Modified clostridium autoethanogenum bacteria, their preparation and use

Abstract

The present invention relates to genetic modifications of Clostridial bacteria, typically solventogenic Clostridial bacteria, to enable said bacteria to produce ethanol. Methods, tools and kits for obtaining said bacteria, the obtained genetically modified bacteria and uses thereof are also described.

Description

Technical Field

[0001] This invention relates to the genus Clostridium ( Clostridium Genetic modification of bacteria (typically solvent-producing bacteria of the genus Clostridium). In particular, the inventors describe methods, tools, and kits for removing or modifying one or more sequences encoding or controlling the transcription of sequences encoding enzymes such as: an enzyme having 3-hydroxybutyryl-CoA dehydrogenase (Hbd) activity and a hydrogenase (HydA) or two thiolases (ThlA and ThlB). Genetically modified bacteria thus obtained and their uses, particularly for the production of ethanol, are also described, possibly for the production of ethanol alone. Background Technology

[0002] Bacteria belonging to the genus *Clostridium* are strictly anaerobic, Gram-positive bacilli capable of forming endospores and belong to the phylum *Firmicutes*. This genus contains many species studied for their pathogenic properties or their industrial and medical value.

[0003] Industrially valuable Clostridium species are nonpathogenic and capable of producing compounds of interest, such as acids and solvents from a variety of sugars ranging from glucose to cellulose and substrates. The growth of solvent-producing (“solvent-producing” or “acid-producing”) Clostridium species is described as two-stage. Acids are produced during the acid-producing stage, which corresponds to the exponential growth phase. When cell growth ceases and the bacteria enter the stationary phase, they enter the solvogenic stage, re-assimilate the produced acids, and convert them into solvents.

[0004] Clostridium acetonebutanol ( Clostridium acetobutylicum A mixture of ethanol, acetone, and n-butanol can be naturally produced during a fermentation process known as ABE fermentation. Figure 1 ).

[0005] Due to the relatively high titers achieved during fermentation, *Clostridium acetone-butanol* is considered a model organism for solvent-producing microorganism research. Numerous genetic tools have been specifically developed to perform metabolic engineering, allowing for the reorientation of its metabolic pathways. For example, in 2011, Lehmann and Lütke-Eversloh used ClosTron (Heap JT...) to... et al (., 2007) describes a Δ hbd The construction of mutants that no longer produce butyrate or butanol as final fermentation products. The following year, Cooksley... et al. (2012) also used the same tools to construct many mutants, among which different genes in the central metabolism ( adhE1 , adhE2 , bdhA , bdhB ,ptb , ack , ctfA , ctfB , adc CA_P0059) is inactivated. In this study, thlA The gene is one of two copies encoding thiolytic enzymes and is targeted in two different ways, but cannot be inactivated, leading these authors to conclude that the gene is indispensable. However, it is interesting to note, however, that the mutant Cac- obtained in the same study... ptb 505a::CT has a nucleotide insertion, which leads to thlA Shifting of the reading frame within the gene leads to its inactivation. Due to the contradictory nature of the impossibility of deleting this gene using the ClosTron tool, these authors conclude the following: thlA and ptb Double inactivation is possible, but it may be... thlA Single gene inactivation may not be possible (Cooksley) et al Similarly, it has been shown that when with hydA Inactivation occurs simultaneously. thlA Inactivation is possible. hydA The product is hydrogenase (US20170240869). These independent results appear to indicate that the thlA gene is indispensable. However, as the inventors have taught in the context of this invention, Clostridium acetobutane contains a second copy of the gene encoding thiolase on the pSOL giant plasmid ( thlB Whether in acid formation or solvent formation, its expression is related to thlA Compared to the relatively weaker (K. Winzer) et al ., Differential regulation of twothiolase genes from Clostridium acetobutylicum DSM 792. ; J. Mol. Microbiol. Biotechnol. (2000) 2(4): 531-541).

[0006] In the context of this invention, the inventors have for the first time described a novel Clostridium acetone-butanol mutant that no longer produces or almost no longer produces n-butanol, butyric acid, and acetone, and whose main (and possibly only) fermentation product is ethanol. In one of these variants, the enzyme encoding thiolase... thlA Genes and thlB The gene is inactivated, with no other visible changes in the genome, contrary to what scientific literature and existing technology teach. In the second mutant, hbd (Enzymes that encode 3-hydroxybutyryl-CoA dehydrogenase activity) andhydA The gene encoding hydrogenase is inactivated. Summary of the Invention

[0007] In the context of this invention, the inventors describe bacteria of the genus Clostridium, typically solvent-producing bacteria, particularly Clostridium acetone-butanol mutants, which no longer or almost no longer produce n-butanol, butyrate, and acetone, and whose main (and possibly only) fermentation product is ethanol.

[0008] The inventors specifically described genetically modified bacteria belonging to the genus Clostridium. ， Especially solvent-producing bacteria, hbd and hydA The gene is nearly inactivated. This genetically modified bacterium is one that does not express... hbd and hydA The product of the gene (especially the product of the gene with sequences SEQ ID NO: 32 and SEQ ID NO: 69), or a nonfunctional version thereof expressing the product.

[0009] The inventors also describe genetically modified bacteria belonging to the genus Clostridium, particularly solvent-producing bacteria, whose thlA Gene or thlA and thlB The gene has been inactivated. This genetically modified bacterium is one that does not express... thlA The product of the gene (especially the product of the gene with sequence SEQ ID NO: 67), or thlA and thlB The product of the gene (especially the product of the gene with sequences SEQ ID NO: 67 and SEQ ID NO: 68), or a nonfunctional version thereof expressing the product.

[0010] The genetically modified bacteria particularly preferred according to the present invention correspond to strain IFP 974 and strain IFP 973 identified in this specification. Strain IFP 974 was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMGP-33549 (also identified herein as "Δ"). hbd Δ hydA IFP strain 973 was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33548 (also referred to as "Δ" in this article). thlA Δ thlB The strain identified as IFP 972 in this specification was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33547 (also identified as "Δ" in this document).thlA This invention relates to any derived bacteria, clones, mutants, or genetically modified versions thereof, characterized in that, like strain IFP 972, it produces more ethanol than the wild-type Clostridium acetone-butanol. The invention also relates to any derived bacteria, clones, mutants, or genetically modified versions of the bacteria according to the invention, characterized in that, like strains IFP 973 and IFP 974, it produces ethanol and does not produce acetone, butanol, and / or butyric acid.

[0011] Another genetically modified bacterium described by the inventors is strain IFP 969, which was registered at the BCCM-LMG Collection Center on February 17, 2023, with accession number LMG P-32993 (also referred to herein as "Δ"). hbd This specification can be used to prepare strains equivalent to strain IFP 974. This specification also covers any derived bacteria, clones, mutants, or genetically modified versions thereof.

[0012] The inventors also describe a method for producing recombinant bacteria as described herein, particularly including deletion or inactivation of... hbd Genes and hydA Genes, missing or inactivated thlA Genes, either missing or inactivated. thlA Genes and thlB Genes, and optionally another gene of interest, for preventing or reducing the expression of the corresponding functional protein, preferably involving a method of CRISPR-Cas technology, and genetically modified bacteria that can be obtained by the method, wherein bacteria IFP969, IFP974 and IFP973 are examples.

[0013] The inventors also describe the use of a genetically modified bacterium belonging to the genus Clostridium, characterized in that the bacterium does not express... hbd Genes and hydA The product of genes, or thlA The product of genes, or thlA Genes and thlB The product of the gene, and optionally, the product of another gene of interest, which the bacterium expresses, or... hbd Genes and hydA Non-functional versions of gene products, thlA A non-functional version of the gene product, or thlA gene and thlBA nonfunctional version of the gene product, and optionally a nonfunctional version of the product expressing another gene of interest, used for the preparation of genetically modified bacteria according to the invention, which do not express... hbd Genes and hydA genes or thlA Genes and thlB The product of a gene, or the expression of one or more non-functional versions of it.

[0014] The inventors also described the plasmid pGRNA-Δ with sequence SEQ ID NO: 29. hbd pGRNA-Δ sequence SEQ ID NO: 64 hydA pGRNA-Δ sequence SEQ ID NO: 60 thlA and pGRNA-Δ of sequence SEQ ID NO: 62 thlB Furthermore, they have one, more, or all of their uses for transforming, and preferably for genetically modifying, Clostridium bacteria to enhance their ability to produce ethanol.

[0015] A method for transforming (and preferably genetically modified) Clostridium bacteria is described, and genetically modified Clostridium bacteria obtained by the method are also described. The method includes the step of transforming the bacteria by introducing a plasmid selected from plasmids containing the sequence SEQ ID NO: 29, pGRNA-Δ. hbd pGRNA-Δ sequence SEQ ID NO: 64 hydA pGRNA-Δ sequence SEQ ID NO: 60 thlA and pGRNA-Δ of sequence SEQ ID NO: 62 thlB .

[0016] The inventors also describe the use of the bacteria according to the invention for producing bio-based molecules, such as solvents, biofuels, or any (bio)chemical intermediates, particularly ethanol. Such bacteria can be advantageously used to produce solvents or solvent mixtures (especially on an industrial scale).

[0017] The present invention also relates to a fermentation method that involves the use of genetically modified bacteria as described herein.

[0018] Finally, the inventors describe kits, particularly kits for transforming and preferably for genetically modifying bacteria belonging to the genus Clostridium, and kits for producing bio-based molecules using bacteria belonging to the genus Clostridium, such as solvents, biofuels, or any (bio)chemical intermediates, particularly ethanol, said kits comprising i) genetically modified bacteria belonging to the genus Clostridium according to the invention and ii) a preservation or culture medium for said bacteria, particularly a suitable culture medium containing sugars or mixtures of sugars (preferably hexoses and / or pentoses, more preferably glucose and / or arabinose and / or xylose). Attached Figure Description

[0019] Figure 1 Figure 1 The central metabolism of *Clostridium acetobutanol* is illustrated. During acid production, *Clostridium acetobutanol* produces acetic acid, butyrate, and possibly lactate. In the solvation phase, butyrate and acetic acid are re-assimilated, and carbon flow is redirected to the production of acetone, ethanol, and n-butanol. Ack, acetate kinase; Adc, acetoacetate decarboxylase; Adh, alcohol dehydrogenase; Ald, aldehyde dehydrogenase; Aldc, acetolactate decarboxylase; Als, acetolactate synthase; Bcd, butyryl-CoA dehydrogenase; Buk, butyrate kinase; CtfA-CtfB, butyrate-acetoacetate coenzyme A-transferase (A and B subunits); Crt, crotonic acid oxidase; EtfA-EtfB, electron transport flavoprotein (α and β subunits); Fnor, ferroredoxin-NAD(P). + Oxidoreductases; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; HydA, hydrogenase; Ldh, lactate dehydrogenase; Pdc, pyruvate decarboxylase; Pfor, pyruvate ferroreductase; Pta, phosphoacetyltransferase; Ptb, phosphobutyryltransferase; Thl, thiolase. Detailed Implementation

[0020] Although bacteria belonging to the genus Clostridium, especially solvent-producing bacteria, have been used in industry for over a century, very little is known about them.

[0021] Clostridium acetone-butanol is now considered a representative model of solvent-producing Clostridium. Despite the difficulties known to those skilled in the art in genetically modifying bacteria belonging to the genus Clostridium, the inventors have, for the first time in the context of this invention, successfully obtained Clostridium acetone-butanol, which is capable of producing ethanol uniquely or almost exclusively. In a preferred embodiment, these mutants no longer produce or almost no longer produce n-butanol, butyric acid, and acetone.

[0022] The inventors have discovered and, therefore, for the first time disclosed, an enzyme encoding 3-hydroxybutyryl-CoA dehydrogenase activity. hbd Genes, and those encoding hydrogenase hydA The combined inactivation of genes, or both encoding thiolasases. thlA and thlB The combined inactivation of genes makes it possible to obtain strains that no longer produce or almost no longer produce n-butanol, butyric acid, and acetone, and whose main (and possibly only) fermentation product is ethanol, as demonstrated in the experimental section of this specification. thlA Individual gene inactivation also makes it possible to obtain strains that produce more ethanol than wild-type Clostridium acetone-butanol.

[0023] Therefore, one subject matter described by the inventors relates to genetically modified bacteria belonging to the genus *Clostridium*, particularly industrially interesting *Clostridium* species, especially *Clostridium acetone-butanol*, characterized in that, when said bacteria is *Clostridium acetone-butanol*, it does not express genes. hbd (The preferred sequence SEQ ID NO: 32 of the gene) hbd The product of ), and when the bacteria is Clostridium acetone-butanol, it does not express the gene. hydA (Preferred sequence SEQ ID NO: 69) hydA The product of a gene, or the expression of a non-functional version thereof; or characterized in that, when the bacterium is Clostridium acetone-butanol, it does not express the gene. thlA (The preferred sequence is the gene SEQ ID NO: 67) thlA The product of ), and when said bacteria is Clostridium acetonebutanol, it does not express the gene. thlB (The preferred sequence SEQ ID NO: 68 of the gene) thlB The product of ).

[0024] In the context of this invention, the term " hbd "Gene" specifically refers to the sequence SEQ ID NO: 32 (CA_C2708). The term "gene" hbd "Gene" also refers to variants of the sequence SEQ ID NO: 32, particularly variants that show sequence homology with the sequence SEQ ID NO: 32.

[0025] In the context of this invention, the term " hydA "Gene" specifically refers to the sequence SEQ ID NO: 69 (CA_C0028) or a variant of the sequence, particularly a variant that shows sequence homology with the sequence SEQ ID NO: 69.

[0026] In the context of this invention, the term " thlA"Gene" specifically refers to the sequence SEQ ID NO: 67 (CA_C2873). The term "gene" thlA "Gene" also refers to variants of the sequence SEQ ID NO: 67, particularly variants that show sequence homology with the sequence SEQ ID NO: 67.

[0027] In the context of this invention, the term " thlB "Gene" specifically refers to the sequence SEQ ID NO: 68 (CA_P0078). The term "gene" thlB gene "Also refers to variants of the sequence SEQ ID NO: 68, particularly variants that show sequence homology with the sequence SEQ ID NO: 68."

[0028] Typical examples of variants of the invention show sequence homology with sequences SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67, or SEQ ID NO: 68, with homology between 95% and 100%, and preferably between 96% and 100%. Sequences SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67, or SEQ ID NO: 68 and their variants are, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous. According to a preferred embodiment, sequences SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67, or SEQ ID NO: 68 and their variants have sequences with at least 96%, at least 97%, or at least 98% homology.

[0029] the term" hbd "Gene" also refers to the sequence of a functional variant or fragment that encodes (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) protein (enzyme), especially a protein that can / is capable of performing (S)-3-hydroxybutyryl-CoA dehydrogenase activity.

[0030] In a specific implementation scheme, the term " hbd "Gene" also refers to the sequence that encodes a functional variant or fragment of the Hbd protein, especially a protein that can / may exert (S)-3-hydroxybutyryl-CoA dehydrogenase activity.

[0031] the term" hydA "Gene" also refers to the sequence of a functional variant or fragment that encodes a HydA protein (enzyme), especially a protein that can / may exert hydrogenase activity.

[0032] In a specific implementation scheme, the term " hydA"Gene" also refers to the sequence of a functional variant or fragment that encodes a HydA protein, especially a protein that can / may exert hydrogenase activity.

[0033] the term" thlA "Gene" also refers to the sequence of a functional variant or fragment that encodes Th1A protein (enzyme), especially a protein that can / may exert acetyl-CoA acetyltransferase activity.

[0034] In a specific implementation scheme, the term " thlA "Gene" also refers to the sequence that encodes a functional variant or fragment of the Th1A protein, especially a protein that can / may exert the activity of acetyl-CoA acetyltransferase.

[0035] the term" thlB "Gene" also refers to the sequence of a functional variant or fragment that encodes a Th1B protein (enzyme), especially a protein that can / may exert the activity of acetyl-CoA acetyltransferase.

[0036] In a specific implementation scheme, the term " thlB "Gene" also refers to the sequence that encodes a functional variant or fragment of the Th1B protein, especially a protein that can / may exert the activity of acetyl-CoA acetyltransferase.

[0037] When this text refers to genetically modified bacteria belonging to the genus Clostridium, it is characterized by the fact that they do not express genes. hbd , hydA , thlA and / or thlB The product of, or an expression of its non-functional version, a representation hbd , hydA , thlA or thlB “Non-functional version of the product of a gene” refers to a protein, typically identified in this document as “Hbd”, “HydA”, “ThlB”, or “ThlA”, which is non-functional, meaning it cannot perform the function of the protein encoded by the wild-type version of the target gene.

[0038] The term "Clostridium bacteria" is specifically understood to refer to species of industrial interest, typically solvent-producing or acetic acid-producing bacteria of the genus *Clostridium*. The expression "Clostridium bacteria" includes wild-type bacteria, as well as genetically modified strains derived from them to improve their properties.

[0039] The phrase “clostridium species of industrial interest” or “clostridium bacteria of industrial interest” should be understood as referring to species capable of producing solvents (such as ethanol, butanol, acetone, or isopropanol) and / or acids (such as butyric acid, acetic acid, or lactic acid) from sugars or monosaccharides (typically sugars containing 5 carbon atoms, such as xylose, arabinose, or fructose, or sugars containing 6 carbon atoms, such as glucose or mannose)), polysaccharides (such as cellulose or hemicellulose), and / or any other carbon source (such as CO, CO2, and methanol) that can be absorbed and used by Clostridium bacteria. Examples of solvent-producing bacteria of interest in the context of this invention are Clostridium species that produce acetone, butanol, ethanol, and / or isopropanol (propan-2-ol), such as those identified in the literature as “ABE strains” [strains capable of fermenting to produce acetone, butanol, and ethanol], “IBE strains” [strains capable of fermenting to produce isopropanol (or propan-2-ol) (by reducing acetone), butanol, and ethanol], and “AIBE strains” [strains capable of fermenting to produce acetone, isopropanol (or propan-2-ol), butanol, and ethanol]. Solvent-producing bacteria of the genus Clostridium can be selected, for example (this list is not limiting) Clostridium acetone-butanol, Clostridium fibrinolyticum (… C. cellulolyticum ), plant fermentation clostridium ( C. phytofermentans Clostridium beyerridis ( ), C. beijerinckii Clostridium butyricum ( ) C. saccharobutylicum ), Clostridium glycoacetate polybutanol ( C. saccharoperbutylacetonicum Clostridium sporogenes ( C. sporogenes Clostridium butyricum ( C. butyricum Clostridium butyricum ( C. aurantibutyricum ) and Clostridium butyricum ( C. Tyrobutyricum ) ， Preferred strains include *Clostridium acetonebutanol*, *Clostridium beyere*, *Clostridium butyricum ... and *Clostridium fibrinolyticum*. Furthermore, it is even more preferably derived from Clostridium acetonebutanol and Clostridium baileyi.

[0040] Acetogenic bacteria of interest are bacteria that produce acid and / or solvent from CO2 and H2. Acetogenic bacteria of the genus *Clostridium* can be selected, for example, from *Clostridium acetylcholine* (…). C. aceticum ), C. drakei Clostridium thermoacetate ( C. thermoaceticum Clostridium lagus ( C. ragsdalei Clostridium yangii ( C. ljungdahlii ), autotrophic ethanol-producing Clostridium ( C. autoethanogenum Clostridium difficile ( C. difficile ), Clostridium difficile ( C. scatologenes ) and carbon monoxide-eating Clostridium ( C. carboxidivorans ).

[0041] In a particular embodiment, the Clostridium species is Clostridium acetobutyricum, such as strain DSM 792 (also known as strain ATCC 824 or LMG 5710).

[0042] In a particular embodiment, the Clostridium species is Clostridium beyerii, such as strains of Clostridium beyerii NCIMB8052, DSM 6423, LMG 7814, LMG 7815, NRRL B-593, or NCCB 27006.

[0043] Therefore, according to a preferred embodiment, the bacteria belonging to the genus Clostridium according to the invention are industrially interesting Clostridium bacteria, particularly solvogenic bacteria in the wild-type state capable of producing solvents and / or acids by fermentation from a carbon source, such as sugars, CO, CO2, alcohols, and organic acids. In a preferred embodiment, the carbon source is a sugar, particularly a sugar containing 5 carbon atoms (e.g., xylose or arabinose), a sugar containing 6 carbon atoms (e.g., glucose, fructose, or mannose), or a polysaccharide (e.g., cellulose or hemicellulose).

[0044] As described above, the particularly preferred bacteria belong to the Clostridium acetobutyricum species and do not express the following products, or express the non-functional form of the expressed products: i) the gene of sequence SEQ ID NO: 32 (CA_C2708) (or a sequence that is at least 95%, for example, at least 96%, at least 97%, or at least 98% homologous to it), and the gene of sequence SEQ ID NO: 69 (CA_C0028) (or a sequence that is at least 95%, for example, at least 96%, at least 97%, or at least 98% homologous to it), ii) the gene of sequence SEQ ID NO: 67 (CA_C2873) (or a sequence that is at least 95%, for example, at least 96%, at least 97%, or at least 98% homologous to it), or iii) the genes of sequences SEQ ID NO: 67 and sequence SEQ ID NO: 68 (CA_P0078) (or sequences that are at least 95%, for example, at least 96%, at least 97%, or at least 98% homologous to them).

[0045] Particularly preferred are the bacteria modified according to options i) and iii) above, which exhibit the above-mentioned technical capabilities, namely, they are able to produce ethanol uniquely or almost uniquely, in contrast to no longer or almost no longer producing n-butanol, butyric acid and acetone.

[0046] This type of strain has been characterized for the first time in the context of this application.

[0047] One of the strains was registered at the BCCM-LMG Collection on February 21, 2024, with accession number LMG P-33549 (IFP 974, "Δ"). hbdΔ hydA In this strain, hbd and hydA The gene has been (jointly) inactivated. This specification also covers any derived bacteria, clones, mutants, or genetically modified versions thereof, which typically lack [the gene's name / characteristic]. hbd and hydA Genes, or the genes mentioned therein, have also been inactivated.

[0048] The second strain was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33548 (IFP 973, "Δ"). thlA Δ thlB In this strain, thlA and thlB The gene has been (jointly) inactivated. This specification also covers any derived bacteria, clones, mutants, or genetically modified versions thereof, which typically lack [the gene's name / characteristic]. thlA and thlB Genes, or the genes mentioned therein, have also been inactivated.

[0049] The third strain was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33547 (IFP 972, "Δ"). hydA In this strain, hydA The gene has been inactivated. This specification also covers any derived bacteria, clones, mutants, or genetically modified versions thereof, which typically lack [the gene's characteristics]. hydA Genes, or the genes mentioned therein, have also been inactivated.

[0050] Another strain was registered at the BCCM-LMG Collection on February 17, 2023, with accession number LMG P-32993 (IFP 969, "Δ"). hbd In this strain, hbd The gene is inactivated. This specification also covers any derived bacteria, clones, mutants, or genetically modified versions thereof, which typically lack [the gene's name / characteristic]. hbd Genes, or the genes mentioned therein, have also been inactivated.

[0051] The genetically modified bacteria according to the invention can be advantageously used to produce solvents, such as biofuels (preferably ethanol) or mixtures of solvents, such as mixtures of biofuels (preferably mixtures containing ethanol and one or more other alcohols), particularly on an industrial scale.

[0052] The present invention also relates to fermentation methods, typically industrial methods, which involve the use of bacteria according to the invention.

[0053] This invention provides a desirable solution for optimizing ethanol production.

[0054] The genetically modified bacteria according to the invention can also be advantageously used to produce bio-based molecules from biomass or dedicated energy crops, such as solvents, biofuels or any (bio)chemical intermediates.

[0055] In the context of this invention, the term "bio-based molecule" should be understood to refer to alcohol or ketone molecules, specifically characterized by the fact that the raw materials used for their production must be derived from plant biomass (e.g., lignocellulose biomass), rather than from fossil-derived resources (e.g., petroleum, coal, or natural gas). For example, these are alcohols derived from the fermentation of sugary beverages, particularly C5 sugars (having 5 carbons) and / or C6 sugars (having 6 carbons), produced by "solvent-producing" strains of the genus *Clostridium*. Examples of bio-based molecules that can be produced by means of this invention are primarily ethanol (which can be used as a biofuel), or n-propanol and 2,3-butanediol.

[0056] The present invention also relates to a method for producing recombinant bacteria according to the invention, comprising deletion or inactivation. hbd and hydA Gene, thlA Genes, or thlA and thlB Genes that inhibit or reduce the expression of Hbd and HydA, ThlA, or ThlA and ThlB proteins.

[0057] The known modification methods that yield the highest performance from genetically modified strains are based on homologous recombination events, which enable precise and stable modification of the genome.

[0058] A specific production method according to the present invention includes the step of transforming the bacteria by introducing the nucleic acid of interest into the bacteria.

[0059] For the purposes of this invention, the term "nucleic acid" should be understood to mean any natural, synthetic, semi-synthetic, or recombinant DNA or RNA molecule that has optionally been chemically modified (i.e., containing non-natural bases, modified nucleotides containing, for example, modified bonds, modified bases, and / or modified sugars), or optimized such that the codons of the transcript synthesized from the coding sequence are the most common codons in Clostridium bacteria, with the aim of its use therein. In the case of Clostridium, optimized codons are typically codons rich in adenine ("A") and thymine ("T") bases.

[0060] In the peptide sequences described herein, amino acids are represented by their single-letter codes 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 the specific embodiments described, the nucleic acid of interest comprises at least two regions, each complementary to the target sequence, and is 100% identical or at least 80% identical to the target DNA region / part / sequence within the bacterial genome, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%. These regions are capable of hybridizing with all or part of the complementary sequence of the region / part / sequence, typically with a sequence comprising at least one nucleotide, preferably at least 100 nucleotides, typically between 100 and 1000 nucleotides, as described above. Regions within the nucleic acid of interest that are complementary to the target sequence can be identified and preferably targeted, specifically the 5' and 3' flanking regions of the target sequence in gene-modification tools known to those skilled in the art (typically any tool based on homologous recombination).

[0062] In a preferred embodiment, a portion of the nucleic acid of interest is also recognized (at least partially bound), and preferably targeted, i.e., in the genome of the Clostridium of interest, to recognize and allow breakage of at least one strand of i) a target sequence, ii) a sequence controlling transcription of the target sequence, or iii) a sequence flanking the target sequence. The recognized sequence is also defined herein as a “target sequence” or “targeting sequence”.

[0063] In the same preferred embodiment, the nucleic acid of interest comprises at least one region complementary to a target sequence that is 100% identical or at least 80% identical to a target DNA region / part / sequence within the bacterial genome, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and is capable of hybridizing with all or part of the complementary sequence of said region / part / sequence (typically with sequences containing 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 containing 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides).

[0064] The nucleic acids of interest described in the context of this invention are capable of deleting the target sequence from the bacterial genome or modifying its expression, for example, regulating / regulating them, particularly inhibiting them, preferably modifying them, so that the bacteria cannot express one or more proteins from the sequence, particularly one or more functional proteins.

[0065] According to another aspect of the invention, the nucleic acid of interest, as described in the context of the invention, can introduce a sequence of interest into the genome of a bacterium so that the bacterium can express one or more proteins from the sequence, particularly one or more functional proteins.

[0066] "Nucleic acid of interest," typically an expression cassette or vector, can be constructed using conventional techniques well known to those skilled in the art and may include one or more promoters, a bacterial origin of replication (ORI sequence), a termination sequence, a selection gene (e.g., an antibiotic resistance gene), and sequences that enable the cassette or vector to be inserted in a targeted manner ("flanking regions"). For example, the ORI sequence of interest may be selected from pIP404, pAMβ1, repH (the origin of replication in Clostridium acetobutyricum), ColE1, or rep (the origin of replication in Escherichia coli). E. coli The origin of replication (ROR) in a bacterium (typically a plasmid), or any other origin of replication that enables the vector (typically a plasmid) to be maintained within a bacterial cell belonging to the genus Clostridium. For example, the termination sequence of interest can be selected from the following sequences: adc and thl Gene, bcs An operon or any other terminator known to those skilled in the art allows transcription to be stopped within bacterial cells belonging to the genus *Clostridium*. The selection gene of interest (resistance gene) can be selected from... ermB , catP , bla , tetA , tetM And / or resistance genes to ampicillin, erythromycin, chloramphenicol, thiamphenicol, spectinomycin, tetracycline, or any other antibiotic known to those skilled in the art that can be used to select Clostridium bacteria.

[0067] The nucleic acid of interest can be natural or synthetic RNA or RNA produced via recombinant technology. The nucleic acid of interest can be prepared by any method known to those skilled in the art, such as chemical synthesis, in vivo transcription, or amplification techniques. When the nucleic acid of interest is introduced directly into cells in the form of RNA molecules (mature or precursor), such as guide RNA (gRNA), these molecules may contain modified nucleotides or chemical modifications, for example, allowing them to increase their resistance to nucleases, thereby extending their lifespan in the cell. They may specifically contain at least one modified nucleotide or non-natural nucleotide, for example, nucleotides containing modified bases such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine, or any other modified base that allows hybridization.

[0068] The nucleic acids of interest used according to the present invention can also be modified at the nucleotide internucleotide level (e.g., phosphate thioides, H-phosphonates, or alkylphosphonates), or at the backbone level (e.g., α-oligonucleotides, 2'-O-alkylribose, or PNA (peptide nucleic acid)). et al ., 1992).

[0069] In the context of this specification, a specific example of a nucleic acid of interest for the transformation and / or genetic modification of an interest bacterium is a DNA fragment that i) recognizes a sequence encoding the enzyme described below, ii) controls the transcription of a sequence encoding the enzyme described below, or iii) is located flanking a sequence encoding the enzyme described below: enzyme Hbd (3-hydroxybutyryl-CoA dehydrogenase), enzyme HydA (hydrogenase), thiolase ThlA, or thiolase ThlB (acetyl-CoA acetyltransferase).

[0070] The specific nucleic acid of interest described by the inventors is, for example, a vector, preferably a plasmid, such as the plasmid pGRNA-Δ of sequence SEQ ID NO:29. hbd plasmid pGRNA-Δ with sequence SEQ ID NO: 64 hydA plasmid pGRNA-Δ with sequence SEQ ID NO: 60 thlA , or plasmid pGRNA-Δ with sequence SEQ ID NO: 62 thlB The embodiments are described in the embodiment section of this specification.

[0071] The identified sequence (target sequence) or one of these sequences is preferably one of the following sequences: sequence SEQ ID NO:39, sequence SEQ ID NO:36, or sequence SEQ ID NO:37, corresponding to hbd, hydA, thlA and thlBGenes encoding Hbd, HydA, ThlA, and ThlB proteins, respectively, or amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the proteins, or sequences containing all or at least 95%, 96%, 97%, 98%, or 99% of sequences SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67, or SEQ ID NO: 68. In other words, the identified sequence can be a sequence containing at least one nucleotide, preferably at least one, two, three, four, five, ten, fifteen, twenty, twenty, twenty, thirty, thirty, or forty nucleotides, typically between one and forty nucleotides, preferably containing 14, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twelve, twenty ...

[0072] According to another specific instance, the target sequence can also be a sequence that controls the transcription of the coding sequence as described above, typically a promoter sequence, such as " hbd” The promoter sequence of the gene (CA_C2708, SEQ ID NO: 32), hydA” The promoter sequence of the gene (CA_C0028, SEQ ID NO: 69), thlA "The promoter sequence of the gene (CA_C2873, SEQ ID NO: 67), or " thlB The promoter sequence of the gene “CA_P0078, SEQ ID NO: 68”. The nucleic acids of interest are then recognized and, therefore, typically able to bind to the sequence that controls transcription of the coding sequence as described above.

[0073] According to another specific example, the target sequence can be a sequence located on the flank of the coding sequence as described above, such as a sequence located on the flank of sequence SEQ ID NO: 32, SEQ ID NO: 69, SEQ ID NO: 67 or SEQ ID NO: 68, or a sequence that is at least 70% identical to it. Such flanking sequences typically contain 1, 10, or 20 to 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] Depending on the specific aspect, the target sequence corresponds to a sequence pair located flanking the coding sequence of this type, each flanking sequence typically containing at least 20 nucleotides, usually between 100 and 1000 nucleotides, preferably between 200 and 800 nucleotides.

[0075] Preferably, the method for generating recombinant bacteria according to the invention includes transforming bacterial cells with at least one nucleic acid of interest, such as one, two, three, or four nucleic acids of interest as described above, said nucleic acid of interest being i) capable of recognizing (and at least partially binding) a sequence encoding an Hbd protein, controlling the transcription of the sequence encoding an Hbd protein, or located flanking a sequence encoding an Hbd protein; ii) capable of recognizing (and at least partially binding) a sequence encoding a HydA protein, controlling the transcription of the sequence encoding a HydA protein, or located flanking a HydA protein; iii) capable of recognizing (and at least partially binding) a sequence encoding a ThlA protein, controlling the transcription of the sequence encoding a ThlA protein, or located flanking a ThlA protein; and preferably iv) capable of recognizing (and at least partially binding) a sequence encoding a ThlB protein, controlling the transcription of the sequence encoding a ThlB protein, or located flanking a ThlB protein.

[0076] The nucleic acid of interest described in the context of this invention is preferably capable of deleting the target sequence from the bacterial genome or a genome modified thereon, for example, regulating / modifying it, particularly inhibiting it, and preferably modifying it so that the bacteria cannot express proteins (typically Hbd, HydA, ThlA, or ThlB proteins), particularly functional proteins. In a particularly preferred embodiment, the nucleic acid of interest is capable of modifying the bacteria to prevent it from expressing one and / or another of the proteins Hbd, HydA, ThlA, or ThlB.

[0077] Depending on the specific implementation scheme, this specification relates more specifically to the plasmid pGRNA-Δ of sequence SEQ ID NO: 29. hbd The plasmid pGRNA-Δ with sequence SEQ ID NO: 64 hydA plasmid pGRNA-Δ with sequence SEQ ID NO: 60 thlA , and / or plasmid pGRNA-Δ with sequence SEQ ID NO: 62 thlB The purpose of the transformation is to modify Clostridium bacteria to improve their ability to produce ethanol, and, depending on the specific embodiment, also to improve their ability to produce n-propanol and / or 2,3-butanediol.

[0078] The present invention also relates to a method for transforming, and preferably for gene modification, Clostridium bacteria, characterized in that the method comprises the step of transforming the bacteria by introducing a plasmid as described herein into the bacteria, preferably a plasmid selected from the plasmid pGRNA-Δ of sequence SEQ ID NO: 29. hbd plasmid pGRNA-Δ with sequence SEQ ID NO: 64 hydA plasmid pGRNA-Δ with sequence SEQ ID NO: 60 thlA , or plasmid pGRNA-Δ with sequence SEQ ID NO: 62 thlB This specification also relates to any genetically modified Clostridium bacteria obtained by such methods.

[0079] Introducing any nucleic acid of interest into bacteria can be done by any method known to those skilled in the art, whether direct or indirect, such as by transformation, conjugation, microinjection, transfection, electroporation, etc., and preferably by electroporation (Mermelstein). et al ., 1993).

[0080] Furthermore, these nucleic acids of interest (such as DNA fragments, RNA fragments, expression cassettes, or expression vectors) can be integrated into the bacterial genome using techniques well known to those skilled in the art.

[0081] In certain embodiments, the methods for transformation, and preferably for genetic modification of bacteria as described herein, include the step of transforming bacteria by introducing the nucleic acid of interest according to the invention as described above, and involve gene modification tools, such as those selected from CRISPR tools, insertional mutagenesis tools (e.g., based on the use of type II introns (e.g., Targetron® tools or ClosTron® tools)) and allele exchange tools (e.g., ACE® tools).

[0082] For transformation, and preferably also for gene modification, the method for Clostridium bacteria may additionally include the steps of obtaining, recovering, selecting or isolating the transformed bacteria (i.e., bacteria exhibiting the desired recombinant / modified / optimized characteristics).

[0083] In certain embodiments, the method according to the invention is based on the use of CRISPR (clustered regularly spaced short palindromic repeats) gene tools / techniques, particularly CRISPR-Cas (CRISPR-associated protein) gene tools. The invention can be implemented using conventional CRISPR-Cas gene tools that utilize nucleases, gRNA, and repair templates (such as Wang). et al A single plasmid as described in (2015). Those skilled in the art can readily determine the sequence and structure of the gRNA based on the targeted chromosomal region or mobile genetic element using known techniques (see, for example, DiCarlo). et al (Article, 2013). The inventors have also developed and described a gene modification tool for bacteria based on the use of two plasmids, applicable to Clostridium species, which can be used in the context of this invention (see WO2017 / 064439, Wasels). et al (2017).

[0084] In another embodiment, the method according to the invention is based on the use of an insertion mutagenesis tool, such as a type II intron, and for example, the use of ClosTron® gene tools / technology or Targetron® gene tools.

[0085] Targetron® technology is based on the use of reprogrammable group II introns (based on Lactococcus lactis). Lactococcus lactis The Ll.ltrB intron in the gene pool can rapidly integrate into the bacterial genome at the desired locus (Chen). et al ., 2005, Wang et al (2013), typically aiming to inactivate target genes. The mechanism for recognizing edited regions and inserting them into the genome via backsplicing is based on both the homology between introns and said regions and the activity of the protein (LtrA).

[0086] ClosTron® technology is based on a similar approach, supplemented by adding selection markers (Heap) to the intron sequence. et al (., 2007). This marker allows for the selective integration of introns into the genome and thus facilitates the acquisition of desired mutants. This genetic system also utilizes type I introns. Specifically, selection markers (called RAMs for reverse transcription activation) are blocked by such gene elements, preventing their expression from the plasmid (for a more precise description of this system, see: Zhong, 2007). et al The splicing of this gene element occurs before integration into the genome, allowing for the acquisition of chromosomes exhibiting the active form of the resistance gene. An optimized version of this system includes FLP / FRT sites upstream and downstream of the gene, enabling the removal of the resistance gene (Heap) using FRT recombinase. et al (., 2010).

[0087] In another embodiment, the method according to the invention is based on the use of an allele exchange tool, and for example employs the ACE® gene tool / technology.

[0088] The ACE® technology is based on the use of auxotrophic mutants (targeting uracil, in Clostridium acetobutyricum ATCC 824, through deletion) pyrE The gene, which also causes resistance to 5-fluoroorotic acid (5-FOA); Heap et al. (2012). This system uses an allele exchange mechanism well known to those skilled in the art. After transformation with a pseudo-suicide (very low copy) vector, the resistance gene initially present on the plasmid can be verified to be integrated into the bacterial chromosome via a first allele exchange event. The integration step can be performed in two different ways, either in pyrE Within a locus or within another locus. In integration... pyrE In the case of a gene locus, pyrE The locus was also placed on the plasmid, but it was not expressed (no functional promoter was present). A second recombination restored functionality. pyrE Genes can then be selected based on nutritional deficiencies (basal culture medium, without uracil). Due to nonfunctionality... pyrE The gene also exhibits selectivity (sensitivity to 5-FOA), and other integrations can then be envisioned on the same model, through... pyrEA continuous alternation between functional and non-functional states. In the case of integration into another locus, the genomic region capable of expressing an anti-selection marker after recombination is targeted (typically, the operon following another gene, preferably the highly expressed gene). This second recombination is then selected using a nutrient auxotroph (basal medium without uracil).

[0089] In the described implementation, the target sequence is typically one of the sequences described herein, based on the use of type II introns and, for example, using ClosTron® gene tools / technology or Targetron® gene tools, or based on the use of allele exchange tools and, for example, using ACE® gene tools / technology.

[0090] This invention also relates to kits for transforming and / or genetically modifying Clostridium bacteria, comprising at least one nucleic acid of interest as described herein (e.g., two or three nucleic acids of interest, typically DNA fragments, each recognizing a target sequence), for transforming and preferentially modifying Clostridium bacteria, and possibly one or more selectable molecules. Specific kits contain elements essential to the function of CRISPR tools (typically at least one nucleic acid that can be used as gRNA, a nucleic acid that can be used as a repair template, at least one primer pair, and an inducer capable of expressing a nuclease (particularly Cas9 or Cas12 nuclease, e.g., MAD7)), elements essential to the function of tools based on the use of type II introns (typically at least one type II intron, at least one primer pair, and an inducer capable of expressing a reverse transcriptase (e.g., LtrA RT or Tel4c RT type), or elements essential to the function of allelic exchange tools (typically at least two nucleic acids that can be used as templates for homologous recombination and at least one primer pair).

[0091] The kit according to the invention may also contain one or more consumables, such as preservation or culture medium, at least one competent Clostridium species (i.e., treated for transformation), or a booklet containing instructions for use.

[0092] This invention typically relates to a kit for carrying out the transformation and / or gene modification methods described herein, using Clostridium bacteria.

[0093] This instruction manual specifically addresses genetically modified bacteria belonging to the genus Clostridium (which possess genes that do not express). hbd and hydA , thlA ,or thlA and thlB The products of, or the basic characteristics expressing their non-functional forms, as well as any derived bacteria, clones, mutants or their genetically modified forms, and their uses.

[0094] This application also describes strain IFP 969 (“Δ hbd ", was registered at the BCCM-LMG Collection Center on February 17, 2023, with accession number LMG P-32993, in which hbd The gene has been inactivated, and any derived bacteria, clones, mutants, or their genetically modified versions are typically missing. hbd Genes, or said genes, have been inactivated, preferably using CRISPR technology.

[0095] This application also describes strain IFP 972 (“Δ thlA ", was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33547, in which thlA The gene has been inactivated, and any derived bacteria, clones, mutants, or their genetically modified versions are typically missing. thlA Genes, or said genes, have been inactivated, preferably using CRISPR technology.

[0096] This application also describes strain IFP 974 (“Δ hbd Δ hydA The document, registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33549, contains... hbd and hydA The gene has been inactivated, and any derived bacteria, clones, mutants, or genetically modified versions thereof have also been inactivated, typically missing. hbd and hydA Genes, or the genes mentioned therein, have also been inactivated.

[0097] This application also describes strain IFP 973 (“Δ thlA Δ thlB ", was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33548, in which thlA and thlB The gene has been inactivated, and any derived bacteria, clones, mutants, or genetically modified versions thereof have also been inactivated, typically missing. thlA and thlB Genes, or the genes mentioned therein, have also been inactivated.

[0098] In particular, the use of genetically modified bacteria belonging to the genus Clostridium is involved, characterized in that the bacteria do not express... hbd The product of the gene, or the expression of the bacteria. hbdA nonfunctional version of the gene product, such as strain IFP 969, was registered at the BCCM-LMG Collection on February 17, 2023, with accession number LMG P-32993, for use in the preparation of products according to the present invention that do not express the following product. hbd and hydA Genes, or genetically modified bacteria that express a non-functional version of them.

[0099] Furthermore, the use of genetically modified bacteria belonging to the genus Clostridium is particularly relevant, characterized in that the bacteria do not express... thlA The product of a gene, or its expression thlA A non-functional version of the gene product, such as strain IFP 972, was registered at the BCCM-LMG Collection Center on February 21, 2024, with accession number LMG P-33547. This is used for the preparation of a non-expressing gene according to the present invention. thlA and thlB The product of a gene, or a genetically modified bacterium that expresses a non-functional version of it.

[0100] The present invention also relates to a kit for using bacteria belonging to the genus Clostridium to produce bio-based molecules (e.g., solvents, biofuels, or any (bio)chemical intermediates), comprising i) genetically modified bacteria belonging to the genus Clostridium according to the invention, such as IFP 969, IFP 972, IFP 973, or IFP 974, and ii) a culture medium, typically a preservation or culture medium for said bacteria. The kit may also include a booklet containing instructions for use.

[0101] The culture medium for preserving or culturing the genetically modified bacteria (belonging to the genus Clostridium) present in the kit according to the present invention is preferably supplemented with a carbon source consisting of glucose and / or at least one pentose, preferably i) glucose and / or ii) arabinose and / or iii) xylose. The culture medium preferably contains 0.1 to 250 g / L, more preferably 1 to 100 g / L of the carbon source (consisting of glucose and / or arabinose and / or xylose).

[0102] The culture medium used for preserving or culturing genetically modified bacteria is preferably RCM medium, more preferably GAPES medium, and even more preferably CGM medium.

[0103] This specification also relates to specific kits for producing solvents or biofuels, or mixtures of solvents or biofuels, particularly ethanol, or mixtures containing ethanol and one or more other alcohols, using bacteria belonging to the genus *Clostridium*, said kits comprising i) genetically modified bacteria (belonging to the genus *Clostridium*) according to the invention, characterized in that it does not express genes. hbd and hydA , thlA ,orthlA and thlB The product, and ii) the culture medium, preferably RCM medium, more preferably GAPES medium, more preferably CGM medium, which contains at least one carbon source, preferably glucose and / or arabinose and / or xylose.

[0104] The present invention also relates to the possible use of the methods or kits according to the invention for transforming and / or genetically modifying Clostridium bacteria, typically solvent-producing Clostridium bacteria, for example, for generating improved variants of said bacteria.

[0105] Finally, this relates to the possible uses of the method, kit, or the transformed and preferably genetically modified Clostridium species according to the invention, particularly for the production, typically on an industrial scale, of bio-based molecules, such as solvents, biofuels, or (bio)chemical intermediates, or mixtures thereof.

[0106] The embodiments and figures below are intended to illustrate the invention more fully, but do not limit the scope of the invention. In particular, these embodiments demonstrate the acquisition and characterization of bacteria according to the invention, in which genes... hbd , hydA , thlA and / or thlB Inactivation, especially on the one hand hbd and hydA and on the other hand thlA and thlB The CRISPR-Cas9 tool is used according to a preferred specific embodiment. The gene can be inactivated or introduced, according to other specific embodiments well known to those skilled in the art, and based on, for example, inactivation or introduction of the gene by homologous recombination or insertional mutagenesis, as described above.

[0107] Example

[0108] Materials and methods

[0109] Strains, plasmids and culture media

[0110] Clostridium acetone-butanol DSM 792 (German Microbial and Cell Culture Collection, DSMZ) was cultured at 34°C under anaerobic conditions (90% N2, 5% CO2, 5% N2) on 2 YTG medium (16 g / L tryptone). -1 Yeast extract 10 g / L -1 5 gL of glucose -1 , NaCl 4 gL -1 In ) Escherichia coli ( Escherichia coliNEB 10-beta (New England Biolabs, NEB) was cultured at 37°C under aerobic conditions in LB medium (10 g / L tryptone). -1 Yeast extract 5g / L -1 10 gL NaCl -1 Cultured in [a specific medium]. By adding 15 g / L [a specific amount of something]. -1 Agarose is added to liquid medium to produce a solid medium. If necessary, erythromycin (Em, 40 mg / L) is added. -1 ) and / or thiamphenicol (Tm, 15 mg / L) -1 ) was used for Clostridium acetone-butanol cultures. Similarly, chloramphenicol (12.5 mg / L in liquid medium) was used. -1 and in solid culture medium 25 mg.L -1 ) and / or tetracycline (20 mg / L) -1 It is used for Escherichia coli culture.

[0111] The plasmids used in this study are listed in Table 1 below.

[0112] Table 1:

[0113]

[0114]

[0115] a ermB Erythromycin resistance gene; catP Genes for resistance to thiamphenicol and chloramphenicol; tetA Tetracycline resistance genes; ColE1 and p15A ori, origins of replication in Escherichia coli; pIP404, pAMB1, and pCB102, origins of replication in Clostridium acetone-butanol.

[0116] plasmid construction

[0117] Nucleic acids were purified using the QIAquick PCR Purification Kit (Qiagen), the QIAprep Spin Miniprep Kit (Qiagen), and the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich). PCR amplification was performed using Q5 high-fidelity DNA polymerase (NEB). The oligonucleotides used to construct the plasmids are shown in Tables 2A and 2B.

[0118] Table 2A:

[0119]

[0120] plasmid pGRNA- hbd(SEQ ID NO: 28) was created by cloning the hybrid products of oligonucleotides PO1 and PO2 into pGRNA using BsaI and T4 DNA ligase (NEB). ind Constructed from (SEQ ID NO: 25). The fragment obtained by overlapping PCR of the amplified material (obtained from gDNA of DSM 792 using oligonucleotide pairs PO3-PO4 and PO5-PO6) was cloned into pGRNA- hbd pGRNA is obtained from the BamHI and SalI sites. Δhbd (SEQ ID NO: 29). Plasmid pGRNA-C Δhbd (SEQ ID NO: 30) was created by cloning the hybrid products of oligonucleotides P13 and P14 into pGRNA using BsaI and T4 DNA ligase (NEB). ind It was constructed from pGRNA-C plasmid. hbd (SEQ ID NO: 31) Obtained by HiFi assembly (NEB) of the following: pGRNA-C digested with BamHI and SalI Δhbd And the use of oligonucleotide pairs P15-P16 and P17-P18 to obtain amplification products from gDNA of DSM 792.

[0121] Table 2B:

[0122]

[0123]

[0124] plasmid pGRNA- thlA (SEQ ID NO: 59) is obtained by cloning the hybrid products of oligonucleotides PO1 and PO2 into pGRNA using BsaI and T4 DNA ligase (NEB). ind Constructed from (SEQ ID NO: 25). Fragments obtained by PCR from gDNA of DSM 792 using oligonucleotide pairs PO3-PO4 and PO5-PO6 were cloned into pGRNA- via HiFi DNA assembly (NEB). thlA pGRNA is obtained from the BamHI and SalI sites. ΔthlA (SEQ ID NO: 60). Plasmid pGRNA- thlB (SEQ ID NO: 61) By cloning the hybridization product of oligonucleotides P07 and P08 into pGRNA ind(SEQ ID NO:25) was used for construction. Fragments obtained from DSM 792 gDNA by PCR using oligonucleotide pairs P09-P10 and P11-P12 were cloned into pGRNA using HiFi DNA assembly. thlB pGRNA is obtained from the BamHI and SalI sites. ΔthlB (SEQ ID NO: 62). Plasmid pGRNA- hydA (SEQ ID NO: 63) By cloning the hybrid product of oligonucleotides P13 and P14 into pGRNA ind The fragments obtained from DSM 792 gDNA by PCR using oligonucleotide pairs P15-P16 and P17-P18 were cloned into pGRNA via HiFi DNA assembly. hydA To obtain pGRNA, use the BamHI and SalI sites. ΔhydA (SEQ ID NO: 64). Plasmid pFW01- thlA (SEQ ID NO:66) was constructed by cloning fragments obtained by PCR from gDNA of DSM 792 using oligonucleotide pairs P19-P20 into the XhoI and SalI sites of pFW01 (SEQ ID NO:65) via HiFi DNA assembly.

[0125] Gene editing

[0126] For example, Mermelstein LD et al The plasmid construct was introduced into Clostridium acetone-butanol, such as Wasels F. et al (2020) and Wasels F. et al The gene editing events selected in (2017) are listed below in Table 3 (Table 3A and Table 3B).

[0127] Table 3A:

[0128]

[0129] Table 3B:

[0130]

[0131] Using other genetic tools, such as those based on homologous recombination or the insertion of mobile genetic elements, it becomes possible to obtain mutants with equivalent genotypes (i.e., those that no longer express the gene). hbd , thlA , thlB and / or hydA It became possible to produce (or express its non-functional version) the product of.

[0132] Fermentation

[0133] The fermentation performance of the microorganisms described in this study was evaluated in batch mode. Pre-culture was performed in 1 mL of CGM medium (KH₂PO₄ 0.75 g / L). -1 0.75 g / L K2HPO4 -1 MgSO4 H2O 0.4 gL -1 MnSO4 H2O 0.01 gL -1 FeSO4 7H2O 0.01 gL -1 NaCl 1.0 gL -1 Asparagine 2.0 g / L -1 Yeast extract 5.0 gL -1 (NH4)2SO4 2.0 gL -1 80 g / L of glucose -1 The process was carried out in an anaerobic incubator. After 18 h of incubation, 500 µL of these pre-cultures were inoculated into 9.5 mL of CGM medium in vials. Once inoculated, these vials were then incubated at 34 °C and 100 rpm for 96 h. At the end of fermentation, the samples were centrifuged at 5000 g for 5 min and incubated with an internal standard (final concentration of 0.5 g / L). -1 The supernatant was diluted with n-propanol, filtered through a 0.22 µm filter, and then analyzed by chromatography.

[0134] Solvents were detected by gas chromatography using a flame ionization detector on a PoraBOND-Q (Agilent Technologies) column. Helium was used as the carrier gas at a flow rate of 1.6 mL / min. -1 The column was heated from 50°C to 250°C during a 30-minute run. Acids were detected by high-performance liquid chromatography (HPLC) on an Aminex HPX-87H (Biorad) column coupled to a Spectra System RI-150 refractometer and a Waters 2487 dual-λ UV detector (adjusted to 210 nm). The mobile phase consisted of 0.1 M sulfuric acid solution, and the column temperature was set at 60°C. Results represent the average of at least three independent determinations.

[0135] Genomic DNA sequencing

[0136] ΔthlA ΔthlBThe genomic DNA of the double mutant was sequenced using INVIEW resequencing (Next Gen Sequencing 2x151 nt, Illumina) from Eurofins Genomics. A total of 2 x 13,957,557 reads were obtained and aligned to the Clostridium acetobutyricum DSM792 genome using Geneious Prime 2023.0.4 (https: / / www.geneious.com). The average chromosome coverage was 819 x 1000, and the average pSOL giant plasmid coverage was 1868 x 1000.

[0137] result

[0138] create Δhbd mutant

[0139] Δ hbd The mutant was constructed using Clostridium acetone-butanol strain DSM 792. The fermentation performance of this mutant compared to the wild-type strain is shown in Table 4.

[0140] Table 4:

[0141]

[0142] This mutant is no longer able to produce n-butanol or butyric acid. It allows for the production of large amounts of ethanol, which becomes its dominant fermentation product. Acetone production is reduced by more than 65% compared to the wild-type strain.

[0143] To reduce the amount of ethanol produced by this mutant, the CRISPR-Cas9 tool was used to target the gene containing an enzyme encoding acetaldehyde dehydrogenase (Ald) activity. adhE1 and adhE2 The pSOL giant plasmid (described in patent application WO2017 / 064439), this CRISPR-Cas9 tool is used to create Δ hbd Mutants. Loss of giant plasmids is easily achieved in wild-type strains and only requires the introduction of plasmids that enable the expression of gRNAs targeting that nucleic acid (Wasels). et al (., 2017).

[0144] Obtain plasmid pCas9 acr The frequency of transformants (using plasmid pEC750C) from the wild-type strain was 10¹ to 10² cfu·μg. DNA - The order of magnitude of ¹.

[0145] Obtain plasmid pCas9 acr Transformants of wild-type strains (using pGRNA-derived transformants) conPlasmids used for expression cas9 The frequency of targeting microbial chromosomes (without containing editing templates for the target loci) in the presence of the inducer anhydrous tetracycline (aTc) is zero.

[0146] In addition, plasmid pCas9 was obtained. acr Transformants of wild-type strains (using pGRNA-derived transformants) con The frequency of the plasmid (which targets the pSOL giant plasmid in the presence of aTc) is 10. 1 Up to 10 2 cfu·µg DNA -1 The order of magnitude. Analysis of the obtained transformants showed that they had lost the pSOL giant plasmid.

[0147] Obtain plasmid pCas9 acr Δ hbd The frequency of transformants of the mutant (using plasmid pEC750C) is typically 10. 3 cfu.µg DNA -1 The order of magnitude. Surprisingly, this mutant (using pGRNA derived from...) con The transformation frequency of the plasmid (which targets the pSOL giant plasmid in the presence of aTc and does not contain an editable template for the target locus) is zero.

[0148] This result indicates that pSOL giant plasmids in Δ hbd This is indispensable in the mutant and cannot be eliminated, contrary to what is observed in the wild-type strain. Therefore, using CRISPR-Cas9 tools in Δ hbd Precise editing within giant plasmids in mutants has become possible.

[0149] For example, plasmid pGRNA-Δ pdc Used to inactivate what was found on pSOL pdc Gene. Transforming wild-type strain DSM 792 with this plasmid induced the loss of the pSOL giant plasmid in the obtained transformants. However, it is possible to obtain Δ with fermentation performance as shown in Table 5 below. hbd Δ pdc mutant .

[0150] Table 5:

[0151]

[0152] After various gene editing operations are performed on the pSOL giant plasmid using CRISPR-Cas9 tools, the pSOL giant plasmid can be used for reintroduction. hbdGenes, thereby preserving other modifications located on giant plasmids and / or on chromosomes. This complementation at the gene locus can, for example, be achieved using the plasmid pGRNA-C. hbd (SEQ ID NO: 31) was performed, which made it possible to reintroduce functional genes that did not affect the performance of the microorganisms compared to the wild-type strain.

[0153] Derived from Δhbd DSM 792 mutant hbd C The performance of the mutants is shown in Table 6.

[0154] Table 6:

[0155]

[0156] The results obtained show that hbd The gene complement is functional, and the mutant, in which the gene has been reintroduced to its original locus, behaves in the same way as the wild-type strain.

[0157] Create Δ thlA Δ thlB Double mutant

[0158] In Clostridium acetone-butanol strain DSM 792, the CRISPR-Cas9 tool (Wasels) was used. et al A mutant was constructed (in 2020), in which... thlA The gene (CA_C2873, SEQ ID NO: 67) has been inactivated. The fermentation performance of this mutant compared to the wild-type strain is shown in Table 7.

[0159] Table 7:

[0160]

[0161] Ethanol becomes the dominant fermentation product in this mutant, which retains the ability to produce n-butanol and butyric acid. A second copy of the gene encoding an enzyme with thiolytic activity (…) thlB The presence of CA_P0078, SEQ ID NO: 68 (located on pSOL) can explain this phenomenon. Now, as in Δ hbd The mutant observed (patent application FR2303203 filed March 31, 2023), pSOL became thlA Essential genetic elements in mutants, which allow for editing using tools such as CRISPR-Cas9. Therefore, Δ was constructed. thlA Δ thlB The double mutants and their fermentation performance in batch mode are shown in Table 8.

[0162] Table 8:

[0163]

[0164] This double mutant no longer produces acetone, butanol, or butyrate, consistent with the expectation that microorganisms lack the enzyme activity of thiolytic enzymes. The genome of this mutant has been sequenced, and in addition to... thlA and thlB It does not exhibit any other modifications besides gene deletion.

[0165] Δ thlA Δ thlB Completion of double mutants

[0166] To confirm that the observed phenotype (100% ethanol) was indeed due to the double deletion, the corresponding mutant was augmented with plasmid to express the expression. thlA Genes. The performance of the corresponding transformants is shown in Table 9.

[0167] Table 9:

[0168]

[0169] Indeed, the re-establishment of the wild-type phenotype was observed.

[0170] Create Δ hbd Δ hydA Double mutant

[0171] As thlA Same, hydA (SEQ ID NO: 69) has been described as indispensable and cannot be missing (Cooksley) et al (., 2012). However, as mentioned above, using CRISPR-Cas9 tools (Wasels) et al (2020), able to obtain from Δ hbd Mutant construction Δ hbd Δ hydA Double mutant. The properties of this mutant are shown in Table 10.

[0172] Table 10:

[0173]

[0174] For example, Lehmann D. et al As reported in (2011), hbd The absence of this component makes it possible to eliminate the production of butyric acid and butanol. Δ hbd Δ hydAThe double absence has the effect of reducing the amount of acetone produced to a negligible level, making ethanol the sole fermentation product. Therefore, the obtained fermentation profile is comparable to that obtained using the Δ method according to the invention. thlA Δ thlB The fermentation profiles obtained from the mutants were similar.

[0175] in conclusion

[0176] In the course of this work, the inventors have demonstrated the possibility and advantages of using the CRISPR-Cas9 tool to perform precise gene modification within the pSOL megapmid of Clostridium acetone-butanol strain DSM 792, provided that the strain no longer expresses [the desired gene]. hbd Genes and hydA Genes, or thlA Genes and thlB Gene.

[0177] In the context of this invention, the inventors describe two Clostridium acetone-butanol double mutants, Δ thlA Δ thlB mutants and Δ hbd Δ hydA The mutant no longer produces acetone, butanol, or butyric acid, and therefore only produces ethanol.

[0178] References

[0179] - 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.

[0180] - DiCarlo, JE, Norville, JE, Mali, P., Rios, X., Aach, J., &Church, GM (2013). Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research , 41 (7), 4336-4343.

[0181] - 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 (7658), 436-439.

[0182] - Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. TheClosTron: 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.

[0183] - Joseph RC, Kim NM, Sandoval NR. 2018. Recent Developments of theSynthetic Biology Toolkit for Clostridium . Front. Microbiol. 9:154. doi:10.3389 / fmicb.2018.00154.

[0184] - Lehmann D, Lütke-Eversloh T. 2011. Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate / butanolfermentative pathway. Metabolic Engineering 13:464–473. doi:10.1016 / j.ymben.2011.04.006.

[0185] - 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 typeI and type V CRISPR-Cas inhibitors. Science , 362 (6411), 240-242.

[0186] - Mermelstein LD, Papoutsakis ET. 1993. In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase toprotect 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

[0187] - Nölling 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. Genomesequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum . J Bacteriol 183:4823–4838. doi:10.1128 / JB.183.16.4823-4838.2001.

[0188] - Pawluk, A., Davidson, A. R., & Maxwell, K. L. (2018). Anti-CRISPR:discovery, mechanism and function. Nature Reviews Microbiology , 16 (1), 12-17.

[0189] - Rauch, B. J., Silvis, M. R., Hultquist, J. F., Waters, C. S.,McGregor, M. J., Krogan, N. J., & Bondy-Denomy, J. (2017). Inhibition ofCRISPR-Cas9 with bacteriophage proteins. Cell , 168 (1-2), 150-158.

[0190] - Wasels F, Jean-Marie J, Collas F, López-Contreras AM, LopesFerreira 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.

[0191] - Wasels F, Chartier G, Hocq R, Lopes Ferreira N. 2020. A CRISPR / Anti-CRISPR Genome Editing Approach Underlines the Synergy of ButanolDehydrogenases in Clostridium acetobutylicum DSM 792. Applied andEnvironmental Microbiology 86. doi:10.1128 / AEM.00408-20.

[0192] - K. Winzeret al ., Differential regulation of two thiolase genes from Clostridium acetobutylicum DSM 792. ; J. Mol. Microbiol. Biotechnol.(2000) 2(4): 531-541. PCT / RO / 134 Table

Claims

1. A genetically modified solvent-producing bacterium belonging to the genus Clostridium, which produces ethanol without producing acetone, butanol, and / or butyric acid, characterized in that... i) The bacteria do not express the gene of sequence SEQ ID NO:

32. hbd Genes with sequence (CA_C2708) and sequence SEQ ID NO: 69 hydA The product of (CA_C0028), or the bacteria expressing its non-functional version, or ii) the bacteria not expressing the gene of sequence SEQ ID NO:

67. thlA Genes with sequence (CA_C2873) and sequence SEQ ID NO: 68 thlB The product of (CA_P0078), or the bacteria expressing its non-functional version.

2. The bacteria according to claim 1, characterized in that, The bacteria in question is Clostridium acetone-butanol.

3. The bacteria according to claim 2, characterized in that, The bacteria is IFP973 strain, registered at the BCCM-LMG Collection Center on February 21, 2024, with the number LMG P-33548, or a genetically modified version thereof, characterized in that they produce ethanol and do not produce acetone, butanol and / or butyric acid.

4. The bacteria according to claim 2, characterized in that, The bacteria is IFP974 strain, registered at the BCCM-LMG Collection Center on February 21, 2024, with the number LMG P-33549, or a genetically modified version thereof, 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 any one of claims 1 to 4 in the production of ethanol, preferably for industrial-scale production of ethanol.

6. A fermentation method involving the use of bacteria as described in any one of claims 1 to 4.

7. A kit for producing ethanol, which uses bacteria belonging to the genus Clostridium, including genetically modified bacteria belonging to the genus Clostridium according to any one of claims 1 to 4 and a culture medium for preserving or culturing said bacteria.

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