Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/12—Methionine; Cysteine; Cystine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)

Definitions

• the present invention relates to a new process for the isolation and / or selection of genes coding for enzymes involved in the bioconversion of a substrate into methionine or its derivatives, such as 2-hydroxy 4- (methylthio) butanoic acid or its salts, in particular hydrolyzing amide groups into carboxylic acids, or involved in the bioconversion of nitrile groups into corresponding carboxylic acids, by means of an appropriate selection screen, and the
• the enzymes catalyzing the hydrolysis of nitrile groups into corresponding carboxylic acids and ammonium ions are nitrilases (Faber, Biotransformations in Organic Chemistry, Springer Verlag, Berlin Heidelberg, 1992, ISBN3-540-55762-8). However, this bioconversion of the nitrile groups into
• corresponding carboxylic acids the final balance of which consists in hydrolysis of the nitrile groups can also be carried out in two stages, the first stage consisting in the conversion of the nitriles into corresponding amides by a nitrile hydratase, the second stage consisting in hydrolizing the amides obtained into corresponding carboxylic acids by amidases.
• Nitrilases 25 characterized in thermophilic bacteria (Cramp et al, 1997, Microbiology. 143: 2313-2320). Nitrilases have specificities of various substrates but can be grouped into three groups according to their specificity: nitrilases specific for aliphatic nitriles, those specific for aromatic nitriles or those specific for arylacetonitriles (Kobayashi et al., 1993, Proc. Natl Salt Academy
• Nitrilases are of interest in biocatalysis because many synthetic processes involve hydrolysis of nitrile groups (Lévy- Schil et al.).
• the nitrilase from Alcaligenes faecalis ATCC9750 and that from Comamonas testosteroni can be used to access the hydroxy analogue of methionine (FR9411301, WO9609403, FR9613077).
• microbiological isolation of microorganisms from specific biotopes using a screen selection based on the presence of the desired enzymatic activity ii) search for an enzymatic activity in microorganisms by culture of the strains and assay of the activity in question, iii) search, in cultivable microorganisms, of silent and homologous genes to a gene of interest then cloning and expression of this gene in a model microorganism, iv) engineering of proteins to improve the characteristics of an available enzyme and finally v) cloning by expression of directly isolated genes of various biotopes without prior microbiological isolation.
• in vivo selection screen is meant a medium and / or growth conditions which allow growth only of microorganisms harboring the desired enzymatic activity.
• strategies iv) and v) are particularly interesting for making better use of biodiversity by targeting the biodiversity available but not accessible by microbiological isolation (strategy v) or by creating more diversity from cultivable strains (strategy iv). It is particularly for these two strategies that the possibility of having an in vivo selection screen which is simple to implement represents a definite advantage which leads to screening a large number of clones in a short time and if necessary with means.
• the invention consists of a simple, rapid and inexpensive method for isolating and / or selecting DNA sequences coding for enzymes involved in the bioconversion of a substrate into methionine salt (AMTBS) or its derivatives such as 2-hydroxy 4- (methyl thio) butanoic acid (hereinafter HMTBS), in particular the ammonium salt, and in particular involved in the bioconversion of 2-amino 4- (methyl thio) butyronitrile (AMTBN) or its derivatives, such as 2-hydroxy 4- (methyl thio) butyronitrile (hereinafter HMTBN) in methionine or its derivatives such as HMTBS, either directly or via 2-amino 4-methyl thio butanamide or its derivatives , such as 2-hydroxy 4-methyl thio butanamide (hereinafter HMTBAmide).
• ATBS methionine salt
• HMTBS 2-hydroxy 4- (methyl thio) butanoic acid
• HMTBN 2-hydroxy 4- (methyl thio) butyronit
• the enzymes are involved in the bioconversion of an appropriate substrate into HMTBS, the suitable substrates being HMTBN or HMTBAmide.
• HMTBS HMTBN or HMTBAmide
• the indications relating to HMTBS, HMTBN or HMTBAmide also apply to methionine or its derivatives, and the corresponding substrates, 2-amino 4- (methyl thio) butyronitrile or its derivatives and 2-amino 4-methyl thio butanamide or its derivatives.
• the present invention therefore relates to a method of selecting and / or isolating DNA sequences coding for enzymes involved in the bioconversion of an appropriate substrate into methionine and its derivatives, such as HMTBS, said method comprising the following steps of 1) cloning of DNA sequences into a vector allowing their expression in an appropriate host microorganism,
• the appropriate substrate 2-hydroxy 4- (methylthio) butyronitrile (hereinafter HMTBN) which is converted into 5 HMTBS, either directly by a nitrilase, or via 2 -hydroxy 4- (methylthio) butanamide (hereinafter HMTBAmide), this second route involving a first conversion of HMTBN to HMTBAmide by a nitrile hydratase, followed by a conversion of HMTBAmide by an amidase.
• HMTBN 2-hydroxy 4- (methylthio) butyronitrile
• HMTBAmide 2 -hydroxy 4- (methylthio) butanamide
• the suitable substrate is HMTBAmide, which is converted into HMTBS by an amidase, coded by the DNA sequence isolated and / or selected by the method according to the invention.
• isolation is essentially meant according to the invention the separation of a particular sequence from a set of varied sequences.
• selection is essentially meant according to the invention the choice of the best sequence having a particular property.
• microorganism suitable auxotrophic for methionine is meant according to the invention any microorganism auxotrophic for methionine capable of being transformed and capable of growing on a methionine-free medium comprising HMTBS. It can be a yeast, a fungus or a bacteria.
• the appropriate microorganism is a bacterium, preferably E. coll.
• the appropriate microorganism useful according to the invention can be naturally auxotrophic for methionine. or modified so as to induce this auxotrophy by mutagenesis.
• the various methods for obtaining auxotrophic mutants are well known to those skilled in the art and widely described in the literature (in particular Roberts CJ, Selker EU.
• the appropriate microorganism transformed with a vector comprising a DNA sequence coding for an enzyme involved in the bioconversion of HMTBN into the corresponding carboxylic acid is capable of converting HMTBN into HMTBS, allowing the auxotrophic microorganism to methionine to develop.
• the microorganism ensures the bioconversion of HMTBS into methionine to allow the strain to grow.
• the transformed with a vector suitable microorganism comprising a DNA sequence encoding an enzyme involved in the bioconversion del 'HMTBAmide corresponding carboxylic acid, is able to convert the HMTBAmide in HMTBS, allowing the microorganism auxotrophic for methionine to develop oneself.
• the microorganism suitable auxotrophic for methionine also comprises a gene coding for an enzyme involved in the bioconversion of HMTBAmide into HMTBS, and when this microorganism is transformed with a vector comprising a DNA sequence coding for an enzyme involved in the bioconversion of HMTBN into HMTBAmide, it is capable of converting HMTBN into HMTBS, allowing it to develop.
• the isolated and / or selected enzyme involved in the bioconversion of HMTBN into HMTBS is a nitrilase allowing the conversion of HMTBN into HMTBS necessary for the growth of microorganisms modified.
• the isolated and / or selected enzyme involved in the bioconversion of HMTBN into HMTBS is a nitrile hydratase allowing the conversion of HMTBN into HMTBAmide.
• the appropriate microorganism also comprising a natural or heterologous gene coding for a complementary amidase ensuring the bioconversion of HMTBAmide into HMTBS necessary for the growth of the modified microorganisms.
• the isolated and / or selected enzyme involved in the bioconversion of HMTBN into HMTBS is an amidase, the appropriate microorganism also comprising a natural or heterologous gene coding for a complementary nitrile hydratase.
• the gene for the complementary enzyme if it is heterologous, is either integrated into the genome of the appropriate microorganism, or carried by a plasmid.
• the isolated and / or selected enzyme involved in the bioconversion of HMTBamide into HMTBS is an amidase, and the appropriate culture medium contains HMTBAmide.
• the DNA sequence is a DNA sequence isolated from one or more genomes, by total or partial restriction of said one or more genomes.
• the DNA sequence can also be a DNA sequence isolated from a part of the genome by total or partial restriction of said part of the genome.
• restriction is meant any means, enzymatic or not, capable of fragmenting DNA, specifically or not.
• the method according to the invention is in this case particularly suitable for the selection of new enzymes according to strategies i) and v) defined above.
• the DNA sequence can also be a DNA sequence isolated from a construct by PCR (Polymerase Chain Reaction).
• the DNA sequence can also be obtained by random or targeted mutagenesis of a sequence coding for a reference enzyme defined above.
• the method according to the invention is particularly suitable for the selection of new enzymes according to strategy iv) defined previously.
• the DNA sequence can also be an isolated DNA sequence having determined homology with a reference enzyme defined above. This determined degree of homology is advantageously greater than 50%, preferably greater than 60%, more preferably greater than 70%.
• the method according to the invention constitutes a rapid screen to be implemented to identify to what extent a nucleic acid sequence having a certain level of homology with a reference sequence, has the same function and / or o the same activity as the reference sequence.
• reference enzyme is meant according to the invention a known enzyme involved in the bioconversion of the appropriate substrate into HMTBS, in particular HMTBN into HMTBS or in the bioconversion of HMTBAmide into HMTBS, preferably a nitrilase, a nitrile hydratase or an amidase, defined above, which will serve as a reference for evaluating the function and activity of the new enzymes isolated and / or selected by the process according to the invention, in particular the enzymes for which new mutants are sought and / or those with a certain degree of homology defined previously.
• the culture of the microorganisms transformed for the isolation and / or selection process according to the invention is carried out by any suitable means known to those skilled in the art. It is in particular a mass culture (batch), in particular by successive cultures, or by continuous cultures, in particular cultures in chemostat (Seegers JF & al., Plasmid, 1995 Jan 33: 1 71 -7; Weikert C & al., Microbiology, 1997 May 143 (Pt 5): 1567-74; Tsen SD & al, Biochem Biophys Res Commun, 1996 Jul 16 224: 2 351-7; Tsen SD, Biochem Biophys Res Commun , 1990 Feb 14 166: 3 1245-50; Berg OG, J Theor Biol, 1995 Apr 7 173: 3 307-20).
• appropriate culture medium comprising a sufficient amount of suitable substrate
• any culture medium suitable for the growth of the microorganism transformed auxotrophic for methionine said medium being substantially free of methionine, and comprising a sufficient amount of suitable substrate to allow the growth of said microorganism transformed after bioconversion of said suitable substrate into HMTBS.
• medium substantially free of methionine is meant a medium free of methionine or optionally comprising an amount of methionine insufficient to allow the growth of the microorganism suitable auxotrophic for methionine.
• the sufficient amount of HMTBN is advantageously between 0 and 60 g / 1 (equivalent of approximately 400 mM), more preferably between 3 mg / 1 and 17 mg / 1 (equivalent of approximately 20 ⁇ M and approximately 100 ⁇ M) , more preferably between 6 mg / 1 and 10 mg / 1 (equivalent of approximately 40 ⁇ M and approximately 60 ⁇ M).
• the sufficient amount of HMTBAmide is advantageously between 0 and 60 g / 1 (equivalent of approximately 400 mM), more preferably between 3 mg / 1 and 17 mg / 1 (equivalent of approximately 20 ⁇ M and approximately 100 ⁇ M), more preferably between 6 mg / 1 and 10 mg / 1 (equivalent of approximately 40 ⁇ M and approximately 60 ⁇ M).
• the essential source of HMTBS necessary for the growth of the transformed microorganisms is constituted by the HMTBS produced by the conversion of the appropriate substrate, in particular HMTBN or HMTBAmide, by the enzyme encoded by the DNA sequence introduced into the transformed microorganism.
• the medium according to the invention may also initially comprise (before and at the time of inoculation with the transformed microorganisms) an appropriate quantity of HMTBS, in particular to allow the growth of microorganisms to be initiated, this quantity of HMTBS being replaced as the microorganisms grow with HMTBS resulting from the conversion of the appropriate substrate, in particular HMTBN or HMTBAmide.
• the HMTBS is present in the medium at a concentration lower than the concentration sufficient to allow the growth of the microorganisms transformed according to the invention.
• This concentration can be determined according to the appropriate culture medium, in particular its form (bacth, continuous liquid, solid, etc.), and will preferably be less than 350 ⁇ g / 1 (equivalent to approximately 2 ⁇ M) more preferably less than 250 ⁇ g / 1 (equivalent of approximately 1.5 ⁇ M), more preferably between 130 ⁇ g / 1 and 170 ⁇ g / 1. (equivalent to approximately 0.8 ⁇ M and approximately 1 ⁇ M).
• the appropriate medium comprises a source of organic nitrogen not comprising traces of methionine.
• the organic nitrogen source is a yeast extract, in particular from Yeats Nitrogen Broth w / o Amino Acids (YNB, Yeats Nitrogen
• the content of organic nitrogen source is between 0 and 15 g / 1, more preferably between 2 and 10 g / 1, even more preferably between 3 and 8 g / 1.
• the appropriate medium according to the invention comprises M9fru described below as the minimum medium.
• the suitable medium according to the invention comprises as carbon source a compound chosen from glucose, fructose, galactose, trehalose, mannose, melibiose, sucrose, raffinose, maltotriose, maltose, lactose , lactulose, arabinose, xylose, rhamnose, fucose, mannitol, sorbitol, malate, saccharate, mucate, mesotartrate. glucuronate. galacturonate and their mixtures in all proportions.
• the carbon source is chosen from glucose or fructose, and their mixtures in all proportions, more preferably fructose.
• the culture medium according to the invention can be liquid or solid, and in this case can contain agar or agarose.
• Figure 1 shows the plasmid RPA-BIOCAT41.
• the sites in parenthesis are sites that were eliminated during cloning.
• Ptrp tryptophan promoter
• nitB nitrilase gene
• TrrnB transcription terminators
• end ROP end of the gene coding for the protein ROP (Chambers et al., 1988. Gene 68: 139-149);
• ORI origin of replication;
• RNAI / II RNA involved in replication (Chambers et al., Cited above); Te: tetracycline resistance gene.
• Figure 2 shows the growth at 37 ° C, 200 rpm and in a 50 ml hermetically closed tube, of the two strains RPA-BIOCAT 610 and 842 by reading the optical densities in microplates at 630nm.
• FIG. 3 shows the growth of the RPA-BIOCAT 841 and 842 strains in selective medium: A. with a limiting HMTBS concentration of 0.8 ⁇ M; B. with a limiting HMTBS concentration of 1 ⁇ M.
• the selective media are constituted by the minimal medium M9YNBfru + IPTG 0.5 mM supplemented with: HMTBS 0.8 ⁇ M (S0.8) or in HMTBS 1 ⁇ M (SI), or in HMTBN 50 ⁇ M + HMTBS 0.8 ⁇ M (NS0.8) or else in HMTBN 50 ⁇ M + HMTBS 1 ⁇ M (NSI).
• FIG. 4 shows the growth of the RPA-BIOCAT 841 and 960 strains in selective medium: A. in the presence of 0.8 ⁇ M HMTBS; B. in the presence of 1 ⁇ M HMTBS.
• the selective media are constituted by the minimal medium M9YNBfru + IPTG 0.5 mM supplemented with: HMTBS 0.8 ⁇ M (S0.8) or in HMTBS 1 ⁇ M (SI), or in HMTBN 50 ⁇ M + HMTBS 0.8 ⁇ M (NS0.8) or else in HMTBN 50 ⁇ M + HMTBS 1 ⁇ M (NSl).
• the minimal medium M9fru has the following composition: Na 2 HPO 4 7g / l, KH 2 PO 4 3g / l, NaCl 0.6 g / 1, NH 4 C1 1 g / 1, fructose, 4 g / 1, MgSO 4 1 mM , CaCl 2 0.1 mM, thiamine 10 ⁇ g / ml.
• the minimal medium M9YNBfru corresponds to the medium M9fru supplemented with YNB (Yeast Nitrogen Base, Difco) at a final concentration of 5 g / 1.
• This medium is prepared from the following mother solutions previously sterilized: M9 salts xlO (Na 2 HPO 4 70g / l, KH 2 PO 4 30g / l, NaCl 6 g / 1, NH 4 C1 10 g / 1, autoclave) , fructose 20% filtered, YNB 20% filtered, MgSO 4 100 mM autoclave. CaCl 2 10 mM autoclave, 1% thiamine filtered.
• the precultures are carried out in 3 ml of LB rich medium (tryptone 10 g / 1, yeast extract 5 g / 1, NaCl 10 g / 1, pH 7.0) in the presence of the appropriate antibiotics, if necessary, by seeding from a glycerol stock of the strain concerned.
• the preculture is incubated at 37 ° C, 200 rpm for 6 to 7 hours.
• the activities are expressed in kg of HMTBS produced / hour / kg of dry cells
• Example 1 Construction of the pRSP-BCAT41 piasmid.
• the 1.27 kb fragment containing the Ptrp promoter, the binding site of the ⁇ phage cili gene (RBScII) ribosome and the alkaligenes faecalis ATCC8750 (nitB) nitrilase gene was extracted from the plasmid pRPA-BCAT6 (Request FR 96/13077) using the restriction enzymes EcoRI and Xbal to be cloned into the vector pXL642 (described in application CIP No. 08 / l 94,588) opened with the same restriction enzymes.
• pRPA-BCAT15 was opened by enzymes Stul and Bsml and the 4.3 kb fragment was ligated with the 136 bp Stul-Bsml fragment purified from pRPA-BCAT4 (Application FR 96/13077) to yield the plasmid pRPA-BCAT19.
• the partial sequencing of pRPA-BCAT19 confirmed the replacement of the codon of the residue Asp279 of nitrilase by the codon of a residue Asn279.
• the vector pRPA-BCAT28 was obtained by ligating the 3.9 kb Sspl-Scal fragment of pXL642 (CIP application N ° 08 / l 94.588) with the 2.1 kb Smal fragment of pHP45 ⁇ Tc (Fellay et al, 1987, Gene 52: 147-154) in order to replace the ampicillin resistance marker with the tetracycline resistance marker.
• the plasmid pRPA-BCAT41 was obtained, a map of which is shown in FIG. 1.
• the sequence of the expression cassette is represented by the sequence identifier No. 1 (S ⁇ Q ID NO 1) .
• nitB gene was amplified by PCR using as matrix the plasmid pRPA-BCAT41, the primers PCRAF1 described in application FR96 / 13077 and NitB 160 described below and the enzyme Pfu (Stratagene).
• NitB 160 5'-GGGGAGAGGT GCTCCCAGCA GCACAGGCCA CCGACGGG-3 'The program used included a 5 min cycle at 95 ° C, 5 cycles of 1 min at 95 ° C; 1 min at 60 ° C; 1 min at 72 ° C, 30 cycles of 30 sec at 95 ° C; 30 sec at 60 ° C; 30 sec at 72 ° C and a 5 min sequence at 72 ° C.
• the fragment of approximately 0.495 bp thus amplified was digested with the enzymes NdeI and Rs / HKAI (New England Biolabs).
• the vector pRPA-BCAT72 was obtained by eliminating from the vector pRPA-BCAT41, by digestion Xcm 1 and religation, the fragment Xcml of approximately 0.55 kb.
• the plasmid resulting from this cloning was called pRPA-BCAT77.
• Example 3 Construction of a strain of E. auxotrophic coli expressing the nitrilase A' ⁇ lcaligenes faecalis from the P lac promoter.
• the alkaligenes faecalis ATCC8750 nitrilase gene was amplified by PCR using, as template, the plasmid pRPA-BCAT77, the primers nitBMN1 and nitBMN2 described below and the polymerase Pfu (Stratagene).
• nitBMNl 5'-TTGTTATCTA AGGAAATACT TA-3 'nitBMN2: 5'-CGACTCTAGA ACTAGTGGAT CC-3'
• the program used included a 5 min cycle at 95 ° C, 5 cycles of 1 min at 95 ° C; 1 min at 50 ° C; 1 min at 72 ° C, 30 cycles of 30 sec at 95 ° C; 30 sec at 50 ° C; 30 sec at 72 ° C and a 5 min sequence at 72 ° C.
• the approximately 1.2 kb fragment obtained was then digested with the enzyme Xbal to be cloned into the vector pbsll ks- (Stratagene) opened by the enzymes EcoRV and Xbal.
• the plasmid obtained, pRPA-BCAT105 was then introduced into the E. coli strain RPA-BIOCAT610.
• This strain contains a deletion in the metA gene and corresponds to the ⁇ 180 strain described in Richaud et al. (J. Biol. Chem. (1993) 268: 26827-26835).
• a clone was selected and cultivated in 3 copies in LB under the conditions of expression described above.
• the nitrilase activity was measured on the cell pellets obtained as described above and found after average at 2.5 kg / h.kg CS against 0 kg / h.kg CS for the strain RPA-BIOCAT 842 described in Example 4 and grown under the same conditions.
• This new strain expressing an active nitrilase was named RPA-BIOCAT 841.
• Example 4 Construction of a strain of E. auxotrophic coli expressing nitrilase ⁇ ' ⁇ lcaligenes faecalis inactive from the P lac promoter.
• the gene for a nitrilase variant NitB was amplified as described in Example 3 using the plasmid pRPA-BCAT69 as a template.
• the plasmid pRPA-BCAT69 corresponds to the vector pRPA-BCAT41 but contains a mutation in the nitB gene which leads to the replacement of the Cysteine 163 residue of the nitrilase NitB with an Alanine residue.
• Plasmid pRPA-BCAT69 was obtained as follows.
• Example 6 Growth of E. mutants auxotrophic coli for methionine in a minimum medium supplemented with HMTBS.
• the RPA-BIOCAT 610 and 842 strains were precultivated in LB medium supplemented, only for the strain 842, with 0.5 mM IPTG and carbenicillin 100 ⁇ g / ml, washed in M9YNBfru medium and taken up in an equal volume of this same medium. The two cultures were then diluted to 1/100 in 10 ml of the following media supplemented with carbenicillin 100 ⁇ g / ml and 0.5 mM IPTG only for the RPA-BIOCAT 842 strain: i) M9YNBfru + HMTBN 50 ⁇ M ii) M9YNBfru + 5 HMTBN 50 ⁇ M + HMTBS 10 ⁇ M. . These cultures were carried out in a hermetically sealed 50 ml tube at 37 ° C. with stirring at 200 rpm and the growth of the strains, measured by optical density of the culture at 630 nm read in microplate, is reported on the figure 2.
• HMTBS can be used as a source of o methionine by strains of E. coli auxotrophs for methionine.
• Example 7 Influence of YNB, HMTBN and HMTBS on the growth, in minimum medium, of a strain of E. coli auxotroph for methionine expressing active nitrilase of ⁇ lcalîgenes faecalis
• the RPA-BIOCAT 841 and 842 strains were precultivated in the presence of 5 carbenicillin 100 ⁇ g / ml and 0.5 mM IPTG, washed as described in Example 6 and diluted to l / 1000 emc in 5 ml media listed in Table 1, all supplemented with carbenicillin 100 ⁇ g / ml and IPTG 0.5 mM. Their growth was estimated after 5 days of incubation at 37 ° C., 200 rpm, in a 50 ml tube hermetically closed, by visual observation of the turbidity of the cultures. These results are collated in Table 1.
• HMTBN of 50 ⁇ M is too low to provide a sufficient source of methionine for the growth of E. coli.
• Example 8 Determination of the minimum concentration of HMTBS allowing the growth of the auxotrophic strains
• the RPA-BIOCAT 841 strain was precultured in the presence of carbenicillin 100 ⁇ g / ml and IPTG 0.5 mM, washed as described in Example 6 and diluted to l / 1000 ee in 5 ml of the media mentioned in table 2, all supplemented with carbenicillin 100 ⁇ g / ml and IPTG 0.5 mM. Their growth was estimated after 5 days of incubation at 37 ° C, 200 rpm. in a 50 ml hermetically sealed tube, by visual observation of the turbidity of the cultures. These results are collated in Table 2. Table 2: Growth of the RPA-BIOCAT 841 strain in the presence of HMTBS at low concentration.
• Example 9 Development of a selective medium allowing the growth of a strain of E. coli auxotrophic for methionine expressing the active nitrilase of callcaligenes faecalis.
• the RPA-BIOCAT 841 and 842 strains were precultivated in the presence of carbenicillin 100 ⁇ g / ml and 0.5 mM IPTG, washed under the conditions described in Example 6 then diluted to l / 100 th in 10 ml of selective medium M9YNBfru + 0.5 mM IPTG containing 0.8 or 1 ⁇ M HMTBS and 50 ⁇ M HMTBN.
• FIG. 3 shows the growth of these two strains at 37 ° C., 200 rpm in hermetically sealed 50 ml tubes, growth measured by reading the optical densities in microplates at 630 nm. The results show that the selective medium M9YNBfru + IPTG 0.5 mM +
• HMTBN 50 ⁇ M + HMTBS 0.8 or 1 ⁇ M makes it possible to differentiate a strain of E. coli expressing an active nitrilase of a strain of E. coli expressing an inactive nitrilase.
• Example 10 Growth of E. strains. coli auxotrophs for methionine expressing active nitrilases on hydroxy-methyl-thio-butyronitrile.
• the RPA-BIOCAT 841 and 960 strains were precultivated in the presence of carbenicillin 100 ⁇ g / ml and 0.5 mM IPTG, washed under the conditions described in Example 6 and then diluted to 1/100 in the selective growth media described. in Example 9.
• FIG. 4 describes the growth of these two strains at 37 ° C., 200 rpm in hermetically sealed 50 ml tubes, growth measured by reading the optical densities in microplates at 630 nm.
• Example 9 The results show that the medium described in Example 9 is selective for auxotrophic strains for methionine expressing nitrilases of two different origins in the expression system using the promoter R / ⁇ c -

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