WO2007010740A1 - Nouvelle endoribonucléase - Google Patents

Nouvelle endoribonucléase Download PDF

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Publication number
WO2007010740A1
WO2007010740A1 PCT/JP2006/313270 JP2006313270W WO2007010740A1 WO 2007010740 A1 WO2007010740 A1 WO 2007010740A1 JP 2006313270 W JP2006313270 W JP 2006313270W WO 2007010740 A1 WO2007010740 A1 WO 2007010740A1
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Prior art keywords
polypeptide
sequence
seq
nucleic acid
amino acid
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PCT/JP2006/313270
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English (en)
Japanese (ja)
Inventor
Masamitsu Shimada
Masanori Takayama
Kiyozo Asada
Ikunoshin Kato
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Takara Bio Inc.
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Priority to JP2007525935A priority Critical patent/JPWO2007010740A1/ja
Publication of WO2007010740A1 publication Critical patent/WO2007010740A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to a novel sequence-specific endoribonuclease useful in the field of genetic engineering.
  • mK is an endoribonuclease that recognizes a specific base of UAH (H is C, A or U) and cleaves mRNA (Patent Document 1, Non-Patent Document 10).
  • RelE and PemK family toxins may be endoribonucleases that cleave mRNA in a base-specific manner.
  • the PemK family of toxins may be endoribonucleases that recognize specific bases and cleave mRNAs independently of ribosomes.
  • Many PemK family toxins are present in prokaryotes, and their sequence comparison has been well studied (Non-patent Documents 1 and 11).
  • Patent Document 1 Pamphlet of International Publication No. 2004Z113498
  • Non-Patent Document 1 Journal 'Ob' Battereriol. (J. Bacteriol.), 182, p5 61-572 (2000)
  • Non-Patent Document 2 Science, No. 301, pl496-1499 (2003)
  • Non-Patent Document 3 Molecular Microbiol., No. 48, pl389-1400 (2003)
  • Non-Patent Document 4 Cell, 122, 131-140 (2003)
  • Non-Patent Document 5 Journal 'Ob' Molequila 'Biology. Mol. Biol.), No. 332, p809-819 (2003)
  • Non-Patent Document 6 Molecular One's Microbiology, No. 51, pl705-1717 (2004)
  • Non-Patent Document 7 Molecular Cell, No. 12, p913-920, 200 3)
  • Non-Patent Document 8 Journal 'Ob' Biological 'Chemistry (J. Biol. Chem.), No. 280, p3143-3150 (2005)
  • Non-Patent Document 9 FEBS Letters, No. 567, p316-320 (2004)
  • Non-Patent Document 10 Journal 'Ob' Biological 'Chemistry, Vol. 279, p20678 -20684 (2004)
  • Non-Patent Document 11 Journal ⁇ Bob ⁇ Molequila 'Microbiology' and Biotechnology (J. Mol. Microbiol. Biotechnol.), No. 1, p295-302 (1999)
  • Non-patent document 12 Genome Biology, IV, R81 (2003)
  • Non-patent document 13 Nucleic Acids Research, Vol. 33, p966-976 (2005)
  • Non-Patent Document 14 Molequila 'Microbiology, No. 56, pi 139— 1148 (2005)
  • Non-Patent Document 15 Method in Enzymology, No. 3 41, p28-41 (2001)
  • An object of the present invention is to provide a novel sequence-specific endoribonuclease in view of the above prior art, and the specificity of the novel sequence-specific endoribonuclease cleavage sequence. Is to provide and use for genetic engineering.
  • the present inventors screened a sequence-specific endoribonuclease, and the polypeptide encoded by each gene of Nostoc s p. A113211, Bacillus subtilis YdcE and Enterococcus faecalis EFO 850 was a novel sequence-specific. Was found to be a typical endoribonuclease. Furthermore, the specificity of the cleavage sequence of the enzyme was identified and the present invention was completed.
  • the present invention provides:
  • nucleic acid encoding a polypeptide capable of being or hybridized under stringent conditions to the nucleic acid of [2] or [3] and having sequence-specific endoribonuclease activity
  • a method for producing a single-stranded RNA degradation product comprising the step of allowing the polypeptide of [1] to act on single-stranded RNA;
  • a method for degrading single-stranded RNA comprising the step of allowing the polypeptide of [1] to act on single-stranded RNA, About.
  • the polypeptide of the present invention is an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3, or an amino acid sequence having at least one deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence. It is characterized by its sequence-specific and sequence-specific endoribonuclease activity.
  • the activity of the polypeptide of the present invention is a single-stranded RNA-specific endoribonuclease activity, and the activity of ribonucleotides in a single-stranded nucleic acid containing ribonucleotides as a constituent base.
  • the phosphodiester bond on the side can be hydrolyzed.
  • the nucleic acid hydrolyzed by the activity has a 3 ′ end having a hydroxyl group and a 5 ′ end having a phosphate group, a 3 ′ end having a phosphate group and a 5 ′ end having a hydroxyl group, or a 2 ′ or 3 ′ site. This produces a 5 'end with click phosphate and hydroxyl groups.
  • the substrate of the polypeptide of the present invention may be a nucleic acid having at least one molecule of ribonucleotide, such as RNA, RNA containing deoxyribonucleotide, DNA containing ribonucleotide, and the like. It is not limited to these.
  • the substrate contains a nucleotide different from that contained in a normal nucleic acid within a range not inhibiting the action of the polypeptide of the present invention, such as deoxyinosine, deoxyuridine, hydroxymethyldeoxyuridine and the like. Well, okay.
  • the polypeptide of the present invention specifically acts on a single-stranded nucleic acid.
  • Double-stranded nucleic acids such as double-stranded RNA, RNA-DNA nobled, etc. cannot be cleaved! /.
  • the polypeptide of the present invention is characterized by having an activity of cleaving a nucleic acid in a specific base sequence.
  • the present invention is not particularly limited, for example, a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having the amino acid sequence shown in SEQ ID NO: 2,
  • the peptide and the polypeptide having the amino acid sequence shown in SEQ ID NO: 3 are located at the 5 ′ end of the sequence when a 5′-UACAU-3 ′ sequence is present in the single-stranded RNA molecule. It hydrolyzes the phosphodiester bond between the residue and the 3 'adjacent A residue.
  • This activity is confirmed as an activity of hydrolyzing a phosphodiester bond between the 14th and 15th bases of the oligoribonucleotide using, for example, mazG30, which is an oligoribonucleotide having the base sequence shown in SEQ ID NO: 13, as a substrate. can do.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 hydrolyzes the phosphodiester bond between the U residue and the A residue for the 5′-UACA-3 ′ sequence in the single-stranded RNA molecule. Can be solved. Since the endoribonuclease activity of the polypeptide of the present invention is expressed without the coexistence of ribosomes, the activity is ribosome-independent.
  • the single-stranded RNA-specific endoribonuclease activity of the polypeptide of the present invention can be measured, for example, using single-stranded RNA as a substrate.
  • a single-stranded RNA transcribed with RNA polymerase in the shape of a DNA or a chemically synthesized single-stranded RNA is allowed to act on a polypeptide whose activity is to be cleaved. It can be measured by examining whether it occurs.
  • RNA degradation can be confirmed, for example, by electrophoresis (agarose gel, acrylamide gel, etc.). If a suitable label (for example, a radioisotope, a fluorescent substance, etc.) is attached to RNA as a substrate, it becomes easy to detect degradation products after electrophoresis.
  • a suitable label for example, a radioisotope, a fluorescent substance, etc.
  • the polypeptide of the present invention has one or more amino acid sequences described in SEQ ID NO: 1, 2 or 3 in the sequence listing as long as it exhibits endoribonuclease activity that hydrolyzes single-stranded RNA in a sequence-specific manner. It includes a polypeptide represented by an amino acid sequence in which at least one of deletion, addition, insertion or substitution of amino acid residues has been made. Examples of the polypeptide having such a mutation include a polypeptide having 50% or more homology to the polypeptide described in SEQ ID NO: 1, 2, or 3, preferably a polypeptide having 70% or more homology, Particularly preferred is a polypeptide having 90% or more homology. Polypeptides having these mutations are included in the present invention even if they recognize and cleave a sequence different from the polypeptide of the amino acid sequence described in SEQ ID NO: 1, 2 or 3.
  • the polypeptide has a peptide region essential for its activity! /,
  • peptides for improving the efficiency of translation for example, peptides for facilitating purification of the polypeptide (for example, histidine tag, dartathione S transferase, maltose binding protein, etc.), chaperones, etc.
  • those added with a tank are included in the polypeptide of the present invention as long as they exhibit RNA-cleaving activity specific to single-stranded RNA.
  • the present invention provides a nucleic acid encoding a polypeptide having sequence-specific endoribonuclease activity.
  • the nucleic acid is not particularly limited, but the amino acid sequence described in SEQ ID NO: 1, 2, or 3 in the sequence listing, or one or more, for example, 1-10 amino acid residues missing from the sequence. Examples thereof include those which are represented by an amino acid sequence having at least one of deletion, addition, insertion or substitution, and which encode a polypeptide having the sequence-specific endoribonuclease activity.
  • amino acid sequence having at least one of deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence described in SEQ ID NO: 1, 2 or 3 for example, SEQ ID NO: 1, 2 or An amino acid sequence having 50% or more homology in the polypeptide described in 3, preferably an amino acid sequence having 70% or more homology, particularly preferably an amino acid sequence having 90% or more homology.
  • the nucleic acid of the present invention includes a nucleic acid that can hybridize to the above-mentioned nucleic acid under stringent conditions and encodes a polypeptide having sequence-specific endoribonuclease activity.
  • the above stringent conditions are as follows: 1989, Cold 'Spring' Nova 1 'Laboratory published, edited by J. Sambrook et al., Molecular ⁇ ⁇ ⁇ Cloning:' Laboratory ⁇ ⁇ ⁇ Examples include conditions described in the second edition of the manual (Molecular Cloning: A Laboratory Manual 2nd ed.). Specifically, for example, conditions of incubation with a probe at 65 ° C.
  • nucleic acid hybridized to the probe can be detected after removing non-specifically bound probe by washing at 37 ° C. in 0.1 ⁇ SSC containing 0.5% SDS, for example.
  • the nucleic acid encoding the polypeptide of the present invention can be obtained, for example, by the following means.
  • a gene having homology in the amino acid sequence to a toxin such as MazF or PemK that has endoribonuclease activity that recognizes a specific base sequence and cleaves mRNA is a polypeptide having sequence-specific ribonuclease activity.
  • Candidate nucleic acids to encode. Such candidate genes can be found, for example, from the bacterial genome.
  • Bacillus subtilis has one PemK family toxin, Nostoc sp. Has four PemK family toxins, and Enterococcus faecalis has three PemK family toxins.
  • Candidate genes can also be isolated from bacterial genomic strength by PCR using primers designed based on nucleotide sequence information, for example. If the entire base sequence is known, the entire sequence of the candidate gene can be synthesized using a DNA synthesizer.
  • Protein expression with candidate gene ability can be carried out in an appropriate host transformed with an expression vector incorporating the candidate gene, for example, E. coli.
  • Expression of sequence-specific ribonucleases that degrade host RNA may be lethal to the host, and the expression of candidate genes must be strictly suppressed until induction.
  • an expression system such as a pET system (manufactured by Novagen) using a T7 polymerase promoter or a cold shock expression control system pCold system (manufactured by Takara Bio Inc.).
  • a peptide such as the histidine tag
  • an expression vector containing such a peptide coding region may be used.
  • the measurement of endoribonuclease activity can be carried out by the above-described method using single-stranded RNA as a substrate.
  • the cleavage site can be identified by a primer extension using a cleaved RNA as a saddle and a primer complementary to the RNA and reverse transcriptase. Since the extension reaction stops at the cleavage site in the primer extension, the cleavage site can be identified by determining the length of the extended strand by electrophoresis. Furthermore, in order to precisely identify the nucleotide sequence specificity, an oligoribonucleosome having an arbitrary sequence is used. After chemically synthesizing the tide and allowing the expression product of the candidate gene to act, the presence or absence of cleavage may be determined by denaturing acrylamide gel electrophoresis or the like.
  • the polypeptide of the present invention is, for example, (1) purified from a culture of a microorganism producing the polypeptide of the present invention, or (2) capable of culturing a transformant containing a nucleic acid encoding the polypeptide of the present invention. It can be produced by a method such as purification.
  • the microorganism that produces the polypeptide of the present invention is not particularly limited, and examples include bacteria belonging to the genera Bacillus, Nostoc and Enterococcus.
  • the polypeptide of the present invention can be obtained from Bacillus suDtilis, Nostoc sp., Enterococcus faecaiis, particularly preferably Baku subtilis 168 strain, Nostoc sp. PCC7120 strain, E. faecalis V583 strain.
  • the culture of the microorganism should be performed under conditions suitable for the growth of the microorganism.
  • the target polypeptide produced in the bacterial cells or the culture solution can be obtained by methods commonly used for protein purification, such as disruption of bacterial cells, fractionation by precipitation (such as ammonium sulfate salting-out), and various chromatography (ion It can be purified by exchange chromatography, affinity chromatography, hydrophobic chromatography, molecular sieve chromatography, etc.) or a combination thereof.
  • the polypeptide of the present invention can be obtained from the transformant transformed with the recombinant DNA containing the nucleic acid encoding the polypeptide of the present invention.
  • the recombinant DNA is preferably provided with a suitable promoter functionally connected upstream of the nucleic acid encoding the polypeptide of the present invention. Since the polypeptide of the present invention may have a lethal action on the host, the expression system including the promoter and promoter described above strictly transcribes the nucleic acid that encodes the polypeptide of the present invention. It is preferably one that can be closely controlled. Examples of such a system include the pET system and the pCold system.
  • the above recombinant DNA may be introduced as it is into a host cell, and may be introduced by being inserted into an appropriate vector such as a plasmid vector, a phage vector, or a virus vector. Furthermore, the above recombinant DNA may be integrated into the host chromosome.
  • the host to be transformed is not particularly limited, for example, E. coli, Bacillus subtilis, yeast, Examples include hosts that are commonly used in the field of recombinant DNA, such as filamentous fungi, plants, animals, plant culture cells, and animal culture cells.
  • the polypeptide of the present invention produced by these transformants can be purified using the purification method as described above.
  • the nucleic acid encoding the polypeptide of the present invention encodes a polypeptide to which a peptide for facilitating the purification of the polypeptide is added, purification becomes very easy.
  • a purification method according to the added peptide for example, using a metal chelate resin for histidine-tag and a dartathione-fixed resin for daltathione S-transferase, respectively, A pure polypeptide can be obtained by a simple operation.
  • RNA degradation product By using the polypeptide of the present invention, single-stranded RNA can be degraded to produce an RNA degradation product. Since the polypeptide of the present invention can cleave RNA in a base sequence-specific manner, the average chain length of the generated RNA degradation product correlates with the appearance frequency of the base sequence recognized by the polypeptide. That is, the present invention provides an RNA degradation product having a certain chain length distribution. Furthermore, it is possible to excise a specific region in RNA using its sequence specificity.
  • single-stranded RNA can be selectively degraded by the polypeptide of the present invention.
  • protein synthesis systems for example, cell-free translation systems and mRNAs in transformants can be degraded with the polypeptide of the present invention to inhibit protein synthesis.
  • the mRNA encoding the desired protein which is artificially prepared so as not to contain the base sequence recognized by the polypeptide of the present invention, is allowed to exist in the above system, so that only the mRNA is degraded.
  • the desired protein is specifically produced in the system.
  • This embodiment is particularly useful for producing highly pure protein.
  • the polypeptide of the present invention has the ability to cleave single-stranded RNA. It does not cleave double-stranded RNA or RNA-DNA hybrids! This property can be used to analyze the secondary structure of RNA.
  • tRNA forms a secondary structure called the cloverleaf model, and the three loop structures are single-stranded.
  • a sequence that can be cleaved by the polypeptide of the present invention present in tRNA is cleaved by the polypeptide of the present invention only when the sequence is located in the loop region. Utilizing this fact, tRNA identification and structural analysis using the polypeptide of the present invention can be carried out.
  • Example 1 Isolation of Bdc subtilis 168 strain YdcE, Nostoc sp. PCC7120 strain All 3211, E. faecalis V583 strain EF0850 isolation and expression plasmid construction
  • amino acid sequence and nucleotide sequence of the polypeptide obtained from ydcE from Bacillus subtilis 168, all3211 from Nostoc sp. PCC7120, and EF0850 from Enterococcus faecalis V583 were obtained from the NCBI database. (Accession No. NP—388347 and NC—000964, NP—487251, and NC—003272, NP—814592 and NC—004668).
  • primer ydcE—F SEQ ID NO: 7
  • primer ydcE —R SEQ ID NO: 7
  • Genomic DNA was prepared from B. subtilis 168 strain and Nostoc sp. PCC7120 strain by the following method.
  • the cells were suspended in 0.5 ml of TE buffer containing 50 U of mutanolysin (manufactured by Nacalai Testa) and 0.1 mg of lysozyme (manufactured by Sigma), incubated at 37 ° C for 2 hours, and then 10% SDS50; zl, 10mg / ml proteinase K5 1 Incubated for an additional hour. Next, phenol extraction and black mouth form extraction were performed, followed by ethanol precipitation to obtain genomic DNA. Genomic DNA was dissolved in TE buffer. E. faecalis V583 strain genomic DNA was obtained from ATCC (ATCC No. 700 802D).
  • PCR using Pyrobest DNA polymerase was performed using 50 ng of B. subtilis 168 genomic DNA, primers ydcE-F and ydcE-R, and an amplified DNA fragment of 371 bp was obtained.
  • This amplified fragment was digested with restriction enzymes Ndel and Xhol and subjected to agarose electrophoresis, and a 350 bp DNA fragment was recovered from the gel after electrophoresis.
  • a 461 bp amplified fragment and a 440 bp restriction enzyme digested DNA fragment were obtained using Nostoc sp.
  • a 398 bp amplified fragment and a 377 bp restriction enzyme digested DNA fragment were obtained using E. faecalis V583 strain genomic DNA and primers EF08 50-F and EF0850-R.
  • Plasmids were prepared from the thus obtained transformant colonies, their nucleotide sequences were confirmed, and these were designated expression vectors pET-ydcE, pET-all3211 and pET-EF0850, respectively.
  • the base sequence encoding the YdcE polypeptide derived from the B. subtilis 168 strain inserted into the expression vector pET-ydcE is shown in SEQ ID NO: 5, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 2, respectively.
  • the nucleotide sequence encoding the A113211 polypeptide derived from Nostoc sp. PC C7120 strain inserted into the expression vector pET-all3211 is shown in SEQ ID NO: 4, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, respectively.
  • the polypeptide having the amino acid sequence of SEQ ID NO: 1, 2 or 3 also has an 8 amino acid residue strength including 6 residues of histidine.
  • a polypeptide with a single histidine tag is expressed Is done.
  • Example 2 Preparation of YdcE from B. subtilis 168 strain, All 3211 polypeptide from Nostoc sp. PCC7120 strain, EF0850 polypeptide from E. faecalis V583 strain
  • Expression vectors pET-ydcE and pET obtained in Example 1 — E. coli BL21 (DE3) strain (Novagen) transformed with all3211 or pET—EF0 85 for expression E. coli pET—ydcEZBL21 (DE3), pET—all321 lZBL21 (DE3) Pleasure—ET0 Got.
  • oligoribonucleotides were synthesized and subjected to cleavage assay.
  • oligoribonucleotides whose base sequences were shown in SEQ ID NOs: 13 to 21 were synthesized as substrates.
  • the reaction product was subjected to 20% denaturing acrylamide gel (20% acrylamide, 7M urea, 0.5 XTBE buffer) electrophoresis, stained with SYBR GREE N II (manufactured by Takara Bio Co., Ltd.), and then fluorescence image analyzer FMBIOII Multi view (Takara Bio). Fluorescence images were analyzed using a Table 1 shows the state of cleavage of each oligoribonucleotide.
  • Cutting indication +++ indicates complete cutting, ++ indicates partial cutting, + indicates very weak cutting.
  • Cut site The cut site is cleaved between -1 and eleven.
  • cleavage was performed using tRNA synthesized by in vitro transcription as a quality.
  • tRNA40 derived from Deinococcus radiodurans having an anticodon (UAC) corresponding to the amino acid parin was obtained from The Genomic tRNA Database (http: ZZlowelab. Ucsc. EduZGtRNAdbZ).
  • tRNA40 DNA sequence with T7 promoter sequence to synthesize tRNA by in vitro transcription The DNA shown in SEQ ID NO: 22 having a sequence and its complementary sequence DNA were synthesized with a DNA synthesizer to prepare a 92 bp double-stranded DNA fragment. Using this DNA fragment and in vitro transcription kit (manufactured by Takara Bio Inc.), in vitro transcription was performed according to the instruction manual of the kit.
  • the resulting reaction solution was treated with DNasel (manufactured by Takara Bio Inc.) at a final concentration of 0.5UZw 1 for 30 minutes at 37 ° C, and then treated with phenol Z chloroform, chloroform, isopropanol precipitation, and RNA.
  • the RNA precipitate was dissolved in sterile distilled water.
  • This tRNA40 contains 10 X Annealing Buffer (lOOmM Tris—HCl, pH 8.0, 500 mM CH COOK, lOmM EDTA) included in the kit in an lZlO capacity.
  • A113211 showed that 75-base tRNA40 was degraded into 33-base and 42-base, and cleavage occurred at the U / AC A site in the anticodon loop of tRNA40. From this result, it was shown that A113211 polypeptide has an activity capable of cleaving tRNA containing anticodon UAC. Since an A residue continues on the 3 ′ side of the site in tRNA40, it was speculated that the site could not be cleaved with YdcE and EF0850 polypeptides.
  • the present invention provides a novel sequence-specific endoribonuclease. Since the enzyme can recognize and cleave specific sequences in RNA, it can analyze RNA molecules.
  • RNA fragments it is useful for preparation of RNA fragments, cell control through RNA cleavage in cells (for example, inhibition of protein production), and the like.
  • SEQ ID NO: 7 PCR primer ydcE— F to amplify a DNA fragment encoding YdcE prote in.
  • SEQ ID NO: 8 PCR primer ydcE-R to amplify a DNA fragment encoding YdcE prote in.
  • PCR primer EF0850 F to amplify a DNA fragment encoding EF0850 protein
  • SEQ ID NO: 22 DNA to transcribe Deinococcus radiodurans tRNA 40.
  • RNA molecule partially equal to RNA sequence of Deinococcus radi odurans tRNA 40.

Abstract

L’invention concerne un polypeptide ayant une activité endoribonucléase nouvelle ; un acide nucléique codant le polypeptide ; un ADN recombiné contenant l’acide nucléique ; un transformant transformé avec l’ADN recombiné ; un procédé de production du polypeptide comprenant les étapes de culture du transformant et de collecte du polypeptide à partir de la culture ; un procédé de production d’un produit de digestion d’ARN simple brin comprenant l’étape de réaction du polypeptide avec l’ARN simple brin et un procédé de digestion d’un ARN simple brin.
PCT/JP2006/313270 2005-07-21 2006-07-04 Nouvelle endoribonucléase WO2007010740A1 (fr)

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Cited By (2)

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WO2008133137A1 (fr) 2007-04-20 2008-11-06 Takara Bio Inc. Vecteur pour une thérapie génique
JP2014161284A (ja) * 2013-02-26 2014-09-08 Kao Corp 変異微生物及びそれを用いた有用物質の生産方法

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WO2005074986A2 (fr) * 2004-02-10 2005-08-18 Genobiotix Aps Especes bioactive capables d'interferer avec un complexe toxine-antitoxine microbien et procedes d'evaluation et d'utilisation de ladite espece bioactive

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Cited By (2)

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WO2008133137A1 (fr) 2007-04-20 2008-11-06 Takara Bio Inc. Vecteur pour une thérapie génique
JP2014161284A (ja) * 2013-02-26 2014-09-08 Kao Corp 変異微生物及びそれを用いた有用物質の生産方法

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