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 the recombinant microorganism recombinantly expresses a polypeptide that facilitates the oxidative maturation of a prFMNH2 precursor into 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.: AK1352 PCT BS

[0004] METHODS AND 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 the recombinant microorganism recombinantly expresses a polypeptide that facilitates the oxidative maturation of a prFMNH2 precursor into 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 cofactorto 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 fortheir 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, prFMNF , 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 characterized as a maturase that catalyzes the oxidation of reduced prFMN (prFMNFh) to its catalytically active form. PhdC utilizes molecular oxygen to oxidize the prFMN semiquinone radical, which is formed through spontaneous air oxidation, into the active cofactor (DiRocco et al., ACS Catalysis, 2024, 14 (13), 10223-10233).

[0013] Concurrently, Gahloth et al. (J. Biol. Chem., 2024, 300(2), 105653) identified the prFMNFh-binding protein LpdD as a chaperone in the activation of UbiD decarboxylase in the presence of molecular oxygen (see e.g., Fig. 14 of Gahloth et al.).

[0014] 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.

[0015] 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.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.

[0016] 76 (2010), 8004-8010) and van Leeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387) and W02010 / 001078.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] SUMMARY OF THE INVENTION

[0021] The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:1. 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 the recombinant microorganism recombinantly expresses a polypeptide that facilitates the oxidative maturation of a prFMNH2 precursor into the active prFMN cofactor.

[0022] 2. The method of embodiment 1, wherein the polypeptide that facilitates the oxidative maturation of the prFMNFh precursor into the active prFMN cofactor is a LpdD-like protein and / or a PhdC-like protein.

[0023] 3. The method of embodiment 2, wherein the LpdD-like protein comprises or consists of (i) an amino acid sequence as set forth in any one of SEQ ID NO:30-34; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO:30-34, wherein the amino acid sequence retains the ability to bind to prFMNF and facilitate the oxidative maturation of prFMNFh into prFMN.

[0024] 4. The method of embodiment 2 or 3, wherein the PhdC-like protein comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:35; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:35, wherein the amino acid sequence retains the ability to bind to prFMNFh and facilitate the oxidative maturation of prFMNFh into prFMN.

[0025] 5. The method of any one of embodiments Ito 4, wherein the recombinant microorganism expresses a flavin prenyltransferase.

[0026] 6. The method of any one of embodiments 1 to 5, 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.

[0027] 7. The method of any one of embodiments 1 to 6, wherein the product is isobutene and, preferably, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity.

[0028] 8. The method of embodiment 7, 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.

[0029] 9. The method of embodiment 7 or 8, wherein the isobutene is produced from 3- methylcrotonic acid.

[0030] 10. The method of any one of embodiments Ito 9, 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.

[0031] 11. The method of any one of embodiments 1 to 10 wherein the recombinant microorganism is an anaerobic microorganism.

[0032] 12. A recombinant microorganism comprising one or more nucleic acids encoding:

[0033] a) a prFMN-dependent enzyme;

[0034] b) a flavin prenyltransferase; and

[0035] c) a polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor.

[0036] 13. The recombinant microorganism of embodiment 12, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity and, preferably, comprises orconsists of (i) an amino acid sequence as set forth in SEQ ID NO:1 or (ii) an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 1, wherein the amino acid sequence retains 3-methylcrotonic acid decarboxylase activity.

[0037] 14. The recombinant microorganism of embodiment 12 or 13, 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.

[0038] 15. The recombinant microorganism of any one of embodiments 12 to 14, wherein the polypeptide that facilitates the oxidative maturation of the prFMNH2precursor into the active prFMN cofactor is an Lpd D-like protein and / or a PhdC-like protein.

[0039] 16. The recombinant microorganism of embodiment 15, wherein the LpdD-like protein comprises or consists of (i) an amino acid sequence as set forth in any one of SEQ ID NQ:30-34; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NQ:30-34, wherein the amino acid sequence retains the ability to bind to prFMNH2and facilitate the oxidative maturation of prFMNH2into prFMN.

[0040] 17. The recombinant microorganism of embodiment 15 or 16, wherein the PhdC-like protein comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:35; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:35, wherein the amino acid sequence retains the ability to bind to prFMNH2and facilitate the oxidative maturation of prFMNH2into prFMN.

[0041] 18. The recombinant microorganism of any one of embodiments 12 to 17, wherein the recombinant microorganism is a bacterium, in particular Escherichia coli, or aClostridium species, or a fungus, in particular wherein the fungus is a yeast, such as Saccharomyces cerevisiae.

[0042] 19. The recombinant microorganism of any one of embodiments 12 to 18, wherein the recombinant microorganism is an anaerobic microorganism.

[0043] 20. Use of the recombinant microorganism of any one of embodiments 12 to 19 in the production of a product, wherein the product is produced by the prFMN-dependent enzyme.

[0044] 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 the recombinant microorganism recombinantly expresses a polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into the active prFMN cofactor.

[0045] The present invention is based on the unexpected finding that the oxidative maturation of the prFMN cofactor from its precursor, prFMNFh, can be achieved in the absence of molecular oxygen. This finding unlocks new possibilities for utilizing prFMN-dependent enzymes in the production of various products under anaerobic conditions. Anaerobic processes are often desirable because they typically consume less energy than aerobic processes and are particularly advantageous for the production of (flammable) gaseous products, such as isobutene, as the absence of air mitigates the risk of explosions and prevents the contamination of the gaseous product with other gases, thereby enhancing both safety and product purity.

[0046] To identify factors that may enable the oxidative maturation of prFMN in the absence of molecular oxygen, the inventors conducted a bioinformatic analysis in which they investigated the genome neighbourhood of a prFMN-dependent enzyme in obligate anaerobic organisms (see Example 1). This analysis revealed that the prFMN-dependent enzyme UbiD is very frequently encoded in close proximity to the enzyme LpdD.Co-expression of LpdD with the prFMN-dependent enzyme FDC under anaerobic conditions demonstrated that LpdD can significantly increase the productivity of the prFMN-dependent enzyme (see Example 6). Similar results were obtained in Example 8 with another enzyme, PhdC.

[0047] These findings are surprising, as both LpdD and PhdC have been recently described to require the presence of molecular oxygen to facilitate the maturation of the prFMN cofactor (DiRocco et al., and Gahloth et al., see above). The present invention uniquely demonstrates that these enzymes can efficiently facilitate the oxidative maturation of the prFMN cofactor in the absence of molecular oxygen, thereby enhancing the productivity of prFMN-dependent enzymes under anaerobic conditions.

[0048] The polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor

[0049] The method of the present invention requires the recombinant expression of a polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor in a cell.

[0050] It has been demonstrated that the recombinant expression of a polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor, such as LpdD or PhdC, drastically enhances the productivity of prFMN-dependent enzymes in a cell under anaerobic conditions, specifically in the absence of atmospheric air. This enhancement can likely be attributed to the increased availability of active, oxidized prFMN cofactor, which is essential for the function of prFMN-dependent enzymes. Importantly, it was found that the oxidative activation of the cofactor can occur in the absence of oxygen, which contrasts with previous reports in the prior art.

[0051] The polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor may be any polypeptide that directly or indirectly assists in theoxidation of prFMNH2 to prFMN, thereby enabling the cofactor's activation and subsequent participation in enzymatic reactions.

[0052] In certain embodiments, the polypeptide that facilitates the oxidative maturation of a prFMNF precursor into an active prFMN cofactor is an enzyme that directly binds to the prFMNF precursor and catalyzes its oxidative maturation into the active prFMN cofactor.

[0053] Preferably, the polypeptide facilitates the oxidative maturation of the prFMNFh precursor into an active prFMN cofactor in the absence of oxygen. That is, the polypeptide that facilitates the oxidative maturation of a prFMNF precursor into an active prFMN cofactor is preferably an enzyme that utilizes an electron acceptor other than oxygen for the oxidization of prFMNFh. In certain embodiments, the polypeptide that facilitates the oxidative maturation of a prFMNF precursor into an active prFMN cofactor utilizes a transition metal as the electron acceptor, such as, without limitation, iron, copper, manganese, cobalt, nickel, molybdenum, or zinc, in any of their biologically relevant oxidation states.

[0054] In certain embodiments, the polypeptide that facilitates the oxidative maturation of a prFMNF precursor into an active prFMN cofactor utilizes iron or manganese as the electron acceptor.

[0055] The skilled person is able to determine whether a polypeptide facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor.

[0056] For instance, a candidate polypeptide may be co-expressed in a cell along with a prFMN-dependent enzyme, preferably a prFMN-dependent decarboxylase, and, optionally, a flavin prenyltransferase. The resulting cells may then be cultured under anaerobic conditions, and a substrate of the prFMN-dependent enzyme may be added at a suitable time point and in a suitable concentration. Subsequently, the conversion of the substrate by the prFMN-dependent enzyme may be quantified. As a control, cells that do not express the candidate polypeptide may be tested in parallel.

[0057] The candidate polypeptide may be determined to facilitate the oxidative maturation of a prFMNF precursor into an active prFMN cofactor if the conversion of the substrate in the presence of the polypeptide is at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% higher than in the absence of the polypeptide.

[0058] Suitable methods are described in Examples 6 and 8.To rule out the possibility that the candidate polypeptide increases the conversion of the substrate by other means, i.e., not by facilitating the oxidative maturation of a prFMNH2 precursor into an active prFMN cofactor, additional tests may be performed to confirm that the candidate polypeptide interacts with prFMN or its precursor. For example, the binding of prFMNF to the candidate polypeptide may be tested using in vitro reconstitution and spectrophotometric analysis. Purified polypeptides may be incubated with prFMNF or prFMN under controlled conditions. The interaction may be confirmed by observing specific spectral changes using UV-Vis spectrophotometry, which indicate the formation of a prFMN-protein complex. Additionally, the specificity of the binding may be assessed by comparing the spectral features with those obtained from control experiments using FMNH2 or oxidized FMN, which should not exhibit similar binding characteristics.

[0059] Accordingly, in certain embodiments, the polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor is a polypeptide that directly interacts with and / or binds to prFMNFh.

[0060] In some embodiments, reference is made to sequence variants of proteins that retain the ability to bind to prFMNF . In such embodiments, the term "bind" is intended to be understood in its broadest sense, encompassing not only the formation of stable complexes but also including transient interactions that may influence the protein's function or structure.

[0061] In a particular embodiment, the invention relates to the method according to the invention, wherein the polypeptide that facilitates the oxidative maturation of the prFMNF precursor into the active prFMN cofactor is a LpdD-like protein.

[0062] Thus, 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 the recombinant microorganism recombinantly expresses a LpdD-like protein.

[0063] A "LpdD-like protein" refers to a protein that shares structural and functional characteristics with LpdD, a known chaperone protein associated with the UbiD-like gallate decarboxylaseLpdC from Lactobacillus plantarum. LpdD specifically binds to reduced prFMNH2 and assists in the activation of UbiD decarboxylase by facilitating the incorporation and oxidative maturation of prFMNH2 into the active prFMN cofactor. This binding and chaperone activity enable the proper functioning of UbiD enzymes, which are crucial for various biochemical processes, including decarboxylation reactions.

[0064] LpdD-like proteins are classified under the InterPro protein family IPR048844. Accordingly, in certain embodiments, the polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into the active prFMN cofactor is a LpdD-like protein belonging to InterPro protein family IPR048844.

[0065] Currently, over 1300 LpdD-like proteins have been classified under InterPro protein family IPR048844 and may be used in the method according to the invention.

[0066] That is, in certain embodiments, the LpdD-like protein may be a LpdD-like protein from Streptococcus gallolyticus (A0A060RIC4), Brevibacillus laterosporus LMG 15441 (A0A075RAR1), Secundilactobacillus oryzae JCM 18671 (A0A081BGA7), Paenibacillus durus (A0A089HPY1), Paenibacillus sp. FSL H7-0357 (A0A089I7X3), Paenibacillus sp. FSL H7-0737 (A0A089IP73), Paenibacillus sp. FSL R5-0912 (A0A089K4J5), Paenibacillus sp. FSL R7-0273 (A0A089KID7), Paenibacillus sp. FSL R7-0331 (A0A089KX11), Paenibacillus borealis (A0A089LII5), Paenibacillus stellifer (A0A089N674), Paenibacillus graminis (A0A089NJA5), Paenibacillus macerans (A0A090ZEA5), Tetragenococcus muriaticus 3MR10-3 (A0A091BYZ1), Flavonifractor plautii l_3_50AFAA (A0A096BCX4), Flavonifractor plautii l_3_50AFAA (A0A096BEA7), Paenibacillus wynnii (A0A098M5D6), Desulfosporosinus sp. HMP52 (A0A099SCS0), Methanococcoides methylutens (A0A099T1T9), Paucilactobacillus hokkaidonensis JCM 18461 (A0A0A1GWI5), Desulfosporosinus sp. Tol-M (A0A0A2TGH9), Desulfosporosinus sp. Tol-M (A0A0A2THP2), Clostridium baratii str. Sullivan (A0A0A7FU23), Levilactobacillus brevis (A0A0C1M2E1), Paucilactobacillus wasatchensis (A0A0D0Y3P7), Dethiosulfatarculus sandiegensis (A0A0D2J2Z3), Dethiosulfatarculus sandiegensis (A0A0D2J7F6), Paenibacillus sp. IHBB 10380 (A0A0D3V8J7), Paenibacillus sp. E194 (A0A0D7KLQ7), Paenibacillus terrae (A0A0D7WZ30), Methanosarcina thermophila (A0A0E3H8Y5), Methanosarcina sp. WH1 (A0A0E3KWI2), Methanosarcina thermophila CHTI-55 (A0A0E3L1C6), Methanosarcina sp. WWM596 (A0A0E3L236), Methanosarcina siciliae T4 / M (A0A0E3L7T9), Methanosarcina siciliae HI350 (A0A0E3LA27), Methanosarcina mazeiWWM610 (A0A0E3LG16), Methanosarcina mazei S-6 (A0A0E3LUX1), Clostridium scatologenes (A0A0E3M5F7), Methanosarcina sp. MTP4 (A0A0E3NYT4), Methanosarcina siciliae C2J (A0A0E3PKH9), Methanosarcina vacuolata Z-761 (A0A0E3Q7L4), Methanosarcina sp. Kolksee (A0A0E3QF87), Methanosarcina barkeri str. Wiesmoor (A0A0E3QQB0), Methanosarcina barkeri MS (A0A0E3QQU1), Methanosarcina barkeri 1 1 (A0A0E3R1M1), Methanosarcina mazei SarPi (A0A0E3RBP2), Methanosarcina mazei LYC (A0A0E3RSV1), Methanosarcina mazei C16 (A0A0E3RZI5), Methanosarcina horonobensis HB-1 = JCM 15518 (A0A0E3SHM0), Methanosarcina barkeri 3 (A0A0E3SM67), Methanococcoides methylutens MM1 (A0A0E3SS54), Methanosarcina lacustris Z-7289 (A0A0E3WQI5), Syntrophomonas zehnderi OL-4 (A0A0E4GBI7), Paenibacillus riograndensis SBR5 (A0A0E4HB17), Clostridium sp. FS41 (A0A0F0CAG4), Peptococcaceae bacterium BRH_c4a (A0A0F2N1Z9), Peptococcaceae bacterium BRH_c8a (A0A0F2PI52), Peptococcaceae bacterium BRH_c4b (A0A0F2PKM8), Clostridiaceae bacterium BRH_c20a (A0A0F2Q316), Brevibacillus laterosporus (A0A0F7BYH2), Paenibacillus durus ATCC 35681 (A0A0F7F973), Methanosarcina sp. 2.H.A.1B.4 (A0A0F8C6J2), Methanosarcina sp. 2.H.T.1A.3 (A0A0F8CD61), Methanosarcina sp. 2.H.T.1A.6 (A0A0F8CU21), Methanosarcina sp. 2.H.T.1A.8 (A0A0F8E9M0), Methanosarcina sp. 2.H.T.1A.15 (A0A0F8EKR5), Methanosarcina mazei (A0A0F8JH87), Methanosarcina mazei (A0A0F8M5F2), Methanosarcina sp. l.H.A.2.2 (A0A0F8QGV7), Methanosarcina sp. 1.H.T.1A.1 (A0A0F8SWM6), marine sediment metagenome (A0A0F9F0E7), marine sediment metagenome (A0A0F9K794), Methanosarcina barkeri CM1 (A0A0G3CAX6), Lactiplantibacillus plantarum (A0A0G9FD85), Vibrio tasmaniensis (A0A0H3ZJJ4), Vibrio sp. 1F_189 (A0A0H4A3L3), Companilactobacillus ginsenosidimutans (A0A0H4QXE4), Peptococcaceae bacterium CEB3 (A0A0J1F6V6), Desulfosporosinus acididurans (A0A0J1FWJ8), Clostridium cylindrosporum DSM 605 (A0A0J8DA06), [Clostridium] citroniae WAL-19142 (A0A0J9C765), Paenibacillus peoriae (A0A0K2FC80), Companilactobacillus heilongjiangensis (A0A0K2LB57), Propionispora sp. 2 / 2-37 (A0A0K8JCH7), Propionispora sp. 2 / 2-37 (A0A0K8JH15), Acetobacterium bakii (A0A0L6TZD1), Thermincola ferriacetica (A0A0L6W2B5), Clostridiales bacterium PH28_bin88 (A0A0M2UCR4), Roseburia inulinivorans (A0A0M6WJL1), Paenibacillus xylanivorans (A0A0M9BR84), Enterococcus sp. RIT-PI-f (A0A0N0KSN0), Oxobacter pfennigii (A0A0P8W3I1), Paenibacillus sp. Leaf72 (A0A0Q4RDL6), Paenibacillus sp. Soil787 (A0A0Q9U585), Loigolactobacillus coryniformis subsp. coryniformis KCTC 3167 = DSM 20001 (A0A0R1FAN9), Levilactobacillus parabrevis ATCC 53295 (A0A0R1GRY4), Loigolactobacillusbifermentans DSM 20003 (A0A0R1H279), Companilactobacillus kimchii DSM 13961 = JCM 10707 (A0A0R1HZY1), Companilactobacillus bobalius DSM 19674 (A0A0R1KJ51), Levilactobacillus acidifarinae DSM 19394 (A0A0R1LHL8), Secundilactobacillus odoratitofui DSM 19909 = JCM 15043 (A0A0R1LVC1), Companilactobacillus paralimentarius DSM 13238 = JCM 10415 (A0A0R1PET7), Lacticaseibacillus manihotivorans DSM 13343 = JCM 12514 (A0A0R1QN66), Companilactobacillus mindensis DSM 14500 (A0A0R1QSC4), Furfurilactobacillus rossiae DSM 15814 (A0A0R1R735), Paucilactobacillus oligofermentans DSM 15707 = LMG 22743 (A0A0R1RGJ5), Companilactobacillus versmoldensis DSM 14857 = KCTC 3814 (A0A0R1SGD5), Ligilactobacillus apodemi DSM 16634 = JCM 16172 (A0A0R1TU65), Lacticaseibacillus pantheris DSM 15945 = JCM 12539 = NBRC 106106 (A0A0R1U2E4), Lactobacillus kalixensis DSM 16043 (A0A0R1U451), Levilactobacillus hammesii DSM 16381 (A0A0R1UJ43), Limosilactobacillus gastricus DSM 16045 (A0A0R1VC73), Paucilactobacillus suebicus DSM 5007 = KCTC 3549 (A0A0R1W7L1), Companilactobacillus nantensis DSM 16982 (A0A0R1WRH1), Agrilactobacillus composti DSM 18527 = JCM 14202 (A0A0R1XSU5), Lentilactobacillus parafarraginis DSM 18390 = JCM 14109 (A0A0R1YNB7), Lacticaseibacillus sharpeae JCM 1186 = DSM 20505 (A0A0R1ZW63), Paucilactobacillus vaccinostercus DSM 20634 (A0A0R2A1S6), Secundilactobacillus collinoides DSM 20515 = JCM 1123 (A0A0R2B9S5), Lapidilactobacillus dextrinicus DSM 20335 (A0A0R2BI65), Lacticaseibacillus thailandensis DSM 22698 = JCM 13996 (A0A0R2C8E7), Liguorilactobacillus cacaonum DSM 21116 (A0A0R2CPQ1), Loigolactobacillus rennini DSM 20253 (A0A0R2CYU4), Secundilactobacillus similis DSM 23365 = JCM 2765 (A0A0R2F5Z0), Lactobacillus selangorensis (A0A0R2FJ51), Liguorilactobacillus mali (A0A0R2FX34), Companilactobacillus kimchiensis (A0A0R2LB47), Lactiplantibacillus xiangfangensis (A0A0R2MGG6), Lacticaseibacillus saniviri JCM 17471 = DSM 24301 (A0A0R2N0R2), Lactiplantibacillus fabifermentans DSM 21115 (A0A0R2NSZ3), Paenibacillus sp. IHB B 3084 (A0A0S2S8P2), Moorella thermoacetica Y72 (A0A0S6UA70), Lactococcus lactis subsp. lactis (A0A0V8CZH0), Paenibacillus etheri (A0A0W1AZZ4), Vibrio sp. MEBiC08052 (A0A0W8JBH2), Turicibacter sp. H121 (A0A0X1TJR8), Desulfovibrio fairfieldensis (A0A0X8JK89), Paenibacillus amylolyticus (A0A100VM65), Moorella sp. 60_41 (A0A101EB29), Desulfitibacter sp. BRH_cl9 (A0A101VFE6), Clostridia bacterium BRH_c25 (A0A101WAQ4), Paenibacillus sp. DMB5 (A0A101Y0K9), Desulfitibacter sp. BRH_cl9 (A0A117S450), Enterococcus faecium (A0A132P798), Paenibacillus riograndensis (A0A132U4G3), candidate division MSBL1 archaeon SCGC-AAA259E19 (A0A133UEF6), candidate division MSBL1archaeon SCGC-AAA259I14 (A0A133UPG7), Candidatus Thorarchaeota archaeon SMTZ1-45 (A0A135VNT9), Candidatus Thorarchaeota archaeon SMTZ1-45 (A0A135VQ11), Candidatus Thorarchaeota archaeon SMTZ-45 (A0A135VVY5), Candidatus Thorarchaeota archaeon SMTZ1-83 (A0A135VYJ6), Methanohalophilus sp. T328-1 (A0A139CM83), Methanolobus sp. T82-4 (A0A139CTN3), Streptococcus gallolyticus (A0A139R5E7), Eubacteriaceae bacterium CHKCI004 (A0A143XEC4), Moorella mulderi DSM 14980 (A0A151B1S6), Sulfurovum riftiae (A0A151CGW8), Thermoplasmatales archaeon SG8-52-4 (A0A151DV21), Thermoplasmatales archaeon SG8-52-1 (A0A151ELT1), Thermoplasmatales archaeon SG8-52-3 (A0A151EXR2), Theionarchaea archaeon DG-70 (A0A151F3M1), Paenibacillus elgii (A0A163Z1D7), Secundilactobacillus collinoides (A0A166G6Z3), Paenibacillus crassostreae (A0A167DYB3), Paenibacillus antarcticus (A0A168JUU3), Paenibacillus glacialis (A0A168KRD8), Clostridium ljungdahlii (A0A168LRC0), Clostridium ljungdahlii (A0A168RAG1), Dorea longicatena (A0A173QUE3), Anaerobutyricum hallii (A0A173TAV3), Roseburia intestinalis (A0A173U797), Clostridium disporicum (A0A173Y7Z3), Fusicatenibacter saccharivorans (A0A173YC25), Collinsella aerofaciens (A0A174BYQ8), Dorea longicatena (A0A174DFI3), Faecalicatena contorta (A0A174DTL0), Dorea longicatena (A0A174F8L4), Hungatella hathewayi (A0A174FTA1), Faecalicatena contorta (A0A174H9T9), Collinsella aerofaciens (A0A174M9Z2), Faecalicatena contorta (A0A174MFG5), Fusicatenibacter saccharivorans (A0A174PFB5), Clostridium baratii (A0A174RTS7), Loigolactobacillus backii (A0A192GZH7), Desulfoplanes formicivorans (A0A194AEZ8), Paenibacillus oryzae (A0A1A5YHJ7), Paenibacillus yonginensis (A0A1B1MZK6), Paenibacillus sp. BIHB 4019 (A0A1B2DMP0), Secundilactobacillus paracollinoides (A0A1B2IX49), Desulfotomaculum copahuensis (A0A1B7LH71), Paenibacillus sp. KS1 (A0A1B8UTF5), Methanohalophilus sp. DALI (A0A1B8X1Q2), Dehalobacter sp. TeCBl (A0A1C2XPB6), Desulfosporosinus sp. BG (A0A1C3FB56), uncultured Blautia sp. (A0A1C5KWE3), uncultured Eubacterium sp. (A0A1C5L649), uncultured Blautia sp. (A0A1C5MAY3), uncultured Blautia sp. (A0A1C5P6V7), uncultured Blautia sp. (A0A1C5SDU0), uncultured Clostridium sp. (A0A1C5SHA5), uncultured Blautia sp. (A0A1C5SSB1), uncultured Eubacterium sp. (A0A1C5XIB7), uncultured Blautia sp. (A0A1C5YDP5), uncultured Roseburia sp. (A0A1C5YII7), uncultured Clostridium sp. (A0A1C5YSF4), uncultured Clostridium sp. (A0A1C6BT97), uncultured Clostridium sp. (A0A1C6CK15), uncultured Blautia sp. (A0A1C6CUM7), uncultured Eubacterium sp. (A0A1C6DIR5), uncultured Clostridium sp. (A0A1C6GX76), uncultured Eubacterium sp. (A0A1C6H7P9), uncultured Clostridium sp.(A0A1C6HXY9), uncultured Clostridium sp. (A0A1C6IIP6), uncultured Clostridium sp. (A0A1C6JZA8), uncultured Clostridium sp. (A0A1C6L844), Methanohalophilus sp. 2-GBenrich (A0A1D2UYM9), Methanosarcina sp. A14 (A0A1D2WIP6), Vulcanibacillus modesticaldus (A0A1D2YWD5), Moorella thermoacetica (A0A1D7X6Q7), Geosporobacter ferrireducens (A0A1D8GMC7), Eisenbergiella tayi (A0A1E3AFA3), Eisenbergiella tayi (A0A1E3ATS8), Lactiplantibacillus plantarum (A0A1E3KPV0), Paenibacillus nuruki (A0A1E3KYJ1), Eisenbergiella tayi (A0A1E3UQ85), Desulfuribacillus alkaliarsenatis (A0A1E5G0U6), Desulfuribacillus stibiiarsenatis (A0A1E5L983), Methanosarcina sp. Anti (A0A1E7G8R0), Enterococcus sp. HMSC077E04 (A0A1E9W0H3), Clostridium sp. HMSC19A10 (A0A1F1KZF0), Acetobacterium wieringae (A0A1F2PNH2), Actinobacteria bacterium RBG_13_35_12 (A0A1F2W7K5), candidate division NC10 bacterium RIFCSPLOWO2_02_FULL_66_22 (A0A1F4Q6H9), candidate division NC10 bacterium RIFCSPLOWO2_12_FULL_66_18 (A0A1F4R434), Candidatus Lambdaproteobacteria bacterium RIFOXYD2_FULL_50_16 (A0A1F6G9H3), Candidatus Lambdaproteobacteria bacterium RIFOXYD2_FULL_56_26 (A0A1F6H2R3), Candidatus Schekmanbacteria bacterium GWA2_38_11 (A0A1F7RIL9), Candidatus Schekmanbacteria bacterium RIFCSPLOWO2_12_FULL_38_15 (A0A1F7SG12), Candidatus Tectomicrobia bacterium RIFCSPLOWO2_12_FULL_69_37 (A0A1F7T3G0), Candidatus Tectomicrobia bacterium RIFCSPLOWO2_02_FULL_70_19 (A0A1F7TFD4), Chloroflexi bacterium RBG_13_51_36 (A0A1F8MI10), Chloroflexi bacterium RBG_16_50_9 (A0A1F8PQH8), Chloroflexi bacterium RBG_16_57_8 (A0A1F8QX36), Deltaproteobacteria bacterium RBG_16_5O_11 (A0A1F9BMH0), Deltaproteobacteria bacterium RBG_16_47_11 (A0A1F9DBC0), Deltaproteobacteria bacterium RBG_16_47_11 (A0A1F9DMN7), Deltaproteobacteria bacterium RIFCSPLOWO2_02_FULL_40_36 (A0A1F9HDN0), Nitrospinae bacterium RIFCSPLOWO2_12_FULL_45_22 (A0A1G1EHG3), Paenibacillus tianmuensis (A0A1G4PP24), Paenibacillus polysaccharolyticus (A0A1G5DWG1), Blautia sp. SF-50 (A0A1G5EB25), Succiniclasticum ruminis (A0A1G6L7L3), Paenibacillus sp. cl6col (A0A1G7FKJ5), Fontibacillus panacisegetis (A0A1G7GHM5), Desulfovibrio legallii (A0A1G7KH09), Desulfosporosinus hippei DSM 8344 (A0A1G8A5G1), Vibrio xiamenensis (A0A1G8GVQ6), Paenibacillus typhae (A0A1G8H100), Paenibacillus jilunlii (A0A1G9K6R8), Lachnospira pectinoschiza (A0A1G9UL54), Desulforhopalus singaporensis (A0A1H0KUD8), Clostridium gasigenes (A0A1H0LBZ2), Evansella caseinilytica (A0A1H3PKY4), Paenibacillus sp.

[0067] 276b (A0A1H4DUM2), Paenibacillus sp. OK003 (A0A1H7NPN3), Paenibacillus sophorae(A0A1H8FUH8), Propionispora vibrioides (A0A1H8ULD2), Propionispora vibrioides (A0A1H8V360), Butyrivibrio fibrisolvens (A0A1H9VG37), Methanococcoides vulcani (A0A1H9Y476), Paenibacillus sp. NFR01 (A0A1I0DG19), Enterocloster lavalensis (A0A1I0GLR4), Succiniclasticum ruminis DSM 9236 (A0A1I2AH55), Paenibacillus algorifonticola (A0A1I2C466), Planifilum fulgidum (A0A1I2N2R0), Desulfotruncus arcticus DSM 17038 (A0A1I2NGI8), Lachnospiraceae bacterium NLAE-zl-G231 (A0A1I2Z3Z4), Methanolobus profundi (A0A1I4QVX1), Oscillibactersp. PC13 (A0A1I5QBI1), Desulfoscipio geothermicus DSM 3669 (A0A1I6DX49), Methanosarcina thermophila (A0A1I6XCY7), Streptococcus gallolyticus (A0A1I7GNZ7), Candidatus Desulfovibrio trichonymphae (A0A1J1DQ35), Moorella thermoacetica (A0A1J5P128), Desulfovibrio piger (A0A1K1LHB0), Loigolactobacillus rennini (A0A1K2I870), Methanohalophilus halophilus (A0A1L3Q246), Enterococcus canis (A0A1L8RH74), Enterococcus hermanniensis (A0A1L8TPX5), Actinomyces glycerinitolerans (A0A1M4RXZ2), Vibrio gazogenes DSM 21264 = NBRC 103151 (A0A1M4SII0), Lactonifactor longoviformis DSM 17459 (A0A1M4U147), Desulforamulus putei DSM 12395 (A0A1M4WWP5), Desulfofundulus australicus DSM 11792 (A0A1M5B3Q1), Clostridium intestinale DSM 6191 (A0A1M5W7Q4), Sporobacter termitidis DSM 10068 (A0A1M5WF95), Vibrio aerogenes CECT 7868 (A0A1M6ASQ0), Parasporobacterium paucivorans DSM 15970 (A0A1M6E3Y1), Propionispora hippei DSM 15287 (A0A1M6G8Q0), Desulfofundulus thermosubterraneus DSM 16057 (A0A1M6J5A0), Propionispora hippei DSM 15287 (A0A1M6KSI8), Caldanaerovirga acetigignens (A0A1M7LPU0), Desulfovibrio litoralis DSM 11393 (A0A1M7SS72), Vibrio guintilis (A0A1M7Z087), Vibrio spartinae (A0A1N6M1U9), Companilactobacillus allii (A0A1P8Q5Z8), Paenibacillus sp. P3E (A0A1Q5XBF4), Paenibacillus sp. P46E (A0A1Q5XQH3), Eggerthella sp. 51_9 (A0A1Q6E264), Collinsella sp. 60_9 (A0A1Q6EWP4), Eubacterium sp. 38_16 (A0A1Q6NFB1), Desulfosporosinus metallidurans (A0A1Q8R1X9), Thorarchaeota archaeon (strain AB_25) (A0A1Q9PAW9), Paenibacillus odorifer (A0A1R0WSC0), Paenibacillus odorifer (A0A1R0Y3H7), Paenibacillus borealis (A0A1R0YLD4), Paenibacillus odorifer (A0A1R0ZMW8), Paenibacillus sp. FSL R5-0765 (A0A1R1FMU1), Vibrio ruber (strain DSM 16370 / JCM 11486 / BCRC 17186 / CECT 7878 / LMG 23124 / VR1) (A0A1R4LG06), Anaerobacillus isosaccharinicus (A0A1S2L7C2), Anaerobacillus arseniciselenatis (A0A1S2LCS6), Anaerobacillus alkalilacustris (A0A1S2LE16), Anaerobacillus alkalidiazotrophicus (A0A1S2M1J3), Clostridium felsineum (A0A1S8KWY8), Clostridium beijerinckii (A0A1S8RZR5), Enterococcus faecium (A0A1S9MD12), Paenibacillusselenitireducens (A0A1T2X7E3), Desulfobaculum bizertense DSM 18034 (A0A1T4X0C2), Maledivibacter halophilus (A0A1T5KGY5), Desulfobacteraceae bacterium 4484_190.3 (A0A1V4S4N5), Ruminiclostridium hungatei (A0A1V4SJD8), Pelotomaculum sp. PtaB.Bin013 (A0A1V4V7Y2), Pelotomaculum sp. PtaB.BinlO4 (A0A1V4VI36), Pelotomaculum sp. PtaB.Bin013 (A0A1V4VLI5), Pelotomaculum sp. PtaB.Binll7 (A0A1V4VM03), Pelotomaculum sp. PtaB.Bin013 (A0A1V4VML7), Pelotomaculum sp. PtaB.Bin013 (A0A1V4W2T2), Pelotomaculum sp. PtaB.BinlO4 (A0A1V4W9Y3), Syntrophorhabdus sp. PtaB.Bin006 (A0A1V4WAH6), Syntrophorhabdus sp. PtaB.BinO47 (A0A1V4WT83), Syntrophus sp. PtaB.Binl38 (A0A1V4XW61), Methanomethylovorans sp. PtaUl.Bin093 (A0A1V4YH94), Methanocella sp. PtaUl.Binl25 (A0A1V4Z4R7), Pelotomaculum sp. PtaUl.Bin065 (A0A1V5BUM7), Pelotomaculum sp. PtaUl.Bin065 (A0A1V5BZL0), Syntrophorhabdus sp. PtaUl.Bin050 (A0A1V5C8R8), Syntrophorhabdaceae bacterium PtaUl.BinO34 (A0A1V5CG07), Syntrophorhabdus sp. PtaUl.Bin002 (A0A1V5CGS8), Syntrophorhabdus sp. PtaUl.Binl53 (A0A1V5D4N3), Syntrophorhabdus sp. PtaUl.BinO58 (A0A1V5D7C0), Syntrophus sp. PtaUl.BinOO5 (A0A1V5DFP7), Firmicutes bacterium ADurb.Bin456 (A0A1V5KRZ5), Deltaproteobacteria bacterium ADurb.Binl35 (A0A1V6CHJ8), Deltaproteobacteria bacterium ADurb.BinO26 (A0A1V6IEG3), hydrothermal vent metagenome (A0A1W1C6Z1), hydrothermal vent metagenome (A0A1W1EG86), Thermanaeromonas toyohensis ToBE (A0A1W1W375), Desulfobacteraceae bacterium 4572_130 (A0A1W9T228), Desulfobacteraceae bacterium 4572_88 (A0A1W9WYW2), Parendozoicomonas haliclonae (A0A1X7ANI5), Paenibacillus uliginis N3 / 975 (A0A1X7HGA4), Paenibacillus aguistagni (A0A1X7J8L9), Methanohalophilus portucalensis FDF-1 (A0A1X7NMC2), Alloscardovia macacae (A0A1Y2SYW5), Pseudoflavonifractor sp. An44 (A0A1Y3Y130), Eubacterium sp. An3 (A0A1Y4B5J5), Blautia sp. An249 (A0A1Y4FHD4), Collinsella sp. An2 (A0A1Y4HR87), Faecalibacterium sp. Anl92 (A0A1Y4JAH8), Pseudoflavonifractor sp. Anl87 (A0A1Y4KKX3), Pseudoflavonifractor sp. Anl76 (A0A1Y4M9J9), Enterococcus cecorum (A0A1Y4R1A3), Lachnoclostridium sp. Anl4 (A0A1Y4RUS4), Faecalibacterium sp. Anl22 (A0A1Y4SU31), Gemmiger sp. Anl20 (A0A1Y4TKK2), Lachnoclostridium sp. Anll8 (A0A1Y4TX11), Eubacterium sp. Anil (A0A1Y4V0T0), Vibrio mangrovi (A0A1Y6IVS8), Levilactobacillus zymae (A0A1Y6JWH6), Vibrio gazogenes (A0A1Z2SLU7), Secundilactobacillus silagincola (A0A1Z5J0S6), Companilactobacillus bobalius (A0A202F882), Companilactobacillus kimchii (A0A210P6U2), uncultured Desulfovibrio sp. (A0A212KK78), uncultured Desulfovibrio sp. (A0A212KYW5),uncultured Sporomusa sp. (A0A212LMB5), Paenibacillus kribbensis (A0A222WK06), Bifidobacterium vansinderenii (A0A229VXA1), Acetobacterium sp. MES1 (A0A231NX72), Desulfovibrio sp. MES5 (A0A231P1G8), Enterococcus faecium (A0A242BBR4), Enterococcus sp.

[0068] 3H8_DIV0648 (A0A242CX04), Enterococcus sp. (strain 3Gl_DIV0629) (A0A242DJJ4), Commensalibacter intestini (A0A251ZSM9), Saccharibacillus sp. 023 (A0A254NWP3), Lactobacillus taiwanensis (A0A256LC69), Thermoplasmatales archaeon ex4572_165 (A0A256XHR8), Thermoplasmatales archaeon ex4572_165 (A0A256XP18), Euryarchaeota archaeon ex4484_162 (A0A256ZXE4), Acidobacteria bacterium 21-70-11 (A0A257TW10), Acidobacteria bacterium 37-71-11 (A0A257VBC0), Alloscardovia macacae (A0A261F780), Helicobacter sp. 11S02629-2 (A0A268TM46), Helicobacter sp. 13S00401-1 (A0A268U406), Methanohalophilus euhalobius (A0A285F4D8), Anaerobutyricum hallii (A0A285PV99), Methanosarcina spelaei (A0A2A2HWF4), Candidatus Reconcilbacillus cellulovorans (A0A2A6DYS6), Desulforamulus profundi (A0A2C6MA64), Enterococcus faecium (A0A2D0BGN4), Collinsella aerofaciens (A0A2D1TYB8), Methanohalophilus portucalensis (A0A2D3C471), Desulfobacter sp. (A0A2D6EU04), Dehalococcoidales bacterium (A0A2D6J0J8), Neomarinimicrobiota bacterium (A0A2E9PN48), Neomarinimicrobiota bacterium (A0A2E9PPI5), Rhodospirillaceae bacterium (A0A2E9Y0P9), Sulfurovum sp. (A0A2G2N2C9), bacterium DOLZORAL124_64_63 (A0A2G6G0P7), Desulfobacterales bacterium (A0A2G6MY32), Deltaproteobacteria bacterium (A0A2G6NBT1), Paenibacillus sp. LK1 (A0A2G7LXT3), Streptococcus macedonicus (A0A2I1YFM9), Desulfuromonas sp. (A0A2J6IYE1), Paenibacillus sp. F4 (A0A2K1EFW7), Clostridium sp. chh4-2 (A0A2K2TT94), Thermoplasmata archaeon M9B1D (A0A2K3IZI5), Thermoplasmata archaeon M8B2D (A0A2K3JCM9), Thermoplasmata archaeon M9B2D (A0A2K3JJA1), Companilactobacillus alimentarius DSM 20249 (A0A2K9HJ87), Deltaproteobacteria bacterium CG03_land_8_20_14_0_80_45_14 (A0A2M7CB41), bacterium CG_4_9_14_3_um_filter_65_15 (A0A2M7YU10), Nitrospirae bacterium CG_4_9_14_0_8_um_filter_70_14 (A0A2M8DJV4), Firmicutes bacterium HGW-Firmicutes-8 (A0A2N2AIG7), Firmicutes bacterium HGW-Firmicutes-4 (A0A2N2BMX4), Firmicutes bacterium HGW-Firmicutes-4 (A0A2N2BPK7), Firmicutes bacterium HGW-Firmicutes-14 (A0A2N2DCM9), Firmicutes bacterium HGW-Firmicutes-11 (A0A2N2DSX1), Actinobacteria bacterium HGW-Actinobacteria-6 (A0A2N3FLA6), Desulfuromonas sp. (A0A2N6FER0), Desulfuromonas sp. (A0A2N6FGF7), Companilactobacillus nuruki (A0A2N7AVI8), Enterococcus avium (A0A2N8PRR7), Companilactobacillus formosensis(A0A2P4R446), Lactobacillus sp. CBA3605 (A0A2R3JR44), Lactobacillus sp. CBA3606 (A0A2R3JXA5), Candidatus Carbobacillus altaicus (A0A2R6Y310), Paenibacillus sp. CAA11 (A0A2S0UGH0), Lactiplantibacillus plantarum subsp. plantarum (A0A2S3U604), Lacrimispora xylanisolvens (A0A2S6HEL0), Lacrimispora xylanisolvens (A0A2S6HTJ8), Clostridium butyricum (A0A2S7F6K6), Marinicaulis flavus (A0A2S7KB80), Enterococcus faecium (A0A2S7M9B7), Veillonella sp. T11011-6 (A0A2S7YVS9), Paenibacillus sp. PCH8 (A0A2S8P0F7), Paenibacillus sp. MYb63 (A0A2S8X9W9), Lactiplantibacillus pentosus (A0A2S9W7C8), Moorella humiferrea (A0A2T0ATV6), Planifilum fimeticola (A0A2T0LGR7), Enterocloster lavalensis (A0A2T3FB26), Brockia lithotrophica (A0A2T5G4I4), Hydrogenibacillus schlegelii (A0A2T5G6K5), Paenibacillus elgii (A0A2T6G7B5), Intestinimonas butyriciproducens (A0A2U1BIY9), Heimdallarchaeota archaeon (strain B3-JM-08) (A0A2U3CIB2), Syntrophobacter sp. SbDl (A0A2U3KYS3), Clostridium drakei (A0A2U8DS61), Clostridia bacterium (A0A2V2CKW8), Bacillota bacterium (A0A2V2GSG4), Hungatella effluvii (A0A2V3YDN3), Paenibacillus barcinonensis (A0A2V4VRU8), Paenibacillus illinoisensis (A0A2W0C151), Paenibacillus silvae (A0A2W6ND21), Faecalicatena orotica (A0A2Y9B8V8), Paenibacillus donghaensis (A0A2Z2KT68), Methanosphaera sp. BMS (A0A2Z4LAE7), Methanohalophilus euhalobius (A0A314ZVY7), Desulfovibrionaceae bacterium (A0A316MIL5), Blautia sp. BCRC 81119 (A0A317UIA9), Dielma fastidiosa (A0A318KMT0), Dielma fastidiosa (A0A318KQV6), Methanosphaera sp. rholeuAM6 (A0A328RY38), Methanosphaera sp. SHI613 (A0A328SD25), Paenibacillus taichungensis (A0A329QMG5), Faecalibacterium prausnitzii (A0A329U641), Clostridium isatidis (A0A343JED7), Deltaproteobacteria bacterium (A0A348ZU29), Desulfovibrio sp. (A0A349HIL4), Lactobacillus sp. (A0A349MYS9), Lachnospiraceae bacterium (A0A349YFG1), Bacillota bacterium (A0A350X4B7), Acidaminococcaceae bacterium (A0A351EA30), Acetobacterium sp. (A0A351HUF1), Syntrophorhabdus aromaticivorans (A0A351U313), Clostridium sp. (A0A352NRP8), Deltaproteobacteria bacterium (A0A352Z5X4), Desulfobacteraceae bacterium (A0A353H9D1), Clostridiaceae bacterium (A0A355S0P4), Neomarinimicrobiota bacterium (A0A355XNN0), Desulfosporosinus sp. (A0A356PFN6), Desulfotomaculum sp. (A0A356UHF0), Paenibacillus sp. (A0A357M7J6), Bacillota bacterium (A0A357NEK7), Peptococcaceae bacterium (A0A357VIK1), Paenibacillus sp. (A0A358GJ07), Desulfosporosinus sp. (A0A358Q2T6), Desulfotomaculum sp. (A0A358QW72), Desulfotomaculum sp. (A0A359B244), Alkalibaculum bacchi (A0A366IAJ6), Enterococcus durans (A0A367CKF2), Blautia obeum (A0A367G4G8), Fontibacillus phaseoli (A0A369BPP6),Blautia sp. AM23-13AC (A0A373GSN9), Blautia sp. AF32-4BH (A0A373MZD5), Blautia sp. AF26-2 (A0A373S1W3), Anaerostipes sp. AF04-45 (A0A374AHE9), Firmicutes bacterium AF12-30 (A0A374AR31), Anaerobutyricum hallii (A0A374NUQ2), Hungatella hathewayi (A0A374P5W9), Collinsella sp. TM06-3 (A0A374QYP9), Collinsella sp. TM05-37 (A0A374RPM9), Collinsella sp. TF11-5AC (A0A374TIG1), Collinsella sp. TF10-11AT (A0A374TP27), Collinsella sp. TF08-11AT (A0A374TZ28), Collinsella sp. TF07-1 (A0A374U509), Collinsella sp. TF06-6AC (A0A374UCK9), Collinsella sp. OM06-18AC (A0A374W142), Collinsella sp. OM04-5 (A0A374X042), Enterococcus durans (A0A377KIQ9), Enterococcus durans (A0A377L0J0), Paenibacillus polymyxa (A0A378Y3D3), Paenibacillus alvei (A0A383RDL3), Companilactobacillus zhachilii (A0A386PPW0), Collinsella sp. AM36-4AA (A0A396A6K3), Collinsella sp. AM17-1 (A0A396CK33), Collinsella sp. AM16-21 (A0A396CUA0), Collinsella sp. AF36-3AT (A0A396F5D1), Blautia sp. TM10-2 (A0A396P6Z8), Lachnospiraceae bacterium OF09-33XD (A0A396QJN0), Lachnospiraceae bacterium OF09-33XD (A0A396QPZ8), Paenibacillus thiaminolyticus (A0A3A3GJ07), Omnitrophica bacterium (A0A3A4NYB8), Erysipelotrichaceae bacterium AF15-26LB (A0A3A6CZZ3), Lachnospiraceae bacterium TF09-5 (A0A3A6GUW2), Ammonifex sp. (A0A3A6N5Y4), Dethiobacter sp. (A0A3A6NQW2), Desulfarculus sp. (A0A3A6NXG1), Parablautia intestinalis (A0A3A9AR96), Thermoanaerobacteraceae bacterium SP2 (A0A3A9JW67), Syntrophobacteraceae bacterium (A0A3B8HMU8), Bacillota bacterium (A0A3B8K340), Treponema sp. (A0A3C0BPD9), Bacillota bacterium (A0A3C1YDA8), Syntrophomonas sp. (A0A3D1QCN1), Acidaminococcaceae bacterium (A0A3D2QAT4), Lachnospiraceae bacterium (A0A3D4XFD7), Ammonifex thiophilus (A0A3D8P3M7), Lacrimispora amygdalina (A0A3E2N8R5), Enterocloster citroniae (A0A3E2VJA1), Hungatella hathewayi (A0A3E2WEM8), Hungatella hathewayi (A0A3E2WXE1), Hungatella hathewayi (A0A3E2X2D2), Hungatella hathewayi (A0A3E3DRL5), Hungatella hathewayi (A0A3E4UCN9), Formimonas warabiya (A0A3G1KXL2), Biomaibacter acetigenes (A0A3G2R978), Paenibacillus sp. M-152 (A0A3G8R7A3), Phascolarctobacterium faecium (A0A3G9GS81), Thorarchaeota archaeon (strain OWC) (A0A3L6JJJ0), Thorarchaeota archaeon (strain OWC) (A0A3L6JKZ2), Thorarchaeota archaeon (strain OWC) (A0A3L6JPJ1), Clostridium autoethanogenum (A0A3M0SDQ6), Nitrospirota bacterium (A0A3M1R4H3), Schekmanbacteria bacterium (A0A3M1RXV5), Methanohalophilus sp. RSK (A0A3M9LKZ7), Enterococcus faecium (A0A3N3V083), Thermodesulfitimonas autotrophica (A0A3N5AWN5), Desulfobacteraceae bacterium (A0A3N5RNJ9), Calditrichota bacterium (A0A3N9MX51),Paenibacillus rhizophilus (A0A3N9Q3K7), Desulfovibrio sp. OH1186_COT-O70 (A0A3P1ZW98), Paenibacillus oralis (A0A3P3UC99), Lactobacillus helveticus (A0A3Q8SUG1), Paenibacillus lutimineralis (A0A3Q9I8R0), Clostridium sp. AF19-22AC (A0A3R6SSU1), Lactiplantibacillus garii (A0A3R8J6T9), Paenibacillus anaericanus (A0A3S1BSB4), Paenibacillus lentus (A0A3S8RX00), Clostridium sp. AWRP (A0A3T0LVS1), Lentilactobacillus curieae (A0A401FHZ9), Collinsella sp. AF23-6 (A0A412HKL1), Collinsella sp. AF23-3LB (A0A412HR45), Collinsella sp. AF19-1LB (A0A412N324), Collinsella sp. AF16-8 (A0A412SW06), Collinsella sp. AF15-51 (A0A412UIE6), Collinsella sp. AF14-35 (A0A412XHD7), Enterocloster bolteae (A0A412ZBL5), Collinsella sp. AF11-11 (A0A413CJD6), Collinsella sp. AF05-8-2 (A0A413DY49), Collinsella sp. AF04-24 (A0A413F3N9), Enterocloster asparagiformis (A0A413F7M2), Anaerotruncus sp. AF02-27 (A0A413G5D8), Collinsella sp. AM44-11 (A0A413RCA7), Collinsella sp. AM42-18AC (A0A413TJT8), Collinsella sp. AM41-2BH (A0A413UIW6), Collinsella sp. AM40-7AC (A0A413UN00), Roseburia intestinalis (A0A413ZBY2), Enterocloster bolteae (A0A414AZB4), Anaerobutyricum hallii (A0A414B1W9), Collinsella sp. AM34-10 (A0A414C6W1), Collinsella sp. AM31-2AC (A0A414EUG0), Collinsella sp. AM24-1 (A0A414PZS5), Collinsella sp. AM23-17 (A0A414RS16), Dorea longicatena (A0A414S0F3), Collinsella sp. AM20-15AC (A0A414VGW4), Collinsella sp. AM 18-10 (A0A414WFX5), Collinsella sp. AM 15-2 (A0A415A444), Collinsella sp. AM13-34 (A0A415BIV8), Collinsella sp. AF39-11AT (A0A415I4Q7), Collinsella sp. AF37-9 (A0A415KRN0), Anaerobutyricum hallii (A0A415UBQ6), Collinsella sp. AF31-11 (A0A415USL8), Collinsella sp. AF29-7AC (A0A415V7T8), Lachnospiraceae bacterium AM21-21 (A0A415W6E8), Lachnospiraceae bacterium AM10-38 (A0A415YIH4), Blautia sp. AF34-10 (A0A416EUK5), Eisenbergiella sp. OF01-20 (A0A416HWH2), Firmicutes bacterium AF25-13AC (A0A416MP43), Blautia sp. AF19-13LB (A0A416RYA9), Blautia sp. AF19-1 (A0A416SWC6), Blautia sp. AF17-9LB (A0A416U3I2), Blautia sp. AF14-40 (A0A416XSX3), Blautia sp. AM47-4 (A0A416ZRP7), Blautia sp. AM28-10 (A0A417HYD7), Lachnospiraceae bacterium AM26-1LB (A0A417JRR5), Firmicutes bacterium TM09-10 (A0A417LFY0), Blautia sp. OM05-6 (A0A417SQQ3), Lachnospiraceae bacterium OM02-26 (A0A417V532), Blautia sp. OF09-25XD (A0A417WVQ6), Ammoniphilus oxalaticus (A0A419SL18), Lacrimispora algidixylanolytica (A0A419SU55), Lacrimispora algidixylanolytica (A0A419T7Q3), Methanosalsum natronophilum (A0A424YWF4), Epsilonproteobacteria bacterium (ex Lamellibrachia satsuma) (A0A426VXC8), Methanohalophilus sp. (A0A427UB02), Bifidobacterium callimiconis (A0A430FBS3), Deltaproteobacteria bacterium (A0A432H9T4), Deltaproteobacteria bacterium(A0A432HU23), Paenibacillus zeisoli (A0A433XR49), Methanosuratincola subterraneus (A0A444L617), uncultured Blautia sp. (A0A448W485), Veillonella tobetsuensis (A0A480B800), Oenococcus oeni (A0A483AZM9), Methanimicrococcus blatticola (A0A484F5N1), anaerobic digester metagenome (A0A485M7X1), Desulfofundulus salinus (A0A494WUH2), Ruminococcus sp. B05 (A0A494ZTL8), Spirochaetota bacterium (A0A496QN71), Deltaproteobacteria bacterium (A0A496ZF69), Chloroflexota bacterium (A0A497C640), Thermoplasmata archaeon (A0A497H7L2), Thermoplasmata archaeon (A0A497H9I6), Candidatus Bathyarchaeota archaeon (A0A497NC14), Thorarchaeota archaeon (strain OWC) (A0A497PGE7), Thorarchaeota archaeon (strain OWC) (A0A497Q0C5), Thorarchaeota archaeon (strain OWC) (A0A497QBB6), Thorarchaeota archaeon (strain OWC) (A0A497QJK6), Anaerotruncus massiliensis (ex Liu et al. 2021) (A0A498CN95), Methanosarcina sp. MSH10X1 (A0A498GTM3), Methanohalophilus sp. WG1-DM (A0A498HBD8), Lucifera butyrica (A0A498R2T8), Methanolobus halotolerans (A0A4E0Q8K0), Moorella sp. E306M (A0A4P6BJQ9), Blautia producta (A0A4P6LYV0), Periweissella cryptocerci (A0A4P6YTI4), Desulfovibrio desulfuricans (A0A4P7UKU2), Anaerostipes rhamnosivorans (A0A4P8I7Y9), Blautia sp. SC05B48 (A0A4P9EGW2), Levilactobacillus suantsaii (A0A4Q0VIG4), Anaerobacillus alkaliphilus (A0A4Q0VPA0), Blautia sp. aa_0143 (A0A4Q5F429), Cuneatibacter caecimuris (A0A4Q7PN73), Mediterraneibacter sp. gm002 (A0A4Q8TWU4), Lactiplantibacillus paraplantarum (A0A4Q9Y6F1), Kineothrix alysoides (A0A4R1QS34), Hydrogenispora ethanolica (A0A4R1S012), Hydrogenispora ethanolica (A0A4R1S9Z5), Tepidibacillus fermentans (A0A4R3KDY1), Dehalobacter sp. 12DCB1 (A0A4R4CVY5), Paenibacillus albiflavus (A0A4R4EC38), Companilactobacillus farciminis (A0A4R5NCW5), Secundilactobacillus malefermentans (A0A4R5NL61), Lachnospiraceae bacterium (A0A4S2H7I4), Desulfopila sp. IMCC35006 (A0A4U1ADQ3), Paenibacillus terrae (A0A4U2PMW2), Paenibacillus sp. CFBP13512 (A0A4U3FAG8), Enterococcus sp. VV15 (A0A4U4EJG1), Ruminiclostridium herbifermentans (A0A4U7JJN9), Blautia faecicola (A0A4V1NSB8), Cohnella fermenti (A0A4V3WGD6), Vibrio inusitatus NBRC 102082 (A0A4Y3HXS6), Vibrio inusitatus NBRC 102082 (A0A4Y3HYT8), Enterococcus sp. NBRC 3427 (A0A4Y3JPK4), Saccharibacillus brassicae (A0A4Y6V065), Pelotomaculum sp. FP (A0A4Y7RLM6), Pelotomaculum propionicicum (A0A4Y7RRI9), Levilactobacillus suantsaiihabitans (A0A4Z0JCB9), Companilactobacillus suantsaicola (A0A4Z0JLB2), Desulfosporosinus sp. Sb-LF (A0A4Z0QR27), Desulfosporosinus fructosivorans (A0A4Z0R2F9), Desulfosporosinus fructosivorans (A0A4Z0R7K3),Lacticaseibacillus rhamnosus (A0A508YKR3), Vibrio superstes NBRC 103154 (A0A511QNH6), Sporomusa termitida (A0A517DVU3), Sporomusa termitida (A0A517DWL3), Brevibacillus laterosporus (A0A518V2L9), Anaerolineaceae bacterium (A0A521JGA9), Acidobacteriota bacterium (A0A522MK01), Candidatus Methanomethylicota archaeon (A0A523BD82), Candidatus Methanomethylicota archaeon (A0A523BDW3), Thorarchaeota archaeon (strain OWC) (A0A523RN51), Thorarchaeota archaeon (strain OWC) (A0A523S594), Dehalococcoidia bacterium (A0A523UK90), Anaerolineales bacterium (A0A524A880), Dehalococcoidia bacterium (A0A524AM18), Thorarchaeota archaeon (strain OWC) (A0A524C3P7), Thorarchaeota archaeon (strain OWC) (A0A524CU89), Thorarchaeota archaeon (strain OWC) (A0A524EBG6), Thorarchaeota archaeon (strain OWC) (A0A524EJV1), Thorarchaeota archaeon (strain OWC) (A0A524FSG5), Thorarchaeota archaeon (strain OWC) (A0A524FV15), Thorarchaeota archaeon (strain OWC) (A0A524G4F7), Thorarchaeota archaeon (strain OWC) (A0A524LBU4), Methanosarcina sp. (A0A524M0W8), Thorarchaeota archaeon (strain OWC) (A0A524MDW3), Chloroflexi bacterium B3_Chlor (A0A532U9R9), bacterium (A0A533SHC4), Lactobacillus sp. LL6 (A0A556UCE9), Paenibacillus vietnamensis (A0A559IZW2), Dorea longicatena (A0A564SR72), Blautia luti (A0A564W529), Paenibacillus sp. B2(2019) (A0A5B0W2P1), Companilactobacillus futsaii (A0A5B7SZ70), Caloramator sp. E03 (A0A5B7TG46), Levilactobacillus brevis (A0A5B7Y2G6), Loigolactobacillus coryniformis (A0A5B8TIN2), Thorarchaeota archaeon (strain OWC) (A0A5C9E313), Paenibacillus faecis (A0A5D0CV43), Acetobacterium wieringae (A0A5D0WJV5), Uncultured archaeon (A0A5E4I4T9), Uncultured archaeon (A0A5E4L9H6), Weizmannia acidilactici (A0A5J4JDK4), Paenibacillus spiritus (A0A5J5GGB7), Collinsella aerofaciens (A0A5K1I8G6), Collinsella aerofaciens (A0A5K1J6P6), Paenibacillus sp. UASWS1643 (A0A5M9TLE7), Paenibacillus amylolyticus (A0A5M9WW81), Enterococcus durans (A0A5N0YY24), Companilactobacillus halodurans (A0A5P0ZQH5), Lacticaseibacillus manihotivorans (A0A5P8JTQ1), Lentilactobacillus parafarraginis (A0A5R9CXK0), bioreactor metagenome (A0A644SUI4), bioreactor metagenome (A0A644UHJ0), bioreactor metagenome (A0A644V1C0), bioreactor metagenome (A0A644VD38), bioreactor metagenome (A0A644ZJG0), bioreactor metagenome (A0A645AB96), candidate division MSBL1 archaeon SCGC-AAA259J03 (A0A656YWJ1), Lactiplantibacillus mudanjiangensis (A0A660E3E1), Methanosarcina flavescens (A0A660HRE3), Brockia lithotrophica (A0A660L3R0), Spirochaetota bacterium (A0A660UF93), Campylobacterota bacterium (A0A661I218), Deltaproteobacteria bacterium(A0A661ICR8), Deltaproteobacteria bacterium (A0A661IHQ3), Deltaproteobacteria bacterium (A0A661IQL0), Deltaproteobacteria bacterium (A0A661JQC0), Deltaproteobacteria bacterium (A0A661K645), Deltaproteobacteria bacterium (A0A661KMH1), Deltaproteobacteria bacterium (A0A661L9S9), Deltaproteobacteria bacterium (A0A661LDL1), Deltaproteobacteria bacterium (A0A661LTM1), Deltaproteobacteria bacterium (A0A661Q240), Deltaproteobacteria bacterium (A0A661SXB7), Candidatus Aminicenantes bacterium (A0A662D2L0), Acidobacteriota bacterium (A0A662DSR6), Acidobacteriota bacterium (A0A662EGP8), Thermoplasmata archaeon (A0A662KB11), Thermoplasmata archaeon (A0A662LKC6), Thermoplasmata archaeon (A0A662LNY5), Thermoplasmata archaeon (A0A662LPR5), Thermococci archaeon (A0A662PET8), Lactobacillus helveticus (A0A6A7K2P3), Alkalibaculum sporogenes (A0A6A7KB05), Ligilactobacillus salivarius (A0A6A8LPM3), Lactobacillus porci (A0A6A8ME57), Weissella muntiaci (A0A6C2CBF8), Paenibacillus sp. SYP-B3998 (A0A6G3ZW22), Desulfovibrio legallii (A0A6H3FED7), Turicibacter sanguinis (A0A6I3NBW0), Moorella glycerini (A0A6I5ZQH4), Clostridium bovifaecis (A0A6I6F6M9), Enterococcus sp. T0101B.F-10 (A0A6I7ZD36), Candidatus Desulfovibrio kirbyi (A0A6L2R4M0), Desulfovibrio porci (A0A6L5XHP4), Roseburia porci (A0A6L5YTY1), Roseburia intestinalis (A0A6L6L7A9), Collinsella aerofaciens (A0A6L8RLT0), Dorea longicatena (A0A6L8S0P0), Blautia massiliensis (ex Durand et al. 2017) (A0A6L8TAZ9), Paenibacillus apii (A0A6M1PCL7), Enterocloster bolteae (A0A6N2S8Q1), Anaerostipes caccae (A0A6N2UYX4), Anaerostipes hadrus (A0A6N2VKP1), Flavonifractor plautii (A0A6N2YAR4), Intestinibacter bartlettii (A0A6N2YSP7), Enterococcus faecium (A0A6N2ZVZ2), Clostridium butyricum (A0A6N3D9A3), Collinsella aerofaciens (A0A6N3EKI1), Flavonifractor plautii (A0A6N3FLI1), Desulfovibrio sp. (A0A6N3I5C5), Hungatella hathewayi (A0A6N3I600), Amedibacterium intestinale (A0A6N4THU5), Desulfofundulus thermobenzoicus (A0A6N7IM37), Lactonifactor sp. BIOML-A7 (A0A6N7SKQ8), Olsenella porci (A0A6N7XSI0), Anaerobutyricum soehngenii (A0A6N7XZK2), Bacillota bacterium (A0A6N8B8F2), Desulfovibrio sp. (A0A6N8BPU7), Paenibacillus macerans (A0A6N8EYG4), Furfurilactobacillus rossiae (A0A6N9I3V1), Ruminococcaceae bacterium BL-6 (A0A6S6XAU3), Thermoplasmata archaeon (A0A6V8F2P9), Desulfobacterales bacterium (A0A7C0UCV4), Pseudomonadota bacterium (A0A7C0VJM7), Methanomicrobia archaeon (A0A7C0X008), Thermoplasmatales archaeon (A0A7C1BAM3), Ammonifex degensii (A0A7C1F3U9), Anaerolineae bacterium (A0A7C1JPX7), Bacillota bacterium (A0A7C1K7E1), Desulfobacterales bacterium (A0A7C1NL38), Chloroflexotabacterium (A0A7C2F4R0), Candidatus Methylomirabilota bacterium (A0A7C2IRK6), Deltaproteobacteria bacterium (A0A7C2VPV6), Candidatus Bathyarchaeota archaeon (A0A7C2VR49), Pseudomonadota bacterium (A0A7C3E6G3), Candidatus Methanomethylicus mesodigestus (A0A7C3J518), Deltaproteobacteria bacterium (A0A7C3N0Z2), Candidatus Methanomethylicus sp. (A0A7C3TTR7), Micrarchaeota archaeon (A0A7C3Z4I1), Pseudomonadota bacterium (A0A7C4ANC5), Hadesarchaea archaeon (A0A7C4AVM4), Candidatus Methanomethylicus sp. (A0A7C4CZM1), Candidatus Bathyarchaeota archaeon (A0A7C4DA31), Candidatus Bathyarchaeota archaeon (A0A7C4H7M4), Candidatus Methanomethylicus sp. (A0A7C4KX05), Candidatus Bathyarchaeota archaeon (A0A7C4ZTQ5), Methanomicrobia archaeon (A0A7C5DWN3), Campylobacterota bacterium (A0A7C5X0E8), Acidobacteriota bacterium (A0A7C5XWJ6), Papillibacter sp. (A0A7C6BTJ2), Deltaproteobacteria bacterium (A0A7C6F7S6), Peptococcaceae bacterium (A0A7C6NBR1), Bacillota bacterium (A0A7C6S7E6), Thermoanaerobacterales bacterium (A0A7C6UL08), Desulfotomaculum sp. (A0A7C6V4H2), Bacillota bacterium (A0A7C7DC84), Candidatus Bathyarchaeota archaeon (A0A7C7UET1), Anaerolineae bacterium (A0A7C7UPE9), Desulfarculaceae bacterium (A0A7C7VGL1), Furfurilactobacillus rossiae (A0A7C9IVT1), Methanolobus zinderi (A0A7D5E953), Clostridium intestinale (A0A7D6ZJ94), Cohnella cholangitidis (A0A7G5C5N6), Wansuia hejianensis (A0A7G9GBA3), Ligilactobacillus saerimneri (A0A7H9EMG0), Anaeromyxobacter diazotrophicus (A0A7I9VJD9), Candidatus Methanomethylicia archaeon (A0A7J2JGU1), Methanomicrobia archaeon (A0A7J2MQU4), Methanobacteriota archaeon (A0A7J2UUB0), Thermoplasmatales archaeon (A0A7J2UVS6), Candidatus Bathyarchaeota archaeon (A0A7J3NLS3), Candidatus Methanosuratincola petrocarbonis (A0A7J3UYI3), Thermoplasmata archaeon (A0A7J4IFC2), Thermoplasmata archaeon (A0A7J4KII9), Methanosarcinaceae archaeon (A0A7J4PHW0), Methanobacteriota archaeon (A0A7J9Q0W3), Thorarchaeota archaeon (strain OWC) (A0A7K4IEU3), Methanosarcinales archaeon (A0A7L4QXH3), Companilactobacillus pabuli (A0A7L7KW16), Sulfurovum indicum (A0A7M1S3T6), Paenibacillus sp. JNUCC-31 (A0A7M2A9A7), Lacticaseibacillus rhamnosus (strain ATCC 53103 / LMG 18243 / GG) (A0A7S7FR92), Clostridiales bacterium (A0A7T7ANA0), Sulfurovum lithotrophicum (A0A7U4RRC4), Clostridium butyricum 60E.3 (A0A7U9D540), Desulfobacteraceae bacterium (A0A7V0JLQ5), Deltaproteobacteria bacterium (A0A7V0P5E6), Pseudomonadota bacterium (A0A7V0U3P4), Spirochaetota bacterium (A0A7V1H3G6), Rhodospirillales bacterium (A0A7V2EYU5),Deltaproteobacteria bacterium (A0A7V2YDE0), Deltaproteobacteria bacterium (A0A7V4A779), Deltaproteobacteria bacterium (A0A7V4PJ75), Acidobacteriota bacterium (A0A7V4Y3Y8), Deltaproteobacteria bacterium (A0A7V4ZR39), Deltaproteobacteria bacterium (A0A7V5K537), Caldithrix abyssi (A0A7V5UDX1), Clostridia bacterium (A0A7V6KVM5), Thermoanaerobacterales bacterium (A0A7V6T981), Thermoanaerobacterales bacterium (A0A7V6UCU8), Clostridia bacterium (A0A7V6WH99), Enterococcus faecium (A0A7V7GKF2), Enterococcus lactis (A0A7W1XHZ2), Thermoactinomyces mirandus (A0A7W1XQG3), Fontibacillus solani (A0A7W3SQ55), Desulfovibrio intestinalis (A0A7W8C1D8), Clostridium gasigenes (A0A7X0SEE1), Lactococcus hircilactis (A0A7X1Z7A5), Paenibacillus monticola (A0A7X2H6U9), Eubacterium sp. BIOML-A2 (A0A7X2JD16), Eubacterium sp. BIOML-A2 (A0A7X2JGE2), Eubacterium sp. BIOML-A2 (A0A7X2JHH0), Phascolarctobacterium faecium (A0A7X2XFR9), Paenibacillus woosongensis (A0A7X2Z4R9), Secundilactobacillus folii (A0A7X3C2E4), Paenibacillus dendrobii (A0A7X3IED7), Vibrio eleionomae (A0A7X4LKE0), Vibrio eleionomae (A0A7X4LM28), Thermoanaerobacterales bacterium (A0A7X6XS24), Desulfobulbaceae bacterium (A0A7X6YVH0), Syntrophomonadaceae bacterium (A0A7X7ADQ9), Peptococcaceae bacterium (A0A7X8DGB0), Syntrophomonadaceae bacterium (A0A7X8FCJ8), Clostridiaceae bacterium (A0A7X8W0Y2), Clostridiaceae bacterium (A0A7X8X6E1), Clostridia bacterium (A0A7X9A2P5), Desulfovibrio sp. (A0A7X9IGK6), Desulfovibrio sp. (A0A7X9II89), Streptococcus ratti (A0A7X9QHG5), Paenibacillus sp. SZ31 (A0A7X9YMP7), Gemmatimonadota bacterium (A0A7Y3E2L3), Desulfobacterales bacterium (A0A7Y3EL34), Paenibacillus xylanilyticus (A0A7Y6C438), Methanolobus vulcani (A0A7Z7AVG5), Paenibacillus sp. OK076 (A0A7Z7CG45), Methanolobus vulcani (A0A7Z8P1N1), Desulfovibrio sp. Gil (A0A807ZIG3), Paenibacillus sp. URB8-2 (A0A810E2C4), Pusillibacter faecalis (A0A810QF25), Turicibacter sp. HGF1 (A0A828RSZ8), Enterococcus faecium EnGen0003 (A0A828ZSX8), Enterococcus faecium EnGen0026 (A0A829A2P6), Enterococcus faecium EnGen0180 (A0A829F992), Enterococcus faecium EnGenO192 (A0A829FA51), Thermanaeromonas sp. C210 (A0A829ZUC9), Desulfobacteraceae bacterium (A0A831KIX3), Paenibacillaceae bacterium (A0A831TGC1), Candidatus Methanomethylicus sp. (A0A832FLU4), Methanosarcina sp. (A0A832L8U9), Methanosarcina acetivorans (A0A832S9X7), Methanosphaera sp. (A0A832SP37), Methanosphaera sp. (A0A832SXE1), Methanosarcina sp. (A0A832TUB5), Methanosarcina sp. (A0A832UAY4), Methanosarcina sp. (A0A832VTZ4), Nanoarchaeota archaeon (A0A833A376), Pediococcus sp. EKM201D(A0A833WQZ0), Lactiplantibacillus plantarum WJL (A0A837P993), Lactiplantibacillus pentosus DSM 20314 (A0A837R7X7), Companilactobacillus crustorum JCM 15951 (A0A837RLG7), Texcoconibacillus texcoconensis (A0A840QTY3), Paenibacillus sp. JGP012 (A0A841F4Y3), Anaerosolibacter carboniphilus (A0A841KQS4), Cohnella thailandensis (A0A841SV09), Methanomethylovorans sp. (A0A842KPR7), Candidatus Methanomethylicales archaeon (A0A842M0L8), Candidatus Methanosuratincola sp. (A0A842MD72), Candidatus Methanosuratincola sp. (A0A842MDR4), Hadesarchaea archaeon (A0A842MSP1), Promethearchaeota archaeon (A0A842QF24), Promethearchaeota archaeon (A0A842WUQ1), Thorarchaeota archaeon (strain OWC) (A0A842Y8L5), Thermoplasmata archaeon (A0A842ZKM6), Thermoplasmata archaeon (A0A843A0F7), Methanosarcinaceae archaeon (A0A843CL69), Methanosphaera sp. (A0A843H816), Thermoplasmatales archaeon (A0A843I3J4), Dorea longicatena (A0A845KPE5), Dehalobacter sp. 4CP (A0A845RQT2), Methanosarcina sp. (A0A847PTN7), Veillonellaceae bacterium (A0A847QH24), Desulfovibrio piger (A0A848CFX7), Methanococcoides sp. (A0A849Q3K3), Thorarchaeota archaeon (strain OWC) (A0A849QF30), Paenibacillus agri (A0A850ETQ4), Paenibacillus pabuli (A0A855Y413), Dehalobacter restrictus (A0A857DJJ7), Enterococcus avium (A0A8B5W089), Blautia sp. AF13-16 (A0A8G1WXX5), Paenibacillus polymyxa (A0A8I1IWE0), Acidaminococcaceae bacterium (A0A8I1WME6), Paenibacillus sp. PvR133 (A0A8I1XY78), Syntrophorhabdus sp. (A0A8I1ZNR4), Lawsonibacter hominis (A0A8J6J5U2), Gorillibacterium sp. (A0A8J6MVU4), Candidatus Sulfomarinibacter kjeldsenii (A0A8J6Y5N9), Thermicanus sp. (A0A8J6YR94), Mailhella sp. (A0A8J7R1U5), Theionarchaea archaeon (A0A8J8BMC6), Theionarchaea archaeon (A0A8J8BRY8), Theionarchaea archaeon (A0A8J8BZ37), Acididesulfobacillus acetoxydans (A0A8S0VXE2), Methanobacteriota archaeon (A0A8T3ZBS0), Thermoplasmatota archaeon (A0A8T4DVW3), Thorarchaeota archaeon (strain OWC) (A0A8T4E8I7), Thorarchaeota archaeon (strain OWC) (A0A8T4EI80), Eubacteriaceae bacterium Marseille-04139 (A0A8T4K5P0), Thermoplasmatota archaeon (A0A8T4VW66), Thorarchaeota archaeon (strain OWC) (A0A8T4W9R8), Thermoplasmatota archaeon (A0A8T4WJV7), Thermoplasmatota archaeon (A0A8T5P877), Thorarchaeota archaeon (strain OWC) (A0A8T5SLD9), Thorarchaeota archaeon (strain OWC) (A0A8T6R9I7), Anaerostipes butyraticus (A0A916VCQ6), Paenibacillus albidus (A0A917FBI1), Paenibacillus antibioticophila (A0A919XSV7), Paenibacillus apis (A0A919Y5N4), Paenibacillus azoreducens (A0A920CRL0), Paenibacillus sp. J45TS6 (A0A920DET2), Mailhella massiliensis (A0A921AUU6),Lapidilactobacillus dextrinicus (A0A921B5Q9), Lapidilactobacillus dextrinicus (A0A921DV37), Levilactobacillus hammesii (A0A921EZY6), Companilactobacillus farciminis (A0A921L9K0), Lachnoclostridium phocaeense (A0A921LDR6), Gemmatimonas sp. (A0A923ICP8), Acetobacterium paludosum (A0A923KXH2), Mediterraneibacter hominis (A0A923LGP5), Lachnospiraceae bacterium (A0A927VGP2), Lachnospiraceae bacterium (A0A927VQ98), Streptococcus gallolyticus (A0A927XAZ3), Eggerthellaceae bacterium (A0A928E894), Solobacterium sp. (A0A930F678), Nitrospinota bacterium (A0A931R2Y9), Tectomicrobia bacterium (A0A932CLE4), Tectomicrobia bacterium (A0A932HYG9), Candidatus Methylomirabilota bacterium (A0A932MHV7), Chloroflexota bacterium (A0A933EI97), Tectomicrobia bacterium (A0A933LQP4), Candidatus Methylomirabilota bacterium (A0A933MMA7), Desulfarculus sp. (A0A933Z2L0), Chloroflexota bacterium (A0A934LQG0), Clostridium aciditolerans (A0A934M4M4), bacterium (A0A935UNL9), bacterium (A0A935V772), bacterium (A0A935YKH9), Desulfobacteraceae bacterium (A0A937Q7F8), Deltaproteobacteria bacterium (A0A938RHY6), Mordavella massiliensis (A0A938XBT0), Collinsella sp. (A0A938Z1R5), Enterococcus sp. (A0A939C9Q3), Fusicatenibacter saccharivorans (A0A939CFX8), Peptococcaceae bacterium (A0A940HZR4), Oscillospiraceae bacterium (A0A940UQ63), Bacillota bacterium (A0A940VR16), Bacillota bacterium (A0A940VXH5), Deltaproteobacteria bacterium (A0A940ZJ23), Bacillota bacterium (A0A941AAT5), Clostridiales bacterium (A0A942WDZ3), Collinsella sp. (A0A943AUY9), Bacillota bacterium (A0A943FPQ0), Fusobacterium sp. (A0A943GED4), Collinsella sp. (A0A943HVW9), Eggerthella sp. (A0A943I1V6), Eggerthella sp. (A0A943J3D8), Eggerthella sp. (A0A943MYE4), Bacillota bacterium (A0A943YP13), Campylobacteraceae bacterium (A0A945DE91), Rhodospirillaceae bacterium (A0A945KRV3), Rhodospirillaceae bacterium (A0A946TRJ0), Gemmatimonadota bacterium (A0A947CD73), bacterium (A0A948Q6Q2), Chloroflexota bacterium (A0A948R9G9), Bacillota bacterium (A0A949BPT2), Desulforudis sp. (A0A949KGK5), Desulfarculus sp. (A0A949KN84), Theionarchaea archaeon (A0A949MZM2), Diplocloster agilis (A0A949NB63), Clostridium thailandense (A0A949TIX7), Clostridium thailandense (A0A949U3C9), Rhodospirillales bacterium (A0A951VP82), Paracoccaceae bacterium (A0A954AW88), bacterium (A0A960VMF7), Rhodobiaceae bacterium (A0A961S997), Clostridiales bacterium (A0A970G4X3), Syntrophomonadaceae bacterium (A0A970NG07), Clostridiales bacterium (A0A970VK92), Acidobacteriota bacterium (A0A970ZGT9), Gaiellales bacterium (A0A971BYG0), Clostridiaceae bacterium (A0A971J8I0),Clostridia bacterium (A0A971L5W0), Syntrophomonadaceae bacterium (A0A971T5B0), Clostridiaceae bacterium (A0A971UY26), Syntrophomonadaceae bacterium (A0A971W3Z8), Bacillota bacterium (A0A972ANV1), Bacillota bacterium (A0A972C309), Clostridiales bacterium (A0A972CF89), Clostridiales bacterium (A0A972CG29), Rhodospirillales bacterium (A0A972UQU9), Syntrophaceae bacterium (A0A972Z9C2), Sedimentibacter hydroxybenzoicus DSM 7310 (A0A974BN63), Paenibacillus sonchi (A0A974PAR7), Clostridia bacterium (A0A974UHZ7), Desulfonema magnum (A0A975BXD5), Vibrio ostreae (A0A975YM87), Gammaproteobacteria bacterium (A0A9C9KN06), Candidatus Scatomorpha intestinavium (A0A9D0ZFN0), Candidatus Choladousia intestinavium (A0A9D1AA45), Candidatus Enterenecusfaecium (A0A9D1CG99), Candidatus Scatomorpha merdipullorum (A0A9D1FEF5), Candidatus Scatomorpha pullistercoris (A0A9D1G3S3), Candidatus Avacidaminococcus intestinavium (A0A9D1MP35), Candidatus Levilactobacillus faecigallinarum (A0A9D1QSK0), Candidatus Blautia stercorigallinarum (A0A9D1TET4), Candidatus Anaerostipes excrementavium (A0A9D1WWZ4), Candidatus Anaerobutyricum stercoripullorum (A0A9D1X517), Candidatus Companilactobacillus pullicola (A0A9D1ZLW9), Candidatus Ligilactobacillus excrementigallinarum (A0A9D2AA64), Candidatus Eubacterium avistercoris (A0A9D2D338), Candidatus Allofournierella merdipullorum (A0A9D2E5G9), Bacillota bacterium (A0A9D2GE07), Candidatus Desulfovibrio intestinavium (A0A9D2HND0), Candidatus Anaerobutyricum stercoris (A0A9D2J7E7), Candidatus Lachnoclostridium pullistercoris (A0A9D2PF78), Candidatus Blautia avicola (A0A9D2QY19), Candidatus Blautia stercoripullorum (A0A9D2RA19), Candidatus Acutalibacter pullicola (A0A9D2SG81), Candidatus Blautia merdigallinarum (A0A9D2SIM3), Candidatus Anaerostipes avistercoris (A0A9D2T9X6), Candidatus Methylomirabilota bacterium (A0A9D6JFK6), Schekmanbacteria bacterium (A0A9D6ZCF2), Gammaproteobacteria bacterium (A0A9D8IZH9), Candidatus Cellulosilyticum pullistercoris (A0A9E2KBU1), Methanococcoides seepicolus (A0A9E5DAY9), Anaerolineae bacterium (A0A9E5NM30), Pelotomaculum sp. (A0A9E5Y7Y3), Lactobacillus helveticus (A0A9Q5G3Q9), Turicibacter bills (A0A9Q9CJ71), Paenibacillus mellifer (A0A9X1XXM4), Paenibacillus mangrovi (A0A9X2B2P1), Pelotomaculum isophthalicicum JI (A0A9X4JUL2), Pelotomaculum isophthalicicum JI (A0A9X4JVK6), Pelotomaculum isophthalicicum JI (A0A9X4JWE6), Turicibacter sanguinis (A0A9X4XFN3), Parablautia muri (A0A9X5BE36), Moorella stamsii (A0A9X7P4M7), Ruminococcus sp. AM26-12LB (A0A9X8GR11), Streptococcus gallolyticus (strain UCN34) (A0AA36JZU0), Oscillibacter sp. KLE1728 (A0AA37A964), Hungatella hathewayi (A0AA37NB55), Oscillospiraceae bacterium (A0AA37NMN9), Paenibacillus sp. PastF-3 (A0AA43L6U3), Paenibacillus sp. PastH-2 (A0AA43LXP4), Lactococcus taiwanensis (A0AA45KGT3), Mesoterricola Silvestris (A0AA48GM36), Mesoterricola sediminis (A0AA48GN15), Brevibacillus aydinogluensis (A0AA48M9X2), Thermoanaerobacterium sp. CMT5567-10 (A0AA49JMT5), Sedimentibacter sp. MB35-C1 (A0AA51DD28), Methanolobus sediminis (A0AA51UKR2), Methanolobus mangrovi (A0AA51YJ27), Lactobacillus paragasseri JV-V03 (A0AA87DJ24), Methanococcoides alaskense (A0AA90TXY2), Desulfovibrio desulfuricans (A0AA94HUY1), Paenibacillus woosongensis (A0AA95HZP7), Candidatus Cohnella colombiensis (A0AA95JBZ7), Paenibacillus sp. G2S3 (A0AA95RP78), Paenibacillus sp. MMS20-IR301 (A0AA96MIA6), Methanimicrococcus hongohii (A0AA96UZN3), Methanolapillus millepedarum (A0AA96V420), Methanolapillus ohkumae (A0AA96V7X7), Methanimicrococcus stummii (A0AA96ZY79), Paenibacillus odorifer (A0AAD0P6Q8), Lactiplantibacillus paraplantarum (A0AAD0X6P8), Candidatus Jordarchaeia archaeon (A0AAE2XS84), Candidatus Jordarchaeia archaeon (A0AAE2Y9V6), Anthropogastromicrobium aceti (A0AAE3E751), Hominifimenecus microfluidus (A0AAE3EAB9), Fusicatenibacter saccharivorans (A0AAE3JRH9), Methanolobus chelungpuianus (A0AAE3KY44), Enterococcus pseudoavium (A0AAE4L1D7), Methanolapillus africanus (A0AAE4MIR0), Brevibacillus sp. 7WMA2 (A0AAE7BTB1), Streptococcus gallolyticus (A0AAE7CVD3), Latilactobacillus sakei (A0AAE8LVX4), Paenibacillus polymyxa (A0AAE9IEC4), Odinarchaeota yellowstonii (strain LCB_4) (A0AAF0D3G1), Latilactobacillus sakei (A0AAF0GSK4), Paenibacillus suaedae (A0AAJ2N510), Paenibacillus polymyxa (A0AAJ3MD50), Levilactobacillus brevis (A0AAJ5FI40), Lactiplantibacillus argentoratensis (A0AAN1UH78), Paenibacillus sp. FSL R5-192 (A0AAN4L5N2), Tetragenococcus koreensis (A0AAN4UDE3), Methanooceanicella nereidis (A0AAP2RDG1), Brevibacillus laterosporus (A0AAP3G983), Paenibacillus polymyxa (A0AAP4EDG2), Paenibacillus amylolyticus (A0AAP5H0F4), Dorea longicatena (A0AAP7AVD6), Paenibacillus alvei (A0AAP7DKT8), Lacticaseibacillus rhamnosus (A0AAP7KKK0), Paenibacillus amylolyticus (A0AAP7Q6I6), Brevibacillus laterosporus (A0AAP8QD85), Paenibacillus thiaminolyticus (A0AAP9DSF3), Clostridium butyricum (A0AAP9RIE0), Lactiplantibacillus plantarum 2025 (A0AAQ0EGP1), Ligilactobacillus salivarius (A0AAQ3INP8), Clostridium sp. JS66 (A0AAU0NIZ8), Pediococcus pentosaceus CGMCC 7049 (A0AAU7NLU7), Paenibacillus sp. AN1007 (A0AAU8NLI1), Desulfofundulus kuznetsovii (strain DSM 6115 / VKM B-1805 / 17) (A0AAU8PAZ8), Desulfoferula mesophila (A0AAU9EVI9),Enterococcus faecium TX0133a01 (A0AAV3DPP6), Enterococcus faecium TX0133a04 (A0AAV3DYX1), Enterococcus faecium R496 (A0AAV3GS39), Enterococcus avium ATCC 14025 (A0AAV3J6P5), Enterococcus faecium 10 / 96A (A0AAV3L4S5), Clostridium diolis (A0AAV3W765), Lachnospiraceae bacterium (A0AAV5H9K3), Lactiplantibacillus plantarum CMPG5300 (A0AAW3FS68), Blautia fusiformis (A0AAW4W4X7), Enterocloster aldenensis (A0AAW5BT51), Blautia massiliensis (ex Durand et al. 2017) (A0AAW5CE83), Clostridium symbiosum (A0AAW5F7Z0), Flavonifractor plautii (A0AAW6CBW3), Flavonifractor plautii (A0AAW6CJQ5), Streptococcus pasteurianus (A0AAW6YKV1), Desulforamulus aquiferis (A0AA\N7ZAZ2), Enterococcus raffinosus (A0AAW8SWH5), Enterococcus raffinosus (A0AAW8T4D0), Enterococcus asini (A0AAW8U4C0), Lactiplantibacillus pentosus (A0AAW8WK60), Enterococcus sp. E5-162 (A0AAW9N135), Hungatella hathewayi (A0AAW9WDB1), Lactiplantibacillus plantarum (A0AAX1K9V9), Limosilactobacillus reuteri (A0AAX2SQL7), Paenibacillus kyungheensis (A0AAX3M037), Lactiplantibacillus pentosus (A0AAX6LDF4), Enterococcus faecium EnGenO265 (A0AB33YMU5), Lactobacillus gasseri (A0AB33ZV40), Companilactobacillus crustorum (A0AB34A8E3), Clostridium sp. IBUN125C (A0AB34SGH3), Lactobacillus paragasseri (A0AB35SG35), Dielma fastidiosa (A0AB35UTZ2), Paenibacillus odorifer (A0AB36JKI0), Enterococcus faecium (A0AB36M9U1), Lactobacillus gasseri (A0AB36X5V7), Lactiplantibacillus pentosus (A0AB37RJ33), Levilactobacillus brevis (A0AB38X5S6), Oenococcus oeni ATCC BAA-1163 (A0NKN9), Desulforamulus reducens (strain ATCC BAA-1160 / DSM 100696 / MI-1) (A4J6J5), Pelotomaculum thermopropionicum (strain DSM 13744 / JCM 10971 / SI) (A5D2C8), Sulfurovum sp. (strain NBC37-1) (A6Q6V6), Enterocloster bolteae (strain ATCC BAA-613 / DSM 15670 / CCUG 46953 / JCM 12243 / WAL 16351) (A8S1T2), Anaerostipes caccae (strain DSM 14662 / CCUG 47493 / JCM 13470 / NCIMB 13811 / Ll-92) (B0MFG8), Desulforudis audaxviator (strain MP104C) (B1I3P8), Desulfovibrio piger ATCC 29098 (B6WQK5), Desulfovibrio desulfuricans (strain ATCC 27774 / DSM 6949 / MB) (B8J0C3), [Clostridium] hylemonae DSM 15053 (C0C543), Blautia hydrogenotrophica (strain DSM 10507 / JCM 14656 / S5a33) (C0CJZ4), [Clostridium] asparagiforme DSM 15981 (C0D6N7), Anaerobutyricum hallii DSM 3353 (C0F061), Anaerobutyricum hallii DSM 3353 (C0F0L5), Lacticaseibacillus rhamnosus (strain LMS2-1) (C2JUS5), Clostridium butyricum E4str. BoNTEBL5262 (C4IC89), Maridesulfovibriosalexigens (strain ATCC 14822 / DSM 2638 / NCIMB 8403 / VKM B-1763) (C6BYZ1), Paenibacillus sp. oral taxon 786 str. D14 (C6J4V7), Clostridium carboxidivorans P7 (C6PZV7), Desulfofarcimen acetoxidans (strain ATCC 49208 / DSM 771 / KCTC 5769 / VKM B-1644 / 5575) (C8VWF5), Ammonifex degensii (strain DSM 10501 / KC4) (C9RC44), Methanocella paludicola (strain DSM 17711 / JCM 13418 / NBRC 101707 / SANAE) (D1YZW3), Hungatella hathewayi DSM 13479 (D3ACT3), Oenococcus oeni AWRIB429 (D3L9Y4), Anaerostipes hadrus (D4N0Y8), Methanohalophilus mahii (strain ATCC 35705 / DSM 5219 / SLP) (D5E7T0), Thermincola potens (strain JR) (D5XAY2), Methanohalobium evestigatum (strain ATCC BAA-1072 / DSM 3721 / NBRC 107634 / OCM 161 / Z-7303) (D7E873), Thermoanaerobacterium thermosaccharolyticum (strain ATCC 7956 / DSM 571 / NCIMB 9385 / NCA 3814 / NCTC 13789 / WDCM 00135 / 2032) (D9TTI7), Streptococcus eguinus ATCC 700338 ( E0PF37), Desulfarculus baarsii (strain ATCC 33931 / DSM 2075 / LMG 7858 / VKM B-1802 / 2stl4) (E1QJ98), Olsenella uli (strain ATCC 49627 / DSM 7084 / CCUG 31166 / CIP 109912 / JCM 12494 / LMG 11480 / NCIMB 702895 / VPI D76D-27C) (E1QWR0), Sediminispirochaeta smaragdinae (strain DSM 11293 / JCM 15392 / SEBR 4228) (E1R2B4), Erysipelotrichaceae bacterium 3 1 53 (E2SRD7), Paenibacillus polymyxa (strain SC2) (E3E5R1), Lachnospiraceae bacterium 5_1_63FAA (E5VJE4), Bilophila wadsworthia (strain 3_1_6) (E5Y2T7), Evansella cellulosilytica (strain ATCC 21833 / DSM 2522 / FERM P-1141 / JCM 9156 / N-4) (E6TXX6), Coprobacillus cateniformis (E7G709), Clostridium symbiosum (strain WAL-14163) (E7GN09), Lachnospiraceae bacterium 6_1_37FAA (E9RXQ9), Pseudodesulfovibrio mercurii (F0JC27), Syntrophobotulus glycolicus (strain DSM 8271 / FIGIyR) (F0T1K0), Clostridium sp. D5 (F0YVY7), Clostridium sp. D5 (F0Z553), Coriobacterium glomerans (strain ATCC 49209 / DSM 20642 / JCM 10262 / PW2) (F2N9Z3), Streptococcus pasteurianus (strain ATCC 43144 / JCM 5346 / CCUG 46074 / CDC 1723-81) (F5X2J5), Desulfotomaculum nigrificans (strain DSM 14880 / VKM B-2319 / CO-l-SRB) (F6B6Q3), Methanotorris igneus (strain DSM 5666 / JCM 11834 / Koi 5) (F6BE91), Desulforam ulus ruminis (strain ATCC 23193 / DSM 2154 / NCIMB 8452 / DL) (F6DRS8), Lactiplantibacillus pentosus MP-10 (F6IW09), Lachnospiraceae bacterium 3_1_57FAA_CT1 (F7KC10), Acetonema longum DSM 6540 (F7NM86), Clostridium sp. (strain SY8519) (F7V4Q6), Methanosalsum zhilinae (strain DSM 4017 / NBRC 107636 / OCM 62 / WeN5) (F7XQS0), Lactiplantibacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (F9UT68), Lactiplantibacillus pentosus IG1 (G0M3V2), Desulfovibrio sp. 6_1_46AFAA (G1UNN0), Bilophila sp. 4_l_30 (G1V5H2), Collinsella tanakaei YIT 12063 (G1WHG8), Desulfosporosinus sp. OT(G2FL85), [Clostridium] citroniae WAL-17108 (G5HGC5), Commensalibacter intestini A911 (G6F359), Paenibacillus terrae (strain HPL-003) (G7VSE7), Desulfosporosinus orientis (strain ATCC 19365 / DSM 765 / NCIMB 8382 / VKM B-1628 / Singapore I) (G7WBN1), Flavonifractor plautii ATCC 29863 (G9YQK8), Lentilactobacillus parafarraginis F0439 (G9ZNW3), Methanotorris formicicus Mc-S-70 (H1KXM1), Caldithrix abyssi DSM 13497 (H1XXS6), Paenibacillus dendritiformis C454 (H3SBW0), Desulfosporosinus youngiae DSM 17734 (H5Y504), Methanocella conradii (strain DSM 24694 / JCM 17849 / CGMCC 1.5162 / HZ254) (H8I7Y8), Enterococcus faecium (strain ATCC BAA-472 / TX0016 / DO) (I3TZ67), Desulfosporosinus acidiphilus (strain DSM 22704 / JCM 16185 / SJ4) (I4D7X5), Lactiplantibacillus pentosus KCA1 (I9AQV2), Liguorilactobacillus mali KCTC 3596 = DSM 20444 (J0URL1), Enterococcus faecium 505 (J7CWD3), Desulfosporosinus meridiei (strain ATCC BAA-275 / DSM 13257 / KCTC 12902 / NCIMB 13706 / S10) (J7IS31), Lactobacillus eguicursoris 66c (K0NQ29), Thermacetogenium phaeum (strain ATCC BAA-254 / DSM 26808 / PB) (K4LD63), Dehalobacter sp. CF (K4LF70), Desulforamulus hydrothermalis Lam5 = DSM 18033 (K8DWV8), Methanomethylovorans hollandica (strain DSM 15978 / NBRC 107637 / DMS1) (L0L1W5), Clostridium celatum DSM 1785 (L1Q572), Clostridium celatum DSM 1785 (L1Q5T3), Clostridium saccharoperbutylacetonicum N1-4(HMT) (M1MNA6), Methanosarcina mazei TucOl (M1P9V5), Levilactobacillus brevis KB290 (M5AGS2), Paenibacillus popilliae ATCC 14706 (M9M5C8), Schaedlerella arabinosiphila (N2A2I5), Pediococcus pentosaceus (strain ATCC 25745 / CCUG 21536 / LMG 10740 / 183-lw) (Q03HH3), Levilactobacillus brevis (strain ATCC 367 / BCRC 12310 / CIP 105137 / JCM 1170 / LMG 11437 / NCIMB 947 / NCTC947) (Q03P28), Oenococcus oeni (strain ATCC BAA-331 / PSU-1) (Q04EQ5), Methanocella arvoryzae (strain DSM 22066 / NBRC 105507 / MRE50) (Q0W0E2), Methanococcoides burtonii (strain DSM 6242 / NBRC 107633 / OCM 468 / ACE-M) (Q12W51), Moorella thermoacetica (strain ATCC 39073 / JCM 9320) (Q2RMA0), Latilactobacillus sakei subsp. sakei (strain 23K) (Q38Y43), Methanosarcina barkeri (strain Fusaro / DSM 804) (Q46C18), Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Gael / Gol / JCM 11833 / OCM 88) (Q8PVT8), Methanosarcina acetivorans (strain ATCC 35395 / DSM 2834 / JCM 12185 / C2A) (Q8TST3), Enterocloster bolteae 90B8 (R0ANH7), Enterocloster bolteae 90A9 (R0BSQ7), Enterococcus faecium EnGenO263 (R2MGT9), Enterococcus malodoratus ATCC 43197 (R2QVB2), Enterococcus raffinosus ATCC 49464 (R2S1P3), Enterococcus asini ATCC 700915 (R2SLT6), Enterococcus pollens ATCC BAA-351 (R2TBP8), Intestinibacter bartlettii CAG.1329 (R5X408), Phascolarctobacterium faecium (R6IEP8), Phascolarctobacterium faecium (R6IHM7), Phascolarctobacterium faecium (R6IIW1), Eggerthella sp. CAG:298 (R7B6N9), Eggerthella sp. CAG:368 (R7BNQ9), Cryptobacterium sp. CAG:338 (R7CLV1), Paenibacillus barengoltzii G22(R9LFX7), Oscillibacter sp. 1-3 (R9LXX7), Paenibacillus alvei TS-15 (S9SKH9), Dehalobacter sp. UNSWDHB (T0HZT5), Clostridiales bacterium oral taxon 876 str. F0540 (U2CLK3), Blautia sp. KLE 1732 (U2EKQ8), Clostridium intestinale URNW (U2NNC8), Levilactobacillus brevis ATCC 14869 = DSM 20054 (U2QXP0), Lactobacillus helveticus CIRM-BIA 953 (U4QFU3), Youngiibacter fragilis 232.1 (V7I3V9), Paenibacillus sp. FSL R7-277 (W4DPI0), Lactiplantibacillus fabifermentans T30PCM01 (W6TAH8), Methanolobus tindarius DSM 2278 (W9DQN3), marine sediment metagenome (X0SCR8), marine sediment metagenome (X1C853), marine sediment metagenome (X1CBH9), marine sediment metagenome (X1ECZ2), marine sediment metagenome (X1MNT9), marine sediment metagenome (X1UJX9), and Paenibacillus sabinae T27 (X4ZK34).

[0069] In Example 6, an advantageous effect on the productivity of a prFMN-dependent enzyme has been demonstrated for five different LpdD-like proteins, namely, LpdD-like proteins from Clostridium aciditolerans, [Clostridium] hylemonae DSM 15053, Clostridium saccharoperbutylacetonicum N1-4(HMT), Lachnoclostridium phocaeense and Lactiplantibacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (Lactobacillus plantarum).

[0070] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the LpdD-like protein is a LdpD-like protein from Clostridium aciditolerans (Uniprot accession number A0A934M4M4; SEQ ID NO:30), [Clostridium] hylemonae DSM 15053 (Uniprot accession number C0C543; SEQ ID NO:31), Clostridium saccharoperbutylacetonicum N1-4(HMT) (Uniprot accession number M1MNA6; SEQ ID NO:32), Lachnoclostridium phocaeense (Uniprot accession number A0A921LDR6; SEQ ID NO:33) or Lactiplantibacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (Lactobacillus plantarum) (Uniprot accession number F9UT68; SEQ ID NO:34).

[0071] In certain embodiments, the LpdD-like protein employed in the method of the present invention is a LpdD-like protein derived from Clostridium aciditolerans (Uniprot accession number A0A934M4M4; SEQ ID NQ:30), [Clostridium] hylemonae DSM 15053 (Uniprot accession number C0C543; SEQ ID NO:31), Clostridium saccharoperbutylacetonicum Nl-4(HMT) (Uniprot accession number M1MNA6; SEQ ID NO:32), Lachnoclostridium phocaeense(Uniprot accession number A0A921LDR6; SEQ ID NO:33) or Lactiplantibacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (Lactobacillus plantarum) (Uniprot accession number F9UT68; SEQ ID NO:34) having an amino acid sequence as shown in SEQ ID NQ:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34, respectively.

[0072] In certain embodiments, the LpdD-like protein is a protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 to 34 or a sequence which is at least n % identical to any of SEQ ID NOs: 30 to 34 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, wherein the protein retains the ability to bind to prFMNH2 and facilitate the oxidative maturation of prFMNFh into prFMN.

[0073] In a particular embodiment, the invention relates to the method according to the invention, wherein the LpdD-like protein comprises or consists of (i) an amino acid sequence as set forth in any one of SEQ ID NQ:30-34; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NQ:30-34, wherein the protein retains the ability to bind to prFMNFU and facilitate the oxidative maturation of prFMNF into prFMN.

[0074] The polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor may also be a PhdC-like protein.

[0075] Thus, 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 the recombinant microorganism recombinantly expresses a PhdC-like protein.

[0076] A PhdC-like protein refers to a type of protein that functions similarly to PhdC, which is involved in the oxidative maturation of prFMNFh to its active coenzyme form, prFMN. PhdC-like proteins were described by DiRocco et al. (ACS Catalysis, 2024, 14, 10223-10233) tofacilitate the conversion of the prFMN semiquinone radical to the fully oxidized, catalytically active prFMN cofactor using molecular oxygen.

[0077] However, it was surprisingly demonstrated herein that PhdC, similarly to LpdD, can facilitate the formation of the active prFMN cofactor even in the absence of molecular oxygen under anaerobic process conditions. This finding expands the potential applications of PhdC-like proteins in anaerobic environments, enhancing the productivity of prFMN-dependent enzymes without the need for oxygen.

[0078] It has been demonstrated in Example 8 that PhdC from Mycolicibacterium fortuitum (Mycobacterium fortuitum) (Uniprot accession number A0AAE5AEH3; SEQ ID NO:35) can enhance the productivity of a prFMN-dependent enzyme.

[0079] Thus, in certain embodiments, the PhdC-like protein employed in the method of the present invention is a PhdC-like protein derived from Mycolicibacterium fortuitum (Mycobacterium fortuitum) (Uniprot accession number A0AAE5AEH3) having an amino acid sequence as set forth in SEQ ID NO:35).

[0080] In a particular embodiment, the PhdC-like protein is a protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NOs:35 ora sequence which is at least n % identical to SEQ ID NO:35 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, wherein the protein retains the ability to bind to prFMNH2 and facilitate the oxidative maturation of prFMNH2 into prFMN.

[0081] It is known, for example from Bloor et al. (The FEBS Journal, 2023, 290, 2232-2245), that the maturation of prFMNH2 to prFMN involves multiple steps and the formation of various intermediates. Without being bound to theory, there is a possibility that LpdD and PhdC catalyze different steps in the maturation process of the prFMN cofactor. For instance, LpdD may be particularly effective at facilitating the initial oxidation of prFMNH2 to a prFMN radical, while PhdC may catalyze the subsequent oxidation of the prFMN radical to prFMNiminium, or vice versa. In such scenarios, the co-expression of LpdD and PhdC may further enhance the maturation of prFMN, thereby increasing the productivity of prFMN-dependent enzymes. This synergistic approach may optimize the efficiency of prFMN cofactor formation, leading to improved enzyme performance in various biotechnological applications.Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the recombinant cell recombinantly expresses a LpdD-like protein and a PhdD-like protein.

[0082] The prFMN-dependent enzyme

[0083] The prFMN cofactor, activated in the presence of a polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into the active prFMN cofactor, can then be utilized by a prFMN-dependent enzyme in the enzymatic production of various products.

[0084] 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.

[0085] In a particular embodiment, the prFMN-dependent enzyme is a prFMN-dependent decarboxylase.

[0086] 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 productsthrough either carboxylation or decarboxylation reactions.

[0087] 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 "prFMNF " or "prFMNFh 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 prFMNF precursor into the catalytically active prFMNiminium cofactor is described in detail by Bloor et al. in The FEBS Journal, 2023, 290, 2232-2245.

[0088] Non-limiting examples of prFMN-dependent carboxylases for use in the method of the present invention are provided herein below:

[0089] 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.

[0090] 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.

[0091] 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).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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.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).

[0099] 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.-.

[0100] 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.

[0101] 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.

[0102] 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).

[0103] 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.

[0104] In certain embodiments, the FDC-like enzyme is a fungal Fdcl (EC 4.1.1.102) from Hypocrea atroviridis (Uniprot accession number G9NLP8).

[0105] 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 N0:5. 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.

[0106] 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.

[0107] 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).

[0108] 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 asset forth in SEQ ID NO:6 ora 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.

[0109] 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.

[0110] 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 anamino 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.

[0111] 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.

[0112] In certain embodiments, the prFMN-dependent decarboxylase is a 4-hydroxy-3- polyprenylbenzoate decarboxylase (UbiD; EC 4.1.1.98).

[0113] 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).

[0114] 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.

[0115] In certain embodiments, the prFMN-dependent decarboxylase is a protocatechuate decarboxylase (EC 4.1.1.63).

[0116] Thus, in certain embodiments, the carboxylation or decarboxylation of a substrate is catalysed by a protocatechuate (PCA) decarboxylase (EC 4.1.1.63).

[0117] 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.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 derived from 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.

[0118] In certain embodiments, the prFMN-dependent decarboxylase is a 2,5-furandicarboxylic acid (FDCA) decarboxylase.

[0119] FDCA decarboxylases have been described to catalyse the carboxylation of 2-furoic acid (Payne et al., ACS Catal., 2019, 9(4):2854-2865).

[0120] 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.

[0121] 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.

[0122] The flavin prenyltransferase

[0123] The method according to the invention requires the presence of a prFMN cofactor. The precursor of the prFMN cofactor, i.e., the reduced form prFMNFh, is preferably produced bythe 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.

[0124] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the recombinant microorganism expresses a flavin prenyltransferase.

[0125] 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.

[0126] 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.

[0127] 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:

[0128] 3-octaprenyl-4-hydroxybenzoate = 2-octaprenylphenol + CO2.

[0129] 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.In Lactiplantibacillus plantarum, the protein LpdB has been shown to be associated to the gallate decarboxylase LpdC that catalyses the reactions:

[0130] 3,4,5-trihydroxybenzoate + H+ = 1,2,3-trihydroxybenzene + CO2; and

[0131] 3,4-dihydroxybenzoate + H+ = catechol + CO2

[0132] Non-limiting examples of flavin prenyltransferases of the PAD1, LpdB or UbiX-type will be provided herein below:

[0133] 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.

[0134] 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.

[0135] 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).

[0136] 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).

[0137] 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 groupconsisting 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.

[0138] In certain embodiments, the modification of FMNH2 into prFMNFh 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.

[0139] In certain embodiments, the flavin prenyltransferase which modifies FMNH2 into prFMNFh 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).

[0140] 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 NQ:20) or Enterobacter sp. DC4 (Uniprot accession number W7P6B1; SEQ ID NO:21).

[0141] 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.

[0142] 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).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.

[0143] In certain embodiments, the modification of FMNH2 into prFMNF is catalysed by an UbiX-like flavin prenyltransferase derived from Lactiplantibacillus plantarum encoded by LpdB (Uniprot accession number F9UT67; SEQ ID NO:25).

[0144] 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.

[0145] In certain embodiments, the modification of FMNH2 into prFMNH2 is catalysed by an UbiX-like flavin prenyltransferase derived from Clostridium aciditolerans (Uniprot accession number A0A934HV38; SEQ ID NO:26), [Clostridium] hylemonae DSM 15053 (Uniprot accession number C0C544; SEQ ID NO:27), Clostridium saccharoperbutylacetonicum N1-4(HMT) (Uniprot accession number M1MM91; SEQ ID NO:28), or Lachnoclostridium phocaeense (Uniprot accession number A0A921LDR2; SEQ ID NO:29).

[0146] In certain embodiments, the UbiX-like flavin prenyltransferase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 29 or a sequence which is at least n % identical to any of SEQ ID NOs: 26 to 29 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, wherein the enzyme retains the enzymatic activity of modifying FMNH2 into prFMNH2.

[0147] In certain embodiments, the recombinant microorganism used in the method of the invention comprises an LpdD variant and a UbiX-like flavin prenyltransferase derived from the same species.In certain embodiments, the recombinant microorganism used in the method of the invention comprises an LpdD variant from Lactiplantibacillus plantarum (Uniprot accession number F9UT68; SEQ ID NO:34) and a UbiX-like flavin prenyltransferase from Lactiplantibacillus plantarum (Uniprot accession number F9UT67; SEQ ID NO:25).

[0148] In certain embodiments, the recombinant microorganism used in the method of the invention comprises an LpdD variant from Clostridium aciditolerans (Uniprot accession number A0A934M4M4; SEQ ID NQ:30) and a UbiX-like flavin prenyltransferase from Clostridium aciditolerans (Uniprot accession number A0A934HV38; SEQ ID NO:26).

[0149] In certain embodiments, the recombinant microorganism used in the method of the invention comprises an LpdD variant from [Clostridium] hylemonae DSM 15053 (Uniprot accession number C0C543; SEQ ID NO:31) and a UbiX-like flavin prenyltransferase from [Clostridium] hylemonae DSM 15053 (Uniprot accession number C0C544; SEQ ID NO:27).

[0150] In certain embodiments, the recombinant microorganism used in the method of the invention comprises an LpdD variant from Clostridium saccharoperbutylacetonicum N1-4(HMT) (Uniprot accession number M1MNA6; SEQ ID NO:32) and a UbiX-like flavin prenyltransferase from Clostridium saccharoperbutylacetonicum N1-4(HMT) (Uniprot accession number M1MM91; SEQ ID NO:28).

[0151] In certain embodiments, the recombinant microorganism used in the method of the invention comprises an LpdD variant from Lachnoclostridium phocaeense (Uniprot accession number A0A921LDR6; SEQ ID NO:33) and a UbiX-like flavin prenyltransferase from Lachnoclostridium phocaeense (Uniprot accession number A0A921LDR2; SEQ ID NO:29).

[0152] Sequence variants of the aforementioned proteins

[0153] The present invention also encompasses sequence variants of the aforementioned proteins, specifically those with a certain degree of sequence identity to the proteins disclosed herein. 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 whichare 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.

[0154] 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.

[0155] 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.

[0156] Preferably, the degree of identity is calculated over the complete length of the sequence.

[0157] 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 35 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 35 and by identifying the positions which correspond to the above indicated positions of any one of SEQ ID NOs:l to 35. 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.

[0158] 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.

[0159] Preferably, the degree of identity is calculated over the complete length of the sequence.

[0160] The sequence variants disclosed herein retain the activity of the proteins with which they share sequence identity. Specifically, a protein is considered to retain the activity of a particular protein disclosed herein if it maintains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity of the original protein. This range of activity retention ensures that the core functional properties of the protein are preserved, allowing the variants to perform effectively in their intended applications.

[0161] Culture conditions

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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 toensure 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.

[0166] The culture medium may be supplemented with various compounds that are essential or beneficial for the production of the target product.

[0167] 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.

[0168] 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 prFMNFh 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 ensures that there is an adequate supply of FMN to support the efficient production of the prFMNFh precursor, thereby facilitating the optimal activity of the prFMN-dependent enzyme.

[0169] The recombinant organism is grown in a culture medium under anaerobic conditions, meaning that atmospheric air is not introduced into the culture vessel. Instead, the critical step of oxidizing the prFMN cofactor is facilitated by a polypeptide that does not require molecular oxygen as an electron acceptor. This method prevents the introduction of atmospheric gases into the culture vessel, thereby avoiding the dilution and / or contamination of gaseous products.

[0170] 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 becontinuously 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.

[0171] 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. 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.

[0172] The recombinant microorganism

[0173] 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.

[0174] 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.

[0175] 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 Bacillussubtilisare 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.

[0176] 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.

[0177] 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. Clostridiumspecies 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.

[0178] 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 anaerobic conditions adds to their versatility, allowing for tailored bioprocesses that can optimize production yields and reduce costs.

[0179] 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. Itsrobustness and adaptability to different fermentation conditions, including both aerobic and anaerobic environments, make it a versatile organism for diverse industrial applications.

[0180] 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.

[0181] In a particular embodiment, the invention relates to the method according to the invention, wherein the recombinant microorganism is an anaerobic microorganism.

[0182] An anaerobic microorganism refers to a microorganism that can grow in the absence of molecular oxygen. These microorganisms are capable of carrying out metabolic processes without the need for oxygen, and may include obligate anaerobes, which are harmed or killed by the presence of oxygen, as well as facultative anaerobes, which can grow in both the presence and absence of oxygen. Anaerobic microorganisms can be found across various domains of life, including bacteria, archaea, and certain eukaryotes, and are utilized in various biotechnological and industrial applications due to their unique metabolic capabilities.

[0183] In certain embodiments, the recombinant microorganism utilized in the method of the invention is an obligate anaerobe, including, without limitation, any of the Clostridium species disclosed herein above.

[0184] In certain embodiments, the recombinant microorganism utilized in the method of the invention is a facultative anaerobe, including, without limitation, Escherichia coli or Saccharomyces cerevisiae.

[0185] 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.

[0186] The recombinant microorganism for use in the method of the invention preferably has been engineered to express a prFMN-dependent enzyme and a polypeptide that facilitates the oxidative maturation of a prFMNH2 precursor into the active prFMN cofactor, as described in more detail elsewhere herein. The genetic engineering process typically involves the insertionof genes encoding the prFMN-dependent enzyme and the polypeptide that facilitates the oxidative maturation of a prFMNH2 precursor into the active prFMN cofactor into the host microorganism's genome or the use of a plasmid or vector to express the genes 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] Promoters for use in connection with the nucleic acid molecule may be homologous or heterologous with regard to its origin and / or with regard to the gene to be expressed. Suitable promoters are for instance promoters which lend themselves to constitutive expression.However, promoters which are only activated at a point in time determined by external influences can also be used. Artificial and / or chemically inducible promoters may be used in this context.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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).

[0199] 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 fortranscription. 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).

[0200] 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.

[0201] 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.

[0202] 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 decarboxylation reaction catalysed by prFMN-dependent decarboxylases

[0203] 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.

[0204] 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.

[0205] Isobutene

[0206] In a particular embodiment, the invention relates to the method according to the invention, wherein the product is isobutene.

[0207] 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.

[0208] 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.

[0209] Conversion of 3-methylcrotonic acid into isobutene may be catalysed by any suitable prFMN-dependent decarboxylases disclosed herein. A prFMN-dependent decarboxylase having affinity forthe 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.

[0210] 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.

[0211] 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 eitherform, 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+.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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:

[0216] (i) conversion of acetyl-CoA into acetoacetyl-CoA,

[0217] (ii) conversion of the resulting acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl- CoA,

[0218] (iii) conversion of the resulting 3-hydroxy-3-methylglutaryl-CoA into 3- methylgl utaconyl-CoA,

[0219] (iv) conversion of the resulting 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA, and

[0220] (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.

[0221] The enzymatic conversion of acetyl-CoA into acetoacetyl-CoA

[0222] 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).

[0223] 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.

[0224] 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.

[0225] 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).

[0226] 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).

[0227] 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 (I PR), 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.

[0228] The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA

[0229] 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).

[0230] 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.-.

[0231] The enzymatic conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA

[0232] 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).

[0233] 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 xanthus 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).

[0234] The enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid

[0235] 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).

[0236] Thus, the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid may be achieved by

[0237] (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);

[0238] (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

[0239] (c) two enzymatic steps comprising

[0240] (i) first enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and

[0241] (ii) then enzymatically converting the thus obtained 3-methylcrotonyl phosphate into said 3-methylcrotonic acid (step Vic as shown in Figure 2).

[0242] 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.

[0243] 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).

[0244] The conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid can, e.g., be achieved by making use of an enzyme which is classified as EC 2.7.2.-, i.e., a phosphotransferase. Such enzymes use a carboxy group as acceptor. Thus, the conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid can, e.g., be achieved by making use of an enzyme with a carboxy group as acceptor (EC 2.7.2.-). In a preferred embodiment, the conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acid is achieved by the use of a propionate kinase (EC 2.7.2.15), an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).As mentioned above, the conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid can also be achieved by two alternative conversions wherein 3-methylcrotonyl-CoA is directly converted into 3-methylcrotonic acid.

[0245] 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.

[0246] 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.

[0247] 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:

[0248] - acetyl-CoA hydrolase (EC 3.1.2.1);

[0249] - palmitoyl-CoA hydrolase (EC 3.1.2.2);

[0250] - 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4);

[0251] - oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);

[0252] - ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);

[0253] - ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19);

[0254] - l,4-dihydroxy-2-naphthoyl-CoA hydrolase (EC 3.1.2.28); and

[0255] - acyl-CoA hydrolase (EC 3.1.2.20).

[0256] 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).

[0257] 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).

[0258] 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).

[0259] 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:

[0260] - propionate:acetate-CoA transferase (EC 2.8.3.1);

[0261] - acetate CoA-transferase (EC 2.8.3.8); and

[0262] - butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

[0263] 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).

[0264] In certain embodiments, methods may be utilized wherein the yield, pool and / or flux 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.

[0265] Styrene

[0266] 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.

[0267] 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).

[0268] 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

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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).

[0273] Hepta-l,3,5-triene

[0274] 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.

[0275] 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.

[0276] 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).

[0277] 4-vinyl uiacol

[0278] 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.

[0279] 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).

[0280] 1,3-pentadiene

[0281] 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.

[0282] 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)

[0283] Products of a carboxylation reaction catalysed by prFMN-dependent decarboxylase

[0284] 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.

[0285] 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.

[0286] 2,5-Furandicarboxylic acid (FDCA)

[0287] 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.

[0288] 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.

[0289] 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).

[0290] 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).

[0291] 2-naphtoic acid

[0292] 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.

[0293] 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.

[0294] 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, ora precursor thereof, such as bicarbonates.

[0295] 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.

[0296] 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 aspects of the invention

[0297] In another aspect, the invention relates to a method of oxidizing prFMNFh into a prenylated flavin mononucleotide (prFMN) cofactor of a decarboxylase in the absence of molecular oxygen, wherein the oxidation of the cofactor is catalysed by a LpdD-like protein or a PhdC-like protein.

[0298] In another aspect, the invention relates to a recombinant microorganism comprising one or more nucleic acids encoding:

[0299] a) a prFMN-dependent enzyme;

[0300] b) a flavin prenyltransferase; and

[0301] c) a polypeptide that facilitates the oxidative maturation of a prFMNFh precursor into an active prFMN cofactor.

[0302] That is, the present invention further relates to a recombinant microorganism that can be used in the method according to the invention.

[0303] 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.

[0304] 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 prFMNH2 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.

[0305] 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 SEQID NO:15, wherein the enzyme retains the enzymatic activity of modifying FMNH2 into prFMNH2.

[0306] Furthermore, the recombinant microorganism comprises a nucleic acid encoding a polypeptide that facilitates the oxidative maturation of a prFMNF precursor into an active prFMN cofactor. Expression of the polypeptide that facilitates the oxidative maturation of the prFMNH2 precursor into the active prFMN cofactor in the recombinant microorganism ensures sufficient levels of the prFMN cofactor under anaerobic conditions.

[0307] In a particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein the polypeptide that facilitates the oxidative maturation of the prFMNH2precursor into the active prFMN cofactor in the absence of oxygen is an LpdD-like protein or a PhdC-like protein.

[0308] In a particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein the LpdD-like protein comprises or consists of (i) an amino acid sequence as set forth in any one of SEQ ID NO:30-34; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO:30-34, wherein the amino acid sequence retains the ability to bind to prFMNFh and facilitate the oxidative maturation of prFMNFh into prFMN.

[0309] In a particular embodiment, the invention relates to the recombinant microorganism of the invention, wherein the PhdC-like protein comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:35; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:35, wherein the amino acid sequence retains the ability to bind to prFMNF and facilitate the oxidative maturation of prFMNF into prFMN.

[0310] In particular embodiments, the invention relates to a recombinant microorganism comprising one or more nucleic acids encoding:

[0311] a) a prFMN-dependent enzyme;

[0312] b) a flavin prenyltransferase; and

[0313] c) a LpdD-like protein or a PhdC-like protein.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.

[0314] 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.

[0315] 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.

[0316] 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.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.

[0317] 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.

[0318] 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.

[0319] In certain embodiments, the recombinant microorganism is an anaerobic microorganism, which may include both facultative and obligate anaerobes, as detailed elsewhere in this document.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.

[0320] In a further aspect, the present invention also relates to the use of any ofthe above-described recombinant microorganisms for the production of a desired product by a prFMN-dependent enzyme. Ina preferred embodiment, the recombinant microorganisms ofthe invention is used for the production of isobutene, wherein the isobutene is produced by a prFMN-dependent decarboxylase having 3-methylcrotonic acid decarboxylase activity.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] BRIEF DESCRIPTION OF THE DRAWINGS

[0328] 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.

[0329] 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.

[0330] Figure 3 shows the sequence similarity network (SSN) of UbiD enzymes from Clostridium species.

[0331] Figure 4 shows the genome neighbourhood network (GNN) created from the SSN of UbiD enzymes from Clostridium species (shown in Figure 3, cluster 1 only).

[0332] Figure 5 shows the genome neighbourhood network (GNN) created from the SSN of all LpdD-like proteins (IPR048844) (cluster 1 and cluster 2 only).

[0333] Figure 6 shows the taxonomy sunburst of LpdD-like proteins (IPR048844).

[0334] Figure 7 shows the increase of volumetric isobutene productivity (mg / L.h) upon addition of 3-methylcrotonic acid in anaerobic conditions with the LpdD-expressing strains (SB2932, SB2933, SB2934, SB2935, SB2936) and the control strain (SB2885).

[0335] Figure 8 shows the increase of volumetric isobutene productivity (mg / L.h) upon addition of 3-methylcrotonic acid in anaerobic conditions with the PhdC-expressing strain (SB2937) and the control strain (SB2885).

[0336] Figure 9 shows the effect of 1 min air injection on the isobutene production of the LpdD-expressing strain SB2935 in anaerobic conditions.EXAMPLES

[0337] 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.

[0338] Example 1. Genome neighbourhood analysis of UbiD enzymes in Clostridium species

[0339] All the oxidative maturation mechanisms of the prFMNH2 cofactor precursor described to date involve the use of molecular oxygen. However, UbiD and UbiX enzymes have been found in obligate anaerobes, which raises the question of the oxidative maturation mechanism in these organisms.

[0340] To elucidate this problem, we decided to analyse the genome neighbourhood of UbiD enzymes in obligate anaerobes to identify potential additional proteins that might be required for the UbiD / UbiX system in the absence of molecular oxygen.

[0341] The online computational tools EFI-EST (https: / / efi.igb.illinois.edu / efi-est / ) and EFI-GNT (https: / / efi.igb.illinois.edu / efi-gnt / ) were used to analyse, respectively, the sequence similarity network (SSN) and the associated genome neighbourhood network (GNN) of UbiD enzymes in the Clostridium genus (Zallot et al. The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways. Biochemistry 201958 (41), 4169-4182; Oberg et al. EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for Genomic Enzymology Tools. J Mol Biol. 2023 Jul 15;435(14):168018).

[0342] The Clostridium genus was used as an archetypal genus of obligate anaerobes. To select the UbiD enzymes of the Clostridium species, the UbiD decarboxylyase InterPro family (IPR002830) was submitted, and a taxonomy filter was used to select the Clostridium genus. An alignment score corresponding to 35% sequence identity was chosen as threshold to separate clusters and no restriction on length was applied. All the other parameters were used as default.Eighty-three UbiD enzymes were identified in Clostridium species, with some species having several UbiD enzymes. Cluster 1 contained 78 sequences and cluster 2 contained 4 sequences annotated as Pyrrole-2-carboxylic acid decarboxylases (FDC-like enzymes) (Figure 3).

[0343] A genome neighbourhood network (GNN) was created from this SSN with a neighbourhood size of 10 and a minimal co-occurrence percentage lower limit of 20 (Figure 4). It appeared that the most frequent neighbour of UbiD in Clostridium species was UbiX (included in the Pfam family PF02441 named "Flavoprotein") (67 / 78 for cluster 1 and 2 / 4 for cluster 2). It also appeared that the second most frequent neighbour of UbiD in Clostridium species was LpdD (included in the Pfam family PF21758 named "PAC_bac") (45 / 78 for cluster 1 and 3 / 4 for cluster 2). This high frequency of LpdD-like proteins in the neighbourhood of UbiD was surprising given there were only 1,419 LpdD sequences (IPR048844) in the InterPro database for 32,437 UbiD sequences (IPR002830). This result suggested a strong bias of LpdD frequency in obligate anaerobes, represented here with Clostridium species, which was surprising given the mechanism of action that was proposed in 2024 involved molecular oxygen (Gahloth et al. The prFMNH2-binding chaperone LpdD assists UbiD decarboxylase activation. J Biol Chem.

[0344] 2024 Feb;300(2):105653; Figure 14).

[0345] Example 2. Genome neighbourhood analysis of LpdD-like proteins (IPR048844)

[0346] A similar SSN / GNN analysis, as the one described in Example 1, was carried out with all LpdD-like proteins present in the InterPro database (IPR048844). The GNN of the two largest clusters (cluster 1 and cluster 2) is shown Figure 5. This analysis confirmed a high co-occurrence of UbiD and UbiX in the neighbourhood of LpdD-like proteins and the taxonomy sunburst tool of EFI-EST also confirmed a strong bias of the presence of LpdD-like proteins in anaerobic organisms (bacteria and archaea) with an absence in eukaryotes (Figure 6).

[0347] All these genomic analyses led us to the hypothesis that LpdD might be directly involved in the anaerobic oxidative maturation of the cofactor precursor prFMNH2 in anaerobic organisms, an open question since the discovery of the prFMN cofactor in 2015.

[0348] To confirm this hypothesis, we tested the LpdD-like proteins of the 4 Clostridium species having a Pyrrole-2-carboxylic acid decarboxylase (Figure 3, cluster 2) since these UbiD enzymes are FDC-like enzymes, sharing 38-40% sequence identity with FDC from Yersiniafrederiksenii (UniParc accession number UPI0005DC25B2, RefSeq WP_050108772.1; SEQ ID NO:1, WO 2022 / 207684). We also included in our tests the LpdD protein from Lactiplantibacillus plantarum (Uniprot accession number F9UT68) described by Gahloth et al. (J Biol Chem. 2024 Feb;300(2):105653). These 5 LpdD-like proteins were co-expressed in Saccharomyces cerevisiae with a mutant of FDC from Yersinia frederiksenii (UniParc accession number UPI0005DC25B2, RefSeq WP_050108772.1; SEQ ID NO:1, WO 2022 / 207684) and PAD1 from Saccharomyces cerevisiae (UniProt Accession number P33751) for isobutene production-tests in anaerobic conditions (Example 6).

[0349] Example 3. Construction of the yeast strain SB2885 converting 3-methylcrotonic acid into isobutene

[0350] 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).

[0351] Saccharomyces cerevisiae strain SB2885 was constructed by transforming the strain YPH499 with two plasmids. The first plasmid with a 2p yeast origin of replication was derived from the pESC-URA vector (Agilent, #217454). In this vector the DNA sequence between the yeast ADH1 terminator and the yeast CYC1 terminator (1885-3227) was replaced by a transcription unit containing the GPD promoter (p415 GPD, ATCC #87358), 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) and the yeast ADH2 terminator. The second plasmid with a CEN6 / ARSH4 origin of replication was derived from the p415 GPD vector (ATCC #87358). In this vector the GPD promoter was replaced by the yeast TEF1 promoter and the PAD1 gene from Saccharomyces cerevisiae (UniProt Accession number P33751) was cloned between the TEF1 promoter and the CYC1 terminator. The FDC and PAD1 gene sequences were codon-optimized for expression in Saccharomyces cerevisiae and synthesized by Twist Bioscience.The YPH499 strain was made chemically competent and transformed with the two plasmids (2pg of each plasmid with lOOpI of competent 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.

[0352] Example 4. Isobutene production in anaerobic conditions by Saccharomyces cerevisiae strain SB2885

[0353] A 1.4 L vessel was filled with 0.5 L of a culture medium containing 1.7 g / L yeast synthetic drop out medium supplement which does not contain uracil and leucine (commercial substance from Merck, catalogue number : Y1771), 3.4 g / L yeast nitrogen base without amino acids (commercial substance from Merck, catalogue number : Y1251), 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 10.8 pM iron II sulphate heptahydrate, 30.5 pM calcium chloride dihydrate, 1.2 pM cobalt dichloride, 5.1 pM manganese chloride, 8.8 pM zinc sulphate, 1.2 pM copper sulphate, 0.6 pM potassium iodide, 1.4 pM sodium molybdate and 16.2 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 the culture decreased below 15 g / L it was added continuously in order to maintain glucose concentration between 10 and 20 g / L.

[0354] The culture medium was inoculated with 30 mL of a preculture of strain SB2885 previously grown in a flask at 30°C in SD-URA-LEU medium: 1.7 g / L yeast synthetic drop out medium supplement which does not contain uracil and leucine (Merck, catalogue number: Y1771), 3.4 g / L yeast nitrogen base without amino acids (Merck, catalogue number: Y1251), 5 g / L ammonium sulphate, 0.32 g / L L-tryptophan. The 600 nm optical density of the preculture was about 0.3. In the bioreactor, pH was set and regulated at pH 5.0 with ammonia 25% andphosphoric acid 5M. Temperature was set at 30 °C for 43 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).

[0355] After 43 hours of culture when the biomass level had reached about 7 to 8 g / L temperature was increased up to 34°C and potassium 3-methylcrotonate was added at a concentration of 1 g / L (from a concentrated solution at 400 g / L) to start the production of isobutene. This concentration of 1 g / L in the bioreactor was then maintained by feeding a concentrated solution of potassium 3-methylcrotonate at 10 g / L. A production of isobutene was detected and isobutene concentration in the exhaust gas was about 0.0103 %mol. The resulting production after 44 hours was about 0.074 g isobutene.

[0356] Example 5. Construction of five yeast strains converting 3-methylcrotonic acid into isobutene and expressing a LpdD-encoding gene (SB2932, SB2933, SB2934, SB2935, SB2936)

[0357] The five strains were constructed as described in Example 3 except that the second plasmid with a CEN6 / ARSH4 origin of replication had an additional transcription unit cloned after the CYC1 terminator. This additional transcription unit contained the GPD promoter (p415 GPD, ATCC #87358), the LpdD-encoding gene and the yeast ADH2 terminator. Five LpdD-encoding genes were tested in five different strains:

[0358] SB2932 with the LpdD-like chaperone-like domain-containing protein from Clostridium aciditolerans (Uniprot accession number A0A934M4M4),

[0359] SB2933 with the LpdD-like chaperone-like domain-containing protein from [Clostridium] hylemonae DSM 15053 (Uniprot accession number C0C543),

[0360] SB2934 with the LpdD-like chaperone-like domain-containing protein from Clostridium saccharoperbutylacetonicum N1-4(HMT) (Uniprot accession number M1MNA6),

[0361] SB2935 with the LpdD-like chaperone-like domain-containing protein from Lachnoclostridium phocaeense (Uniprot accession number A0A921LDR6),

[0362] SB2936 with the LpdD protein from Lactiplantibacillus plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) (Lactobacillus plantarum) (Uniprot accession number F9UT68).The LpdD gene sequences were codon-optimized for expression in Saccharomyces cerevisiae and synthesized by Twist Bioscience.

[0363] The percentage of sequence identity between these five proteins was calculated with the alignment tool Clustal Omega 1.2.2 provided by the software Geneious Prime (default parameters) and is described in Table 1 below.

[0364] Table 1: Percentage of sequence identity between the LpdD proteins described in Example 5 and tested in Example 6

[0365]

[0366] Example 6. Activation of isobutene production in anaerobic conditions by LpdD gene addition in SB2885 strain

[0367] The same experiment as described in Example 4 was performed with the strains SB2932, SB2933, SB2934, SB2935 and SB2936 described in Example 5. A production of isobutene was detected in the exhaust gas for the 5 strains and the resulting production after 44 hours is described in Table 2 below.Table 2: Increase of total isobutene production (g) in anaerobic conditions with the expression of a LpdD-encoding gene in the SB2885 control strain (SB2932, SB2933, SB2934, SB2935, SB2936)

[0368]

[0369] The increase of volumetric isobutene productivity (mg / L.h) upon addition of 3-methylcrotonic acid with the LpdD-expressing strains (SB2932, SB2933, SB2934, SB2935, SB2936) and the control strain (SB2885) is shown in Figure 7.

[0370] Substantial isobutene production increase was observed for the 5 tested strains compared to the control strain, suggesting the activation of FDC by LpdD expression in anaerobic conditions.

[0371] Example 7. Construction of a yeast strain converting 3-methylcrotonic acid into isobutene and expressing a PhdC-encoding gene (SB2937)

[0372] The strain was constructed as described in Example 3 except that the second plasmid with a CEN6 / ARSH4 origin of replication had an additional transcription unit cloned after the CYC1 terminator. This additional transcription unit contained the GPD promoter (p415 GPD, ATCC #87358), the PhdC-encoding gene from Mycolicibacterium fortuitum (Mycobacterium fortuitum) (Uniprot accession number A0AAE5AEH3) and the yeast ADH2 terminator. The resulting strain is SB2937.

[0373] The PhdC gene sequence was codon-optimized for expression in Saccharomyces cerevisiae and synthesized by Twist Bioscience.Example 8. Activation of isobutene production in anaerobic conditions by PhdC gene addition in SB2885 strain

[0374] The same experiment as described in Example 4 was performed with the strains SB2885 and SB2937 except that isobutene production was started after 41 hours of culture and maintained in anaerobic conditions for 5 hours after the addition of 3-methylcrotonic acid. The production of isobutene was detected in the exhaust gas for the two strains and the resulting production after 5 hours is described in Table 3 below.

[0375] Table 3: Increase of total isobutene production (mg) in anaerobic conditions with the expression of a PhdC-encoding gene in the SB2885 control strain (SB2937)

[0376]

[0377] After 5 hours of isobutene production in anaerobic conditions, air was injected at 0.15 L / min for 1 min to activate FDC with molecular oxygen and the isobutene production was continued for 5 more hours in anaerobic conditions with a nitrogen flow of 0.15 L / min.

[0378] The increase of volumetric isobutene productivity (mg / L.h) upon addition of 3-methylcrotonic acid and injection of air with the PhdC-expressing strain (SB2937) and the control strain (SB2885) is shown in Figure 8.

[0379] In anaerobic conditions, before air injection, substantial isobutene production increase was observed for the PhdC-expressing strain compared to the control strain (17 vs 2 mg / L.h, i.e.

[0380] 8.5 fold increase), suggesting the activation of FDC by PhdC expression in anaerobic conditions. After the activation with air injection, isobutene production increased by 13-fold for the control strain SB2885 (from 2 to 26 mg / L.h) and by 2.6-fold for the PhdC-expressing strain SB2937 (from 17 to 45 mg / L.h), suggesting partial FDC anaerobic activation by PhdC expression in the conditions tested.Example 9. Effect of 1 min air injection on the isobutene production of the LpdD-expressing strain SB2935 in anaerobic conditions

[0381] The same experiment as described in Example 8 was performed with the strain SB2935 except that the lmin air injection was carried out at the same time as the addition of 3-methylcrotonic acid and only for 1 bioreactor (BR14). The anaerobic production of isobutene was carried out for 7 hours. The production of isobutene was detected in the exhaust gas for the two bioreactors.

[0382] With SB2935 the volumetric isobutene productivity (mg / L.h) obtained upon addition of 3-methylcrotonic acid was actually slightly higher for the bioreactor that did not have air injection (55 vs 51 mg / L.h) (Figure 9), suggesting that the anaerobic activation of FDC by LpdD is independent of the presence of molecular oxygen.

Claims

New PCT-Patent ApplicationGlobal BioenergiesVossius Ref.: AK1352 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 the recombinant microorganism recombinantly expresses a polypeptide that facilitates the oxidative maturation of a prFMNH2 precursor into the active prFMN cofactor.

2. The method of claim 1, wherein the polypeptide that facilitates the oxidative maturation of the prFMNF precursor into the active prFMN cofactor is a LpdD-like protein and / or a PhdC-like protein.

3. The method of claim 2, wherein the LpdD-like protein comprises or consists of (i) an amino acid sequence as set forth in any one of SEQ ID NO:30-34; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO:30-34, wherein the amino acid sequence retains the ability to bind to prFMNFh and facilitate the oxidative maturation of prFMNF into prFMN.

4. The method of claim 2 or 3, wherein the PhdC-like protein comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:35; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:35, wherein the amino acid sequence retains the ability to bind to prFMNFh and facilitate the oxidative maturation of prFMNFh into prFMN.

5. The method of any one of claims 1 to 4, wherein the recombinant microorganism expresses a flavin prenyltransferase.

6. The method of any one of claims 1 to 5, 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.

7. The method of any one of claims 1 to 6, wherein the product is isobutene and, preferably, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity.

8. The method of claim 7, 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.

9. The method of claim 7 or 8, wherein the isobutene is produced from 3-methylcrotonic acid.

10. The method of any one of claims 1 to 9, 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.

11. The method of any one of claims 1 to 10 wherein the recombinant microorganism is an anaerobic microorganism.

12. A recombinant microorganism comprising one or more nucleic acids encoding:a) a prFMN-dependent enzyme;b) a flavin prenyltransferase; andc) a polypeptide that facilitates the oxidative maturation of a prFMNH2 precursor into an active prFMN cofactor.

13. The recombinant microorganism of claim 12, wherein the prFMN-dependent enzyme has 3-methylcrotonic acid decarboxylase activity and, preferably, comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:1 or (ii) an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 1, wherein the amino acid sequence retains 3-methylcrotonic acid decarboxylase activity.

14. The recombinant microorganism of claim 12 or 13, 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 FMNH2 into prFMNH2.

15. The recombinant microorganism of any one of claims 12 to 14, wherein the polypeptide that facilitates the oxidative maturation of the prFMNFh precursor into the active prFMN cofactor is an LpdD-like protein and / or a PhdC-like protein.

16. The recombinant microorganism of claim 15, wherein the LpdD-like protein comprises or consists of (i) an amino acid sequence as set forth in any one of SEQ ID NQ:30-34; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NQ:30-34, wherein the amino acid sequence retains the ability to bind to prFMNFh and facilitate the oxidative maturation of prFMNFh into prFMN.

17. The recombinant microorganism of claim 15 or 16, wherein the PhdC-like protein comprises or consists of (i) an amino acid sequence as set forth in SEQ ID NO:35; or (ii) an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:35, wherein the amino acid sequence retains theability to bind to prFMNH2 and facilitate the oxidative maturation of prFMNH2 into prFMN.

18. The recombinant microorganism ofany one of claims 12 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. The recombinant microorganism ofany one of claims 12 to 18, wherein the recombinant microorganism is an anaerobic microorganism.

20. Use of the recombinant microorganism of any one of claims 12 to 19 in the production of a product, wherein the product is produced by the prFMN-dependent enzyme.

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