN-dependent enzyme, preferably wherein the product is selected from the group consisting of: 2,5- furandicarboxylic acid (FDCA) and 2-naphthoic acid.

10. The method of any one of claims 1 to 9, wherein the product is isobutene and, preferably, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity.

11. The method of claim 10, wherein the prFMN-dependent enzyme having 3- methylcrotonic acid decarboxylase activity comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQID NO: 1.

12. The method of claim 10 or 11, wherein the isobutene is produced from 3-methylcrotonic acid.

13. The method of any one of claims 1 to 12, wherein the recombinant microorganism is a bacterium, in particular Escherichia coli, or a Clostridium species, or a fungus, in particular wherein the fungus is a yeast, such as Saccharomyces cerevisiae.

14. A recombinant microorganism comprising one or more nucleic acids encoding:a) a prFMN-dependent enzyme;b) a flavin prenyltransferase; andc) a catalase.

15. The recombinant microorganism of claim 14, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity and, preferably, comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 1, wherein the enzyme retains 3-methylcrotonic acid decarboxylase activity.

16. The recombinant microorganism of claim 14 or 15, wherein the flavin prenyltransferase comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:15 or (ii) an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 15, wherein the amino acid sequence retains the enzymatic activity of modifying FMNH2into prFMNH2.

17. The recombinant microorganism of any one of claims 14 to 16, wherein the catalase is a heme-containing catalase or a manganese-containing catalase.

18. The recombinant microorganism of any one of claims 14 to 17, wherein the recombinant microorganism is a bacterium, in particular Escherichia coli, or a Clostridium species, or a fungus, in particular wherein the fungus is a yeast, such as Saccharomyces cerevisiae.

19. Use of the recombinant microorganism of any one of claims 14 to 18 in the production of a product, wherein the product is produced by the prFMN-dependent enzyme.

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