• 专利附录
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  • 权利要求
  • 类似技术
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    • 专利摘要:
    The present invention relates to mutant bacteria of the genus Clostridium beijerinckii, CNCM I-4985, CNCM I-4986, CNCM I-4987 and CNCM I-4988, deposited May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux , F-75724 PARIS Cedex 15, France) and CNCM I-5027, cNCM I-5028 and CNCM I-5029 filed November 26, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15 , France) which are useful in the fermentation of sugars for the production of isopropanol and butanol.
    公开号:FR3037077A1
    申请号:FR1562114
    申请日:2015-12-10
    公开日:2016-12-09
    发明作者:De Gerando Hadrien Mate；Laetitia Rudant；Marcel Ropars；Ferreira Nicolas Lopes；Contreras Ana Lopez
    申请人:IFP Energies Nouvelles IFPEN；Stichting Dienst Landbouwkundig Onderzoek DLO；
    IPC主号:
    专利说明:

    [0001] The present invention relates to bacteria of the genus Clostridium beijerinckii improved by random mutagenesis and to a process for producing a mixture comprising butanol and isopropanol by means of these bacteria. STATE OF THE ART Many chemical molecules called "platforms" composed of 2 to 6 carbon atoms are mainly produced from fossil resources. These base molecules are used for the production of intermediate compounds whose uses are varied, for example for the synthesis of polymers, pharmaceutical molecules, in fuel formulations or in the perfume industry. However, for certain environmental reasons, but especially in response to the unavoidable decrease in petroleum resources, alternative processes are being developed to enable the production of these "platform" molecules from renewable raw material. Propylene (or propene) is the second most used molecule in petrochemicals. It is mainly derived from petroleum and is especially obtained by steam cracking, by catalytic cracking or by the dehydrogenation of propane. One of the possible ways to produce propylene other than from petroleum is to dehydrate the isopropanol obtained by the biological fermentation of sugars extracted from biomass, a fermentation known as IBE (Isopropanol, Butanol, Ethanol) fermentation. ). Fermentations known as "solvent-borne fermentation" are carried out by microorganisms of the genus Clostridium. Clostridia are gram-positive bacilli capable of forming endospores and belonging to the phylum Firmicutes. These bacteria are anaerobic strict and ubiquitous, they can be found in the intestines of animals, soils etc. They can degrade different sugars and produce solvents from a wide variety of substrates. At the end of this "solventogenic" fermentation, the final products can vary depending on whether it is a type ABE fermentation (production of acetone, butanol and ethanol) or IBE (production of Isopropanol, Butanol and Ethanol). The most studied strain "ABE" is Clostridium acetobutylicum ATCC 824, isolated from soil in 1924 in Connecticut (Weyer & Rettger, 1927, J. Bacteriol 14, 399-424). Another highly studied strain is Clostridium beijerinckii DSMZ 6423 (NRRL B593), since it is capable of producing a solvent mixture of isopropanol / butanol / ethanol by reducing acetone to isopropanol by virtue of the presence in its genome of an alcohol dehydrogenase. (adh) primary / secondary dependent NADPH (Ismaiel et al., 1993, J. Bacteriol., 175 (16), 5097-105). The main constraint related to the development of industrial processes for the production of solvents by Clostridium strains is the final title in solvents, a problem which is mentioned as early as the first constructions of industrial units (Jones, DT, Woods, DT, 1986, Microbiological. 50 (4), 484-524). The main cause of the low solvent content is the toxicity of the end products, particularly butanol. A butanol concentration of greater than 10-12 g / L in the culture medium is known to limit the growth of Clostridium strains (Zheng et al., 2009, J. Ind.Microb. & Biotechnol 36: 1127-1138). Most of the scientific work done to date is aimed at improving the synthesis of butanol by Clostridium strains. For this, several approaches have been applied to improve the tolerance to butanol including that based on mutagenesis and the selection of Clostridium strains capable of withstanding higher levels of butanol (US 4,757,010). The technique of random mutagenesis makes it possible to modify the genetic inheritance of the strains to make them evolve towards a particular character by exerting a pressure of environmental selection. The technique thus optimizes and accelerates the phenomenon of natural selection. Another technique that can be employed is that of the "genetic mixing" ("genome shuffling" according to the English terminology) which allows to combine and recombine the DNA of whole genomes of multiple organisms by imitating the reproductive process, but at a larger scale. ladder. The recombination phenomenon is carried out by fusion of protoplasts of strains that have been selected, for example, following a mutagenic treatment and spreading on a selective medium. The combination of the two techniques can promote the production of strains with improved production of metabolites, better assimilation of the substrate, greater tolerance to the products formed.
    [0002] At present, there is still a need for new bacterial strains, which, grown under proper fermentation conditions, provide improved concentrations of a mixture of isopropanol and butanol.
    [0003] SUMMARY OF THE INVENTION The present invention relates to: a bacterium of the genus Clostridium beijerinckii deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-4985 - a bacterium of the genus Clostridium beijerinckii deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under number 10 CNCM 1 -4986 - a bacterium of the genus Clostridium beijerinckii deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-4987 - a bacterium of the genus Clostridium beijerinckii filed on November 26, 2015 at 15 Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-5027 - a bacterium of the genus Clostridium beijerinckii filed on November 26 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the name CNCM 1-5028 20 - a bacterium of the genus Clostridium beijerinckii deposited on November 26, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-5029 Bacteria mutant CMCM 1-4985, CNCM 1-4986, CNCM 1-4987 were obtained from the strain Clostridium beijerinckii DSMZ-6423 after treatment of the latter with a mutagenic agent, N-methyl-N'-nitro-N nitrosoguanidine (NTG), and selection in a medium containing a large amount of isopropanol, or methyl bromobutyrate or ethyl bromobutyrate to select mutants potentially of interest for the production of a mixture of isopropanol and butanol. In comparison with the Clostridium beijerinckii DSMZ-6423 parent strain, the selected bacteria demonstrated a benefit in terms of the final title of isopropanol and butanol solvents after fermentation of sugars.
    [0004] The invention also relates to a mutant bacterium of the genus Clostridium beijerinckii, deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1- 4988. Strain CNMC 1-4988 results from a genetic mixing step carried out with strains CNCM 1-4986 and CNCM 1-4987 followed by a selection step based on the ability of the mutants to tolerate a large concentration of isopropanol. in its environment. The CNCM 1-4988 strain used in fermentation has the capacity to produce a mixture of solvents whose isopropanol and butanol concentration is improved relative to the strain Clostridium beijerinckii DSMZ-6423.
    [0005] The invention also relates to a process for producing a mixture of isopropanol and butanol, by anaerobic fermentation carried out at a temperature between 25 and 37 ° C, in a culture medium containing sugars by means of a bacterium chosen from CNCM 1-4985, CNCM 1-4986, CNCM 1-4987 and CNCM 1-4988 bacteria.
    [0006] The process according to the invention may also use a bacterium chosen from the bacteria CNCM 1-5027, CNCM 1-5028 and CNCM 1-5029. The CNCM 1-5027, CNCM 1-5028 and CNCM 1-5029 bacteria were obtained by genetic mixing with strains mutated with the NTG mutagenic agent.
    [0007] Preferably the culture medium contains sugar as glucose. According to one embodiment, the culture medium contains a hydrolysed starchy substrate. According to the invention, the culture medium may contain carboxylic acid. For example, the culture medium contains acetic acid and / or butyric acid. DETAILED DESCRIPTION OF THE INVENTION To improve the performance of the strain naturally producing IBE Clostridium beijerinckii DSMZ-6423, the inventors proceeded to treatment with a mutagenic agent, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), of said strain to modify its genetic content. The mutagenesis technique used consists in carrying out, in a liquid medium, bringing the mutagenic agent into contact with the bacterial strain, then spreading on different solid culture media, for example in Petri dishes, the strains resulting from the 3037077 treatment. The mutagen. Culture media include levels of isopropanol, or methyl bromobutyrate or ethyl bromobutyrate that are toxic to the native Clostridium beijerinckii strain DSMZ-6423. Strain selection is based on the principle of looking for mutants resistant to isopropanol in the event that the latter would withstand an accumulation of isopropanol in the IBE fermentation tests. The use of methyl bromobutyrate or ethyl bromobutyrate as a screening product is based on the article by Clark, SW, Bennett, GN, & Rudolph, FB ((1989), Isolation and Characterization of Mutants of Clostridium Acetobutylicum ATCC 824 Acetoacetyl-Coenzyme A: Acetate / Butyrate: Coenzyme A-Transferase (EC 2.8.3.9) and Other Solvent Pathway Enzymes, Appl., Environ., Microbiol., 55 (4), 970-6) in which it is described mutants of Clostridium acetobutylicum ATCC824, selected for their resistance to 2-bromobutyrate, and which have modified butyraldehyde and butanol dehydrogenase activities. The inventors have assumed that strains with modified butyraldehyde activity are likely to produce isopropanol in an improved manner since the metabolic reactions involved in the ABE fermentation are close to those of an IBE fermentation. The mutated strains recovered after the culturing step thus saw their genome modified to acquire a resistance to toxic products and potentially better fermentative faculties for the production of solvents (more particularly isopropanol and butanol). The resistant strains were then cultured in a growth medium containing glucose or a hydrolysed starchy substrate to determine their actual ability to produce isopropanol and butanol. At the end of these fermentation steps, three mutant strains of Clostridium beijerinckii could be selected and were the subject of deposits according to the Budapest Treaty, at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F75724 PARIS Cedex 15, France), May 27, 2015, which respectively bear references CNCM 1-4985, CNCM 1-4986 and CNCM 1-4987.
    [0008] According to the invention, a mutant strain CNCM 1-4988 also deposited according to the Budapest Treaty at the Pasteur Institute has been obtained by genetic mixing (or "genome shuffling" according to the English terminology) of mutant strains. To initiate the brewing step, fusions are induced between Clostridium beijerinckii mutants. This genetic exchange between different populations thus mimics the 6 recombinations characteristic of sexual reproduction. The daughter cells obtained after mixing are again selected by looking for an evolution of their fermentation profiles. The "shuffling" process can be repeated over several generations (or brewing tower) until an improved final strain is obtained for its ability to produce solvents, especially isopropanol and butanol, and to tolerate concentrations. important isopropanol in its environment. Thus, the CNCM 1-4988 mutant strain was obtained after a brewing run carried out with mutant strains CNCM 1-4986 and CNCM 1-4987 and with a selection of strains for improved tolerance to isopropanol (beyond 40%). g / L isopropanol).
    [0009] The mutagenesis method used to obtain the strains which are the subject of the invention is described in detail below. The starting strain, also designated by the term "wild-type" according to the Anglo-Saxon terminology, is Clostridium beijerinckii DSMZ-6423 deposited with Leibniz-Institut DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The strain was precultured in a medium called GAPES whose composition is detailed in Table 1. The preculture step was carried out at a temperature of 3537 ° C for 24 hours.
    [0010] Compound Concentration (g / L) Yeast Extract 2.5 KH2PO4 1 K2HPO4 0.6 MgSO4, 7H20 1 FeSO4, 7H20 0.0066 p-Aminobenzoic Acid 0.1 Ammonium Acetate 2.9 Glucose 60 Table 1: Composition This preculture is then used to initiate a culture in CGM medium (Clostridium Growth Medium), the composition of which is given in Table 2.
    [0011] Compound Concentration [g / L] Yeast extract K2HPO4 0.75 KH2PO4 0.75 MgSO4.7H2O 0.4 MnSO4, H2O 0.01 FeSO4.7H2O 0.01 NaCl 1 Asparagine 2 (NH4) 2SO4 2 Cysteine 0.125 Glucose 12.5 Table 2: Composition of CGM Medium 5 When cultured, the wild strain of Clostridium beijerinckii DSMZ-6423 was subjected to different conditions of mutation by contact in a liquid medium CGM with N-methyl-N'- nitro-N-nitrosoguanidine (NTG; Sigma-Aldrich) which is known for its mutagenic properties. In a first series of tests, the wild strain of Clostridium beijerinckii DSMZ-6423 recovered in exponential growth phase, that is to say after 3 to 4 hours of culture, is brought into contact at a temperature of 35-37. C was a test tube with NTG added in CGM medium to obtain a concentration of 50 μg / ml NTG. The contact with the NTG is 1 hour. The cells are washed twice with potassium phosphate buffer solution (pH = 6.6) before being taken up in fresh CGM medium to carry out a regeneration step of 1 to 3 hours at 35 ° C. For the selection operation, the mutated cells are spread on selection boxes after the regeneration step. The selection medium is a CGM medium containing as selection agent ethyl bromobutyrate at a concentration of 0.5 mL / L. Incubation in the selection medium is carried out at a temperature of 35-37 ° C and for 24-48 hours. After 24 to 48 hours of culture, mutants exhibiting resistance to the selection agent were selected.
    [0012] The performances of the different mutants thus selected are then tested in vials under anaerobic conditions and with the aid of two different fermentation media, namely the GAPES medium described in Table 1 and in GAPES medium to which glucose has been replaced. by a hydrolysed starch substrate (GRITZ) corresponding to a concentration of 70 g / L glucose equivalent in said medium. The anaerobic fermentations are conducted at a temperature of 37 ° C for 48 hours and with stirring. In a second series of tests, the wild strain of Clostridium beijerinckii DSMZ-6423 is cultured in the medium CGM and collected in exponential phase, that is to say after 3 to 4 hours of culture, then is contacted with 75 μg / mL NTG. The mutants were then selected under the same conditions as mentioned above but using as selective agent isopropanol at a concentration of 40 g / l. The mutants thus selected which have acquired isopropanol resistance are then tested in fermentation under anaerobic conditions in flasks in the medium described in Table 1 and in GAPES medium, to which was added glucose instead of glucose. Hydrolyzed starch substrate (GRITZ) to provide 70 g / L glucose equivalent. The anaerobic fermentations are conducted at a temperature of 37 ° C for 48 hours and with stirring.
    [0013] In a third series of tests, the wild strain of Clostridium beijerinckii DSMZ-6423 is cultured in the CGM medium and collected in the exponential phase, that is to say after 3 to 4 hours of culture, and is then contacted. with 50 μg / mL NTG. The mutants are selected by means of a CGM culture solution containing in addition 40 g / l of isopropanol.
    [0014] Isopropanol resistant mutants are then fermented under anaerobic vial conditions in the medium described in Table 1 and in GAPES medium containing, in place of glucose, the hydrolysed starchy substrate (GRITZ) providing 70 g / L of glucose equivalent. The anaerobic fermentations are conducted at a temperature of 37 ° C for 48 hours and with stirring.
    [0015] Table 3 below summarizes the results of analysis of the solvents produced after 48 hours of anaerobic fermentation in GAPES medium. The solvents are determined by gas chromatography (Varian® apparatus), with a CP-PoraBOND Q column and a flame ionization detector (FID: Flame Detector according to the English terminology). Propan-1-ol is used as an internal standard. The parameters of the chromatography are as follows: Column: length 25 meters; Internal diameter (ID): 0.32 mm; outer diameter (ED): 0.45 mm; film thickness: 5! IL Injector temperature: from 90 ° C to 250 ° C, 150C / min Flow rate of the carrier gas 1.6 mL / min (6.8 psi) Column temperature: 50 ° C at 250 ° C, 50 ° Chin FID sensor temperature: 80 ° C 10 Injection volume: Ethanol Acetone Isopropanol Butanol [Solvent] in g / L 6423 0.1 0.1 2.4 7.3 C. beijerinckii DSMZ NTG concentration / selection medium No. Strain 50 gg / mL NTG 6 0.1 0.1 2.8 7.2 / 7 0.2 0.1 2.5 7.1 Selection medium 8 0.1 0 2,4 7 containing ethyl 9 * 0.2 0 2.8 10.5 bromobutyrate at 0.5 10 0.1 0.1 2.4 7.2 mUL 1 0.2 0.1 2, 8 8.3 3 0.2 0 0.3 6.6 NTG 75 gg / mL 5 0.1 0 1.9 5.3 / 6 * 0.1 0.1 2.3 5.5 Selection medium 7 0.1 0.1 1.9 7.8 containing isopropanol at 40g / L 8 0.1 0 0.6 0.7 9 0.1 0 2.3 6.5 10 0.1 0 2, 2 6.2 1 0.1 0.1 2.5 7.1 2 0.1 0 2.3 6.6 NTG 50 g / ml 3 0.1 0.1 2.7 6.4 / Medium of Selection 4 0.4 0.1 2.4 7 containing 5 0.1 0 1.9 6.5 isopropanol 40g / L 6 0.2 0 2.4 7.3 7 * 0 0 3.9 9 , 3 3037077 10 Table 3: Concent solvent feed after fermentation on GAPES medium from mutant strains obtained by random mutagenesis using NTG.
    [0016] The mutant strains which have been deposited according to the Budapest Treaty are indicated in Table 3 by an asterisk. The 9 * strain corresponds to the CNCM 1-4985 strain deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France). The 6 * strain corresponds to the CNCM 1-4986 strain deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France). The strain 7 * corresponds to the CNCM 1-4987 strain deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France). Table 4 gives the solvent concentrations produced by mutant strains of Clostridium beijerinckii CNCM 1-4985, CNCM 1-4986 and CNCM 1-4987 by fermentation in GAPES medium to which glucose was replaced by a hydrolysed starchy substrate (GRITZ). ) at 70 g / L glucose equivalent. After anaerobic fermentation for 48 hours, the glucose contents are determined by HPLC (Varian®) with an Aminex0HPX-87P column (Biorad, 300 mm long and 7.8 mm in diameter) at 80 ° C. The eluent used is water with a flow rate of 0.4 ml / min. The detector is a refractometer (Varian® 350 RI). The sample volume injected is 351..11_. The solvents are analyzed by gas chromatography as mentioned above. Concentration (in g / L) Yield Solvent / Glucose Glucose Acetate Butyrate Ethanol Acetone Isopropanol Butanol will be consumed. Solvent consumed Strain 44.8 3.1 3.1 0.18 0.17 6 10.1 16.4 36.7% DSMZ 6423 Strain 47.9 4.4 1.7 0.23 0.21 7.1 10.4 18 37.5% CNCM I-4985 Strain 48.1 4.5 1.7 0.21 0.16 7 10.3 17.7 36.9% CNCM I-4986 Strain 47.3 4 1, Table 4: Concentration of solvents after fermentation in GAPES medium containing a hydrolysed starch substrate (GRITZ) (70 g / L) glucose equivalent) as a replacement for glucose. It is found that the fermentation carried out with strains CNCM 1-4985, CNCM 1-4986 and CNCM 1-4987 produce a fermentation broth having higher concentrations of isopropanol and butanol than that produced by the wild strain Clostridium beijerinckii DSMZ 6423.
    [0017] The mutant strains obtained after the NTG random mutagenesis step and selected for their resistance to 40 g / l isopropanol or 0.5 ml / l ethyl bromobutyrate were used for a brewing step. genetics according to the protocol described by Gao, X., Zhao, H., Zhang, G., He, K. & Jin, Y. (2012). Genome shuffling of Clostridium acetobutylicum CICC 8012 for improved production of acetone-butanol-ethanol (ABE). Curr. Microbiol., 65 (2), 12832. The mutant strains are first separately grown in an exponential phase in a CGM medium and removed after 3 to 4 hours of culture. The cultures are then centrifuged for 10 minutes at 4000 g and washed twice with a solution of sodium maleate monohydrate No. 1 (MMS 1) at pH 6.5 containing 0.5 M sucrose, 20 mM sodium maleate monohydrate, and 20 mM MgCl 2. The collected cells are contacted at 35 ° C. for 1 h with a solution of sodium maleate monohydrate No. 2 which has the following composition: 0.5 M sucrose, 20 mM sodium maleate monohydrate, 20 mM MgC12, 1 g / L cysteine, 1 g / L glutathione to which 15 mg / mL lysozyme was added. At the end of the treatment, the cells are collected, washed with the solution of sodium maleate monohydrate No. 1 and centrifuged at 4000 G for 5 min. The different populations are then mixed in 10 ml of a solution of sodium maleate monohydrate No. 3 (0.5 M sucrose, 20 mM sodium maleate monohydrate, 30 mM MgCl 2, 1 g / l cysteine, 1 g / L glutathione, 50 mM CaCl2) supplemented with 30% w / v (30g per 100mL) PEG 4000 and incubated for 20 min at a temperature of 35-37 ° C to induce protoplast fusion. The fused cells are suspended in CGM medium and regenerated on CGM agar for 40 hours.
    [0018] Several protoplast fusion crosses were made from mutant strains treated with NTG. Genetically brewed strains were then selected for increased tolerance to isopropanol in CGM medium (45-50 g / L isopropanol).
    [0019] Table 5 compares the solvent concentrations of the fermentation must, after 48 hours of fermentation, obtained with the strain CNCM 1-4988, filed May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, France). -75724 PARIS Cedex 15, France) and the wild strain Clostridium beijerinckii DSMZ 6423. The fermentations were carried out in GAPES medium containing hydrolysed starch substrate (GRITZ, 70 g / L equivalent glucose) in place of glucose. The CNCM 1-4988 strain is derived from a single round of genetic mixing with strains CNCM 1-4986 and CNCM 1-4987. Concentration (in g / L) Yield Solvent / Glucose Glucose Acetate Butyrate Ethanol Acetone Isopropanol Butanol Solvent consumed, totals consumes Strain 44.8 3.1 3.1 0.18 0.17 6 10.1 16.4 36.7% DSMZ 6423 Strain 45.9 3.5 0.8 0.21 0.21 6 10.7 17.2 37.4% CNCM I-4988 Table 5: Concentration of solvents after fermentation in GAPES medium containing a hydrolyzed starchy substrate (GRITZ, 70 g / L glucose equivalent) instead of glucose It is noted that the strain CNCM 1-4988 produces more butanol than the wild strain, while maintaining an identical level of isopropanol production. Different mutant strains were also obtained after two rounds of genetic mixing, and were then tested in anaerobic fermentation in a GAPES medium supplemented with a hydrolysed starch substrate (70 g / L glucose equivalent) instead of glucose. The CNCM 1-5027 strain was isolated as described below. A genetic mixing step was carried out with the CNCM 1-4985 strain and with a strain resulting from mutagenesis with a solution of NTG at 75 μg / mL and then selected for its resistance to a medium containing 40 g GMM. / L isopropanol. After the brewing run, mutated cells were selected using a CGM selection medium containing 40 g / L isopropanol. Incubation in the selection medium is carried out at a temperature of 35-37 ° C and for 24 hours. After 24 hours of culture, resistant mutants were selected and again incubated at 35-37 ° C and for 24 hours in CGM medium containing 50 g / L isopropanol. The CNCM 1-5027 strain is the result of this second selection step.
    [0020] As for the CNCM 1-5028 strain, it is the result of a genetic mixing step carried out with the CNCM 1-4985 strain and with two other strains which have undergone a prior step of mutagenesis with NTG (75 μg / mL ) selected in a CGM selection medium containing 40 g / L isopropanol. As above, the CNCM 1-5028 strain is derived from two selection steps using a CGM medium containing 40 g / l of isopropanol and then a CGM selection medium containing 50 g / l of isopropanol. The strain CNCM I-5029 was isolated by applying the protocol described above but in which the genetic mixing step was carried out with two strains resulting from NTG mutagenesis (75 μm / ml) and selected for their resistance in a CGM screening medium containing 40 g / L isopropanol. As before, the isolation of the CNCM 1-5029 strain was carried out in two selection steps using a CGM medium containing 40 g / l of isopropanol and then a CGM selection medium containing 50 g / l of isopropanol.
    [0021] Table 6 gives the solvent concentrations of the fermentation must, after 48 hours of fermentation at 37 ° C, obtained with strains CNCM 1-5027, CNCM 1-5028 and CNCM 1-5029. The fermentations were carried out in GAPES medium, the composition of which is given in Table 1.
    [0022] Concentration (in g / L) Glucose Butyrate Ethanol Acetone Isopropanol Butanol Yield, solvent / consumes Glucose strain 18 0.77 0.24 0.09 1.54 5.69 42% DSMZ 6423 CNC M 23 0.99 0.22 0.07 1.59 4.69 29% 1-5027 CNCM 21 0.60 0.19 0.06 1.9 5.75 38% 1-5028 CNCM 22 0.66 0.22 0.08 1.81 6.24 38% 1-5029 Table 6: Concentration in solvents after fermentation at 37 ° C. in a GAPES medium containing a hydrolysed starch substrate (GRITZ, 70 g / L glucose equivalent) as a replacement for glucose It is observed that strains CNCM 1-5027, CNCM 1-5028 and CNCM 1-5029 are capable of producing more isopropanol than the reference strain DSM 6423. Furthermore CNCM I5028 and CNCM 1-5029 allow to produce more butanol compared to the strain DSMZ 6423. 5 10
    权利要求:
    Claims (12)
    [0001]
    REVENDICATIONS1. Bacterium of the genus Clostridium beijerinckii deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-4985.
    [0002]
    2. Bacterium of the genus Clostridium beijerinckii deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-4986.
    [0003]
    3. Bacterium of the genus Clostridium beijerinckii deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-4987.
    [0004]
    4. Bacterium of the genus Clostridium beijerinckii deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-4988.
    [0005]
    5. Bacteria of the genus Clostridium beijerinckii deposited on November 26, 2015 at the Pasteur Institute (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under number CNCM 1-5027.
    [0006]
    6. Bacterium of the genus Clostridium beijerinckii deposited on November 26, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-5028.
    [0007]
    7. Bacteria of the genus Clostridium beijerinckii deposited on November 26, 2015 at the Institut Pasteur (CNCM, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France) under the number CNCM 1-5029.
    [0008]
    8. Process for producing a mixture of isopropanol and butanol by anaerobic fermentation, at a temperature between 25 and 37 ° C, in a culture medium containing sugars by means of a bacterium according to one of any of claims 1 to 7.
    [0009]
    9. The method of claim 8, wherein the sugars of the culture medium is glucose.
    [0010]
    The method of claims 8 or 9, wherein the culture medium contains a hydrolysed starchy substrate. 10
    [0011]
    11. Method according to one of claims 8 to 10, wherein the culture medium contains carboxylic acid.
    [0012]
    The process according to claim 11, wherein the carboxylic acid medium is acetic acid or butyric acid.
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    同族专利:
    公开号 | 公开日
    KR20180027496A|2018-03-14|
    CN108473942A|2018-08-31|
    EP3303638A1|2018-04-11|
    US20180142311A1|2018-05-24|
    US10253380B2|2019-04-09|
    FR3037077B1|2018-01-19|
    FR3037076A1|2016-12-09|
    FR3037076B1|2018-11-09|
    WO2016192871A1|2016-12-08|
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    法律状态:
    2016-12-09| PLSC| Publication of the preliminary search report|Effective date: 20161209 |
    2016-12-12| PLFP| Fee payment|Year of fee payment: 2 |
    2017-12-14| PLFP| Fee payment|Year of fee payment: 3 |
    2019-12-24| PLFP| Fee payment|Year of fee payment: 5 |
    2021-09-10| ST| Notification of lapse|Effective date: 20210806 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1555105|2015-06-04|
    FR1555105A|FR3037076B1|2015-06-04|2015-06-04|MUTANT STRAINS OF THE GENUS CLOSTRIDIUM BEIJERINCKII|US15/579,453| US10253380B2|2015-06-04|2016-04-01|Mutant strains of the genus Clostridium beijerinckii |
    PCT/EP2016/057236| WO2016192871A1|2015-06-04|2016-04-01|Mutant strains of the genus clostridium beijerinckii|
    EP16714851.9A| EP3303638A1|2015-06-04|2016-04-01|Mutant strains of the genus clostridium beijerinckii|
    CN201680032468.6A| CN108473942A|2015-06-04|2016-04-01|The mutant strain of Clostridium beijerinckii|
    KR1020187000299A| KR20180027496A|2015-06-04|2016-04-01|Mutant strains of the genus clostridium beijerinckii|
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