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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/145—Clostridium
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• 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.
• 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).
• Clostridium acetobutylicum ATCC 824 isolated from soil in 1924 in Connecticut (Weyer & Rettger, 1927, J. Bacteriol 14, 399-424).
• Clostridium beijerinckii DSMZ 6423 (NRRL B593), because it is capable of to produce an Isopropanol / Butanol / Ethanol solvent mixture by reducing acetone to isopropanol by virtue of the presence in its genome of an NADPH-dependent primary / secondary alcohol-dehydrogenase (adh) (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 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: 1,127-1 138)
• the present invention relates to:
• the mutant bacteria CMCM I-4985, CNCM I-4986, CNCM I-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 potentially interesting mutants for the production of a mixture of isopropanol and butanol.
• NTG N-methyl-N'-nitro- N-nitrosoguanidine
• the selected bacteria showed a benefit in terms of the final title in Isopropanol and Butanol solvents after fermentation of sugars.
• 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.
• the invention also relates to a process for producing a mixture of isopropanol and butanol, by anaerobic fermentation carried out at a temperature of between 25 and 37 ° C, in a culture medium containing sugars by means of a selected bacterium among the bacteria CNCM 1-4985, CNCM 1-4986, CNCM 1-4987 and CNCM 1-4988.
• the method according to the invention can also implement a bacterium selected from CNCM 1-5027, CNCM 1-5028 and CNCM 1-5029 bacteria.
• 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.
• the culture medium contains a hydrolysed starchy substrate.
• the culture medium may contain carboxylic acid.
• the culture medium contains acetic acid and / or butyric acid.
• methyl bromobutyrate or ethyl bromobutyrate as a screening product is based on the article by Clark, S.W., Bennett, G.N., & Rudolph, F. B. (1989). Of Acetacetyl-Coenzyme A: Acetate / Butyrate: Coenzyme A-Transferase (EC 2.8.3.9) and Other Solvent Pathway Enzymes. Appl. About. Microbiol., 55 (4), 970-6) in which there are 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 a modified butyraldehyde activity are likely to produce risopropanol in an improved manner insofar as the metabolic reactions involved in the ABE fermentation are close to those of an IBE fermentation.
• the mutated strains recovered after the culturing step have thus seen their genome modified to acquire a resistance to toxic products and potentially better fermentative faculties for the production of solvents (more particularly in 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 risopropanol and butanol.
• a mutant strain CNCM I-4988 also deposited according to the Budapest Treaty at the Pasteur Institute was obtained by genetic mixing (or "shuffling genome” according to the English terminology) of mutant strains.
• the mutagenesis method used to obtain the strains that are the subject of the invention is described in detail below.
• the starting strain also designated by the term "wild-type" according to English terminology, is Clostridium beijerinckii DSMZ-6423 deposited with Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.
• NVG N-methyl-N'-nitro-N-nitrosoguanidine
• 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 ⁇ g / mL of 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 resistant to isopropanol are then tested in fermentation under anaerobic conditions in flasks in the medium described in Table 1 and in GAPES medium containing, in place of glucose, hydrolysed starch substrate (GRITZ) providing 70 g / L glucose equivalent.
• the anaerobic fermentations are conducted at a temperature of 37 ° C for 48 hours and with stirring.
• Table 3 summarizes the results of analysis of 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 Ionization Detector according to the English terminology).
• FID Flame Ionization Detector according to the English terminology.
• Propan-1-ol is used as an internal standard.
• Table 3 Concentration of solvents after fermentation on GAPES medium from mutant strains obtained by random mutagenesis using NTG. Mutant strains that have been deposited under the Budapest Treaty are indicated in Table 3 by an asterisk.
• the 6 * strain corresponds to the CNCM strain I-4986 filed on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Dondel Roux, F-75724 PARIS Cedex 15, France).
• the strain 7 * corresponds to the CNCM strain I-4987 deposited on May 27, 2015 at the Institut Pasteur (CNCM, 25 rue du Do Budapest Roux, F-75724 PARIS Cedex 15, France).
• Table 4 gives the concentrations of solvents produced by mutant strains of Clostridium beijerinckii CNCM 1-4985, CNCM 1-4986 and CNCM 1-4987 by fermentation in GAPES medium to which the glucose was replaced by a hydrolysed starch substrate (GRITZ). at 70 g / L glucose equivalent.
• mutant strains obtained after the random mutagenesis step at NTG and selected for their resistance to isopropanol at 40 g / L or to ethyl bromobutyrate at 0.5 mIJL, were used for a genetic mixing step according to US Pat. 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), 128-32.
• the mutant strains are first brought separately in the exponential phase in a CGM medium and taken 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 .
• MMS 1 sodium maleate monohydrate No. 1
• strain CNCM 1-4988 produces more butanol than the wild strain, while maintaining an identical level of production of isopropanol.
• 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 a mutagenesis with a solution of NTG at 75 ⁇ g / mL and then selected for its resistance to a CGM medium containing 40 g / L of isopropanol.
• 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 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 ⁇ / ⁇ ) and selected for their resistance in a CGM selection medium containing 40 g / l of 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.
• 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.

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