GENETIC TOOL FOR PROCESSING BACTERIA CLOSTRIDIUM

专利摘要:
The present invention relates to a genetic tool comprising at least two distinct nucleic acids allowing transformation by homologous recombination of a bacterium of the genus Clostridium, typically of a solventogenic bacterium.
公开号:FR3042506A1
申请号:FR1559846
申请日:2015-10-16
公开日:2017-04-21
发明作者:François WASELS；Ferreira Nicolas Lopes；Florent Collas；Contreras Ana Maria Lopez
申请人:IFP Energies Nouvelles IFPEN；
IPC主号:

专利说明:

Les résultats de transformation obtenus indiquent que Cas9 est fonctionnel. En effet, lorsque la nucléase est exprimée et que le gène upp est ciblé par l'ARNg, aucun transformant n'est obtenu, en raison de la coupure provoquée dans l'ADN génomique et de l'incapacité de la bactérie à réaliser la réparation du génome (transformation avec p EC5 OOE-mimPthl-ca.vP et pEC750C-gRNA_w/ p) en l'absence de matrice de réparation.
Ciblage de upp
Les résultats obtenus lorsque les vecteurs de ciblage sont introduits dans la souche contenant pEC500E montrent que le génome de la souche ATCC824 ne contient pas d’homologue de cas9. puisque des transformants sont obtenus avec chaque vecteur de ciblage.
Un transformant contenant pEC500E-miniPthl-cav9 et pEC750C-gRNA_w/ / -upp_stop a ensuite été repiqué à plusieurs reprises sur un milieu non sélectif afin qu’il perde les plasmides qu’il contenait. Une fois les colonies curées de leurs plasmides et sensibles aux antibiotiques, le gène upp (SEQ ID NO : 3) a été séquencé (cf. figure 7).
Les modifications désirées sont bien présentes. L’outil génétique CRISPR-Cas9 comprenant l’introduction de deux plasmides et l’expression du gène cas9 par un promoteur fort et constitutif est donc bien fonctionnel.
Ciblage de adhE
Des transformants sont obtenus lors des transformations de la souche sauvage avec les plasmides pEC5 OOE-miniPthl-cas9 et pEC750C_gRNA adhE. Puisque cas9 est actif, ce résultat indique qu’une coupure dans le mégaplasmide pSOL n’affecte pas la viabilité d’ATCC824.
Afin de confirmer l’éventuelle perte de pSOL, différents tests ont été réalisés : - Détection par PCR d’un gène présent sur le pSOL : ctfli
La PCR catP fwd x catPrev permet de détecter le gène de résistance au thiamphénicol, présent sur les plasmides pEC750C. Sa détection confirme que les vecteurs de ciblage sont présents.
La PCR RH_ctfB_R x V-CTFA-CAC2707 R permet de détecter une partie du gène ctfB, présent sur le mégaplasmide pSOL, et permet de savoir si ce dernier est présent dans la cellule. L’amplification semble montrer que le mégaplasmide pSOL n’est plus présent chez les clones transformés avec pEC500E-miniPthl-ca,v9 et pEC750C-gRNA_ai//zL' (cf. figure 8).
- Détection d’une activité enzymatique codée par un gène présent sur le pSOL
Parmi les gènes contenus sur le mégaplasmide pSOL, amyP code une enzyme extracellulaire à activité α-amylase. Cette activité peut être détectée sur un milieu solide contenant de l’amidon et du glucose (Sabathé et al., 2002). Des dilutions de cultures liquides ont été déposées sur une boîte de gélose contenant 0,2% de glucose et 2% d’amidon et incubées 72h à 37°C. L’activité α-amylase est ensuite révélée par une coloration à l’iode. Les halos clairs autours des spots de bactéries indiquent la présence d’une activité α-amylase. L’absence d’activité autour de ATCC824 contenant pEC500E-miniPthl-ca.vO et pEC750C-gRNA_o<7M indique que le gène amyP n’est pas exprimé dans cette souche, confirmant que le mégaplasmide n’est plus présent (cf. Figure 9). - Bilan fermentaire
La souche sauvage ATCC824 ainsi qu’un transformant ont été cultivés pendant 24h dans un milieu GAPES afin d’établir les bilans fermentaires des deux souches. Le bilan fermentaire obtenu montre une diminution de la production d’éthanol ainsi qu’une abolition de la production de butanol et d’acétate en raison de l’absence des gènes adhE, adhEl et adc (présents sur le mégaplasmide pSOL) chez le transformant (cf. Figure 10).
Cas9 est donc capable d’agir sur le chromosome ou sur le plasmide naturel de la souche ATCC824 ce qui permet d’élargir son action au chromosome et à tout matériel génétique extra-chromosomique présent dans la souche (plasmide bactériophage, etc.).
Expression inductible de cas9 dans C. acetobutylicum ATCC824
Afin de permettre les événements de recombinaison homologue entre le génome et les matrices de réparation, il faut augmenter le nombre de cellules dans lesquelles la nucléase est active (jusqu’à 103 lorsque la souche ATCC824 contenant pEC500E-miniPthl-6'a,v9 est transformée avec un vecteur de ciblage). Dans ce but, un système dans lequel l’expression de la nucléase est contrôlée est nécessaire. Deux vecteurs dans lesquels le gène cas9 est placé sous le contrôle d’un promoteur inductible à l’anhydrotétracycline ont été construits, dérivés du vecteur pFWOOOl : • pFWOOOl-Pcm-2tet01-cas9 (cf. Figure 11A + SEQ ID NO:6); • pFWOOOl-Pcm-tet02/l-cas9 (cf. Figure 11B + SEQ ID NO:7);
Les promoteurs contrôlant l’expression de cas9 contiennent des séquences opératrices, tetOl ettet02, sur lesquelles se fixe le répresseur transcriptionnel TetR. Cette répression est levée par la présence d’anhydrotétracycline (aTc). Ce système permet une expression contrôlée, avec peu de fuites. En présence d’aTc, la synthèse est plus importante à partir du promoteur Pcm-2tet01 (cf. Figure 12).
Transformation de C. acetobutylicum ATCC824 :
Les vecteurs d’expression ainsi que le vecteur vide pFWOOO 1 ont été introduits dans ATCC824. Par la suite, les plasmides suivants ont été introduits dans chaque type de transformant (cf. tableau 2) : • pEC750C-gRNA_w/ />, contenant l’ARNg ciblant upp ; • pEC750C-gRNA_w/ / -upp_del, contenant l’ARNg ciblant upp et la matrice upp del ; • pEC750C-gRNA_w/ />-upp_stop, contenant l’ARNg ciblant upp et la matrice upp stop ;
Les colonies transformées ont été étalées sur différents milieux solides, à des dilutions différentes : • 2YTG + érythromycine pour vérifier la viabilité cellulaire, dilution d’un facteur 106 ; • 2YTG + érythromycine + thiamphénicol pour sélectionner les transformants ; • 2YTG + érythromycine + thiamphénicol + aTc (200ng/mL) pour sélectionner les transformants en présence de l’inducteur.
Tableau 2 : nombre de colonies obtenues sur chaque type de milieu pour chaque transformation, ery, érythromycine, thiam, thiamphénicol. aTc, anhydrotétracycline. Entre parenthèses, facteurs de dilutions. ND, non dilué. -, non testé.
On observe un effet toxique de l’aTc, puisque peu de transformants sont obtenus lorsqu'il est présent, même lorsque le vecteur de ciblage vide (pEC750C, contrôle) est utilisé. Conformément à ce qui est attendu, aucun transformant n’est obtenu lorsqu’un pEC750C contenant la cassette gRNAjipp est introduite dans une cellule exprimant cas9, sur un milieu contenant de l’aTc. En revanche, de nombreux transformants sont obtenus pour chaque combinaison de plasmides sur milieu sans aTc, indiquant que cas9 n’est pas exprimée.
Expression de cas9 en présence d’aTc :
Les différents transformants obtenus sur boîtes contenant de l’érythromycine et du thiamphénicol ont été repiqués sur le même type de milieu, puis utilisés pour ensemencer des précultures liquides contenant les deux antibiotiques. Ces précultures ont ensuite été utilisées pour ensemencer d’autres cultures liquides contenant des concentrations variables d’aTc afin de déterminer si le système est fonctionnel.
Induction de cas9 :
Trois transformants ont été utilisés pour analyser la capacité d’induction de l’expression de cas9 en présence d’aTc : ATCC824 contenant pFWOOOl et pEC750C-gRNA_«/j/j ; ATCC824 contenant pFWOOO 1 -Pcm-2tetO 1 -cas9 et pEC750C-gRNA_«/j/j ; ATCC824 contenant pFWOOO 1 -Pcm-tet02/1 -cas9 et pEC750C-gRNA_«/j/j ;
Des milieux liquides contenant de l’érythromycine, du thiamphénicol et des concentrations croissantes d’aTc ont été ensemencés à partir de précultures liquides de ces transformants (cf. figure 13). La capacité de croissance des cellules est évaluée par mesure des densités optiques après 72h de culture. Le transformant n’exprimant pas la nucléase n’est pas ou peu affecté par la présence d’aTc. En revanche, même à des faibles concentrations d’aTc, le transformant contenant le plasmide exprimant cas 9 via le promoteur Pcm-2tet01 (pFWOOO l-Pcm-2tet01-cas9) et le plasmide contenant uniquement le gRNA (pEC750C-gRNA_upp) présentent un important retard de croissance. Le transformant contenant le plasmide exprimant cas9 via le promoteur Pcm-tet02/l (pFWOOO 1 -Pcm-tct02/l -6a.v9) et le plasmide contenant uniquement le gRNA (pEC750C-gRNA_w/ p) n’est pas affecté à de faibles concentrations d’aTc. Cependant, à partir de 150 ng/mL un fort retard de croissance est observée, et aucune croissance n’est observée à 300ng/mL. Le promoteur Pcm-tet02/l semble donc permettre une meilleure répression de l’expression que Pcm-2tet01 en absence d’inducteur. Génération de mutants
Des cultures liquides des transformants contenant les plasmides de ciblage permettant la réparation des cassures double brin ont également été réalisées, en absence ou en présence (lOOng/mL) d’aTc. Les transformants utilisés contenaient l’une des douze combinaisons de plasmides apparaissant dans le tableau 3.
Tableau 3 : Combinaisons de plasmides présents dans les transformants.
Après 72h de culture, des aliquots ont été déposés sur différents milieux solides: • 2YTG contenant du thiamphénicol et de l’érythromycine ; • 2YTG contenant du thiamphénicol, de l’érythromycine, et lOOng/mL d’aTc ; • 2YTG contenant du 5-fluorouracile.
Seuls les transformants dans lesquels des événements de recombinaison homologue ont permis l’insertion de la matrice de réparation sont capables de pousser sur 2YTG + 5-FU (cf. figure 14).
Analyse des transformants upp del :
Les clones isolés sur 2YTG + 5-FU ont été analysés par PCR (cf. figure 15).
La PCR catP fwd x catP rev permet de détecter le gène de résistance au thiamphénicol, présent sur les plasmides pEC750C. Sa détection confirme que les vecteurs de ciblage sont présents.
La PCR LHA upp fwd x RHA upp rev permet d’amplifier le gène upp ainsi que les régions flanquantes. Les amorces apparaissant ci-dessous ont servi à la construction de la matrice de réparation upp_del (cf. figure 15 + SEQ ID NO : 14-21) :
Tableau n°4 :
La PCR upp matrice fwd x upp matrice rev permet d’amplifier un fragment interne du gène upp, absent de la matrice de réparation upp del.
Les résultats obtenus confirment la délétion à l’intérieur du gène upp dans les transformants analysés. Analyse des mutants upp stop :
Le gène upp a été séquencé chez trois clones isolés sur 2YTG+5-FU après exposition à l’aTc (cf. Figure 16) :
• Un contenant le plasmide exprimant cas9 via le promoteur Pcm-2tet01 (pFW0001-Pcm-2tet01- cas9) et le plasmide contenant le gRNA ainsi que la matrice de réparation upp_stop (pEC750CgRNA_w/ / -upp_stop) ; • Deux contenant le plasmide exprimant cas9 via le promoteur Pcm-tet02/l (pFW0001-Pcm-tet02/l- cas9) et le plasmide contenant le gRNA ainsi que la matrice de réparation upp stop (pEC7 5 OCgRNAwpp-uppstop).
La stratégie visant à développer un système de modification génétique par l’utilisation du gène cas9 sous contrôle d’un promoteur inductible et du gRNA présent dans un second plasmide est donc fonctionnelle. Par rapport à l’utilisation du promoteur constitutif miniPthl, l’induction du gène cas9 permet de maîtriser l’action de l’enzyme et de faciliter la sélection de transformants ayant subis les modifications souhaitées.
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GENETIC TOOL FOR PROCESSING BACTERIA CLOSTRIDIUM
The present invention relates to a genetic tool comprising at least two distinct nucleic acids allowing transformation by homologous recombination of a bacterium of the genus Clostridium, typically of a solventogenic bacterium of the genus Clostridium.
BACKGROUND
Bacteria belonging to the genus Clostridium are strict anaerobic bacilli with gram positive staining, capable of forming endospores and belonging to the phylum Firmicutes. This genus contains many species studied because of their pathogenicity or their industrial and medical interest. Clostridium tetani, Clostridium botulinum, Clostridium perfringens and Clostridium difficile are the agents responsible for tetanus, botulism, gas gangrene and pseudomembranous colitis respectively. At the same time, other species such as Clostridium acetobutylicum, Clostridium butyricum and Clostridium beijerinckii, which are non-pathogenic for humans, are used in fermentation. Finally, Clostridium novyi and Clostridium sporogenes have recently been used in studies aimed at the development of anti-cancer therapies. The so-called Clostridium species of industrial interest are capable of producing solvents from a wide variety of sugars and substrates ranging from glucose to cellulose. The growth of solvent-producing Clostridium bacteria is called biphasic. Acids (acetic and butyric) are produced during the exponential phase of growth. Then, when the cell growth stops and the bacteria enter the stationary phase, they produce solvents.
The majority of the solventogenic strains of Clostridium produce acetone, butanol and ethanol as end products. These strains are referred to as "ABE strains". This is for example the case of strains ATCC824 of C. acetobutylicum and NCIMB 8052 of C. beijerinckii. Other strains are also capable of reducing acetone to isopropanol, and are referred to as IBE strains. This is the case, for example, of the C. beijerinckii strain DSM 6423 (NRRL B593) which has in its genome an adh gene coding for a primary / secondary alcohol-dehydrogenase which allows the reduction of acetone to isopropanol.
The production of isopropanol by C. acetobutylicum ATCC824 is possible after introduction of a plasmid allowing the expression of the adh gene of C. beijerinckii DSM 6423. Such a genetically modified strain has performances equivalent to the DSM 6423 strain. be improved after overexpression within an operon structure of the ct / A, clfB and adc genes respectively encoding the CoA-transferases involved in the re-assimilation of acids and the acetoacetate decarboxylase (Collas et al, 2012). The introduction of a plasmid containing these genes alters the fermentative profile of the ATCC824 strain to produce an IBE mixture. However, the maintenance of this genetic construct requires the presence of antibiotic in the growth medium, making it impossible to use this strain for industrial applications.
Despite the undeniable interest of bacteria of the genus Clostridium, their metabolism is still little studied and / or modified because of the difficulties in obtaining genetically modified strains. The most efficient systems are based on homologous recombination events allowing the precise modification of the genome in a stable manner. The frequencies of homologous recombination observed in Clostridium being, however, very low, selection markers (eg an antibiotic resistance gene) and counter-selection markers (eg a gene encoding a toxin) have proved necessary. Two tools have recently been developed (Al Hinai et al., 2012; Cartman et al., 2012), each based on the use of a single plasmid. Although innovative, these systems have disadvantages. The first system has the defect of leaving at the site of modification a FRT cassette (used during the excision of the selection marker) which can alter the genetic context of the mutant, and preventing the reuse of the tool to make many modifications . These defects are well known to those skilled in the art. The second system, requiring two sequential homologous recombination events, can not be used to modify essential regions of the genome. A tool involving the sequential use of two plasmids, one of them encoding I-SceI meganuclease capable of causing double-strand DNA cleavage at specific target sites and promoting homologous recombination events, was then developed (Zhang et al., 2015). Here again, the modification of certain essential genes is made impossible by the need to perform two sequential homologous recombination events. The latest generation of tools developed to date (Wang et al., 2015; Xu et al., 2015) uses CRISPR (Clustered Regularly Interspaced Short Palindrome Repeats) that works in a manner analogous to RNA interference. observed in eukaryotes (Barrangou et al, 2007). The tools described by Wang et al. and Xu et al., respectively adapted to C. beijerinckii and C. cellulolyticum, rely on the use of a single plasmid. Xu et al. use a modified version of the Cas9 enzyme creating single-strand breaks instead of double-strand breaks. The two available tools based on CRISPR technology have the major disadvantage of significantly limiting the size of the nucleic acid of interest (and therefore the number of coding sequences or genes) that can be inserted into the bacterial genome (1, About 8 kb at best according to Xu et al.).
SUMMARY OF THE INVENTION The object of the present invention is to provide a genetic tool adapted to the genus Clostridium as a whole, in particular to the solventogenic bacteria of the genus Clostridium, allowing for the first time to modify the bacterial genome in order to allow the exploitation of Clostridium bacteria on an industrial scale. The invention thus relates to a genetic tool for transformation by homologous recombination of a bacterium of the genus Clostridium, preferably a bacterium of the genus Clostridium, characterized in that it comprises at least two distinct nucleic acids. This tool is for example capable of modifying a region of the genome of the Clostridium bacterium including a sequence essential for bacterial survival, or of allowing the insertion of large fragments of nucleic acid sequences impossible using the existing tools. .
A first subject of the invention thus relates to a genetic tool for the transformation by homologous recombination of a solventogenic bacterium of the genus Clostridium characterized in that it comprises: a first nucleic acid encoding at least Cas9, in which the coding sequence Cas9 is placed under the control of a promoter, and - at least one second nucleic acid containing a repair matrix allowing, by a homologous recombination mechanism, the replacement of a portion of the bacterial DNA targeted by Cas9 with a sequence of interest, and i) at least one of said nucleic acids further codes for one or more guide RNAs (gRNAs), or ii) the genetic tool further comprises one or more guide RNAs, each guide RNA comprising a RNA binding structure to the enzyme Cas9 and a sequence complementary to the targeted portion of the bacterial DNA. The invention also relates to a method for transforming and / or genetically modifying by homologous recombination a bacterium of the genus Clostridium, typically a solventogenic bacterium of the genus Clostridium, characterized in that it comprises a step of introduction into the bacterium of a genetic tool according to the invention. The invention also relates to bacteria of the genus Clostridium thus transformed and / or genetically modified.
The inventors also describe a kit for transforming and / or genetically modifying a bacterium of the Clostridium genus, or for producing at least one solvent, for example a mixture of solvents, using a bacterium of the genus Clostridium comprising the elements of the genus Clostridium. genetic tool according to the invention, and optionally, in particular, one or more inducers adapted to the inducible promoter (s) selected (s) used (s) within the tool.
Also described are the uses of the genetic tool according to the invention, the method for transforming and / or modifying genetically by homologous recombination a bacterium of the genus Clostridium, the bacterium thus transformed and / or genetically modified to allow the production of a solvent or a mixture of solvents on an industrial scale, preferably acetone, butanol, ethanol, isopropanol or a mixture thereof, typically an isopropanol / butanol mixture.
DETAILED DESCRIPTION OF THE INVENTION CRISPR (Clustered Regularly Interspaced Short Palindrome Repeats) designates in bacteria and archaea gene loci having a role of immune defense against phages and plasmids. The CRISPR-Cas9 system is essentially based on the association of a Cas9 protein producing double-strand breaks in the target genome and a guide RNA (gRNA) responsible for the specificity of the cleavage site. This ability to create targeted double-strand breaks in DNA helps to promote the homologous recombination events necessary to introduce mutations into the genome of the strains of interest. Cell viability depends on the integrity of the genome. The bacterium will have to repair any cleavage in its DNA, either by a non-homologous end joining mechanism, or by a homologous recombination mechanism requiring a repair matrix. By providing the cell with such a matrix, it is then possible to modify the region corresponding to the cutoff site (FIG. 2). The ability of CRISPR to perform double-strand breaks in DNA molecules has allowed its recent use as a genetic tool in different organisms, particularly Streptococcus pyogenes, in which it was first characterized, in E. coli, and within eukaryotic cells (Jiang et al 2013, Cong et al 2013, Hwang et al 2013, Hsu et al 2013). It has recently been used in Clostridium beijerinckii and Clostridium cellulolyticum using genetic tools that have allowed only limited modification of the bacterial genome, unusable on an industrial scale (Wang et al., 2015, Xu et al. ., 2015).
A genetic tool for transformation by homologous recombination of a bacterium of the genus Clostridium and comprising at least two distinct nucleic acids is described for the first time in the present text. The inventors have demonstrated that this tool makes it possible to transform and / or genetically modify the solventogenic bacteria of the Clostridium genus, in a manner that is sufficiently effective to render them particularly industrially exploitable, thereby meeting a long-standing need.
A particular genetic tool according to the invention, allowing transformation by homologous recombination of a bacterium of the genus Clostridium, comprises: a first nucleic acid encoding at least Cas9, in which the coding sequence Cas9 is placed under the control of a promoter and at least one second nucleic acid containing a repair matrix, allowing, by a homologous recombination mechanism, the replacement of a portion of the bacterial DNA targeted by Cas9 by a sequence of interest, it being understood that: least one of said nucleic acids further codes for one or more guide RNAs (gRNAs) or ii) the genetic tool further comprises one or more guide RNAs. In this tool, each guide RNA comprises an RNA structure for binding to the Cas9 enzyme and a sequence complementary to the targeted portion of the bacterial DNA.
By "bacterium of the genus Clostridium" is meant in particular the species of Clostridium said to be of industrial interest, typically the solventogenic bacteria of the genus Clostridium. The term "Clostridium bacterium" encompasses wild-type bacteria and genetically-engineered strains thereof with the aim of improving their performance (eg overexpressing the ctfA, ctfB and adc genes) without having been exposed to the system. CRISPR.
By "species of Clostridium of industrial interest" is meant species capable of producing, by fermentation, solvents from sugars such as glucose, xylose, fructose or mannose, polysaccharides such as cellulose or hemicelluloses, acids such as butyric acid or acetic acid, or any other source of assimilable carbon that can be used by bacteria of the Clostridium genus (CO, CO2, and methanol, for example). Examples of the solventogenic bacteria of interest are the bacteria of the Clostridium genus producing acetone, butanol, ethanol and / or isopropanol, such as the strains identified in the literature as "ABE strain" [strains producing fermentations allowing the production of acetone, butanol and ethanol] and "IBE strain" [strains carrying out fermentations allowing the production of isopropanol (by reduction of acetone), butanol and ethanol]. Solventogenic bacteria of the genus Clostridium can be selected from C. acetobutylicum, C. cellulolyticum, C. phytofermentans, C. beijerinckii, C. saccharobutylicum, C. saccharoperbutylacetonicum, C. sporogenes, C. butyricum, C. aurantibutyricum and C. tyrobutyricum, preferably from C. acetobutylicum, C. beijerinckii, C. butyricum and C. tyrobutyricum and C. cellulolyticum and even more preferably from C. acetobutylicum and C. beijerinckii.
In a particular embodiment, the bacterium of the Clostridium genus concerned is an "ABE strain", preferably C. acetobutylicum strain ATCC824 or C. beijerinckii strain NCIMB 8052.
In another particular embodiment, the bacterium of the Clostridium genus in question is an "IBE strain", preferably the strain DSM 6423 (also identified as strain NRRL B593) of C. beijerinckii.
The CRISPR system contains two distinct elements, ie i) an endonuclease, in this case the CRISPR associated nuclease (Cas or "CRISPR associated protein"), Cas9, and ii) a guide RNA. The guide RNA is in the form of a chimeric RNA that consists of the combination of a CRISPR (RNAcr) bacterial RNA and a RNAtracr (RI-CRISPR) (Jinek et al., Science 2012 - see Figure 3). The gRNA combines the targeting specificity of the cRNA corresponding to the "spacing sequences" that serve as guides to the Cas proteins, and the conformational properties of the Rtracr in a single transcript. When gRNA and Cas9 protein are expressed simultaneously in the cell, the target genomic sequence can be permanently modified or interrupted. The modification is advantageously guided by a repair matrix. The genetic tool according to the invention comprises a first nucleic acid encoding at least Cas9. By "Cas9" is meant a protein Cas9 (also called Csn1 or Csx12) or a functional protein fragment thereof, ie capable of interacting with the guide RNA (s) and of exerting the enzymatic activity (nucleases) which allows the double strand cleavage of the DNA of the target genome. "Cas9" can thus denote a modified protein, for example truncated to remove domains of the protein that are not essential to the predefined functions of the protein, in particular the domains that are not necessary for interaction with the gRNA (s).
The sequence encoding Cas9 (the entire protein or a fragment thereof) as used in the context of the invention can be obtained from any known Cas9 protein (Makarova et al., 2011). Examples of Cas9 proteins useful in the present invention include, but are not limited to, Cas9 proteins of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Campylobacter jejuni, Francisella novicida and Neisseria meningitidis. Other Cas9 proteins that can be used in the present invention are also described in the article by Fonfara et al., 2013.
In a particular embodiment, the Cas9 protein encoded by one of the nucleic acids of the genetic tool according to the invention comprises, or consists of, the amino acid sequence SEQ ID NO: 1, or any other sequence of amino acids having at least 50%, preferably at least 60% identity therewith, and containing at least the two aspartic acids ("D") occupying positions 10 ("D10") and 840 (" D840 ") of the amino acid sequence SEQ ID NO: 1.
In a preferred embodiment, Cas9 comprises, or consists of, the Cas9 protein (NCBI accession number: WP_010922251.1, SEQ ID NO: 1), encoded by the cas9 gene of Streptococcus pyogenes strain Ml GAS (Number NCBI: NC 002737.2 SPy_1046, SEQ ID NO: 2) or an optimized version thereof ("optimized version") at the origin of a transcript containing the codons used preferentially by the bacteria of the genus Clostridium, typically the codons rich in adenine ("A") and thymine ("T") bases, allowing a facilitated expression of the Cas9 protein within this bacterial genus. These optimized codons respect the codon usage bias, well known to those skilled in the art, specific to each bacterial strain. In the peptide sequences described in this document, the amino acids are represented by their one-letter code according to the following nomenclature: C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan and Y: tyrosine.
According to a particular embodiment, the Cas9 domain consists of an entire Cas9 protein, preferably the Streptococcus pyogenes protein Cas9 or an optimized version thereof.
The sequence encoding Cas9 present in one of the nucleic acids of the genetic tool according to the invention is placed under the control of a promoter. This promoter can be a constitutive promoter or an inducible promoter. In a preferred embodiment, the promoter controlling expression of Cas9 is an inducible promoter.
Examples of constitutive promoters that may be used in the context of the present invention may be selected from the promoter of the gene th1, the gene ptb, the gene adc, the operon bcs, or a derivative thereof, preferably a functional derivative but shorter (truncated) such as the "miniPthl" derivative of the promoter of the thl gene of C. acetobutylicum (Dong et al., 2012), or any other promoter, well known to those skilled in the art, allowing the expression of a protein within Clostridium.
Examples of inducible promoters that may be used in the context of the present invention may be selected for example from a promoter the expression of which is controlled by the TetR transcriptional repressor, for example the promoter of the A gene (the originally present tetracycline resistance gene). on the transposon Tn10 of E. coli); a promoter whose expression is controlled by L-arabinose, for example the ptk gene promoter (see Zhang J. et al., 2015), preferably in combination with the araR cassette for regulating the expression of C acetobutylicum to construct an ARAi system (see Zhang J. et al., 2015); a promoter whose expression is controlled by laminaribiose (β-1,3 glucose dimer), for example the promoter of the celC gene, preferably immediately followed by the glyR3 repressor gene and the gene of interest (see Meals EB and al. (2015)) or the promoter of this IC gene (see Newcomb M. et al., 2011); a promoter whose expression is controlled by lactose, for example the promoter of the bgaL gene, preferably immediately followed by the AdhEl (aldehyde / alcohol dehydrogenase) gene (see Baneqee et al., 2014); a promoter whose expression is controlled by xylose, for example the xylB gene promoter (see Nariya H et al., 2011); and a promoter whose expression is controlled by UV exposure, for example the ben promoter (see Dupuy et al., 2005).
A promoter derived from one of the promoters described above, preferably a functional derivative but shorter (truncated) may also be advantageously used in the context of the invention. Other inducible promoters that can be used in the present invention are also described, for example, in the articles by Ransom EM et al. (2015), Currie DH et al. (2013) D'Urzo N et al. (2013) and Hartman AH et al. (2011).
A preferred inducible promoter is a tetA-derived, anhydrotetracycline inducible (aTc) -induced promoter (less toxic than tetracycline and capable of raising TetR-lowering transcriptional repressor inhibition), selected from Pcm-2tet01 and Pcm-2tet02 / l (Dong et al., 2012).
The inducible promoters as described in the present invention make it possible to advantageously control the action of the enzyme and to facilitate the selection of transformants having undergone the desired genetic modifications.
The term "guide RNA" or "gRNA" denotes within the meaning of the invention an RNA molecule capable of interacting with "Cas9" in order to guide it towards a target region of the bacterial chromosome. The specificity of cleavage is determined by the gRNA. As previously explained, each gRNA comprises two regions:
a first region (commonly referred to as "SDS" region), at the 5 'end of the gRNA, which is complementary to the target chromosomal region and mimics the endogenous CRISPR CRRR, and - a second region (commonly called the "handle" region), at the 3 'end of the gRNA, which mimics the base-pairing interactions between the trans-activating crRNA (rtracr) and the endogenous CRISPR CRRR and presents a double-strand structure in stem and loop ending in 3 'by an essentially single strand sequence. This second region is essential for gRNA binding to Cas9.
The first region of the gRNA ("SDS" region) varies according to the targeted chromosomal sequence.
The "SDS" region of the gRNA which is complementary to the target chromosomal region, comprises at least 1 nucleotide, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, typically between 1 and 40 nucleotides. Preferably, this region has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
The second region of the gRNA (handle region) has a stem and loop structure (or hairpin structure). The "handle" regions of the different gRNAs do not depend on the chromosomal target chosen.
According to a particular embodiment, the "handle" region comprises, or consists of, a sequence of at least 1 nucleotide, preferably at least 1, 50, 100, 200, 500 and 1000 nucleotides, typically between 1 and 1000 nucleotides. . Preferably, this region is 40 to 120 nucleotides in length.
The total length of a gRNA is generally from 50 to 1000 nucleotides, preferably from 80 to 200 nucleotides, and more preferably from 90 to 120 nucleotides. According to a particular embodiment, a gRNA as used in the present invention has a length of between 95 and 110 nucleotides, for example a length of about 100 or about 110 nucleotides. Those skilled in the art can easily define the sequence and structure of the gRNAs according to the chromosomal region to be targeted using well known techniques (see for example the article by Di Carlo et al., 2013).
The region / portion / DNA sequence targeted within the bacterial chromosome may be a portion of non-coding DNA or a portion of coding DNA. In a particular embodiment, the targeted portion of the bacterial DNA comprises one or more genes or portion (s) of essential gene (s) for bacterial survival or one or more genes or DNA sequences whose inactivation allows the selection of bacteria having integrated the nucleic acid (s) of interest.
Specific examples of targeted DNA portion within a bacterium of the genus Clostridium are the sequences used in the experimental part. These are for example sequences encoding the genes upp (SEQ ID NO: 3) and adhEl (SEQ ID NO: 4).
The targeted region / portion / DNA sequence is followed by a "PAM"("protospacer adjacent motif") sequence that intervenes in the Cas9 binding.
The "SDS" region of a given gRNA is identical (at 100%) or at least 80% identical, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% to the region / portion / DNA sequence targeted within the bacterial chromosome and is capable of hybridizing to all or part of the sequence complementary to said region / portion / sequence, typically to a sequence comprising at least 1 nucleotide, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides, typically between 1 and 40 nucleotides, preferably at a sequence comprising 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29 or 30 nucleotides.
In the process according to the invention, one or more gRNAs can be used simultaneously. These different gRNAs can target identical or different chromosomal regions, preferably different.
The gRNAs can be introduced into the bacterial cell in the form of gRNA molecules (mature or precursors), in the form of precursors or in the form of one or more nucleic acids encoding said gRNAs. The gRNAs are preferably introduced into the bacterial cell in the form of one or more nucleic acids encoding said gRNAs.
When the gRNA (s) are introduced into the cell directly in the form of RNA molecules, these gRNAs (mature or precursor) may contain modified nucleotides or chemical modifications allowing them, for example, to increase their resistance to nucleases and thus to increase their life in the cell. They may in particular comprise at least one modified or non-natural nucleotide such as, for example, a nucleotide comprising a modified base, such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine or diamino. -2,6-purine, bromo-5-deoxyuridine or any other modified base for hybridization. The gRNAs used according to the invention can also be modified at the level of the internucleotide linkage, for example phosphorothioates, H-phosphonates or alkylphosphonates, or at the level of the backbone such as for example the alpha-oligonucleotides, the 2'- 0-alkyl ribose or PNA (Peptid Nucleic Acid) (Egholm et al., 1992).
The gRNAs can be natural, synthetic RNA or produced by recombinant techniques. These gRNAs can be prepared by any method known to those skilled in the art such as, for example, chemical synthesis, in vivo transcription or amplification techniques.
When the gRNAs are introduced into the bacterial cell in the form of one or more nucleic acids, the sequence or sequences coding for the gRNA (s) are placed under the control of an expression promoter. This promoter can be constitutive or inducible.
When multiple gRNAs are used, the expression of each gRNA can be controlled by a different promoter. Preferably, the promoter used is the same for all gRNAs. The same promoter may in a particular embodiment be used to allow the expression of several, for example only a few, gRNAs to be expressed.
In a preferred embodiment, the promoter (s) controlling the expression of the gRNA (s) is / are constitutive promoters.
Examples of constitutive promoters that may be used in the context of the present invention may be selected from the promoter of the thl gene, the ptb gene or the bcs operon, or a derivative thereof, preferably miniPthl, or any other promoter, well known to those skilled in the art, allowing the synthesis of an RNA (coding or non-coding) within Clostridium.
Examples of inducible promoters that may be used in the context of the present invention may be selected from the promoter of the A gene, the xylA gene, the lac1 gene or the bgaL gene, or a derivative thereof, preferably 2tet01 or tet02. / l. A preferred inducible promoter is tetO 2/1.
Promoters controlling expression of Cas9 and gRNA (s) may be the same or different and constitutive or inducible. In a particular and preferred embodiment of the invention, only one of the promoters respectively controlling the expression of Cas9 or gRNA is an inducible promoter.
For the purpose of the invention, the term "nucleic acid" is intended to mean any molecule of natural, synthetic, semi-synthetic or recombinant DNA or RNA, optionally chemically modified (ie comprising non-natural bases, modified nucleotides comprising, for example, a modified binding, modified bases and / or modified sugars), or optimized so that the codons of transcripts synthesized from the coding sequences are the codons most frequently found in a bacterium of the genus Clostridium for its use in this one. As explained above, in the case of the Clostridium genus, the optimized codons are typically codons rich in adenine ("A") and thymine ("T") bases.
Each of the nucleic acids present in the genetic tool according to the invention, typically the "first" nucleic acid and the "second" nucleic acid, consists of a separate entity and corresponds, for example, to i) an expression cassette ( or "construct") such as a nucleic acid comprising at least one transcriptional promoter operatively linked (as understood by those skilled in the art) to one or more (coding) sequences of interest, typically to an operon comprising a plurality of coding sequences of interest whose expression products contribute to the realization of a function of interest within the bacterium, or such as a nucleic acid further comprising an activating sequence and / or a transcription terminator ; or ii) a vector, circular or linear, single or double-stranded, for example a plasmid, a phage, a cosmid, an artificial or synthetic chromosome, comprising one or more expression cassettes as defined above. Preferably, the vector is a plasmid.
The cassettes and expression vectors may be constructed by standard techniques well known to those skilled in the art and may comprise one or more promoters, origins of bacterial replication (ORI sequences), termination sequences, selection genes, for example antibiotic resistance genes, and sequences ("flanked regions") allowing targeted insertion of the cassette or vector. Moreover, these cassettes and expression vectors can be integrated into the genome by techniques well known to those skilled in the art.
ORI sequences of interest may be chosen from pIP404, ρΑΜβΙ, repH (origin of replication in C. acetobutylicum), ColEl or rep (origin of replication in E. coli), or any other origin of replication allowing the maintenance of the vector, typically of the plasmid, within a cell of
Clostridium.
Termination sequences of interest may be chosen from those of the adc, thl genes of the b es operon. or any other terminator, well known to those skilled in the art, allowing the stop of transcription within Clostridium.
Selection genes of interest can be selected from ermB, catP, bla, tetA, tetM, and any other ampicillin, erythromycin, chloramphenicol, thiamphenicol, tetracycline or other resistance gene. antibiotic that can be used to select Clostridium bacteria well known to those skilled in the art.
A particular vector comprises one or more expression cassettes, each cassette encoding a gRNA.
In a particular embodiment, the invention relates to a genetic tool comprising, as "first" nucleic acid as identified in the claims, a plasmid vector whose sequence is selected from one of the sequences SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
In a particular embodiment, the invention relates to a genetic tool comprising as a "second" or "nth" nucleic acid a plasmid vector whose sequence is selected from one of the sequences SEQ ID NO: 8, SEQ ID NO : 9, SEQ ID NO: 10 and SEQ ID NO: 11.
The sequence of interest is introduced into the bacterial genome via a homologous recombination mechanism guided by a selected repair matrix (according to CRISPR technology). The sequence of interest replaces the targeted portion within the bacterial genome. The recombination process thus makes it possible to modify or delete (le) or partially (le) the targeted portion within the genome of the bacterium or to allow the insertion of fragments (in a particular embodiment of large fragments). nucleic acid in the genome of the bacterium. The selected repair matrix can indeed comprise all or part of the targeted sequence of the bacterial genome or a more or less modified version thereof depending on the nature of the desired transformation. Like the targeted DNA portion, the matrix can itself thus comprise one or more nucleic acid sequences or nucleic acid sequence portions corresponding to natural and / or synthetic, coding and / or non-coding sequences. The matrix may, for example, comprise one or more sequences or portion (s) of sequences corresponding to a gene essential for bacterial survival, in particular for the survival of bacteria of the Clostridium genus, or for one or more genes or DNA sequences. whose inactivation allows the selection of bacteria of the Clostridium genus having integrated the nucleic acid (s) of interest. The matrix may also comprise one or more "foreign" sequences, ie naturally absent from the genome of bacteria belonging to the genus Clostridium or from the genome of particular species of the genus. The matrix may also include a combination of sequences as described above.
Particular examples of sequences of interest are the sequences used in the experimental part. These are for example sequences upp del (SEQ ID NO: 12) and upp stop (SEQ ID NO: 13). The genetic tool according to the invention allows the repair matrix to guide the incorporation into the bacterial genome of Clostridium bacteria of a nucleic acid of interest, typically a sequence or portion of a DNA sequence. comprising at least 1 base pair (bp), preferably at least 1, 2, 3, 4, 5, 10, 15, 20, 50, 100, 1000, 10,000, 100,000 or 1,000,000 bp, typically between 1 bp and 20 kb or between 1 bp and 10 kb, preferably between 10 bp and 20 kb or between 10 bp and 10 kb, for example between 1bb and 2 kb.
In a particular embodiment, the DNA sequence of interest encodes at least one product of interest, preferably a product promoting the production of solvent, typically at least one protein of interest, for example an enzyme; a membrane protein such as a transporter; a transcription factor; or a combination thereof.
In a preferred embodiment, the DNA sequence of interest promotes solvent production and is typically selected from a sequence encoding i) an enzyme, preferably an enzyme involved in the conversion of aldehydes to alcohol, for example selected among a coding sequence for an alcohol dehydrogenase (for example a sequence selected from among adh, adhE, adhEl, adhE2, bdhA, and bdhB), a sequence coding for a transferase (for example a sequence selected from ctfA, ctfB, atoA and atoB) , a coding sequence for a decarboxylase (for example adc), a sequence encoding a hydrogenase (for example a sequence selected from etfA, andfB and hydA), and a combination thereof, ii) a membrane protein, for example a sequence encoding a phosphotransferase (e.g., a sequence selected from glcG, bglC, cbe4532, cbe4533, cbe4982, cbe4983, cbe0751), and iii) a transcactor ription (for example a sequence selected from sigE, sigF, sigG, sigH, sigK).
The present invention also relates to a method for transforming and / or genetically modifying by homologous recombination a bacterium of the genus Clostridium, preferably a solventogenic bacterium of the genus Clostridium. This method comprises a step of introducing into the bacterium a genetic tool according to the invention as described in the present application. The method may further comprise a step of obtaining the transformed bacterium, ie the bacterium having the desired recombination (s) / optimization (s).
A particular method according to the invention for genetically transforming and / or modifying by a homologous recombination a solventogenic bacterium of the genus Clostridium, comprises, in order, the following steps: a) the introduction into the bacterium of a genetic tool according to the invention as described in the present application comprising at least one inducible promoter, and b) inducing the expression of the inducible promoter for genetically modifying the bacterium. The introduction into the bacterium of the elements (nucleic acids or gRNA) of the genetic tool according to the invention is carried out by any method, direct or indirect, known to those skilled in the art, for example by transformation, conjugation, microinjection, transfection, electroporation, etc., preferably by transformation (Lütke-Eversloh, 2014). The induction step when it is necessary can be carried out by any method known to those skilled in the art after introduction into the target bacterium of the genetic tool according to the invention. It is for example carried out by contacting the bacterium with a suitable substance, present in sufficient quantity or by exposure to UV light. This substance makes it possible to eliminate the expression inhibition linked to the selected inducible promoter. When the selected promoter is an inducible anhydrotetracycline (aTc) promoter selected from Pcm-2tet01 and Pcm-tet02 / 1, aTc is preferably used at a concentration of between about 1 ng / ml and about 5000 ng / ml. ml, preferably from about 100 ng per ml to about 500 ng / ml or from about 200 ng per ml to about 300 ng / ml, for example about 250 ng / ml.
In a particular embodiment, the method comprises one or more additional steps, subsequent to step b) when it is present, of introducing an uth, for example third, fourth, fifth, etc., nucleic acid. coding i) a repair matrix distinct from the one (s) already introduced and ii) one or more guide RNAs allowing their integration into a targeted zone of the genome of the bacterium, each additional step preferably being advantageously preceded by a step of removing the nucleic acid encoding the previously introduced repair matrix, the bacterial cell then being considered as "cured" of said nucleic acid, and preferably of a step of eliminating the guide RNA or sequences encoding the the guide RNAs introduced beforehand.
Particularly advantageously and unlike the tools available in the prior art, the genetic tool according to the invention allows the introduction of sequences of interest of small sizes as well as large sizes, in one step, ie to the using a single nucleic acid (typically the "second" nucleic acid as described herein) or in several steps, ie using several nucleic acids (typically the "second" and the "nth") nucleic acids as described herein), preferably in one step.
In a particular embodiment of the invention, this "nth" nucleic acid makes it possible to eliminate the targeted portion of the bacterial DNA or to replace it with a shorter sequence (for example by a deleted sequence of at least one pair of bases) and / or non-functional. In a particular preferred embodiment of the invention, the "second" or "nth" nucleic acid advantageously makes it possible to introduce into the bacterial genome a nucleic acid of interest comprising at least one base pair, and up to 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 kb.
The nucleic acids of interest may be inserted into the bacterial chromosome at identical or different regions depending on the gRNAs used, and if this proves useful in a portion of the bacterial genome comprising a gene essential for bacterial survival, for example one of the genes gyrA, pflcA, hydA, crt, thl, hbd, or any other gene known to those skilled in the art as essential for the survival of a bacterium of the genus Clostridium, and / or in a gene or DNA sequence whose inactivation allows the selection of bacteria having integrated the nucleic acid (s) of interest, for example the upp gene.
Thanks to the invention, typically thanks to the genetic tool and to the method according to the invention, it is now possible to modify efficiently (high-performance homologous recombination frequency) substantially (incorporation possible within the genome of the bacterium). a large nucleic acid of interest) and stably (the maintenance of transformed bacteria in contact with antibiotics is not necessary), bacteria of the genus Clostridium so as to obtain transformed bacteria of interest, for example improved variants having a genotypic or phenotypic difference with respect to the bacterium from which it is derived, typically industrially exploitable bacteria, for example bacteria usable in the production of solvents or biofuels.
Another subject of the invention relates to a bacterium of the genus Clostridium, typically a solventogenic bacterium of the genus Clostridium, transformed using the method and / or the genetic tool according to the invention. Such a bacterium expresses the nucleic acid (s) of interest introduced into its genome by homologous recombination using the repair matrix. Such a bacterium may comprise all or part of the genetic tool according to the invention, typically Cas9 or a nucleic acid encoding Cas9.
A bacterium of the particular genus Clostridium according to the invention, for example an ATCC824 bacterium, transformed using the method and the genetic tool according to the invention, no longer contains the pSOL megaplasmid.
In a particular embodiment, the bacterium of the genus Clostridium according to the invention, transformed using the method and the genetic tool according to the invention, is able to produce one or more solvents only by virtue of the expression of the nucleic acid (s) of interest introduced voluntarily into its genome. The invention also relates to a kit (kit) for genetically transforming and / or modifying a bacterium of the genus Clostridium comprising all or part of the elements of the genetic tool as described herein, typically i) a first nucleic acid encoding Cas9 wherein the sequence coding for Cas9 is under the control of a promoter, and ii) at least one second nucleic acid encoding a repair matrix allowing, by a homologous recombination mechanism, the replacement of a portion of the DNA bacterial targeted by Cas9 by a sequence of interest, and optionally one or more inducers adapted to the (x) promoters) inducible (s) selected (s) optionally used (s) within the tool.
A particular kit according to the invention allows the expression of a Cas9 protein comprising a tag (or "tag").
The kits according to the invention may further comprise one or more consumables such as a culture medium, at least one competent bacterium of the Clostridium genus (ie packaged for the purpose of processing), at least one gRNA, one Cas9 protein, one or several selection molecules, or an explanatory note. The invention typically relates to a kit for carrying out the transformation method described herein or for producing solvent (s) (at least one solvent) using a bacterium of the genus Clostridium. The invention also relates to the possible uses of the genetic tool, or the method, or the kit according to the invention for transforming and / or genetically modifying a bacterium of the genus Clostridium, typically a solventogenic bacterium of the genus Clostridium, for example to generate improved variants of a bacterium of the genus Clostridium. The invention finally relates to the possible uses of the genetic tool, the method, the kit or a bacterium of the genus Clostridium transformed according to the invention, in particular to allow the production of solvents or biofuels, or mixtures thereof. ci, typically on an industrial scale. Solvents that can be produced are typically acetone, butanol, ethanol, isopropanol or a mixture thereof, typically an ethanol / isopropanol, butanol / isopropanol or ethanol / butanol mixture, preferably a isopropanol / butanol mixture.
In a particular embodiment, the ratio of the ethanol / isopropanol mixture is at least equal to 1/4. This ratio is preferably between 1/3 and 1, and is even more preferably equal to 1. In a particular embodiment, the ratio of the ethanol / butanol mixture is at least equal to 1/4. This ratio is preferably between 1/3 and 1, and is even more preferably equal to 1.
In a particular embodiment, the ratio of the isopropanol / butanol mixture is at least equal to 1/4. This ratio is preferably between 1/3 and 1, and is even more preferably equal to 1. The use of transformed bacteria according to the invention typically allows the production per year on an industrial scale of at least 100. tonnes of acetone, not less than 100 tonnes of ethanol, not less than 1 000 tonnes of isopropanol, not less than 1 800 tonnes of butanol, or not less than 40 000 tonnes of a mixture thereof .
The examples and figures below are intended to illustrate more fully the invention without limiting its scope.
FIGURES
Figure 1: Metabolism of solvent-borne Clostridium strains. ABE strains produce acetone, ethanol and butanol while IBE strains have the adh gene converting acetone to isopropanol. Modified by Lee et al., 2012.
Figure 2: CRISPR action mode. Mali et al.
Figure 3: Using CRISPR-Cas9 for genome editing. The double-strand break is performed by Cas9 nuclease, directed by gRNA. Repair of this cleavage by homologous recombination makes it possible to introduce into the genome the modifications contained in the repair matrix. Figure modified since Ann Ran et al., 2013.
Figure 4: Targeting Plasmids of upp. pIP404, origin of replication in C. acetobutylicum. ColEl, origin of replication in E. coli. catP, chloramphenicol acetyltransferase gene (chloramphenicol / thiamphenicol resistance gene). CDS, coding sequence.
Figure 5: pSOL targeting plasmid. pIP404, origin of replication in C. acetobutylicum. ColEl, origin of replication in E. coli. catP, chloramphenicol acetyltransferase gene (chloramphenicol / thiamphenicol resistance gene). CDS, coding sequence.
Figure 6: map of the vector pEC500E-miniPthl-Cas9. ρΑΜβΙ, origin of replication in C. acetobutylicum. rep, origin of replication in E. coli. bla, β-lactamase gene (rampicillin resistance). ermB, methylase (erythromycin resistance). CDS, coding sequence.
Figure 7: sequencing of the zone targeted by case9 in the wild strain and in the transformant obtained. NC_003030, Clostridium acetobutylicum ATCC824 sequence (GenBank); crRNA, a site recognized by the ARNG; P AM, protospacer adjacent motif, involved in the fixation of Cas9. CDS, coding sequence. SEQ ID NO: 22 corresponds to the fragment of NC_003030 appearing in FIG. 7 and SEQ ID NO: 23 corresponds to the fragments appearing in FIG. 7 of the sequences identified respectively as "upp_stop repair matrix", "ATCC824" and "ATCC824 upp-" . Figure 8: Amplification results. A: catP_fwd x catP rev (expected size: 709pb) B: RHctfBR x V-CTFA-CAC2707 R (expected size: 351pb) 1: 2-Log marker (NEB). 2: H 2 0, negative control. 3: ATCC824 not transformed. 4: ATCC824 transformed with pEC500E-miniPthl-cav9, 5 &amp; 6: ATCC824 transformed with pEC500E-miniPthl-cas'9 and pEC750C (2 independent transformants). 7 &amp; 8: ATCC824 transformed with pEC500E-miniPthl-cas9 and pEC750C-gRNA_adhE (2 independent transformants).
Figure 9: Detection of α-amylase activity of strains derived from ATCC824. 1: ATCC824;
2: ATCC824 transformed with pEC500E and pEC750C; 3: ATCC824 transformed with pEC500E-miniPthl-cavP and pEC750C; 4: ATCC824 transformed with pEC500E and pEC750C-gRNA_a £ / ri £ '; 5: ATCC824 transformed with pEC5 () 0E-miniPth1-6a, v9 and p EC75 OC-gRN AadhE
Figure 10: Fermental balances of the wild-type strain and a transformant (two technical replicates). Figure 11: A / B. Inducible expression plasmids of cas9. repH, origin of replication in C. acetobutylicum. ColEl, origin of replication in E. coli. ermB, methylase (erythromycin resistance). tetR, a gene encoding the TetR transcriptional repressor. CDS, coding sequence.
Figure 12: Effect of induction on expression from the promoters Pcm-2tet01 and Pcm-tet02 / 1. Promoters were placed downstream of the gus A gene, and GusA activity was measured in C. acetobutylicum ATCC824 cells in the absence or presence of 100 ng / ml of aTc. Modified from Dong et al., 2012.
Figure 13: Effect of aTc concentration on the viability of transformants containing pEC750C-gRNA_upp.
Figure 14: Generation of mutants resistant to 5-FU. Serial dilutions of liquid cultures are deposited on different media. Only transformants in which homologous recombination events allowed insertion of the repair matrix are able to grow on 2YTG + 5-FU. The white arrows indicate the colonies selected for the following experiments. ND, undiluted.
Figure 15: Analysis of upp del transformants by PCR. A. Genetic organization around the upp gene. The coding sequences are indicated by arrows. The gray rectangle indicates the region absent from the matrix upp del. The primers used are represented by triangles. CDS, coding sequence. PAM, protospacer adjacent motif, involved in the fixation of Cas9. B. Results of the amplification. M: 2-Log marker (NEB). 1: H 2 0, negative control. 2: ATCC824 not transformed. 3: ATCC824 transformed with pFWOOO 1 -Pcm-2tetO 1 -cas9 and pEC750CgRNA _ "/ j '/> upp_dcl before exposure to aTc. 4 &amp; 5: ATCC824 transformed with pFWOOO 1 -Pcm-2tetO 1 -cas9 and pEC750CgRNA_w / P-upp_del before exposure to aTc, isolated on 2YTG + 5-FU (2 independent transformants).
Figure 16: sequencing of the case-targeted area9 in isolated colonies on 2YTG + 5-FU. NC 003030, Clostridium acetobutylicum ATCC824 sequence (GenBank); crRNA, a site recognized by the ARNG; PAM, protospacer adjacent motif, involved in the fixation of Cas9. CDS, coding sequence. SEQ ID NO: 24 corresponds to the fragment of the genomic sequence of the ATCC824 strain appearing in FIG. 16 and SEQ ID NO: 25 corresponds to the fragments appearing in FIG. 16 of the sequences identified respectively as "upp stop matrix", "clone pFWOOO 1 -Pcm-2tctO 1 -cas9 -pEC750C-gRNA_w / P-upp_stopl "and" clone pFWOOO 1 -Pcm-tct02 / l-ca, S'9 -pEC750C-gRNA_w / P-upp_stopl "and" clone pFWOOO 1 - Pcm-tct02 / l-6a.v9 - pEC75OC-gRNA upp-upp_stop2 ".
EXAMPLES
The inventors have tested the genetic tool described and claimed in the present text on two targets: the upp and adhE genes.
Inactivation of the uvv gene
The first chosen target validates the genetic modification technique designed by simple screening. The upp gene encodes an uracil phosphoribosyltransferase. This enzyme allows the formation of uracil monophosphate (UMP) from uracil, but also the formation of 5-fluorouracil monophosphate (5-FUMP), from 5-fluorouracil (5-FU). 5-FUMP is a cell-toxic compound that blocks RNA synthesis. Therefore, a bacterium containing in its genome the upp gene can not grow on a medium containing 5-FU, unlike a strain not expressing this gene.
Targeting this gene makes it possible to determine simply and rapidly, by simple phenotypic observation, whether the modification strategy is effective. Three plasmids for targeting upp were constructed (see Figure 4 + SEQ ID NO: 9, 10 and 11). All three contain the same gRNA targeting the gene, and two of them also contain different repair matrices to show the capabilities of the tool to perform deletions or point mutations: • The uppdel matrix (SEQ ID NO: 12) contains two fragments of 500 nucleotides (nt) located at 150nt on both sides of the cleavage site determined by the gRNA. The use of this template to repair the cleavage causes a deletion of 300nt within the coding sequence of the upp gene so that the latter will then encode an inactive protein. Uppstop matrix (SEQ ID NO: 13) contains two fragments of 650nt located on both sides of the cleavage site, modified at the site of recognition of the gRNA by the presence of nonsense mutations (inducing the replacement of a codon encoding an amino acid by a stop codon) so that Cas9 can no longer target the gene that will encode an incomplete and inactive protein.
Loss of plasmid pSOL
The second target chosen is of interest in the fermentation process: all the genes involved in the solventogenesis, in particular adhE, are localized on the pSOL megaplasmid, and it has been shown that its loss abolishes the production of acetone and butanol. After removing these fermentative pathways using the pSOL targeting plasmid (see Figure 5), it is possible to reintroduce the genes of interest directly into the genome. In order to obtain a strain no longer containing pSOL, a plasmid making it possible to target adhE was constructed. The inventors have demonstrated that pSOL does not contain essential functions for the cell so that the cell can survive without its presence.
Constitutive Expression of Case9 in C. acetobutvlicum ATCC824
The chosen strategy requires the concomitant use of two plasmids: the nuclease expression vector constitutively, derived from plasmid pEC500E: pEC500E-miniPth1-Cas9 (see FIG. 6 + SEQ ID NO: 5); one of the targeting vectors, which determine the nuclease cleavage site and possibly allow the repair of cleavage, derived from pEC750C: pEC750C-gRNA_wp / (SEQ ID NO: 9), containing the targeting gRNA upp; • pEC750C-gRNA "/ i /> upp_del (SEQ ID NO: 10), containing the upp-targeting gRNA and the upp del matrix; • pEC750C-gRNA "/ i /> upp_stop (SEQ ID NO: 11), containing the upp-targeting gRNA and the upp stop matrix; • pEC750C-gRNA_adM '(SEQ ID NO: 8), containing the gRNA targeting adhE.
The expression vector pEC500E-miniPth1-Cas9 was introduced into the ATCC824 strain, as well as a control plasmid corresponding to an empty vector (pEC500E). The two strains obtained were then transformed with the targeting vectors, derived from the vector pEC750C, serving as a control. The results of this second transformation step are shown below in Table 1.
Table 1: Transformation results. ++, many transformants obtained (between 102 and 103 colonies obtained / transformation); -, no transformants obtained.
The transformation results obtained indicate that Cas9 is functional. Indeed, when the nuclease is expressed and the upp gene is targeted by the gRNA, no transformant is obtained, because of the cut induced in the genomic DNA and the inability of the bacterium to perform the repair. of the genome (transformation with p EC5 OOE-mimPth1-ca.vP and pEC750C-gRNA_w / p) in the absence of a repair matrix.
Targeting of upp
The results obtained when the targeting vectors are introduced into the strain containing pEC500E show that the genome of the ATCC824 strain does not contain a case homolog9. since transformants are obtained with each targeting vector.
A transformant containing pEC500E-miniPth1-cav9 and pEC750C-gRNA_w / / - upp_stop was then subcultured several times to nonselective medium to lose the plasmids it contained. Once the colonies were cured of their plasmids and antibiotic-sensitive, the upp gene (SEQ ID NO: 3) was sequenced (see Figure 7).
The desired modifications are present. The CRISPR-Cas9 genetic tool comprising the introduction of two plasmids and the expression of the cas9 gene by a strong and constitutive promoter is therefore very functional.
Targeting of adhE
Transformants are obtained during transformations of the wild-type strain with the plasmids pEC5 OOE-miniPthl-cas9 and pEC750C_gRNA adhE. Since case9 is active, this result indicates that a cleavage in meSaplasmid pSOL does not affect the viability of ATCC824.
In order to confirm the possible loss of pSOL, various tests were performed: - PCR detection of a gene present on pSOL: ctfli
PCR catP fwd x catPrev makes it possible to detect the thiamphenicol resistance gene present on plasmids pEC750C. Its detection confirms that the targeting vectors are present.
The RH_ctfB_R x V-CTFA-CAC2707 R PCR makes it possible to detect a portion of the ctfB gene, present on the pSOL megaplasmid, and makes it possible to know if the latter is present in the cell. The amplification seems to show that meSplasmid pSOL is no longer present in clones transformed with pEC500E-miniPth1-ca, v9 and pEC750C-gRNA_ai // zL '(see Figure 8).
- Detection of enzymatic activity encoded by a gene present on pSOL
Among the genes contained on the pSOL megaplasmid, amyP encodes an extracellular enzyme with α-amylase activity. This activity can be detected on a solid medium containing starch and glucose (Sabathé et al., 2002). Dilutions of liquid cultures were plated on an agar plate containing 0.2% glucose and 2% starch and incubated 72h at 37 ° C. The α-amylase activity is then revealed by iodine staining. The light halos around the spots of bacteria indicate the presence of α-amylase activity. The lack of activity around ATCC824 containing pEC500E-miniPthl-ca.vO and pEC750C-gRNA_o <7M indicates that the amyP gene is not expressed in this strain, confirming that the megaplasmid is no longer present (see Figure 9). - Fermentation report
The wild strain ATCC824 and a transformant were cultured for 24 hours in a GAPES medium in order to establish the fermental balance of the two strains. The fermentation balance obtained shows a decrease in ethanol production as well as an abolition of the butanol and acetate production due to the absence of the adhE, adhEl and adc genes (present on the megasplasmid pSOL) in the transformant (see Figure 10).
Cas9 is therefore capable of acting on the chromosome or on the natural plasmid of the ATCC824 strain, which makes it possible to extend its action to the chromosome and to any extrachromosomal genetic material present in the strain (bacteriophage plasmid, etc.).
Inducible expression of cas9 in C. acetobutylicum ATCC824
In order to allow homologous recombination events between the genome and the repair templates, the number of cells in which the nuclease is active must be increased (up to 103 when the ATCC824 strain containing pEC500E-miniPth1-6'a, v9 is transformed with a targeting vector). For this purpose, a system in which the expression of nuclease is controlled is necessary. Two vectors in which the cas9 gene is placed under the control of an anhydrotetracycline inducible promoter have been constructed, derived from the vector pFWOOO1: • pFWOOO1-Pcm-2tet01-cas9 (see Figure 11A + SEQ ID NO: 6) ; • pFWOOO1-Pcm-tet02 / l-cas9 (see Figure 11B + SEQ ID NO: 7);
Promoters controlling cas9 expression contain the tet01 and ett02 operator sequences on which the TetR transcriptional repressor binds. This repression is lifted by the presence of anhydrotetracycline (aTc). This system allows controlled expression, with few leaks. In the presence of aTc, the synthesis is greater from the Pcm-2tet01 promoter (see Figure 12).
Transformation of C. acetobutylicum ATCC824:
The expression vectors as well as the empty vector pFWOOO 1 were introduced into ATCC824. Subsequently, the following plasmids were introduced into each type of transformant (see Table 2): • pEC750C-gRNA_w / />, Containing the upp-targeting gRNA; • pEC750C-gRNA_w / / - upp_del, containing upp-targeting gRNA and upp del matrix; • pEC750C-gRNA_w / /> - upp_stop, containing the gRNA targeting upp and the upp stop matrix;
The transformed colonies were plated on different solid media, at different dilutions: • 2YTG + erythromycin to check cell viability, dilution by a factor of 106; • 2YTG + erythromycin + thiamphenicol to select transformants; • 2YTG + erythromycin + thiamphenicol + aTc (200ng / mL) to select the transformants in the presence of the inducer.
Table 2: number of colonies obtained on each type of medium for each transformation, ery, erythromycin, thiam, thiamphenicol. aTc, anhydrotetracycline. In parentheses, diluting factors. ND, undiluted. -, not tested.
A toxic effect of aTc is observed, since few transformants are obtained when present, even when the empty targeting vector (pEC750C, control) is used. As expected, no transformant is obtained when a pEC750C containing the gRNAjipp cassette is introduced into a cell expressing cas9, on a medium containing aTc. In contrast, many transformants are obtained for each plasmid combination on medium without aTc, indicating that cas9 is not expressed.
Expression of case9 in the presence of aTc:
The different transformants obtained on plates containing erythromycin and thiamphenicol were subcultured on the same type of medium and then used to seed liquid precultures containing both antibiotics. These precultures were then used to inoculate other liquid cultures containing varying concentrations of αTc to determine if the system is functional.
Induction of cases9:
Three transformants were used to analyze the ability to induce the expression of cas9 in the presence of aTc: ATCC824 containing pFWOOO1 and pEC750C-gRNA; ATCC824 containing pFWOOO 1 -Pcm-2tetO 1 -cas9 and pEC750C-gRNA; ATCC824 containing pFWOOO 1 -Pcm-tet02 / 1 -cas9 and pEC750C-gRNA;
Liquid media containing erythromycin, thiamphenicol and increasing concentrations of αTc were seeded from liquid precultures of these transformants (see Figure 13). The growth capacity of the cells is evaluated by measuring the optical densities after 72 hours of culture. The transformant not expressing nuclease is not or only slightly affected by the presence of αTc. On the other hand, even at low concentrations of αTc, the transformant containing the plasmid expressing case 9 via the Pcm-2tet01 promoter (pFWOOO 1-Pcm-2tet01-cas9) and the plasmid containing only gRNA (pEC750C-gRNA_upp) exhibit a significant growth retardation. The transformant containing the plasmid expressing cas9 via the Pcm-tet02 / 1 promoter (pFWOOO 1 -Pcm-tct02 / 1-6a.v9) and the plasmid containing only gRNA (pEC750C-gRNA_w / P) is not assigned to low concentrations of aTc. However, from 150 ng / mL, a strong growth retardation is observed, and no growth is observed at 300 ng / mL. The Pcm-tet02 / 1 promoter therefore seems to allow a better repression of the expression than Pcm-2tet01 in the absence of an inducer. Generation of mutants
Liquid cultures of the transformants containing the targeting plasmids for the repair of double-strand breaks were also performed in the absence or presence (100 ng / ml) of aTc. Transformants used contained one of the twelve plasmid combinations shown in Table 3.
Table 3: Combinations of plasmids present in the transformants.
After 72 hours of culture, aliquots were deposited on various solid media: • 2YTG containing thiamphenicol and erythromycin; • 2YTG containing thiamphenicol, erythromycin, and 100ng / mL of aTc; • 2YTG containing 5-fluorouracil.
Only transformants in which homologous recombination events allowed insertion of the repair matrix are able to grow on 2YTG + 5-FU (see Figure 14).
Analysis of upp del transformants:
Clones isolated on 2YTG + 5-FU were analyzed by PCR (see Figure 15).
PCR catP fwd x catP rev makes it possible to detect the thiamphenicol resistance gene present on plasmids pEC750C. Its detection confirms that the targeting vectors are present.
The LHA PCR upp fwd x RHA upp rev makes it possible to amplify the upp gene as well as the flanking regions. The primers shown below were used to construct the upp_del repair matrix (see Figure 15 + SEQ ID NO: 14-21):
Table n ° 4:
PCR upp matrix fwd x upp matrix rev makes it possible to amplify an internal fragment of the upp gene, absent from the upp del repair matrix.
The results obtained confirm the deletion within the upp gene in the transformants analyzed. Analysis of mutants upp stop:
The upp gene was sequenced in three clones isolated on 2YTG + 5-FU after exposure to aTc (see Figure 16):
• One containing the plasmid expressing cas9 via the Pcm-2tet01 promoter (pFW0001-Pcm-2tet01-cas9) and the plasmid containing the gRNA as well as the upp_stop repair matrix (pEC750CgRNA_w / / - upp_stop); Two containing the plasmid expressing cas9 via the Pcm-tet02 / 1 promoter (pFW0001-Pcm-tet02 / l-cas9) and the plasmid containing the gRNA as well as the upp stop repair matrix (pEC7 OCgRNAwpp-uppstop).
The strategy for developing a genetic modification system by using the cas9 gene under the control of an inducible promoter and the gRNA present in a second plasmid is therefore functional. Compared with the use of the miniPth1 constitutive promoter, induction of the cas9 gene makes it possible to control the action of the enzyme and to facilitate the selection of transformants having undergone the desired modifications.
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权利要求:
Claims (15)
[1" id="c-fr-0001]
1. Genetic tool allowing the transformation by homologous recombination of a solventogenic bacterium of the genus Clostridium characterized in that it comprises: a first nucleic acid encoding at least Cas9, in which the sequence coding for Cas9 is placed under the control of a promoter , and at least one second nucleic acid containing a repair matrix allowing, by a homologous recombination mechanism, the replacement of a portion of the bacterial DNA targeted by Cas9 by a sequence of interest, and in that i) at least one of said nucleic acids further codes for one or more guide RNAs (gRNAs), or ii) the genetic tool further comprises one or more guide RNAs, each guide RNA comprising an enzyme-binding RNA structure Cas9 and a sequence complementary to the targeted portion of the bacterial DNA.
[2" id="c-fr-0002]
2. Tool according to claim 1, characterized in that the sequence encoding said one or more guide RNAs is preceded by a promoter and in that said promoter or the promoter controlling Cas9 is an inducible promoter.
[3" id="c-fr-0003]
3. Tool according to claim 1 or 2, characterized in that the targeted portion of the bacterial DNA comprises an essential gene or a portion of gene essential for bacterial survival.
[4" id="c-fr-0004]
4. Tool according to one of claims 1 to 3, characterized in that the solventogenic bacterium of the genus Clostridium is selected from C. acetobutylicum, C. cellulolyticum, C. phytofermentans, C. beijerinckii, C. saccharobutylicum, C. saccharoperbutylacetonicum, C. sporogenes, C. butyricum, C. aurantibutyricum and C. tyrobutyricum.
[5" id="c-fr-0005]
5. Tool according to claim 4, characterized in that when the solventogenic bacterium is C. acetobutylicum said bacterium C. acetobutylicum is the strain ATCC824, and when the solvent bacterium is C. beijerinckii said bacterium C. beijerinckii is the strain DSM 6423.
[6" id="c-fr-0006]
6. Tool according to one of claims 1 to 5, characterized in that the Cas9 protein comprises the sequence SEQ ID NO: 1.
[7" id="c-fr-0007]
7. Tool according to one of claims 1 to 6, characterized in that the promoter of Cas9 is an inducible promoter.
[8" id="c-fr-0008]
8. Tool according to one of claims 1 to 7, characterized in that the DNA sequence of interest encodes at least one product promoting the production of solvent, typically at least one enzyme involved in the conversion of aldehydes to alcohol, a membrane protein, a transcription factor, or a combination thereof.
[9" id="c-fr-0009]
9. Tool according to one of claims 1 to 8, characterized in that each of the nucleic acids present in the tool belongs to a separate expression cassette or to a vector, for example a separate plasmid.
[10" id="c-fr-0010]
10. Process for transforming by homologous recombination a solventogenic bacterium of the genus Clostridium, characterized in that it comprises a step of introduction into the bacterium of a genetic tool according to one of claims 1 to 9.
[11" id="c-fr-0011]
11. The method of claim 10, characterized in that it comprises the following steps: a) the introduction into the bacterium of a genetic tool according to one of claims 1 to 9 comprising at least one inducible promoter, and b ) induction of the expression of the inducible promoter for genetically modifying the bacterium.
[12" id="c-fr-0012]
12. The method of claim 10 or 11, characterized in that it comprises one or more additional steps, subsequent to step b) when it is present, introducing an uth nucleic acid encoding i) a matrix distinct from the one (s) already introduced and (ii) one or more guide RNAs allowing their integration into a targeted zone of the genome of the bacterium, each additional step being preceded by an acid elimination step nucleic acid encoding the previously introduced repair matrix, and preferably removal of the guide RNAs or sequences encoding the guide / RNAs previously introduced.
[13" id="c-fr-0013]
13. Solventogenic bacterium of the genus Clostridium transformed with the method according to one of claims 10 to 12.
[14" id="c-fr-0014]
14. Kit for transforming a bacterium of the genus Clostridium or for producing at least one solvent using a bacterium of the genus Clostridium comprising the elements of the genetic tool as described in one of Claims 1 to 9 and optionally one or more inductors adapted to the selected inducible promoter (s) used within the tool.
[15" id="c-fr-0015]
15. Use of the genetic tool according to one of claims 1 to 9, the method according to one of claims 10 or 11, or a transformed bacterium according to claim 12, to allow the production of a solvent or a mixture of solvents on an industrial scale, preferably acetone, butanol, ethanol, isopropanol or a mixture thereof, typically an isopropanol / butanol mixture.

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US11268082B2|2017-03-23|2022-03-08|President And Fellows Of Harvard College|Nucleobase editors comprising nucleic acid programmable DNA binding proteins|

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FR3081881A1|2018-06-04|2019-12-06|IFP Energies Nouvelles|OPTIMIZED GENETIC TOOL FOR MODIFYING CLOSTRIDIUM BACTERIA|

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FR3090691A1|2018-12-20|2020-06-26|IFP Energies Nouvelles|GENETICALLY MODIFIED CLOSTRIDIUM BACTERIA, PREPARATION AND USES THEREOF|

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FR3096373A1|2019-05-24|2020-11-27|IFP Energies Nouvelles|OPTIMIZED GENETIC TOOL TO MODIFY BACTERIA|

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WO2021123391A1|2019-12-18|2021-06-24|Exomnis Biotech B.V.|Genetically modified clostridium strains and uses thereof|

法律状态:
2016-10-03| PLFP| Fee payment|Year of fee payment: 2 |

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2017-04-21| PLSC| Publication of the preliminary search report|Effective date: 20170421 |

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2017-09-25| PLFP| Fee payment|Year of fee payment: 3 |

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2017-11-03| TQ| Partial transmission of property|Owner name: IFP ENERGIES NOUVELLES, FR Effective date: 20170928 Owner name: STICHTING DIENST LANDBOUWKUNDIG ONDERZOEK, NL Effective date: 20170928 |

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2018-04-27| CD| Change of name or company name|Owner name: IFP ENERGIES NOUVELLES, FR Effective date: 20180321 Owner name: STICHTING WAGENIONGEN RESEARCH, NL Effective date: 20180321 |

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2018-10-22| PLFP| Fee payment|Year of fee payment: 4 |

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2019-10-21| PLFP| Fee payment|Year of fee payment: 5 |

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2020-10-13| PLFP| Fee payment|Year of fee payment: 6 |

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2021-10-12| PLFP| Fee payment|Year of fee payment: 7 |

优先权:

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申请号 | 申请日 | 专利标题

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FR1559846A|FR3042506B1|2015-10-16|2015-10-16|GENETIC TOOL FOR PROCESSING BACTERIA CLOSTRIDIUM|

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FR1559846|2015-10-16|FR1559846A| FR3042506B1|2015-10-16|2015-10-16|GENETIC TOOL FOR PROCESSING BACTERIA CLOSTRIDIUM|

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US15/768,018| US20180305680A1|2015-10-16|2016-10-14|Genetic tool for the transformation of clostridium bacteria|

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DK16795118.5T| DK3362559T3|2015-10-16|2016-10-14|GENETIC TOOL FOR TRANSFORMATION OF CLOSTRIDIUM BACTERIA|

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PCT/FR2016/052663| WO2017064439A1|2015-10-16|2016-10-14|Genetic tool for the transformation of clostridium bacteria|

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BR112018007622-8A| BR112018007622A2|2015-10-16|2016-10-14|Clostridium Bacterial Transformation Genetic Tool|

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CA3001815A| CA3001815A1|2015-10-16|2016-10-14|Genetic tool for the transformation of clostridium bacteria|

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KR1020187013946A| KR20180081527A|2015-10-16|2016-10-14|Genetic tools for transformation of Clostridium bacteria|

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EP16795118.5A| EP3362559B1|2015-10-16|2016-10-14|Genetic tool for the transformation of clostridium|

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CN201680068812.7A| CN108431221A|2015-10-16|2016-10-14|Genetic tool for converting Clostridium bacterium|