Identification of carbamate esterase based on growth selection using 4-aminobenzoic acid (PABA)

Abstract

The present invention relates to a biological assay for identifying microorganisms expressing a carbamate enzyme gene. Growth on a medium lacking folate and supplemented with u-aminobenzoic acid is used for the assay.

Description

[0001] This invention relates to a bioassay for identifying microorganisms expressing carbamate enzyme genes. Growth on a folic acid-deficient medium supplemented with u-aminobenzoic acid is used for this assay.

[0002] In bacteria, 4-aminobenzoic acid (PABA) is a precursor in folic acid biosynthesis. PABA itself is derived from cladonic acid through two consecutive reactions: in the first reaction, cladonic acid is converted to 4-amino-4-deoxycladonic acid. This reaction requires the deamination of glutamine to provide the necessary NH4. In the second step, aminodeoxycladonic acid lyase (PabC) converts this intermediate to PABA. In *Escherichia coli* and other bacterial species, the first reaction is catalyzed by a heterodimer of two enzymes: PabA converts glutamine to glutamate, thereby providing the NH4 required by 4-amino-4-deoxycladonic acid synthase PabB.

[0003] Inactivation of any of these enzymes renders the microbial cell a PABA auxotroph. However, in the case of PabA, this lack of enzyme function can be compensated for by increasing the ammonia concentration in the culture medium, allowing the HN4 required by PabB to be provided from an external source.

[0004] Polyurethanes are an important class of polymers produced in large quantities. The transition to a circular economy requires recycling methods that enable the reuse of their carbon components. Enzymatic degradation of carbamate bonds via carbamate enzymes has been disclosed, for example in WO 2019 / 243293. However, additional carbamate enzymes with novel or improved properties are still needed.

[0005] WO 2019 / 243293 describes an assay that requires the expression of a putative carbamate enzyme gene. The gene product is then incubated with a test substrate, which, upon cleavage by the enzyme, releases a fluorescent dye that can be detected by chromatography. Because this system requires culturing a single clone of a microorganism, the throughput is limited. An assay that does not rely on culturing a single clone to identify candidate enzymes is desired. This problem is solved by the embodiments defined in the claims and the description below.

[0006] In a first embodiment, the present invention relates to a method for identifying carbamate enzyme genes in microorganisms, comprising the following steps:

[0007] a) Microorganisms incubated in a culture medium containing compounds according to general formula (I) as 4-aminobenzoic acid (PABA) auxotrophs and transformed with nucleic acid molecules containing at least one open reading frame under the control of a promoter that enables gene expression in the microorganisms.

[0008]

[0009] (I)

[0010] Where R is any organic molecule, provided that an oxygen atom is attached to a carbon atom of the organic molecule.

[0011] Furthermore, the culture medium contains no more than 1 nM of PABA and no more than 1 nM of folic acid.

[0012] b) Identify microorganisms carrying carbamate enzyme genes based on their ability to grow in culture media.

[0013] The culture medium may be any medium that meets the requirements defined above and is suitable for the proliferation of the microorganisms discussed. Suitable culture media for different microorganisms are well known to those skilled in the art and are documented in the literature. It is preferred to use culture media containing only compounds with well-defined chemical compositions, as the concentrations of PABA and folic acid are easier to control in these media. Furthermore, these media do not contain folic acid-derived metabolites, so growth in such media depends on the supply of PABA by cleaving the molecule defined by formula (I).

[0014] The culture medium can be used in liquid form. However, in a preferred embodiment of the invention, the culture medium is solidified using a suitable thickener, most preferably agar. A culture medium containing the correct concentration of microbial cells can then be spread on the solidified medium, allowing the microbial cells capable of growing in the medium to produce well-separated individual colonies. Therefore, in this embodiment, the step preceding method step a) is the provision of a solidified culture medium and the spreading of a liquid composition containing microbial cells onto the solidified medium.

[0015] If the carbamate bond present in the compound defined by formula (I) is cleaved, PABA is released and can be used as an intermediate in the biosynthesis of folic acid, thereby enabling microorganisms to grow.

[0016] The structure of the compound according to formula (I) can be adjusted according to the type of carbamate enzyme to be identified.

[0017] In a preferred embodiment of the invention, R is a residue selected from branched or unbranched alkyl residues, branched or unbranched alkenyl residues, branched or unbranched aralkyl residues, and aryl residues. Preferably, these residues have a molecular weight of 27 to 100,000 g / mol, more preferably 27 to 10,000 g / mol, and most preferably 27 to 2,000 g / mol. The residues may contain heteroatoms, preferably nitrogen, oxygen, or sulfur. It is also contemplated that one or more hydrogen atoms of the residues may be substituted with different atoms or functional groups. Preferred substituents are halogen atoms, carboxyl groups, thiols, hydroxyl groups, nitro groups, nitrile groups, carbonyl groups, and carboxyl groups.

[0018] In a preferred embodiment of the invention, R is a branched or unbranched alkyl or alkenyl residue having 2 to 12 carbon atoms, preferably 2 to 6 carbon atoms. At least one hydrogen atom of the residue may be substituted by a member selected from halogen, carboxyl, thiol, hydroxyl, nitro, nitrile, carbonyl, and carboxyl groups.

[0019] In a more preferred embodiment of the invention, R is an alkenyl residue comprising 2 to 12, preferably 2 to 6, carbon atoms. At least one hydrogen atom of the residue may be substituted with a member selected from halogen, carboxyl, thiol, hydroxyl, nitro, nitrile, carbonyl, and carboxyl groups. However, R is preferably unsubstituted. In this embodiment, R preferably comprises a double bond located next to the terminal oxygen atom of formula (I).

[0020] In a particularly preferred embodiment, the compound according to formula (I) is 4-[(allyloxycarbonyl)amino]benzoic acid.

[0021] If the aim is to identify a carbamate enzyme capable of cleaving polyurethane, then R is preferably a polyurethane residue with a number-average molecular weight of 2,000 to 100,000 g / mol. Such a molecule can be readily prepared by synthesizing an isocyanate-functionalized prepolymer and end-capping said prepolymer with PABA. The term "polyurethane" covers all compounds having the aforementioned molecular weight and containing at least three, preferably at least five, and most preferably at least ten carbamate groups. The polyurethane may be a polyether polyurethane, a polyester polyurethane, or a polycarbonate polyurethane. Those skilled in the art will appreciate that carbamates can be obtained by reacting a polyisocyanate with any mixture of polyether polyols, polyester polyols, and polycarbonate polyols, such that polyurethanes comprising any combination of polyether, polyester, and polycarbonate are also covered by the term "polyurethane".

[0022] The concentration of the compound according to formula (I) is preferably from 1 μM to 500 μM.

[0023] The term "incubation" refers to bringing microorganisms into contact with a culture medium under conditions suitable for microbial growth, particularly temperature.

[0024] The microorganism may be bacteria or yeast. Preferably, it is bacteria, more preferably Escherichia coli.

[0025] Crucially, the microorganism is a 4-aminobenzoic acid (PABA) auxotroph, meaning it cannot synthesize the compound and thus depends on supplementation. This can be achieved by inactivating the enzyme that provides 4-amino-4-deoxyclique acid and / or by inactivating aminodeoxyclique acid lyases that convert 4-amino-4-deoxyclique acid to PABA.

[0026] If the microorganism is Escherichia coli, this nutritional deficiency can be achieved by inactivating at least one of an enzyme selected from PabA, PabB and PabC, preferably PabA and / or PabB, and most preferably PabA.

[0027] The term "inactivation" refers to any genetic modification that prevents the expression of a corresponding enzyme or results in a defective enzyme. Enzyme expression can be prevented by any method known in the field of molecular biology, particularly by deleting the gene encoding the enzyme or by modifying the regulatory sequence of the corresponding gene to suppress gene expression. Defective enzymes are caused by introducing a stop codon into the gene in question, resulting in the expression of a truncated protein, or by introducing a gene mutation or partial gene deletion that affects the substrate binding site or active site.

[0028] The methods defined above are particularly suitable for identifying carbamate enzyme genes in isolated nucleic acid sequences. If microbial growth is restored by transforming microorganisms with DNA containing at least one open reading frame under the control of a suitable promoter, then that open reading frame likely encodes a carbamate enzyme. A "suitable promoter" as described above is a promoter capable of expressing the gene in the microorganism in question. In this case, the identification of the carbamate enzyme gene is the same as the identification of microorganisms that can grow under the culture conditions described above in this application. If transformation of microorganisms with at least one open reading frame under the control of a suitable promoter is sufficient to allow the microorganisms to grow in the culture medium, then it must be concluded that the open reading frame encodes the gene responsible for that effect.

[0029] Escherichia coli is a particularly suitable microorganism for identifying carbamate enzyme genes in recombinant DNA because its genetics is well-known and a large number of tools for genetic manipulation are available. Preferred E. coli strains for this purpose are strains that do not express at least one gene selected from pabA, pabB, and pabC. More preferred are strains that do not express pabA and / or pabC. Particularly preferred are strains that do not express pabA.

[0030] Obviously, the method of the present invention is particularly useful for screening carbamate genes in gene libraries, especially metagenomic libraries. Those skilled in the art are familiar with the means and methods used to construct such libraries.

[0031] If the aim is to screen for the presence of an open reading frame encoding a carbamate enzyme in a DNA sequence, the method of the present invention preferably includes an additional method step prior to step a). In this method step, a microorganism is transformed with a nucleic acid carrying at least one open reading frame under the control of a promoter sequence capable of expressing the open reading frame in the microorganism.

[0032] Those skilled in the art will recognize that the method of the present invention may produce false positive results in certain situations, i.e., the growth of the microorganism may resume even in the absence of carbamate enzymes. This occurs if the nucleic acid molecules introduced into the microorganism contain functional forms of one or more enzymes that are inactivated to produce PABA auxotrophic effects. Clearly, such genes restore growth simply by repairing the disrupted metabolic pathways.

[0033] To eliminate such false positive results, in another embodiment of the invention, method step b) is followed by an additional method step to confirm carbamate enzyme activity. This can be done by any biochemical method known to those skilled in the art. Preferably, the microorganism whose growth has been restored, or a lysate of such a microorganism, is incubated with a suitable test substrate containing at least one carbamate group, which generates a signal if the carbamate group cleaves. Suitable test substrates and detection methods are disclosed in WO 2019 / 243293. In a more preferred embodiment, the cells detected in method step b) are grown in two parallel cultures, one supplemented with PABA and the other without PABA. Neither culture contains the compound defined by formula (I). If growth is observed in the PABA-free culture, it indicates that the microorganism has simply regained a component of the biosynthetic pathway that produces PABA.

[0034] In another embodiment of the invention, the confirmation step is performed during method step b). Preferably, this is achieved by adding a suitable test substrate to the culture medium or agar plate, such that the cleavage of the carbamate groups of the test substrate induces a color change in the culture medium or agar.

[0035] In another embodiment, the present invention relates to the use of compounds having the general formula (I) as defined above for the identification of carbamate enzyme genes in microorganisms.

[0036] The following examples are intended to illustrate the invention only. They should not be used to limit the scope of the claims in any way. Example

[0037] 1. Development of auxotrophic strains

[0038] The auxotrophic Escherichia coli strains ΔpabA (JW3323-KC; CGSC strain #10483) and ΔpabB (JW1801-KC; CGSC strain #9507), derived from the Escherichia coli K-12 Keio strain collection, were purchased from the National Bioresource Project E. coli Strain Office, Mishima, Japan (https: / / shigen.nig.ac.jp / ecoli / strain / ). The received strains were attached to small pieces of filter paper. The paper was added to 5 mL of LB medium containing 50 μg / mL kanamycin sulfate and incubated overnight at 30°C. A glycerol preservation solution was prepared by adding 750 µL of the culture to 750 µL of sterile 50% glycerol prior to storage at 80°C.

[0039] Selection of ΔpabA strains

[0040] M9 glycerol basal medium (M9 medium for short) contains only 18.69 mM ammonium chloride and clearly demonstrates that strain ΔpabA requires at least 10 nm PABA for growth and will not grow without the addition of PABA. No such data were reported for strain ΔpabB. Strain ΔpabA also grew slightly faster than ΔpabB in M9 medium supplemented with 10 μM PABA. Therefore, strain ΔpabA was chosen for further work, despite its conditional trophic deficiency (the ammonium concentration in M9 medium was far below the 100 mM required for ΔpabA growth).

[0041] 2. Basic culture medium used in the study

[0042] Folic acid (and therefore PABA) is essential for the biosynthesis of metabolites that can be derived from compound media. Therefore, PABA auxotrophy is conditional, and PABA auxotrophic strains can grow in compound media. Thus, M9 glycerol basal medium was used for culture experiments. Two x M9 basal salts from Gibco (Thermo Fisher catalog number A1374401) were used as the base of the medium to avoid PABA contamination. Glycerol used as the carbon source was obtained from an unopened bottle. All other components were kept as clean as possible but not prepared from unopened bottles. The basal medium (50 mL) contained 1 x M9 salt (25 mL 2x stock solution), 1% glycerol (1 mL 50% solution), 100 µM calcium chloride (5 µL 1 M solution), and 2 mM magnesium sulfate (100 µL 1 M solution). Because the Keio strains were resistant to kanamycin, 50 µg / mL kanamycin sulfate (50 µL 50 mg / mL solution) was added to avoid contamination by wild-type *E. coli*, which was then grown in M9 glycerol basal medium without PABA. For strains transformed with the pBAD-based vector, 100 µg / mL ampicillin (50 µL 100 mg / mL solution) was also added to the medium. To induce protein expression from the pBAD vector, 0.01% arabinose was added to the medium unless otherwise specified (actual assays were performed without arabinose).

[0043] For PABA growth assay agar plates, use approximately 1.5% (w / w) washed agar (catalog number 5210.4, CarlRoth GmbH & Co. KG). Due to PABA contamination, the agar must be washed before use. To perform washing, autoclave and solidify a 5x concentrated agar solution (75 g / L). Crush the agar gel into small pieces and wash for two days in 5 L of MilliQ water with stirring at 4°C, performing several water exchanges (2-3 times daily). Then weigh the PABA-free agar to estimate the amount needed for 1x agar solution and divide into 1x portions, freezing at -20°C. For fresh M9 medium agar plates, thaw these portions and autoclave them together with M9 basal medium. After cooling to approximately 60°C, before preparing the plate, ampicillin (100 μg / mL), kanamycin sulfate (50 μg / mL), and a suitable substrate (no substrate, 5 μM PABA, or 10 μM alloc-PABA) are added to the solution.

[0044] 3. Incubation

[0045] The culture typically consists of 1 mL of M9 medium in a 15 mL centrifuge tube. To inoculate with PABA-free M9 medium, the starting culture is grown to saturation (48 hours at 37°C) in medium containing 5 mM PABA. OD600 is normalized to the lowest value when comparing different cultures. The culture is diluted 100,000 times. This is approximated by twice serially diluting 3 µL of culture to 997 µL of PABA-free M9 glycerol basal medium (111,111-fold dilution). This high dilution is important for two reasons: first, the residual PABA concentration should be below the 5 nM required for growth (0.063 nM in our case); second, cells can divide up to 7 times without synthesizing new PABA, meaning that even without PABA, cultures containing 50,000 cells / mL or more will become turbid. Therefore, using a smaller inoculum is crucial. A 100,000-fold dilution of saturated culture (OD600≈1) yields approximately 5,000 cells / mL.

[0046] 4. Transform the ΔpabA strain of Escherichia coli into chemically competent cells.

[0047] To transform the ΔpabA *E. coli* strain, chemocompetent cells were prepared using the Mix & Go! *E. coli* Transformation Buffer Kit (ZymoResearch catalog number T3002). The manufacturer's competent cell preparation and transformation protocol was followed. Single transformed colonies were picked from LB agar plates containing 50 μg / mL kanamycin sulfate and 100 μg / mL ampicillin. The culture was inoculated into M9 glycerol basal medium containing 5 mM PABA and grown to saturation (37°C for 48 h). A glycerol preservation solution was prepared by mixing 750 µL of the culture with 750 µL of 50% glycerol and stored at -80°C. This glycerol preservation solution was used to inoculate all subsequent cultures. The strains (*E. coli* ΔpabA empty and other controls) failed to grow after being diluted approximately 100,000 times to PABA-free M9 medium, confirming their suitability for our carbamate enzyme growth selection experiment.

[0048] 5. Preparation of Escherichia coli ΔpabA electrocompetent cells

[0049] To prepare electrocompetent cells, a preculture obtained from *E. coli* ΔpabA glycerol preservation medium was prepared in 4 mL LB medium containing ampicillin (100 μg / mL) and kanamycin sulfate (50 μg / mL) and incubated at 37°C and 200 rpm for 16–18 h. Subsequently, 100 μL of the preculture was seeded into 100 mL of preheated SOC medium in a 1 L flask. The flask was incubated in a rotary shaker at 37°C and 140 rpm. When the culture reached OD600 ≈ 0.5, the flask was rapidly transferred to an ice-water bath for 15–30 min. The culture was occasionally swirled to ensure uniform cooling. To prepare for the next step, 50 mL centrifuge tubes were placed in an ice-water bath. The culture was transferred to the ice-cold centrifuge tubes. Cells were harvested by centrifuging the mixture at 1,500 g for 15 min at 4°C. Discard the supernatant and carefully resuspend the cell pellet in 50 mL of ice-cold 10% sterile glycerol. Repeat centrifugation and washing in glycerol at least three times until the pellet is resuspended in 1 mL of 10% sterile glycerol. When not used directly, aliquot the cells into 50 μL portions, freeze in liquid nitrogen, and store at 80°C.

[0050] 6. PABA growth selection assay (and electroporation) on agar plates

[0051] To transform DNA into electrocompetent cells via electroporation, the *E. coli* cells were kept at room temperature until the bacterial suspension thawed, and then placed on ice. 25 µL of the suspension was pipetted into ice-cold microcentrifuge tubes, and 1 µL of DNA was added (1000 ng per transformation). Electroporation was then performed on the 25 µL bacterial / DNA solution (2.1 kV, 100 Ω, and 25 μF). The typical time constant was approximately 2.6 ms. For cell resuscitation, 1 mL of SOC medium was added at room temperature and the cells were gently incubated at 37°C for 1 h. After resuscitation, the cells were harvested by centrifugation at 2,000–3,000 g for 5 min at 4°C and carefully resuspended in 2 mL of M9 medium. The cells were then centrifuged at 2,000 g for 5 min and gently resuspended in 2 mL of M9 medium. Finally, the culture was spread in 50 µL portions onto PABA growth assay agar plates containing 10 μM alloc-PABA. The plate was incubated at 37°C for two days.

[0052] Synthesis and washing of 7,4-[(allyloxycarbonyl)amino]benzoic acid alloc-PABA

[0053] The Alloc-PABA used in the growth assay was prepared as follows: PABA (1 g, 7.29 mmol) was added to a stirred H₂O / dioxane (10 mL / 10 mL) solution in a glass reaction flask at room temperature. N,N-diisopropylethylamine (2.6 mL, 14.9 mmol) and NaHCO₃ (1.8 g, 21.8 mmol) were added, and the mixture was stirred for several minutes. Approximately 3 mL of dioxane was added to counteract evaporation. Finally, 1 mL of chloroformate was added dropwise to the stirred reaction mixture. The reaction mixture was stirred for 15 minutes while a gas stream was passed through to evaporate some of the dioxane, thus reducing the solubility of alloc-PABA. The reaction was then quenched by adding 100 mL of 1.2 M hydrochloric acid (HCl). During this step, a large amount of foam was formed due to the reaction of HCl with bicarbonate (CO₂ production), so HCl was added in several steps. Subsequently, approximately 100 mL of 1.2 M HCl was added again to ensure that all alloc-PABA precipitated and PABA dissolved. Stir the reaction mixture for approximately 15 minutes. Vacuum filter the alloc-PABA and wash with an additional 100 mL of 1.2 M HCl. Wash the reaction flask and filter glass with approximately 60 mL of acetone to recover most of the alloc-PABA. Precipitate alloc-PABA with 60 mL of 1.2 M HCl, and evaporate the acetone through an air stream for approximately 20 minutes, then add another 140 mL of 1.2 M HCl. Vacuum filter all the material again and wash with 1.2 M HCl. To improve the purity of the product, dissolve the remaining alloc-PABA (approximately 0.7 g) in 25 mL of ethyl acetate. First, filter the solution through a PFTE filter to remove cellulose fibers from the vacuum filtration, then fill the solution into a separatory funnel and add 60 mL of 1.2 M HCl. Mix the two phases thoroughly and discard the lower aqueous phase. Add fresh 1.2 M HCl, and repeat this process four times. Finally, place the ethyl acetate phase in a glass beaker and evaporate overnight under a ventilated hood. Following this washing process, alloc-PABA with PABA contamination of <0.01% was obtained.

[0054] 8. Establish the measurement layout

[0055] Since the amount of PABA required for the growth of *E. coli* ΔpabA is extremely small, the negative variants in the library are highly likely to utilize PABA diffused from PABA-producing positive strains for growth. Therefore, an agar plate apparatus was chosen because it was expected to have advantages over liquid culture in the diffusion and isolation of positive candidate clones. To investigate the suitability of this apparatus, the extent to which the cell density of the negative control (ΔpabA_empty) could be increased without obtaining a bacterial colony was investigated. Therefore, 50 mL of ΔpabA_empty culture was cultured in LB medium, washed once with M9 medium, and finally resuspended in approximately 10 mL of M9 medium. Different dilutions of the culture were plated on screening plates containing M9 medium and 10 μM alloc-PABA and control plates containing LB to estimate the cell density. The LB-control showed a cell density of approximately 6 billion colony-forming units (CFU) / mL for the culture (washed and subsequently resuspended in 10 mL of M9 medium). Therefore, it was concluded that even with a cell density >6 million cells / plate (using E. coli ΔpabA_empty as a negative control), cells could not grow on our M9 plates supplemented with 10 μM alloc-PABA.

[0056] 9. Screening of metagenomic libraries

[0057] After establishing the assay, it was applied to a metagenomic library constructed from soil (supplied by Covestro of Leverkusen, Germany) that had been exposed to polyurethane for several years. For this purpose, the library was transformed into *E. coli* ΔpabA electrocompetent cells. After 1 hour of resuscitation in SOC medium, the cells were first washed and then resuspended in M9 medium before being plated on the screening plates. After two days at 37°C, numerous single colonies formed. However, when screening metagenomic libraries, it is important to note that homologs of pabA are highly likely to be found within the library, as it is a ubiquitous gene. Therefore, this assay serves well as an effective pre-screen for metagenomic screening, but positive results from this assay subsequently require rescreening to distinguish between pabA homologs and actual carbamate positivity. For this purpose, carbamate esterase activity was confirmed on the model substrate 7-ethoxycarbonyl-4-methylcoumarin. Following rescreening, an open reading frame encoding a hydrolase (named: UMG-SP-4) that had never been previously documented was obtained. The closest homolog (80.0% identity) was an amidase from bacteria of the family Hyphomonadaceae. UMG-SP-4 showed 43.64%, 44.24%, and 46.10% identity with previously discovered and characterized carbamate enzymes UMG-SP-1, UMG-SP-2, and UMG-SP-3, respectively (WO2023 / 194440).

[0058] 10. Rescreening metagenomically positive samples from PABA growth selection assays

[0059] Since the PABA growth selection assay selects not only carbamates but also PabA homologs frequently found in metagenomics, it is necessary to rescreen the positives in the assay to distinguish between carbamates and PabA homologs. Colonies from the assay plate are inoculated into microtiter plates containing M9 glycerol basal medium (with or without 5 μM PABA), with wells without PABA inoculated first. Only E. coli positives that grow in 5 µM PABA but not in PABA-free medium necessarily contain the desired true carbamate positives, as they cannot produce PABA de novo. Alternatively, positives from the pre-screening can be incubated on the model substrate 7-ethoxycarbonyl-4-methylcoumarin to verify carbamate activity.

Claims

1. A method for identifying carbamate enzyme genes in microorganisms, comprising the following steps: a) Microorganisms incubated in a culture medium containing compounds according to general formula (I) as 4-aminobenzoic acid (PABA) auxotrophs and transformed with nucleic acid molecules containing at least one open reading frame under the control of a promoter that enables gene expression in the microorganisms. (I) Where R is any organic molecule, provided that an oxygen atom is attached to a carbon atom of the organic molecule; Furthermore, the culture medium contains no more than 1 nM of PABA and no more than 1 nM of folic acid. b) Identify microorganisms carrying carbamate enzyme genes based on their ability to grow in culture media.

2. The method according to claim 1, wherein the concentration of the compound according to formula (I) in the culture medium is from 1 μM to 500 μM.

3. The method according to claim 1 or 2, wherein R is a residue selected from branched or unbranched alkyl residues, branched or unbranched alkenyl residues, branched or unbranched aralkyl residues and aryl residues with a molecular weight of 27 to 100,000 g / mol.

4. The method according to any one of claims 1 to 3, wherein the culture medium is a mineral culture medium without a complex carbon source.

5. The method according to any one of claims 1 to 4, wherein the microorganism is bacteria.

6. The method according to claim 5, wherein the bacteria is Escherichia coli.

7. The method of claim 6, wherein *Escherichia coli* does not express at least one gene selected from pabA, pabB, and pabC.

8. The method according to any one of claims 1 to 7, wherein the presence of carbamate enzyme is confirmed by at least one method selected from: (i) Assess carbamate enzyme activity using a suitable test substrate, which may be performed during method step b) by adding the test substrate to the culture medium, or in a separate culture after method step b); and (ii) Test the growth of the microorganism in a culture medium that does not contain PABA and the compound according to formula (I).

9. Use of a compound having general formula (I) as defined in claim 1 in the identification of carbamate enzyme genes in microorganisms.

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