Lactobacillus n-deoxyribosyl transferases, corresponding nucleotide sequences and their uses

Abstract

The invention concerns novel polypeptides and their fragments, isolated from Lactobacillus , having at least a N-deoxyribosyl transferase activity, the polynucleotides encoding said polypeptides, cloning and/or expression vectors including said polynucleotides, cells transformed by said vectors and specific antibodies directed against said polypeptides. The invention also concerns a method for enzymatic synthesis of deoxyribonucleosides.

1 . Purified or isolated polynucleotide of  Lactobacillus  having at least one N-deoxyribosyltransferase activity with an amino acid sequence chosen from the sequences SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12. 
     
     
         2 . Polypeptide isolated according to  claim 1  characterized in that the polypeptide of SEQ ID No.2 is coded by the N-deoxyribosyltransferase coded by the ntd Lh gene of  Lactobacillus helveticus.    
     
     
         3 . Polypeptide isolated according to  claim 1  characterized in that the polypeptide of SEQ ID No.4 is coded by the N-deoxyribosyltransferase coded by the ptd Lh gene of  Lactobacillus helveticus.    
     
     
         4 . Polypeptide isolated according to  claim 1  characterized in that the polypeptide of SEQ ID No.6 is coded by the N-deoxyribosyltransferase coded by the ntd Lf gene of  Lactobacillus fermentum.    
     
     
         5 . Polypeptide isolated according to  claim 1  characterized in that the polypeptide of SEQ ID No.8 is coded by the N-deoxyribosyltransferase coded by the ntd gene of  Lactobacillus crispatus.    
     
     
         6 . Polypeptide isolated according to  claim 1  characterized in that the polypeptide of SEQ ID No.10 is coded by the N-deoxyribosyltransferase coded by the ntd gene of  Lactobacillus amylovorus.    
     
     
         7 . Polypeptide isolated according to  claim 1  characterized in that the polypeptide of SEQ ID No.12 is coded by the N-deoxyribosyltransferase coded by the ntd gene of  Lactobacillus acidophilus.    
     
     
         8 . Isolated polypeptide characterized in that it comprises a polypeptide chosen from:
 (a) a polypeptide of sequence SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12.   (b) a polypeptide variant of a polypeptide with an amino acid sequence defined in a);   c) a polypeptide homologous to the polypeptide defined in a) or b) and comprising at least 80% identity with said polypeptide of a);   d) a fragment of at least 15 consecutive amino acids of a polypeptide defined in a);   (e) a biologically active fragment of a polypeptide defined in a), b) or c).   
     
     
         9 . Polypeptide according to  claims 1  to  8  characterized in that it makes it possible to satisfy the guanine requirement of the PAK6 strain deposited at the CNCM on 2 May 2001 under No. I-2664. 
     
     
         10 . Purified or isolated polynucleotide characterized in that it codes for a polypeptide according to  claims 1  to  9 . 
     
     
         11 . Polynucleotide according to  claim 10  of sequence SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13. 
     
     
         12 . a Isolated polynucleotide characterized in that it comprises a polynucleotide chosen from:
 (a) SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13;   (b) the sequence of a fragment of at least 15 consecutive nucleotides of the sequence SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13;   (c) a nucleic sequence having a percentage identity of at least 70% after optimal alignment with a sequence defined in a) or b);   (d) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a), b) or c).   
     
     
         13 . Polynucleotide according to  claims 10  to  12  characterized in that its expression in the PAK6 strains makes it possible to satisfy the guanine requirement of said strain. 
     
     
         14 . Use of a polynucleotide according to  claim 12  as a primer for the amplification or polymerization of nucleic sequences of N-deoxyribosyltransferases. 
     
     
         15 . Use of a polynucleotide according to  claims 10  to  13  as a probe for the detection of nucleic sequences of N-deoxyribosyltransferases. 
     
     
         16 . Recombinant cloning and/or expression vector comprising a polynucleotide according to one of  claims 10  to  13  or expressing a polypeptide according to any one of  claims 1  to  9 . 
     
     
         17 . Recombinant vector called pLH2 comprising the polynucleotide SEQ ID No.1 as present in the bacterial strain deposited at the CNCM on 30 May 2001 under No.I-2676; 
     
     
         18 . Recombinant vector called pLH4 comprising the polynucleotide SEQ ID No.3 as present in the bacterial strain deposited at the CNCM on 30 May 2001 under No.I-2677; 
     
     
         19 . Recombinant vector called pLF6 comprising the polynucleotide SEQ ID No.5 as present in the bacterial strain deposited at the CNCM on 30 May 2001 under No.I-2678; 
     
     
         20 . Recombinant vector called pLA comprising the polynucleotide SEQ ID No.20 as present in the bacterial strain deposited at the CNCM on 21 Jun. 2001 under No.I-2689; 
     
     
         21 . Host cell, characterized in that it is transformed by a vector according to  claims 16  to  20 . 
     
     
         22 . Bacterium transformed by the vector pLH2 comprising the polynucleotide SEQ ID No.1, as deposited at the CNCM on 30 May 2001 under No.I-2676; 
     
     
         23 . Bacterium transformed by the vector pLH4 comprising the polynucleotide SEQ ID No.3, as deposited at the CNCM on 30 May 2001 under No.I-2677; 
     
     
         24 . Bacterium transformed by the vector pLF6 comprising the polynucleotide SEQ ID No.5, as deposited at the CNCM on 30 May 2001 under No.I-2678; 
     
     
         25 . Bacterium transformed by the vector pLA comprising the polynucleotide SEQ ID No.9, as deposited at the CNCM on 21 Jun. 2001 under No.I-2689; 
     
     
         26 . Metazoic, plant or animal organism, except human, characterized in that it comprises a cell according to  claim 21 . 
     
     
         27 . Process for preparing a recombinant polypeptide characterized in that a host cell according to  claim 21  or a bacterium according to  claims 22  to  25  is cultured under conditions allowing the expression and optionally the secretion of said recombinant polypeptide and that said recombinant polypeptide is recovered. 
     
     
         28 . Recombinant polypeptide capable of being obtained by a process according to  claim 27 . 
     
     
         29 . Monoclonal or polyclonal antibody and its fragments characterized in that it binds selectively a polypeptide according to one of  claims 1  to  9  or  28 . 
     
     
         30 . Process for in vitro or in vivo enzymatic synthesis of deoxyribonucleotides characterized in that it comprises at least one reaction stage catalyzed by one N-deoxyribosyltransferase according to any one of  claims 1  to  9 . 
     
     
         31 . Process according to  claim 30  characterized in that said N-deoxyribosyltransferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second nucleobase. 
     
     
         32 . Process according to  claim 31  characterized in that said second nucleobase is selected from the group composed of the purines bound by N9, pyrimidines bound by N1, azines bound by N1, imidazoles bound by N1, said second nucleobases being able to carry substitutions of the hydrogens at the non-bound positions. 
     
     
         33 . Process according to  claim 32  characterized in that said second nucleobase is selected from the group composed of 6-methyl purine, 2-amino-6-methylmercaptopurine, 6-dimethylaminopurine, 5-azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6-chloropurine, 6-aza-thymine, 5-fluoro-uracile, ethyl-4-amino-5-imidazole carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide. 
     
     
         34 . Process according to  claim 31  characterized in that said first nucleobase is selected from the group composed of adenine, guanine, thymine, uracile and hypoxanthine. 
     
     
         35 . In vivo process according to  claims 31  to  34  characterized in that it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside. 
     
     
         36 . In vivo process according to  claims 31  to  34  characterized in that it moreover comprises the stage of introducing into the host cell the second nucleobase present in a deoxyribonucleoside. 
     
     
         37 . In vivo process according to  claims 31  to  34  characterized in that it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside and the second nucleobase simultaneously and/or staggered over time.