WO2021260322A1

WO2021260322A1 - Strains of clostridium bacteria resistant to 5-fluorouracil, genetic tools and uses thereof

Info

Publication number: WO2021260322A1
Application number: PCT/FR2021/051143
Authority: WO
Inventors: Nicolas Lopes Ferreira; François WASELS; Rémi HOCQ
Original assignee: IFP Energies Nouvelles
Priority date: 2020-06-23
Filing date: 2021-06-23
Publication date: 2021-12-30
Also published as: FR3111642A1

Abstract

The present invention relates to recombinant bacteria of the genus Clostridium made resistant to 5-fluorouracil (5-FU), the tools and methods enabling them to be obtained, as well as the uses of the recombinant strains and the tools, in particular for genetically modifying a bacterium of the same genus. The invention also relates to the methods for genetic modification of a bacterium of the genus Clostridium, comprising a step of transforming a recombinant bacterium according to the invention.

Description

5-FLUOROURACIL RESISTANT CLOSTRIDIUM BACTERIAL STRAINS, GENETIC TOOLS AND USES OF THEM

The present invention relates to the genetic modification of bacteria, more particularly bacteria belonging to the genus Clostridium, for example solvent-forming bacteria. It relates more particularly to recombinant bacteria made resistant to 5 -fluorouracil (5 -FU) belonging to the Clostridium genus, the tools and methods allowing them to be obtained, as well as the uses of said recombinant strains and of said tools, in particular for genetically modifying a bacterium of the same. kind. The invention also relates to the use, as marker gene (s) in a method of genetic modification of a bacterium of the genus Clostridium, of several genes chosen from among the upp, udp and / or udk genes, and the methods of genetic modification of a bacterium of the genus Clostridium comprising a step of transformation of a recombinant bacterium according to the invention.

TECHNOLOGICAL BACKGROUND

The genus Clostridium contains Gram-positive bacteria, strictly anaerobic and sporulating, belonging to the phylum Firmicutes. Clostridia are an important group to the scientific community for several reasons. The first is that a number of serious diseases (e.g. tetanus, botulism) are due to infections of pathogenic members of this family (John & Wood, 1986; Gonzales et al., 2014). The second is the possibility of using so-called acidogenic or solventogenic strains in biotechnology (Moon et al., 2016). These Clostridia, non-pathogenic, naturally have the capacity to convert a large variety of sugars to produce chemical species of interest, and more particularly acetone, butanol, and ethanol (John & Wood, 1986) in during a process called ABE fermentation. Similarly, IBE fermentation is possible in certain particular species, capable of reducing acetone in variable proportions to isopropanol (Chen et al., 1986, George et al., 1983) thanks to the presence in the genome of these strains genes encoding secondary alcohol dehydrogenases (s-ADH; Ismael et al., 1993, Hiu et al., 1987).

Although used in industry for over a century, knowledge of these bacteria has long been limited by the difficulties encountered in genetically modifying them. Various genetic tools have been designed in recent years to optimize strains of this genus, the latest generation being based on the use of CRISPR (Clustered Regularly Interspaced Short Palindromie Repeats) -Cas (CRISPR-associated protein) technology.

C. beijerinckii DSM 6423 is a solventogenic strain of the Clostridium genus which has the particularity of producing F isopropanol, mixed with n-butanol and ethanol (IBE metabolism). This capacity makes it interesting for the production of advanced biofuels or for green chemistry. Until recently, little information was available on this strain, which limited its use. The inventors have recently developed tools and methods described in patent EP3 362 559 and in applications EP3 578 662, PCT / FR2019053227 and FRI 9/05485 making it possible to modify in a targeted manner the genome of bacteria belonging to the genus Clostridium, including this microorganism. One of the proofs of concept employed by the inventors to demonstrate the effectiveness of their tools and methods consisted in deleting the upp gene (CIBE 0562 - SEQ ID NO: 1) encoding a uracil phosphoribosyltransferase. This gene is an ortholog of the CA C2879 gene (SEQ ID NO: 5) from C. acetobutylicum ATCC 824, a related species. According to the patent application WO2008 / 040387 (2006), the inactivation of CA C2879 causes in C. acetobutylicum a resistance to 5-fluorouracil (5-FU), an organofluorine analogue of uracil, because Upp allows the conversion 5-FU in 5-Fluorouridine monophosphate (5-FUMP), a compound toxic to the bacterial cell which interferes in particular with the synthesis of RNA. It is thus possible to envisage replacing the upp gene with a nucleic acid sequence of interest, the recombinant strain becoming capable of growing in liquid or agar medium containing 5-FU. However, as the inventors were able to observe, in C. beijerinckii DSM 6423, the inactivation of the CIBE 0562 gene does not make it possible to obtain a strain resistant to 5-FU (cf. FR 19/05485 and example 1 of part experimental).

Few counter-selection markers have been developed for bacteria of the genus Clostridium, in particular for C. beijerinckii (Joseph et al., 2018; Little et al., 2018; Wen et al., 2020), and none have was for the naturally isopropanol-producing strain DSM 6423. The identification of such a marker and the development of new tools allowing its use would be particularly useful to facilitate the selection of recombinant strains and, in a manner general modification of the genome of bacteria belonging to the genus Clostridium.

The present invention describes recombinant bacteria resistant to 5-FU as well as the tools and methods for their production and manipulation. It thus considerably and very advantageously facilitates the selection of the recombinant strains of interest, and therefore the exploitation, in particular on an industrial scale, of bacteria belonging to the genus Clostridium.

SUMMARY OF THE INVENTION

The inventors describe, in the context of the present invention, recombinant bacteria belonging to the genus Clostridium, in particular to the species Clostridium beijerinckii, advantageously capable of growing in the presence of 5-fluorouracil (5-FU).

They thus describe for the first time a recombinant bacterium of the genus Clostridium resistant to 5- FU characterized in that it lacks at least two genes responsible for the production of 5- Fluorouridine monophosphate (5-FUMP) from 5 -FU, or in other words a bacterium of the genus Clostridium modified by inactivation of several genes (at least two, for example three genes) responsible for the production of 5-FUMP from 5 -FU, in particular a recombinant bacterium belonging to the species Clostridium beijerinckii.

They also describe the process making it possible to obtain such a recombinant bacterium and the use of said recombinant bacterium in a process for the genetic modification of a bacterium of the genus Clostridium, for example to manufacture a derived bacterium, genetically modified, preferably usable in industrial scale to produce a solvent or a mixture of solvents.

A particular process described by the inventors allows the genetic modification of a bacterium of the genus Clostridium. It comprises one or more steps of transforming a bacterium of the genus Clostridium using one or more nucleic acids comprising a sequence targeting and inactivating the upp and udp genes, making it possible to make said bacterium 5 -FU resistant. Also described are recombinant bacteria of the genus Clostridium resistant to 5-FU characterized in that they have been obtained by such a process.

In a particular embodiment, the nucleic acid used to transform a bacterium of the genus Clostridium comprises a sequence targeting and inactivating, by a homologous recombination mechanism, the upp gene and the udp gene and / or the udk gene of the bacterium, said bacterium of the genus Clostridium comprising, in the wild state, preferably at least two genes responsible for the production of 5-FUMP from 5-FU and being preferably selected from C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum , C. botulinum, C. perfringens, C. diolis, C. taeniosporum and C. butyricum.

In another particular embodiment, the nucleic acid used to transform a bacterium of the genus Clostridium comprises a sequence allowing the insertion of an intron into the upp gene, the udp gene and / or the udk gene of the bacterium, said bacterium of the Clostridium genus comprising, in the wild state, preferably at least two genes responsible for the production of 5-FUMP from 5-FU and being preferably selected from C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, C. botulinum, C. perfringens, C. diolis, C. taeniosporum and C. butyricum.

In the context of the present invention, the method of genetic modification of the bacterium is preferably one of those developed by the inventors described in patent EP 3 362 559 and in applications EP 3 578 662 and FRI 8 73492 .

The present description is also aimed at the use of the upp, udp and / or udk genes as counter-selection marker (s), typically in the presence of 5-FU, in a process for the genetic modification of a recombinant bacterium of the genus Clostridium. according to the invention, typically from a bacterium of the genus Clostridium comprising, in the wild state, several genes responsible for the production of 5-FUMP from 5-FU.

The inventors also describe an advantageous process for the genetic modification of a bacterium of the genus Clostridium, preferably of a (strain of) bacterium comprising in the wild state several genes responsible for the production of 5-FUMP from 5-FU. , including a transformation step of a recombinant bacterium of the genus Clostridium as described in the present text using a nucleic acid comprising a sequence allowing the modification, by a homologous recombination mechanism, of the genetic material of the bacterium by mutation, insertion or deletion of a sequence of interest.

The inventors also describe a kit for transforming, and preferably genetically modifying, a bacterium belonging to the genus Clostridium, preferably a (strain of) bacterium comprising in the wild state several genes responsible for the production of 5-FUMP from of 5 -FU, or to produce at least one solvent, for example a mixture of solvents, using such a bacterium. They also describe the use of such a kit or of one or more of the elements of this kit, for the implementation of a process described in the present text of transformation, and ideally of genetic modification, of a bacterium belonging to the genus Clostridium, preferably a bacterium comprising in the wild state several genes responsible for the production of 5-FUMP from 5 -FU, and / or for the production of solvent (s) or biofuel (s), or mixtures thereof, preferably on an industrial scale, using a recombinant bacterium according to the invention. This kit preferably comprises a recombinant bacterium as described in the present text and 5 -FU.

DETAILED DESCRIPTION OF THE INVENTION

Classical methods of genetic modification based on the integration into the genome of linear DNA fragments and which work well in many microorganisms such as E. coli or yeast are not feasible in bacteria of the genus Clostridium. which considerably limited the possibilities of genetic modification of these microorganisms. Indeed, in addition to the low transformation efficiencies of these bacteria, the half-life of a nucleic acid to be recombined is low due to the presence of nucleases degrading the exogenous nucleic acids, while the efficiency (frequency) of the recombination in this bacterial genus is particularly weak. For example, the C. beijerinckii DSM 6423 strain naturally suffers from a very low transformation efficiency (less than 5 colonies / pg of plasmid prepared in an A'Escherichia coli Dam + Dcm + strain), and it would be futile to attempt the integration direct by double homologous recombination event of a linear DNA fragment in the genome of this microorganism.

Very recently, the inventors have developed tools and methods, described in patent EP3 362 559 (incorporated by reference) and in application EP3 578 662 (incorporated by reference) as well as in applications PCT / FR2019053227 and FR19 / 05485 , allowing targeted modification of the genome of this microorganism. They have in particular described a tool involving CRISPR-Cas technology comprising two nucleic acids, one containing the genetic elements allowing the controlled expression of the Cas nuclease (which produces a double-stranded cleavage within a molecule of nucleic acid), and the other at least one guide RNA (gRNA) targeting the region of DNA to be modified as well as a repair template. Preferably (cf. EP 3,578,662), at least one of said acids nucleic acid further comprises a sequence encoding an anti-CRISPR protein placed under the control of an inducible promoter. The genetic tool may otherwise further comprise a third nucleic acid encoding an anti-CRISPR protein preferably placed under the control of an inducible promoter.

The inventors constructed the C. beijerinckii IFP962 Aupp strain which is a mutant of the DSM 6423 strain in which the upp gene (CIBE 0562, SEQ ID NO: 1) has been inactivated (cf. FIG. 1). The inventors were able to observe that, contrary to what is observed in C. acetobutylicum ATCC 824 following the inactivation of the upp gene (patent WO2008 / 040387), the C. beijerinckii DSM 6423 Aupp mutants obtained remain sensitive to 5 -fluorouracil ( 5 -FU) (cf. FIG. 8), due to the accumulation of 5-Fluorouridine monophosphate (5-FUMP) which causes a significant cytotoxic effect leading to the arrest of cell growth (cell death). A diagram illustrating this toxic metabolic pathway is detailed in Figure 2.

Uracil is a metabolic intermediate in the pyrimidine rescue pathway which recycles the pyrimidine bases Cytosine (C) and Uracil (U).

The inventors have very advantageously succeeded in developing a recombinant bacterium of the Clostridium genus resistant to 5-fluorouracil (5 -FU) characterized in that it lacks several, ie at least two genes, responsible for the production of 5-FUMP from 5 -FU, or in other words in which several genes responsible for the production of 5-FUMP from 5-FU have been inactivated.

This recombinant bacterium of the Clostridium genus is typically a strain comprising in the wild several genes, ie, at least two genes, responsible for the production of 5-FUMP from 5-FU, for example two, three, four or five genes responsible for such production.

In a particular embodiment, the recombinant bacterium of the genus Clostridium is the bacterium C. botulinum or the bacterium C. perfringens.

In a preferred embodiment, the recombinant bacterium of the genus Clostridium is selected from C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, C. botulinum, C. perfringens, C. diolis, C. taeniosporum and C. butyricum. It is preferably selected from C. beijerinckii, C. saccharoperbutylacetonicum, C. diolis and C. butyricum.

In yet another preferred embodiment, the recombinant bacterium of the genus Clostridium is a bacterium sensitive to an antibiotic belonging to the class of amphenicols such as, for example, chloramphenicol, thiamphenicol, azidamphenicol or florfenicol, preferably chloramphenicol and / or thiamphenicol, for example a bacterium no longer expressing an amphenicol-O-acetyltransferase, for example a bacterium expressing the catB gene in the wild, and lacking said catB gene or incapable of expressing said catB gene.

A particularly preferred bacterium belongs to the species C. beijerinckii. The C. beijerinckii bacterium can be a bacterium sensitive to thiamphenicol such as for example the C. beijerinckii strain registered on December 6, 2018 under the deposit number LMG P-31151 with the Belgian Co-ordinated Collections of Micro-organisms (“BCCM », KL Ledeganckstraat 35, B-9000 Gent - Belgium), or the C. beijerinckii strain registered on February 20, 2019 under the deposit number LMG P-31277 with the BCCM.

In a particular embodiment, the bacterium genetically modified by means of a method described in the present text, is a bacterium of the genus Clostridium not expressing (due to said genetic modification) the upp gene and one and / or l another of the udp and udk genes, characterized in that it is also sensitive to an antibiotic belonging to the class of amphenicols as described above. The description relates to, besides the recombinant bacteria according to the invention, any bacterium derived, clone, mutant or genetically modified version thereof, for example any bacterium derived, clone, mutant or genetically modified version remaining resistant to 5 -FU.

The inventors have discovered the existence of several enzymes capable of metabolizing 5-FU in bacteria belonging to the genus Clostridium and have succeeded in developing a method for obtaining recombinant Clostridium bacteria resistant to 5-FU.

A particular method of genetic modification of a bacterium of the genus Clostridium described by the inventors comprises a step of transforming a bacterium of the genus Clostridium using a nucleic acid making it possible, by a homologous recombination mechanism, to make said bacteria resistant to 5-fluorouracil (5-FU). This method typically comprises the steps of a method described in patent EP3 362559 or in application EP3 578 662.

The term "transformation" refers to the incorporation of exogenous nucleic acid by a cell, this acquisition of new genes being transient (if the exogenous nucleic acid carrying the genes can subsequently be eliminated) or permanent (in the case of where the exogenous nucleic acid is integrated into the DNA of the host cell).

The term "homologous recombination" refers to the event of substitution of a segment of DNA by another which has identical (homologous) or nearly identical regions.

A preferred method comprises a step of transforming a bacterium of the genus Clostridium as described in the present text, preferably a bacterium comprising in the wild state several genes responsible for the production of 5-FUMP from 5- FU, using all or part of a modification tool (also identified in the present text as “exogenous sequence” or “exogenous nucleic acid”) by homologous recombination, typically characterized i) in that it comprises at least : a "first" nucleic acid encoding at least one DNA endonuclease, for example the Cas9 enzyme, in which the sequence encoding the DNA endonuclease 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 targeted bacterial DNA ("target DNA sequence") by the endonuclease by a “Sequence of interest”, in that ii) at least one of said nucleic acids further encodes one or more guide RNAs (gRNA) or in that the genetic tool further comprises one or more guide RNAs, each RNA guide comprising an RNA structure for binding to the DNA endonuclease and a sequence complementary to the targeted portion of the bacterial DNA, and preferably iii) in that at least one of said nucleic acids further comprises a coding sequence an anti-CRISPR protein placed under the control of an inducible promoter, or in that the genetic tool further comprises a third nucleic acid encoding an anti-CRISPR protein placed under the control of an inducible promoter. The expression “target DNA sequence” denotes any gene, intergenic region, promoter and / or regulatory sequence of interest chosen by a person skilled in the art. They are in particular genes encoding proteins of interest, for example enzymes involved in cell metabolism. The expression “sequence of interest” denotes a coding sequence or not. It can be a mutated sequence of the target DNA sequence, for example of the target gene, of the promoter or of the regulatory sequence and / or of a marker such as an antibiotic resistance gene or an enzyme. color generator.

The sequence of interest may be longer or shorter than the replaced sequence ("target DNA sequence"), depending on the distance between the two homologous regions.

The anti-CRISPR protein is a protein capable of inhibiting or preventing / neutralizing the action of DNA endonuclease, typically from Cas, and / or a protein capable of inhibiting or preventing / neutralizing the action of a CRISPR-Cas system, for example of a CRISPR-Cas type II system when the nuclease is a Cas9 type nuclease, preferably during the phase of introduction of the nucleic acid sequences of the genetic tool in the bacterial strain of interest.

This sequence is typically placed under the control of an inducible promoter different from the promoters controlling expression of DNA endonuclease and / or gRNA (s), and is inducible by another inducing agent. This promoter can for example be selected from the Pbgal promoter (inducible to lactose), the promoter of the tetA gene, of the xylA gene or of the lad gene, or a derivative thereof. The promoter controlling the expression of the anti-CRISPR protein makes it possible to advantageously control the action of the DNA endonuclease, for example of the Cas9 enzyme, and thus to facilitate the transformation of bacteria and the production of transformants. having undergone the desired genetic modifications.

The anti-CRISPR protein is typically an “anti-Cas9” protein or an “anti-MAD7” protein, ie a protein capable of inhibiting or preventing / neutralizing the action of Cas9 or MAD7. The anti-CRISPR protein is advantageously an “anti-Cas9” protein, for example selected from AcrlIA1, AcrIIA2, AcrIIA3, AcrIIA4, AcrIIA5, AcrIIC1, AcrIIC2 and AcrIIC3 (Pawluk et al, 2018). Preferably, the “anti-Cas9” protein is AcrIIA2 or AcrIIA4. Even more preferably, the “anti-Cas9” protein is AcrIIA4. Such a protein is typically capable of very significantly limiting, ideally preventing, the action of Cas9, for example by binding to the enzyme Cas9 (Dong et al, 2017; Rauch et al, 2017). Another advantageously usable anti-CRISPR protein is an “anti-MAD7” protein, for example the AcrVA1 protein (Marino et al, 2018).

The inventors describe in particular such a genetic tool comprising at least:

- a "first" nucleic acid encoding at least one DNA endonuclease, in which the sequence encoding the DNA endonuclease is placed under the control of a promoter, and

- an “other” nucleic acid comprising, or consisting of, an “OREP nucleic acid” sequence, ie comprising, or consisting of, i) all or part of the sequence SEQ ID NO: 41 and ii) a sequence allowing the modification of the genetic material of a bacterium by mutation, insertion or deletion of a sequence of interest and / or the expression within said bacterium of a DNA sequence partially or totally absent from the genetic material present within the wild version of said bacteria.

In a particular embodiment, the “second nucleic acid containing a repair template” as described above comprises this “other nucleic acid”.

The "OREP nucleic acid" sequence comprises a nucleotide sequence (SEQ ID NO: 41) encoding a protein involved in the replication of an OPT nucleic acid of interest. This protein involved in replication is also identified in the present text as the “REP” protein (SEQ ID NO: 42

"MNNNNTESEELKEQSQLLLDKCTKKKKKNPKFSSYIEPLVSKKLSERIKECGDFLQMLSDLN LENSKLHRASFCGNRFCPMCSWRIACKDSLEISILMEHLRKEESKEFIFLTLTTPNVKGADLDN SIKAYNKAFKKLMERKEVKSIVKGYIRKLEVTYNLDKSSKSYNTYHPHFHVVLAVNRSYFKK QNLYINHHRWLSLWQESTGDYSITQVDVRKAKINDYKEVYELAKYSAKDSDYLINREVFTVF YKSLKGKQVLVFSGLFKDAHKMYKNGELDLYKKLDTIEYAYMVSYNWLKKKYDTSNIRELT EEEKQKFNKNLIEDVDIE"). The REP protein has a domain conserved in firmicutes, called “COG 5655” (Plasmid rolling circle replication initiator protein REP), of sequence SEQ ID NO: 43. It facilitates the transformation of bacteria by improving the maintenance within said bacteria of the all the genetic material introduced.

A particular method advantageously comprises a step of transforming the bacterium by introducing into said bacterium all or part of a genetic tool as described in the present text, in particular of a nucleic acid comprising, or consisting of, i) all or part of the sequence SEQ ID NO: 41 and ii) a sequence allowing the modification of the genetic material of a bacterium and / or the expression within said bacterium of a DNA sequence partially or totally absent from the genetic material present within the wild version of said bacterium.

In the context of the present description, the nucleic acid used to transform a bacterium, or a population of bacteria, denotes an entity (P “exogenous nucleic acid”) typically in the form of an expression cassette (or “Construct”) such as for example a nucleic acid comprising at least one transcriptional promoter or a regulatory sequence of interest, operably linked (as understood by those skilled in the art) to one or more sequences (coding or not) of interest, for example to an operon comprising several coding sequences of interest whose expression products contribute to the realization of a function of interest within the bacterium, or a nucleic acid comprising in in addition to an activator sequence and / or a transcription terminator. It is typically in the form of 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 herein. -above. Preferably, the vector is a plasmid.

The nucleic acids of interest, typically the cassettes or expression vectors, can be constructed by standard techniques well known to those skilled in the art and can comprise one or more promoters, origins of bacterial replication (ORI sequences) , termination sequences, selection genes, eg antibiotic resistance genes, and sequences ("flanked regions") allowing targeted insertion of the cassette or vector. Furthermore, these expression cassettes and vectors can be integrated within the bacterial genome by techniques well known to those skilled in the art.

ORI sequences of interest can be chosen from pIP404, rAMbI, repH (origin of replication in C. acetobutylicum), ColE1 or rep (origin of replication in E. coli), or any other origin of replication allowing the maintenance of the vector, typically from the plasmid, within a Clostridium cell. Termination sequences of interest can be chosen from those of the adc, th1 genes, of the bcs operon, or of any other terminator, well known to those skilled in the art, allowing the stopping of transcription within of a bacterial cell. Selection genes (resistance genes) of interest can be chosen from ermB, catP, bla, tetA, tetM, and / or any other gene for resistance to ampicillin, erythromycin, chloramphenicol, thiamphenicol, to spectinomycin, to tetracycline or to any other antibiotic which can be used to select bacteria of the Clostridium genus well known to those skilled in the art.

In a particular embodiment, the method advantageously comprises the following steps: a) introduction into the bacteria of a modification tool by homologous recombination as described above, preferably in the presence of an agent inducing l. 'expression of the anti-CRISPR protein, and b) culture of the transformed bacterium obtained at the end of step a) on a medium not containing (or under conditions not involving) the agent inducing the expression of the anti-CRISPR protein, and typically allowing the expression of the ribonucleoprotein endonuclease complex of DNA / gRNA, for example Cas9 / gRNA (in order to stop the production of said anti-CRISPR protein and to allow the action of the endonuclease).

A particular method as described above aims, in the context of the present invention, to render a bacterium of the genus Clostridium (for example a bacterium as described in the present text comprising at least two genes responsible for the production of 5- FUMP from 5 -FU) resistant to 5 -FU. In such a method, the sequence of interest used typically comprises, or consists of, a sequence targeting and inactivating the upp gene, and one and / or the other of the udp and udk genes, and allows, by a mechanism of homologous recombination, to render the bacteria resistant to 5-fluorouracil (5 -FU). In a particular embodiment, the nucleic acid used in the method comprises, or consists of, a sequence targeting and inactivating the upp and udp genes, the upp and udk genes, or the upp, udp and udk genes.

In a preferred embodiment, the nucleic acid used in the method comprises, or consists of, a sequence targeting and inactivating the upp and udp genes.

In another preferred embodiment, the nucleic acid used in the method comprises, or consists of, a sequence targeting and inactivating the upp and udk genes.

Two particular nucleic acids described by the inventors make it possible, by homologous recombination mechanisms, to make resistant to 5-fluorouracil (5 -FU) a bacterium of the genus Clostridium comprising, in the wild state, at least two genes responsible for the production. from 5- FUMP from 5 -FU. These nucleic acids comprise, in a particular embodiment, a sequence targeting and inactivating the upp gene, and the udp gene and / or the udk gene. In a particular embodiment, the bacterium is selected from C. beijerinckii, C. diolis, C. saccharobutylicum, C. taeniosporum, C. botulinum, C. saccharoperbutylacetonicum, C. butyricum and C. perfringens, preferably from C. beijerinckii , C. diolis, C. saccharoperbutylacetonicum and C. butyricum. In another particular embodiment, the bacterium is the bacterium C. botulinum or the bacterium C. perfringens.

The upp gene is the gene of sequence SEQ ID NO: 1 present in C. beijerinckii or an analogous gene encoding or exhibiting the activity of a uracil phosphoribosyl-transferase, the sequence of which is at least 70%, 75% or more identical. 80%, preferably at least 81%, 82%, 83% or 84% identical, even more preferably at least 85% identical, to the sequence SEQ ID NO: 1.

The udp gene is the gene of sequence SEQ ID NO: 2 present in C. beijerinckii or an analogous gene encoding or exhibiting the activity of a uridine phosphorylase, the sequence of which is identical to at least 70%, 75% or 80%, preferably identical to 81%, 82%, 83% or 84%, even more preferably identical to at least 85%, to the sequence SEQ ID NO: 2.

The udk gene is the gene of sequence SEQ ID NO: 3 present in C. beijerinckii or an analogous gene encoding or exhibiting the activity of a uridine kinase, the sequence of which is at least 70%, 75% or 80% identical. , preferably 81%, 82%, 83% or 84% identical, even more preferably at least 85% identical, to the sequence SEQ ID NO: 3, or the gene of sequence SEQ ID NO: 4 or a analogue gene encoding or exhibiting the activity of a uridine kinase, the sequence of which is identical at least 70%, preferably at least 80%, even more preferably at least 85%, to the sequence SEQ ID NO : 4. In the context of the present description, a recombinant bacterium of the genus Clostridium which has been modified by inactivation of the upp and udk genes and is presented as resistant to 5-FU, denotes a bacterium which does not express any copy of the upp and genes. udk, typically a bacterium which does not express any of the sequences SEQ ID NO: 1, 3 and 4.

Genes functionally equivalent to the upp, udp and udk genes are present in other Clostridia (see Figures 3, 4, 5 and 6). The phylogenetic analysis of C. beijerinckii strains carried out by the inventors indicates that this metabolic pathway is widespread in this species. For example, almost identical sequences are found, for example within the species C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, C. botulinum, C. perfringens, C. diolis, C. taeniosporum and C. butyricum.

The inventors have noticed that the recombinant bacteria obtained by this process, which are also subjects of the present invention, were, surprisingly and very advantageously, resistant to 5-fluorouracil. 5-FU can therefore be used as an agent for the counter-selection of bacteria exhibiting a genetic modification of interest, derived (/ obtained from) said recombinant bacteria according to the invention, typically as an agent for the counter-selection of bacteria exhibiting one or more mutations in the upp, udp and / or udk gene (s), or comprising an exogenous sequence of interest inserted into one of said genes.

A preferred recombinant bacterium according to the invention is a bacterium of the genus Clostridium lacking the upp and udp genes. A recombinant bacterium according to the invention, devoid of the upp and udp genes, which is particularly preferred is the bacterium C. beijerinckii DSM 6423 AcatB Aupp Audp (“IFP965”) registered on June 9, 2020 under the deposit number LMG P-31854 with the Belgian Co-ordinated Collections of Micro-organisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium). Such a bacterium can typically be used as a laboratory tool for obtaining genetically improved strains of bacteria of the genus Clostridium. A preferred object of the present description is thus aimed at the reuse of a recombinant bacterium according to the invention, preferably the bacterium C. beijerinckii DSM 6423 AcatB Aupp Audp (“IFP965”) registered on June 9, 2020 under the deposit number LMG P- 31854 from the Belgian Co-ordinated Collections of Microorganisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium), in a process for the modification / genetic improvement of a bacterium of the genus Clostridium.

Another preferred recombinant bacterium according to the invention is a bacterium of the genus Clostridium lacking the upp and udk genes.

A particular recombinant bacterium according to the invention, devoid of the upp gene and of a copy of the udk gene, is the bacterium C. beijerinckii DSM 6423 AcatB Aupp Audk (“IFP966”) registered on June 9, 2020 under the deposit number LMG P -31855 from the Belgian Co-ordinated Collections of Microorganisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium). Such a bacterium can typically be used as a laboratory tool for obtaining genetically improved strains of bacteria of the genus Clostridium.

Another subject of the present description is thus the use of a recombinant bacterium according to the invention, preferably the bacterium C. beijerinckii DSM 6423 AcatB Aupp Audk (“IFP966”) registered on June 9, 2020 under the deposit number LMG P-31855 from the Belgian Co-ordinated Collections of Microorganisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium), in a process for the modification / genetic improvement of a bacterium of the genus Clostridium.

A preferred recombinant bacterium according to the invention is a bacterium of the genus Clostridium lacking the upp gene and the udk gene, ie all copies of the udk gene (for example the sequences identified in the figures and the experimental part as CIBE 1796 and CIBE 3382 at C. beijerinckii DSM 6423). Such a bacterium can typically be used as a laboratory tool for obtaining genetically improved strains of bacteria of the genus Clostridium.

Another preferred recombinant bacterium according to the invention is a bacterium of the genus Clostridium lacking (all copies) of the upp, udp and udk genes. Such a bacterium can typically be used as a laboratory tool for obtaining genetically improved strains of bacteria of the genus Clostridium.

Another preferred object of the present description is thus aimed at the use of a recombinant bacterium as described in the context of the present description in a process for the modification / genetic improvement of a bacterium of the genus Clostridium. Such a method is typically similar to the methods of genetic modification of a bacterium of the genus Clostridium described in the present text comprising a step of transforming a bacterium using an exogenous genetic tool (“exogenous nucleic acid”) comprising a sequence allowing the modification, as explained above, by a mechanism of homologous recombination, of the genetic material of the bacterium, typically by mutation, insertion or deletion of a sequence of interest. This method of genetic modification aims to obtain a recombinant bacterium exhibiting improved properties allowing it to be exploited directly on an industrial scale, for example to produce a biofuel. The improved properties of the bacterium obtained by such a process, preferably of a bacterium of the genus Clostridium comprising, in the wild state, several genes responsible for the production of 5-FUMP from 5 -FU, modified by inactivation of d 'at least two of said genes, for example three genes, are directly linked to the modification (s) made to its genome, and therefore to the selected sequence (s) of interest, introduced in a stable manner into its genome.

In the method according to the invention, the exogenous nucleic acid advantageously comprises all or part of the sequence encoding Upp, Udp and / or Udk and makes the bacteria in which it is present sensitive to 5-FU.

According to one particular aspect, the inventors thus describe, as explained below, the use as counter-selection marker (s) of the upp, udp and / or udk genes in a process for the genetic modification of a recombinant bacterium of the genus Clostridium according to the invention. They preferably describe their use as counter-selection marker (s) in the presence of 5-FU.

The expression "marker gene" refers to a sequence encoding a marker protein under the control of functional regulatory elements allowing the synthesis of said protein in Clostridia. The selection marker genes can be divided into several subcategories depending on whether they confer positive or negative selection, and whether the selection is conditional or unconditional on the presence of external substrates.

The so-called “positive selection” marker genes promote the growth of cells whose genome has been genetically modified. The detection of these marker genes with positive selection is subordinated to the use of toxic agents (antibiotics, herbicides or drugs). These genes are, for example, genes for resistance to an antibiotic such as the nptl and nptll genes conferring resistance to the antibiotic kanamycin; in this case, all the cells having acquired the transgene will resist the antibiotic while the others will be killed. The use of positive selection marker genes of this type has the double "advantage" of allowing both the identification and rapid sorting of genetically modified cells (the only ones to survive), compared to those which have not acquired the gene. transgene. Conversely, the so-called “counter-selection” or “negative selection”, “negatively selectable” marker genes cause the death of genetically modified cells or organisms under certain conditions that are controllable and known to the experimenter. Conventionally, once the recombination event has occurred in the context of the genetic modification process according to the invention, the exogenous nucleic acid (preferably in the form of a replicative vector, for example d 'a plasmid) must be removed. Elimination of a replicative vector generally occurs with successive cultures of clones, followed by negative or positive selection of clones which have eliminated this vector. The present invention advantageously makes it possible to dispense with these successive cultures. It makes it possible in fact to very simply eliminate (/ purge), in the presence of 5 -FU, the bacterial cells of the exogenous nucleic acid (typically in the form of a vector) thanks to the presence within said exogenous nucleic acid of the sequence encoding Upp, Udp and / or Udk which makes the bacteria still comprising the exogenous nucleic acid sensitive to 5-FU unlike the bacteria which have eliminated it. The strains which no longer express the genes upp, upp and udp, upp and udk, or upp, udp and udk, can thus be selected on this characteristic.

In a preferred embodiment, the method of genetic modification of a bacterium of the genus Clostridium described by the inventors thus comprises a step of transforming a population of recombinant bacteria of the genus Clostridium according to the invention, and further comprises i) a step of selecting the only bacteria that have been modified by mutation, insertion or deletion of the sequence of interest, and ii) a step of selecting said modified bacteria no longer possessing the nucleic acid containing the counter-selection marker (s), said bacteria multiplying in the presence of 5-FU unlike bacteria which have not lost said nucleic acid.

According to one particular aspect, the recombinant bacteria targeted by the transformation step of the method according to the invention does not express the upp gene and does not also express the udp gene and / or the udk gene. Said bacterium is advantageously transformed, in the context of a particular process for the genetic modification of said bacterium, using a nucleic acid comprising the upp gene or the upp and udp genes or the upp and udk genes or the upp genes. , udp and udk, said / said genes being used as counter-selection marker (s).

Typically, the recombinant bacterium is transformed using an exogenous nucleic acid comprising the, or more, gene (s) (typically upp, udp and / or udk) whose genome has been deleted in accordance with the teaching provided. by the inventors in the present text.

The use of the counter-selection marker is particularly useful because the recombinant Clostridia according to the invention are capable of growing on a medium comprising 5-FU before the transformation and after the elimination of the exogenous genetic tool ("nucleic acid exogenous'), typically of the vector. The strains which have eliminated said exogenous tool can thus be selected positively.

In a preferred method, said bacterium is transformed with the aid of a nucleic acid comprising the upp gene, said gene being used as a counter-selection marker.

In another preferred method, said bacterium is transformed with the aid of a nucleic acid comprising the udp and / or udk gene (s), said gene (s) being used as marker (s) against -selection. In another preferred method, said bacterium is transformed with the aid of a nucleic acid comprising the genes upp and udp and / or udk, said gen (s) being used as counter-selection marker (s).

According to a particular aspect of the invention, the inventors describe the use of a recombinant bacterium according to the invention to manufacture a genetically modified derivative bacterium, which can be used on an industrial scale to produce a solvent or a mixture of solvents, as well as the derivative recombinant bacteria thus obtained. They also describe the use of the derivative recombinant bacterium thus obtained, to produce, thanks to the expression of the nucleic acid (s) of interest voluntarily introduced into its genome, one or more solvents, preferably at least isopropanol, of preferably on an industrial scale.

The description also relates to a kit (kit) for transforming, and preferably genetically modifying, a bacterium belonging to the genus Clostridium to produce at least one solvent, for example a mixture of solvents, using such a bacterium. This kit preferably comprises a recombinant bacterium as described in the present text and 5 -FU. It can also comprise a nucleic acid as described in the present text, all or part of the elements of a genetic tool as described in the present text making it possible to transform, and typically genetically modify such a bacterium, in order to produce a improved variant of said bacterium, and / or an inducer suitable for an inducible promoter present within the tool, for example an inducible promoter of the expression of the selected anti-CRISPR protein.

A preferred nucleic acid described in the present text comprises a sequence targeting and inactivating the upp gene, and the udp gene and / or the udk gene of a bacterium of the genus Clostridium comprising in the wild state at least two genes responsible for the production. of 5-FUMP from 5-FU selected from C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, C. botulinum, C. perfringens, C. diolis, C. taeniosporum and C. butyricum.

The kit according to the invention can also comprise one or more consumables such as a culture medium, at least one gRNA, a nuclease, one or more selection molecules, or even an explanatory leaflet.

The description also relates to the use of a kit according to the invention, or of one or more of the elements of this kit, for the implementation of a method described in the present conversion text, and ideally of genetic modification, of a bacterium belonging to the genus Clostridium and / or for the production of solvent (s) or biofuel (s), or mixtures thereof, preferably on an industrial scale, using 'such a bacterium.

Solvents capable of being produced are typically acetone, butanol, ethanol, isopropanol or a mixture of these, typically an ethanol / isopropanol, butanol / isopropanol or ethanoFbutanol mixture, preferably an isopropanol mixture. / butanol. The use of bacteria transformed according to the invention typically allows the production per year on an industrial scale of at least 100 tonnes of acetone, of at least 100 tonnes of ethanol, of at least 1000 tonnes of isopropanol. , at least 1,800 tonnes of butanol, or at least 40,000 tonnes of a mixture thereof.

The examples and figures below are intended to illustrate the invention more fully without limiting its scope.

FIGURES

[Fig 1 A] Figure 1 A depicts a genetic construct.

[Fig IB] Figure IB shows the verification of the presence of mutant strains of C. beijerinckii DSM 6423 in which the upp gene (CIBE 0562) is deleted (cf. figure 19 of patent FR1905485).

[Fig 2] Figure 2 represents the 5-FU metabolic pathway described for C. acetobutylicum via the Upp enzyme encoded by the CA C2879 gene, as well as the metabolic pathways in C. beijerinckii DSM 6423 involving the enzyme Upp encoded by the CIBE 0562 gene or an alternative route passing through 5-Flurouridine involving the Udp and Udk enzymes encoded respectively by the CIBE 3715 (Udp), and CIBE 1796 and CIBE 3382 (Udk) genes. 5-FUMP: 5-fluorouracil monophosphate.

[Fig 3] Figure 3 represents the phylogenetic tree Blast grouping the Clostridium strains exhibiting a udp gene 90% identical to the amino acid sequence of the CIBE 3715 gene. (Position of the udp gene of C. beijerinckii surrounded by a solid line ).

[Fig 4] Figure 4 represents the phylogenetic tree grouping the Clostridium strains exhibiting a udk gene 85% identical to the amino acid sequence of the CIBE 1796 gene. (Position of the udk gene of C. beijerinckii surrounded by a solid line) .

[Fig 5] Figure 5 represents the phylogenetic tree grouping the Clostridium strains exhibiting a udk gene 85% identical to the amino acid sequence of the CIBE 3382 gene. (Position of the udk gene of C. beijerinckii surrounded by a solid line) .

[Fig 6] FIG. 6 represents the phylogenetic tree grouping the Clostridium strains exhibiting an upp gene 85% identical to the amino acid sequence of the CIBE 0562 gene. (Position of the upp gene of C. beijerinckii surrounded by a solid line) .

[FIG. 7] FIG. 7 represents the verification by PCR on a colony of the size of the loci targeted in each type of strain obtained. Sizes of expected amplicons, per target: upp (primers RH10 x RH11) - WT: 1,680 kb, Aupp: 1,080 kb; udp (RH171 x RH172 primers) - WT: 1,732 kb, Audp: 1,099 kb; udk (RH175 x RH176 primers) - WT: 1.848 kb, Audk: 1.225 kb.

[Fig 8] Figure 8 shows the growth on agar medium of C. beijerinckii DSM 6423 (WT) and of the various mutants constructed during this study. [Fig 9] Figure 9 shows the map of the plasmid pNF3C-ABsaI.

[Fig 10] Figure 10 shows the map of the plasmid pNF3C-GRNAind.

[Fig 11] Figure 11 shows the Map of the plasmid pNF3C-GRNAind-udp-HR.

[Fig 12] Figure 12 shows the Map of the plasmid pNF3C-GRNAind-udk-HR.

[Fig 13] Figure 13 shows the Map of the plasmid pNF3C -Audp.

[Fig 14] Figure 14 shows the Map of the plasmid pNF3C -Audk.

EXAMPLES

EXAMPLE n ° l

As described in application FRI 9/05485, the inventors studied the inactivation of the upp gene in C. beijerinckii. To do this, they used the CRISPR-Cas9 tool described in application EP 3,578,662 (incorporated by reference).

A clone of the C. beijerinckii DSM 6423 AcalB strain was first transformed with the vector pCas9 _acr not exhibiting methylation at the levels of the motifs recognized by the methyltransferases of the dam and dcm type (prepared from an Escherichia coli bacterium exhibiting the half demj genotype. An erythromycin resistant clone was then transformed with previously demethylated pEC750C-Aw /? /? The colonies thus obtained were selected on medium containing erythromycin (20 μg / ml), thiamphenicol (15 pg / mL) and lactose (40 mM).

Several of these clones were then resuspended in 100 μL of culture medium (2YTG) and then serially diluted in culture medium (up to a dilution factor of 10 ⁴ ). Five μl of each dilution were placed on a Petri dish containing erythromycin, thiamphenicol and anhydrotetracycline (200 ng / mL). For each clone, two colonies resistant to aTc were tested by PCR colony with primers intended to amplify the upp locus (cf. FIG. 1A). The resistance of these clones to 5-FU (50 mg / L) was then tested, and all the recombinant bacteria C. beijerinckii DSM 6423 AcatB Aupp were sensitive to this compound. The growth of one of these recombinant bacteria [“IFP962 Aupp” or “IFP964” - strain registered on June 9, 2020 under the deposit number LMG P-31853 with the Belgian Co-ordinated Collections of Micro-organisms (“BCCM ", KL Ledeganckstraat 35, B-9000 Gent - Belgium), designated in the figure as" Aupp "] as well as the bacterium C. beijerinckii DSM 6423 AcatB [" IFP962 "- strain registered on December 6, 2018 under the deposit number LMG P-31151 from the Belgian Co-ordinated Collections of Microorganisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium), designated in the figure as “WT”] on 2YTG medium and on supplemented 2YTG medium in 5-FU is presented in Figure 8. Contrary to what is observed in the recombinant bacterium C. acetobutylicum MGC ACA_C1502 Aupp (Croux et al, 2016), the deletion of the upp gene in the bacterium IFP964 does not lead to resistance to 5-FU. EXAMPLE n ° 2

MATERIALS & METHODS

The inventors studied the inactivation of the udp and udk genes in C. beijerinckii.

To do this, they also used the CRISPR-Cas9 tool described in application EP 3,578,662 (incorporated by reference).

As described below, the inventors have succeeded in eliminating the two target genes (udp and udk genes) within the C. beijerinckii DSM 6423 AcatB strains [“IFP962” - strain registered on December 6, 2018 under the deposit number LMG P -31151 from Belgian Co-ordinated Collections of Micro-organisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium)] and C. beijerinckii DSM 6423 AcatB Aupp [“IFP962 Aupp” or “IFP964” - strain registered on June 9, 2020 under the deposit number LMG P-31853 with Belgian Co-ordinated Collections of Micro-organisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium)].

_{The method described in application EP 3,578,662} comprises a step of introduction into the strain of a first plasmid, “pCas9 aCT”, which makes it possible to express the Cas9 enzyme under the control of several regulatory elements (inducible promoter and transient presence. anti-CRISPR AcrIIA4 protein). In a second step, a second so-called “targeting” plasmid is introduced into said strain. The targeting plasmid comprises:

- the “pNF3C” plasmid, used as base vector;

- a repair matrix corresponding to the areas of homology surrounding the region to be deleted by homologous recombination; and

- an expression cassette for a guide RNA targeting the region of the chromosome to be modified.

STRAINS AND CULTIVATION CONDITIONS

The E. coli NEB 10-beta strain (New England Biolabs, Ref C3019) is used for the various clonings. For the preparation of the plasmids before transformation in C. beijerinckii DSM 6423, a strain of Έ. coli Dam Dcm expressing the products of the genes encoding certain methyl transferases from C. beijerinckii (CIBE 3327, CIBE 3328, CIBE 3322) placed under the control of an arabinose-inducible promoter (concentration used: 1 mM) was used . The different E. coli strains were cultured on LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, agar 0 or 15 g / L if liquid or solid, respectively).

For the construction of the Audp and Audk strains, the strain “IFP 962” [strain C. beijerinckii registered on December 6, 2018 under the deposit number LMG P-31151 with the Belgian Co-ordinated Collections of Micro-organisms (“BCCM” , KL Ledeganckstraat 35, B-9000 Gent - Belgium) - patent application FR 1905485] was used. For the construction of the strains A upp Auclp and A upp Audk, the strain “IFP 962 A upp” [or “IFP964” - strain registered on June 9, 2020 under the deposit number LMG P-31853 with Belgian Co-ordinated Collections of Micro -organisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium)] obtained in example n ° l was used. All these strains are cultured on 2YTG medium (tryptone: 16 g / L, yeast extract: 10 g / L, glucose: 5 g / L, NaCl: 4 g / L) liquid (balanced at pH 5.2) or solid (supplemented with 15 g / L of agar).

Erythromycin (at concentrations of 20 or 500 mg / L respectively in 2YTG or LB medium), chloramphenicol (25 or 12.5 mg L ¹ respectively in solid or liquid LB medium), thiamphenicol (15 mg / L in 2YTG medium) and ampicillin (100 mg / L in solid or liquid LB medium) were used, if necessary. For the 5-fluorouracil (5-FU) sensitivity tests, the solid 2YTG medium was supplemented with 50 mg / L of 5-fluorouracil (Sigma-Aldrich).

PRIMERS AND PLASMIDS

Primers

The primers used in this study are described below, in order of appearance: Name DNA sequence

RH 159 5 ’-GTGGGTCACGCGGTATCATTGC-3’ RH 160 5 ’-GCAATGATACCGCGTGACCCAC-3’ M13-rev 5 ’-CAGGAAACAGCTATGAC-3’ pECGAAACAGCTATGAC-3 ’pECAGATA750C-fwd 5’-CAATATTCCATACATACATTACAT 3’-CAATATTCCATACATACATTACAT 3 ’-CAATATTAC ’TCAGATACATTAC’ 3 ’’ATTACATTAC’ ’’ pECAGATTAC ’’ CAATATTAC ’’ATTACATTACATTAC’ 3 ’pECAGATA’ ’TCAGACATTAC’ ’3’ CAATATTATT ’TCAGACATTACATTAC’ ’3’ T ’ACATTACAT

3 ’

RH 164 5 "-CGAAAAAGAAAAACTGCCGGGATCCGGTAATCTACTTCCTTCTGG-3"

RH161 5'-

ATCATAGTTACATTAATACGGATCCGGAGTATGTTTTTCTAATAATTCAAC-3 '

RH 162 5 ’-GTAGGTTCAGTATTTCTAGTTGTTG-3’

RH 165 5 ’- ATCATAGTTACATTAATACGGATCCGCTTTAAAAAATATAACTTTCAGG-

3 ’

RH 166 5 '-CCTCCATAAGTAATCTAATATAATTATAC-3'

3 ’

RH 168 5 '-CGAAAAAGAAAAACTGCCGGGATCCCTTTCCAATAATTCAAGAGC-3' udk-guide-fwd 5 '-TCATCGTTGATCCGAGTAAGAGAA-3' udk-guide-rev 5 '- AAACTT CT CTTACTCGGAT C AACG- 3' fATTG-udTAAC-guide -guide-rev 5'-AAACTGGTAATATTACGTATTTAC-3 ' RH010 5 '-CGGATATTGCATTACCAGTAGC-3'

RH011 5 "-TTATCAATCTCTTACACATGGAGC-3"

RH171 5 ’-AAAGGACATTGGTAATAATTCTTCG-3’

RH172 5 ’-CACCAATTAATTTAGATAGTTCAGG-3’

RH175 5 ’-GTAAAGTGGGTGATATGTTAAATG-3’

RH176 5 "-TCAACTTGATCTCCACAAAC-3"

Plasmids

Plasmids described in patent application EP 3,578,662 were used, in particular the plasmids pCas9acr (SEQ ID NO: 32 - cf. sequence SEQ ID NO: 23 and FIG. 4 of EP 3,578,662), pGRNAind (SEQ ID NO: 33 - cf. sequence SEQ ID NO: 82 of EP3 578 662) and pNF3C (SEQ ID NO: 34 - cf. sequence SEQ ID NO: 125 and FIG. 29 of EP3 578 662).

In the present study, several subclonings made it possible to produce the plasmids pNF3 C-udp of sequence SEQ ID NO: 39 and pNF3C -Audk of sequence SEQ ID NO: 40.

First, the plasmid pNF3C had to be modified to remove a Bsal restriction site. To do this, a “Quickchange” type method was used. The vector was amplified with the primers RH159 and RH160, then digested with DpnI. The PCR product was then introduced into E. coli, which made it possible to obtain the vector pNF3C-ABsaI of sequence SEQ ID NO: 35 (see Figure 9). In order to obtain a vector similar to pGRNAind with the origin of replication of pNF2, this vector was then modified to incorporate the region for expressing a guide RNA with an easily modifiable protospacer sequence. To do this, this zone was amplified by PCR with the primers M13-rev and pEC750C-fwd from pGRNAind. The PCR product was then digested with SacI then cloned by ligation (T4 DNA Ligase, NEB) in pNF3C-ABsaI, previously digested with SacI and dephosphorylated with the enzyme Antarctic Phosphatase (NEB), which made it possible to obtain pNF3C-GRNAind of sequence SEQ ID NO: 36 (cf. FIG. 11).

In this new vector, areas of upstream and downstream homology of the CIBE 3715 and CIBE 1796 genes were integrated in order to obtain the plasmids pNF3C-GRNAind-udp-HR of sequence SEQ ID NO: 37 (cf. FIG. 11) and pNF3C-GRNAind-udk-HR of sequence SEQ ID NO: 38 (cf. FIG. 12). To do this, the targeted areas were amplified by PCR from the genomic DNA of strain DSM 6423 (udp: upstream with RH 163 and RH 164, downstream with RH161 and RH 162; udk: upstream with RH 165 and RH 166, downstream with RH 167 and RH 168). For each target, the two PCR products were assembled by PCR fusion. The two repair matrices thus obtained were then cloned in BamHI in pNF3C-gRNAind, previously dephosphorylated with the enzyme Antarctic Phosphatase (NEB).

The last step consisted in introducing into these two vectors the variable sequences (protospacer) of the guide RNAs targeting udp and udk. These sequences were determined in silico using the Geneious RIO software (https://www.geneious.com). For each protospacer, a pair of primers is hybridized in a thermal cycler (thermal denaturation at 95 ° C, then gradually lowering the temperature to room temperature). The udp-guide-fwd / udp-guide-rev and udk-guide-fwd / udk-guide-rev pairs were used to target the udp and udk genes respectively. The hybridization products were then integrated into the vectors pNF3C-GRNAind-udp-HR and pNF3C-GRNAind-udk-HR by Golden Gâte assembly in Bsal, which made it possible to obtain the final vectors pNF3C -Audp of sequence SEQ ID NO: 39 (cf. Figure 13) and pNF3C -Audk of sequence SEQ ID NO: 40 (cf. Figure 14).

Bacterial transformation

The same protocol as previously described (Diallo et al, 2020) was used to transform the strain with 20 µg of DNA. The main difference is the use of DNA prepared in a particular strain of E. coli (see sub-section “Strains and culture conditions”).

Inactivation of the udp and udk genes

After introduction of the plasmid pCas9 _aCr into the IFP 962 and IFP 962 Aupp strains, the resulting strains are again transformed with the vectors pNF3C -Audp and pNF3C -Audk. The transformants are selected on selective medium in the presence of lactose (40 mM) to induce the anti-CRISPR system described in applications EP3 578 662 and FR 19/05485. Individual colonies are suspended in water, then serial dilutions are deposited on selective solid medium supplemented with anhydrotetracycline (200 ng / mL).

Colonies resistant to anhydrotetracycline are subcultured and then their genotypes are tested by PCR on a colony and their phenotypes by exposure on a dish to 5-fluorouracil (50 mg / L).

Colony PCR

An isolated colony of C. beijerinckii DSM 6423 is suspended in 100 μL of 10 mM Tris pH 7.5 5 mM EDTA. This solution is heated at 98 ° C. for 10 min without stirring. 900 μL of water are added, then the tubes are centrifuged at maximum speed for 3 min. 0.5 µL of the supernatant from this bacterial lysate can then be used as a PCR template in 10 µL reactions with Phusion polymerase (Thermo Scientific).

The pairs of primers RH10 / RH11, RH171 / RH172 and RH175 / RH176 are used to verify the inactivation of the upp, udp and udk genes, respectively. Each of these pairs makes it possible to amplify a region comprising the zones of homologies used to inactivate each of these genes, so that the PCR product can only come from an amplification of the genomic DNA at the targeted locus. RESULTS

Two targeting plasmids were constructed, pNF3C -Audp and pNF3C -Audk (respectively targeting the udp and udk genes), which were then introduced into the IFP 962 strains [C. beijerinckii strain registered on December 6, 2018 under the deposit number LMG P-31151 from the Belgian Co- ordinated Collections of Micro-organisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium] and IFP 962 Aupp [or “IFP964” - strain registered on June 9, 2020 under the LMG deposit number P-31853 with Belgian Co-ordinated Collections of Micro-organisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium)].

After induction of the CRISPR system, correct mutants were obtained with a frequency rate of 100% (data not shown). FIG. 7 shows the typical profiles obtained by PCR on a colony for each type of strain obtained.

The growth of the various mutants was then tested in the presence of 5-FU on agar medium (cf. FIG. 8).

The results (size and number of colonies, speed of growth) indicate that only the genetic contexts Aupp Audp, and, to a significantly lesser extent, Audp alone, make it possible to obtain growth on 5-FU agar medium, and therefore resistance. to this compound. The deletion of the upp gene alone does not make it possible to obtain a recombinant bacteria resistant to 5-FU, unlike what has been observed in C. acetobutylicum. Furthermore, the activation of the udk gene of sequence SEQ ID NO: 3, does not result in resistance to 5-fluorouracil, even for a strain in which the upp gene has been inactivated. A second copy of this gene (SEQ ID NO: 4) has been identified in the genome of strain DSM 6423 (cf. FIG. 5) which explains the sensitivity phenotype observed (partial deletion of the udk gene). The deletion of this second gene, in addition to the gene of sequence SEQ ID NO: 3, i.e., the complete deletion of the udk gene, is able to confer resistance to 5-FU on the recombinant bacteria.

CONCLUSIONS

With a view to having new genetic tools dedicated to Clostridia, C. beijerinckii DSM 6423 AcatB Aupp Audp and C. beijerinckii DSM 6423 AcatB Aupp Audk strains have been obtained by genetic engineering. The inventors deduce from the experiments carried out that once at least two genes involved in the formation of 5-FUMP from 5-FU are deleted, the recombinant bacterium of the Clostridium genus is made resistant to 5-FU. These phenotypes are advantageous in that they allow the use of a counter-selection marker (the upp gene, the udp gene and / or the udk gene) to obtain strains of C. beijerinckii not possessing any other. genetic modification than desired. Such a marker can thus be used to accelerate the phase of elimination of the exogenous plasmids after a given genetic modification, a step which, before the present invention, required numerous subcultures on non-selective medium. The metabolic pathways responsible for the assimilation of 5-FU in C. beijerinckii DSM 6423 are also present in a number of clostridia, ie C. beijerinckii, C. diolis, C. saccharoperbutylacetonicum, C. saccharobutylicum, C. taeniosporum, C botulinum, C. butyricum, C. perfringens, C. saccharobutylicum, in which it is also possible to delete at least two of the genes upp, udp and udk.

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Claims

1. Recombinant bacterium of the genus Clostridium characterized in that it has been modified by inactivation of at least two genes responsible for the production of 5-Fluorouridine monophosphate (5-FUMP) from 5-FU.

2. Recombinant bacterium of the genus Clostridium according to claim 1, characterized in that the bacterium of the genus Clostridium is a strain comprising in the wild state at least two genes responsible for the production of 5-FUMP from 5-FU, of preference selected from C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, C. botulinum, C. perfringens, C. diolis, C. taeniosporum and C. butyricum.

3. Recombinant bacterium of the Clostridium genus according to claim 1 or 2, characterized in that the at least two genes responsible for the production of 5-FUMP from 5-FU are selected from the upp, udp and udk genes.

4. Recombinant bacterium of the Clostridium genus according to claim 1 or 2, characterized in that it has been modified by inactivation of the upp and udp genes.

5. Recombinant bacterium of the Clostridium genus according to one of claims 1 to 4, characterized in that the recombinant bacterium is the bacterium C. beijerinckii DSM 6423 AcatB Aupp Audp registered on June 9, 2020 under the deposit number LMG P-31854 with of the Belgian Co-ordinated Collections of Micro-organisms ("BCCM", KL Ledeganckstraat 35, B-9000 Gent - Belgium) or the bacterium C. beijerinckii DSM 6423 AcatB Aupp Audk registered on June 9, 2020 under the deposit number LMG P -31855 from the Belgian Co-ordinated Collections of Micro-organisms (“BCCM”, KL Ledeganckstraat 35, B-9000 Gent - Belgium).

6. Use of a recombinant bacterium according to one of claims 1 to 5, in a process for the genetic modification of a bacterium of the genus Clostridium, or to manufacture a derived bacterium genetically modified, which can be used on an industrial scale to produce a solvent or a mixture of solvents.

7. Use of the upp, udp and / or udk genes as counter-selection marker (s), in the presence of 5-FU, in a process for the genetic modification of a recombinant bacterium of the genus Clostridium according to one of claims 1. at 5.

8. A method of genetic modification of a bacterium of the genus Clostridium comprising a step of transforming a recombinant bacterium of the genus Clostridium as described in one of claims 1 to 5 using a nucleic acid comprising a sequence. allowing the modification, by a mechanism of homologous recombination, of the genetic material of the bacterium by mutation, insertion or deletion of a sequence of interest.

9. The method of claim 8 for genetic modification of a bacterium of the genus Clostridium, characterized in that the recombinant bacteria targeted by the transformation step does not express the upp gene and does not further express the udp gene or the udk gene, and in that said bacterium is transformed to using a nucleic acid comprising the upp gene or the upp and udp genes or the upp and udk genes, used as counter-selection marker (s).

10. The method of claim 9, characterized in that it comprises a step of transforming a population of recombinant bacteria of the genus Clostridium as described in one of claims 1 to 5, and in that it comprises in besides i) a step of selecting the only bacteria which have been modified by mutation, insertion or deletion of the sequence of interest, and ii) a step of selecting said modified bacteria no longer possessing the nucleic acid containing the counter marker (s). - selection, said bacteria multiplying in the presence of 5-FU unlike bacteria which have not lost said nucleic acid.

11. A method of genetic modification of a bacterium of the genus Clostridium comprising a step of transforming a bacterium of the genus Clostridium using a nucleic acid making it possible to make said bacterium resistant to 5 -fluorouracil (5-FU) by a mechanism of homologous recombination, in which said nucleic acid comprises a sequence targeting and inactivating the upp and udp genes.

12. Recombinant bacterium of the genus Clostridium resistant to 5-fluorouracil (5-FU) characterized in that it has been obtained by a process according to claim 11.

13. Method according to one of claims 8 to 11, characterized in that the nucleic acid used to transform the bacterium or the population of bacteria is an expression cassette or a vector, for example a plasmid.

14. Nucleic acid comprising a sequence targeting and inactivating the upp gene, and the udp gene and / or the udk gene of a bacterium of the genus Clostridium comprising in the wild state at least two genes responsible for the production of 5-FUMP at from 5-FU selected from C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, C. botulinum, C. perfringens, C. diolis, C. taeniosporum and C. butyricum.