AU1568900A

AU1568900A - Method for isolating and selecting genes coding for enzymes, and suitable culture medium

Info

Publication number: AU1568900A
Application number: AU15689/00A
Authority: AU
Inventors: Nadine Batisse Debitte; Olivier Favre-Bulle; Jerome Pierrard
Original assignee: Aventis Animal Nutrition SA
Current assignee: Adisseo Ireland Ltd
Priority date: 1998-12-11
Filing date: 1999-12-10
Publication date: 2000-07-03
Anticipated expiration: 2019-12-10
Also published as: EP1137784A1; AU773130B2; FR2787121A1; FR2787121B1; JP2002532096A; WO2000036120A1

Description

WO 00/36120 1 PCT/FR99/03089 METHOD FOR ISOLATING AND SELECTING GENES ENCODING ENZYMES, AND SUITABLE CULTURE MEDIUM The present invention relates to a novel 5 process for isolating and/or selecting genes encoding enzymes which are involved in the bioconversion of a substrate to methionine or derivatives thereof, such as 2-hydroxy-4-(methylthio)butanoic acid or salts thereof, in particular which hydrolyze amide groups to 10 carboxylic acids, or which are involved in the bioconversion of nitrile groups to the corresponding carboxylic acids, by means of a suitable selection screen, and the culture medium suitable for carrying out said process. 15 The enzymes which catalyze the hydrolysis of nitrile groups to the corresponding carboxylic acids and ammonium ions are nitrilases (Faber Biotransformations in Organic Chemistry, Springer Verlag, Berlin Heidelberg, 1992, ISBN3-540-55762-8). 20 However, this bioconversion of nitrile groups to the corresponding carboxylic acids, the final result of which consists of hydrolysis of the nitrile groups, can also be carried out in two steps, the first step consisting of the conversion of the nitriles to the 25 corresponding amides with a nitrile hydratase, the second step consisting in hydrolyzing the amides 2 obtained to the corresponding carboxylic acids with amidases. Nitrilases were first discovered in plants (Thimann and Mahadevan, 1964, Arch. Biochem. Biophys. 5 105: 133-141) and then isolated in many representatives of the microflora of the soil (Kobayashi and Shimizu, 1994, FEMS Microbiology Letters 120: 217-224): Pseudomonas, Nocardia, Arthrobacter, Fusarium, Rhodoccocus, Alcaligenes. More recently, nitrilases 10 have been characterized in thermophilic bacteria (Cramp et al., 1997, Microbiology, 143: 2313-2320). Nitrilases have varied substrate specificities but can be grouped together in three groups as a function of their specificity: nitrilases specific for aliphatic 15 nitriles, those specific for aromatic nitriles or those specific for arylacetonitriles (Kobayashi et al., 1993, Proc. Natl. Acad. Sci. USA 90: 247-251; Kobayashi and Shimizu, 1994, mentioned above; L6vy-Schil et al., 1995, Gene 161: 15-20). Nitrilases are of value in 20 biocatalysis since many synthetic processes involve nitrile group hydrolyses (L6vy-Schil et al.). In particular the nitrilase from Alcaligenes faecalis ATCC9750 and that of Comamonas testosteroni can be used to obtain the hydroxy analog of methionine (FR9411301, 25 W09609403 and FR9613077). It is common practice, when searching for novel enzymes, to use several strategies (Dalboge and Lange, 1998, TibTech 16: 265-272): i) the 3 microbiological isolation of microorganisms, on the basis of specific biotopes, using a selection screen based on the presence of the enzymatic activity being sought, ii) the search for an enzymatic activity in 5 microorganisms by culturing strains and assaying the activity in question, iii) the search, in microorganisms which can be cultured, for genes which are silent and homologous to a gene of interest, and then the cloning and expression of this gene in a model 10 microorganism, iv) the engineering of proteins in order to improve the characteristics of an available enzyme and, finally, v) the cloning by expression of genes directly isolated from varied biotopes without prior microbiological isolation. Strategies i), iii), iv) and 15 v) are considerably accelerated and simplified if an in vivo selection screen is available. The expression "in vivo selection screen" is intended to mean a growth medium and/or growth conditions which allow only microorganisms harboring the enzymatic activity being 20 sought to grow. In addition, given the very low proportion of microorganisms which can be cultured in the laboratory (Amann et al., 1995, Microbiol Rev., 59. 143), strategies iv) and v) are of particular value for 25 taking better advantage of biodiversity by targeting biodiversity which is available, but which is not accessible through microbiological isolation (strategy v), or by creating greater diversity using strains 4 which can be cultured (strategy iv). It is particularly in the case of these two strategies that the possibility of having an in vivo selection screen which is simple to implement represents a definite advantage 5 which leads to the screening of a considerable number of clones in a short period of time and, where appropriate, with limited means (Minshull, 1998, "Evolution of enzyme activities and substrate preferences by DNA shuffling", IBC's second 10 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 15 Industrial Enzymes, Sept. 14-15, San Diego). The invention consists of a simple, rapid and relatively inexpensive method which makes it possible to isolate and/or select DNA sequences encoding enzymes which are involved in the bioconversion of a substrate 20 to to a methionine salt (AMTBS) or derivatives thereof such as 2-hydroxy-4-(methylthio)butanoic acid (hereinafter HMTBS), especially the ammonium salt, and in particular which are involved in the bioconversion of 2-amino-4-(methylthio)butyronitrile (AMTBN) or 25 derivatives thereof, such as 2-hydroxy-4 (methylthio)butyronitrile (hereinafter HMTBN), to methionine or derivatives thereof such as HMTBS, either directly or via 2-amino-4-(methylthio)butanamide or 5 derivatives thereof, such as 2-hydroxy-4 (methylthio)butanamide (hereinafter HMTBAmide). According to a preferential embodiment of the invention, the enzymes are involved in the 5 bioconversion of a suitable substrate to HMTBS, the suitable substrates being HMTBN or HMTBAmide. In the interest of clarity, the remainder of the description has been written with HMTBS, HMTBN or HMTBAmide. However, in the description which follows, 10 the indications relating to HMTBS, HMTBN or HMTBAmide also apply to methionine or derivatives thereof, and the corresponding substrates, 2-amino-4 (methylthio)butyronitrile or derivatives thereof and 2 amino-4-(methylthio)butanamide or derivatives thereof. 15 By cloning such sequences into a plasmid which allows the expression of these nitrilases in a mutant of a microorganism which is auxotrophic for methionine, it is then possible to select the only strains which have integrated an active enzyme involved 20 in the bioconversion of the substrate to HMTBS, the microorganism which is 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 produces cultures 25 enriched in clones expressing active enzymes, or even cultures enriched in clones expressing enzymes for which the catalytic properties have been improved by site-directed or random mutagenesis.

6 The present invention therefore relates to a process for selecting and/or isolating DNA sequences encoding enzymes which are involved in the bioconversion of a suitable substrate to methionine and 5 derivatives thereof, such as HMTBS, said process comprising the following steps of 1) cloning DNA sequences into a vector which allows their expression in a suitable host microorganism, 10 2) transforming a suitable microorganism which is auxotrophic for methionine, by introducing the vectors obtained above into said suitable microorganism, 3) culturing the transformed microorganisms 15 obtained above in a suitable culture medium comprising a sufficient amount of suitable substrate, and 4) selecting the transformed microorganisms capable of growing in the suitable medium, 20 and 5) isolating and, where appropriate, identifying the DNA sequences involved in the bioconversion of the suitable substrate. 25 According to a preferential embodiment of the invention, the suitable substrate [lacuna] 2-hydroxy-4 (methylthio)butyronitrile (hereinafter HMTBN) which is converted to HMTBS, either directly by a nitrilase or 7 via 2-hydroxy-4-(methylthio)butanamide (hereinafter HMTBAmide), this second pathway involving a first conversion of the HMTBN to HMTBAmide by a nitrile hydratase, followed by conversion of the HMTBAmide by 5 an amidase. In the first case, the DNA sequence isolated and/or selected using the process according to the invention encodes a nitrilase. In the second case, the DNA sequence isolated and/or selected using the process according to the invention encodes a nitrile 10 hydratase or an amidase. According to another preferential embodiment of the invention, the suitable substrate is HMTBAmide, which is converted to HMTBS by an amidase, which is encoded by the DNA sequence isolated and/or selected 15 using the process according to the invention. According to the invention, the term "isolation" is essentially intended to mean the separation of a particular sequence from a set of varied sequences. According to the invention, the term 20 "selection" is essentially intended to mean 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 conventional culture medium so as to increase the number thereof and 25 facilitate the isolation and identification of the DNA sequences involved in the bioconversion of the suitable substrate.

8 According to the invention, the expression "suitable microorganism which is auxotrophic for methionine" is intended to mean any microorganism which is auxotrophic for methionine, which can be 5 transformed, and which is capable of growing on a methionine-free medium comprising HMTBS. It may be a yeast, a fungus or a bacterium. According to a preferential embodiment of the invention, the suitable microorganism is a bacterium, preferably E. coli. 10 The suitable microorganism which is useful according to the invention may be naturally auxotrophic for methionine or alternatively modified by mutagenesis so as to induce this auxotrophy. The various methods for obtaining auxotrophic mutants are well known to 15 those skilled in the art and widely described in the literature (in particular Roberts CJ, Selker EU, Nucleic Acids Res, 1995 Dec. 11 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; 20 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 suitable microorganism transformed with a vector comprising a DNA sequence encoding an enzyme 25 involved in the bioconversion of HMTBN to the corresponding carboxylic acid is capable of converting HMTBN to HMTBS, allowing the microorganism which is auxotrophic for methionine to develop. In this case, 9 the microorganism carries out the bioconversion of HMTBS to methionine so that the strain can grow. The suitable microorganism transformed with a vector comprising a DNA sequence encoding an enzyme 5 involved in the bioconversion of HMTBAmide to the corresponding carboxylic acid is capable of converting HMTBAmide to HMTBS, allowing the microorganism which is auxotrophic for methionine to develop. When the suitable microorganism which is 10 auxotrophic for methionine also comprises a gene encoding an enzyme involved in the bioconversion of HMTBAmide to HMTBS, and when this microorganism is transformed with a vector comprising a DNA sequence encoding an enzyme involved in the bioconversion of 15 HMTBN to HMTBAmide, it is capable of converting HMTBN to HMTBS, allowing it to develop. According to a first preferential embodiment of the invention, the isolated and/or selected enzyme involved in the bioconversion of HMTBN to HMTBS is a 20 nitrilase which allows the conversion of HMTBN to the HMTBS required for the growth of the modified microorganisms. According to another preferential embodiment of the invention, the isolated and/or selected enzyme 25 involved in the bioconversion of HMTBN to HMTBS is a nitrile hydratase which allows the conversion of HMTBN to HMTBAmide, the suitable microorganism also comprising a gene, which may be natural or 10 heterologous, encoding a complementary amidase for carrying out the bioconversion of the HMTBAmide to the HMTBS required for the growth of the modified microorganisms. 5 According to another preferential embodiment of the invention, the isolated and/or selected enzyme involved in the bioconversion of HMTBN to HMTBS is an amidase, the suitable microorganism also comprising a gene, which may be natural or heterologous, encoding a 10 complementary nitrile hydratase. In the two cases above, the gene of the complementary enzyme, if it is heterologous, is either integrated into the genome of the suitable microorganism or carried by a plasmid. 15 According to another preferential embodiment of the invention, the isolated and/or selected enzyme involved in the bioconversion of HMTBamide to HMTBS is an amidase and the suitable culture medium contains HMTBAmide. 20 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 genome(s). The DNA sequence may also be a DNA sequence isolated from a portion of 25 genome by total or partial restriction of said portion of genome. The term "restriction" is intended to mean any means, which may or may not be enzymatic, capable of fragmenting DNA specifically or nonspecifically. In 11 this case, the process according to the invention is particularly suitable for selecting novel enzymes according to strategies i) and v) defined above. The DNA sequence may also be a DNA sequence 5 isolated using a construction by PCR (polymerase chain reaction). The DNA sequence may also be obtained by random or targeted mutagenesis of a sequence encoding a reference enzyme defined previously. In this case, the 10 process according to the invention is particularly suitable for selecting novel enzymes according to strategy iv) defined above. The DNA sequence may also be an isolated DNA sequence having a given homology with a reference 15 enzyme defined previously. This given degree of homology is advantageously greater than 50%, preferably greater than 60%, more preferably greater than 70%. In this case, the process according to the invention constitutes a rapid screen to be used for identifying 20 to what extent a nucleic acid sequence having a certain degree of homology with a reference sequence has the same function and/or the same activity as the reference sequence. According to the invention, the term 25 "reference enzyme" is intended to mean a known enzyme involved in the bioconversion of the suitable substrate to HMTBS, in particular of HMTBN to HMTBS, or in the bioconversion of HMTBAmide to HMTBS, preferably a 12 nitrilase, a nitrile hydratase or an amidase, defined above, which will be used as a reference for evaluating the function and the activity of the novel enzymes isolated and/or selected using the process according to 5 the invention, in particular the enzymes for which novel mutants are being sought and/or those having a certain degree of homology defined above. The culturing of the transformed microorganisms for the isolation and/or selection 10 process according to the invention is carried out by any suitable means known to those skilled in the art. It especially involves batchwise culturing, in particular using successive culturing, or using continuous culturing, particularly chemostat culturing 15 (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 20 Biol, 1995 Apr 7 173:3 307-20). According to the invention, the expression "suitable culture medium comprising a sufficient amount of suitable substrate" is preferably intended to mean any culture medium suitable for the growth of the 25 transformed microorganism which is auxotrophic for methionine, said medium being substantially methionine free and comprising a sufficient amount of suitable substrate to allow the growth of said transformed 13 microorganism after bioconversion of said suitable substrate to HMTBS. The expression "substantially methionine-free medium" is intended to mean a medium which is free of 5 methionine or possibly comprises an amount of methionine which is insufficient to allow the suitable microorganism which is auxotrophic for methionine to grow. The sufficient amount of HMTBN is 10 advantageously between 0 and 60 g/l (equivalent to approximately 400 mM), more preferably between 3 mg/l and 17 mg/l (equivalent to approximately 20 ,iM and approximately 100 pM), more preferably between 6 mg/l and 10 mg/l (equivalent to approximately 40 M and 15 approximately 60 pM). The sufficient amount of HMTBAmide is advantageously between 0 and 60 g/l (equivalent to approximately 400 mM), more preferably between 3 mg/l and 17 mg/l (equivalent to approximately 20 jiM and 20 approximately 100 pM), more preferably between 6 mg/l and 10 mg/l (equivalent to approximately 40 pM and approximately 60 ptM). It is understood that, for the process and the medium according to the invention, the essential 25 source of HMTBS required for the growth of the transformed microorganisms consists of the HMTBS produced from the conversion of the suitable substrate, in particular of the HMTBN or of the HMTBAmide, by the 14 enzyme encoded by the DNA sequence introduced into the transformed microorganism. However, depending on the microorganism under consideration and on the degree of selection desired, the medium according to the 5 invention may also initially (prior to and at the time of inoculation with the transformed microorganisms), comprise a suitable amount of HMTBS, in particular in order to initiate the growth of the microorganisms, this amount of HMTBS being replaced, as the 10 microorganisms grow, with the HMTBS derived from the conversion of the suitable 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 15 transformed microorganisms according to the invention. This concentration may be determined as a function of the suitable culture medium, in particular of its form (batch, continuous liquid, solid, etc.), and will preferably be less than 350 pg/l (equivalent to 20 approximately 2 pM), more preferably less than 250 ptg/l (equivalent to approximately 1.5 pM), more preferably between 130 pg/l and 170 pg/l (equivalent to approximately 0.8 pM and approximately 1 p-M). According to a preferential embodiment of the 25 invention, the suitable medium comprises an organic nitrogen source which does not contain any traces of methionine.

15 Advantageously, the organic nitrogen source is a yeast extract, in particular Yeast Nitrogen Broth w/o Amino Acids (YNB, Yeast Nitrogen Broth w/o Amino Acids, DIFCO, composition given in the DIFCO Manual, 5 Tenth edition, ISBN 9-9613169-9-3, 1984, page 1136) or Casamino acids (Casamino Acids, Difco, composition given in the DIFCO Manual, Tenth edition, ISBN 9-9613169-9-3, 1984, page 208). According to a preferential embodiment of the 10 invention, the content of organic nitrogen source, more preferably of YNB, is between 0 and 15 g/l, more preferably between 2 and 10 g/l, even more preferably between 3 and 8 g/l. Advantageously, the suitable medium according 15 to the invention comprises M9fru, described later on, as minimum medium. Advantageously, the suitable medium according to the invention comprises, as a carbon source, a compound chosen from glucose, fructose, galactose, 20 trehalose, mannose, melibiose, sucrose, raffinose, maltotriose, maltose, lactose, lactulose, arabinose, xylose, rhamnose, fucose, mannitol, sorbitol, malate, saccharate, mucate, mesotartrate, glucuronate, galacturonate and mixtures thereof in any proportions. 25 Preferably, the carbon source is chosen from glucose or fructose, and mixtures thereof in any proportions, more preferably fructose.

16 The culture medium according to the invention can be liquid or solid and, in this case, can contain agar or agarose. The examples hereinafter make it possible to 5 illustrate the invention without however seeking to limit the scope thereof. All the methods or procedures described below in these examples are given by way of examples and correspond to a choice made between the various methods available for achieving the same 10 result. Most of the methods for engineering DNA fragments are described in "Current Protocols in Molecular Biology" Volumes 1 and 2, Ausubel F.M. et al., published by Greene Publishing Associates and Wiley-Interscience (1989) or in Molecular cloning, 15 T. Maniatis, E.F. Fritsch and J. Sambrook (1982). Legend of the Figures Figure 1 represents the plasmid RPA-BIOCAT41. The sites between brackets are sites which have been 20 eliminated during cloning. Ptrp: tryptophan promoter; nitB: nitrilase gene; TrrnB: transcription terminators; end ROP: end of the gene encoding the ROP protein (Chambers et al., 1988, Gene 68: 139-149); ORI: origin of replication; RNAI/II: RNA involved in replication 25 (Chambers et al., mentioned above); Tc: tetracyclin resistance gene. Figure 2 shows the growth, at 37*C and 200 rpm and in hermetically sealed 50 ml tubes, of the 17 two strains RPA-BIOCAT 610 and 842, by reading optical densities in microplates at 630 nm. In A): growth of the strains RPA-BIOCAT 610 and 842 in M9YNBfru minimum medium + 50 pM HMTBN free of HMTBS; in B): growth of 5 the strains RPA-BIOCAT 610 and 842 in M9YNBfru minimum medium + 50 jiM HMTBN supplemented with 10 pM HMTBS. Figure 3 shows the growth of the strains RAP-BIOCAT 841 and 842 in selective medium: A. with a limiting concentration of HMTBS of 0.8 pM; B. with a 10 limiting concentration of HMTBS of 1 pM. The selective media consist of the M9YNBfru minimum medium + 0.5 mM IPTG supplemented with: 0.8 M HMTBS (SO.8) or with 1 pM HMTBS (Sl), or with 50 pM HMTBN + 0.8 pM HMTBS (NSO.8) or with 50 pM HMTBN + 1 pM HMTBS (NS1). 15 Figure 4 shows the growth of the strains RPA-BIOCAT 841 and 960 in selective medium: A. in the presence of 0.8 jiM HMTBS; B. in the presence of 1 pM HMTBS. The selective media consist of the M9YNBfru minimum medium + 0.5 mM IPTG supplemented with: 0.8 M 20 HMTBS (SO.8) or with 1 M HMTBS (Si), or with 50 pM HMTBN + 0.8 jiM HMTBS (NSO.8) or with 50 pM HMTBN + 1 FM HMTBS (NS1). Methods: 25 The techniques uses are conventional techniques of molecular biology and microbiology, which are known to those skilled in the art and described, for example, by Ausubel et al., 1987 (Current Protocols 18 in Molecular Biology, John Willey and Sons, New York), Maniatis et al., 1982, (Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). 5 The M9fru minimum medium has the following composition: 7 g/l Na 2

HPO

4 , 3 g/l KH 2

PO

4 , 0.6 g/l NaCl, 1 g/l NH 4 Cl, 4 g/l fructose, 1 mM MgSO 4 , 0.1 mM CaCl 2 and 10 pg/ml thiamine. The M9YNBfru minimum medium corresponds to the M9fru medium supplemented with YNB 10 (Yeast Nitrogen Base, Difco) to a final concentration of 5 g/l. This medium is made up from the following presterilized stock solutions: 10 x M9 salts (70 g/l Na 2

HPO

4 , 30 g/l KH 2

PO

4 , 6 g/l NaCl, 10 g/l NH 4 Cl, autoclaved), filtered 20% fructose, filtered 20% YNB, 15 autoclaved 100 mM MgSO 4 , autoclaved 10 mM CaCl 2 and filtered 1% thiamine. The stock solutions of HMTBN (59 mM), of HMTBS (1.3 mM) and of HTBAmide (100 mM) used for making up the selective medium are produced by diluting, in 20 M9YNBfru, stock solutions of these products at respective concentrations of 5.9 M (Rh6ne-Poulenc batch reference: PHM 439 J) and 1.3 M (Rh6ne-Poulenc batch reference: BFR 81). These solutions are prepared according to a protocol described in patents and patent 25 applications EP 330 521, EP 142 488, US 3 773 927 and US 4 353 924. The HMTBamide is obtained by hydrolysis with sulfuric acid of the amino cyanohydrin 19

(CH

3

SCH

2

CH

2

CHNH

2 CN) described in the same patents and patent applications. The precultures are produced in 3 ml of LB rich medium (10 g/l tryptone, 5 g/l yeast extract, 5 10 g/l NaCl, 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 0 C and 200 rpm for 6 to 7 hours. The cultures for expressing the strains 10 RPA-BIOCAT 841 and 842 are produced by diluting a preculture to 1

/

1 0 0 th in 10 ml of LB + 100 pg/ml of carbenicillin + 0.5 mM IPTG. These cultures are incubated at 37 0 C and 200 rpm and in hermetically sealed 50 ml tubes for 16 h. The biomasses are 15 estimated by measuring the optical density at 660 nm (OD660), using the equation biomass in grams of dry weight per liter of culture = OD660 x 0.35. The assaying of nitrilase activity of the cultures is carried out as follows: the expression 20 cultures are centrifuged and the cell pellet is washed in 100 mM phosphate buffer. It is resuspended in this buffer and 200 pl of cell suspension are incubated with 200 pl of 200 mM HMTBN (in solution in 100 mM phosphate buffer) for 2 h at 37 0 C. A 5 pl sample of the reaction 25 medium is taken at time points of 0, 30, 60, 90 and 120 min, and mixed with 50 p1 of 200 mM H 3

PO

4 . 105 pl of a 5.1% phenol/2.54 N NaOH solution are added to these 55 pl, followed, after mixing, by 40 pl of a solution 20 of bleach 47/50 0 C (sodium hypochlorite in solution at 12.5%, Dousselin & Geoffray Jacquet r6unis, Couzon aux Monts d'Or, France), diluted 50/50. After shaking followed by a 10 min incubation at room temperature, 5 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. Sample [HMTBS] [HMTBN] 1 0 mM 100 mM 2 1 mM 99 mM 3 5 mM 95 mM 4 10 mM 90 mM 5 20 mM 80 mM 6 50 mM 50 mM 7 80 mM 20 mM 8 100 mM 0 mM 10 The activities are expressed in kg of HMTBS produced/hour/kg of dry cells (DC). Examples Example 1: Construction of the plasmid pRPA-BCAT41. 15 The 1.27 kb fragment containing the Ptrp promoter, the ribosome binding site of the X phage cII gene (RBScII) and the nitrilase gene from Alcaligenes faecalis ATCC8750 (nitB) was extracted from the plasmid 21 pRPA-BCAT6 (Application FR 96/13077) using the EcoRI and XbaI restriction enzymes, in order to be cloned into the vector pXL642 (described in CIP Application No. 08/194,588) opened with the same restriction 5 enzymes. The resulting plasmid, pRPA-BCAT15 was opened with the StuI and BsmI enzymes and the 4.3 kb fragment was ligated with the 136 bp StuI-BsmI fragment purified from pRPA-BCAT4 (Application FR 96/13077), to produce the plasmid pRPA-BCAT19. The partial sequencing of 10 pRPA-BCAT19 confirmed the replacement of the codon of the Asp279 residue of the nitrilase with the codon of an Asn279 residue. The 1.2 kb EcoRI-XbaI fragment of pRPA-BCAT19 containing the Ptrp::RBScII::nitB fusion was then cloned into the vector pRPA-BCAT28 opened with the 15 same enzymes, to produce the 6.2 kb plasmid pRPA-BCAT29. The vector pRPA-BCAT28 was obtained by ligating the 3.9 kb SspI-ScaI fragment of pXL642 (CIP Application No. 08/194,588) with the 2.1 kb SmaI fragment of pHP45QTc (Fellay et al., 1987, Gene 52 20 147-154) in order to replace the ampicillin resistance marker with the tetracyclin resistance marker. Destruction of the NdeI site close to the origin of replication of the plasmid pRPA-BCAT29 by partial NdeI digestion and the action of E. coli polymerase I 25 (Klenow fragment) produce the plasmid pRPA-BCAT41, a map of which is represented in Figure 1. The sequence of the expression cassette is represented by the sequence identifier No. 1 (SEQ ID No. 1).

22 Example 2: Construction of the plasmid pRPA-BCAT77. A portion of the nitB gene was amplified by PCR using the plasmid pRPA-BCAT41 as matrix, the primer 5 PCRAFl described in application FR 96/13077 and the primer NitB160 described below, and the enzyme Pfu (Stratagene). NitB160: 5'-GGGGAGAGGT GCTCCCAGCA GCACAGGCCA CCGACGGG-3' The program used comprised one cycle of 5 10 minutes at 95*C; 5 cycles of 1 min at 950C; 1 min at 60*C; 1 min at 720C; 30 cycles of 30 sec at 950C; 30 sec at 60*C; 30 sec at 72 0 C and a sequence of 5 min at 720C. The approximately 0.495 bp fragment thus amplified was digested with the NdeI and BsiHKAI 15 enzymes (New England Biolabs). It was then ligated to the 0.261 kb BsiHKAI-StuI fragment purified from digestion of the plasmid pRPA-BCAT41, and to the 5.43 kb NdeI-StuI fragment purified from digestion of pRPA-BCAT72. The vector pRPA-BCAT72 was obtained by 20 removing from the vector pRPA-BCAT41, by Xcml digestion and religation, the approximately 0.55 kb Xcm1 fragment. The plasmid resulting from this cloning was named pRPA-BCAT77. It corresponds to the plasmid pRPA-BCAT41, but allows the expression of a NitB 25 nitrilase carrying a substitution of the alanine residue at position 160 with a glycine residue.

23 Example 3: Construction of an auxotrophic E. coli strain expressing the nitrilase from Alcaligenes faecalis using the Plac promoter. The nitrilase gene from Alcaligenes faecalis 5 ATCC8750 was amplified by PCR using the plasmid pRPA-BCAT77, as a matrix, the primers nitBMNl and nitBMN2 described below and the polymerase Pfu (Stratagene). nitBMNI 5'-TTGTTATCTA AGGAAATACT TA-3' nitBMN2 : 5'-CGACTCTAGA ACTAGTGGAT CC-3' 10 The program used comprised one cycle of 5 min at 950C; 5 cycles of 1 min at 95*C; 1 min at 50*C; 1 min at 72*C; 30 cycles of 30 sec at 950C; 30 sec at 500C; 30 sec at 720C and a sequence of 5 min at 720C. The approximately 1.2 kb fragment obtained was then 15 digested with XbaI enzyme in order to be cloned into the vector pbsII ks- (Stratagene) opened with the EcoRV and XbaI enzymes. The plasmid obtained, pRPA-BCAT105, was then introduced into the E. coli strain RPA-BIOCAT610. This strain contains a deletion in the 20 metA gene and corresponds to the 180 strain described in Richaud et al. (J. Biol. Chem. (1993) 268: 26827 26835). One clone was selected and cultured in triplicate in LB under the expression conditions described above. The nitrilase activity was measured on 25 the cell pellets obtained as described above and was found, after averaging, to be 2.5 kg/h.kg DC against 0 kg/h.kg DC for the strain RPA-BIOCAT 842 described in 24 Example 4 and cultured under the same conditions. This novel strain expressing an active nitrilase was named RPA-BIOCAT 841. 5 Example 4: Construction of an auxotrophic E. coli strain expressing the inactive nitrilase from Alcaligenes faecalis using the Pac promoter. The gene of a variant of the NitB nitrilase was amplified as described in Example 3 using, as a 10 matrix, the plasmid pRPA-BCAT69. 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 NitB nitrilase with an alanine residue. The plasmid 15 pRPA-BCAT69 was obtained as follows. After amplification by PCR on the matrix pRPA-BCAT41 with the primer PCRAF1 described in application FR96/13077 and the primer NitBl described above, the amplified product was digested with NdeI and BanI to obtain an 20 approximately 0.476 kb insert. NitBi : 5'-GCAGCACAGG GCACCGACGC-3' Similarly, after amplification by PCR on the matrix pRPA-BCAT41 with the primers NitB2 and SR described below, the amplified product was digested 25 with BanI and StuI to obtain an approximately 0.34 kb insert. NitB2 :5'-CGCGTCGGTG CCCTGTGCGC CTGGGAGC-3' SR:5'-CGGCAATGAT CAGGCCTTCG GC-3' 25 After digestion of the vector pRPA-BCAT72 with NdeI and StuI, the 5.43 kb fragment was ligated to the 0.476 kb NdeI-BanI and 0.34 kb BanI-StuI inserts described above, so as to form the vector pRPA-BCAT69. 5 The amplification product obtained with the primers primers nitBMNl and nitBMN2 and the matrix pRPA-BCAT69 was then cloned into the vector pbsII ks- (Stratagene) as described in Example 3. The resulting plasmid, named pRPA-BCAT107, was introduced into the strain 10 RPA-BIOCAT610 so as to obtain the novel strain RPA-BIOCAT 842. Example 5: Construction of an E. coli strain which is auxotrophic for methionine and which expresses the 15 active nitrilase from Comamonas testosteroni using the

P

1 c promoter. The PCR amplification of a 1.35 kb fragment containing the nitA gene from Comamonas testosteroni was carried out using the matrix plasmid pXL2158 20 (FR 96/13077), 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 25 using the QIAEX II kit (QIAGEN) and digestion with the HindIII enzyme, the PCR fragment was ligated to the plasmid pbsII ks- digested beforehand with the HindIII 26 and HincII enzymes, so as to produce the plasmid pBCAT145. The latter was introduced into the strain RP-BIOCAT 610 according to the method of Chung et al. (Proc. Natl. Acad. Sci. USA (1988) 86: 2172-2175). The 5 novel strain thus obtained was named RPA-BIOCAT 960. Example 6: Growth of E. coli mutants which are auxotrophic for methionine, in minimum medium supplemented with HMTBS. 10 The strains RPA-BIOCAT 610 and 842 were precultured in LB medium supplemented, only for strain 842, with 0.5 mM IPTG and 100 pg/ml of carbenicillin, washed in M9YNBfru medium and taken up in an equal volume of this same medium. The two cultures were then 15 diluted to 1

/

1 0 0 th in 10 ml of the following media supplemented with 100 pg/ml of carbenicillin and 0.5 mM IPTG only for the strain RPA-BIOCAT 842: i) M9YNBfru + 50 pM HMTBN, ii) M9YNBfru + 50 pM HMTBN + 10 pM HMTBS. These cultures were prepared in hermetically sealed 20 50 ml tubes at 370C with shaking at 200 rpm, and the growth of the strains, measured by optical density of the culture at 630 nm and read in microplates, is given in Figure 2. The results show that the HMTBS can be used 25 as a methionine source by E. coli strains which are auxotrophic for methionine.

27 Example 7: Influence of YNB, of HMTBN and of HMTBS on the growth, in minimum medium, of an E. coli strain which is auxotrophic for methionine and which expresses the active nitrilase from Alcaligenes faecalis 5 The strains RPA-BIOCAT 841 and 842 were precultured in the presence of 100 pg/ml of carbenicillin and 0.5 mM IPTG, washed as described in Example 6 and diluted to 1

/

1 0 0 0 th in 5 ml of the media cited in Table 1, all supplemented with 100 pg/ml of 10 carbenicillin and 0.5 mM IPTG. Their growth was estimated after 5 days of incubation at 37*C and 200 rpm and in hermetically sealed 50 ml tubes, by visual observation of the turbidity of the cultures. These results are given in Table 1.

28 Table 1: Growth of the strains RPA-BIOCAT 841 and 842 in the presence and absence of YNB Media RPA-BIOCAT 841 RPA-BIOCAT 842 M9fru M9fru + 50 pM HMTBS M9fru + 50 pM HMTBS+ 50 jM HMTBN M9fru + 50 pM HMTBN M9YNBfru M9YNBfru + 50 iM HMTBS + + M9YNBfru + 50 pM HMTBS + + + 50 iM HMTBN M9YNBfru + 50 pM HMTBN -: absence of growth; +: growth. 5 These results show that the YNB constitutes a supplement which is essential for the growth of the E. coli strain which is auxotrophic for methionine, under the conditions described in the example. 10 These results also show that the selective media tested do not make it possible to differentiate a AmetA E. coli strain expressing an active nitrilase from a strain expressing this enzyme in inactive form. The HMTBN concentration of 50 pM is too low to provide 15 a methionine source sufficient for the growth of E. coli.

29 Example 8: Determination of the minimum concentration of HMTBS which allows the auxotrophic strains to grow The strain RPA-BIOCAT 841 was precultured in 5 the presence of 100 ptg/ml of carbenicillin and 0.5 mM IPTG, washed as described in Example 6 and diluted to 1/ 1 0 0 0 th in 5 ml of the media cited in Table 2, all supplemented with 100 pg/ml of carbenicillin and 0.5 mM IPTG. Their growth was estimated after 5 days of 10 incubation at 370C and 200 rpm and in hermetically sealed 50 ml tubes, by visual observation of the turbidity of the cultures. These results are given in Table 2. 15 Table 2: Growth of the strain RPA-BIOCAT 841 in the presence of HMTBS at low concentration. Media RPA-BIOCAT 841 M9YNBfru + 0 pM HMTBS M9YNBfru + 0.8 pM HMTBS M9YNBfru + 1 FM HMTBS M9YNBfru + 2 pM HMTBS + M9YNBfru + 4 ptM HMTBS + M9YNBfru + 6 jM HMTBS + M9YNBfru + 10 pM HMTBS + M9YNBfru + 50 JIM HMTBN + -: absence of growth; +: growth.

30 These results show that the minimum concentration of HMTBS required for the growth of the strain RPA-BIOCAT 841 is greater than or equal to 2 pM. 5 Example 9: Development of a selective medium which allows the growth of an E. coli strain which is auxotrophic for methionine and which expresses the active nitrilase from Alcaligenes faecalis 10 The strains RPA-BIOCAT 841 and 842 were precultured in the presence of 100 pg/ml of carbenicillin and 0.5 mM IPTG, washed under the conditions described in Example 6 and then diluted to 1

/

1 0 0 th in 10 ml of selective medium M9YNBfru + 0.5 mM 15 IPTG containing 0.8 or 1 M HMTBS and 50 iM HMTBN. Figure 3 shows the growth of these two strains at 370C and 200 rpm and in hermetically sealed 50 ml tubes, this growth being measured by reading the optical densities at 630 nm in microplates. 20 The results show that the selective medium M9YNBfru + 0.5 mM IPTG + 50 M HMTBN + 0.8 or 1 pM HMTBS makes it possible to differentiate an E. coli strain which expresses an active nitrilase from an E. coli strain which expresses an inactive nitrilase.

31 Example 10: Growth of E. coli strains which are auxotrophic for methionine and which express nitrilases which are active on hydroxymethylthiobutyronitrile. The strains RPA-BIOCAT 841 and 960 were 5 precultured in the presence of 100 pg/ml of carbenicillin and 0.5 mM IPTG, washed under the conditions described in Example 6 and then diluted to 1

/

1 0 0 th in the selective growth media described in Example 9. Figure 4 shows the growth of these two 10 strains at 37 0 C and 200 rpm and in hermetically sealed 50 ml tubes, this growth being measured by reading the optical densities at 630 nm in microplates. The results show that the medium described in Example 9 is selective for strains which are 15 auxotrophic for methionine and which express nitrilases of two different origins in the expression system using the Plac promoter.

Claims

1. Process for selecting and/or isolating DNA sequences encoding enzymes which are involved in the bioconversion of a suitable substrate to methionine 5 and derivatives thereof, such as HMTBS, characterized in that it comprises the following steps of 1) cloning DNA sequences into a vector which allows their expression in a suitable host microorganism, 10 2) transforming a suitable microorganism which is auxotrophic for methionine, by introducing the vectors obtained above into said suitable microorganism, 3) culturing the transformed microorganisms 15 obtained above in a suitable culture medium comprising a sufficient amount 4) of suitable substrate, 5) selecting and/or isolating the transformed microorganisms capable of growing in the 20 suitable medium, and 6) isolating and, where appropriate, identifying the DNA sequences involved in the bioconversion of the suitable substrate.

2. Process according to claim 1, 25 characterized in that the suitable substrate is 2-amino-4-(methylthio)butyronitrile or derivatives thereof, such as 2-hydroxy-4-(methylthio)butyronitrile 33 (HMTBN).

3. Process according to claim 2, characterized in that the suitable substrate is converted directly to methionine or derivatives 5 thereof, such as HMTBS, by a nitrilase.

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

5. Process according to claim 2, 10 characterized in that the 2-amino-4-(methylthio) butyronitrile or derivatives thereof, such as HMTBN, is converted to methionine or derivatives thereof, such as HMTBS, via 2-amino-4-(methylthio)butanamide or derivatives thereof, such as 2-hydroxy-4-(methylthio) 15 butanamide (HMTBAmide), by a first conversion of the 2-amino-4-(methylthio)butyronitrile or derivatives thereof, such as HMTBN, to 2-amino-4-(methylthio) butanamide or derivatives thereof, such as HMTBAmide, by a nitrile hydratase, followed by conversion of the 20 2-amino-4-(methylthio)butanamide or derivatives thereof, such as HMTBAmide, to methionine or derivatives thereof, such as HMTBS, by an amidase.

6. Process according to claim 5, characterized in that the isolated and/or selected DNA 25 sequence encodes a nitrile hydratase or an amidase.

7. Process according to claim 6, characterized in that the isolated and/or selected DNA sequence encodes a nitrile hydratase, the suitable 34 microorganism also comprising a gene, which may be natural or heterologous, encoding a complementary amidase.

8. Process according to claim 6, 5 characterized in that the isolated and/or selected DNA sequence encodes an amidase, the suitable microorganism also comprising a gene, which may be natural or heterologous, encoding a complementary nitrile hydratase. 10

9. Process according to claim 1, characterized in that the suitable substrate is 2-amino-4-(methylthio)butanamide or derivatives thereof, such as HMTBAmide, which is converted to methionine or derivatives thereof, such as HMTBS, by an 15 amidase.

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

11. Process according to one of claims 1 to 20 10, characterized in that the suitable microorganism which is auxotrophic for methionine is a microorganism which is auxotrophic for methionine, which can be transformed and which is capable of growing in a methionine-free medium comprising HMTBS. 25

12. Process according to claim 11, characterized in that the suitable microorganism is chosen from yeasts, fungi and bacteria.

13. Process according to claim 12, 35 characterized in that the bacteria is E. coli.

14. Process 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 5 partial restriction of said genome(s).

15. Process according to one of claims 1 to 13, characterized in that the DNA sequence is a DNA sequence isolated from a portion of genome by total or partial restriction of said portion of genome. 10

16. Process according to one of claims 1 to 13, characterized in that the DNA sequence is a DNA sequence isolated using a construction by PCR.

17. Process according to one of claims 1 to 13, characterized in that the DNA sequence is obtained 15 by random mutagenesis.

18. Process according to one of claims 1 to 17, characterized in that the culturing of the transformed microorganisms is batchwise culturing, in particular by successive culturing, or continuous 20 culturing, in particular chemostat culturing.

19. Process according to one of claims 1 to 18, characterized in that the suitable culture medium comprises an insufficient amount of methionine to ensure the growth of the microorganism and comprises a 25 sufficient amount of suitable substrate to allow the growth of the transformed microorganism after bioconversion to methionine or derivatives thereof, such as HMTBS. 36

20. Process according to claim 19, characterized in that the sufficient amount of 2-amino 4-(methylthio)butyronitrile or derivatives thereof, such as HMTBN, is between 0 and 60 g/l, preferably 5 between 3 mg/l and 17 mg/l, more preferably between 6 mg/l and 10 mg/i.

21. Process according to claim 19, characterized in that the sufficient amount of 2-amino 4-(methylthio)butanamide or derivatives thereof, such 10 as HMTBAmide, is advantageously between 0 and 60 g/l, preferably between 3 mg/l and 17 mg/l, more preferably between 6 mg/l and 10 mg/i.

22. Process according to one of claims 1 to 21, characterized in that the suitable culture medium 15 comprises a suitable amount of methionine or derivatives thereof, such as HMTBS.

23. Process according to claim 22, characterized in that the suitable amount of methionine or derivatives thereof, such as HMTBS, is less than the 20 concentration sufficient to allow the growth of transformed microorganisms.

24. Process according to either of claims 22 and 23, characterized in that the amount of HMTBS is less than 350 jg/i, preferably less than 250 pg/l, more 25 preferably between 130 and 170 pg/i.

25. Process according to one of claims 1 to 24, characterized in that the suitable culture medium comprises an organic nitrogen source. 37

26. Process according to one of claims [lacuna) 25, characterized in that the content of organic nitrogen source is between 0 and 15 g/l, preferably between 2 and 10 g/l, more preferably 5 between 3 and 8 g/l.

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

28. Process according to one of claims 1 to 10 27, characterized in that the suitable culture medium is a liquid or solid medium.

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

30. DNA sequence encoding an enzyme involved 15 in the bioconversion of a suitable substrate to methionine or derivatives thereof, such as HMTBS, characterized in that it is selected and/or isolated using the process according to the invention.