EP1137784A1

EP1137784A1 - Method for isolating and selecting genes coding for enzymes, and suitable culture medium

Info

Publication number: EP1137784A1
Application number: EP99958292A
Authority: EP
Inventors: Olivier Favre-Bulle; Jérôme PIERRARD; Nadine Batisse Debitte
Original assignee: Aventis Animal Nutrition SA
Current assignee: Adisseo France SAS
Priority date: 1998-12-11
Filing date: 1999-12-10
Publication date: 2001-10-04
Also published as: AU773130B2; WO2000036120A1; JP2002532096A; FR2787121B1; AU1568900A; FR2787121A1

Abstract

The invention concerns a method for selecting and/or isolating DNA sequences coding for enzymes involved in the biological conversion of an appropriate substrate consisting of methionine and its derivatives, such as HMTBS, said method comprising the following steps: 1) cloning DNA sequences in a carrier enabling their expression in an appropriate host micro-organism; 2) transforming a suitable auxotroph micro-organisms for methionine by introducing carriers previously obtained in said suitable micro-organism; 3) growing the transformed micro-organisms previously obtained in a suitable culture medium comprising a sufficient amount of appropriate substrate; and 4) selecting the transformed micro-organisms capable of growing in the appropriate culture medium; and 5) isolating and as the case may be identifying the DNA sequences involved in the biological conversion of the appropriate substrate.

Description

METHOD OF ISOLATING AND SELECTING GENES ENCODING ENZYMES, AND APPROPRIATE CULTURE MEDIUM

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

10 culture medium suitable for the implementation of said method.

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

15 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.

20 Nitrilases were first discovered in plants (Thimann and

Mahadevan. 1964, Arch. Biochem. Biophys. 105: 133-141) then isolated from many representatives of the soil micro flora (Kobayashi and Shimizu. 1994, FEMS Microbiology Letters 120: 217-224): Pseudomonas, Nocardia, Arthrobacter, Fusarium, Rhodoccocus, Alcaligenes. More recently, nitrilases have been

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

USA 90: 247-251; Kobayashi and Shimizu, 1994, supra; Lévy-Schil et al, 1995, Gene 161: 15-20). Nitrilases are of interest in biocatalysis because many synthetic processes involve hydrolysis of nitrile groups (Lévy- Schil et al.). In particular, the nitrilase from Alcaligenes faecalis ATCC9750 and that from Comamonas testosteroni can be used to access the hydroxy analogue of methionine (FR9411301, WO9609403, FR9613077).

It is common, when looking for new enzymes, to use several strategies (Dalboge and Lange, 1998, TibTech 16: 265-272): i) 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. Strategies i), iii), iv), and v) are considerably accelerated and simplified if an in vivo selection screen is available. By in vivo selection screen is meant a medium and / or growth conditions which allow growth only of microorganisms harboring the desired enzymatic activity.

In addition, given the very small proportion of microorganisms that can be cultivated in the laboratory (Amann et al., 1995, Microbiol Rev., 59, 143), 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. limited (Minshull, 1998, "Evolution of enzyme activities and substrate preferences by DNA shuffling", IBC 's second International Symposium on Directed Evolution of industrial Enzymes, Sept 14-15, San Diego; Grayling, 1998, "Concepts and Strategies in high throughput screening for improved enzyme variants ”, IBC's second International Symposium on Directed Evolution of Industrial Enzymes, Sept 14-15, San Diego). 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).

According to a preferred embodiment of the invention, the enzymes are involved in the bioconversion of an appropriate substrate into HMTBS, the suitable substrates being HMTBN or HMTBAmide.

For clarity, the rest of the description has been written with HMTBS, HMTBN or HMTBAmide. However, in the description which follows, 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.

By cloning such sequences into a plasmid allowing the expression of these nitrilases in a mutant of an auxotrophic microorganism for methionine, it is then possible to select the only strains which have integrated an active enzyme involved in the bioconversion of the substrate into HMTBS , the microorganism auxotrophic for methionine being capable of growing in the presence ^of HMTBS obtained by bioconversion, in the absence of methionine in the substrate. This strategy therefore leads to cultures enriched in clones expressing active enzymes, or even to cultures enriched in clones expressing enzymes whose catalytic properties have been improved by site-directed or random mutagenesis.

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,

2) transformation of a microorganism suitable auxotrophic for methionine by introduction of the vectors previously obtained in said

5 suitable microorganism,

3) culture of the transformed microorganisms obtained previously in an appropriate culture medium comprising a sufficient quantity of suitable substrate, and

4) selection of the transformed microorganisms capable of growing in the appropriate medium, and

5) isolation and, where appropriate, identification of the DNA sequences involved in the bioconversion of the appropriate substrate.

According to a preferred embodiment of the invention, 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. In the first case, the DNA sequence isolated and / or selected by the method according to the invention codes for a nitrilase. In the second case, the DNA sequence isolated and / or selected by the method according to the invention codes for a nitrile hydratase or an amidase.

According to another preferred embodiment of the invention, 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. By isolation is essentially meant according to the invention the separation of a particular sequence from a set of varied sequences. By selection is essentially meant according to the invention the choice of the best sequence having a particular property.

Once the microorganisms have been selected and / or isolated, they can be cultured on a usual culture medium to increase the number and facilitate the isolation and identification of the DNA sequences involved in the bioconversion of the appropriate substrate. By 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. According to a preferred embodiment of the invention, 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. Nucleic Acids Res, 1995 Dec 1 1 23:23 4818-26; McAdam RA & al., Infect Immun, 1995 Mar 63: 3 1004-12; Manning M & al., Can J Microbiol 1984 Jan 30: 1 31-5; Yamagata S, J Bacteriol, 1987 Aug 169: 8 3458-63; Wabiko H & al., J Bacteriol, 1988 Jun 170: 6 2705-10; Frank P & al., J Biol Chem, 1985 May 10 260: 9 5518-25).

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. In this case, 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.

When 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. According to a first preferred embodiment of the invention, 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. According to another preferred embodiment of the invention, 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.

According to another preferred embodiment of the invention, 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. In the two cases above, 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.

According to another preferred embodiment of the invention, the isolated and / or selected enzyme involved in the bioconversion of HMTBamide into HMTBS is an amidase, and the appropriate culture medium contains HMTBAmide.

According to a particular embodiment of the invention, 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. By 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. In this case, 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%. In this case, 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.

By 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).

By appropriate culture medium comprising a sufficient amount of suitable substrate, is preferably meant according to the invention 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.

By 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 μM and approximately 100 μM) , more preferably between 6 mg / 1 and 10 mg / 1 (equivalent of approximately 40 μM and approximately 60 μM).

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 μM and approximately 100 μM), more preferably between 6 mg / 1 and 10 mg / 1 (equivalent of approximately 40 μM and approximately 60 μM).

It is understood that for the method and the medium according to the invention, 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. However, depending on the microorganism considered and the level of selection sought, 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. Advantageously, 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 μg / 1 (equivalent to approximately 2 μM) more preferably less than 250 μg / 1 (equivalent of approximately 1.5 μM), more preferably between 130 μg / 1 and 170 μg / 1. (equivalent to approximately 0.8 μM and approximately 1 μM).

According to a preferred embodiment of the invention, the appropriate medium comprises a source of organic nitrogen not comprising traces of methionine.

Advantageously, the organic nitrogen source is a yeast extract, in particular from Yeats Nitrogen Broth w / o Amino Acids (YNB, Yeats Nitrogen

Broth w / o Amino Acids, DIFCO, composition given in DIFCO Manual, Tenth edition, ISBN 9-9613169-9-3, 1984, page 1 136) or Casamino acids (Casamino Acids, Difco, composition given in DIFCO Manual, Tenth edition, ISBN 9-

9613169-9-3, 1984, page 208).

According to a preferred embodiment of the invention, the content of organic nitrogen source, more preferably in YNB, is between 0 and 15 g / 1, more preferably between 2 and 10 g / 1, even more preferably between 3 and 8 g / 1.

Advantageously, the appropriate medium according to the invention comprises M9fru described below as the minimum medium.

Advantageously, 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. Preferably, 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.

The examples below illustrate the invention, without however seeking to limit its scope. All the methods or operations described below in these examples are given by way of examples and correspond to a choice made from among the different methods available to achieve the same result.

Most methods of engineering DNA fragments are described in "Current Protocols in Molecular Biology" Volumes 1 and 2, Ausubel FM et al, published by Greene Publishing Associates and Wiley -Interscience (1989) or in Molecular cloning, T. Maniatis, EFFritsch, J. Sambrook (1982).

Legend of figures

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. In A): growth of the RPA-BIOCAT 610 and 842 strains in a minimum medium M9YNBfru + HMTBN 50 μM free of HMTBS; in B): growth of the RPA-BIOCAT 610 and 842 strains in minimum medium M9YNBfru + HMTBN 50 μM supplemented with HMTBS 10 μM.

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 μM; B. with a limiting HMTBS concentration of 1 μM. The selective media are constituted by the minimal medium M9YNBfru + IPTG 0.5 mM supplemented with: HMTBS 0.8 μM (S0.8) or in HMTBS 1 μM (SI), or in HMTBN 50 μM + HMTBS 0.8 μM (NS0.8) or else in HMTBN 50 μM + HMTBS 1 μM (NSI).

FIG. 4 shows the growth of the RPA-BIOCAT 841 and 960 strains in selective medium: A. in the presence of 0.8 μM HMTBS; B. in the presence of 1 μM HMTBS. The selective media are constituted by the minimal medium M9YNBfru + IPTG 0.5 mM supplemented with: HMTBS 0.8 μM (S0.8) or in HMTBS 1 μM (SI), or in HMTBN 50 μM + HMTBS 0.8 μM (NS0.8) or else in HMTBN 50 μM + HMTBS 1 μM (NSl).

Methods : The techniques used are conventional techniques of molecular biology and microbiology, known to those skilled in the art and described for example by Ausubel et al., 1987 (Current Protocols in Molecular Biology, John Willey and

Sons, New York), Maniatis et al., 1982, (Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Sring Harbor, New York).

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 μg / 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 stock solutions of HMTBN (59 mM), HMTBS (1.3 mM) and HTBAmide (100 mM) used for the preparation of the selective medium are carried out by dilutions in M9YNBfru of stock solutions of these products at respective concentrations 5.9 M (Rhône-Poulenc reference for the lot: PHM 439 J) and 1.3 M (Rhône-Poulenc reference for the lot: BFR 81). These solutions are prepared according to a protocol described in patents and patent applications EP 330 521, EP 142 488, US 3 773 927 and US 4 353 924. HMTBamide is obtained by hydrolysis with sulfuric acid of the amino cyanhydrin ( CH3SCH2CH2CHNH2CN) described in the same patents and patent applications.

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 expression cultures of the RPA-BIOCAT 841 and 842 strains are carried out by dilution to ^1/100 of a preculture in 10 ml of LB + carbenicillin 100 μg / ml + 0.5 mM IPTG. These cultures are incubated at 37 ° C., 200 rpm in a 50 ml hermetically closed tube for 16 h. Biomass is estimated by measuring the optical density at 660 nm (DO660) using the biomass relationship in grams of dry weight per liter of culture = DO660 x 0.35. The assay of nitrilase activity of the cultures is carried out as follows: the expression cultures are centrifuged and the cell pellet washed in 100 mM phosphate buffer. It is resuspended in this same buffer and 200 μl of cell suspension are incubated with 200 μl of 200 mM HMTBN (in solution in 100 mM phosphate buffer) for 2 h at 37 ° C. At a time interval of 0, 30, 60, 90 and 120 min, a 5 μl sample of the reaction medium is taken and mixed with 50 μl of 200 mM H ₃ PO ₄ . To these 55 μl are added 105 μl of a 5.1% phenol solution -NaOH 2.54 N, then, after mixing, 40 μl of a bleach solution 47/50 ° C (Sodium hypochloryte in 12.5% solution, Dousselin & Geoffray Jacquet together, Couzon aux Monts d'Or, France), diluted by half. After shaking followed by a 10 min incubation at room temperature, the optical densities are read at 630 nm. The amount of HMTBS released is calculated by comparison with a standard range of HMTBS prepared in 100 mM phosphate buffer in the presence of HMTBN according to the table below.

The activities are expressed in kg of HMTBS produced / hour / kg of dry cells

(CS).

Examples

Example 1: Construction of the pRSP-BCAT41 piasmid. The 1.27 kb fragment containing the Ptrp promoter, the binding site of the λ 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. The resulting plasmid, 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 1.2 kb EcoRl-Xbal fragment of pRPA-BCAT19 containing the Ptrp:: RBScII:: nitB fusion was then cloned into the vector pRPA-BCAT28 opened by the same enzymes to yield the plasmid pRPA-BCAT29 of 6.2 kb. 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 pHP45ΩTc (Fellay et al, 1987, Gene 52: 147-154) in order to replace the ampicillin resistance marker with the tetracycline resistance marker. By destroying the Ndel site close to the origin of replication of the plasmid pRPA-BCAT29 by partial digestion Ndel and action of polymerase I of E. coli (Klenow fragment), 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 (SΕQ ID NO 1) .

Example 2: Construction of the plasmid pRPA-BCAT77.

A portion of the 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). It was then ligated to the 0.261 kb fragment BsiHKAl-Slul purified from a digestion of the plasmid pRPA-BCAT41 and to the 5.43 kb Ndel-Stul fragment purified from a digestion of pRPA-BCAT72. 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. It corresponds to the plasmid pRPA-BCAT41 but makes it possible to express a nitrilase NitB carrying a substitution of the Alanine residue in position 160 by a glycine residue Example 3: Construction of a strain of E. auxotrophic coli expressing the nitrilase A'Αlcaligenes 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 β180 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 ά ^' Αlcaligenes 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. After amplification by PCR on the pRPA-BCAT41 matrix with the primers PCRAF1 described in application FR96 / 13077 and NitBl described below, the amplified product was digested with Ndel and Banl to obtain an insert of approximately 0.476 kb. NitBl: 5'-GCAGCACAGG GCACCGACGC-3 ' Similarly, after amplification by PCR on the pRPA-BCAT41 matrix with the primers NitB2 and SR described below, the amplified product was digested with Banl and Stwl to obtain an insert of approximately 0.34 kb. NitB2: 5'-CGCGTCGGTG CCCTGTGCGC CTGGGAGC-3 'SR: 5'-CGGCAATGAT CAGGCCTTCG GC-3'

After digestion of the vector pRPA-BCAT72 with Ndel and Stwl, the 5.43 kb fragment was ligated to the 0.476 kb Ndel-Banl and 0.34 kb Banl-Stul inserts described above to form the vector pRPA-BCAT69 . The amplification product obtained with the primers primers nitBMNl and nitBMN2 and the matrix pRPA-BCAT69 was then cloned into the vector pbsll ks- (Stratagene) as described in Example 3. The resulting plasmid, named pRPA-BCAT107, a was introduced into the strain RPA-BIOCAT610 to obtain the new strain RPA-BIOCAT 842. Example 5: Construction of a strain of E. auxotrophic coli for methionine expressing the active nitrilase of Comamonas testosteroni from the P _lac promoter.

Amplification by PCR of a 1.35 kb fragment containing the nitA gene of Comamonas testosteroni was carried out using the template plasmid pXL2158 (FR9613077), the primers NitAl and NitA2 described below and the DNA polymerase Pfu. NitAl: 5'-GGGCATACAT TCAATCAATT G-3 'NitA2: 5'-AGGTGGGACC CAAGCTTGCA-3'

After purification with phenol / chloroform / isoamyl alcohol (25: 24: 1), desalting using the QIAΕX II kit (QIAGΕN) and digestion with the enzyme HindIII, the PCR fragment was ligated to the plasmid pbsll ks- previously digested by the enzymes Hindlll and Hincll to lead to the plasmid pBCAT145. The latter was introduced according to the method of Chung et al. (Proc. Natl. Acad. Sci. USA (1988) 86: 2172-2175) in the strain RP-BIOCAT 610. The new strain thus obtained was named RPA-BIOCAT 960.

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 μg / 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 μg / ml and 0.5 mM IPTG only for the RPA-BIOCAT 842 strain: i) M9YNBfru + HMTBN 50 μM ii) M9YNBfru + 5 HMTBN 50 μM + HMTBS 10 μM. . 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.

The results show that 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 Αlcalîgenes faecalis

The RPA-BIOCAT 841 and 842 strains were precultivated in the presence of 5 carbenicillin 100 μg / 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 μg / 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.

Table 1: Growth of RPA-BIOCAT 841 and 842 strains in the presence and absence of YNB

-: absence of growth; T: growth.

These results show that YNB constitutes an essential supplement for the growth of the strain of E. coli auxotrophic for methionine under the conditions described in the example.

These results also show that the selective media tested do not make it possible to differentiate a strain of E. coli ΔmetA expressing an active nitrilase of a strain expressing this enzyme in inactive form. The concentration in

HMTBN of 50 μM 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 μg / 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 μg / 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.

-: absence of growth; T: growth.

These results show that the minimum HMTBS concentration necessary for the growth of the RPA-BIOCAT 841 strain is greater than or equal to 2 μM. 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 μg / 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 μM HMTBS and 50 μM 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 μM + HMTBS 0.8 or 1 μM 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 μg / 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.

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 / _αc -

Claims

claims

1. A method of selecting and / or isolating DNA sequences coding for enzymes involved in the appropriate substrate bioconversion into methionine and its derivatives, such as HMTBS, characterized in that it comprises the following steps:

1) cloning of DNA sequences into a vector allowing their expression in an appropriate host microorganism,

2) transformation of an auxotrophic microorganism suitable for methionine by introduction of the vectors obtained previously into said appropriate microorganism,

3) culture of the transformed microorganisms obtained previously in an appropriate culture medium comprising a sufficient amount

4) appropriate substrate, 5) selection and / or isolation of the transformed microorganisms capable of growing in the appropriate medium, and 6) isolation and if necessary identification of the DNA sequences involved in the bioconversion of the appropriate substrate.

2. Method according to claim 1, characterized in that the suitable substrate is 2-amino 4- (methylthio) butyronitrile or its derivatives, such as 2-hydroxy 4- (methylthio) butyronitrile (HMTBN).

3. Method according to claim 2, characterized in that the appropriate substrate is directly converted into methionine or its derivatives, such as HMTBS, by a nitrilase.

4. Method according to claim 3, characterized in that the isolated and / or selected DNA sequence codes for a nitrilase.

5. Method according to claim 2, characterized in that 2-amino 4-

(methylthio) butyronitrile or its derivatives, such as HMTBN, is converted to methionine or its derivatives, like HMTBS via 2-amino 4- (methyl thio) butanamide or its derivatives, such as 2-hydroxy 4 - (methylthio) butanamide (HMTBAmide), by a first conversion of 2-amino 4- (methylthio) butyronitrile or its derivatives, such as HMTBN into 2-amino 4- (methyl thio) butanamide or its derivatives, such as HMTBAmide by a nitrile hydratase, followed by conversion of 2-amino 4- (methyl thio) butanamide or its derivatives, such as HMTBAmide to methionine or its derivatives, such as HMTBS, an amidase.

6. Method according to claim 5, characterized in that the isolated and / or selected DNA sequence codes for a nitrile hydratase or an amidase.

7. Method according to claim 6, characterized in that the isolated and / or selected DNA sequence codes for a nitrile hydratase, the appropriate microorganism also comprising a natural or heterologous gene coding for a complementary amidase.

8. Method according to claim 6, characterized in that the isolated and / or selected DNA sequence codes for an amidase, the appropriate microorganism also comprising a natural or heterologous gene coding for a complementary nitrile hydratase.

9. Method according to claim 1, characterized in that the suitable substrate is 2-amino 4- (methyl thio) butanamide or its derivatives, such as HMTBAmide, which is converted to methionine or its derivatives, such as HMTBS by an amidase.

10. Method according to claim 9, characterized in that the DNA sequence isolated and / or selected is an amidase.

1 1. Method according to one of claims 1 to 10, characterized in that the microorganism suitable auxotrophic for methionine, is an auxotrophic microorganism for methionine capable of being transformed and capable of growing in an environment free methionine comprising HMTBS.

12. Method according to claim 1 1, characterized in that the appropriate microorganism is chosen from yeasts, fungi and bacteria.

13. Method according to claim 12, characterized in that the bacterium is E. coli.

14. Method according to one of claims 1 to 13, characterized in that the DNA sequence is a DNA sequence isolated from one or more genomes, by total or partial restriction of said genome or genomes.

15. Method according to one of claims 1 to 13, characterized in that the DNA sequence is a DNA sequence isolated from a part of the genome by total or partial restriction of said part of the genome.

16. Method according to one of claims 1 to 13, characterized in that the DNA sequence is a DNA sequence isolated from a construct by

PCR.

17. Method according to one of claims 1 to 13, characterized in that the DNA sequence is obtained by random mutagenesis.

18. Method according to one of claims 1 to 17, characterized in that the culture of the transformed microorganisms is a mass culture (batch), in particular by successive cultures, or a continuous culture, in particular a cultures in chemostat.

19. Method according to one of claims 1 to 18, characterized in that the appropriate culture medium comprises an amount of methionine insufficient to ensure the growth of the microorganism and comprises a sufficient amount of suitable substrate to allow the growth of the micro -organism transformed after bioconversion into methionine or its derivatives, such as HMTBS.

20. The method of claim 19, characterized in that the sufficient amount of 2-amino 4- (methylthio) butyronitrile or its derivatives, such as HMTBN, is between 0 and 60 g / 1, preferably between 3 mg / 1 and 17 mg / 1, more preferably between 6 mg / 1 and 10 mg / 1.

21. The method of claim 19, characterized in that the sufficient amount of 2-amino 4- (methyl thio) butanamide or its derivatives, such as HMTBAmide is advantageously between 0 and 60 g / 1, preferably between 3 mg / 1 and 17 mg / 1, more preferably between 6 mg / 1 and 10 mg / 1.

22. Method according to one of claims 1 to 21, characterized in that the appropriate culture medium comprises an appropriate amount of methionione or its derivatives, such as HMTBS.

23. The method of claim 22, characterized in that the appropriate amount of methionine or its derivatives, such as HMTBS is less than the sufficient concentration to allow the growth of transformed microorganisms.

24. Method according to one of claims 22 or 23, characterized in that the amount of HMTBS is less than 350 μg / 1, preferably less than 250 μg / 1, more preferably between 130 and 170 μg / 1.

25. Method according to one of claims 1 to 24, characterized in that the appropriate culture medium comprises a source of organic nitrogen.

26. Method according to one of claims 25, characterized in that the content of organic nitrogen source is between 0 and 15 g / 1, preferably between 2 and 10 g / 1, more preferably between 3 and 8 g / 1.

27. Method according to one of claims 1 to 26, characterized in that the appropriate culture medium comprises M9fru as minimum medium.

28. Method according to one of claims 1 to 27, characterized in that the appropriate culture medium is a liquid or solid medium.

29. Culture medium as defined in one of claims 19 to 28.

30. DNA sequence coding for an enzyme involved in the bioconversion of an appropriate substrate into methionine or its derivatives, such as HMTBS, characterized in that it is selected and / or isolated by the method according to the invention.