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

Abstract

The present invention relates to a bio assay which identifies microorganisms expressing a urethanase gene. Growth on a medium deficient in folic acid an supplemented with u- aminobenzoic acid is used in this assay.

Description

[0001] Identification of urethanases based on growth selection using 4-aminobenzoic acid (PABA)

[0002] The present invention relates to a bio assay which identifies microorganisms expressing a urethanase gene. Growth on a medium deficient in folic acid an supplemented with u- aminobenzoic acid is used in this assay.

[0003] In bacteria 4-aminobenzoic acid (PABA) is a precursor in the biosynthesis of folic acid. PABA itself is derived from chorismate by two subsequent reactions: chorismate is converted to 4-amino-4-deoxychorismate in a first reaction. This reaction required deamination of glutamine in order to supply the required NH4. In a second step aminodeoxychorismate 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, thus providing the NH4 needed by the 4-amino-4-deoxychorismate synthase PabB.

[0004] The inactivation of any of these enzyme functions renders a microbial cell auxotrophic for PABA. The inactivation However, in the case of PabA, a lack of this enzyme function may be compensated by increased ammonia concentrations in the culture medium so that the HN4 required by PabB is supplied by an external source.

[0005] Polyurethanes are an important class of polymers produced in large quantities. The transition to a circular economy requires recycling method which enable the reuse of their carbon content. Enzymatic degradation of urethane bonds by urethanases has been disclosed, e.g. in WO 2019 / 243293. However, there remains the need for additional urethanases with new or improved properties.

[0006] WO 2019 / 243293 describes an assay which requires the expression of putative urethanase genes. The gene products are then incubated with a test substrate which - when cleaved by an enzyme - releases a fluorescent dye which can be detected by chromatography. As such a system requires cultivation of individual clones of microbes, the throughput is limited. It would be desirable to have an assay which does not rely on the cultivation of individual clones in order to identify candidate enzymes. This problem is solved by the embodiments defined in the claims and the description below.

[0007] In a first embodiment, the present invention relates to a method for identifying of urethanase genes in microorganisms comprising the steps of a) Incubating a microorganism which is auxotrophic for 4-amino benzoic acid (PABA) and which has been transformed with a nucleic acid molecule comprising at least one open reading frame under the control of a promotor enabling gene expression in said microorganism in a culture medium comprising a compound according to general formula (I)

[0008] Wherein R is any organic molecule with the proviso that the oxygen atom is linked to a carbon atom of said organic molecule. and comprising not more than 1 nM PABA and not more than 1 nM folic acid, b) identifying the microorganism carrying an urethanase gene based on its ability to grow in the culture medium.

[0009] The culture medium may be any medium meeting the requirements defined above and suitable for proliferation of the microorganism in question. Suitable culture media for different microorganisms are well known to the person skilled in the art and described in the literature. It is preferred to use culture media only containing chemically defined compounds because in those media it is easier to control the concentrations of PABA and folic acid. Moreover, these media are free of metabolites derived from folic acid so that growth in such a medium is dependent on the provision of PABA by cleavage of the molecule defined by formula (I).

[0010] The medium may be used in liquid form. However, in a preferred embodiment of the present invention, the medium is solidified with a suitable thickener, most preferably agar-agar. Culture medium comprising microbial cells in the correct concentration may then be spread on the solidified medium so that microbial cells capable of growth in the medium give rise to well separated individual colonies. Thus, in this embodiment, method step a) is preceded by the steps of providing the culture medium in solidified form and spreading a liquid composition comprising the microbial cells on the solidified medium. If the urethane bond present in the compound defined by formula (I) is cleaved, PABA is released and can be used as intermediate for the biosynthesis of folate, thus enabling the microorganism to grow.

[0011] The structure of the compound according to formula (I) may be adapted depending on the type of urethanase that is to be identified.

[0012] In a preferred embodiment of the present invention, R is a residue selected from the group consisting of branched or non-branched alkyl residues, branched or non-branched alkenyl residues, branched or non-branched aralkyl residues and aryl residues. Preferably these residues have a molecular weight between 27 and 100,000 g / mol, more preferably between 27 and 10,000 g / mol and most preferably between 27 and 2,000 g / mol. Said residues may comprise hetero atoms, preferably nitrogen, oxygen or sulphur. It is also envisaged that one or more hydrogen atoms of said residues may be substituted by a different atom or a functional group. Preferred substituents are halogen atoms, carboxyl groups, thiol groups, hydroxyl groups, nitro groups, nitrile groups, carbonyl groups and carboxyl groups.

[0013] In a preferred embodiment of the present invention, R is a branched or non-branched alkyl or alkenyl residue with 2 to 12 carbon atoms, preferably 2 to 6 carbon atoms. At least one hydrogen atom of said residue may be substituted by a member of the group consisting of halogen atoms, carboxyl groups, thiol groups, hydroxyl groups, nitro groups, nitrile groups, carbonyl groups and carboxyl groups.

[0014] In a more preferred embodiment of the present invention R is an alkenyl residue comprising 2 to 12, preferably 2 to 6, carbon atoms. At least one hydrogen atom of said residue may be substituted by a member of the group consisting of halogen atoms, carboxyl groups, thiol groups, hydroxyl groups, nitro groups, nitrile groups, carbonyl groups and carboxyl groups. However, R is preferably unsubstituted. In this embodiment i is preferred that R comprises one double bond which is located next to the terminal oxygen atom of formula (I).

[0015] In a particularly preferred embodiment, the compound according to formula (I) is 4- [(allyloxycarbonyl)amino]benzoic acid.

[0016] If the identification of urethanases capable of cleaving polyurethanes is intended, R is preferably polyurethane residue having a number-average molecular weight from 2,000 to 100,000 g / mol. Such molecules may be easily prepared by synthesizing an isocyanate- functional prepolymer and capping said prepolymer with PABA. The term “polyurethane” encompasses all compounds having the aforementioned molecular weight and comprising at least three, preferably at least five and most preferably at least ten urethane groups. The polyurethane may be a polyether polyurethane, a polyester polyurethane or a polycarbonate polyurethane. It is clear to the person skilled in the art that urethanes may be obtained by reacting polyisocyanates with any arbitrary mixture of polyether polyols, polyester polyols and polycarbonate polycarbonate polyols so that polyurethanes comprising any combinations of polyethers, polyesters and polycarbonates are also encompassed by the term “polyurethane”.

[0017] It is preferred that the concentration of the compound according to formula (I) is between 1 pM and 500 pM.

[0018] The term “incubating” refers to the contacting of the microorganism with the culture medium under conditions, particularly temperature, that are suitable for growth of the microorganism.

[0019] The microorganism may be a bacterium or a yeast. Preferably, it is a bacterium, more preferably Escherichia coli.

[0020] It is essential that the microorganism is auxotrophic for 4-amino benzoic acid (PABA), i.e. that it is not capable of synthetizing said compound so that it is dependent on supplementation. This can be achieved by inactivating the enzymatic function providing 4- amino-4-deoxychorismate and / or the aminodeoxychorismate lyase which converts 4- amino-4-deoxychorismate to PABA.

[0021] If the microorganism is E. coli, this auxotrophy may be achieved by the inactivation of at least one of the enzymes selected from the group consisting of PabA, PabB and PabC, preferably PabA and / or PabB, most preferably PabA.

[0022] The term “inactivation” relates to any genetic modification which either prevents the expression of the respective enzyme or which leads to production of a functionally deficient enzyme. Expression of an enzyme may be prevented by any method known in the art of molecular biology, particularly by deletion of the gene encoding the enzyme or by modifying the regulatory sequence of the respective gene to suppress gene expression. Functionally deficient enzymes result from the introduction of a stop codon into the gene question leading to the expression of a truncated protein or they result from the introduction of mutations or deletions of parts of the gene which affect the substrate binding site or the active center.

[0023] The method defined above is particularly suited for the identification of urethanase genes in isolated nucleic acid sequences. If growth of the microorganism is restored by transformation of the microorganism with DNA comprising at least one open reading frame under control of a suitable promotor, then it is likely that this open reading frame encodes a urethanase. A “suitable promotor” as referred to above, is a promotor which enables expression of a gene in the microorganism in question. In that case the identification of the urethanase gene is identical to identifying a microorganism that can grow under the culture conditions set forth above in this application. If the transformation of the microorganism with at least one open reading frame under control of a suitable promotor is sufficient to allow growth of the microorganism in the medium, then it must be concluded that the open reading frame encodes the gene responsible for that effect.

[0024] A particularly suitable microorganism for the identification of urethanase genes in recombinant DNA is E. coli because its genetics are well understood and a large array of tools for genetic manipulation is available. Preferred strains of E. coli for this use are those not expressing at least one gene selected from the group consisting of of pabA, pabB and pabC. More preferred are strains not expressing pabA and / or pabC. Particularly preferred is a strain not expressing pabA.

[0025] It is evident that the method of the present invention is particularly useful for the screening for urethanase genes in genetic libraries, particularly in metagenomic libraries. The person skilled in the art is well aware of means and methods for establishing such libraries.

[0026] If the screening of a DNA sequence for the presence of open reading frames encoding an urethanase is intended, the method of the present invention preferably comprises a further method step preceding step a). In this method step, the microorganism is transformed with a nucleic acid carrying at least one open reading frame under the control of a promoter sequence enabling expression of the open reading frame in said microorganism.

[0027] The person skilled in the art is aware that the method of the present invention may - in certain circumstances - yield false-positive results, i.e. growth of the microorganism is restored even though no urethanase is present. This is the case if a nucleic molecule introduced into the microorganism comprises a functional version of the enzyme or enzymes which were inactivated to create the PABA-auxotrophy. Obviously, such genes simply restore growth by repairing the disrupted metabolic pathway.

[0028] In order to eliminate such false-positive results, in one further embodiment of the present invention, method step b) is followed by an additional method step of confirming urethanase activity. This may be done by all biochemical methods known to the person skilled in the art. Preferred is the incubation of microorganisms with restored growth or lysates of such microorganisms together with a suitable test substrate comprising at least on urethane group which generates a signal if said urethane group is cleaved. Suitable test substrates and detection methods are disclosed in WO 2019 / 243293. In a more preferred embodiment, cells detected in method step b) are grown in two parallel cultures, one supplemented with PABA and the other one without PABA. Both cultures are free from compounds as defined by formula (I). If growth is observed in the PABA-free culture, it is shown that the microorganism simply reacquired the constituents of the biosynthetic pathway leading to PABA. In another embodiment of the present invention, the confirmation takes place during method step b). Preferably, this is achieved by adding a suitable test substrate to the culture medium or the agar plate so that cleavage of the urethane group of the test substrate induces a colour change of the medium or agar. In another embodiment, the present invention relates to the use of a compound having the general formula (I) as defined above for the identification of urethanase genes in microorganisms.

[0029] The following examples are merely intended to illustrate the present invention. They shall not limit the scope of the claims in any way.

[0030] Examples

[0031] 1. Development of auxotrophic strains

[0032] The Escherichia coli auxotrophs ApabA (JW3323-KC; CGSC Strain #10483) and ApabB (JW1801-KC; CGSC Strain #9507) from the E. coli K-12 Keio Collection were ordered from The National Bioresource Project E. coli Strain Office at the National Institute of Genetics in Mishima, Japan (https: / / shigen.nig.ac.jp / ecoli / strain / ). The strains were received on small pieces of filter paper. The pieces of paper were added to 5 mL of LB medium containing 50 pg / mL kanamycin sulfate and incubated overnight at 30 °C. Glycerol stocks were made by adding 750 pL culture to 750 pL sterile 50% glycerol before storage at 80 °C.

[0033] Selection of the ApabA strain

[0034] The M9 glycerol minimal medium (short: M9 medium) contained only 18.69 mM ammonium chloride and it was clearly demonstrated that at least 10 nm PABA is required for growth of the ApabA strain, with no growth in the absence of added PABA. No such data were reported for the ApabB strain. The ApabA strain also grew slightly faster than ApabB in M9 media supplemented with 10 pM PABA. Therefore, the ApabA strain was selected for further work, despite the conditional auxotrophy of this strain (the ammonium concentration in M9 medium is far below the 100 mM required for ApabA to grow).

[0035] 2. Minimal medium used in the study

[0036] Folate (and therefore PABA) is required for the biosynthesis of metabolites that may be derived from complex media. Therefore, PABA auxotrophy is conditional and PABA auxotrophs can grow in complex media. Therefore, an M9 glycerol minimal medium was used for cultivation experiments. 2x M9 Minimal Salts from Gibco (Thermo Fisher Catalogue NumberA1374401)was used as the base for the medium to avoid contamination with PABA. The glycerol used as carbon source was taken from an unopened bottle. All other components were as clean as possible but could not be prepared from unopened bottles. The basal medium (50 mL) contained 1x M9 salts (25 mL of the 2x stock), 1 % glycerol (1 mL of a 50% solution), 100 pM calcium chloride (5 pL of a 1 M solution), and 2 mM magnesium sulfate (100 pL of a 1 M solution). Because the strains from the Keio Collection are kanamycin resistant, 50 pg / mL kanamycin sulfate (50 pL of a 50 mg / mL solution) was added to avoid contamination with wild-type E. coli, which would grow in M9 glycerol minimal medium without added PABA. For strains transformed with pBAD-based vectors, 100 pg / mL ampicillin (50 pL of a 100 mg / mL solution) was also added to the medium. For induction of protein expression from pBAD vectors, 0.01% arabinose was added to the medium if not mentioned otherwise (the actual assay was performed without arabinose).

[0037] For PABA-growth assay agar plates, -1.5% (w / w) washed Agar-Agar (Art.-Nr. 5210.4, Carl Roth GmbH & Co. KG) was used. It was essential to wash the agar prior to use due to PABA contamination. For washing a 5x concentrated agar solution (75 g / L) was autoclaved and solidified. The agar gel was crushed into small pieces and washed in 5 L MilliQ water under stirring at 4 °C for two days with several water exchanges (2-3 times a day). The PABA-free agar was then and weighted to estimate the amount necessary for 1x agar solution and frozen at -20 °C in 1x portions. For fresh M9 medium agar plates, these portions were thawed and autoclaved with M9 minimal medium. After cooling down to -60 °C, ampicillin (100 pg / mL), kanamycin sulfate (50 pg / mL) and the appropriate substrate (no substrate, 5 pM PABA, or 10 pM alloc-PABA) were added to the solution before preparing the plates.

[0038] 3. Incubation

[0039] Cultures generally consisted of 1 mL of M9 medium in a 15 mL centrifuge tube. For inoculation of PABA-free M9 medium, starter cultures were grown to saturation (48 h at 37 °C) in medium containing 5 mM PABA. If different cultures were compared, the GD600 was normalised to the lowest value. The cultures were diluted 100,000-fold. This was approximated by two serial dilutions of 3 pL of culture into 997 pL of PABA-free M9 glycerol minimal 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 seven times without synthesising new PABA, meaning that cultures containing 50,000 cells / mL or more become turbid even in the absence of PABA. Therefore, it is critical that smaller inocula are used. A 100,000-fold dilution of a saturated culture (GD600 » 1) results in approximately 5,000 cells / mL.

[0040] 4. Transformation of the ApabA E. coli strain into chemically competent cells

[0041] For transformation of the ApabA E. coli strain, chemically competent cells were made using the Mix & Go! E. coli Transformation Buffer Set (Zymo Research Catalogue Number T3002). The manufacturer's protocols for competent cell preparation and transformation were followed. Single transformed colonies were picked from LB agar plates containing 50 pg / mL kanamycin sulfate and 100 pg / mL ampicillin. M9 glycerol minimal media containing 5 mM PABA were inoculated, and the cultures (1 mL) were grown to saturation (37 °C for 48 h). Glycerol stocks were prepared by mixing 750 pL of culture with 750 pL of 50% glycerol and stored at -80 °C. These glycerol stocks were used for inoculation of all subsequent cultures. The strains (E. coli ApabA empty and other controls) could not grow after -100,000-fold dilution into PABA-free M9 medium, confirming that they could be used for our urethanase growth selection experiments.

[0042] 5. Preparation of electrocom petent cells of E. coli ApabA

[0043] To prepare electrocom petent cells, a preculture from a glycerol stock of E. coli ApabA was prepared in 4 mL LB medium with ampicillin (100 pg / mL) and kanamycin sulfate (50 pg / mL) and this was incubated at 37 °C and 200 rpm for 16-18 h. Subsequently, 100 pL of preculture was inoculated into 100 mL of prewarmed SOC medium in a 1 L flask. The flask was incubated at 37 °C and 140 rpm in a rotary shaker. When the culture reached an OD600 » 0.5, the flask was quickly transferred to an ice-water bath for 15-30 min. The culture was occasionally swirled to ensure homogeneous cooling. In preparation 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. The supernatant was discarded, and the cell pellet was carefully resuspended in 50 mL of ice-cold 10% sterile glycerol. Centrifugation and washing in glycerol were repeated at least three times until the pellet was resuspended in 1 mL of 10% sterile glycerol. When not used directly, the cells were aliquoted to 50 pL, frozen in liquid nitrogen, and stored at 80 °C.

[0044] 6. PABA-growth selection assay on agar plates (and electroporation)

[0045] For transformation of DNA via electroporation into electrocompetent cells, the E. coli cells were kept at room temperature until the bacterial suspension was thawed and then placed on ice. 25 pL of the suspension was pipetted into an ice-cold microcentrifuge tube and 1 pL of the DNA (1000 ng per transformation) was added. Then, 25 pL of the bacteria / DNA solution was electroporated (2.1 kV,100 Q, and 25 pF). The typical time constant was ~2.6 ms. For cell recovery, 1 mL of SOC medium was added at room temperature and incubated at 37 °C for 1 h with slight rotation. After recovery, the cells were harvested by centrifugation at 4 °C for 5 min at 2,000-3,000 g and carefully resuspended in 2 mL M9 medium. The cells were subsequently centrifuged at 2,000 g for 5 min and gently resuspension in 2 mL M9 medium. Finally, the culture was plated out on PABA-growth assay agar plates containing 10 pM alloc-PABA in 50 pL fractions. The plates were incubated at 37 °C for two days.

[0046] 7. Synthesis and washing of 4-[(allyloxycarbonyl)amino]benzoic acid alloc-PABA

[0047] Alloc-PABA used in the growth assay was prepared as follows: PABA (1 g, 7.29 mmol) was added to a stirred solution of FLO / dioxane (10 mL / 10 mL) in a reaction bulb glass at room temperature. N,N-diisopropylethylamine (2.6 mL, 14.9 mmol) and NaHCCh (1.8 g, 21.8 mmol) were added and the mixture was stirred for several minutes. -3 mL dioxane was added to counteract evaporation. Eventually, 1 mL of chloroformate was added dropwise to the stirring reaction. The reaction was stirred for 15 min with air flow to evaporate some dioxane so that the alloc-PABA will be less soluble. The reaction was then quenched by addition of 100 mL of 1.2 M aqueous hydrochloric acid (HCI). During this step lots of foam built up due to the reaction of HCI with bicarbonate (CO2 production) so that the HCI was added in several steps. Subsequently another -100 mL of 1.2 M HCI was added to ensure that all alloc-PABA was precipitated and PABA solubilized. The reaction was stirred for -15 min. The alloc-PABA was vacuum-filtered and washed with another 100 mL of 1 .2 M HCI. Reaction bulb and filter glass were washed with -60 mL acetone to recover most of the alloc-PABA. The alloc-PABA was precipitated with 60 mL 1 .2 M HCI and acetone was evaporated by air flow for -20 min before another 140 mL of 1.2 M HCI was added. Everything was vacuum-filtered again and washed with 1.2 M HCI. In order to improve the purity of the product the remaining alloc-PABA (-0.7 g) was dissolved in 25 mL ethyl acetate. First, the solution was filtered with a PFTE filter to get rid of cellulose fibers from the vacuum filtration, then the solution was filled into a separation funnel and 60 mL of 1.2 M HCI were added. The two-phase system was thoroughly mixed and the lower water fraction was discarded. Fresh 1.2 M HCI was added and this process was repeated 4-times. Finally, the ethyl acetate fraction was placed into a glass beaker and evaporated overnight under a fume hood. After this washing process alloc-PABA with a PABA contamination of <0.01% was obtained.

[0048] 8. Establishing the assay layout

[0049] Because of the extreme small amounts of PABA that are necessary for growth of E. coli ApabA, it is very likely that negative variants of a library could grow using PABA which is diffused from PABA-producing hits. Therefore it was decided to use an agar plate setup since it was expected to provide an advantage over liquid cultivation in terms of diffusion and separation of hit candidate clones. To investigate the applicability of the setup, it was investigated to what extent the cell density of the negative control (ApabA_empty) could be increased without obtaining a cell lawn. Therefore 50 mL culture of ApabA_empty were cultivated in LB medium, washed with M9 medium once, and finally resolved in -10 mL of M9 medium with plated out different dilutions on both screening plates containing M9 medium and 10 pM alloc-PABA and control plates containing LB to estimate the cell density. The LB-controls revealed the cell density of the culture (washed and subsequently resolved in 10 mL M9 medium) to be -6 billion colony forming units (CFUs) / mL. It was therefore concluded that even cell densities of >6 million cells / plate (using E. coli ApabA_empty as negative control) can not grow on our M9 plates supplemented with 10 pM alloc-PABA. 9. Screening of a metagenome library

[0050] After establishing the assay, it was applied on a metagenome library constructed from soil (supplied by Covestro, Leverkusen, Germany) that had been exposed to polyurethane for many years. For this, the library was transformed into electrocompetent E. coli ApabA cells. After recovery in SOC medium for 1 h, the cells were first washed and subsequently resuspended in M9 medium before plating them out on the screening plates. After two days at 37 °C, many single colonies developed. However, when screening a metagenome library, it must be kept in mind that it is very likely to find homologues of pabA within this library, because it is a ubiquitous gene. Therefore, for metagenome screening this assay served well as a potent pre-screening but the hits derived from this assay subsequently needed to be re-screened to distinguish between pabA homologues and actual urethanase hits. For this, the urethanase activity was confirmed on the model substrate 7-carbethoxy-4- methylcoumarin. After re-screening, one open reading frame encoding a hydrolase (named: UMG-SP-4) was obtained which has not been described before. The closest homologue (80.0% identity) is an amidase from a Hyphomonadaceae bacterium. UMG-SP-4 exhibits 43.64%, 44.24%, and 46.10% identity to the urethanases UMG-SP-1 , UMG-SP-2, and UMG-SP-3, respectively, which had been found and characterized before (WO 2023 / 194440).

[0051] 10. Re-screening of metagenome hits from the PABA-growth selection assay

[0052] Since the PABA-growth selection assay does not only select urethanases but also PabA homologues which are frequently found in metagenomes, a re-screening of the hits from the assay was necessary to distinguish between urethanases and PabA homologues. Colonies from the assay plates were inoculated in microtiter plates containing M9 glycerol minimal medium either with or without 5 pM PABA whereas the wells without PABA were inoculated first. Only E. coli hits that grew in 5 pM PABA but not in PABA-free medium must contain desired true urethanase hits because they were not able to produce PABA from scratch. Alternatively, the hits from the prescreening were incubated on the model substrate 7-carbethoxy-4-methylcoumarin to verify urethanase activity.

Claims

Claims1 . Method for identifying of urethanase genes in microorganisms comprising the steps of a) Incubating a microorganism which is auxotrophic for 4-amino benzoic acid (PABA) and which has been transformed with a nucleic acid molecule comprising at least one open reading frame under the control of a promotor enabling gene expression in said microorganism in a culture medium comprising a compound according to general formula (I)Wherein R is any organic molecule with the proviso that the oxygen atom is linked to a carbon atom of said organic molecule; and comprising not more than 1 nM PABA and not more than 1 nM folic acid, b) identifying the microorganism carrying an urethanase gene based on its ability to grow in the culture medium.

2. The method according to claim 1 , wherein the concentration of the compound according to formula (I) in the medium is between 1 pM and 500 pM.

3. The method according to claim 1 or 2, wherein R is a residue selected from the group consisting of branched or non-branched alkyl residues, branched or nonbranched alkenyl residues, branched or non-branched aralkyl residues and aryl residues having a molecular weight between 27 and 100,000 g / mol.

4. The method according to any one of claims 1 to 3, wherein the medium is a mineral medium free of complex sources of carbon.

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

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

7. The method according to claim 6, wherein E. coli does not express at least one genes selected from the group consisting of pabA, pabB and pabC.

8. The method according to any one of claims 1 to 7, wherein the presence of a urethanase is confirmed by at least one method selected from the group consisting of(i) determining urethanase activity using as suitable test substrate which may be performed during method step b) by adding the test substrate to the culture medium or subsequently to method step b) in a separate culture; and (ii) testing the microorganism for growth in a medium free of PABA and the compound according to formula (I).

9. Use of a compound having the general formula (I) as denied in claim 1 for the identification of urethanase genes in microorganisms.

Images