NZ532081A

NZ532081A - N-deoxyribosyltransferases of lactobacilli, corresponding nucleotide sequences and their applications

Info

Publication number: NZ532081A
Application number: NZ532081A
Authority: NZ
Inventors: Pierre-Alexandre Kaminski; Patrick Tailliez; Philippe Marliere; Pascal Quenee; Rachel Cotaya
Original assignee: Agronomique Inst Nat Rech; Pasteur Institut
Priority date: 2001-09-14
Filing date: 2002-09-12
Publication date: 2005-08-26
Also published as: FR2829774A1; AU2002362377B2; WO2003025163A3; EP2177606A2; EP2177606A3; JP2009136291A; MXPA04002339A; EP1427819A2; CA2460372A1; EP1427819B1; FR2829774B1; JP2005503159A; DE60234847D1; ATE452970T1; WO2003025163A2

Abstract

Purified or isolated polynucleotide of Lactobacillus having at least one N-deoxyribosyltransferase activity. The polypeptide may be encoded by ntd Lh or ptd LH gene of Lactobacillus helveticus, ntd Lf gene of Lactobacillus fermentum, ntd gene of Lactobacillus crispatus, ntd gene of Lactobacillus amylovorus or the ntd gene of Lactobacillus acidophilus.

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 532081 532 0 81 "N-BEOXYRIBOSYLTRANSFERASES OF LACTOBACILLI, CORRESPONDING NUCLEOTIDE SEQUENCES AND THEIR APPLICATIONS" 5 The present invention relates to the field of biology, and more particularly to the microbiological production of base analogues. The present invention relates to new polypeptides and their fragments, isolated from Lactobacillus, having at least one N-10 deoxyribosyltransferase activity, the polynucleotides coding said polypeptides, the cloning and/or expression vectors including said polynucleotides, the cells transformed by said vectors and the specific antibodies directed against said polypeptides. The invention also 15 relates to a process for enzymatic synthesis of deoxyribonucleosides. The analogues of nucleosides the structure of which comprises alterations of the sugar or heterocyclic base, form a family of molecules active in the treatment of 20 numerous bacterial, viral, parasitic and fungal infections as well as in antitumour chemotherapy [Perigaud et al., 1992]. Moreover the insecticidal and herbicidal properties of certain nucleoside antibiotics make them potential agents in the sector of agrichemicals 25 and the environment [Isono, 1988] . The industry uses two methods for producing these analogues, organic synthesis and biocatalytic conversion (enzymatic conversion and microbiological conversion), which have advantages and, conversely, drawbacks. Organic synthesis makes it 30 possible to achieve extremely widely varied chemical structures but requires multiple stages and is expensive in terms of reagents and solvents. On the other hand, the biocatalytic processes allow easy production in an aqueous medium but limited to a small number of possible 2 compounds due to the specificity of the enzymes, which allow a limited range of analogues in the place of their physiological substrates. The phosphorylase nucleosides and N-deoxyribosyltransferase, which result from the 5 purine and pyrimidine salvage pathways in the bacteria, are the enzymes most used for these enzymatic conversions (Krenisky et al., 1981). There is therefore an urgent requirement to obtain enzymes for conversion of nucleosides and their 10 derivatives, having a broadened enzyme activity in order to diversify the industrial production of these compounds. This is the technical problem which the inventors of the present invention propose to resolve. The N-deoxyribosyltransferase of Lactobacillus 15 leichmannii as well as that of L. helveticus, partially purified or not purified, is shown to be the best glycosyl group donor and tolerates a considerable number of structural variations on the base. This enzyme has been used for producing a certain number of analogues 20 among which should be cited 2-chloro,2'-deoxyadenoside (Carson et al., 1984), 2',3'-dideoxynucleosides of natural bases (Carson and Wasson, 1988), or several pyrazolo(3,4-d)pyrimidine and triazolo(4,5-d)pyrimidine derivatives of 2',3'-dideoxycytidine and the 25 corresponding base (Fischer et al., 1990). With the aim of making available recombinant enzymes capable of treating the widest possible variety of deviant substrates either by the base or by the sugar, the inventors have isolated genes coding for an N-30 deoxyribosyltransferase activity of different strains of lactobacilli. This variety of N-deoxyribosyltransferase enzymes makes it possible to increase the chances of obtaining enzymes with specificity broadened by mutations 3 in the wild-type genes or by chimeras of these wild-type genes. Two classes of N-deoxyribosyltransferase have been distinguished (Danzin and Cardinaud, 1976), the first 5 (Class I) designated ptd (for purine transdeoxyribosylase) catalyzing exclusively the exchange of deoxyribose between two purines: dR-Pur + Pur' <-> dR-Pur' + Pur and the second (Class II) designated ntd (for 10 nucleoside transdeoxyribosylase) , the exchange of deoxyribose between a purine and a pyrimidine, between two pyrimidines or between two purines: dR-Pyr + Pur <-> dR-Pur + Pyr dR-Pyr + Pyr' <-> dR-Pyr' + Pyr 15 dR-Pur + Pur' <-> dR-Pur' + Pur. Only two genes specifying Class II enzymes, designated ntd, have been reported to date (Hiick, 1997; dbj|BAA92683.2| (AB039914)). A subject of the present invention is therefore an 20 isolated or purified polypeptide of Lactobacillus having at least one N-deoxyribosyltransferase activity with a sequence of amino acids 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, SEQ ID No.14. 25 According to a preferred embodiment, the polypeptide according to the invention is the N-deoxyribosyltransferase of SEQ ID No.2 (or SEQ ID No. 14) coded by the ntd Lh gene of Lactobacillus helveticus. According to a second embodiment, the polypeptide 30 according to the invention is the N-deoxyribosyltransferase of SEQ ID No. 4 coded by the ptd Lh gene of Lactobacillus helveticus. According to a third embodiment, the polypeptide according to the invention is the N- 4 deoxyribosyltransferase of SEQ ID No. 6 coded by the ntd Lf gene of Lactobacillus fermentum. According to a fourth embodiment, the polypeptide according to the invention is the N-5 deoxyribosyltransferase of SEQ ID No.8 coded by the ntd gene of Lactobacillus crispatus. According to a fifth embodiment, the polypeptide according to the invention is the N-deoxyribosyltransferase of SEQ ID No.10 coded by the ntd 10 gene of Lactobacillus amylovorus. According to a sixth embodiment, the polypeptide according to the invention is the N-deoxyribosyltransferase of SEQ ID No.12 coded by the ntd gene of Lactobacillus acidophilus. 15 The isolated polypeptide according to the invention is 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, SEQ ID No. 14; (b) a polypeptide variant of a v 20 polypeptide with amino acid sequences defined in a); (c) a polypeptide homologous to the polypeptide defined in (a) or (b) and comprising at least 80% identity, preferably 85%, 87%, 90%, 95%, 97%, 98%, 99% identity with said polypeptide of a) ; (d) a fragment of at least 25 15 consecutive amino acids, preferably 17, 20, 23, 25, 30, 40, 50, 100, 250 consecutive amino acids of a polypeptide defined in a), b) or c); and (e) a biologically active fragment of a polypeptide defined in a), b) and c). 30 The polypeptide according to the invention is characterized in that it makes it possible to satisfy the guanine requirement of certain bacterial strains such as PAK6 which is a strain of E. coli the two genes of the guaBA operon of which, which control the conversion of ••.a#*?-. IMP to XMP then to GMP, as well as those of the deoCABD operon which control the degradation of the deoxynucleosides, have been deleted. In fact, these strains, in order to survive or grow, require a supply of 5 deoxyguanosine (dR-G) to the culture medium and the presence of a N-deoxyribosyltransferase activity of a polypeptide according to the invention in order to carry out the exchange: dR-G + A <-> dR-A + G. In the present description, the term polypeptide 10 will be used equally to designate a protein or a peptide. By variant polypeptide is meant all of the mutated polypeptides which can exist naturally, in particular in the human being, and which correspond in particular to truncations, substitutions, deletions and/or additions of 15 amino acid residues. By homologous polypeptide is meant the polypeptides having, relative to the natural deoxyribosyltransferases of Lactobacillus according to the invention, certain modifications such as in particular a deletion, addition 20 or substitution of at least one amino acid, a truncation, an elongation and/or chimeric fusion. Among the homologous polypeptides, those are preferred, the amino acid sequence of which has at least 80% identity, preferably at least 85%, 87%, 90%, 93%, 95%, 97%, 98%, 25 99% identity with the amino acid sequences of the polypeptides according to the invention. In the case of a substitution, one or more consecutive or non-consecutive amino acids can be replaced by "equivalent" amino acids. The expression "equivalent" amino acid is here intended 30 to designate any amino acid capable of being substituted for one of the amino acids of the basic structure, without however modifying the characteristics or essential functional properties, such as their biological activities (i.e. of deoxyribosyltransferase), 6 corresponding polypeptides such as the in vivo induction of antibodies capable of recognizing the polypeptide the amino acid sequence of which is comprised in the amino acid sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ 5 ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14 or one of its fragments. These equivalent amino acids can be determined either on the basis of their homology of structure with the amino acids for which they are substituted, or on the basis of the results of tests for 10 cross-species reactivity to which the different polypeptides are capable of giving rise. By way of example, there will be mentioned the possibilities of substitutions capable of being carried out without resulting in a more profound modification of biological 15 activities of the corresponding modified polypeptides, the replacements, for example of leucine by valine or isoleucine, of aspartic acid by glutamic acid, of glutamine by asparagine, of arginine by lysine etc., it being naturally possible to envisage the reverse 20 substitutions under the same conditions. By polypeptide fragment is meant a polypeptide comprising at least 15 consecutive amino acids, preferably 17, 20, 23, 25, 30, 40, 50, 100, 250 consecutive amino acids. The polypeptide fragments 25 according to the invention obtained by cleavage of said polypeptide by a proteolytic enzyme, by a chemical reagent, or by placing said polypeptide in a very acid environment, also form part of the invention. By biologically active fragment is meant in 30 particular a fragment of an amino acid sequence of a polypeptide according to the invention, having at least one of the characteristics or functional properties of the polypeptide according to the invention, in particular in that it comprises an N-deoxyribosyltransferase 7 activity. The variant polypeptide, the homologous polypeptide or the polypeptide fragment according to the invention has at least 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the N-5 deoxyribosyltransferase activity. Different protocols known to a person skilled in the art have been described for introducing mutations into the polypeptides. Among these, there should be mentioned by way of example, the polymerase chain reaction (PCR) in 10 the presence of manganese (Cadwell et al., 1992). The mutations can be introduced either randomly - in this case the mutagenesis stage is following by a stage of screening the mutant of interest - i.e. in targeted a manner. In the latter case, the mutations are preferably 15 introduced at the level of the catalytic site of the N-deoxyribosyltransferases according to the invention. Preferably a polypeptide according to the invention is a polypeptide constituted by the sequence SEQ ID No.2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ 20 ID No.12, SEQ ID No.14 or a sequence having at least 80%, preferably at least 85%, 90%, 95%, 98% and 99% identity with the SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14 after optimal alignment. By polypeptide the amino acid sequence 25 of which having a percentage identity of least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with a reference sequence, is meant the polypeptides having certain modifications relative to the reference polypeptide, such as in particular one or more 30 deletions, truncations, an elongation, a chimeric fusion, and/or one or more substitutions. Among the polypeptides the amino acid sequence of which has a percentage identity of least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal 8 alignment with 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, SEQ ID No.14 or with one of their fragments according to the invention, the variant polypeptides coded by the variant 5 peptide sequences as previously defined are preferred, in particular the polypeptides, the amino acid sequence of which has at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one amino acid residue relative to 10 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, SEQ ID No.14 or with one of their fragments; more preferably, the variant polypeptides having at least one mutation which reduces the specificity of the polypeptide according to the 15 invention for its substrate, such that the variant polypeptides according to the invention are capable of catalyzing a larger variety of substrate, in order to obtain a wider range of base analogues. The invention also relates to a purified or isolated 20 polynucleotide characerized in that it codes for a polypeptide as defined previously and preferably for 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, SEQ ID No.14. Preferably, the polynucleotide according to the 25 invention has the sequence SEQ ID No.l, SEQ ID No. 3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13. The isolated or purified polynucleotide according to the invention is characterized in that it comprises a 30 polynucleotide chosen from (a) SEQ ID No.l, 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, preferably at least 18, 21, 24, 27, 30, 35, 40, 50, 75, 100, 150, 200 consecutive 9 nucleotides of the sequence SEQ ID No.l, 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%, preferably at least 75%, 80%, 5 85%, 90%, 95%, 98%, 99% 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). The polynucleotide according to the invention is 10 also characterized in that its expression in the host cells, in particular the bacterial strains such as PAK6, make it possible to satisfy the guanine requirement of said strain. The PAK6 strain was deposited at the CNCM on 2nd May 2001 under No. 1-2664. The PAK6 strain 15 corresponds to the bacterial strain of Escherichia coli MG 1655 deleted of two guaA and guaB genes, as well as of the deoC, deoA, deoB, deoD genes. The PAK6 strain (AguaBA::gm Adeo-11) is auxotrophic for guanine in minimal glucose medium. 20 By nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is meant a precise chain of nucleotides, modified or unmodified, 25 making it possible to define a fragment or a region of a nucleic acid, comprising or not comprising non-natural nucleotides, and being able to correspond equally well to a double-stranded DNA, a single-stranded DNA and transcription products of said DNAs, and/or a fragment of 30 RNA. It must be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, i.e. in the natural state. They are sequences which have been isolated or 10 purified, i.e. they have been collected, directly or indirectly, for example by copying, their environment having been at least partially modified. Thus the nucleic acids obtained by chemical synthesis are also meant. 5 By polynucleotide of complementary sequence is meant any DNA the nucleotides of which are those of SEQ ID No.l, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 or part of SEQ ID No.l, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID 10 No. 11, SEQ ID No. 13, and the orientation of which is reversed. By "percentage identity" between two nucleic acid or amino acid sequences within the meaning of the present invention, is meant a percentage of nucleotides or amino 15 acid residues identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed at random and throughout their length. By "best alignment" or 20 "significant alignment" is meant the alignment for which the percentage identity determined as follows is the highest. The comparisons of sequences between two nucleic acid or amino acid sequences are carried out in standard manner by comparing these sequences after having aligned 25 them in significant manner, said comparison having been carried out by segment or by "window of comparison" in order to identify and compare the local regions of sequence similarity. The significant alignment of the sequences for the comparison can be carried out, apart 30 from manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988), by means of computer software using these 11 algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) . In order to obtain the significant alignment, the BLAST 5 program is used, with the BLOSUM 62 matrix. The PAM or PAM250 matrices can also be used. The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned in significant manner, the nucleic acid 10 or amino acid sequence to be compared being able to comprise additions or deletions relative to the reference sequence for a significant alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which 15 the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two 20 sequences. By nucleic sequences having a percentage identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% after significant alignment with a reference sequence, is meant the nucleic sequences 25 having, relative to the reference nucleic sequence, certain modifications such as in particular a deletion, truncation, elongation, chimeric fusion, and/or a substitution, in particular a point substitution, and the nucleic sequence of which has at least 70%, preferably at 30 least 75%, 80%, 85%, 90%, 95%, 98% and 99% identity after significant alignment with the reference nucleic sequence. These are preferably sequences the complementary sequences of which are capable of being hybridized specifically with the sequences SEQ ID No.l, 12 SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 of the invention. Preferably, the specific conditions of hybridization or of high stringency will be such that they ensure at least 70%, 5 preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% identity after significant alignment between one of the two sequences and the complementary sequence of the other. A hybridization under conditions of high stringency signifies that the conditions of temperature 10 and ionic force are chosen in such a manner that they allow the maintenance of the hybridization between two fragments of complementary DNA. By way of illustration, conditions of high stringency of the hybridization stage for the purposes of defining the polynucleotide fragments 15 described above, are advantageously the following: the DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42°C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate 20 solution), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10 x Denhardt's, 5% dextran sulphate and 1% salmon sperm DNA; (2) hybridization per se for 20 hours at a temperature depending on the size of the probe (i.e.: 42°C, for a probe of size > 100 nucleotides) followed by 25 two 20-minute washings at 20°C in 2 x SSC + 2% SDS, one 20-minute washing at 20°C in 0.1 x SSC + 0.1% SDS. The last washing is carried out in 0.1 x SSC + 0.1% SDS for 30 minutes at 60°C, for a probe of size > 100 nucleotides). The hybridization conditions of high 30 stringency described above for a polynucleotide of defined size, can be adapted by a person skilled in the art for oligonucleotides of a larger or smaller size, according to the teaching of Sambrook et al., 1989. 13 Among the nucleic sequences having a percentage identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% after significant alignment with the sequence according to the invention, the variant 5 nucleic sequences of SEQ ID No.l, SEQ ID No. 3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 or their fragments are also preferred, i.e. all of the nucleic sequences corresponding to allelic variants, i.e. individual variations of the sequences SEQ ID No.l, 10 SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No.11, SEQ ID No.13. More particularly, the invention relates to a purified or isolated nucleic acid according to the present invention, characterized in that it comprises or 15 is consituted by one of the sequences SEQ ID No.l, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No.11, SEQ ID No.13, their complementary sequences or RNA sequences corresponding to SEQ ID No.l, SEQ ID No.3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID 20 No.13. The primers or probes, characterized in that they comprise a nucleic acid sequence according to the invention, also form part of the invention. Thus, the primers or probes according to the invention are useful 25 for the detection, identification, assay or amplification of the nucleic acid sequence. In particular, they can make it possible to demonstrate or distinguish between the variant nucleic sequences, or to identify the genome sequence of new eukaryotic or prokaryotic genes, in 30 particular bacterial, and more precisely, of Lactobacillus bacteria, coding for an N-deoxyribosyltransferase, by using in particular an amplification method such as the PCR method, or a related method. According to the invention, the polynucleotides 14 which can be used as probes or primers in processes for the detection, identification, assay or amplification of the nucleic sequence, have a minimal size of 10 bases, preferably at least 15, 18, 20, 25, 30, 40, 50 bases. 5 According to one embodiment, the primers according to the invention are chosen from the sequences SEQ ID No.15 and SEQ ID No.16. The polynucleotides according to the invention can thus be used as primers and/or probes in processes 10 implementing in particular the PCR (polymerase chain reaction) technique (Rolfs et al., 1991). This technique requires the choice of pairs of oligonucleotide primers framing the fragment which has to be amplified. Reference can, for example, be made to the technique described in 15 US Patent No. 4,683,202. The amplified fragments can be identified, for example according to agarose or polyacrylamide gel electrophoresis, or according to a chromatographic technique such as gel filtration or ion exchange chromatography, then sequenced. The specificity 20 of the amplification can be controlled by using as primers the nucleotide sequences of polynucleotides of the invention and as matrices, plasmids containing these sequences or derived amplification products. The amplified nucleotide fragments can be used as reagents in 25 hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments. The invention also relates to the nucleic acids capable of being obtained by amplification 30 using primers according to the invention. Other techniques for amplification of the target nucleic acid can be advantageously used as an alternative to the PCR (PCR-like) using primer pairs of nucleotide sequences according to the invention. By PCR-like is 15 meant any methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the marking systems have been amplified, these techniques being of course known. Generally, it is a matter of 5 amplification of the DNA by a polymerase; when the original sample is an RNA, reverse transcription should be carried out beforehand. There are currently very numerous processes allowing this amplification, for example the SDA (Strand Displacement Amplification) 10 technique (Walker et al., 1992), the TAS (Transcription-based Amplification System) technique described by Kwoh et al. (1989), the 3SR (Self-Sustained Sequence Replication) technique described by Guatelli et al. (1990), the NASBA (Nucleic Acid Sequence Based 15 Amplification) technique described by Kievitis et al. (1991), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by Landegren et al. (1988), the RCR (Repair Chain Reaction) technique described by Segev (1992), the 20 CPR (Cycling Probe Reaction) technique described by Duck et al. (1990), the Q-(3-replicase amplification technique described by Miele et al. (1983). Certain of these techniques have since been perfected. In the case where the target polynucleotide to be 25 detected is an mRNA, it is advantageous to use, prior to the implementation of an amplification reaction using primers according to the invention, or the implementation of a detection process using probes of the invention, a reverse transcriptase type enzyme in order to obtain a 30 cDNA from the mRNA contained in the biological sample. The cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection process according to the invention. 16 The probes hybridization technique can be carried out in various ways (Matthews et al. 1988) . The most general method consists of immobilizing the nucleic acid extracted from the cells of different tissues or cells in 5 culture on a support (such as nitrocellulose, nylon, polystyrene) in order to produce for example DNA chips, then incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After the hybridization, the probe excess is eliminated and the 10 hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, fluorescence or enzyme activity linked to the probe). ; According to another method of using nucleic probes according to the invention, the latter can be used as 15 capture probes. In this case, a probe, called a "capture probe", is immobilized on a support and serves to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected using a second 20 probe, called a "detection probe", marked by an easily detectable element. Among the fragments of useful nucleic acids, there should moreover be mentioned in particular the anti-sense oligonucleotides, i.e. the structure of which ensures, by 25 hybridization with the target sequence, an inhibition of the expression of the corresponding product. Sense oligonucleotides should also be mentioned, which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce 30 either an inhibition, or an activation of this expression. The oligonucleotides according to the invention have a minimum size of 9 bases, preferably at least 10, 12, 15, 17, 20, 25, 30, 40, 50 bases. 17 The probes, primers and oligonucleotides according to the invention can be marked directly or indirectly by a radioactive or non-radioactive compound, by methods well known to a person skilled in the art, in order to 5 obtain a detectable and/or quantifiable signal. The non-marked polynucleotide sequences according to the invention can be used directly as probes or primers. The sequences are generally marked in order to obtain sequences which can be used for numerous 10 applications. The marking of the primers or probes according to the invention is carried out by radioactive elements or by non-radioactive molecules. Among the radioactive isotopes used, there can be mentioned 32P, 33P, 35S, 3H, or 125I. The non-radioactive entities are selected 15 from the ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, colouring agents, luminescent agents such as the radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents.- The invention also comprises a method for detection 20 and/or assay of a polynucleotide according to the invention, in a biological sample, characterized in- that it comprises the following stages: (i), isolation of the DNA from the biological sample to be analyzed, or obtaining of a cDNA from the RNA of the biological 25 sample; (ii) specific amplification of the DNA coding for the polypeptide according to the invention using primers; (iii) analysis of the amplification products. A subject of the invention is also to provide a kit for the detection and/or assay of a nucleic acid according to the 30 invention, in a biological sample, characterized in that it comprises the following elements: (i) a pair of nucleic primers according to the invention, (ii) the reagents necessary to carry out a DNA amplification reaction, and optionally (iii) a component making it 18 possible to verify the sequence of the amplified fragment, more particularly a probe according to the invention. The invention also comprises a method for detection 5 and/or assay of a nucleic acid according to the invention, in a biological sample, characterized in that it comprises the following stages: (i) bringing a polynucleotide according to the invention into contact with a biological sample; (ii) detection and/or assay of 10 the hybrid formed between said polynucleotide and the nucleic acid of the biological sample. A subject of the invention is also to provide a kit for the detection and/or assay of a nucleic acid according to the invention, in a biological sample, characterized in that 15 it comprises the following elements: (i) a probe according to the invention, (ii) the reagents necessary for implementing a hybridization reaction, and/or, if appropriate, (iii) a pair of primers according to the invention, as well as the reagents necessary for a DNA 20 amplification reaction. Preferably, the biological sample according to the invention in which the detection and assay are carried out, is constituted by a culture medium, a cell homogenate, a body fluid, for example a human or animal 25 serum, blood, milk. The present invention also relates to the recombinant cloning and/or expression vectors comprising a polynucleotide according to the invention and/or expressing a polypeptide according to the invention. Such 30 a host cell is also a subject of the invention. Preferably, the recombinant vectors according to the invention are: the vector called pLH2 comprising the polynucleotide SEQ ID No.l as present in the bacterial strain 19 deposited at the CNCM on 30th May 2001 under No.I-2676; the pLH2 plasmid contains an Alu I fragment of 1.4 kb containing the gene coding for the type II N-deoxyribosyltransferase of Lactobacillus helveticus CNRZ32 cloned in the Smal site of the pBAM3 plasmid; the pLH2 plasmid, which expresses this enzyme, is propagated in the PAK6 strain which is auxotrophic for guanine; the vector called pLH4 comprising the polynucleotide SEQ ID No.3 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I-2677; the pLH4 plasmid contains an Alu I fragment of 1.6 kb containing the gene coding for the type I N-deoxyribosyltransferase of Lactobacillus helveticus CNRZ32 cloned in the Smal site of the pBAM3 plasmid; the pLH4 plasmid, which expresses this enzyme, is propagated in the PAK6 strain which is auxotrophic for guanine; the vector called pLF6 comprising the polynucleotide SEQ ID No.5 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I-2678; the pLF6 plasmid contains an Alu I fragment of 1.36 kb containing the gene coding for the type II N-deoxyribosyltransferase of Lactobacillus fermentum CIP102780T. The pLF6 plasmid, which expresses this enzyme, is propagated in the PAK6 strain which is auxotrophic for guanine; the vector called pLA comprising the polynucleotide SEQ ID No.11 as present in the bacterial strain deposited at the CNCM on 21st June 2001 under No.I-2689; the pLA plasmid corresponds to the pSUl9 plasmid, at the sites PstI and BamHI an insert is cloned containing the gene coding for the type II N-deoxyribosyltransferase of Lactobacillus acidophilus 20 CNRZ 1296. The plasmid is propagated in the strain of Escherichia coli TG-I. The vectors according to the invention comprise the elements necessary for the expression and in particular, 5 preferably a promoter, of the translation initiation and termination signals, as well as appropriate regions for regulation of the transcription. They must be able to be maintained in stable fashion in the cell and can optionally have particular signals specifying the 10 secretion of the translated protein. The different control signals are chosen as a function of the host cell used. To this end, the nucleic acid sequences according to the invention can be inserted into autonomous replication vectors inside the chosen 15 host, or integrative vectors of the chosen host. Among the autonomous replication systems, as a function of the host cell, "plasmid", "cosmid", "phagemid" or "mini-chromosome" type systems or viral type systems are preferably used, the viral vectors being able in 20 particular to be adenoviruses (Perricaudet et al., 1992), retroviruses, lentiviruses, poxviruses, or herpetic viruses (Epstein et al., 1992). A person skilled in the art knows the technologies which can be used for each of these systems. When it is desired to integrate the 25 sequence into the chromosomes of the host cell, it is possible to use, for example, plasmidic or viral type systems; such viruses are, for example, the retroviruses (Temin, 1986), or the AAVs (Carter, 1993). Among the non-viral vectors, naked polynucleotides 30 such as naked DNA or naked RNA are preferred according to the technique developed by VICAL, the bacterial artificial chomosomes (BACs), yeast artificial chromosomes (YACs) for expression in yeast, mouse artificial chromosomes (MACs) for expression in murine 21 cells and in preferred manner human artificial chromosomes (HACs) for expression in human cells. Such vectors are prepared according to the methods currently used by a person skilled in the art and the 5 resultant clones can be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion. 10 The invention comprises moreover the host cells, in particular the eukaryotic and prokaryotic cells transformed by the vectors according to the invention. Among the cells which can be used within the meaning of the present invention, bacteria and yeasts can be 15 mentioned. According to a preferred embodiment of the invention, the bacterium is chosen from the group composed of Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus helveticum, Lactobacillus 20 lactis, Escherichia coli, Bacillus subtilus, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria 25 meningitis. According to a preferred embodiment of the invention, the bacterium is Lactobacillus. According to a preferred embodiment it is: - the bacterium transformed by the pLH2 plasmid comprising the polynucleotide SEQ ID No.l, as 30 deposited at the CNCM on 30th May 2001 under No.I- 2676; - the bacterium transformed by the pLH4 plasmid comprising the polynucleotide SEQ ID No.3, as 22 deposited at the CNCM on 30th May 2001 under No.I-2677; - the bacterium transformed by the pLF6 plasmid comprising the polynucleotide SEQ ID No.5, as 5 deposited at the CNCM on 30th May 2001 under No.I- 2678; the bacterium transformed by the pLA plasmid comprising the polynucleotide SEQ ID No.11, as deposited at the CNCM on 21st June under No.1-2689; 10 According to another preferred embodiment the bacterium is Escherichia coli. According to another embodiment of the invention, the cell is a yeast which is preferably Saccharomyces cerevisiae, Saccharomyces pombe, Candida albicans. 15 Among the host cells according to the invention, there should also be mentioned the cells of insects, animal or plant cells. Preferably, the cell according to the invention is free from any enzyme activity capable of degrading said 20 precursor deoxyribonucleoside or said deoxyribonucleoside obtained by bioenzymatic reaction catalyzed by a polypeptide according to the invention. Alternatively, said host cell can be free from additional bioenzymatic activities intended to transform the precursor 25 deoxyribonucleoside and/or deoxyribonucleoside obtained by the bioenzymatic reaction catalyzed by the polypeptide according to the invention. Among these additional bioenzymatic activities, there should be mentioned phosphorylation, sulphatation, acetylation, 30 succinylation, methylation. The nucleic acid sequence coding for the N-deoxyribosyltransferases according to the invention is either naturally present in said cell or is introduced into said cell by the recombinant DNA techniques known to 23 a person skilled in the art. According to a preferred embodiment, the nucleic acid sequence introduced into said cell by the recombinant DNA techniques and which codes for an N-deoxyribosyltransferase according to the 5 invention is heterologous. By heterologous nucleic acid sequence is meant a nucleic acid sequence which is not naturally present in the cell according to the invention. The present invention also relates to metazoic, plant or animal organisms, preferably mammals, except 10 humans, comprising one of said cells transformed according to the invention. Among the animals according to the invention, rodents are preferred, in particular mice, rats or rabbits, expressing at least one polypeptide according to the invention. 15 The cells, preferably bacterial, or fungal, in particular of yeast, as well as the metazoic organisms according to the invention can be used in a method for producing N-deoxyribosyltransferase according to the invention. The method for producing a polypeptide of the 20 invention in recombinant form, itself included in the present invention, is characterized in that the transformed cells are cultured, in particular the cells of the present invention, under conditions allowing the expression and optionally the secretion of a recombinant 25 polypeptide coded by a nucleic acid sequence according to the invention, and said recombinant polypeptide is recovered. The recombinant polypeptides capable of being obtained by this production method also form part of the invention. They can be presented in glycosylated or non-30 glycosylated form, and may or may not have the tertiary structure of the natural protein. The recombinant polypeptide sequences can also be modified in order to improve their solubility, in particular in aqueous solvents. Such modifications are known to a person 24 skilled in the art such as for example the deletion of hydrophobic domains or the substitution of hydrophobic amino acids by hydrophilic amino acids. These polypeptides can be produced from the nucleic 5 acid sequences defined above, according to the techniques for producing recombinant polypeptides known to a person skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a host cell. 10 An effective system for producing a recombinant polypeptide requires a vector and a host cell according to the invention. These cells can be obtained by the introduction into host cells of a nucleotide sequence inserted into a vector as defined above, then the culture 15 of said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence. The processes used for purifying a recombinant polypeptide are known to a person skilled in the art. The recombinant polypeptide can be purified starting with 20 lysates and cell extracts, culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific monoclonal or polyclonal antibodies, etc. A preferred variant consists of 25 producing a recombinant polypeptide fused to a "carrier" protein (chimera protein). The advantage of this system is that it allows a stabilization and reduction of the proteolysis of the recombinant product, an increase in solubility during in vitro renaturation and/or 30 simplification of the purification when the fusion partner has an affinity for a specific ligand. The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the numerous known peptide syntheses, for example the 25 techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by a synthesis in standard solution. The polypeptides obtained by chemical synthesis and being able to comprise 5 corresponding non-natural amino acids are also included in the invention. The polypeptides according to the invention make it possible to prepare monoclonal or polyclonal antibodies. It is therefore also one of the subjects of the present 10 invention to provide a monoclonal or polyclonal antibody and its fragments, characterized in that they selectively and/or specifically bind a polypeptide according to the invention. The chimeric antibodies, humanized antibodies and single-chain antibodies also form part of the 15 invention. The antibody fragments according to the invention are preferably Fab, F(ab')2, Fc or Fv fragments. The polyclonal antibodies can be prepared, for example by immunization of an animal, in particular a mouse, with a polypeptide according to the invention 20 combindd with an immune response adjuvant, then purification of the specific antibodies contained in the serum of the animals immunized on an affinity column on which the polypeptide having served as antigen has been fixed beforehand. The polyclonal antibodies according to 25 the invention can also be prepared by purification on an affinity column on which a polypeptide according to the invention has been immobilized beforehand. The monoclonal antibodies can advantageously be prepared from hybridomas according to the technique described by Kohler and 30 Milstein in 1975. According to a particular embodiment of the invention, the antibody is capable of inhibiting the interaction between the N-deoxyribosyltransferase of the invention and its substrate in order to alter the 26 physiological function of said N-deoxyribosyltransferase polypeptide. The invention also relates to methods for the detection and/or purification of a polypeptide according 5 to the invention, characterized in that they use an antibody according to the invention. The invention moreover comprises purified polypeptides, characterized in that they are obtained by a method according to the invention. 10 Moreover, apart from their use for the purification of the polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, can also be used for the detection of these polypeptides in a biological sample. 15 For these different uses, the antibodies of the invention can also be marked in the same manner as described previously for the nucleic probes of the invention and in preferred manner with an enzymatic, fluorescent or radioactive marking. 20 The antibodies of the invention also constitute a means of analysis of the polypeptide expression according to the invention, for example by immunofluorescence, marking with gold, enzyme immunoconjugates. More generally, the antibodies of the invention can be 25 advantageously used in any situation where the expression of a polypeptide according to the invention, normal or mutated, must be observed, and more particularly in immunocytochemistry or immunohistochemistry or in "western" blotting experiments. Thus, a process for 30 detecting a polypeptide according to the invention in a biological sample, comprising the stages of bringing the biological sample into contact with an antibody according to the invention and demonstrating the antigen-antibody complex formed is also a subject of the invention. 27 It is also one of the subjects of the present invention to provide a process for in vitro or in vivo enzymatic synthesis of deoxyribonucleotides characterized in that it comprises at least one reaction stage 5 catalyzed by at least one N-deoxyribosyltransferase according to the invention. The process according to the invention is characterized in that said N-deoxyribosyltransferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second 10 nucleobase. According to a preferred embodiment of the invention, said second nucleobase is selected from the group composed of the purines bound by N9, pyrimidines bound by Nl, azines bound by Nl, imidazoles bound by Nl, 15 said second nucleobases being able to carry substitutions of the hydrogens at the non-bound positions. Preferably, said second nucleobase is selected from the group composed of 6-methyl purine, 2-amino-6- methylmercaptopurine, 6-dimethylaminopurine, 5- 20 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. Said first nucleobase is itself preferably selected from 25 the group composed of adenine, guanine, thymine, uracile and hypoxanthine. These lists are not exhaustive, and it is evident that natural or non-natural analogues of nucleobases can be used in the present invention as substrate of an N-deoxyribosyltransferase of the 30 invention. Optionally, the in vivo process according to the invention is characterized in that it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside. 28 Optionally, the in vivo process according to the invention is characterized in that it moreover comprises the stage of introducing into the host cell the second nucleobase present in a deoxyribonucleoside. 5 Optionally, the in vivo process according to the invention is 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 one after the 10 other. The deoxyribonucleosides capable of being produced in large quantities and inexpensively by the biosynthesis method according to the invention therefore constitute compounds of interest intended for the preventive or 15 curative treatment of human or animal, tumorous, viral pathologies such as AIDS (acquired human immunodeficiency syndrome), bacterial, parasitic or fungal pathologies. Alternatively, these deoxyribonucleosides capable of being produced in large quantities and inexpensively by 20 the biosynthesis method according to the invention constitute herbicides and insecticides. The present invention also provides a process for nutritional screening intended to isolate deoxyribosyltransferases, preferably the polypeptides 25 according to the invention but also their homologues or their mutants. This first screening according to the invention comprises the stages: (i) (optionally) obtaining a bacterial strain, such as Escherichia coli, auxotrophic for guanine. 30 Preferably this strain is incapable of growing in the presence of deoxyguanosine as a source of guanine. In preferred manner, it is the PAK 6 strain. 29 (ii) transfer of exogenous DNA, preferably in the form of an expression vector, into the bacterium, the exogenous DNA being capable of comprising a sequence coding for a 5 deoxyribosyltransferase. (iii) culture of the bacteria obtained in Stage (ii) on a medium containing deoxyguanosine. (iv) isolation of the exogenous DNA transferred into the bacteria of Stage (iii) which have developed 10 on the medium containing deoxyguanosine. The present invention also provides a nutritional screening for distinguishing the deoxyribosyltransferase I and II activities, preferably in particular for distinguishing between the ntd and ptd polypeptides 15 according to the invention. This second screening comprises the stages of: obtaining a bacterial strain such as for example Escherichia coli, auxotrophic for guanine and thymidine. Preferably, this strain is incapable of growing in the presence of guanine and thymidine. In preferred manner, it is the PAK 26 strain (A guaBguaA :: Adeo-11 AthyA :: erm A (udp-metE)zif9 :: TnlO) is auxotrophic for methionine, guanine and thymidine. transfer of the exogenous DNA, preferably in the form of an expression vector, into the bacterium, the exogenous DNA being capable of comprising a sequence coding for a deoxyribosyltransferase I or II. culture of the bacteria obtained in Stage (ii) on a medium containing deoxyguanosine, then determination of whether the bacteria are growing or not. If the bacteria are growing, then the exogenous DNA codes for a 20 25 (ii) 30 (iii) 30 5 deoxyribosyltransferase II activity which is expressed in said bacterium. If the bacteria are not growing, then the exogenous DNA is capable of coding for a deoxyribosyltransferase I activity. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising" and the like, are to be construed in an 10 inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to". Other characteristics and advantages of the invention are 15 clear from the rest of the description, with examples represented hereafter. 31 EXAMPLES 1. MATERIAL AMD METHODS 5 1.1 Strains and culturing conditions The strains of lactic bacteria used originate from the CNRZ (Centre National de Recherche Zootechnique) collection, Unit6 de Recherches Laiti^res et G6netique 10 Appliqu^e, INRA, Jouy en Josas. They are cultured in MRS medium (from Man et al., J. Appl. Bacterid., 23: 130-135, 1960) and incubated at 30°C, 37°C or 42°C according to the species. The Escherichia coli TGI strain, provided by Stratagene, is cultured in LB (Luria broth base lOg/L, 15 Agar-agar 16g/L) under agitation and at 37°C. 1.2. Preparation of total callular DNA of lactic bactaria: The cultures at the end of the exponential phase are 20 centrifuged for 5 minutes at 13,000 g. The pellet corresponding to a culture of 2 ml is taken up in 200 pi of TES (50 mM Tris, pH8, 10 mM EDTA, pH8, 250 mM saccharose) containing 20 pg/ml of lysozyme and 50 U/ml of mutanolysine (Sigma). After an incubation of one hour 25 at 37°C, the clarification of the preparation is obtained by adding 60 pi of 20% SDS. The extraction of the nucleic acids is carried out by adding to the lysate 500 pi of saturated phenol in water, pH8, to which 0.1% hydroxyquinoline and 100 pi of 30 a mixture of isoamylic chloroform-alcohol (24/1, V/V) has been added. The solution is homogenized then centrifuged for 10 minutes at 13,000 g and at ambient temperature. The upper, limpid phase containing the nucleic acids is retained. The extraction is repeated three times on the 32 latter in order to eliminate the undesired cellular constituents. The phenol traces are eliminated by adding 500 pi of isoamylic chloroform-alcohol to the aqueous phase. After homogenization and centrifuging for 3 5 minutes at 15,000 g and at 4°C, the nucleic acids contained in the upper aqueous phase are precipitated by the addition of a volume of cold isopropanol. After an incubation of one hour at -20 °C, a centrifugation is carried out for 20 minutes at 15, 000 g and at 4°C. The 10 isopropanol is eliminated and replaced by 500 pi of 70% ethanol. A final centrifugation of 10 minutes at 15,000 g and at 4°C allows a pellet of nucleic acids to be recovered. This is left to dry in an evaporator and resuspended in 200 pi of sterile water containing 10 pi 15 of RNase at 10 pg/pl. After 15 minutes of incubation at 37°C to agitate the enzyme degrading the RNAs, 10 pi of the DNA solution is migrated by electrophoresis in an 0.8% agarose gel in order to evaluate the concentration and the quality. 20 1.3. Polymaraae chain reaction of DMA (PCR): Polymerase chain reactions (PCR) are carried out in a reaction volume of 100 pi containing 20 to 100 ng of DNA, 0.5 pM primers, 200 pM dNTPs (dATP, dCTP, dGTP, 25 dTTP) in a 10 mM Tris-HCl buffer pH 9, 50 mM KC1, 1.5 mM MgCl2, 0.002% BSA as well as 2.5 units of Taq polymerase. Thirty amplification cycles were used (Gene Amp PCR systems 2400, Perkin Elmer). The inventors defined two ntdl (SEQ ID no. 15) and ntd2 primers (SEQ ID no. 16) 30 starting from the ntd sequence of Lactobacillus leichmanii described by Huck (personal communication): 33 ntdl 5' -AGA CGA TCT ACT TCG GIGS' 18 bases Tm= 54°C ntd2 5' -ACG GCA CCT TCG TAG AAG-3' 18 bases Tm= 56°C 1.4. Southern-type hybridization: Enzymatic restriction o£ the DNAs. The total DNAs 5 are digested by one or more restriction enzymes. The enzymes used are: BamHI, Bglll, Clal, EcoRI, Hindlll, Hpal, Ncol, NotI, PstI, Xbal, Xhol (Bio-Lab). Digestion is carried out in a final volume of 40 pi containing 70 U of enzyme, 4 pi of 10X NEB buffer (Bio-Lab) and 4 to 8 pg 10 of DNA. The incubation is carried out for 1 hour 30 minutes at 37°C. Transfer of thm UNA to a meembrana. The total DNA fragments resulting from the enzymatic digestion are 15 separated using a 0.7% agarose gel. After migration, the agarose gel is placed under agitation in a depurination solution (0.25N HC1) for 30 minutes. This process thus allows the transfer of DNA fragments larger than 10 kbs. After rinsing with water, the DNA is denatured by placing 20 the gel for 40 minutes into a solution of 5M NaCl, 0.5M NaOH. The gel is rinsed with water then incubated again for 30 minutes in a neutralization solution, 1.5M NaCl, 0.5M Tris HC1; pH 7.5. The DNAs are transferred by capillarity onto a positively charged nylon membrane 25 (Hybond N+, Amersham) . They are eluted by a rising flux of 20X SSC (0.3M trisodium citrate; 3M NaCl; pH7). After the transfer, the DNAs are covalently bound onto the membrane using a UV Stratalinker 2400 apparatus (Stratagene). 30 34 Preparation of thm probm. The probe used is an internal fragment of the ntd gene of Lactobacillus helveticus amplified by PCR. The probe is purified using the Wizard kit (Promega) in order to eliminate the PCR 5 primers. The necessary concentration of the probe is 10 ng/pl. The DNA is marked using the ECL marking kit (Amersham) . To do this, the DNA is denatured by heating for 5 minutes at 100°C and immediately recooled in ice. A volume of marking reagent (peroxidase) then a volume of 10 glutaraldehyde solution are added. This solution is incubated for 10 minutes at 37°C for covalently binding the peroxidase to the DNA. Hybridization and. davmlopmant. After a 15 prehybridization of one hour at 42 °C in hybridization buffer, the membrane is hybridized for 16 hours at 42°C in the presence of the marked probe. In order to eliminate the probe bound in non-specific manner, the membrane is washed for 20 minutes at 42°C in two 20 successive baths of buffer: 6M urea - 0.4% SDS - 0.5X SSC, then rinsed for 5 minutes in two successive baths of buffer: 0.3M sodium citrate - 3M NaCl pH 7. The development is carried out by autoradiography according to the protocol of the ECL kit. A first development 25 reagent containing hydrogen peroxide is reduced by peroxidase bound to the probe. Then the luminol contained in a second development reagent is oxidized, producing light which exposes the autoradiographic film. 30 1.5. Cloning the PCT fragments: The homologous ntd genes amplified by PCR are inserted into the plasmid vector pBluescript II SK+ of E. coli. TGI (Stratagene). This plasmid is first restricted in its single site by the EcoRV enzyme (Gibco-BRL) which 35 creates free ends. The digestion mixing is carried out in a volume of 30 pi, containing 4 pi of DNA. The DNA fragments amplified to be cloned should have their 5' and 3' ends free in order to allow cloning. The preparation 5 of the free ends of 50 pi of PCR products purified using the Wizard kit (Promega) is carried out in a reaction volume of 100 pi containing 3.6 units of polymerase DNA of the T4 phage (Bio-Lab) and 6 units of polymerase I DNA (or Klenow fragment) (Bio-Lab), not having a 5' >3' 10 exonucleic activity. The polymerization is carried out for 20 minutes at 11 °C then the enzymes are deactivated after 10 minutes at 75°C. The DNA is then precipitated in the presence of two volumes of 100% ethanol, glycogen and 10% 3M sodium acetate, pH 4.8. The mixture is placed for 15 one hour at -20 °C then centrifuged for 20 minutes at 15,000 g. The pellet is rinsed with 250 pi of 70% ethanol, centrifuged again for 10 minutes at 15,000 g, dried in an evaporator and resuspended in 20 pi of sterile water. 20 The DNA restricts the pBS-SK+ plasmid and the amplified fragment is comigrated on a 0.7% agarose gel in order to evaluate their respective concentrations: the number of molecules of the fragment to be cloned should 25 be three to four times greater than that of the plasmid. The ligation is carried out in a volume of 10 pi containing 60 ng of insert, 26 ng of restricted pBS-SK+ plasmid, 2 units of ligase (T4 DNA ligase, Boehringer-Mannheim), overnight at 16°C. The ligation products are 30 dialyzed on a 0.025 pm millipore filter so as to eliminate the salts and to thus avoid electric arcs during electroporation. 1.6. Transformation: 36 Preparation of mlmctrocoupmtmnt calls of thm TGI strain of E. coli. Starting from 5 ml of an overnight culture at 37°C under agitation, 500 ml of LB medium are inoculated. The culture is placed under agitation at 37°C 5 until an O.D.6oonm = 1 is reached. It is then recooled for 2 hours in ice then cold-centrifuged for 10 minutes at 5,000 rpm. The supernatant is eliminated, the pellet is taken up in 400 ml of cold water. This preparation is cold-centrifuged for 10 minutes and at 5,000 rpm. The 10 pellet obtained is taken up again in 250 ml of cold water. Following a centrifugation of 10 minutes, the pellet is taken up in 25 ml of cold water then the cells are suspended in a final volume of 1 ml of 10% glycerol, and aliquotted before being rapidly frozen in liquid 15 nitrogen. Transformation by mlmctroporation and smlmction of clonms. The electrocompetent cells preserved at -80°C are thawed in ice then brought into contact with 5 pi of 20 ligature mixture in an electroporation flask. The Gene-Pulser (Bio-Rad) is regulated at 200 volts, 25 mF, 250 ohms. The cells are then subjected to electroporation. 1 ml of an SOC solution is added (20g/L bactopeptone, 5g/L yeast extract, 2ml/L 5M NaCl, 2.5 ml/L 1M KC1, 10 ml/L 1M 25 MgCl2, 10 ml/L 1M MgS04) , containing 0.4% glucose in cell suspension which is incubated at 37°C for one hour. The cells are then plated on an LB selective medium -Xgal (5-bromo-4-chloro-3-indolyl-|3-D-galactoside, lpg/ml) -IPTG (isopropythio-p-D-galactoside, lpg/ml) -ampicillin (50 30 pg/ml) and incubated at 37°C overnight. Rapid mxtraction of pla.ami.dic SNA of recombinant E. coli clonms by alkalinm lysis. The E. coli cells transformed and cultivated in LB medium containing 37 ampicillin (100 pg/ml) are harvested by centrifugation at 15,000 for 10 minutes at 4°C. They are resuspended in 100 pi of a 50 mM saccharose solution, 25 mM Tris-HCl, pH 8, 10 mM EDTA, pH 8. The alkaline lysis and the denaturation 5 of the DNA is carried out by addition of 200 pi 0.2N NaOH, 1% SDS. The reaction medium is left for 1 minute at ambient temperature after having added 200 pi of chloroform. Then 150 pi of a solution of 5M potassium acetate, glacial acetic acid are added. The reaction 10 medium is centrifuged for 15 minutes at 13,000 g at 4°C. The aqueous phase containing the DNA is precipitated in the presence of 2 volumes of 100% ethanol then centrifuged for 20 minutes at 13,000 g at 4°C. The pellet is washed in 70% ethanol, centrifuged for 10 minutes at 15 13,000 g then resuspended in 30 pi of sterile water containing RNase at 10 ng/ml. 1.7. Inverse PCR Inverse PCR allows the regions flanking a fragment 20 of DNA of known sequence to be amplified. This technique takes place in three steps: DlgBStion of the UNA matrix. The DNA matrix is digested by one or two restriction enzymes chosen such 25 that they do not cleave in the known gene sequence and they allow a fragment of suitable size (1 to 3 kb) to be obtained. To choose a suitable enzyme, the total DNA is digested separately by several enzymes. Then Southern-type hybridizations are carried out using the DNA 30 fragment of known sequence as a probe. The digestions for which the probe hybridizes with a fragment of 1 to 3 kb are used for the inverse PCR. The DNA fragments obtained by digestion are circularized. For this purpose, 100 units of T4 DNA ligase and 100 pi of ligation buffer are 38 added to 4 to 8 jag of DNA in a final volume of 1 ml. The ligation mixture is incubated at 15°C overnight. The ligated DNA is then precipitated with 100 pi of 3M sodium acetate, pH 4.8, 700 pi of isopropanol and 2 pi of 5 glycogen, then centrifuged for 30 minutes at 13,000 g at 4°C. The pellet is rinsed in 300 pi of 70% ethanol and centrifuged for 10 minutes at 13,000 g at 4°C. The pellet is taken up in 25 pi of ultrapure water. 10 Ainplification of circularized DNAs using different pximszs. The polymerase chain reactions are carried out in a reaction volume of 100 pi containing 20 to 100 ng of DNA, 0.5 pM primers, 200 pM dNTPs (dATP, dCTP, dGTP, dTTP) in a 10 mM Tris-HCl buffer pH 9, 50 mM KC1, MgCl2 15 with 1.5 mM BSA at 0.002% and 2.5 units of Taq polymerase. The amplification is carried out under the following conditions: 20 94°C 3 minutes 94°C 30 seconds 50 to 60°C (according to the Tm of the primers used) 1 minute 72°C 3 minutes 25 (Gene Amp PCR systems 2400, Perkin Elmer). Sequencing. The PCR fragments are purified by the Wizard kit (Promega) in order to eliminate the non-incorporated oligonucleotides, the salts and the Taq 30 polymerase. The sequencing is carried out using a 373A automatic sequencer (Applied Biosystems-Perkin Elmer) using an ABI PRISM Dye Terminator kit (Perkin Elmer) based on the incorporation of fluorescent phosphate deoxynucleotides during the elongation phase of the >» 25 cycles 39 primers. The sequence reactions are carried out in a reaction volume of 20 pi containing 30 ng of DNA, 4 pi of DyeT Mix (Perkin Elmer Biosystems) and 0.1 mM of primer. 5 Cycle: 96°C 96°C 50°C 10 60°C 20 pi of 3M sodium acetate, pH 4.6, 50 pi of 95% ethanol and 1 pi of glycogen are added to each sequence 15 reaction. The solution is left for 15 minutes at ambient temperature then centrifuged for 20 minutes at 13,000 g. The pellet is then rinsed with 250 pi of 70% ethanol then centrifuged for 10 minutes at 13,000 g. The pellet is then taken up in 6 pi of sequence blue (83% formamide, 20 8.3 mM EDTA, 0.5% dextran blue 2, 000,000 (Sigma)). The samples are denatured for 3 minutes at 90% and 3 pi deposited on 4% acrylamide gel. 2. A SINGLE NUTRITIONAL SCREENING FOR THE TWO 25 CLASSES OF N-DEOXYRIBOSYLTRANSFERASE IN ESCHERICHIA COLI A functional screening allowing the production of guanine to be selected was established on E. coli by deleting the two genes of the guaBA operon which controls 30 the conversion of IMP into XMP then into GMP and also those of the deoCABD operon which controls the degradation of deoxynucleosides to give the PAK 6 strain. The E. coli genome specifies an activity allowing the base G to be converted into GMP (guanine 1 minute 10 seconds 5 seconds 4 minutes 25 cycles 40 phosphoribosyltransferase encoded by the gpt gene), and also an activity allowing the base G to be released from the dR-G deoxynucleoside (purine nucleoside phosphorylase encoded by the deoD gene in the deo operon) . The PAK 6 5 strain thus has a requirement for guanine (G) which cannot be satisfied by the addition of deoxyguanosine (dR-G) . The use of deoxyguanosine (dR-G) can however be selected if an N-deoxyribosyltransferase activity is expressed in the PAK6 strain in order to carry out the 10 exchange: dR-G + A <-> dR-A + G. This exchange between two purine bases can be catalyzed by the two enzyme classes. In fact, the introduction of the ntd gene of L. leichmannii into the 15 PAK 6 strain allows the requirement for guanine to be satisfied using deoxyguanosine (dR-G) and adenine (A). 3. FUNCTIONAL CLONING OF THE PPT GENE OF L. HELVETICUS. 20 DNA fragments of a size comprised between 1 and 2kb obtained by partial digestion (Alul) of L. helveticus CNRZ 32 were ligated in a ColEl plasmid (of pUC type digested by Smal and desphosphorylated) and the mixture 25 is used to transform the PAK6 strain. The transforming clones were selected in a glucose mineral medium to which deoxyguanosine (dR-G) and adenine (A) have been added to the final concentration of 0.3 mM. 30 One of the transforming clones proved to propagate a plasmid containing an insert controlling a Class I N-deoxyribosyltransferase activity and deviating from the restriction profile of the ntd gene of L. helveticus. The sequence of this insert develops a gene specifying a 41 i polypeptide of 167 amino acids with a molecular weight of 18790.70 Daltons having a similarity of 28.6% with the NTD polypeptide of L. leichmanii. The sequence of this gene called ptd deviates from that of the ntd genes 5 making them impossible to hybridize (7.5% identity). Incidentally the ntd gene of L. helveticus controlling a Class II N-deoxyribosyltransferase activity could be isolated once again among the transforming 10 clones selected. 4. functional cloning of the ntd gene of l. FKHMMHTUM. 15 The same nutritional cloning and selection operations were carried out starting from genomic DNA of the L. fermentum CIP 102980T strain. The transforming clones selected proved to propagate a plasmid the inserts of which, having similar restriction profiles, controlled 20 a Class II N-deoxyribosyltransferase activity. The sequence of one of these inserts developed a gene specifying a polypeptide of 168 amino acids with a molecular weight of 18878.20 Daltons having a similarity of 32.9% with the NTD polypeptide of L. leichmanii and 25 36.7% with the PTD polypeptide of L. helveticus. The sequence of this gene deviates from that of the ntd and ptd genes which makes them impossible to hybridize. The NTD polypeptide of L. fermentum has a more marked evolutive relationship for the enzyme of Class I (PTD of 30 L. helveticus) and a functional affinity to Class II, suggesting an early evolutive divergence in the evolution of these enzymes. Its enzymatic activity could prove to be different to those of other species and be suitable 42 for the preparation of a very large spectrum of nucleosides. 5. INVERSE PCR CLONING OF FOUR NTD GENES. 5 Using degenerated oligonucleotides starting from regions of the amino acid sequence of the NTD polypeptide of L. leichmanii (Hiick, 1997) an internal fragment on the ntd gene of L. helveticus was amplified. Starting from this fragment, oligonucleotides were synthesized so as to 10 obtain all the gene by inverse PCR. Starting from the two ntd sequences of L. leichmannii and L. helveticus, we redefined the consensus primers by isolating the ntd genes from other species of lactobacilli such as L. acidophilus, L. crispatus, L. 15 amylovorus with the same result as that described above. 6. A nutritional screening to distinguish the two activities of deoxyribosyltransferase I and II. 20 To distinguish between the two deoxyribosyltransferase activities, the plasmidic DNA of different selected colonies was extracted then used to transform the auxotrophic PAK 26 strain for guanine and thymidine. In the PAK 26 strain, the dTMP cannot be 25 synthesized starting from dUMP because the thymidylate synthase encoded by the thyA gene has been deactivated. Moreover, the thymine cannot be a source of thymidine because the thymidine phosphorylase encoded by the deoA gene and uridine phosphorylase encoded by the udp gene 30 have been deleted. Deoxyguanosine (dR-G) and thymine (T) will be the sources of guanine and thymidine only if an N-deoxyribosyltransferase II activity is expressed in the PAK 26 strain to catalyze the exchange reaction dG + T o dT + G. Only the colonies expressing an N- 43 deoxyribosyltransferase II activity can grow on a mineral glucose medium supplemented by deoxyguanosine and thymine as sources of guanine and thymidine. This second screening for example allowed the N- deoxyribosyltransferase II (ntd) activity to be correlated with the pLH2 plasmid containing the polynucleotide of SEQ ID No. 1 and coding for the ntd enzyme of lactobacillus helveticus and the N-deoxyribosyltransferase I (ptd) activity with the pLH4 plasmid containing the polynucleotide of SEQ ID No. 3 and coding for the ptd enzyme of lactobacillus helveticus. 44 Table 1: Growth of the PAK 6 strain expressing or not eaqpressing an N-deoxyribosyltransferase activity on a glucose mineral medium (in vivo) and enzymatic activity of corresponding raw extracts (in vitro) in in vivo vitro A G dG dG + A dC + T dG + A dC +A PAK 6 - + ± - — — — pSU19 ntd 6 PAK - + ± + + + + LI ntd 6 PAK - + ± + + + + Lh ptd 6 PAK - + ± + — + — Lh ntd 6 PAK - + ± + + + + (+) growth (-) absence of growth PAK 6: MG1655 AguaBA::Apra, Adeo 10 ntd LI: gene coding for the N-deoxyribosyltransferase of Lactobacillus leichmannii ntd Lh: gene coding for the N-deoxyribosyltransferase of Lactobacillus helveticus ntd Lf: gene coding for the N-deoxyribosyltransferase of 15 Lactobacillus fermentum ptd Lh: gene coding for the purine deoxyribosyltransferase of Lactobacillus helveticus A: adenine; G: guanine; T: thymine; dG: deoxyguanosine; dC: deoxycytidine 45 references Cadwell et al. (1992) Research 2 : 28-33. Carson & Wasson (1988) Biochem. Biophys. Res. Comm. 155, 5 829-834. Carson et al., (1990) Proc. Natl. Acad.Sci. USA 81, 2232-2236. Carter (1993) Curr. Op. Biotechnology 3, 533. Danzin & Cardinaud (1976) Eur. J. Biochem. 62, 365-372. 10 Duck et al. (1990), Biotechniques, 9, 142. Epstein (1992) Medecine/Sciences, 8, 902. Fischer et al., (1990) Ger. Offen. DE 3840160. Guatelli et al. (1990), Proc. Natl. Acad. Sci. USA 87: 1874. 15 Isono (1988) J. Antibiotics 12, 1711-1739. Kievitis et al., (1991) J. Virol. Methods, 35, 273. K6hler et Milstein. (1975) Nature 256, 495. Krenitsky et al., (1981) Biochemistry 20, 3615-3621. Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 1173. 20 Landegren et al. (1988) Science 241, 1077. Matthews et al. (1988) Anal. Biochem., 169, 1-25. Miele et al., (1983) J. Mol. Biol., 171, 281-285. Neddleman et Wunsch (1970) J. Mol. Biol. 48 : 443 Pearson et Lipman (1988) Proc. Natl. Acad. Sci. USA 85 : 25 2444 P^rigaud et al., (1992) Annales de l'Institut Pasteur Actualit^s 3, 179-215. Perricaudet et al. (1992), La Recherche 23 : 471. Rolfs, A. et al. (1991), Berlin : Springer-Verlag. 30 Sambrook et al. (1989) Molecular cloning : a laboratory manual second edition - Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. USA Segev, (1992), Kessler C. Springer Verlag, Berlin, New York, 197-205. </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 46 Smith et Waterman (1981) Ad. App. Math. 2 : 482 Temin, (1986) Retrovirus vectors for gene transfer. In Kucherlapati R., ed. Gene Transfer. Walker et al. (1992) EMBO J. 1 : 945-951 5 Wilson et al. (1995) Synthesis 1465-1479. Wong C.H. et al. (1995) Angew. Chem. Int. Ed. Engl. 34, 412-432 and 521-546. 47 CLAIMS 1. Purified or isolated polynucleotide of Lactobacillus having at least one N-5 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. 10 2. Polypeptide isolated according to claim 1 wherein the polypeptide of SEQ ID No.2 is coded by the N- deoxyribosyltransferase coded by the ntd Lh gene of Lactobacillus helveticus. 15 3. Polypeptide isolated according to claim 1 wherein the polypeptide of SEQ ID No.4 is coded by the N- deoxyribosyltransferase coded by the ptd Lh gene of Lactobacillus helveticus. 20 4. Polypeptide isolated according to claim 1 wherein the polypeptide of SEQ ID No.6 is coded by the N-deoxyribosyltransferase coded by the ntd Lf gene of Lactobacillus fermentum. 25 5. Polypeptide isolated according to claim 1 wherein the polypeptide of SEQ ID No.8 is coded by the N- deoxyribosyltransferase coded by the ntd gene of Lactobacillus crispatus. 30 6. Polypeptide isolated according to claim 1 wherein the polypeptide of SEQ ID No. 10 is coded by the N-deoxyribosyltransferase coded by the ntd gene of Lactobacillus amylovorus. ,NTn-C-<7lUAIL PROPERTY OrflcE OF |\j.z. 21 SEP 2004 S1ECEI V£Oj 48 7. Polypeptide isolated according to claim 1 wherein the polypeptide of SEQ ID No. 12 is coded by the N-deoxyribosyltransferase coded by the ntd gene of Lactobacillus acidophilus. 5 8. Isolated polypeptide wherein 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 10 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 95% identity 15 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). 20 9) Polypeptide according to claims 1 to 8 wherein it makes it possible to satisfy the guanine requirement of the PAK6 strain deposited at the CNCM on 2nd May 2001 under No. 1-2664. 25 10) Purified or isolated polynucleotide wherein it codes for a polypeptide according to claims 1 to 9. 11) Polynucleotide according to claim 10 of 30 sequence SEQ ID No.l, 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) Isolated polynucleotide wherein it comprises a polynucleotide chosen from: 'WTE^HUAL PROPERTY o,-HlE of n.z. 2 2 SEP 2004 R ECEIV£q J 49 (a) SEQ ID No.l, 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.l, SEQ 5 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 95% after optimal alignment with a sequence defined in a) or b) ; 10 (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 15 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 20 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. 25 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. 30 17. Recombinant vector called pLH2 comprising the polynucleotide SEQ ID No.l as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I- 2676, intellectual property j OFFiCE OF N 7. I 2 t SEP 2004 RECEIVED ' '■■III* 50 18. Recombinant vector called pLH4 comprising the polynucleotide SEQ ID No. 3 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I-2677; 5 19. Recombinant vector called pLF6 comprising the polynucleotide SEQ ID No. 5 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I-2678; 10 20. Recombinant vector called pLA comprising the polynucleotide SEQ ID No.20 as present in the bacterial strain deposited at the CNCM on 21st June 2001 under No.I-2689; 15 21. A host cell, excluding a human cell in situ, wherein it is transformed by a vector according to claims 16 to 20. 20 22. Bacterium transformed by the vector pLH2 comprising the polynucleotide SEQ ID No.l, as deposited at the CNCM on 30th May 2001 under No.1-2676; 23. Bacterium transformed by the vector pLH4 25 comprising the polynucleotide SEQ ID No.3, as deposited at the CNCM on 30th May 2001 under No.1-2677; 24. Bacterium transformed by the vector pLF6 comprising the polynucleotide SEQ ID No.5, as deposited at 30 the CNCM on 30th May 2001 under No.1-2678; 25. Bacterium transformed by the vector pLA comprising the polynucleotide SEQ ID No.9, as deposited at the CNCM on 21st June 2001 under No.1-2689; 35 r .. I 16 HO V iuwi 1 u/CH 51 26. Metazoic, plant or animal organism, except human, wherein it comprises a cell according to claim 21. 5 27. Process for preparing a recombinant polypeptide wherein 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 10 polypeptide is recovered. 28. Recombinant polypeptide capable of being obtained by a process according to claim 27. 15 29. Monoclonal or polyclonal antibody and its fragments wherein it binds selectively a polypeptide according to one of claims 1 to 9 or 28. 30. Process for in vitro or in vivo enzymatic 20 synthesis of deoxyribonucleotides wherein it comprises at least one reaction stage catalyzed by one N-deoxyribosyltransferase according to any one of claims 1 to 9. 25 31. Process according to claim 30 wherein said N- deoxyribosyltransferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second nucleobase. 30 32. Process according to claim 31 wherein said second nucleobase is selected from the group composed of the purines bound by N9, pyrimidines bound by Nl, azines bound by Nl, imidazoles bound by Nl, said second intellectual property OFRCE OF N.Z. 2 2 SEP 2004 RECEIVE® I 52 nucleobases being able to carry substitutions of the hydrogens at the non-bound positions. 33. Process according to claim 32 wherein said 5 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, 10 imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide. 34. Process according to claim 31 wherein said first nucleobase is selected from the group composed of adenine, guanine, thymine, uracile and hypoxanthine. 15 35. In vitro process according to claims 31 to 34 wherein it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside. 20 36. In vitro process according to claims 31 to 34 wherein it moreover comprises the stage of introducing into the host cell the second nucleobase present in a deoxyribonucleoside. 25 37. In vitro process according to claims 31 to 34 wherein it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside and the second nucleobase 30 simultaneously and/or staggered over time. INTELLECTUAL PROPERTY office of n.z. 22 SEP 2004 RECEIVED 1 SEQUENCE LISTINGS <110> Institut PASTEUR / Institut National de la Recherche Agronomique INRA <120> N-desoxyribosyl transferases of Lactobacillus, corresponding nucleotidic sequences and their applications <130> D19532 <160> 16 <170> Patentln Ver. 2.1 <210> 1 <211> 477 <212> DNA <213> Lactobacillus helveticus NTD <220> <221> CDS <222> (1).. (477) <400> 1 atg aac aag aaa aag act tta tat ttt ggt gcc ggt tgg ttt aat gaa 48 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn Glu 15 10 15 aag caa aac aaa get tac aaa gaa gca atg gca get tta aaa gaa aat 96 Lys Gin Asn Lys Ala Tyr Lys Glu Ala Met Ala Ala Leu Lys Glu Asn 20 25 30 cca aca gtt gat tta gaa aat agt tat gtg ccc ctt gaa aac caa tac 144 Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gin Tyr 35 40 45 aag ggt att cgc att gat gaa cat cca gaa tac ttg cac aac att gaa 192 Lys Gly lie Arg lie Asp Glu His Pro Glu Tyr Leu His Asn lie Glu 50 55 60 tgg get tct gca ace tac cac aat gat tta gta gga att aag act tct 240 Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly lie Lys Thr Ser 65 70 75 80 gat gtc atg ctt ggc gta tat ttg cca gaa gaa gaa gac gtc ggc tta 288 Asp Val Met Leu Gly Val Tyr Leu Pro Glu Glu Glu Asp Val Gly Leu 85 90 95 ggc atg gaa ctg ggc tac gca tta tct caa gga aaa tat att tta ttg 336 Gly Met Glu Leu Gly Tyr Ala Leu Ser Gin Gly Lys Tyr lie Leu Leu 100 105 110 gtt ate cca gat gaa gat tac ggc aag cca ate aac tta atg age tgg 384 Val lie Pro Asp Glu Asp Tyr Gly Lys Pro He Asn Leu Met Ser Trp 115 120 125 ggc gtt tgt gac aat gcc ate aag ate agt gaa tta aaa gac ttc gac 432 Gly Val Cys Asp Asn Ala lie Lys lie Ser Glu Leu Lys Asp Phe Asp 130 135 140 2 ttt aac aag cct cgc tac aat ttc tac gac gga get gta tat taa 477 Phe Asn Lys Pro Arg Tyr Asn Phe Tyr Asp Gly Ala Val Tyr 145 150 155 <210> 2 <211> 158 <212> PRT <213> Lactobacillus helveticus NTD <400> 2 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn Glu 1 5 10 15 Lys Gin Asn Lys Ala Tyr Lys Glu Ala Met Ala Ala Leu Lys Glu Asn 20 25 30 Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gin Tyr 35 40 45 Lys Gly lie Arg lie Asp Glu His Pro Glu Tyr Leu His Asn lie Glu 50 55 60 Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly lie Lys Thr Ser 65 70 75 80 Asp Val Met Leu Gly Val Tyr Leu Pro Glu Glu Glu Asp Val Gly Leu 85 90 95 Gly Met Glu Leu Gly Tyr Ala Leu Ser Gin Gly Lys Tyr lie Leu Leu 100 105 110 Val lie Pro Asp Glu Asp Tyr Gly Lys Pro lie Asn Leu Met Ser Trp 115 120 125 Gly Val Cys Asp Asn Ala lie Lys lie Ser Glu Leu Lys Asp Phe Asp 130 135 140 Phe Asn Lys Pro Arg Tyr Asn Phe Tyr Asp Gly Ala Val Tyr 145 150 155 <210> 3 <211> 504 <212> DNA <213> Lactobacillus helveticus PTD <220> <221> CDS <222> (1)..(504) <400> 3 atg aaa gca gta gtt cca aca gga aaa att tat tta ggc tea cca ttt 48 Met Lys Ala Val Val Pro Thr Gly Lys lie Tyr Leu Gly Ser Pro Phe 1 5 10 15 tac age gat get caa aga gaa aga gca get aag gca aaa gag ttg tta 96 Tyr Ser Asp Ala Gin Arg Glu Arg Ala Ala Lys Ala Lys Glu Leu Leu 20 25 30 3 gca aaa aat eta age ate gcg cac gtc ttc ttc ccc ttt gat gat ggt 144 Ala Lys Asn Leu Ser lie Ala His Val Phe Phe Pro Phe Asp Asp Gly 35 40 45 ttc acc gat cct gat gaa aag aat cct gaa att ggc ggc ate aga age 192 Phe Thr Asp Pro Asp Glu Lys Asn Pro Glu lie Gly Gly lie Arg Ser 50 55 60 atg gtt tgg egg gat gca act tac caa aat gat tta act ggt att teg 240 Met Val Trp Arg Asp Ala Thr Tyr Gin Asn Asp Leu Thr Gly lie Ser 65 70 75 80 aat gcc act tgt ggc gtc ttc tta tat gat atg gat caa tta gat gac 288 Asn Ala Thr Cys Gly Val Phe Leu Tyr Asp Met Asp Gin Leu Asp Asp 85 90 95 ggc tct gcc ttt gaa att ggc ttc atg cgt gcg atg cat aag ccg gtg 336 Gly Ser Ala Phe Glu lie Gly Phe Met Arg Ala Met His Lys Pro Val 100 105 110 ate ttg gtg cca ttc act gag cat ccc gaa aaa gaa aag aaa atg aac 384 lie Leu Val Pro Phe Thr Glu His Pro Glu Lys Glu Lys Lys Met Asn 115 120 125 ctg atg ate gca caa ggc gta acc acc ate att gat ggc aat act gaa 432 Leu Met lie Ala Gin Gly Val Thr Thr lie lie Asp Gly Asn Thr Glu 130 135 140 ttt gaa aaa eta get gat tat aac ttc aac gaa tgt cct ttt aat cca 480 Phe Glu Lys Leu Ala Asp Tyr Asn Phe Asn Glu Cys Pro Phe Asn Pro 145 150 155 160 gtt cgc ggt tac ggt ate tat taa 504 Val Arg Gly Tyr Gly lie Tyr 165 <210> 4 <211> 167 <212> PRT <213> Lactobacillus helveticus PTD <400> 4 Met Lys Ala Val Val Pro Thr Gly Lys lie Tyr Leu Gly Ser Pro Phe 15 10 15 Tyr Ser Asp Ala Gin Arg Glu Arg Ala Ala Lys Ala Lys Glu Leu Leu 20 25 30 Ala Lys Asn Leu Ser lie Ala His Val Phe Phe Pro Phe Asp Asp Gly 35 40 45 Phe Thr Asp Pro Asp Glu Lys Asn Pro Glu lie Gly Gly lie Arg Ser 50 55 60 Met Val Trp Arg Asp Ala Thr Tyr Gin Asn Asp Leu Thr Gly lie Ser 65 70 75 80 Asn Ala Thr Cys Gly Val Phe Leu Tyr Asp Met Asp Gin Leu Asp Asp 85 90 95 4 Gly Ser Ala Phe Glu lie Gly Phe Met Arg Ala Met His Lys Pro Val 100 105 110 lie Leu Val Pro Phe Thr Glu His Pro Glu Lys Glu Lys Lys Met Asn 115 120 125 Leu Met lie Ala Gin Gly Val Thr Thr lie lie Asp Gly Asn Thr Glu 130 135 140 Phe Glu Lys Leu Ala Asp Tyr Asn Phe Asn Glu Cys Pro Phe Asn Pro 145 150 155 160 Val Arg Gly Tyr Gly lie Tyr 165 <210> 5 <211> 516 <212> DNA <213> Lactobacillus fermentum NTD <220> <221> CDS <222> (1).. (504) <400> 5 ttg aaa aat acc gac cca gtt get aac act aaa att tac ctg get acc 48 Leu Lys Asn Thr Asp Pro Val Ala Asn Thr Lys lie Tyr Leu Ala Thr 15 10 15 age ttc ttc aac gaa gaa caa cgt gcc cgc ate cct caa get eta gcc 96 Ser Phe Phe Asn Glu Glu Gin Arg Ala Arg lie Pro Gin Ala Leu Ala 20 25 30 caa eta gaa gcc aac ccg act gtc ggc gtt gtt cac cag cca ttc gat 144 Gin Leu Glu Ala Asn Pro Thr Val Gly Val Val His Gin Pro Phe Asp 35 40 45 ttc caa tat aaa gat gca cgc gta gac tcc gat cct gcc ggc gtc ttt 192 Phe Gin Tyr Lys Asp Ala Arg Val Asp Ser Asp Pro Ala Gly Val Phe 50 55 60 ggc age etc gaa tgg caa att gcc act tac aat aac gac ctc aac gcg 240 Gly Ser Leu Glu Trp Gin lie Ala Thr Tyr Asn Asn Asp Leu Asn Ala 65 70 75 80 gta gga act tcc gat gtc tgc gtt get tta tac gat atg gac caa att 288 Val Gly Thr Ser Asp Val Cys Val Ala Leu Tyr Asp Met Asp Gin lie 85 90 95 gac gaa gga att tgt atg gaa ate ggc atg ttc gtc gcc ctc cat aaa 336 Asp Glu Gly lie Cys Met Glu lie Gly Met Phe Val Ala Leu His Lys 100 105 110 cct ate gtt tta eta cct ttt act aag aaa gat aag tct get tat gaa 384 Pro lie Val Leu Leu Pro Phe Thr Lys Lys Asp Lys Ser Ala Tyr Glu 115 120 125 get aac eta atg eta gca egg ggt gta act acc tgg ttg gaa cct aat 432 Ala Asn Leu Met Leu Ala Arg Gly Val Thr Thr Trp Leu Glu Pro Asn 5 130 135 140 gac ttt agt ccc tta aaa gac ttt aac ttt aac cac cca atg get caa 480 Asp Phe Ser Pro Leu Lys Asp Phe Asn Phe Asn His Pro Met Ala Gin 145 150 155 160 cct ttc cca cca ttc aag gtt ttc taactaacct aa 516 Pro Phe Pro Pro Phe Lys Val Phe 165 <210> 6 <211> 168 <212> PRT <213> Lactobacillus fermentum NTD <400> 6 Leu Lys Asn Thr1 Asp Pro Val Ala Asn Thr Lys lie Tyr Leu Ala Thr 15 10 15 Ser Phe Phe Asn Glu Glu Gin Arg Ala Arg lie Pro Gin Ala Leu Ala 20 25 30 Gin Leu Glu Ala Asn Pro Thr Val Gly Val Val His Gin Pro Phe Asp 35 40 45 Phe Gin Tyr Lys Asp Ala Arg Val Asp Ser Asp Pro Ala Gly Val Phe 50 55 60 Gly Ser Leu Glu Trp Gin lie Ala Thr Tyr Asn Asn Asp Leu Asn Ala 65 70 75 80 Val Gly Thr Ser Asp Val Cys Val Ala Leu Tyr Asp Met Asp Gin lie 85 90 95 Asp Glu Gly lie Cys Met Glu lie Gly Met Phe Val Ala Leu His Lys 100 105 110 Pro lie Val Leu Leu Pro Phe Thr Lys Lys Asp Lys Ser Ala Tyr Glu 115 120 125 Ala Asn Leu Met Leu Ala Arg Gly Val Thr Thr Trp Leu Glu Pro Asn 130 135 140 Asp Phe Ser Pro Leu Lys Asp Phe Asn Phe Asn His Pro Met Ala Gin 145 150 155 160 Pro Phe Pro Pro Phe Lys Val Phe 165 <210> 7 <211> 255 <212> DNA <213> Lactobacillus crispatus NTD <220> <221> CDS <222> (3)..(254) 6 <400> 7 ac aac cag tac aag ggt ate cgc gtt gat gaa cac cct gaa tac ttg 47 Asn Gin Tyr Lys Gly lie Arg Val Asp Glu His Pro Glu Tyr Leu 1 5 10 15 cac gac att gaa tgg gca tea get acc tac cat aac gac tta gta ggg 95 His Asp lie Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly 20 25 30 att aag tcc age gac ate atg ctt ggc gtt tac ttg cct gaa gaa gaa 143 lie Lys Ser Ser Asp lie Met Leu Gly Val Tyr Leu Pro Glu Glu Glu 35 40 45 gat gtt ggt ctg gga atg gaa ctt ggc tat gcc ctt tea aaa ggc aag 191 Asp Val Gly Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser Lys Gly Lys 50 55 60 tac ate ttg ttg gta att cct gat gaa gat tac ggt aag cca ate aac 239 Tyr lie Leu Leu Val lie Pro Asp Glu Asp Tyr Gly Lys Pro lie Asn 65 70 75 tta atg age tgg ggc a 255 Leu Met Ser Trp Gly 80 <210> 8 <211> 84 <212> PRT <213> Lactobacillus crispatus NTD <400> 8 Asn Gin Tyr Lys Gly lie Arg Val Asp Glu His Pro Glu Tyr Leu His 15 10 15 Asp lie Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly lie 20 25 30 Lys Ser Ser Asp lie Met Leu Gly Val Tyr Leu Pro Glu Glu Glu Asp 35 40 45 Val Gly Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser Lys Gly Lys Tyr 50 55 60 lie Leu Leu Val lie Pro Asp Glu Asp Tyr Gly Lys Pro lie Asn Leu 65 70 75 80 Met Ser Trp Gly <210> 9 <211> 399 <212> DNA <213> Lactobacillus amylovorus NTD <220> <221> CDS <222> (1)..(399) 7 <400> 9 atg gaa get tta aag aag aac cct act gtt gac tta gaa aac agt tac 48 Met Glu Ala Leu Lys Lys Asn Pro Thr Val Asp Leu Glu Asn Ser Tyr 1 5 10 15 gtc cca ctt gat aac caa tac aaa ggc ate cgc gtt gat gaa cac cca 96 Val Pro Leu Asp Asn Gin Tyr Lys Gly lie Arg Val Asp Glu His Pro 20 25 30 gaa tat tta cac gac att gaa tgg gca tea tct acc tac cac aat gac 144 Glu Tyr Leu His Asp lie Glu Trp Ala Ser Ser Thr Tyr His Asn Asp 35 40 45 tta gtt ggt att aag tct tea gac gta atg ctc ggt gtt tat tta cct 192 Leu Val Gly He Lys Ser Ser Asp Val Met Leu Gly Val Tyr Leu Pro 50 55 60 gaa gaa gaa gat gtt ggc ctt ggg atg gaa ctt ggc tac gca ttg tct 240 Glu Glu Glu Asp Val Gly Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser 65 70 75 80 caa ggt aaa tac ate ttg ctt gtc ate cct gac gaa gac tat ggt aag 288 Gin Gly Lys Tyr lie Leu Leu Val lie Pro Asp Glu Asp Tyr Gly Lys 85 90 95 cca ate aac ttg atg age tgg ggc gtt tgc gac aac gta ate aag ate 336 Pro lie Asn Leu Met Ser Trp Gly Val Cys Asp Asn Val lie Lys lie 100 105 110 age gaa ttg aaa gac ttc gac ttt aac aga cct cgc ttc aac ttt tac 384 Ser Glu Leu Lys Asp Phe Asp Phe Asn Arg Pro Arg Phe Asn Phe Tyr 115 120 125 gat ggt get gtc tat 399 Asp Gly Ala Val Tyr 130 <210> 10 <211> 133 <212> PRT <213> Lactobacillus amylovorus NTD <400> 10 Met Glu Ala Leu Lys Lys Asn Pro Thr Val Asp Leu Glu Asn Ser Tyr 15 10 15 Val Pro Leu Asp Asn Gin Tyr Lys Gly lie Arg Val Asp Glu His Pro 20 25 30 Glu Tyr Leu His Asp lie Glu Trp Ala Ser Ser Thr Tyr His Asn Asp 35 40 45 Leu Val Gly lie Lys Ser Ser Asp Val Met Leu Gly Val Tyr Leu Pro 50 55 60 Glu Glu Glu Asp Val Gly Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser 65 70 75 80 Gin Gly Lys Tyr lie Leu Leu Val lie Pro Asp Glu Asp Tyr Gly Lys 85 90 95 8 Pro lie Asn Leu Met Ser Trp Gly Val Cys Asp Asn Val lie Lys lie 100 105 110 Ser Glu Leu Lys Asp Phe Asp Phe Asn Arg Pro Arg Phe Asn Phe Tyr 115 120 125 Asp Gly Ala Val Tyr 130 <210> 11 <211> 480 <212> DNA <213> Lactobacillus acidophilus NTD <220> <221> CDS <222> (1)..(480) <400> 11 atg atg gca aaa aca aaa act tta tat ttc ggc get ggt tgg ttt aat 48 Met Met Ala Lys Thr Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn 15 10 15 gaa aag caa aat aag get tat aaa gca get atg gaa get tta aaa caa 96 Glu Lys Gin Asn Lys Ala Tyr Lys Ala Ala Met Glu Ala Leu Lys Gin 20 25 30 aac cct act gtt gat ttg gaa aat agt tat gtt cca ctt gaa aat caa 144 Asn Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gin 35 40 45 tat aaa gat att cgt gtt gat gaa cat cct gaa tac tta cac gac att 192 Tyr Lys Asp lie Arg Val Asp Glu His Pro Glu Tyr Leu His Asp lie 50 55 60 gaa tgg gca tct get act tat cac aac gac tta att ggt ate aaa tct 240 Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu lie Gly lie Lys Ser 65 70 75 80 tea gat att atg tta ggg gtt tac tta cct gaa gaa gaa gat gtt ggt 288 Ser Asp lie Met Leu Gly Val Tyr Leu Pro Glu Glu Glu Asp Val Gly 85 90 95 ctt ggt atg gaa ctt ggc tac gca tta tea caa ggc aaa tat ate tta 336 Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser Gin Gly Lys Tyr lie Leu 100 105 110 cte gtt att cct gac gaa gat tat ggc aag cct ate aac ttg atg agt 384 Leu Val lie Pro Asp Glu Asp Tyr Gly Lys Pro lie Asn Leu Met Ser 115 120 125 tgg ggt gta tgt gat aac get att aag ate age gaa ttg aag gac ttc 432 Trp Gly Val Cys Asp Asn Ala lie Lys lie Ser Glu Leu Lys Asp Phe 130 135 140 gac ttc aat aag cca cgc ttt aac ttc tat gat ggc get gta tat taa 480 Asp Phe Asn Lys Pro Arg Phe Asn Phe Tyr Asp Gly Ala Val Tyr 145 150 155 160 9 <210> 12 <211> 159 <212> PRT <213> Lactobacillus acidophilus NTD <400> 12 Met Met Ala Lys Thr Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn 15 10 15 Glu Lys Gin Asn Lys Ala Tyr Lys Ala Ala Met Glu Ala Leu Lys Gin 20 25 30 Asn Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gin 35 40 45 Tyr Lys Asp lie Arg Val Asp Glu His Pro Glu Tyr Leu His Asp lie 50 55 60 Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu lie Gly lie Lys Ser 65 70 75 80 Ser Asp lie Met Leu Gly Val Tyr Leu Pro Glu Glu Glu Asp Val Gly 85 90 95 Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser Gin Gly Lys Tyr lie Leu 100 105 110 Leu Val lie Pro Asp Glu Asp Tyr Gly Lys Pro lie Asn Leu Met Ser 115 120 125 Trp Gly Val Cys Asp Asn Ala lie Lys lie Ser Glu Leu Lys Asp Phe 130 135 140 Asp Phe Asn Lys Pro Arg Phe Asn Phe Tyr Asp Gly Ala Val Tyr 145 150 155 <210> 13 <211> 795 <212> DNA <213> Lactobacillus helveticus NTD <220> <221> CDS <222> (140)..(616) <220> <223> n signifies any nucleotide : a, g, c or t/u <400> 13 aaaaaaattt tcagtattag tcattgaatt ttaccttcca ttatggaatt actattttta 60 gcgtaagtta acaagacgtt tttttcaatc gaaaatatgt taaagttaat tcgtcagcaa 120 tttttatggg ganaaaatt atg aac aag aaa aag act tta tat ttt ggt gcc 172 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala 15 10 10 ggt tgg ttt aat gaa aag caa aac aaa get tac aaa gaa gca atg gca 220 Gly Trp Phe Asn Glu Lys Gin Asn Lys Ala Tyr Lys Glu Ala Met Ala 15 20 25 get tta aaa gaa aat cca aca gtt gat tta gaa aat agt tat gtg ccc 268 Ala Leu Lys Glu Asn Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro 30 35 40 ctt gaa aac caa tac aag ggt att cgc att gat gaa cat cca gaa tac 316 Leu Glu Asn Gin Tyr Lys Gly lie Arg lie Asp Glu His Pro Glu Tyr 45 50 55 ttg cac aac att gaa tgg get tct gca acc tac cac aat gat tta gta 364 Leu His Asn lie Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val 60 65 70 75 gga att aag act tct gat gtc atg ctt ggc gta tat ttg cca gaa gaa 412 Gly lie Lys Thr Ser Asp Val Met Leu Gly Val Tyr Leu Pro Glu Glu 80 85 90 gaa gac gtc ggc tta ggc atg gaa ctg ggc tac gca tta tct caa gga 460 Glu Asp Val Gly Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser Gin Gly 95 100 105 aaa tat att tta ttg gtt ate cca gat gaa gat tac ggc aag cca ate 508 Lys Tyr lie Leu Leu Val lie Pro Asp Glu Asp Tyr Gly Lys Pro lie 110 115 120 aac tta atg age tgg ggc gtt tgt gac aat gcc ate aag ate agt gaa 556 Asn Leu Met Ser Trp Gly Val Cys Asp Asn Ala lie Lys lie Ser Glu 125 130 135 tta aaa gac ttc gac ttt aac aag cct cgc tac aat ttc tac gac gga 604 Leu Lys Asp Phe Asp Phe Asn Lys Pro Arg Tyr Asn Phe Tyr Asp Gly 140 145 150 155 get gta tat taa aaaataagca aactaaatga cctatcgctt aaaaattgcg 656 Ala Val Tyr ataggtcatt ttttaatatt atctgtcatg tataaaatct ttcttaataa atatactcca 716 agtgattttc caaaaaaatt attattctat acccacttca tatggaagtc cgagtcactt 776 atgtaaatca tatatcact 795 <210> 14 <211> 158 <212> PRT <213> Lactobacillus helveticus NTD <400> 14 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn Glu 15 10 15 Lys Gin Asn Lys Ala Tyr Lys Glu Ala Met Ala Ala Leu Lys Glu Asn 20 25 30 Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gin Tyr 35 40 45 11 5320 Lys Gly lie Arg lie Asp Glu His 50 55 Trp Ala Ser Ala Thr Tyr His Asn 65 70 Asp Val Met Leu Gly Val Tyr Leu 85 Gly Met Glu Leu Gly Tyr Ala Leu 100 Val lie Pro Asp Glu Asp Tyr Gly 115 120 Gly Val Cys Asp Asn Ala lie Lys 130 135 Phe Asn Lys Pro Arg Tyr Asn Phe 145 150 Pro Glu Tyr Leu His Asn lie Glu 60 Asp Leu Val Gly lie Lys Thr Ser 75 80 Pro Glu Glu Glu Asp Val Gly Leu 90 95 Ser Gin Gly Lys Tyr lie Leu Leu 105 110 Lys Pro lie Asn Leu Met Ser Trp 125 lie Ser Glu Leu Lys Asp Phe Asp 140 Tyr Asp Gly Ala Val Tyr 155 <210> 15 <211> 18 <212> DNA <213> Lactobacillus leichmannii NTD1 <400> 15 agacgatcta cttcggtg <210> 16 <211> 18 <212> DNA <213> Lactobacillus leichmannii NTD2 <400> 16 acggcacctt cgtagaag </div>