Modified clostridium bacteria capable of producing ethanol, preparation and uses thereof

Abstract

The invention relates to the genetic modification of bacteria of the genus Clostridium, typically solventogenic bacteria of the genus Clostridium, so as to allow the production of ethanol by said bacteria. Additionally described are methods, tools and kits for obtaining said bacteria, the genetically modified bacteria obtained, and the uses of same.

Description

[0001] MODIFIED CLOSTRIDIUM BACTERIA PRODUCING ETHANOL, PREPARATION AND USES THEREOF

[0002] The present invention relates to the genetic modification of bacteria of the genus Clostridium, typically solvent-forming bacteria of the genus Clostridium. The inventors describe in particular methods, tools and kits for the elimination or modification of sequence(s) coding for, or controlling the transcription of sequence(s) coding for, an enzyme with 3-hydroxybutyryl-CoA dehydrogenase (Hbd) activity and a hydrogenase (HydA) or two thiolases (ThlA and ThlB). The genetically modified bacteria thus obtained are also described, as are their uses, in particular for producing ethanol, possibly exclusively.

[0003] TECHNOLOGICAL BACKGROUND

[0004] Bacteria belonging to the genus Clostridium are strict anaerobic Gram-positive bacilli, capable of forming endospores and belonging to the phylum Firmicutes. This genus contains many species studied for their pathogenic nature or their industrial and medical interest.

[0005] Clostridium species of industrial interest, non-pathogenic, are capable of producing compounds of interest such as acids and solvents from a wide variety of sugars and substrates ranging from glucose to cellulose. The growth of solvent-producing Clostridium bacteria (called "solventogenic" or "acidogenic") is said to be biphasic. Acids are produced during the acidogenesis phase, which corresponds to the exponential growth phase. When cell growth ceases and the bacteria enter the stationary phase, they enter the solventogenesis phase, reassimilate the produced acids and transform them into solvents.

[0006] Clostridium acetobutylicum is naturally capable of producing a mixture of ethanol, acetone and n-butanol during a so-called ABE fermentation (Figure 1).

[0007] C. acetobutylicum is considered a model organism for the study of solventogenic microorganisms, due to the relatively high titers obtained during fermentations. Many genetic tools have been specifically developed to perform metabolic engineering allowing the reorientation of its metabolic pathways. Lehmann and Lütke-Eversloh described in 2011 the construction, using ClosTron (Heap JT, et al. , QQTI), of an hbd mutant that no longer produces butyrate or butanol as fermentation end products. The same tool was used a year later by Cooksley et al. (2012) to construct numerous mutants in which different genes (adhEl, adhE2, bdhA, bdhB, ptb, ack, ctfA, ctfB, adc, CA_P0059) of central metabolism are inactivated.In this study, the thlA gene, one of the two copies of the gene encoding thiolase, was also targeted in two different ways but could not be inactivated, leading the authors to suggest that this gene is essential. Interestingly, however, the Cac-p / b505a::CT mutant obtained in this same study had a frameshift nucleotide insertion in the thlA gene, resulting in gene inactivation. Due to the contradiction with the inability to delete the gene using the ClosTron tool, the authors concluded that double inactivation of thlA and ptb was possible, but probably not single inactivation of the thlA gene (Cooksley et al.). It has been shown in a similar manner that inactivation of thlA is possible when carried out simultaneously with inactivation of hydA, the product of which is a hydrogenase (US20170240869).These independent results seem to indicate that the thlA gene is essential. However, as the inventors teach in the context of the present invention, C. acetobutylicum has on the megaplasmid pSOL a second copy of the gene encoding thiolase (thlB) whose expression is relatively low compared to thlA, whether in acidogenesis or in solventogenesis (K. Winzer et al., Differential regulation of two thiolase genes from Clostridium acetobutylicum DSM 792.; J. Mol. Microbiol. Biotechnol. (2000) 2(4): 531-541).

[0008] The inventors describe in the context of the present invention, and for the first time, new mutants of C. acetobutylicum which no longer produce, or produce almost no more, n-butanol, butyrate and acetone, and whose major (possibly exclusive) fermentation product is ethanol. In one of these mutants, the thlA and thlB genes encoding thiolase are inactivated, without any other modification being visible in the genome, contrary to what the scientific literature and the prior art suggest. In a second mutant, the hbd genes (encoding an enzyme with 3-hydroxybutyryl-CoA dehydrogenase activity) and hydA genes (encoding a hydrogenase) are inactivated.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors describe, in the context of the present invention, bacteria of the genus Clostridium, typically solvent-forming bacteria, in particular mutants of C. acetobutylicum which no longer produce, or produce almost no more, n-butanol, butyrate and acetone, and whose major (possibly exclusive) fermentation product is ethanol.

[0011] The inventors describe in particular a genetically modified bacterium, belonging to the genus Clostridium, in particular a solventogenic bacterium, whose hbd and hydA genes have been inactivated. This genetically modified bacterium is a bacterium not expressing the products of the hbd and hydA genes, in particular the products of the genes of sequences SEQ ID NO: 32 and SEQ ID NO: 69, or expressing non-functional versions of said products.

[0012] The inventors also describe a genetically modified bacterium, belonging to the genus Clostridium, in particular a solventogenic bacterium, in which the thlA gene, or the thlA and thlB genes have been inactivated. This genetically modified bacterium is a bacterium not expressing the product of the thlA gene, in particular the product of the gene of sequence SEQ ID NO: 67, or the products of the thlA and thlB genes, in particular the products of the genes of sequences SEQ ID NO: 67 and SEQ ID NO: 68, or expressing non-functional versions of said products.

[0013] Particularly preferred genetically modified bacteria according to the invention correspond to the strain identified in the present description as IFP 974 as registered on February 21, 2024 under deposit number LMG P-33549 with the BCCM-LMG collection (also identified in the present text as "hbd AhydA"), and to the strain identified in the present description as IFP 973 as registered on February 21, 2024 under deposit number LMG P-33548 with the BCCM-LMG collection (also identified in the present text as "AthlA thlB"). The strain identified in this description as IFP 972 as registered on February 21, 2024 under deposit number LMG P-33547 with the BCCM-LMG collection (also identified herein as "AthlA") may be used to prepare a strain equivalent to strain IFP 973.The description also relates to any bacteria derived, cloned, mutant or genetically modified version thereof, in particular characterized in that, like the IFP 972 strain, it produces more ethanol than a wild C. acetobutylicum bacterium. The invention also relates to any bacteria derived, cloned, mutant or genetically modified version of the bacteria according to the invention characterized in that, like the IF 973 and IFP 974 strains, it produces ethanol and does not produce acetone, butanol and / or butyric acid.

[0014] Another genetically modified bacterium described by the inventors is the strain identified in the present description as IFP 969 as registered on February 17, 2023 under deposit number LMG P-32993 with the BCCM-LMG collection (also identified herein as “Ahhd”), usable for preparing a strain equivalent to strain IFP974. The description also relates to any bacteria derived, cloned, mutant or genetically modified version thereof.

[0015] The inventors further describe methods for producing a recombinant bacterium as described herein, in particular methods comprising the deletion or inactivation of the hbd and hydA genes, the thlA gene, or the thlA and thlB genes, and optionally furthermore of another gene of interest, so as to prevent or decrease the expression of corresponding functional proteins, preferably a method involving CRISPR-Cas technology, as well as the genetically modified bacteria obtainable by this method, of which the bacteria IFP 969, IFP 974 and IFP 973 are examples.

[0016] They also describe the use of a genetically modified bacterium belonging to the genus Clostridium, characterized in that it does not express the product of the hbd and hydA genes or of the thlA gene or of the thlA and thlB genes, and optionally in addition of another gene of interest, or expresses a non-functional version of the products of the hbd and hydA genes, of the product of the thlA gene, or of the products of the thlA and thlB genes, and optionally in addition of another gene of interest, for preparing a genetically modified bacterium according to the invention not expressing the products of the hbd and hydA or thlA and thlB genes, or expressing one or more non-functional versions thereof.The inventors further describe the plasmids pGRNA-A / / bd of sequence SEQ ID NO: 29, pGRNA- hydA of sequence SEQ ID NO: 64, pGRN A- AthlA of sequence SEQ ID NO: 60, and pGRNA-AtWB of sequence SEQ ID NO: 62, as well as the use of one, several or all of them to transform, and preferably genetically modify, a bacterium of the genus Clostridium so as to improve its ethanol production capacity.

[0017] A method for transforming, and preferably genetically modifying, a bacterium of the genus Clostridium, as well as the genetically modified bacterium of the genus Clostridium obtained using the method are also described. This method comprises a step of transforming the bacterium by introducing into said bacterium a plasmid selected from plasmid pGRNA-A / / b<7 of sequence SEQ ID NO: 29, plasmid pGRNA-A / / ycM of sequence SEQ ID NO: 64, plasmid pGRNA-A / / / M of sequence SEQ ID NO: 60, and plasmid pGRNA-AtWB of sequence SEQ ID NO: 62.

[0018] The inventors also describe the use of a bacterium according to the invention to produce a bio-sourced molecule, for example a solvent, a biofuel or any (bio)chemical intermediate product, in particular ethanol.

[0019] Such bacteria can be advantageously used to produce a solvent or a mixture of solvents, particularly on an industrial scale.

[0020] The invention also relates to a fermentation process involving the use of a genetically modified bacterium as described in the present text.

[0021] The inventors finally describe kits, in particular a kit for transforming and preferably genetically modifying a bacterium belonging to the genus Clostridium, and a kit for producing a biosourced molecule, for example a solvent, a biofuel or any (bio)chemical intermediate product, using a bacterium belonging to the genus Clostridium, in particular ethanol, said kit comprising i) a genetically modified bacterium belonging to the genus Clostridium according to the invention and ii) a preservation medium or a culture medium for said bacterium, in particular a suitable culture medium containing a sugar or a mixture of sugars, preferably a hexose and / or a pentose, even more preferably glucose and / or arabinose and / or xylose.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Although used in industry for over a century, knowledge of bacteria belonging to the genus Clostridium, particularly solvent-forming bacteria, remains very limited.

[0024] The bacterium Clostridium acetobutylicum is today considered a model representative of solvent-forming Clostridia. Despite the difficulties, well known to those skilled in the art, encountered in genetically modifying bacteria belonging to the genus Clostridium, the inventors have succeeded, for the first time in the context of the present invention, in obtaining C. acetobutylicum bacteria capable of producing ethanol exclusively or almost exclusively. In a preferred embodiment, these mutants no longer produce, or almost no longer produce, n-butanol, butyrate and acetone.

[0025] The inventors have discovered and thus show for the first time that the joint inactivation of the hbd genes encoding an enzyme with 3-hydroxybutyryl-CoA dehydrogenase activity and hydA encoding a hydrogenase or the joint inactivation of the thlA and thlB genes both encoding a thiolase, makes it possible to obtain a strain which no longer produces, or produces almost no more, n-butanol, butyrate and acetone, and whose major (possibly exclusive) fermentation product is ethanol, as demonstrated in the experimental part of the present description. The inactivation of the thlA gene alone also makes it possible to advantageously obtain a strain producing more ethanol than a wild C. acetobutylicum bacterium.

[0026] An object described by the inventors thus relates to a genetically modified bacterium belonging to the genus Clostridium, in particular a species of Clostridium of industrial interest, in particular C. acetobutylicum, characterized in that it does not express the products of the genes hbd, preferably of sequence SEQ ID NO: 32 when the bacterium considered is C. acetobutylicum, and hydA, preferably of sequence SEQ ID

[0027] NO: 69 when the bacterium considered is C. acetobutylicum, or expresses non-functional versions thereof, or in that it does not express the products of the thlA genes, preferably of sequence SEQ ID

[0028] NO: 67 when the bacterium considered is C. acetobutylicum, and thlB, preferably of sequence SEQ ID

[0029] NO: 68 when the bacteria considered is C. acetobutylicum.

[0030] In the context of the present invention, the term "hbd gene" refers in particular to the sequence SEQ ID NO: 32 (CA_C2708). The term "hbd gene" also refers to variants of said sequence SEQ ID NO: 32, in particular variants having sequence homology with said sequence SEQ ID NO: 32. In the context of the present invention, the term "hydA gene" refers in particular to the sequence SEQ ID NO: 69 (CA_C0028) or a variant of said sequence, in particular a variant having sequence homology with said sequence SEQ ID NO: 69.

[0031] In the context of the present invention, the term "thlA gene" refers in particular to the sequence SEQ ID NO: 67 (CA_C2873). The term "thlA gene" also refers to the variants of said sequence SEQ ID NO: 67, in particular the variants having a sequence homology with said sequence SEQ ID NO: 67. In the context of the present invention, the term "thlB gene" refers in particular to the sequence SEQ ID NO: 68 (CA_P0078). The term "thlB gene" also refers to the variants of said sequence SEQ ID NO: 68, in particular the variants having a sequence homology with said sequence SEQ ID NO: 68.

[0032] A typical example of a variant according to the invention has a sequence homology with the sequence SEQ ID NO: 32, with SEQ ID NO: 69, with SEQ ID NO: 67 or with the sequence SEQ ID NO: 68 of between 95% and 100%, and preferably between 96% and 100%. The sequence SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67, or SEQ ID NO: 68 and its variant are for example homologous to at least 95%, for example to at least 96%, to at least 97%, to at least 98%, or to at least 99%. According to a preferred embodiment, the sequence SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67 or SEQ ID NO: 68 and its variant have sequences that are at least 96%, at least 97%, or at least 98% homologous.

[0033] The term "hbd gene" also refers to a sequence encoding a functional fragment or variant of the protein (enzyme) (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd), in particular a protein capable of / capable of exerting (S)-3-hydroxybutyryl-CoA dehydrogenase activity.

[0034] In a particular embodiment, the term “hbd gene” also refers to a sequence encoding a functional fragment or variant of the Hbd protein, in particular a protein capable of / capable of exerting (S)-3-hydroxybutyryl-CoA dehydrogenase activity.

[0035] The term "hydA gene" also refers to a sequence encoding a functional fragment or variant of the HydA protein (enzyme), in particular a protein capable of / able to exert hydrogenase activity.

[0036] In a particular embodiment, the term “hydA gene” also refers to a sequence encoding a functional fragment or variant of the HydA protein, in particular a protein capable of / capable of exercising a hydrogenase activity.

[0037] The term "thlA gene" also refers to a sequence encoding a functional fragment or variant of the ThlA protein (enzyme), in particular a protein capable of / able to exert acetyl-CoA acetyltransferase activity.

[0038] In a particular embodiment, the term “thlA gene” also refers to a sequence coding a functional fragment or variant of the ThlA protein, in particular a protein capable of / capable of exercising an acetyl-CoA acetyltransferase activity.

[0039] The term "thlB gene" also refers to a sequence encoding a functional fragment or variant of the ThlB protein (enzyme), in particular a protein capable of / able to exert acetyl-CoA acetyltransferase activity.

[0040] In a particular embodiment, the term “thlB gene” also refers to a sequence encoding a functional fragment or variant of the ThlB protein, in particular a protein capable of / capable of exercising an acetyl-CoA acetyltransferase activity.

[0041] When this text refers to a genetically modified bacterium belonging to the genus Clostridium, characterized in that it does not express the products of the hbd, hydA, thlA and / or thlB genes, or expresses non-functional versions thereof, the expression "non-functional version of the gene product" hbd, hydA, thlA or thlB means a protein, typically a protein identified in this text as "Hbd", "HydA", "ThlB" or "ThlA", which is non-functional, i.e., incapable of carrying out the function of the protein encoded by the wild-type version of the gene in question. By "bacteria of the genus Clostridium" is meant in particular the species of Clostridium said to be of industrial interest, typically the solventogenic or acetogenic bacteria of the genus Clostridium. The term "bacteria of the genus Clostridium" includes wild bacteria as well as strains derived from them, genetically modified for the purpose of improving their performance.

[0042] By "Clostridium species of industrial interest" or "Clostridium bacteria of industrial interest" is meant species capable of producing, by fermentation, solvents such as ethanol, butanol, acetone or isopropanol and / or acids such as butyric acid, acetic acid or lactic acid, from sugars or oses, typically from sugars comprising 5 carbon atoms such as xylose, arabinose or fructose, from sugars comprising 6 carbon atoms such as glucose or mannose, from polysaccharides or polysaccharides such as cellulose or hemicellulose, and / or from any other source of carbon assimilable and usable by bacteria of the genus Clostridium (CO, CO2, and methanol for example).Examples of solvent-forming bacteria of interest in the context of the present invention are bacteria of the genus Clostridium producing acetone, butanol, ethanol and / or isopropanol (propan-2-ol), such as the strains identified in the literature as "ABE strain" [strains carrying out fermentations allowing the production of acetone, butanol and ethanol], "IBE strain" [strains carrying out fermentations allowing the production of isopropanol (or propan-2-ol) by reduction of acetone, butanol and ethanol] and "AIBE strain" [strains carrying out fermentations allowing the production of acetone, isopropanol (or propan-2-ol), butanol and ethanol]. Solvent-forming bacteria of the genus Clostridium may be selected, for example, from, but not limited to, C. acetobutylicum, C. cellulolyticum, C. phytofermentans, C. beijerinckii, C. saccharobutylicum, C. saccharoperbutylacetonicum, C. sporogenes, C. butyricum, C.aurantibutyricum and C. tyrobutyricum, preferably among C. acetobutylicum, C. beijerinckii, C. butyricum, C. tyrobutyricum and C. cellulolyticum, and even more preferably among C. acetobutylicum and C. beijerinckii.

[0043] The acetogenic bacteria of interest are bacteria producing acids and / or solvents from CO2 and H2. Acetogenic bacteria of the genus Clostridium can be selected for example from C. aceticum, C. drakei, C. thermoaceticum, C. ragsdalei, C. ljungdahlii, C. autoethanogenum, C. difficile, C. scatologenes and C. carboxidivorans.

[0044] In a particular embodiment, the bacterium of the genus Clostridium concerned is the bacterium C. acetobutylicum, for example the strain DSM 792 (also designated strain ATCC 824 or LMG 5710) of C. acetobutylicum.

[0045] In another particular embodiment, the bacterium of the genus Clostridium concerned is the bacterium C. beijerinckii, for example the strain NCIMB 8052, DSM 6423, LMG 7814, LMG 7815, NRRL B-593 or NCCB 27006 of C. beijerinckii. Thus, according to a preferred embodiment, the bacterium according to the invention belonging to the genus Clostridium is a Clostridium bacterium of industrial interest, in particular a solventogenic bacterium, capable, in the wild state, of producing solvents and / or acids by fermentation from a carbon source, said carbon source being chosen for example from a sugar, CO, CO2, an alcohol, and an organic acid.In a particular preferred embodiment, the carbon source is a sugar, in particular a sugar comprising 5 carbon atoms such as xylose or arabinose, a sugar comprising 6 carbon atoms such as glucose, fructose or mannose, or a polysaccharide or polysaccharide such as cellulose or hemicellulose.

[0046] A particularly preferred bacterium belongs to the species C. acetobutylicum and, as explained above, does not express i) the products of the genes of sequence SEQ ID NO: 32 (CA_C2708) (or a sequence homologous to at least 95%, for example at least 96%, at least 97% or at least 98% thereof) and of sequence SEQ ID NO: 69 (CA_C0028) (or a sequence homologous to at least 95%, for example at least 96%, at least 97% or at least 98% thereof), ii) of sequence SEQ ID NO: 67 (CA_C2873) (or a sequence homologous to at least 95%, for example at least 96%, at least 97% or at least 98% thereof), or iii) of sequence SEQ ID NO: 67 and of sequence SEQ ID NO: 68 (CA_P0078) (or a sequence at least 95% homologous, for example at least 96%, at least 97% or at least 98% homologous thereto), or expresses non-functional versions of said expression products.The bacteria modified according to options i) and iii) described above, particularly preferred, have the technical abilities described above, i.e., they are capable of producing exclusively, or almost exclusively, ethanol, and on the contrary of no longer producing, or almost no longer producing, n-butanol, butyrate and acetone.

[0047] Such strains are characterized for the first time in the context of the present application.

[0048] One of these strains was registered on 21 February 2024 under the deposit number LMG P-33549 in the BCCM-LMG collection (IFP 974, “Ahbd hydA”). In this strain, the hbd and hydA genes have been (jointly) inactivated. The description also relates to any bacterial derivative, clone, mutant or genetically modified version thereof, typically lacking the hbd and hydA genes, or in which said genes have also been inactivated.

[0049] A second strain was registered on 21 February 2024 under the deposit number LMG P-33548 in the BCCM-LMG collection (IFP 973, “AthlA AthlB”). In this strain, the MA and MB genes have been (jointly) inactivated. The description also concerns any derived bacteria, clones, mutants or genetically modified versions thereof, typically lacking the MA and MB genes, or in which said genes have also been inactivated.

[0050] A third strain was registered on February 21, 2024, under the deposit number LMG P-33547 in the BCCM-LMG collection (IFP 972, “AhydA”). In this strain, the hydA gene has been inactivated. The description also relates to any bacterial derivative, clone, mutant or genetically modified version thereof, typically lacking the hydA gene, or in which said gene has also been inactivated.

[0051] Another strain was registered on February 17, 2023 under the deposit number LMG P-32993 in the BCCM-LMG collection (IFP 969, “Ahbd”). In this strain, the hbd gene has been inactivated. The description also relates to any bacterial derivative, clone, mutant or genetically modified version thereof, typically lacking the hbd gene, or in which said gene has also been inactivated.

[0052] The genetically modified bacterium according to the invention can be advantageously used to produce a solvent, for example a biofuel, preferably ethanol, or a mixture of solvents, for example a mixture of biofuels, preferably a mixture comprising ethanol and one or more other alcohols, in particular on an industrial scale.

[0053] The invention also relates to a fermentation process, typically an industrial process, involving the use of a bacterium according to the invention.

[0054] The present invention provides a welcome solution to optimize ethanol production.

[0055] A genetically modified bacterium according to the invention can also be advantageously used to produce a bio-sourced molecule, for example a solvent, a biofuel or any (bio)chemical intermediate product, from biomass or from a dedicated energy crop.

[0056] In the context of the present invention, the term "bio-sourced molecule" means a molecule, of the alcohol or ketone type, the specificity of which is that the raw material used for its production must necessarily come from plant biomass, for example lignocellulosic biomass, and not from resources of fossil origin such as oil, coal or natural gas. These are, for example, alcohols resulting from the fermentation of sugary juices, in particular C5 (with 5 carbons) and / or C6 (with 6 carbons) carried out by "solventogenic" strains of the genus Clostridium. Examples of bio-sourced molecules capable of being produced using the present invention are, firstly, ethanol (usable as biofuel), or n-propanol and 2,3-butanediol.

[0057] The invention further relates to a method for producing a recombinant bacterium according to the invention, comprising the deletion or inactivation of the hbd and hydA genes, the thlA gene, or the thlA and thlB genes, so as to prevent or reduce the expression of Hbd and HydA, ThlA, or ThlA and ThlB proteins.

[0058] The most successful known modification processes for obtaining genetically modified strains are based on homologous recombination events, which allow the genome to be modified in a precise and stable manner.

[0059] A particular production method according to the invention comprises a step of transforming the bacterium by introducing into said bacterium a nucleic acid of interest. For the purposes of the invention, the term "nucleic acid" means any natural, synthetic, semi-synthetic or recombinant DNA or RNA molecule, optionally chemically modified (i.e. comprising non-natural bases, modified nucleotides comprising, for example, a modified bond, modified bases and / or modified sugars), or optimized so that the codons of the transcripts synthesized from the coding sequences are the codons most frequently found in a bacterium of the genus Clostridium for use therein. In the case of the genus Clostridium, the optimized codons are typically codons rich in adenine ("A") and thymine ("T") bases.

[0060] 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.

[0061] In a particular embodiment described, the nucleic acid of interest comprises at least two complementary regions each of a target sequence, 100% identical or at least 80% identical, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said targeted DNA region / portion / sequence within the bacterial genome. These regions are capable of hybridizing to all or part of the complementary sequence of said region / portion / sequence, typically to a sequence as described above comprising at least 1 nucleotide, preferably at least 100 nucleotides, typically between 100 and 1000 nucleotides. The complementary regions of the target sequence present within the nucleic acid of interest can recognize, preferably target, the 5' and 3' flanking regions of the targeted sequence in a genetic modification tool known to those skilled in the art, typically any tool based on homologous recombination.

[0062] In a particular preferred embodiment, a portion of the nucleic acid of interest further recognizes (at least partially binds), and preferably targets, i.e., recognizes and allows the cleavage, in the genome of a Clostridium bacterium of interest, of at least one strand i) of a target sequence, ii) of a sequence controlling the transcription of a target sequence, or iii) of a sequence flanking a target sequence. The recognized sequence is also identified herein as a "target sequence" or "targeted sequence".

[0063] In this same preferred particular embodiment, the nucleic acid of interest comprises at least one complementary region of the target sequence which is 100% identical or at least 80% identical, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the targeted DNA region / portion / sequence within the bacterial genome and is capable of hybridizing to all or part of the complementary sequence of said region / portion / sequence, typically to a sequence comprising at least 5 nucleotides, preferably at least 5, 10, 14, 15, 20, 25, 30, 35 or 40 nucleotides, typically between 15 and 30 nucleotides, preferably to a sequence comprising 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0064] The nucleic acid(s) of interest as described in the context of the present invention are capable of deleting said target sequence(s) from the genome of the bacterium or of modifying their expression, for example of modulating / regulating them, in particular of inhibiting them, preferably of modifying them in such a way as to render said bacterium incapable of expressing one or more proteins, in particular one or more functional proteins, from said sequence(s).

[0065] According to another aspect of the invention, the nucleic acid(s) of interest as described in the context of the present invention are capable of introducing a sequence of interest into the genome of the bacterium so as to make said bacterium capable of expressing one or more proteins, in particular one or more functional proteins, from said sequence(s).

[0066] The “nucleic acids of interest”, typically expression cassettes or vectors, may be constructed by conventional techniques well known to those skilled in the art and may comprise one or more promoters, bacterial origins of replication (ORI sequences), termination sequences, selection genes, for example antibiotic resistance genes, and sequences (“flanked regions”) allowing the targeted insertion of the cassette or vector. ORI sequences of interest may for example be chosen from pIP404, pAMpi, ​​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 the plasmid, within a bacterial cell belonging to the genus Clostridium.Termination sequences of interest may be chosen, for example, from those of the adc, thl genes, the bcs operon, or any other terminator, well known to those skilled in the art, allowing the termination of transcription within a bacterial cell belonging to the genus Clostridium. Selection genes (resistance genes) of interest may be chosen from ermB, catP, bla, tetA, tetM, and / or any other gene for resistance to ampicillin, erythromycin, chloramphenicol, thiamphenicol, spectinomycin, tetracycline, or any other antibiotic, well known to those skilled in the art, which can be used to select bacteria of the genus Clostridium.

[0067] The nucleic acid of interest may be a natural, synthetic or recombinantly produced RNA. This nucleic acid of interest may 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 nucleic acid(s) of interest are introduced into the cell directly in the form of RNA molecules (mature or precursors), for example guide RNA (gRNA), these molecules may contain modified nucleotides or chemical modifications enabling them, for example, to increase their resistance to nucleases and thus increase their lifespan 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, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base allowing hybridization.

[0068] The nucleic acids of interest used according to the invention can also be modified at the level of the internucleotide bond as are for example phosphorothioates, H-phosphonates or alkyl-phosphonates, or at the level of the skeleton as are for example alpha-oligonucleotides, 2'-O-alkyl riboses or PNA (Peptide Nucleic Acids) (Egholm et al., 1992).

[0069] In the context of the present description, a particular example of a nucleic acid of interest, used to transform and / or genetically modify a bacterium of interest, is a DNA fragment i) recognizing a coding sequence, ii) controlling the transcription of a coding sequence, or iii) flanking a coding sequence, the enzyme Hbd (3-hydroxybutyryl-CoA dehydrogenase), the enzyme HydA (hydrogenase), the thiolase ThlA or the thiolase ThlB (acetyl-CoA acetyltransferases).

[0070] A nucleic acid of particular interest described by the inventors is for example a vector, preferably a plasmid, for example the plasmid pGRNA-A / / b<7 of sequence SEQ ID NO: 29, the plasmid pGRNA-hydA of sequence SEQ ID NO: 64, the plasmid pGRNA-A / / / M of sequence SEQ ID NO: 60 or the plasmid pGRNA-AtWB of sequence SEQ ID NO: 62, described in the experimental part of the present description.

[0071] The or one of the recognized sequences (target sequence(s)) is preferably one of the sequences SEQ ID NO: 39, SEQ ID NO: 36 or SEQ ID NO: 37 corresponding to the hbd, hydA, thlA and MB genes respectively encoding the Hbd protein, the HydA protein, the ThlA protein and the ThlB protein, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to said protein, or a sequence comprising all or at least 95%, 96%, 97%, 98% or 99% of the sequence SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67 or SEQ ID NO: 68. In other words, the recognized sequence may be 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 a sequence comprising 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides of the sequence SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67 or SEQ ID NO: 68.

[0072] According to another particular example, the target sequence may also be a sequence controlling the transcription of a coding sequence as described above, typically a promoter sequence, for example the promoter sequence of the “hbd” gene (CA_C2708, SEQ ID NO: 32), that of the “hydA” gene (CA_C0028, SEQ ID NO: 69), that of the “MA” gene (CA_C2873, SEQ ID NO: 67) or that of the “MB” gene (“CA_P0078” (SEQ ID NO: 68). The nucleic acid of interest then recognizes, and is therefore typically capable of binding to, a sequence controlling the transcription of a coding sequence as described above.

[0073] According to another particular example, the target sequence may be a sequence flanking a coding sequence as described above, for example a sequence flanking the sequence SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67, or SEQ ID NO: 68, or a sequence at least 70% identical thereto. Such a flanking sequence typically comprises 1, 10 or 20 and 1000 nucleotides, for example between 1, 10 or 20 and 900, 800, 700, 600, 500, 400, 300 or 200 nucleotides, between 1, 10 or 20 and 100 nucleotides, between 1, 10 or 20 and 50 nucleotides, or between 1, 10 or 20 and 40 nucleotides, for example between 10 and 40 nucleotides, between 10 and 30 nucleotides, between 10 and 20 nucleotides, between 20 and 30 nucleotides, between 15 and 40 nucleotides, between 15 and 30 nucleotides or between 15 and 20 nucleotides.

[0074] According to a particular aspect, the target sequence corresponds to the pair of sequences flanking such a coding sequence, each flanking sequence typically comprising at least 20 nucleotides, typically between 100 and 1000 nucleotides, preferably between 200 and 800 nucleotides.

[0075] Preferably, the method according to the invention for producing a recombinant bacterium comprises transforming the bacterial cell using at least one nucleic acid of interest, for example one, two, three or four nucleic acids of interest as described above, said nucleic acid(s) of interest being capable i) of recognizing (and capable of binding at least in part) a coding sequence, controlling the transcription of a coding sequence, or flanking a coding sequence, the Hbd protein, ii) of recognizing (and capable of binding at least in part) a coding sequence, controlling the transcription of a coding sequence, or flanking a coding sequence, the HydA protein, iii) of recognizing (and capable of binding at least in part) a coding sequence, controlling the transcription of a coding sequence, or flanking a coding sequence, the ThlA protein, and preferably iv) of recognizing (and capable of binding at least in part) a coding sequence,controlling the transcription of a sequence coding for, or flanking a sequence coding for, the ThlB protein.,

[0076] The nucleic acid of interest as described in the context of the present invention is preferably capable of deleting said target sequence from the genome of the bacterium or of modifying its expression, for example of modulating / regulating it, in particular of inhibiting it, preferably of modifying it so as to render said bacterium incapable of expressing a protein (typically an Hbd, HydA, ThlA or ThlB protein), in particular a functional protein, from said sequence. In a particularly preferred embodiment, the nucleic acid of interest is capable of modifying the bacterium so as to render it incapable of expressing one and / or the other of the Hbd, HydA, ThlA or ThlB proteins.

[0077] According to a particular embodiment, the description relates more particularly to the use of the plasmid pGRNA-A / / b<7 of sequence SEQ ID NO: 29, of the plasmid pGRNA-A / zydA of sequence SEQ ID NO: 64, of the plasmid pGRNA-At / z / A of sequence SEQ ID NO: 60 and / or of the plasmid pGRNA-At / z / B of sequence SEQ ID NO: 62 for transforming, and preferably genetically modifying, a bacterium of the genus Clostridium so as to improve its capacity for producing ethanol in particular, and according to a particular embodiment also n-propanol and / or 2,3-butanediol.

[0078] It also relates to a method for transforming, and preferably genetically modifying, a bacterium of the genus Clostridium, characterized in that it comprises a step of transforming the bacterium by introducing into said bacterium a plasmid as described in the present text, preferably a plasmid selected from the plasmid pGRNA-A / / b<7 of sequence SEQ ID NO: 29, the plasmid pGRNA-hydA of sequence SEQ ID NO: 64, the plasmid pGRNA-AtWA of sequence SEQ ID NO: 60 or the plasmid pGRNA-AtWB of sequence SEQ ID NO: 62. The description also relates to any genetically modified bacterium of the genus Clostridium obtained using such a method.

[0079] The introduction into the bacteria of any nucleic acid of interest can be 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 electroporation (Mermelstein et al, 1993).

[0080] Furthermore, these nucleic acids of interest (for example DNA fragments, RNA fragments, expression cassettes or expression vectors) can be integrated into the bacterial genome by techniques which are also well known to those skilled in the art.

[0081] In a particular embodiment, the method for transforming, and preferably genetically modifying, a bacterium as described in the present text, comprises a step of transforming the bacterium by introducing into said bacterium a nucleic acid of interest according to the invention as described above and involves a genetic modification tool, for example a genetic modification tool selected from a CRISPR tool, an insertional mutagenesis tool, for example based on the use of type II introns (for example the Targetron® tool or the ClosTron® tool) and an allelic exchange tool (for example the ACE® tool).

[0082] The method for transforming, and preferably further genetically modifying, a Clostridium bacterium may further comprise a step of obtaining, recovering, selecting or isolating the transformed bacterium, i.e. the bacterium exhibiting the desired recombination(s) / modification(s) / optimization(s).

[0083] In a particular embodiment, the method according to the invention is based on the use of (implements) the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology / genetic tool, in particular the CRISPR-Cas (CRISPR-associated protein) genetic tool. The present invention can be implemented using a conventional CRISPR-Cas genetic tool using a single plasmid comprising a nuclease, a gRNA and a repair template as described by Wang et al. (2015). The person skilled in the art can easily define the sequence and structure of the gRNAs according to the chromosomal region or the mobile genetic element to be targeted using well-known techniques (see for example the article by DiCarlo et al., 2013).The inventors have also developed and described a genetic tool for modifying bacteria, adapted to bacteria of the genus Clostridium, usable in the context of the present invention, based on the use of two plasmids (cf. WO2017 / 064439, Wasels et al., 2017).

[0084] In another embodiment, the method according to the invention is based on the use of insertional mutagenesis tools, for example the use of type II introns, and for example implements the ClosTron® technology / genetic tool or the Targetron® genetic tool. The Targetron® technology is based on the use of a reprogrammable group II intron (based on the Ll.ltrB intron of Lactococcus lactis), capable of integrating the bacterial genome rapidly at a desired locus (Chen et al., 2005, Wang et al., 2013), typically with the aim of inactivating a targeted gene. The mechanisms of recognition of the edited area as well as insertion into the genome by back-splicing are based on homology between the intron and said area on the one hand, and on the activity of a protein (LtrA) on the other hand.

[0085] ClosTron® technology is based on a similar approach, supplemented by the addition of a selection marker in the intron sequence (Heap et al., 2007). This marker allows for the selection of the intron's integration into the genome, and therefore facilitates the production of the desired mutants. This genetic system also exploits type I introns. Indeed, the selection marker (called RAM for retrotransposition-activated marker) is interrupted by such a genetic element, which prevents its expression from the plasmid (a more precise description of the system: Zhong et al.). Splicing of this genetic element occurs before integration into the genome, resulting in a chromosome with an active form of the resistance gene. An optimized version of the system includes FLP / FRT sites upstream and downstream of this gene, which allows the use of FRT recombinase to eliminate the resistance gene (Heap et al., 2010).

[0086] In another embodiment, the method according to the invention is based on the use of an allelic exchange tool, and for example implements the ACE® genetic technology / tool.

[0087] The ACE® technology is based on the use of an auxotrophic mutant (for uracil in C. acetobutylicum ATCC 824 by deletion of the pyrE gene, which also causes resistance to 5-fluoroorotic acid (5-FOA); Heap et al., 2012). This system uses the allelic exchange mechanism, well known to those skilled in the art. Following transformation with a pseudo-suicide vector (with very low copies), the integration of the latter into the bacterial chromosome by a first allelic exchange event can be verified using the resistance gene initially present on the plasmid. The integration step can be carried out in two different ways, either within the pyrE locus or within another locus. In the case of integration at the pyrE locus, the pyrE gene is also placed on the plasmid, but without being expressed (no functional promoter).The second recombination restores a functional pyrE gene and can then be selected by auxotrophy (minimal medium, not containing uracil). Since the non-functional pyrE gene also has a selectable trait (sensitivity to 5-FOA), other integrations can then be envisaged on the same model, successively alternating the state of pyrE between functional and non-functional. In the case of integration at another locus, a genomic area allowing expression of the counter-selection marker after recombination is targeted (typically, in an operon after another gene, preferably a highly expressed gene). This second recombination is then selected by auxotrophy (minimal medium not containing uracil).

[0088] In the described embodiments based on the use of type II introns, and for example implementing the ClosTron® technology / genetic tool or the Targetron® genetic tool, or based on the use of an allelic exchange tool, and for example implementing the ACE® technology / genetic tool, the targeted sequence is typically one of the sequences described in the present text. The invention further relates to a kit for transforming and / or genetically modifying a bacterium of the genus Clostridium comprising at least one nucleic acid of interest as described in the present text (for example two or three nucleic acids of interest, typically a DNA fragment, each recognizing a target sequence) for transforming and preferably genetically modifying a bacterium of the genus Clostridium, and optionally one or more selection molecules.A particular kit comprises the essential elements for the operation of a CRISPR tool (typically at least one nucleic acid usable as gRNA, one nucleic acid usable as a repair template, at least one pair of primers, and an inducer allowing the expression of a nuclease, in particular a Cas9 or Cas 12 type nuclease, for example MAD7), the essential elements for the operation of a tool based on the use of type II introns (typically at least one type II intron, at least one pair of primers and an inducer allowing the expression of a reverse transcriptase, for example type LtrA RT or Tel4c RT), or the essential elements for the operation of an allelic exchange tool (typically at least two nucleic acids usable as a homologous recombination template, and at least one pair of primers).

[0089] The kits according to the invention may further comprise one or more consumables such as a preservation medium or a culture medium, at least one competent bacterium of the genus Clostridium (i.e. conditioned for transformation), or even an explanatory leaflet.

[0090] The invention typically relates to a kit for implementing a method of transformation and / or genetic modification described in the present text using a bacterium of the genus Clostridium.

[0091] The description relates in particular to the genetically modified bacterium belonging to the genus Clostridium having the essential characteristic of not expressing the products of the genes hbd and hydA, thlA, or thlA and thlB, or of expressing non-functional versions thereof, as well as any bacteria derived, cloned, mutant or genetically modified version thereof, and their uses.

[0092] The application also describes strain IFP 969 (“Ahbd”), registered on February 17, 2023 under deposit number LMG P-32993 with the BCCM-LMG collection, in which the hbd gene has been inactivated, as well as any bacterial derivative, clone, mutant or genetically modified version thereof, typically lacking the hbd gene, or in which said gene has also been inactivated preferably using CRISPR technology.

[0093] The application also describes strain IFP 972 (“AthlA”), registered on February 21, 2024 under deposit number LMG P-33547 with the BCCM-LMG collection, in which the thlA gene has been inactivated, as well as any bacteria derived, cloned, mutant or genetically modified version thereof, typically lacking the thlA gene, or in which said gene has also been inactivated preferably using CRISPR technology. The application also describes strain IFP 974 (“Ahbd AhydA”), registered on February 21, 2024 under deposit number LMG P-33549 with the BCCM-LMG collection, in which the hbd and hydA genes have been inactivated, as well as any bacteria derived, cloned, mutant or genetically modified version thereof, typically lacking the hbd and hydA genes, or in which said genes have also been inactivated.The application also describes strain IFP 973 (“AthlA AthlB”), registered on February 21, 2024 under deposit number LMG P-33548 with the BCCM-LMG collection, in which the MA and MB genes have been inactivated, as well as any derived bacteria, clones, mutants or genetically modified versions thereof, typically lacking the MA and MB genes, or in which said genes have also been inactivated. It relates in particular to the use of a genetically modified bacterium belonging to the genus Clostridium, characterized in that it does not express the products of the hbd gene, or expresses a non-functional version of the products of the hbd gene, for example the strain IFP 969, registered on February 17, 2023 under the deposit number LMG P-32993 with the BCCM-LMG collection, to prepare a genetically modified bacterium according to the invention not expressing the products of the hbd and hydA genes, or expressing non-functional versions thereof.

[0094] It also relates in particular to the use of a genetically modified bacterium belonging to the genus Clostridium, characterized in that it does not express the products of the MA gene, or expresses a non-functional version of the products of the MA gene, for example the strain IFP 972, registered on February 21, 2024 under the deposit number LMG P-33547 with the BCCM-LMG collection, to prepare a genetically modified bacterium according to the invention not expressing the products of the MA and MB genes, or expressing non-functional versions thereof.

[0095] The invention further relates to a kit for producing a bio-sourced molecule, for example a solvent, a biofuel or any (bio)chemical intermediate product, using a bacterium belonging to the genus Clostridium, comprising i) a genetically modified bacterium belonging to the genus Clostridium according to the invention, for example IFP 969, IFP 972, IFP 973 or IFP 974, and ii) a medium, typically a preservation medium or a culture medium for said bacterium. The kit may further comprise an explanatory leaflet.

[0096] The medium for preserving or culturing the genetically modified bacterium (belonging to the genus Clostridium) according to the invention present in the kit is preferably supplemented with a carbon source composed of glucose and / or at least one pentose, preferably i) glucose and / or ii) arabinose and / or i) xylose. This medium preferably comprises between 0.1 and 250 g / L, more preferably between 1 and 100 g / L, of said carbon source (composed of glucose and / or arabinose and / or xylose).

[0097] The preservation or culture medium for the genetically modified bacteria is preferably an RCM type culture medium, more preferably a GAPES type culture medium, even more preferably a CGM type culture medium.The description also relates to a particular kit for producing a solvent or a biofuel, or a mixture of solvents or biofuels, in particular ethanol, or a mixture comprising ethanol and one or more other alcohols, using a bacterium belonging to the genus Clostridium, said kit comprising i) a bacterium (belonging to the genus Clostridium) genetically modified according to the invention, characterized in that it does not express the products of the genes hbd and hydA, thlA, or thlA and thlB, or expresses non-functional versions thereof, and ii) a culture medium, preferably an RCM-type culture medium, more preferably a GAPES-type culture medium, even more preferably a CGM-type culture medium, containing at least one carbon source, preferably glucose and / or arabinose and / or xylose.

[0098] The invention further relates to the possible uses of the method or kit according to the invention for transforming and / or genetically modifying a bacterium of the genus Clostridium, typically a solvent-forming bacterium of the genus Clostridium, for example for generating improved variants of said bacterium.

[0099] Finally, it concerns the possible uses of the process, the kit or a bacterium of the genus Clostridium transformed and preferably genetically modified according to the invention, in particular to enable the production of biosourced molecules, for example solvent(s), biofuel(s) or (bio)chemical intermediate product(s), or mixtures thereof, typically on an industrial scale.

[0100] The examples and the figure below are intended to illustrate the invention more fully without limiting its scope. In particular, these examples present the production and characterization of bacteria according to the invention, in which the inactivation of hbd, hydA, thlA and / or thlB genes, in particular hbd and hydA on the one hand and thlA and thlB on the other hand, is carried out according to a particular preferred embodiment using a CRISPR-Cas9 tool. Said genes can be inactivated, or introduced, according to other particular embodiments, well known to those skilled in the art, based for example on the inactivation, or introduction, of gene by homologous recombination or by insertional mutagenesis, as explained above.

[0101] FIGURE

[0102] [Fig 1] Figure 1 represents the central metabolism of C. acetobutylicum. C. acetobutylicum produces acetate, butyrate, and eventually lactate during acidogenesis. During the solventogenesis phase, butyrate and acetate are reassimilated, and the carbon flux is redirected toward the production of acetone, ethanol, and n-butanol. Ack, acetate kinase; Adc, acetoacetate decarboxylase; Adh, alcohol dehydrogenase; Aid, aldehyde dehydrogenase; Aide, acetolactate decarboxylase; Als, acetolactate synthase; Bed, butyryl-CoA dehydrogenase; Buk, butyrate kinase; CtfA-CtfB, butyrate-acetoacetate CoA-transferase (subunits A and B); Crt, crotonase; EtfA-EtfB, electron transfer flavoprotein (subunits a and (3); Fnor, ferredoxin-NAD(P) +oxidoreductase; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; HydA, hydrogenase; Ldh, lactate dehydrogenase; Pdc, pyruvate decarboxylase; Pfor, pyruvate ferredoxin oxidoreductase; Pta, phosphate acetyltransferase; Ptb, phosphate butyryltransferase; Thl, thiolase.

[0103] EXAMPLES

[0104] Materials and methods

[0105] Strains, plasmids and culture media

[0106] C. acetobutylicum DSM 792 (Deutsche Sammlung von Mikroagencen und Zellkulturen, DSMZ) was grown at 34°C under anaerobic conditions (90% N2, 5% CO2, 5% N2) in 2YTG medium (tryptone 16 gL 1 , yeast extract 10 gL 1 , glucose 5 gL 1 , NaCl 4 gL 1 ). Escherichia coli NEB 10-beta (New England Biolabs, NEB) was grown at 37°C under aerobic conditions in LB medium (tryptone 10 gL 1 , yeast extract 5 gL 1 , NaCl 10 gL 1). Solid media were made by adding 15 gL 1 agarose to liquid media. If necessary, erythromycin (Em, 40 mg.L 1 ) and / or thiamphenicol (Tm, 15 mg.L 1 ) were used for C. acetobutylicum cultures. Similarly, chloramphenicol (12.5 mg.L 1 in liquid medium and 25 mg.L 1 in solid medium) and / or tetracycline (20 mg.L 1 ) were used for E. coli cultures.

[0107] The plasmids used in this study are shown in Table 1 below.

[0108] Table 1: a ermB, erythromycin resistance gene; catP, thiamphenicol and chloramphenicol resistance gene; tetA, tetracycline resistance gene; ColEl and p15A ori, origins of replication in E. coli; pIP404, pAMBl and pCB102, origins of replication in C. acetobutylicum. Plasmid construction

[0109] Nucleic acids were purified using the QIAquick PCR Purification Kit (Qiagen), QIAprep Spin Miniprep Kit (Qiagen), and GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich). PCR amplifications were performed with Q5 High-Fidelity DNA Polymerase (NEB). The oligonucleotides used for plasmid construction are shown in Tables 2 A and B below.

[0110] Table 2 A:

[0111] Plasmid pGRNA- / / b<7 (SEQ ID NO: 28) was constructed by cloning the hybridization product of oligonucleotides P01 and P02 into pGRNAmd (SEQ ID NO: 25) using BsaI and T4 DNA ligase (NEB). The fragment obtained by overlapping PCR of amplifications obtained from DSM 792 gDNA using oligonucleotide pairs P03-P04 and P05-P06 was cloned at the BamHI and SalI sites into pGRNA- / / b<7 to obtain pGRNA-A / / b<7 (SEQ ID NO: 29). Plasmid pGRNA-CA / / b<7 (SEQ ID NO: 30) was constructed by cloning the hybridization product of oligonucleotides P13 and P14 into pGRNAmd using Bsal and T4 DNA ligase (NEB). pGRNA-C / / b<7 (SEQ ID NO: 31) was obtained by HiFi assembly (NEB) of pGRNA-CA / / b<7 digested with BamHI and SalI and the amplification products obtained from DSM 792 gDNA using oligonucleotide pairs P15-P16 and P17-P18. Table 2B:

[0112] Plasmid pGRNA-thlA (SEQ ID NO: 59) was constructed by cloning the hybridization product of oligonucleotides P01 and P02 into pGRNAmd (SEQ ID NO: 25) using BsaI and T4 DNA ligase (NEB). PCR fragments obtained from DSM 792 gDNA using oligonucleotide pairs P03-P04 and P05-P06 were cloned at BamHI and SalI sites in pGRNA-thlA by HiFi DNA Assembly (NEB) to obtain pGRNA-A / / / M (SEQ ID NO: 60). Plasmid pGRNA-tWB (SEQ ID NO: 61) was constructed by cloning the hybridization product of oligonucleotides P07 and P08 into pGRNAmd (SEQ ID NO: 25). The fragments obtained by PCR from DSM 792 gDNA using oligonucleotide pairs P09-P10 and P11-P12 were cloned at BamHI and SalI sites in pGRNA-t / zZB by HiFi DNA Assembly to obtain pGRNA-At / zZB (SEQ ID NO: 62).Plasmid pGRNA-ZzydA (SEQ ID NO: 63) was constructed by cloning the hybridization product of oligonucleotides P13 and P14 into pGRNAhd. The fragments obtained by PCR from DSM 792 gDNA using oligonucleotide pairs P15-P16 and P17-P18 were cloned at BamHI and SalI sites in pGRNA-ZzydA by HiFi DNA Assembly to obtain pGRNA-A / zyrZA (SEQ ID NO: 64). Plasmid pFWOl-t / zZA (SEQ ID NO: 66) was constructed by cloning at the XhoI and SalI sites the fragment obtained by PCR from F gDNA of DSM 792 using the oligonucleotide pair P19-P20 in pFWOl (SEQ ID NO: 65) by HiFi DNA Assembly.

[0113] Genetic editing

[0114] Plasmid constructs were introduced into C. acetobutylicum as described by Mermelstein LD et al.. Gene editing events were selected as described by Wasels F. et al. (2020) and Wasels F. et al. (2017). Oligonucleotides used for confirmation of gene editing are shown in Table 3 (A and B) below.

[0115] Table 3 A:

[0116] Table 3B:

[0117] The use of other genetic tools, such as those based on homologous recombination or the insertion of mobile genetic elements, can make it possible to obtain mutants with equivalent genotypes, i.e. no longer expressing the products of the hbd, thlA, thlB and / or hydA genes, or expressing a non-functional version of these. Fermentation

[0118] The fermentation performances of the microorganisms described in this study were evaluated in batch. Precultures were carried out in an anaerobic chamber in a volume of 1 mL of CGM medium (KH2PO4 0.75 gL 1 , K2HPO40.75 gL 1 , MgSO4H2O 0.4 gL 1 , MnSO4H2O 0.01 gL 1 , FeSO4-7H2O 0.01 gL 1 , NaCl 1.0 gL 1 , Asparagine 2.0 gL 1 , Yeast extract 5.0 gL 1 , (NIL^SCL 2.0 gL 1 , Glucose 80 gL 1 ). After an 18-hour incubation, a volume of 500 pL of these precultures was used to inoculate 9.5 mL of CGM medium into flasks. Once crimped, the flasks were then incubated for 96 hours at 34°C, 100 rpm. At the end of fermentation, samples were centrifuged at 5000 g for 5 minutes, and the supernatants were diluted using an internal standard (final concentration of 0.5 gL 1of n-propanol) then filtered at 0.22 pm before being analyzed by chromatography.

[0119] Solvent detection was performed by gas chromatography on a PoraBOND-Q column (Agilent Technologies) with a flame ionization detector. Helium was used as the carrier gas at a flow rate of 1.6 mL.min 1 , and the column heated from 50 to 250°C during a 30-min run. Acid detection was performed by high-performance liquid chromatography on an Aminex HPX-87H column (Biorad) coupled with a Spectra System RI-150 refractometer and a Waters 2487 dual X UV detector set at 210 nm. The mobile phase consisted of a 0.1 M sulfuric acid solution, and the column temperature was set at 60°C. The results presented are the average of at least three independent runs.

[0120] Genomic DNA sequencing

[0121] Genomic DNA of the thlA thlB double mutant was sequenced by INVIEW Resequencing (Next Gen Sequencing 2x151 nt, Illumina) at Eurofins Genomics. A total of 2 x 13,957,557 reads were obtained and aligned to the C. acetobutylicum DSM 792 genome using Geneious Prime 2023.0.4 (https: / / www.geneious.com). Chromosome coverage averaged 819x, and megaplasmid pSOL coverage averaged 1868x.

[0122] Results

[0123] Creating a mutant \hbd

[0124] A Mibd mutant was constructed in C. acetobutylicum strain DSM 792. The fermentation performance of this mutant compared to the wild-type strain is shown in Table 4 below. Table 4:

[0125] This mutant is no longer capable of producing n-butanol or butyrate. It allows the production of significant quantities of ethanol, which becomes its predominant fermentation product. The amount of acetone produced is reduced by more than 65% compared to the wild-type strain.

[0126] To reduce the amount of ethanol produced by this mutant, the pSOL megaplasmid containing the adhEl and adhE2 genes encoding enzymes with acetaldehyde dehydrogenase (Aid) activity was targeted to F using the CRISPR-Cas9 tool (described in application WO2017 / 064439) used for the creation of the 2Jibd mutant. The loss of the megaplasmid is easily achievable in the wild-type strain, and only requires the introduction of a plasmid allowing the expression of a gRNA targeting this nucleic acid (Wasels et al., 2017).

[0127] The frequency of obtaining transformants of the wild strain containing the plasmid pCas9 acr with plasmid pEC750C is of the order of 101 at 10 2 ufc.pgDNA -1 .

[0128] The frequency of obtaining transformants of the wild strain containing the plasmid pCas9 acr with a plasmid derived from pGRNA con targeting the microorganism's chromosome and not containing an editing template for the targeted locus, in the presence of anhydrotetracycline (aTc), the inducer of Cas9 expression, is zero.

[0129] In addition, the frequency of obtaining transformants of the wild strain containing the plasmid pCas9 acr with a plasmid derived from pGRNA con targeting the pSOL megaplasmid in the presence of aTc is of the order of 10 1 at 10 2 ufc.pgDNA -1 Analysis of the transformants obtained shows that they have lost the pSOL megaplasmid.

[0130] The frequency of obtaining transformants of the Jibd mutant containing the pCas9 plasmid acr with plasmid pEC750C is usually in the order of 10 3ufc.pgDNA 1 . Surprisingly, the transformation frequency of this mutant with a plasmid derived from pGRNA con targeting the pSOL megaplasmid in the presence of aTc and not containing an editing template for the targeted locus is zero. This result indicates that the pSOL megaplasmid is essential in the hbd mutant and cannot be eliminated, unlike what is observed in the wild-type strain. It therefore becomes possible to use the CRISPR-Cas9 tool to make precise modifications within the megaplasmid in the Jibd mutant.

[0131] As an example, the plasmid pGRNA-Apdc was used to inactivate the pdc gene located on pSOL. Transformation of the wild-type strain DSM 792 with this plasmid induces the loss of the pSOL megaplasmid in the resulting transformants. On the other hand, it was possible to obtain bJibd pdc mutants whose fermentation performances are presented in Table 5 below.

[0132] Table 5:

[0133] Following the various genetic edits that can be carried out in the pSOL megaplasmid at T using the CRISPR-Cas9 tool, the latter can be used to reintroduce the hbd gene, and thus retain only the other modifications, located on the megaplasmid and / or on the chromosome. This complementation at the locus can be carried out for example with the plasmid pGRNA-C / / b<7 (SEQ ID NO: 31) which makes it possible to reintroduce a functional gene that does not impact the performance of the microorganism compared to the wild-type strain.

[0134] The performance of DSM 792 hbdc mutants derived from the hbd mutant is shown in Table 6 below. Table 6:

[0135] The results obtained show that the complementation of the hbd gene is functional and that the mutant in which the gene has been reintroduced at its original locus behaves in the same way as the wild-type strain.

[0136] Creation of a double mutant AthlA AthlB

[0137] A mutant in which the thlA gene (CA_C2873, SEQ ID NO: 67) was inactivated was constructed in C. acetobutylicum strain DSM 792 with a CRISPR-Cas9 tool (Wasels et al., 2020). The fermentation performance of this mutant compared to the wild-type strain is shown in Table 7.

[0138] Table 7:

[0139] Ethanol becomes the major fermentation product of this mutant, which retains the ability to produce n-butanol and butyrate. The presence of a second copy of a gene encoding an enzyme with thiolase activity (thlB, CA_P0078, SEQ ID NO: 68), located on the pSOL, may explain this phenomenon. However, just as was observed in the hbd mutant (application FR2303203 filed on March 31, 2023), the pSOL becomes an essential genetic element in the thlA mutant, allowing its editing, for example, with a CRISPR-Cas9 tool. A thlA thlB double mutant was therefore constructed, and its batch fermentation performances are presented in Table 8.

[0140] Table 8:

[0141] This double mutant no longer produces acetone, butanol, or butyrate, as would be expected from a microorganism that no longer possesses a thiolase enzyme. The genome of this mutant has been sequenced and shows no changes other than the deletions of the thlA and thlB genes.

[0142] Complementation of the double mutant AthlA AthlB

[0143] To confirm that the observed phenotype (100% ethanol) is indeed due to the double deletion performed, the corresponding mutant was complemented with a plasmid expressing the thlA gene. The performances of the corresponding transformants are presented in Table 9. Table 9:

[0144] We clearly observe a restoration of the wild phenotype. Creation of a double mutant \hbd \hydA

[0145] Like thlA, hydA (SEQ ID NO: 69) has been described as essential and cannot be deleted (Cooksley et al., 2012). However, a double Ahbd AhydA mutant could be constructed from an Ahbd mutant as described above, using the CRISPR-Cas9 tool (Wasels et al., 2020). The performance of this mutant is shown in Table 10. Table 10:

[0146] The deletion of hbd suppresses the production of butyrate and butanol, as reported by Lehmann D. et al. (2011). The double deletion Ahbd AhydA has the effect of reducing the amount of acetone produced to negligible quantities, making ethanol the only fermentation product. The fermentation profile is therefore similar to that obtained with the AthlA AthlB mutant according to the invention.

[0147] Conclusions During this work, the inventors demonstrate the possibility and the interest of using a CRISPR-Cas9 tool to carry out precise genetic modifications within the pSOL megaplasmid of the C. acetobutylicum DSM 792 strain, since the said strain no longer expresses, at choice, the hbd gene and the hydA gene, or the thlA gene and the thlB gene.

[0148] The inventors describe, within the framework of the present invention, two double mutants of C. acetobutylicum, the AthlA AthlB mutant and the Ahbd AhydA mutant, which no longer produce acetone, butanol or butyrate and therefore only produce ethanol. REFERENCES

[0149] - Cooksley CM, Zhang Y, Wang H, Redl S, Winzer K, Minton NP. 2012. Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway. Metabolic Engineering 14:630-641. doi:10.1016 / j.ymben.2012.09.001.

[0150] - DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M. (2013). Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research, 47(7), 4336-4343.

[0151] - Dong, D., Guo, M., Wang, S., Zhu, Y., Wang, S., Xiong, Z., ... & Huang, Z. (2017). Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein. Nature, 546(165 ), 436-439.

[0152] - Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal gene knock-out system for the genus Clostridium. Journal of Microbiological Methods 70:452-464. doi:10.1016 / j.mimet.2007.05.021.

[0153] - Joseph RC, Kim NM, Sandoval NR. 2018. Recent Developments of the Synthetic Biology Toolkit for Clostridium. Front. Microbiol. 9:154. doi:10.3389 / fmicb.2018.00154.

[0154] - Lehmann D, Lütke-Eversloh T. 2011. Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate / butanol fermentative pathway. Metabolic Engineering 13:464-473. doi:10.1016 / j.ymben.2011.04.006.

[0155] - Marino, N. D., Zhang, J. Y., Borges, A. L., Sousa, A. A., Leon, L. M., Rauch, B. J., ... & Bondy-Denomy, J. (2018). Discovery of widespread type I and type V CRISPR-Cas inhibitors. Science, 362(6411), 240- 242.

[0156] - Mermelstein LD, Papoutsakis ET. 1993. In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Applied and Environmental Microbiology 59:1077-1081. doi:10.1128 / aem.59.4.1077-1081.1993

[0157] - Nôlling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, Wolf YI, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, Smith DR. 2001. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183:4823—4838. doi:10.1128 / JB.183.16.4823-4838.2001.

[0158] - Pawluk, A., Davidson, A. R., & Maxwell, K. L. (2018). Anti-CRISPR: discovery, mechanism and function. Nature Reviews Microbiology, 16(1), 12-17.

[0159] - Rauch, B. J., Silvis, M. R., Hultquist, J. F., Waters, C. S., McGregor, M. J., Krogan, N. J., & Bondy- Denomy, J. (2017). Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell, 168(1-2), 150-158.

[0160] - Wasels F, Jean-Marie J, Collas F, Lopez-Contreras AM, Lopes Ferreira N. 2017. A two-plasmid inducible CRISPR / Cas9 genome editing tool for Clostridium acetobutylicum. Journal of Microbiological Methods 140:5-11. doi:10.1016 / j.mimet.2017.06.010. - Wasels F, Chartier G, Hocq R, Lopes Ferreira N. 2020. A CRISPR / Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792. Applied and Environmental Microbiology 86. doi:10.1128 / AEM.00408-20.

[0161] - K. Winzer et al. , Differential regulation of two thiolase genes from Clostridium acetobutylicum DSM 792. ; J. Mol. Microbiol. Biotechnol. (2000) 2(4) : 531-541.

Claims

CLAIMS 1. Genetically modified solvent-producing bacterium belonging to the genus Clostridium producing ethanol without producing acetone, butanol and / or butyric acid, characterized in that i) it does not express the products of the genes hbd (CA_C2708) of sequence SEQ ID NO: 32 and hydA (CA_C0028) of sequence SEQ ID NO: 69, or expresses non-functional versions thereof, or ii) it does not express the products of the genes MA (CA_C2873) of sequence SEQ ID NO: 67 and thlB (CA_P0078) of sequence SEQ ID NO: 68, or expresses non-functional versions thereof.

2. Bacterium according to claim 1, characterized in that the bacterium is C. acetobutylicum.

3. Bacteria according to claim 2, characterized in that the bacterium is the strain IFP973 registered on February 21, 2024 under the number LMG P-33548 with the BCCM-LMG collection, or genetically modified version thereof, said bacteria and genetically modified version thereof being characterized in that they produce ethanol and do not produce acetone, butanol and / or butyric acid.

4. Bacteria according to claim 2, characterized in that the bacterium is the strain IFP974 registered on February 21, 2024 under the number LMG P-33549 with the BCCM-LMG collection or genetically modified version thereof, said bacterium and genetically modified version thereof being characterized in that they produce ethanol and do not produce acetone, butanol and / or butyric acid.

5. Use of the genetically modified bacteria according to one of claims 1 to 4, for producing ethanol, preferably on an industrial scale.

6. A fermentation process involving the use of a bacterium as described in any one of claims 1 to 4.

7. Kit for producing ethanol, using a bacterium belonging to the genus Clostridium, comprising a genetically modified bacterium belonging to the genus Clostridium according to any one of claims 1 to 4 and a medium for preserving or culturing said bacterium.

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