WO2002101003A2 - Preparation stereoselective de l-acides amines cycliques - Google Patents
Preparation stereoselective de l-acides amines cycliques Download PDFInfo
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- WO2002101003A2 WO2002101003A2 PCT/FR2002/001983 FR0201983W WO02101003A2 WO 2002101003 A2 WO2002101003 A2 WO 2002101003A2 FR 0201983 W FR0201983 W FR 0201983W WO 02101003 A2 WO02101003 A2 WO 02101003A2
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- 0 *[C@]1N(*)*CCCCCCC1 Chemical compound *[C@]1N(*)*CCCCCCC1 0.000 description 2
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- C12N9/88—Lyases (4.)
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- 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
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
Definitions
- the present invention relates to the stereoselective preparation of cyclic L-amino acids or their salts and derivatives.
- L-proline has been elucidated and described. It can be done in two ways:
- N-acylase (WO 95/10604, WO 99/07873), amidase (EP-A-0 686 698), amino acid oxidase (JP 06030789), nitrilase (JP 06038781, JP 11127885) or esterase (WO 00/12745) acting on suitable substrates.
- the Applicant has now shown that it is possible to convert, with high yields, concentrated solutions of di-amino acids into solutions of -imino-cyclic acids, in particular in aqueous solution of ammonium salts of ⁇ - acids.
- imino-cyclical by implementing an enzyme encoded by the omithine cyclodeaminase gene (EC 4.3.1.12) of the Agrobacterium C58 strain or by a gene homologous to it, or recombinant microorganisms overproducing such enzymes.
- the subject of the invention is a process for producing a cyclic L-amino acid of formula (I) or of a salt or derivative of an amino acid of formula (I):
- R 1 is chosen from the hydrogen atom, a linear or branched alkyl radical comprising from 1 to 6 carbon atoms and a linear or branched acyl radical comprising from 1 to 6 carbon atoms;
- X represents a saturated, or partially or completely unsaturated, linear or branched C 1 -C 9 , preferably C 2 -C 4 , hydrocarbon chain optionally comprising in the chain and / or at the chain end one or more heteroatoms or heterogroups chosen from O, S,
- R 'and R " identical or different, representing hydrogen or a hydrocarbon radical, linear or branched, saturated, or totally or partially unsaturated, and comprising from 2 to 20 carbon atoms, it being understood that R' and R" can form a cycle with the atom which carries them, characterized in that: a) a L-diamine acid of formula (II) is reacted:
- X and R-i are as defined above; or a salt or derivative thereof, or an enantiomeric mixture comprising an L-diamine acid of formula (II) and a corresponding D-diamine acid, their salts or derivatives in variable proportions, preferably in an aqueous medium, in the presence of 'an ornithine cyclodeaminase, or of a polypeptide homologous to the omithine cyclodeaminase, the enzyme or the homologous polypeptide being obtained from a recombinant expression vector expressing said enzyme or said homologous polypeptide, b) the cyclic L-amino acid of formula (I) or a salt or derivative thereof is recovered in an enantiomeric excess of at least 80%.
- amino acid derivative of formula (I) or (II) means an amide or ester thereof.
- the subject of the present invention is a process for the production of a cyclic L-amino acid of formula (I) or of a salt or derivative of an amino acid of formula (I):
- R 1 represents H or a C 1 -C 6 alkyl group or C 1 -C 6 acyl and X represents a linear saturated hydrocarbon chain or branched in C 2 -C 9 , preferably in C 2 -C 4 , optionally interrupted by one or more heteroatoms or heterogroups chosen from O, S, NR 2 , R 2 representing H or a C 1 -C alkyl or acyl group 4 , and / or optionally substituted by one or more hydroxy, amino or halogen groups, characterized in that a) a L-diamino acid of formula (II) is reacted:
- X and Ri are as defined above; or a salt or derivative thereof, or an enantiomeric mixture comprising an L-diamine acid of formula (II) and a corresponding D-diamine acid, their salts or derivatives in variable proportions, preferably in an aqueous medium, in the presence of 'an ornithine cyclodeaminase or of a polypeptide homologous to the omithine cyclodeaminase, the enzyme or the homologous polypeptide being obtained from a recombinant expression vector expressing said enzyme or said homologous polypeptide, and b) the the cyclic L-amino acid of formula I or a salt or derivative thereof is recovered in an enantiomeric excess of at least 80%.
- hydrocarbon chain comprises one or more unsaturations of ethylenic and / or acetylenic nature, these are preferably not carried by the carbon atoms situated in position ⁇ of the nitrogen atoms forming the amino functions of the diamine acid of formula (II).
- the compounds of formula (I) obtained according to the process of the present invention most often comprise a cycle with 3, 4, 5, 6 or 7 links which are the cycles most frequently encountered in the field of organic chemistry.
- the compounds of formula (I) for which the cycle has 5, 6 or 7 links are preferred and are those which appear the most accessible to those skilled in the art.
- the process of the present invention cannot however be limited to the synthesis of these compounds with cycles of 5, 6 or 7 links.
- (I) comprises a six-link cycle, X representing a four-link hydrocarbon chain. More specifically still, the process is implemented for obtaining a compound of formula (I) in which X is a linear or branched alkylene chain.
- X is a linear or branched alkylene chain.
- hydrocarbon chain a chain comprising carbon and hydrogen atoms.
- the L-diamine acid of formula (II), as defined above is brought into the presence of at least one enzyme and / or at least one polypeptide homologous to the omithine cyclodeaminase, obtained from a recombinant expression vector expressing these enzyme (s) or these homologous polypeptide (s).
- ornithine cyclodeaminase any enzyme capable of cyclizing a diamine acid, more precisely an amino- ⁇ -amino acid and in particular omithine and lysine.
- the omithine cyclodeaminase to which reference is made in the remainder of the text is preferably the omithine cyclodeaminase (EC 4.3.1.12) of the Agrobacterium C58 strain.
- the omithine cyclodeaminase of the C58 ⁇ 'Agrobacterium strain, encoded by a gene carried by the plasmid TiC58, is described by N. Sans et al., Eur. J. Biochem., 173, (1988), 123-130, and its polypeptide sequence is also available on Genbank (gi: 68365).
- polypeptide homologous to the omithine cyclodeaminase is meant the polypeptides having an amino acid sequence homologous to that of an ornithine cyclodeaminase, in particular that of the C58 strain of Agrobacte um.
- homologous sequences can be defined as the sequences similar to at least 25% of the amino acid sequence of the ocd ⁇ 'Agrobacterium gene and having ornithine cyclodeaminase activity as described by R. N. Costiiow et al., J. Biol. Chem., 246 (21), (1971), 6655-60.
- similar refers to the perfect resemblance or identity between the amino acids compared but also to the non-perfect resemblance which is called similarity.
- This search for similarities in a polypeptide sequence takes into account conservative substitutions which are amino acid substitutions of the same class, such as amino acid substitutions for the uncharged side chains (such as asparagine, glutamine, serine, threonine, and tyrosine), amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid); amino acids with apolar side chains (such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine).
- amino acid substitutions for the uncharged side chains such as asparagine, glutamine, serine, threonine, and tyrosine
- homologous amino acid sequence any amino acid sequence which differs from the amino acid sequence of omithine cyclodeaminase encoded by the ocd ⁇ 'Agrobacterium gene by substitution, deletion and / or insertion of an amino acid or of a reduced number of amino acids, in particular by substitution of natural amino acids with non-natural amino acids or pseudo-amino acids at positions such that these modifications do not significantly affect the biological activity of the encoded polypeptide.
- a homologous amino acid sequence is similar to at least 35% of the sequence of the polypeptide encoded by the ocd gene of Agrobacterium, preferably at least 45%.
- Homology is generally determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705, U.S.A.). Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e. identity or similarity, as defined above). To this end, it may be necessary to artificially introduce spaces ("gaps") in the sequence. Once the optimal alignment has been achieved, the degree of homology is established by recording all the positions for which the amino acids of the two sequences compared are identical, relative to the total number of positions.
- sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705, U.S.A.
- polypeptides have in common to exhibit an activity of ⁇ - ⁇ - or ⁇ - ⁇ -diamines-deaminase activity, without this having necessarily been established up to now.
- homologous sequences are included the sequences of the polypeptides homologous to the omithine cyclodeaminase of Agrobacterium found in microorganisms belonging to the genera Agrobacterium, Aeropyrum, Archaeoglobus, Brucella, Corynebacterium, Halobacterium, Mesorhizobium, Methanobacterium, Pseudomonas, Rizobium Sinorhizobium, Schizosaccharomyces, Sulfolobus, Thermoplasma, Staphylococcus, and Streptomyces.
- Streptomyces lo ⁇ densis ATCC-11415
- polypeptides homologous to the omithine cyclodeaminase of Agrobacterium found in eukaryotic organisms such as Arabidopsis thaliana, Drosophila melanogaster, Homo sapiens, Macropus fuliginosus, Mus musculus, and Rattus norvegicus and listed in table 2 below:
- these polypeptides are encoded by genes homologous to that of the omithine cyclodeaminase of the C58 strain of Agrobacterium.
- gene homologous to that of the omithine cyclodeaminase of the C58 strain of Agrobacterium is meant any gene having: a) a nucleotide sequence similar to the coding sequence of the omithine cyclodeaminase gene of the C58 strain of Agrobacterium; or b) a nucleotide sequence hybridizing with the coding sequence of the omithine cyclodeaminase gene of the C58 strain of Agrobacterium or its complementary sequence, under stringent hybridization conditions; or c) a nucleotide sequence coding for a polypeptide homologous to the omithine cyclodeaminase of Agrobacterium as defined above or having an activity of ornithine cyclodeaminase.
- such a homologous nucleotide sequence according to the invention is similar to at least 75% of the sequence of the ocd gene of Agrobacterium or of the pipA, rapL, pipA * gene of sequence SEQ ID No. 1, rapL * of sequence SEQ ID No. 2 and rapL * L * of sequence SEQ ID No. 5 defined below, more preferably at least 85%, or at least 90%.
- such a homologous nucleotide sequence hybrid specifically to the sequence complementary to the sequence of the ocd gene of Agrobacterium or of the pipA, rapJL gene, pipA of sequence SEQ ID No. 1, rapL * of sequence SEQ ID No. 2 and rapL * L * of sequence SEQ ID No. 5 defined below, under stringent conditions.
- the parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
- the hybridization temperature can preferably be 5 to 10 ° C below Tm, and the hybridization buffers used are preferably force solutions high ionic content such as 6xSSC solution for example.
- similar sequences used above refers to the perfect resemblance or identity between the nucleotides compared but also to the non-perfect resemblance which is called similarity. This search for similarities in the nucleic sequences distinguishes, for example, purines and pyrimidines.
- a homologous nucleotide sequence therefore includes any nucleotide sequence which differs from one of the identified sequences, by mutation, insertion, deletion or substitution of one or more bases, or by the degeneration of the genetic code.
- Such homologous sequences can be obtained from microorganisms belonging to the genera Agrobacterium, Aeropyrum, Archaeoglobus, Brucella, Corynebacterium, Halobacterium, Mesorhizobium, Methanobacterium, Pseudomonas, Rhizobium, Rhodobacter, Sinorhizobium, Schizosaccharomyces, Sulfolobus. Thermoplasma, Staphylococcus and Streptomyces.
- Mention may in particular be made of the sequences of the genes coding for polypeptides homologous to the omithine cyclodeaminase of Agrobacterium found in the species or strains listed in Table 1 above.
- rapL genes of Streptomyces hygroscopicus and pipA of Streptomyces pristinaespirales whose sequence is described respectively by Schwecke et al., Proc. Natl. Acad. Sci. U.S.A., 92 (17), (1995), 7839-43 and in WO-A1 -9601901.
- the compound of general formula (II) or the enantiomeric mixture comprising an L-10 amino acid of formula (II) and a corresponding D-amino acid, their salts or derivatives in variable proportions is placed in the presence of a suspension of the microorganism producing the polypeptide homologous to the omithine cyclodeaminase.
- Preferred homologous genes as described above are synthetic genes obtained by site-directed mutagenesis as described below.
- the compound of formula (II) or the enantiomeric mixture comprising an L-diamine acid of formula (II) and a corresponding D-diamine acid in variable proportions is brought into contact with an enzyme in a purified state.
- the compound of formula (II) or the enantiomeric mixture comprising an L-diamine acid of formula (II) and a corresponding D-diamine acid in variable proportions is
- polypeptides as described above allow, under the conditions of the invention, the stereospecific synthesis of the compounds of formula (I) of the invention without requiring the exogenous addition of NAD to the reaction medium.
- a compound of formula (II) or a mixture of enantiomers of the compound of formula (II) and of D-diamine acid is added to the enzymatic preparation or to the optionally permeabilized cell suspension. corresponding to a concentration greater than approximately 0.05 M, preferably greater than approximately 01 M, this same concentration being less than approximately and 3M, preferably less than 2.5 M, their salts or derivatives and they are left to incubate at a temperature between 10 ° C and 100 ° C, advantageously between 20 ° C and 70 ° C, preferably between 25 ° C and 45 ° C, for a period between a few hours and several days, with stirring.
- the compound of formula (I) is in the form of an ammonium salt.
- the product obtained can be collected by precipitation, crystallization or ion exchange chromatography.
- L-lysine or a mixture of L- and D-lysine L-thialysine (S-2-aminoethyl-L-cysteine), L-ornithine, a mixture of L- and D- 5- (R, S) -hydroxylysine, a mixture of L- and D - azalysin ( ⁇ -N- 2 aminoethyldiaminopropionic acid).
- the overproduction of the polypeptide with ornithine cyclodeaminase activity is particularly advantageous.
- the gene of interest coding for a polypeptide as defined above is introduced into a host microorganism, preferably overexpressing the polypeptide.
- a vector containing a nucleic acid comprising one of the nucleotide sequences as defined above or a homologous sequence is transferred into a host cell which is cultured under conditions allowing expression, preferably overexpression of the corresponding polypeptide.
- the nucleic acid sequence of interest can be inserted into an expression vector, in which it is operably linked to elements allowing the regulation of its expression, such as in particular promoters, activators and / or transcription terminators. .
- the signals controlling the expression of the nucleotide sequences are chosen as a function of the cell host used.
- the nucleotide sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or vectors integrative of the chosen host.
- vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods.
- the plasmids pTZ18 Sigma, St-Louis
- pQE70 Qiagen, Kunststoff
- PQE60 Qiagen, Kunststoff
- the QIAGEN plasmids When used, the QIAGEN plasmids had the major drawback of involving the formation of polyhistidines in the enzymes obtained. Also, these plasmids must be modified to overcome this drawback. The coding sequences must thus be modified or disrupted. These modifications did not result in a difference in terms of level of expression of the gene coding for cyclodeaminase.
- the expression vectors are introduced into the host cells and these are cultured under conditions allowing replication and / or expression of the transfected nucleotide sequence.
- Examples of host cells include in particular E. coli, preferably the Escherichia coli DH5 ⁇ , Escherichia coli ⁇ 2033, and Escherichia co // K12 (MG1655) strains.
- the heterologous gene is modified so as to comprise a proportion of less than 65%, advantageously less than 55% of G + C bases, while coding for the same polypeptide as the native gene or a homologous polypeptide as defined above.
- the third base of the codon, or even the second base of the codon when the latter is G or C is replaced if appropriate by A or T, when the codon obtained corresponds to the same amino acid as that coded by the native codon.
- the modified genes which have made it possible to obtain the best levels of expression and also the highest yields of ⁇ -imino-cyclic acids are those in which the native codons have been replaced for a given amino acid of the polypeptide having the activity amino acid cyclodeaminase by the following respective codons shown in Table 3.
- the modified genes were obtained by assembling sections obtained by PCR and directed mutagenesis from the corresponding native genes.
- modified pipA * and rapL ** genes having the sequences SEQ ID No. 1 and SEQ ID No. 5 respectively, shown in the appendix to the present application.
- Another object of the invention is the polynucleotides corresponding to these modified genes.
- the pipA * gene was obtained from the sequence of the polypeptide coded by the pipA gene from Streptomyces pristinaespiralis shown in the appendix as sequence SEQ ID No. 3.
- the rapL ** gene was obtained from the sequence of the polypeptide encoded by the rapL gene from Streptomyces hygroscopicus shown in the appendix as sequence SEQ ID No. 4.
- the modified genes are inserted into cloning vectors as described above for the native genes transfected into recombinant hosts.
- the host microorganisms, where appropriate, recombinant comprising a gene existing in the native state or modified as described above are cultured, optionally in the presence of an expression inducer, for example IPTG.
- the cells When the cells reach a density of the order of at least one absorbance unit at 600 nm for standard cultures and of the order of a few tens or more of such units for high density cultures, they are separated from their culture medium and subjected to a permeabilization treatment which may be physical (for example, alternative freezing and thawing treatments, ultrasound or French press treatments, mechanical grinding) or chemical (for example, addition of solvent or complexing agents such as EDTA), or enzymatic (for example, addition of lysozyme).
- a permeabilization treatment which may be physical (for example, alternative freezing and thawing treatments, ultrasound or French press treatments, mechanical grinding) or chemical (for example, addition of solvent or complexing agents such as EDTA), or enzymatic (for example, addition of lysozyme).
- the cell suspension can then be used to prepare the products of formula (II) as described above where the protein of interest produced can then be recovered and purified.
- concentrations expressed in grams of dry cells per liter of cell suspension, are obtained greater than approximately 10 g / L, advantageously greater than 30 g / L. These same concentrations are, as a general rule, less than 60 g / L, or even less than 50 g / L, without this however indicating an impassable limit.
- concentrations expressed in grams of dry cells per liter of cell suspension, are obtained greater than approximately 10 g / L, advantageously greater than 30 g / L. These same concentrations are, as a general rule, less than 60 g / L, or even less than 50 g / L, without this however indicating an impassable limit.
- the purification methods used are known to those skilled in the art.
- the recombinant polypeptide obtained can be purified from lysates and cell extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques at using specific mono- or polyclonal antibodies, etc.
- the host cells producing the polypeptide having the desired enzymatic activity are destroyed so that their cytoplasmic content is released in the medium containing the diamino acid substrate whose two amino functions are distant (by the shortest route when several paths are possible) of four or five links, in L form or in the form of a mixture of enantiomers and so as to allow the enzyme and its substrate to be brought into contact.
- Example 1 PCR amplification of the Streptomyces pristinaespiralis pipA gene from the total DNA of the strain.
- the Streptomyces pristinaespiralis ATCC 25486 strain was cultured at 28 ° C for 24 h in TSB medium (DIFCO), then 10 ml of the culture thus obtained were centrifuged for 5 min at 11000 g. The centrifugation pellet was taken up in 1 ml of a 10 mM Tris pH 8.0 solution containing 2 mM EDTA and 5 mg / ml of lysozyme. The suspension thus obtained was allowed to stir for 30 min at 37 ° C, then 100 ⁇ L of a 20% SDS solution was added. After incubation at 30 ° C for a few minutes, 125 ⁇ L of a proteinase K solution at 2 mg / ml was further added.
- TSB medium DIFCO
- the DNA extraction was continued using the reagents of the DNeasy Tissue Kit (QIAGEN, Kunststoff) and respecting the supplier's instructions.
- the DNA thus extracted was purified by phenol / chloroform extraction, precipitated with ethanol and taken up in 100 ⁇ l of water.
- the pipA gene was amplified by PCR from the total DNA thus prepared using, for the synthesis of the primers, its sequence described in patent WO-A1 -96/01901.
- the reaction was carried out in a volume of 50 ⁇ L of 20 mM Tris-HCI buffer pH 8.8 containing 10 mM KCI, 10 mM (NH 4 ) 2 S0 4 , 2 mM MgS0 4 , 0.1% Triton X-100, 2 ⁇ L of total DNA solution, 250 ⁇ M of each of the four dNTPs, 1 unit of Vent DNA Polymerase (BioLabs) and 500 nM of each of the following two oligodeoxynucleotides: 5 'CCCGAATTCCGACACCACGCACGGACGAGAAG 5' CCCTGCAGGCATCTCTCTCCTCGCGAGGGC.
- the PCR protocol applied started with a denaturation step at 97 ° C for 4 min followed by an incubation at 80 ° C for 1 min, and continued with 5 cycles characterized by a 1 min denaturation sequence at 97 ° C, followed by 1 min of hybridization at 55 ° C, then 1 min 30 sec of elongation at 72 ° C. Then 40 cycles characterized by a sequence of 1 min of denaturation at 97 ° C, followed by 1 min of hybridization at 50 ° C, then 1 min 30 sec of elongation at 72 ° C were carried out. The protocol ended with an elongation step at 72 ° C for 10 min.
- the fragment thus amplified was purified on the column of the Qiaquick PCR purification kit (QIAGEN), then digested with EcoRI and Pstl and cloned in the vector pTZ18 (SIGMA, St-Louis, MO) previously cut.
- Two plasmid constructs (pKT 35, pKT 36) obtained following independent PCR amplifications were sequenced. The sequences of these two fragments thus cloned were identical.
- the specified protein, of sequence SEQ ID No. 3 however differs from an amino acid (alanine) at position 87 relative to the sequence published in WO-A1 -96/01901 (glycine).
- SEQ ID No. 3 differs from an amino acid (alanine) at position 87 relative to the sequence published in WO-A1 -96/01901 (glycine).
- the Streptomyces hygroscopicus ATCC 29253 strain was cultured at 28 ° C for 24 h in TSB medium (DIFCO). 50 ⁇ L of the cell suspension thus obtained were subjected to the following heating and cooling stages: 30 sec at 65 ° C, 30 sec at 8 ° C, 90 sec at 65 ° C, 180 sec at 97 ° C, 60 sec at 8 ° C, 180 sec at 65 ° C, 60 sec at 97 ° C, 60 sec at 65 ° C, 20 min at 80 ° C.
- the amplification of the rapL gene was carried out by PCR from a suspension of cells using for the synthesis of the primers its sequence described (Schwecke et al., 1995).
- the reaction was carried out in a volume of 50 ⁇ L of 20 mM Tris-HCI buffer pH 8.8 containing 10 mM KCI, 10 mM (NH 4 ) 2 S0 4d 2 mM MgS0 4 , 0.1% Triton X-100, 10 ⁇ L of the suspension of cells previously treated, 250 ⁇ M of each of the four dNTPs, 1 unit of Vent DNA Polymerase (BioLabs) and 500 nM of each of the following two oligodeoxynucleotides: 5'CCCGAATTCGAGGTTGCATGCAGACCAAGGTTCTGTGCCAG 5'CCCAAGCTTAGATCTTTATTACAGCGAGAGAC
- the PCR protocol applied started with a denaturation step at 97 ° C for 4 min followed by an incubation at 80 ° C for 1 min, and continued first with 5 cycles characterized by a sequence of 1 min denaturation at 97 ° C, followed by 1 min hybridization at 60 ° C, then 1 min 30 sec elongation at 72 ° C, then by 5 new cycles characterized by a sequence of 1 min denaturation at 97 ° C, followed by 1 min of hybridization at 52 ° C, then 1 min 30 sec of elongation at 72 ° C.
- the fragment thus amplified was purified on the column of the Qiaquick PCR purification kit (QIAGEN), then digested with EcoRI and Hindlll and cloned in the vector pTZ18 previously cut.
- Three plasmid constructs obtained (pKT30, pKT31, pKT34) following independent PCR amplifications were sequenced. The sequences of these three fragments thus cloned were identical.
- the protein of the invention contains in positions 50, 84, 102, 127 and 129 respectively a residue of threonine, glutamine, aspartic acid, alanine and alanine.
- the pipA gene with a size of 1066 bp derived from Streptomyces pristinaespiralis and whose sequence is described in patent WO-A1 -96/01901, is composed of a G + C proportion of 69.6%.
- This gene has been rewritten according to the optimized genetic code for expression in Escherichia coli described in Table 3.
- the rewritten gene pipA *, of sequence SEQ ID No. 1 is now composed of a G + C proportion of 38% .
- the threonine residue 97 was coded by ACC instead of ACT provided by the code in Table 3.
- Two TAA translation termination codons were also added at positions 15 and 1080 ( 1st codon nucleotide), respectively of the sequence SEQ ID No. 1, and unique restriction sites EcoRI and Ncol on the one hand, Hindlll on the other hand, were introduced respectively 5 'and 3' of the gene thus allowing its insertion into a cloning vector.
- the synthesis of the pipA * gene was carried out by assembling two sections EcoRI - Kpnl (section 1 of 305 bp - nucleotide 1 to nucleotide 304) and Kpnl - Hindlll (section 2 of 798 bp - nucleotide 305 to nucleotide 1092). Each section was synthesized by extension of single-stranded oligonucleotides with a length of 50 to 60 overlapping nucleotides ("sense" strands and "anti-sense” strands), followed by amplification with 5 'end oligonucleotides and 3 '.
- the PCR reaction was carried out in a volume of 100 ⁇ L of 20 mM Tris-HCl buffer pH 8.8 containing 10 mM KCI, 10 mM (NH 4 ) 2 S0 4 , 2 mM MgS0 4 , 0.1% Triton X-100, 0.01 ⁇ M of the oligonucleotides listed below, 0.3 mM of each of the four dNTPs and 2 units of Vent DNA Polymerase (BioLabs).
- the PCR protocol applied was composed of 30 cycles characterized by a 30 sec sequence of denaturation at 96 ° C, followed by 30 sec of hybridization at 55 ° C, then 30 sec of elongation at 72 ° C.
- cycle number 10 the oligonucleotides of the 5 '(PIPAUP) and 3' (PIPAD1) ends were added at a final concentration of 0.5 ⁇ M. At the end of cycle 30, an elongation period of 2 minutes at 72 ° C was added.
- oligonucleotides were used for the reaction: PipaVO, PipaV2, PipaV4, PipaV6, PipaV ⁇ , PipaD4, PipaD5, PipaD ⁇ and PipaD7.
- the sequences of these oligonucleotides are described in Table 4.
- the PCR product thus obtained was purified on the column of the Qiaquick PCR purification kit (QIAGEN), then digested successively with the restriction enzymes Kpnl and EcoRI, and again purified on agarose gel. An apparent size band of 300 bp was extracted from the gel using the Qiaquick gel extraction kit (QIAGEN).
- This purified PCR product was then ligated to the vector pTZ18, previously digested with the enzymes EcoRI and Kpnl and purified on agarose gel, by T4 DNA ligase (BioLabs). The ligation reaction was carried out overnight at 16 ° C in the ligation buffer provided with the enzyme.
- the ligation product was then transformed into strain E, dialJi DH5 ⁇ CIP104738 (D. Hanahan, J. Mol. Biol., 166, (1983), 557-580) by thermal shock and selected recombinant clones on LB medium. (DIFCO) agar supplemented with ampicillin (100 mg / L).
- the plasmids contained in the ampicillin-resistant transformants were isolated using the QIAprep Spin Miniprep Kit (QIAGEN) plasmid preparation system. For four of these plasmids whose analysis by appropriate restriction enzymes showed an insert of expected size, the EcoRI-Kpn1 fragments were sequenced. Each of the sequences of the four constructs contained between one and three deviations from the expected sequence.
- Plasmid pSP39 was thus obtained.
- the sequencing of the EcoRI-Kpn1 fragment of the plasmid pSP39 was carried out and the sequence obtained was found to be in conformity with that expected.
- section 2 The PCR reaction for the synthesis of the Kpnl-Hindlll section was carried out as described for section 1 with the exception of the elongation period, the duration of which was extended to 1 minute for the 30 cycles and to 4 min at the end of the protocol.
- cycle number 10 the oligonucleotides of the 5 '(PIPAV29) and 3' (PIPAD3) ends were added to a final concentration of 0.5 ⁇ M.
- oligonucleotides were used for the reaction: PipaV ⁇ , PipaVIO, PipaV12, PipaV14, PipaV16, PipaV18, PipaV20, PipaV22, PipaV24, PipaV26, PipaV28, PipaD8, PipaD9, PipaDIO, PipaD11, PipaDa, PipaD12, PipaDa PipaD17.
- the sequences of these oligonucleotides are described in Table 4.
- the PCR product was purified on the column of the Qiaquick PCR purification kit (QIAGEN), then digested with the restriction enzymes Kpnl and Hindlll and ligated to the vector pTZ18 previously digested with the same enzymes.
- the transformation of the E. coli DH5 ⁇ strain, and the selection of the transformants and the analysis of the plasmids were carried out as described above for section 1.
- the plasmid pSP25 for which the sequence of Kpnl-Hindlll fragment had five deviations from the expected sequence was subjected to several stages of site-directed mutagenesis (Ansaldi et al., 1996, ibid.) to correct these errors. Plasmid pSP42 was thus obtained.
- PipaV4 5 'CAGAAATTGGTCGTGGTGAACGTCATTTATCTCCATTACGTGGTGGTTTA
- Example 4 Total synthesis by ligation of the rapL * gene, and obtaining the rapL ** variant
- the rapL gene 1029 bp in size, derived from Streptomyces hygroscopicus and the sequence of which has been published (Schwecke et al., 1995, ibid.), Is composed of a G + C proportion of 68%.
- This gene was rewritten by following the genetic code used for the synthesis of the pipA * gene in Example 3.
- the rewritten gene rapL *, of sequence SEQ ID No. 2 is now composed of a G + C proportion of 38 %.
- the threonine 97 residue was coded by ACC instead of ACT as provided by the code shown in Table 3.
- Two TAA translation termination codons were also added and restriction sites unique EcoRI and SphI on the one hand, Hindlll on the other hand were introduced respectively in 5 ′ and 3 ′ of the gene thus allowing its insertion into different cloning vectors.
- the total synthesis of the rapL * gene was carried out by assembling the three sections EcoRI-Kpnl (section 1 of 305 bp), Kpnl-Pstl (section 2 of 316 bp), and Pstl-Hindlll (section 3 of 440 bp). Each section was synthesized by ligation of phosphorylated single stranded oligonucleotides in 5 ′ with a length of 50 nucleotides on average, covering the entire sequence to be cloned (“sense” strands and “antisense” strands) and overlapping . The sequences and positions of these oligonucleotides are described in Table 5.
- oligonucleotides mixed equimolarly (1 ⁇ M) in the overlap buffer (20 mM Tris-HCl, pH 8; 0.08 M NaCl) in a final volume of 20 ⁇ L, were heated 10 minutes to 70 ° C and kept in the heating block until the temperature returned to room temperature.
- the product thus obtained was then ligated by T4 DNA ligase overnight at 16 ° C in the presence of vector pTZ18 (vector / oligonucleotide molar concentration ratio 1/100) previously digested with the appropriate restriction enzymes.
- the ligation product was used to transform by electroporation the ⁇ 2033 strain (E. çoli K12, pjro, thj, rpsL, hsdS, ⁇ laçZ, F '( ⁇ lacZM15. Iacl q , traD36. ProA +, proB +)) and the recombinant clones have were selected on LB agar medium containing 100 ⁇ g / ml ampicillin, 0.5 mM IPTG and 30 ⁇ g / ml X-Gal.
- Plasmids contained in several ampicillin-resistant dual transformants were isolated using the QIAprep Spin Miniprep Kit (QIAGEN) plasmid preparation system. For each section, the inserts of two to four plasmids whose analysis by appropriate restriction enzymes showed a fragment of expected size, were sequenced.
- section 1 The plasmid pTZ18 cut with EcoRI and Kpnl was ligated with the following oligonucleotides (whose sequence is illustrated in Table 5): lcdV-1, lcdV-2, lcdV-3, lcdV-4, lcdV-5 , lcdV-6, lcdVrev-16, lcdVrev-17, IcdVrev- 18, lcdVrev-19, lcdVrev-20, lcdVrev-21, lcdVrev-22.
- the plasmid pSP3 the insert sequence of which had only two deviations from the expected sequence, was retained. Both errors were corrected by site-directed mutagenesis according to a published method (Ansaldi et al., 1996, ibid.) And the corrections confirmed by sequencing the insert of the resulting plasmid pSP14.
- the plasmid pSP8 the insert sequence of which included only a deviation from the expected sequence, was retained.
- the error was corrected by site-directed mutagenesis (Ansaldi et al., 1996, ibid.) And the correction confirmed by sequencing the insert of the resulting plasmid pSP15.
- the plasmid pSP12 the insert sequence of which included only a deletion of 25 bp and a point deviation was retained.
- the two errors were corrected by site-directed mutagenesis (Ansaldi et al., 1996, ibid.) And the corrections confirmed by sequencing the insert of the resulting plasmid pSP26.
- the rapL ** variant was obtained from the rapL * gene of the plasmid pSP33 by introducing by directed mutagenesis (Ansaldi et al., 1996, ibid.)
- the five changes necessary so that the protein coded by the rapL ** gene is the same as that coded by the amplified gene and sequence described in Example 2 and corresponds to the sequence SEQ ID No. 4, while respecting the genetic code described in Table 3.
- a plasmid pSP36 derived from plasmid pTZ18 and containing the rapL ** gene was thus obtained.
- Anti-sense" strand lcdVrev-16: 5 'p-CAACAATAG TTGGTAAATTAAAA-3' lcdVrev-17: 5 'p-CGTTCAAAATTTTCTGGAGAATAAGAAACAGTTTTCATAGTAACACCAAT- 3' lcdVrev-18: 5 '
- GCA GTT GCA TCT GTT A -3 'lcdV-9 5' p-CT ACT CGT TTATTAGCACGTCCAGGT TCT ACT ACT TTA GCA TTA ATT GGT -3 'lcdV-10: 5' p- GCA GGT GCACAA GCAGTTACT CAA GCA CAT
- TGTGCTTGAGTAACTGCTTGTGCACCTGCACCAATTAATGCTAAAGTAGTA-3 lcdVrev-13: 5 'p- GAACCTGGACGTGCTAATAAACGAGTAGTAACGA-TAG' ACT-ACGATATAT ' Pstl-Hindlll fragment
- AAAATGTCTATT GAAT -3 'lcdV-21 5' p-CTACT CCA GAA GAT GTT TTA GAT CCA TAT
- Example 5 Overexpression of the pipA * gene in Escherichia coli.
- the pipA * gene was cloned into the vector pQE60 (QIAGEN) (plasmid whose coding sequences have been previously modified as indicated above), between the Ncol and HindIII restriction sites from the plasmid pSP43 constructed in Example 3.
- the resulting plasmid pSP47 was introduced into a strain of E. coli K12 MG1655 (Vidal et al., 1998) expressing the lacl gene from the helper plasmid pREP4 (QIAGEN).
- the +75 strain thus obtained was cultured in LB medium and the expression of the pipA * gene was induced by the addition of IPTG according to the supplier's protocol.
- Total cell proteins were separated by polyacrylamide / SDS gel electrophoresis and stained with Coomassie blue. A protein with a molecular weight of 36 kD was detected and its expression level was estimated at approximately 5% of total proteins.
- Example 6 Overexpression of the pipA, rapL, rapL *, and rapL ** genes in Escherichia coli.
- a strain of E. coli overexpressing the pipA gene (strain +353) was obtained by introducing the plasmid pKT37.
- This plasmid was obtained from the plasmid pKT36, described in Example 1, by cloning of the gene of interest between the Ncol and HindIII restriction sites of the vector pQE60.
- strains of E. coli overexpressing the rapL (strain +38), rapL * (strain +60), or rapL ** (strain +73) genes were obtained by respectively introducing the plasmids pSP32, pSP37, or pSP45.
- These plasmids pSP32, pSP37, or pSP45 were obtained respectively from the plasmids pKT30, pSP33, or pSP36, described in Examples 2 and 4, by cloning of the gene of interest between the SphI and Hindlll restriction sites of the vector pQE70.
- Example 7 Amplification by PCR and overexpression of the ocd gene Agrobacterium tumefaciens in Escherichia coli.
- CIP 104333 was carried out as described elsewhere (Hayman et al., Mol. Gen. Genêt, 223, (1990), 465-473).
- the oçd gene (Sans et al., (1988), ibid.) was amplified by PCR from this preparation of the plasmid Ti-C58.
- the reaction was carried out in a volume of 50 ⁇ L of 20 mM Tris-HCI buffer pH 8.8 containing 10 mM KCI, 10 mM (NH 4 ) 2 S0 4 , 2 mM MgS0 4 , 0.1% Triton X-100, 2 ng of plasmid Ti-C58, 200 ⁇ M of each of the four dNTPs, 1 unit of Vent DNA Polymerase (BioLabs) and 500 nM of each of the following two oligodeoxynucleotides: 5'CCCGCATGCCTGCACTTGCCAACC 5'CCCAGATCTTAACCACCCAAACGTCGGAAA
- the PCR protocol applied started with a denaturation step at 94 ° C for 2 min followed by 30 cycles characterized by a 30 sec sequence of denaturation at 94 ° C, followed by 30 sec hybridization at 52 ° C, then 1 min 30 sec of elongation at 72 ° C.
- the fragment thus amplified was digested with Ncol and Bg] ll and cloned into the vector pQE70 (QIAGEN) (plasmid whose coding sequences have been previously modified as indicated above) previously cut.
- the Ncol-Bqll1 fragment of the plasmid pKR7 thus obtained was sequenced.
- the specified protein differs by two amino acids from the published sequence (Sans et al., (1988), ibid.), Namely the residue 212Gln replaced by the lysine and residue 29711e replaced by leucine. The two deviations were confirmed by sequencing a PCR amplification product obtained independently.
- strain +78 was obtained by introducing the plasmid pKR7.
- the level of expression of the protein encoded by the oçd gene was determined as described in Example 5.
- a protein of molecular weight conforming to that expected was detected with an expression level of approximately 5% of the total protein.
- the +75 strain constructed in Example 5, is cultivated in the MS mineral medium (Richaud et al., J. Biol. Chem., 268, (1993), 26827-35) containing 2 g / L glucose, 50 mg / L ampicillin and 30 mg / L kanamycin. After 14 hours of culture, 400 mL of MS mineral medium containing 2 g / L glucose are seeded 1/10 with the preculture of the +75 strain. This culture is placed at 37 ° C. with stirring until reaching an OD 6 6 ohm of 0.7. The pipA * gene is then induced for 4 hours at 37 ° C by the addition of 1 mM IPTG to 200 ml of culture.
- a control culture of 200 ml without adding IPTG is also carried out.
- the two cultures are centrifuged at 13,000 g and the cell pellets are taken up in MS mineral medium so as to concentrate the cells 100 times.
- the cells are then permeabilized by two stages of freezing / thawing at -20 ° C promoting access of the substrate to the enzyme.
- the enzymatic reaction is carried out at 37 ° C. with stirring.
- a 300 ⁇ L sample of each culture is taken and centrifuged at 13,000 g so as to recover the supernatant.
- a diluted solution supernatants is then deposited on a thin layer of silica and in parallel dosed by HPLC.
- HPLC assay (conditions described below) indicates a concentration of 40 g / L of L-pipecolate after 20 hours. The enantiomeric excess is greater than 95%.
- L-lysine were produced with the strains +38, +60, +73, +78 and +353 constructed in Examples 6 and 7, under the same conditions as those applied above for the strain +75.
- Analysis by chromatography of the respective supernatants on a thin layer of silica after staining with ninhydrin only revealed production of L-pipecolic acid with the strains
- L-thiomorpholine-2-carboxylic acid was carried out from a cell suspension of the strain +75 prepared as described in Example 8.
- L-thialysine S- (2-aminoethyl) -L -cysteine) (SIGMA) is added to a final concentration of 1 M.
- L-pipecolic was produced from a cell suspension of the +75 strain prepared as described in Example 8.
- 5-R, S-hydroxy-D, L-lysine (SIGMA) is added to 1 M final .
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CA2449836A CA2449836C (fr) | 2001-06-08 | 2002-06-10 | Preparation stereoselective de l-acides amines cycliques |
EP02747526A EP1409691A2 (fr) | 2001-06-08 | 2002-06-10 | Preparation stereoselective de l-acides amines cycliques |
JP2003503755A JP3943540B2 (ja) | 2001-06-08 | 2002-06-10 | 環状l−アミノ酸の立体選択的製造 |
US10/479,719 US7425530B2 (en) | 2001-06-08 | 2002-06-10 | Stereoselective preparation of cyclic L-amino acids |
KR1020037016066A KR100731597B1 (ko) | 2001-06-08 | 2002-06-10 | 환형 l-아미노산의 입체-선택적 제조 |
AU2002317920A AU2002317920A1 (en) | 2001-06-08 | 2002-06-10 | Stereoselective preparation of cyclic l-amino acids |
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FR0107559A FR2825717B1 (fr) | 2001-06-08 | 2001-06-08 | Preparation stereoselective de l-acides amines cycliques |
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JP2005095167A (ja) * | 2003-08-26 | 2005-04-14 | Mitsubishi Chemicals Corp | 光学活性環状アミノ酸の製造方法 |
US7452704B2 (en) | 2002-02-28 | 2008-11-18 | Mitsubishi Chemical Corporation | Dehydrogenase and a gene encoding the same |
WO2014129459A1 (fr) * | 2013-02-19 | 2014-08-28 | 株式会社エーピーアイ コーポレーション | Procédé de production de la l-lysine hydroxylase et de la hydroxy-l-lysine à l'aide de celle-ci, et procédé de production de l'acide hydroxy-l-pipécolique |
WO2020218624A1 (fr) | 2019-04-25 | 2020-10-29 | 株式会社エーピーアイ コーポレーション | Procédé de production d'acides aminés l-cycliques |
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CN101423861B (zh) * | 2008-07-11 | 2011-06-15 | 浙江大学 | L-氨基酸衍生物的生物催化制备方法 |
JP5395395B2 (ja) * | 2008-10-10 | 2014-01-22 | 北興化学工業株式会社 | 微生物による環状アミノ酸の製造法 |
WO2015098774A1 (fr) * | 2013-12-26 | 2015-07-02 | 株式会社カネカ | Procédé de production d'imino acide cyclique optiquement actif |
CN107287256B (zh) * | 2016-03-31 | 2021-06-08 | 南京诺云生物科技有限公司 | 全细胞催化合成l-2-哌啶甲酸的方法 |
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CN107286227B (zh) * | 2016-03-31 | 2021-01-05 | 南京诺云生物科技有限公司 | Streptomyces hirsutus ATCC 19091蛋白的新用途 |
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CN108752253B (zh) * | 2018-06-27 | 2020-11-24 | 深圳市茵诺圣生物科技有限公司 | 一种多元氮杂环状非天然手性氨基酸及其合成方法 |
CN113512571B (zh) * | 2021-07-13 | 2023-02-24 | 浙江华睿生物技术有限公司 | 鸟氨酸环化脱氨酶催化合成l-哌啶酸的方法 |
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US7452704B2 (en) | 2002-02-28 | 2008-11-18 | Mitsubishi Chemical Corporation | Dehydrogenase and a gene encoding the same |
JP2005095167A (ja) * | 2003-08-26 | 2005-04-14 | Mitsubishi Chemicals Corp | 光学活性環状アミノ酸の製造方法 |
WO2014129459A1 (fr) * | 2013-02-19 | 2014-08-28 | 株式会社エーピーアイ コーポレーション | Procédé de production de la l-lysine hydroxylase et de la hydroxy-l-lysine à l'aide de celle-ci, et procédé de production de l'acide hydroxy-l-pipécolique |
CN104685061A (zh) * | 2013-02-19 | 2015-06-03 | 株式会社Api | L-赖氨酸羟化酶和利用了该l-赖氨酸羟化酶的羟基-l-赖氨酸的制造法及羟基-l-2-哌啶酸的制造法 |
EP2889378A4 (fr) * | 2013-02-19 | 2016-03-09 | Api Corp | Procédé de production de la l-lysine hydroxylase et de la hydroxy-l-lysine à l'aide de celle-ci, et procédé de production de l'acide hydroxy-l-pipécolique |
US9512452B2 (en) | 2013-02-19 | 2016-12-06 | Api Corporation | L-lysine hydroxylase and production method for hydroxy-L-lysine and hydroxy-L-pipecolic acid using same |
JPWO2014129459A1 (ja) * | 2013-02-19 | 2017-02-02 | 株式会社エーピーアイ コーポレーション | L−リジン水酸化酵素およびそれを利用したヒドロキシ−l−リジンの製造法およびヒドロキシ−l−ピペコリン酸の製造法 |
JP2019062927A (ja) * | 2013-02-19 | 2019-04-25 | 株式会社エーピーアイ コーポレーション | L−リジン水酸化酵素およびそれを利用したヒドロキシ−l−リジンの製造法およびヒドロキシ−l−ピペコリン酸の製造法 |
WO2020218624A1 (fr) | 2019-04-25 | 2020-10-29 | 株式会社エーピーアイ コーポレーション | Procédé de production d'acides aminés l-cycliques |
Also Published As
Publication number | Publication date |
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JP2004537296A (ja) | 2004-12-16 |
US7425530B2 (en) | 2008-09-16 |
CA2449836C (fr) | 2012-08-14 |
EP1409691A2 (fr) | 2004-04-21 |
CA2449836A1 (fr) | 2002-12-19 |
KR20040026660A (ko) | 2004-03-31 |
FR2825717B1 (fr) | 2005-02-18 |
US20050038255A1 (en) | 2005-02-17 |
JP3943540B2 (ja) | 2007-07-11 |
KR100731597B1 (ko) | 2007-06-22 |
AU2002317920A1 (en) | 2002-12-23 |
WO2002101003A3 (fr) | 2004-02-26 |
CN1529756A (zh) | 2004-09-15 |
CN100482800C (zh) | 2009-04-29 |
FR2825717A1 (fr) | 2002-12-13 |
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