WO2018224712A1 - Mutants de clostridium beijerinckii hyper reproducteurs de butanol - Google Patents

Mutants de clostridium beijerinckii hyper reproducteurs de butanol Download PDF

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WO2018224712A1
WO2018224712A1 PCT/ES2018/070402 ES2018070402W WO2018224712A1 WO 2018224712 A1 WO2018224712 A1 WO 2018224712A1 ES 2018070402 W ES2018070402 W ES 2018070402W WO 2018224712 A1 WO2018224712 A1 WO 2018224712A1
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cbei
gene
instead
butanol
strain
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PCT/ES2018/070402
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Spanish (es)
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Elena PUERTA FERNÁNDEZ
María HIDALGO GARCÍA
José David MONTOYA SOLANO
Almudena ESCOBAR NIÑO
Juan Luis RAMOS MARTÍN
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Abengoa Research, S.L
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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P7/06—Ethanol, i.e. non-beverage
    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C12P7/28—Acetone-containing products
    • 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

  • the present invention falls within the field of industrial processes for the production of butanol, preferably acetone, butanol and ethanol (ABE), by fermentation with strains of Clostridium.
  • the invention relates to mutant strains of Clostridium beijerinckii capable of producing more butanol than the parental strain from which they originate, using different substrates as a carbon source.
  • the invention also relates to the use of said mutant strains in methods of production of ABE by fermentation and to a method of obtaining said mutant strains.
  • Butanol is an important industrial chemical. Compared to ethanol, butanol is more miscible in gasoline and diesel, has a lower vapor pressure and is less miscible in water. It also contains about 22% oxygen, which means that when used as a biofuel it results in a more complete combustion producing a smaller amount of smoke. These qualities make it a fuel extender superior to ethanol. Butanol is also used as a chemical in the plastics industry and as a food extractant in the food and flavor industry.
  • ABE fermentation is the most widely studied among all anaerobic fermentation processes. It is known that some strains of Clostridium produce butanol from carbohydrates through the process called ABE fermentation. During this fermentation three solvents, acetone, butanol and ethanol, are produced in a 3: 6: 1 ratio.
  • the most commonly used strain of Clostridium for ABE fermentation is Clostridium acetobutylicum, which is the model bacterium of Clostroidios solventogenic. However, this strain has the genes involved in the production of butanol in a megaplasmid, and genetic instabilities of the strain can arise.
  • C. beijer ⁇ nckii Another very relevant solvent isogenic Clostridio is C. beijer ⁇ nckii, which has the butanol production genes on the chromosome, which makes it a more robust line for industrial use. In addition, this lineage is less likely to suffer the phenomenon known as "acid crash", which causes cells to stop producing butanol and begin to sporulate.
  • the advantages of using C. beijer ⁇ nckii instead of C. acetobutylicum include that the former can use a wider range of substrates and carry out fermentation in a better pH range, has the ability to produce butanol during its exponential growth phase and it has greater stability when it comes to microbial degeneration.
  • the solvent genes in C. beijer ⁇ nckii are located on the chromosome, while in C. acetobutylicum these genes are located in a plasmid, which makes C. beijer ⁇ nckii more genetically stable.
  • the cells first pass through a phase known as the "acidogenic phase", in which the acids (acetic and butyric) are produced that will be converted into the solvents (butanol, acetone and ethanol) during the solvent phase. Both phases are very connected, and the genes that are expressed during the solvent are regulated for expression after acidogenesis.
  • acids acetic and butyric
  • solvents butanol, acetone and ethanol
  • This mutant strain BA101 is described as a hyperproductive strain of butanol obtained by mutagenesis of the wild strain C. beijer ⁇ nckii NCIMB 8052 (Annous, BA, HP Blaschek. 1991. Appl. Environ. Microbiol. 57: 2544-2548; Formanek, J. , R. Mackie, HP Blaschek. 1997. Appl. Environ. Microbiol. 63: 2306-2310).
  • patent application AU2014216024 describes genetically modified Clostridium solvent strains comprising an altered gene expression or structure that results in increased butanol production efficiency.
  • the modified Clostridium species as described herein is C. beijer ⁇ nckii.
  • the present invention provides three mutants of the Clostr ⁇ dium beijer ⁇ nckii species obtained by random chemical mutagenesis of the parental strains C. beijer ⁇ nckii BA101 and NCIMB 8052, which are capable of producing more butanol or producing it more efficiently than their parental strains using different substrates as a source of carbon and under various conditions.
  • the present invention relates to three mutant strains of C. beijer ⁇ nckii that produce significantly more butanol, or that are capable of producing it more rapidly, than the parental strains from which they originate, thus increasing the productivity of the fermentation process for ABE production.
  • mutants are also capable of maintaining a higher pH, within the acidic range, during their growth. That is, these mutants have a lower acidity or acidogenesis than their parental strains, so they reduce the risk of the effect known as "acid crash" that causes cells to stop producing butanol and start sporulating.
  • causing less acidity in the culture medium means that acids may be drifting to solvents more quickly, so that they can produce a greater amount of these (butanol, preferably ABE) in less time.
  • the mutants of the invention can also use both C6 (for example, glucose) and C5 (for example, xylose and arabinose) sugars as fermentation carbon, and consume them more efficiently than their corresponding parental strains. These mutants can even, under certain conditions, consume both types of sugars, C5 and C6, simultaneously. They can also use complex substrates of industrial interest, such as urban or plant wastes, which favor the recycling and use of waste products derived from other industries or from urban activity. These three mutant strains have been referred to in the present invention as BP1 1, BP25 and BP31, where the first strain comes from the parental strain C. beijer ⁇ nckii NCIMB 8052 and the last two strains are derived from the parental strain C. beijer ⁇ nckii BA101.
  • C6 for example, glucose
  • C5 for example, xylose and arabinose
  • the inventors have developed and developed a method of induced mutagenesis in parental cells of C. beijer ⁇ nckii.
  • the optimization of this procedure involves, among other factors, identifying the appropriate starting parental cells, the appropriate mutagenic agent and defining the appropriate concentration thereof to achieve an optimal ratio of mutagenesis / cell death in C. beijer ⁇ nckii.
  • Other mutants of the C. beijer ⁇ nckii species that exhibit low acidogenesis and / or improved butanol production capacity other than those described in the present invention could also be obtained by this procedure.
  • the present invention relates to a method for obtaining or generating mutant strains of the species C. beijer ⁇ nckii with low acidogenesis and / or with improved butanol production capacity, preferably ABE, from carbohydrates by anaerobic fermentation, where said method comprises the steps of: a. induce chemical mutagenesis in a cell of C. beijer ⁇ nckii, preferably in the strain C. beijer ⁇ nckii NCIMB 8052 or C. beijer ⁇ nckii BA101, more preferably in C. beijer ⁇ nckii NCIMB 8052, and
  • stage (a) select those mutants produced after stage (a) that have low acidogenesis, where said selection is made by culturing the mutants in selective plates comprising a pH indicator, where the chemical mutagenesis of stage (a) is performed by incubating the cell, or a cell culture comprising the same, with the chemical mutagen ethyl methanesulfonate (EMS) at a concentration of, preferably 15 ⁇ of EMS per ml of culture, for preferably 1 hour, and
  • EMS chemical mutagen ethyl methanesulfonate
  • the pH indicator of step (b) is preferably Bromocresol Purple.
  • "Low or lower acidity or acidogenesis” mutants are understood as those cells of the species C. beijerinckii that lower the pH of the culture medium during its growth to a lesser extent than the parental strain from which they originate, after the same or similar period of time in culture, in the presence of a culture medium of identical or similar composition and under culture conditions (T a , pH at the beginning of the culture, anaerobiosis, agitation, volume of the medium, saturation, optical density, etc.) identical or similar.
  • the mutants that have "low or lower acidity or acidogenesis” according to the invention are those that maintain the pH of the culture medium above 5 during growth. This property of the cells can be determined, for example but not limited to, by in vitro culture of the same in selective plates with pH indicator as indicated above.
  • Mutants with "improved butanol production capacity” are understood as those mutants that produce more butanol, preferably in g / l, than the parental strain of C. beijerinckii from which they originate, after the same or similar period of time fermentation, under the same or similar fermentation conditions (T a , pH, anaerobiosis, agitation, volume of the medium, saturation, optical density, etc.) and in the presence of a culture medium comprising a source of carbon or carbohydrates thereof or similar composition.
  • T a pH, anaerobiosis, agitation, volume of the medium, saturation, optical density, etc.
  • Mutants with improved butanol production capacity therefore use the carbon source used as a substrate more efficiently.
  • the increased efficiency or improved production capacity of butanol can be determined by a variety of methods known in the state of the art such as, but not limited to, by measuring the concentration (weight / volume) of the solvent produced in the fermentation medium, or the yield (weight / weight) of the solvent per amount of substrate, or the ratio of solvent formation (weight / volume / time).
  • these product or solvent measurements can be carried out by HPLC, mass spectrometry, immunoassay, activity tests or any other method known to those skilled in the art.
  • the reasoning for assuming that the mutants are hyper- or over-producing butanol or that they have improved butanol production capacity based on the low acidity they cause in the culture medium is that the acidifying cells less the medium may be drifting acids to solvents more quickly, so that they can produce a greater amount of these (butanol, preferably ABE) in less time.
  • the "Purple Bromocresol", preferably used in step (b) of this method of mutant generation, is an organic indicator for acid-base titration whose pH transition range ranges from 5.2 to 6.8, turning from Yellow to purple in the mentioned range. Thus, it is purple at basic pH and turns yellow at acidic pH.
  • step (b) When Bromocresol Purple is used as a pH indicator for the selection of stage (b), the cells cannot be sown directly in said selective medium because their toxicity does not allow the growth of healthy colonies. Therefore, first of all the mutants obtained after step (a) must be sown in a standard culture medium for the growth of C. beijer ⁇ nckii, for example, but not limited to a medium comprising yeast extract, tryptone, acetate ammonium, magnesium acetate, iron sulfate, potassium phosphate and / or magnesium sulfate, and a carbon source, for example, glucose. Thus, colonies with sufficient biomass are obtained to then reseed the selective plates. Subsequently, the mutants thus grown are isolated and re-plated on the plates comprising Purple Bromocresol to finally isolate and select those mutants that generate a lower yellow halo in relation to their parental strain or an absence thereof.
  • a standard culture medium for the growth of C. beijer ⁇ nckii for example, but not
  • chemical mutagen refers to a chemical agent that increases the frequency of mutation above the rate of spontaneous mutation.
  • Another aspect of the invention relates to a mutant strain of the species C. beijer ⁇ nckii with low acidity or acidity and / or with improved butanol production capacity, preferably ABE, from carbohydrates by anaerobic fermentation, produced, obtainable or obtained by the mutant generation method described above in the present invention.
  • Another aspect of the invention relates to a mutant strain or cell of C. beijerinckii (preferably obtained by chemical mutagenesis of strain C. beijerinckii NCIMB 8052) characterized in that said mutant strain comprises in its genome the following mutations with respect to the genome of the parental strain C. beijerinckii NCIMB 8052:
  • mutant BP1 1 comprises all the mutations indicated in (a) to (g) above.
  • This mutant has preferably been obtained by the mutant generation method described above.
  • Another aspect of the invention relates to a strain or mutant cell of C. beijer ⁇ nckii (preferably obtained by chemical mutagenesis of strain C. beijer ⁇ nckii BA101) characterized in that said mutant strain comprises in its genome the following mutations with respect to the genome of the strain parental C. beijer ⁇ nckii BA101: a.
  • this strain will be referred to as a "mutant BP31", and comprises all the mutations indicated in (a) to (f) above.
  • This mutant has preferably been obtained by the mutant generation method described above.
  • the so-called "BP25 mutant” is preferred, since it has ABE production yields, especially butanol, higher than the other mutants in the tested substrates.
  • Fig. 3A shows that the BP25 mutant produces more butanol in the presence of glucose, not only in comparison to its parental strain, but also in comparison to the BP31 mutant that also comes from the same parental strain.
  • Fig. 7 shows that mutant BP25 produces butanol more rapidly in presence of a complex substrate, such as an urban waste hydrolyzate, than the BP31 mutant that is not able to improve butanol production with respect to its parental strain in the presence of this particular substrate.
  • Fig. 3A shows that the BP25 mutant produces more butanol in the presence of glucose, not only in comparison to its parental strain, but also in comparison to the BP31 mutant that also comes from the same parental strain.
  • Fig. 7 shows that mutant BP25 produces butanol more rapidly in presence of a complex substrate, such as an urban waste hydrolyzate, than
  • BP25 mutant is capable of producing more butanol in the presence of corn straw hydrolyzate than the BP31 mutant.
  • Fig. 12 shows that, although the BP1 1 mutant initially consumes arabinose more rapidly, from 48h it slows its consumption of arabinose and butanol production, unlike the BP25 mutant that continues to produce butanol during 72h The fermentation analysis was followed, which means that the BP25 mutant reaches a higher final production yield, consuming more arabinose in the process.
  • the BP25 mutant has an improved butanol production capacity compared to its parental strain, in various substrates.
  • Fig. 3A demonstrates a higher production of butanol by this mutant in the presence of glucose in relation to its parental C. beijerinckii BA101.
  • Fig. 4A shows that this mutant produces butanol more quickly, and therefore more efficiently, in the presence of ground corn than its parental strain, reaching high levels of butanol in a shorter time.
  • Fig. 7A and 10 show that BP25 produces higher levels of butanol in the presence of urban solid waste hydrolyzate and corn straw hydrolyzate, respectively, than its parental strain.
  • Fig. 3A demonstrates a higher production of butanol by this mutant in the presence of glucose in relation to its parental C. beijerinckii BA101.
  • Fig. 4A shows that this mutant produces butanol more quickly, and therefore more efficiently, in the presence of ground corn than its parental strain, reaching high levels of butanol in a shorter
  • FIG. 1 1 B demonstrates a higher production of butanol and a more efficient and faster consumption of arabinose and glucose by BP25 in the presence of a medium comprising these two sugars compared to their parental strain.
  • Fig. 12 demonstrates a higher production of butanol by BP25 in the presence of a medium comprising arabinose above that obtained with the other two mutants (BP1 1 and BP31) and with the parental strain C. beijerinckii BA101.
  • another aspect of the invention relates to a strain or mutant cell of C. beijerinckii (preferably obtained by chemical mutagenesis of strain C. beijerinckii BA101) characterized in that said mutant strain comprises in its genome the following mutations with respect to the genome of the parental strain C. beijerinckii NCIMB 8052: a.
  • T instead of C at position 151 of the Cbei_0769 gene (SEQ ID NO: 2), which comprises positions 935299 to 936627 of the genome of C. beijerinckii NCIMB 8052,
  • NCIMB 8052 n. T instead of C at position 160 of the Cbei_3757 gene (SEQ ID NO: 14), which comprises positions 4310570 to 4310908 of the genome of C. beijerinckii NCIMB 8052,
  • this mutant comprises all the mutations indicated in (a) to (y) above.
  • This mutant has preferably been obtained by the mutant generation method described above.
  • This mutant BP25 has also been deposited in the Spanish Type Culture Collection (CECT), Pare Cient ⁇ fic Universitat de Valencia, Professor Agust ⁇ n Escardino 9, 46980 Paterna (Valencia, Spain), under the accession number CECT 9306 dated 21.03.2017. Therefore, in a preferred embodiment of this aspect of the invention, this mutant strain of Clostridium beijerinckii is that deposited in the Spanish Type Culture Collection under the accession number CECT 9306.
  • the strain C. beijerinckii BA101 is a hyperproductive butanol mutant obtained by mutagenesis from the wild strain C. beijerinckii NCIMB 8052.
  • Said mutant strain BA101 thus comprises the genome of the wild strain NCIMB 8052 in addition to the mutations indicated above for genes Cbei_0769, Cbei_1854, Cbei_1935, Cbei_1975, Cbei_3078, Cbei_4308, Cbei_4400 and Cbei_4761. Therefore, of all the mutations indicated in (a) to (and) above, mutations in these specific genes are shared between mutant BP25 and strain C. beijerinckii BA101.
  • the term "mutants of the invention" refers to any mutant strain of the species C.
  • the parental strain C. beijerinckii NCIMB 8052 is available at, for example, but not limited to, the International Depository Authority National Collection of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Ab2 1 RY, Scotland, England. The complete genome of this strain is described, for example, in the GenBank under accession number CP000721 .1.
  • the parental strain C. beijerinckii BA101 can be prepared as described in Annous, BA, et al., Appl. Environ. Microbiol., 1991, 57: 2544-2548.
  • the genome of this strain is the genome of C. beijerinckii NCIMB 8052 which also comprises the mutations indicated in the present invention in the genes Cbei_0769, Cbei_1854, CbeiJ 935, CbeiJ 975, Cbei_3078, Cbei_4308, Cbei_4400 and Cbei_4761.
  • Another aspect of the invention relates to the use of mutants of the invention, preferably of mutants BP1 1, BP31 and BP25, more preferably of mutant BP25, for the production of solvents.
  • solvents means any solvent known in the state of the art, including mixtures of acetone, butanol, isopropanol and / or ethanol.
  • the solvent is butanol, that is, the main component of the solvent mixture obtained is butanol.
  • the solvents are acetone, butanol and ethanol (ABE), that is, the solvents produced comprise a mixture of these three.
  • ABE acetone, butanol and ethanol
  • the production of solvents by the mutants of the invention is carried out by means of an anaerobic fermentation process in the presence of a culture medium comprising a source of carbon or carbohydrates.
  • “Culture media” suitable for use in the present invention are all those known in the state of the art as appropriate for the growth, Kirtative activity and maintenance in cultivation of C. beijerinckii strains.
  • these culture media are, for example, but not limited to, P2, TGY, PT or TYA, preferably TYA.
  • the culture medium further comprises at least one organic acid, such as acetate and / or butyrate.
  • This organic acid can come from the acidogenic phase of the microorganism used in the fermentation or it can be added externally.
  • the amount of organic acid added to the culture is between 20 mM and 80 mM.
  • the addition of one or more organic acids increases the amount of solvents recovered after fermentation. In addition, it prevents the degeneration of the strain during the fermentation process, favoring its stability and avoiding the degeneration of the crop.
  • the culture medium may further comprise salts and / or buffers.
  • the culture medium is also supplemented with a carbon source, which may comprise, for example but not limited to, C6 sugars (such as glucose), C5 sugars (such as xylose and / or arabinose), or a mixture of both types of sugars
  • a carbon source may comprise, for example but not limited to, C6 sugars (such as glucose), C5 sugars (such as xylose and / or arabinose), or a mixture of both types of sugars
  • the carbon source comprises glucose, maltodextrin, xylobious, cellobiose, xylose and / or arabinose, or any combination thereof. More preferably, the carbon source comprises glucose, cellobiose, xylose and / or arabinose.
  • the carbon source comprises xylose, glucose or arabinose or any combination thereof, even more preferably it comprises glucose and arabinose, since the mutants of the present invention, especially the mutant BP25, are particularly useful for consuming these two sugars and in the case of BP25 even when both are present together in the medium (see Figs. 1 1 B and 12), thus producing high amounts of butanol.
  • the carbon source comprises xylose and glucose.
  • Examples of carbon sources that could be used in the present invention are, but are not limited to, waste products from the timber, forestry, paper, agricultural, livestock, fisheries, sugar, aquaculture, rice processing or the like, as well as products Solid urban waste. Therefore, the source of carbohydrates can be urban waste products, preferably urban organic waste products or "waste syrup", or vegetable biomass, such as corn, sugarcane, starch, wheat, soybeans, nutshells such as nuts , almonds or fatty fruits such as the fruit of the palm tree or avocado, etc. In another preferred embodiment the carbon source is selected from the list consisting of: glucose, ground corn, urban organic waste hydrolyzate, or vegetable biomass hydrolyzate.
  • glucose when used as the carbon source in the present invention, it is found in the culture medium at a concentration between, preferably, 0.5 and 100 g / l, more preferably 60 g / l.
  • ground corn or "corn mash" When ground corn or "corn mash" is used as the carbon source in the present invention, it is in the culture medium at a concentration of preferably between 0.1 and 50% (v / v), more preferably 25% (v / v), of the total volume of the medium.
  • the ground corn is pretreated with hydrolase enzymes, preferably amylases, to increase the soluble glucose and subsequently clarified (centrifuged) before being added to the culture medium as a source of carbon for fermentation.
  • the carbon source is hydrolyzed from urban organic waste.
  • urban organic waste hydrolyzate When urban organic waste hydrolyzate is used as a carbon source in the present invention, it is found in the culture medium at a concentration of between 0.1 and 25% (v / v), preferably 5% (v / v) or less, of the total volume medium.
  • the "idolized urban organic waste” is the product resulting from the enzymatic hydrolysis of the organic part of the solid urban waste.
  • the vegetable biomass hydrolyzate is corn straw hydrolyzate.
  • corn straw hydrolyzate is used as the carbon source in the present invention, it is found in the culture medium at a concentration between 0.1 and 25% (v / v), preferably 16% (v / v) or less, of the total volume of the medium.
  • the "corn straw hydrolyzate" is the product resulting from the enzymatic hydrolysis of the corn straw subjected to a preferably acid and vapor explosion pretreatment.
  • the source of carbon comprised in the culture medium is pretreated before being added to said medium. The objective is to transform it into a more accessible way for the fermentation process.
  • Pretreatment uses various techniques that include, but are not limited to, explosion of the fiber with ammonium, chemical treatment, steam explosion at elevated temperatures to alter the structure of cellulosic biomass and make cellulose more accessible, acid hydrolysis and / or Enzymatic hydrolysis, or any combination thereof.
  • the culture medium further comprises one or more of the following elements: yeast extract, tryptone, ammonium acetate, magnesium acetate, iron sulfate, potassium phosphate and / or magnesium sulfate.
  • the volume of culture medium used for fermentation in the present invention is preferably between 10 and 90% of the reactor capacity.
  • the amount of mutant strain employed for the fermentation in the present invention is preferably between 10 3 and 10 10 cells / ml, more preferably between 10 7 and 10 8 cells / ml.
  • Another aspect of the invention relates to a method for the production of solvents, hereinafter "method of the invention for the production of solvents", which comprises the following steps: to. fermenting the mutant strain of the invention, preferably BP1 1, BP31 or BP25, more preferably BP25, in the presence of a culture medium comprising a carbon source, and
  • step (a) is an anaerobic fermentation, that is, it is carried out in the absence of oxygen.
  • the culture medium suitable for the fermentation stage, as well as the carbon source included therein, are those described previously as useful for the present invention.
  • This fermentation of step (a) can occur, for example but not limited to, in batch, continuous, discontinuous, feed-batch or a combination of at least two of these processes.
  • step (a) takes place inside a bioreactor, preferably of industrial size, which more preferably has suitable means and systems coupled for monitoring and supply of, for example, culture medium, carbohydrates, producing strain and / or water, to the reaction.
  • step (a) is carried out at a temperature between 30 and 40 e C, preferably 37 e C, for a time between 30 and 275 hours, preferably for 72 hours, more preferably while stirring.
  • This agitation is preferably low agitation, that is, about 50 rpm.
  • the pH during the fermentation stage is maintained at or above 5, even more preferably the pH is between 5.5 and 6.
  • the solvent produced is butanol.
  • the solvents produced are acetone, butanol and ethanol (ABE).
  • the carbon source comprised in the culture medium employed in this method of the invention comprises glucose, maltodextrin, xylobiose, cellobiose, xylose and / or arabinose, or any combination thereof. More preferably, the carbon source comprises glucose, cellobiose, xylose and / or arabinose. More preferably, the carbon source comprises xylose, glucose or arabinose or any combination thereof, even more preferably it comprises glucose and arabinose. In another preferred embodiment, the carbon source comprises xylose and glucose. In a particular embodiment, the arabinose is L-arabinose and the xylose is D-xylose.
  • the source of carbon comprised in the culture medium employed in this method of the invention is selected from the list consisting of: glucose, ground corn, hydrolyzate of urban organic solid waste, or hydrolyzate of vegetable biomass, such and as described above herein.
  • the carbon source is hydrolyzed from urban organic solid waste.
  • the vegetable biomass hydrolyzate is corn straw hydrolyzate.
  • the concentration of urban organic solid waste hydrolyzate in the culture medium employed in this method of the invention is 5% (v / v) or less.
  • the concentration of corn straw hydrolyzate in the culture medium employed in this method of the invention is 16% (v / v) or less.
  • the culture medium employed in this method of the invention further comprises at least one of the following elements: yeast extract, tryptone, ammonium acetate, magnesium acetate, iron sulfate, potassium phosphate and / or sulfate of magnesium
  • the bioreactor in which the method for the production of solvents of the invention is taking place preferably further comprises one or more coupled systems for the extraction of the solvents produced, preferably butanol, by way of example, but not limited to, pervaporation, perstraction. , distillation, solvent extraction, reverse osmosis, adsorption, membrane separation, liquid-liquid extraction, gas stream, sweeping gas or the like.
  • step (b) of the solvent production method of the invention refers to the collection of the solvents obtained after the fermentation of step (a). Said recovery can be carried out by any method known in the art, including mechanical and / or manual methods, preferably those described in the previous paragraph.
  • FIG. 1 Analysis of the mortality of C. beijerinckii in the presence of different concentrations of ethyl methanesulfonate (EMS).
  • EMS is the mutagenic chemical agent used for the isolation of mutants of C. beijerinckii.
  • Mortality is determined by quantifying the number of colony forming units (CFUs) per milliliter of culture.
  • the cells were grown in a bottle, under conditions of anaerobiosis
  • the medium used for the culture was TYA-glucose, previously gasified with N 2 to ensure anaerobiosis.
  • FIG. 3 Butanol production by low acid mutants.
  • FIG. 4 Production of butanol from ground corn (corn mash).
  • B) The BP1 1 mutant was tested, against its parental strain, under the conditions described in A). The mutant has higher productivity and final butanol production.
  • FIG. 5 Growth of C. beijerinckii BA101 in urban solid waste hydrolyzate (waste syrup). The growth of the cells was monitored over time in a TYA medium with different concentrations of urban solid waste hydrolyzate. As seen in the figure, C. beijerinckii can grow in a medium containing up to 5% of the complex substrate. We also observe that increasing the concentration of urban solid waste hydrolyzate up to 10% is lethal to cells, which do not form colonies after the first 12 hours of culture.
  • TYA (-) TYA medium without glucose.
  • FIG. 6 Butanol production using hydrolyzate of urban solid waste as a carbon source.
  • the butanol production of the BA101 line in a TYA medium with different concentrations of urban solid waste hydrolyzate was analyzed in bottles of 50 ml of working volume.
  • BA101 can produce butanol in this culture medium, the production in the medium being maximum with 5% urban solid waste hydrolyzate.
  • Virtually all glucose present in this medium is consumed in the first 50 hours, with high productivity in reference to the production of butanol from this glucose.
  • No production is observed in TYA medium without glucose.
  • the results shown are the average of two independent experiments, with error bars representing the standard deviation.
  • FIG. 7 Production of butanol in a medium containing hydrolyzate of urban solid waste and the salts present in the TYA. Production was tested in a new culture medium containing only the salts present in the TYA (magnesium acetate, iron sulfate, potassium phosphate and magnesium sulfate) and 5% hydrolyzate of urban solid waste as a carbon source. The three strains tested produce butanol in this medium, and the production of the BP25 mutant is better than that of the parental strain BA101 (A). However, the production of the BP31 mutant does not improve the production of the parental strain in this medium (B). The data is the average of two independent experiments.
  • FIG. 8 Production of butanol of mutant BP11 in a medium containing hydrolyzate of urban solid waste (waste syrup).
  • Butanol production of the BP1 1 mutant was analyzed in a medium containing waste syrup as a carbon source.
  • the concentration of urban solid waste hydrolyzate present is approximately 1%, with an initial glucose concentration of around 4 g / l (A).
  • the cells were cultured to saturation in TYA medium with glucose, and this culture was used to inoculate bottles of medium containing TYA and the urban solid waste hydrolyzate as a carbon source.
  • the mutant BP1 1 produces more butanol than strain BA101 under the conditions tested, and both strains consume glucose in the first 24 hours (A).
  • FIG. 9 A) Viability of C. beijerinckii BA101 in corn straw hydrolyzate. The viability of BA101 in a medium containing TYA salts and different concentrations of corn straw hydrolyzate was determined. The cells were grown to saturation in TYA medium with glucose, and this culture was used to inoculate medium containing TYA salts and different concentrations of corn straw hydrolyzate. The viability was determined as CFUs / ml, and as seen in the figure, the cells normally grow when the corn hydrolyzate is in a concentration up to 16%.
  • FIG. 10 Butanol production using corn straw hydrolyzate as a carbon source.
  • all strains can use this substrate as a carbon source, and produce butanol.
  • the BP25 mutant produces more butanol than the parental strain, and it is observed that all the glucose in the medium is consumed in the first 48 hours.
  • the data shown are the average of two independent experiments.
  • FIG. 11 Butanol production using arabinose as a carbon source.
  • Strains BA101 (wt) and BP25 were analyzed for their ability to produce butanol in a TYA medium with arabinose (A) or arabinose and glucose (B) as a carbon source.
  • Butanol production was followed over time, and, as shown in A, the two strains can use arabinose to produce butanol.
  • glucose is consumed in the first 24 hours, while only the BP25 mutant consumes arabinose after the use of glucose.
  • Butanol production is better for the BP25 mutant than for its parental strain BA101 in case both sugars are present in the medium.
  • FIG. 12 Butanol production of strains BP11, BP25 and BP31 using arabinose as a carbon source.
  • Butanol production was followed over time, and, as seen in the figure, low acid mutants produce butanol from this C5 sugar more efficiently than strain BA101.
  • the mutant BP1 1 achieves higher production titres in the first hours, using arabinose more efficiently; however, both the BP25 mutant and the BP31 mutant reach a higher final production yield, also consuming more arabinose in the process.
  • FIG. 13 Butanol production of low acid mutants using xylose as a carbon source.
  • the butanol production of BA101 (wt) and the two low acid mutants BP25 and BP31 were analyzed in a medium containing xylose (A) or xylose and glucose (B) as a carbon source.
  • Butanol production was analyzed over time, and the results show that the three strains can use xylose as a carbon source to produce butanol (A), and that when there is glucose and xylose in the middle, the two sugars are used simultaneously and, in the case of mutants, they are consumed in the first 24 hours.
  • the mutants were generated by mutagenesis with the chemical ethyl methanesulfonate (EMS).
  • EMS chemical ethyl methanesulfonate
  • the appropriate EMS concentration was first defined to achieve a good proportion of mutagenesis / cell death in Clostridium beijerinckii.
  • the concentration of EMS for Clostridium beijerinckii mutagenesis was set at 15 ⁇ for 1 ml of culture with an OD 660 of 1. This treatment causes the death of about 90% of the cells.
  • Mutagenesis was carried out by culturing the cells overnight in anaerobiosis.
  • TYA composition detailed in example 8
  • 60 g / l glucose supplemented with 60 g / l glucose, and gasified with N 2 was used as culture medium to remove all 0 2 and ensure anaerobic conditions.
  • An overnight culture was used to inoculate a bottle of TYA medium at an optical density at 660 (OD 660 ) of 0.1.
  • OD 660 optical density at 660 nm
  • one milliliter of culture is collected, centrifuged and treated with 15 ⁇ of EMS for one hour.
  • the cells are washed three times with TYA, and finally incubated in 5 ml of fresh medium for two hours (regeneration time). After regeneration, the cells are seeded in a suitable medium to proceed with the selection.
  • the cells that cause less acidity in the culture medium may be diverting the acids to solvents more quickly, so that They can produce more solvents (butanol).
  • the cells that cause a lower acidity of the culture medium selective plates are used, with a pH indicator, Bromocresol Purple, which is purple at basic pH, and which turns yellow at acidic pH. In this way, the cells that most acidify the medium form colonies with a yellow halo around them (Fig. 2). Less acidic cells (higher pH in the acidic range) do not change the color of the plates, keeping them purple.
  • BA101 is a mutant of 8052, obtained by chemical mutagenesis, and selected for its greater amylolytic capacity, which has been described to produce more butanol than the wild strain. Referring to the selection used, say that both strains produce the yellow halo around the colony in purple bromocresol plates, although the halo of strain 8052 is more pronounced.
  • mutants For selection, after mutagenesis, cells cannot be sown directly in the selective medium with Bromocresol Purple, due to the toxicity of this medium, which does not allow the growth of healthy colonies.
  • the mutants were first seeded in TYA-glucose medium, and already isolated mutants were plated with Purple Bromocresol plates to isolate those that had a lower yellow halo. More than 5000 mutants were seeded on the selective plates, and about 50 mutants were selected lacking yellow halo around the colony, for a subsequent test of their butanol-producing capacity.
  • the cultures were grown in TYA with glucose (60 g / l), at 37 e C and for 72 hours. In each case, aliquots were taken at different times (at least four points) to analyze the production of butanol.
  • the samples, of 1.5 ml, were taken using a syringe and needle, through a septum, so that the anaerobic atmosphere of the culture was not interrupted.
  • Acidic water (0.05M H 2 S0 4 ) is used as the mobile phase, and the retention times (in minutes) for the different compounds is 8.6 for glucose, 21.6 for butyrate, and 36.2 for butanol .
  • an internal standard was used that included known concentrations of the different metabolites.
  • the results obtained show that three of the low acidity mutants (mutants BP1 1, BP25 and BP31) produce more butanol than their respective parents.
  • the parental strain of the mutant BP1 1 is the C. beijerinckii 8052 strain, while the parental strain of the BP25 and BP31 mutants is the C. beijerinckii BA101 strain (Fig. 3).
  • wild strain 8052 does not produce butanol, since the pH of the crop falls below 5 during growth, giving rise to the phenomenon known as "acid crash", which leads to a decrease in the growth of cells and an inhibition of butanol production.
  • Solid urban waste hydrolyzate is the resulting product after enzymatic hydrolysis of the organic part of urban solid waste.
  • a feasibility study was first carried out with increasing concentrations (0%, 1.5%, 5% and 10%). Growth was analyzed by determining the number of colony forming units (CFU / ml) per milliliter of culture, over time. To determine the CFUs / ml, aliquots of the culture were taken at different times, and, after diluting properly, they were plated on TY A-glucose plates, so that the colonies were sufficiently isolated to proceed to count them. These tests were done with strain BA101, and the results show that C.
  • the sugar composition of this medium was analyzed by chromatography (HPLC), using an Aminex HPX-87H column (300 x 7.8 mm).
  • HPLC chromatography
  • the analysis method uses acidulated water as a mobile phase, a column temperature of 60 e C, and a detector temperature of 40 e C.
  • the analysis of sugars after Fermentation indicated that the strains used can consume the C6 and C5 sugars present in this medium.
  • mutant BP1 1 produces butanol using this complex substrate as a carbon source, and produces it with better titres than strain BA101.
  • glucose present in this medium was consumed in the first 24 hours, and that both strains consume it equally.
  • the second of the waste substrates used was the corn straw hydrolyzate. This is the result of enzymatic hydrolysis of corn straw undergoing acid treatment and steam explosion.
  • Corn straw is a type of agricultural waste that has been widely studied as a substrate (after enzymatic hydrolysis) for the production of second generation bio-ethanol.
  • this substrate as a carbon source (composition of the medium in example 8)
  • the viability of strain BA101 was analyzed.
  • Example 6 Production of butanol from C5 sugars with the generated mutants.
  • the results obtained in fermentations with complex substrates showed that C. beijer ⁇ nckii strains can consume the C5 present in these substrates.
  • both strain BA101 and mutants BP25 and BP31 were grown in TYA medium containing different C5 sugars as substrates, and butanol production was analyzed.
  • the C5 chosen for the test were L-arabinose and D-xylose.
  • liquid chromatography HPLC
  • mutants produce more butanol than strain BA101, and mutant BP1 1, although initially consumes arabinose faster, after 48 hours slows down its consumption of arabinose and butanol production, on the contrary than the BP25 and BP31 mutants that continue to produce butanol during the 72 hours that the fermentation analysis was followed.
  • the genomic DNA of the three low acid mutants was sequenced, and the genome sequence was compared with that of their corresponding parental strain. Thus, the genes that appear mutated in each strain were identified. The results showed that there are several biological processes that may be altered in the mutants, and lead to the phenotype of increased butanol production.
  • the BP1 1 mutant shows a mutation in the Cbei_1540 gene, which encodes a pppGpp synthase, and seems the most likely to explain the overproducer phenotype of this mutant, compared to its parental strain.
  • pppGpp is an intracellular signal that causes a stress response by activating the RpoS transcriptional factor.
  • the BP25 mutant shows 25 mutations. Among the mutations that could cause the phenotype studied, several have been identified that could affect the butanol production process. There are several genes involved in chemotaxis that appear mutated, which suggests that chemotaxis may be important for the butanol production process. On the other hand, maintaining an adequate intracellular redox state is essential for the production of butanol, as there are several cofactors that change their redox state during the production process. This mutant shows three genes (Cbei_0316, Cbei_1206 and Cbei_1472) that are involved in the maintenance of the cellular redox state; Mutations in these genes may be responsible for the observed phenotype.
  • the BP31 mutant shows six mutations with respect to its parental strain, however, only three of them are non-conservative, being therefore responsible for the observed phenotype.
  • the one that seems to have more relevance is the one that appears in the Cbei_2475 gene, a gene that is also mutated in the mutant BP1 1, and whose function, and relevance with respect to the butanol production phenotype has already been described.
  • the other two mutations appear in the genes Cbei_0436 and Cbei_1397.
  • the first codes for a histidine kinase, involved in signal transduction processes.
  • the butanol production process is closely linked to the sporulation process, which is highly regulated by histidine kinase-mediated phosphorylations. However, this one in particular is not described as implied in these processes
  • the second of these genes codes for a transposase, which a priori does not seem involved in the butanol production process.
  • Example 8 Composition of the different culture media used in the tests.

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Abstract

La présente invention concerne des souches mutantes de Clostridium beijerinckii, en particulier produites par mutagenèse chimique des souches mères C. beijerinckii BA101 et NCIMB 8052. Lesdits mutants sont capables de produire davantage de butanol, au moyen de processus de fermentation anaérobie, que la souche mère de laquelle ils sont issus à l'aide de différents substrats comme source de carbone. Par conséquent, l'invention concerne également l'utilisation desdites souches mutantes dans des procédés de production d'acétone, butanol et éthanol (ABE) par fermentation et un procédé d'obtention desdites souches mutantes.
PCT/ES2018/070402 2017-06-07 2018-06-01 Mutants de clostridium beijerinckii hyper reproducteurs de butanol WO2018224712A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2016038A6 (es) * 1988-07-13 1990-10-01 Inst Francais Du Petrole Un procedimiento de produccion de butanol y de acetona por fermentacion a partir de melaza de cana de azucar.
WO1998051813A1 (fr) * 1997-05-14 1998-11-19 The Board Of Trustees Of The University Of Illinois PROCEDE DE PREPARATION DE BUTANOL UTILISANT UNE SOUCHE MUTANTE DE $i(CLOSTRIDIUM BEIJERINCKII)
WO2012035420A1 (fr) * 2010-09-16 2012-03-22 Eni S.P.A. Clostridium beijerinckii dsm 23638 et son utilisation dans la production de butanol
WO2016192871A1 (fr) * 2015-06-04 2016-12-08 IFP Energies Nouvelles Souches mutantes du genre clostridium beijerinckii

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2016038A6 (es) * 1988-07-13 1990-10-01 Inst Francais Du Petrole Un procedimiento de produccion de butanol y de acetona por fermentacion a partir de melaza de cana de azucar.
WO1998051813A1 (fr) * 1997-05-14 1998-11-19 The Board Of Trustees Of The University Of Illinois PROCEDE DE PREPARATION DE BUTANOL UTILISANT UNE SOUCHE MUTANTE DE $i(CLOSTRIDIUM BEIJERINCKII)
WO2012035420A1 (fr) * 2010-09-16 2012-03-22 Eni S.P.A. Clostridium beijerinckii dsm 23638 et son utilisation dans la production de butanol
WO2016192871A1 (fr) * 2015-06-04 2016-12-08 IFP Energies Nouvelles Souches mutantes du genre clostridium beijerinckii

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