WO2018224712A1 - Mutants of clostridium beijerinckii that are butanol hyper-producers - Google Patents

Abstract

The present invention relates to mutant strains of Clostridium beijerinckii, particularly produced by chemical mutagenesis of the parent strains C. beijerinckii BA101 and NCIMB 8052. Said mutants can produce more butanol, by means of anaerobic fermentation processes, than the parent strain from which same originate, using different substrates as a carbon source. Therefore, the invention also relates to the use of said mutant strains in methods for producing acetone, butanol and ethanol (ABE) by means of fermentation, and to a method for obtaining said mutant strains.

Description

 MUTANTS OF CLOSTRIDIUM BEIJERINCKII HYPERPRODUCERS OF

 BUTANOL

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. Specifically, 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.

STATE OF THE TECHNIQUE 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.

Therefore, there is a growing interest in achieving an efficient production of this liquid biofuel from cheap and renewable biomass, which would be a solution to several problems simultaneously: ensuring the global supply of biofuels, because they could be produced locally in suitable systems, reduce greenhouse gases, and recycle waste products that are produced as a result of the activity of other industries or even cities.

In recent years, fermentation processes for the production of acetone / butanol / ethanol (ABE) have received considerable attention because they are processes that allow the production of basic chemicals from biomass. 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.

Another very relevant solvent isogenic Clostridio is C. beijerínckii, 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. beijerínckii 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. In addition, as already indicated, the solvent genes in C. beijerínckii are located on the chromosome, while in C. acetobutylicum these genes are located in a plasmid, which makes C. beijerínckii more genetically stable.

During the course of ABE fermentation, 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.

Therefore, the fermentation of carbohydrates to acetone, butanol and ethanol by solvent microorganisms, including microorganisms of the genus Clostridium, is known. Thus, for example, US5192673 describes an improved fermentation process to produce high levels of butanol using a mutant strain of C. acetobutylicum called Clostridium acetobutylicum ATCC 55025. US6358717 describes the production of ABE solvents by anaerobic fermentation of a mutant strain of C. beijerínckii called Clostrídium beijerínckii BA101 ATCC No. PTA-1550 in a nutrient medium with assimilable carbohydrates and the subsequent recovery of butanol, acetone and ethanol. This mutant strain BA101 is described as a hyperproductive strain of butanol obtained by mutagenesis of the wild strain C. beijerínckii 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). On the other hand, patent application AU2014216024 describes genetically modified Clostridium solvent strains comprising an altered gene expression or structure that results in increased butanol production efficiency. In some embodiments, the modified Clostridium species as described herein is C. beijerínckii.

The route of butanol biosynthesis in the Clostridia producing these solvents has also been studied and some of the enzymes involved in the process have been purified and characterized (Boynton et al., 1996, Journal of Bacteriology 178, 3015-3024; Durre et al., 1995, Fems Microbiol Rev 17, 251 262).

However, there is still a need to design improved solvent-forming microorganisms that have an increased efficiency in the production of butanol from various substrates, which are stable strains and with an improved production yield with respect to the currently existing strains. In addition, one of the problems associated with ABE fermentation by C. acetobutylicum and C. beijerínckii is butanol toxicity in the culture. This toxicity requires the continuous elimination of toxic products during the process to achieve maximum solvent production. Thus, there is a need to develop improved solvent-forming microorganisms capable of producing solvents, in particular ABE, more particularly butanol, with higher yield and greater tolerance to the product and preferably also to the substrate.

Therefore, for the production of butanol at the industrial level to be profitable, it is necessary to have microorganisms capable of increasing the levels of butanol produced during fermentation, or capable of producing butanol more quickly, as well as using a variety of substrates of departure.

DESCRIPTION OF THE INVENTION The present invention provides three mutants of the Clostrídium beijerínckii species obtained by random chemical mutagenesis of the parental strains C. beijerínckii 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.

Therefore, the present invention relates to three mutant strains of C. beijerínckii 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.

These 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. In addition, 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. beijerínckii NCIMB 8052 and the last two strains are derived from the parental strain C. beijerínckii BA101. These three mutants described in the invention therefore represent a solution to the need for improved solvent-forming microorganisms that have increased efficiency and yield in the production of butanol by fermentation from different types of carbohydrates. It is therefore proposed in the present invention the use of these three mutants for the production of solvents by anaerobic fermentation, preferably for the production of butanol, more preferably for the production of acetone, butanol and ethanol (ABE).

For the generation of the three specific mutants described in the present invention, the inventors have developed and developed a method of induced mutagenesis in parental cells of C. beijerínckii. 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. beijerínckii. Other mutants of the C. beijerínckii 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.

Therefore, in a first aspect the present invention relates to a method for obtaining or generating mutant strains of the species C. beijerínckii 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. beijerínckii, preferably in the strain C. beijerínckii NCIMB 8052 or C. beijerínckii BA101, more preferably in C. beijerínckii NCIMB 8052, and

 b. 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 μΙ of EMS per ml of culture, for preferably 1 hour, and

where 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. Preferably, 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. Thus, mutants with improved butanol production capacity result in a higher yield of the fermentation process for the production of butanol, compared to the yield obtained when the parental strain of C. beijerinckii is used. 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). By way of example, 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. Thus, cells grown in the presence of this compound that most acidify the medium form colonies with a yellow halo around, while the less acidic cells (i.e., those that maintain the medium at a higher pH, preferably within the acidic range ) do not change the color of the plates, keeping them purple. Therefore, when this compound is used in step (b) of the described method, those mutants that have a smaller or less yellow halo are selected, maintaining the purple color of the culture plates.

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. beijerínckii, 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.

The term "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. beijerínckii 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:

T instead of C at position 82 of the Cbei_0583 gene (SEQ ID NO: 26), which comprises positions 693796 to 694347 of the genome of C. beijerinckii NCIMB 8052,

 A instead of G at position 1 108 of the Cbei_1540 gene (SEQ ID NO: 27), which comprises positions 1814444 to 1816660 of the genome of C. beijerinckii NCIMB 8052,

 T instead of G at position 255 of the Cbei_2475 gene (SEQ ID NO: 28), which comprises positions 2865706 to 2866320 of the genome of C. beijerinckii NCIMB 8052,

 T instead of G at position 740 of the Cbei_2489 gene (SEQ ID NO: 29), which comprises positions 2884426 to 2885343 of the genome of C. beijerinckii NCIMB 8052,

 AA instead of GT at positions 506 and 507 of the Cbei_R01 12 gene (SEQ ID NO: 30), which comprises positions 5742945 to 5741441 of the genome of C. beijerinckii NCIMB 8052,

 T instead of C at position 490 of the Cbei_R01 12 gene (SEQ ID NO: 30), which comprises positions 5742945 to 5741441 of the genome of C. beijerinckii NCIMB 8052, and

 C instead of A at position 703 of the Cbei_5101 gene (SEQ ID NO: 31), which comprises positions 5998873 to 5999637 of the genome of C. beijerinckii NCIMB 8052.

From now on this strain will be referred to as "mutant BP1 1", and 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. beijerínckii (preferably obtained by chemical mutagenesis of strain C. beijerínckii BA101) characterized in that said mutant strain comprises in its genome the following mutations with respect to the genome of the strain parental C. beijerínckii BA101: a. A instead of G at position 567 of the Cbei_0436 gene (SEQ ID NO: 32), which comprises positions 528599 to 530101 of the genome of C. beijerínckii BA101,

 b. T instead of C at position 391 of the Cbei_0529 gene (SEQ ID NO: 33), which comprises positions 636881 to 637588 of the genome of C. beijerínckii BA101,

 C. A instead of G at position 14 of the Cbei_1397 gene (SEQ ID NO: 34), which comprises positions 1644433 to 1644753 of the genome of C. beijerínckii BA101,

 d. A instead of G at position 435 of the Cbei_2084 gene (SEQ ID NO: 35), which comprises positions 2427766 to 2428383 of the genome of C. beijerínckii BA101,

 and. C instead of T at position 1 1 1 of the Cbei_21 13 gene (SEQ ID NO: 36), which comprises positions 2461021 to 2461281 of the genome of C. beijerínckii BA101, and

 F. T instead of G at position 28 of the Cbei_2475 gene (SEQ ID NO: 37), which comprises positions 2865706 to 2866320 of the genome of C. beijerínckii BA101.

From now on, 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.

However, among all the mutants described in the present invention, the so-called "BP25 mutant" is preferred, since it has ABE production yields, especially butanol, higher than the other mutants in the tested substrates. For example, 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. 10 shows that the BP25 mutant is capable of producing more butanol in the presence of corn straw hydrolyzate than the BP31 mutant. Finally, 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.

In addition, the BP25 mutant has an improved butanol production capacity compared to its parental strain, in various substrates. For example, 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. 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.

Therefore, 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. A instead of G at position 235 of the Cbei_0083 gene (SEQ ID NO: 1), which comprises positions 107490 to 108176 of the genome of C. beijerinckii NCIMB 8052, b. 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,

C. A instead of G at position 54 of the Cbei_R0027 gene (SEQ ID NO: 3), which comprises positions 144366 to 147274 of the genome of C. beijerinckii

NCIMB 8052,

d. A instead of G at position 186 of the Cbei_0123 gene (SEQ ID NO: 4), which comprises positions 159137 to 16161 1 of the genome of C. beijerinckii NCIMB 8052,

and. A instead of G at position 201 of the Cbei_0196 gene (SEQ ID NO: 5), which comprises positions 222653 to 224272 of the genome of C. beijerinckii NCIMB 8052,

F. A instead of G at position 871 of the Cbei_0316 gene (SEQ ID NO: 6), which comprises positions 381944 to 382879 of the genome of C. beijerinckii NCIMB 8052,

g. T instead of C at position 731 of the Cbei_1206 gene (SEQ ID NO: 7), which comprises positions 1427189 to 1428124 of the genome of C. beijerinckii NCIMB 8052,

h. T instead of C at position 437 of the Cbei_1472 gene (SEQ ID NO: 8), which comprises positions 1734309 to 1734908 of the genome of C. beijerinckii

NCIMB 8052,

i. C instead of T at position 968 of the Cbei_1854 gene (SEQ ID NO: 9), which comprises positions 2148319 to 2150055 of the genome of C. beijerinckii NCIMB 8052,

j. A instead of C at position 139 of the Cbei_1935 gene (SEQ ID NO: 10), which comprises positions 2234469 to 2235623 of the genome of C. beijerinckii NCIMB 8052,

k. T instead of G at position 31 of the Cbei_1975 gene (SEQ ID NO: 1 1), which comprises positions 2295746 to 2297452 of the genome of C. beijerinckii NCIMB 8052,

 I. T instead of G at position 21 13 of the Cbei_3078 gene (SEQ ID NO: 12), which comprises positions 3591316 to 3593580 of the genome of C. beijerinckii NCIMB 8052,

m. G instead of A at position 88 of the Cbei_3625 gene (SEQ ID NO: 13), which comprises positions 4172987 to 4174981 of the genome of C. beijerinckii

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,

or. A instead of G at position 325 of the Cbei_4026 gene (SEQ ID NO: 15), which comprises positions 4626006 to 4627097 of the genome of C. beijerinckii

NCIMB 8052,

p. A instead of G at position 1079 of the Cbei_4207 gene (SEQ ID NO: 16), which comprises positions 4851 1 18 to 4852431 of the genome of C. beijerinckii NCIMB 8052,

q. T instead of G at position 628 of the Cbei_4256 gene (SEQ ID NO: 17), which comprises positions 4915930 to 4916709 of the genome of C. beijerinckii NCIMB 8052,

r. G deletion at position 672 of the Cbei_4308 gene (SEQ ID NO: 18), which comprises positions 4961979 to 4962692 of the genome of C. beijerinckii NCIMB 8052,

s. T instead of G at position 1766 of the Cbei_4400 gene (SEQ ID NO: 19), which comprises positions 5075330 to 5077168 of the genome of C. beijerinckii NCIMB 8052,

t. A instead of G at position 1732 of the Cbei_4548 gene (SEQ ID NO: 20), which comprises positions 5265930 to 5267861 of the genome of C. beijerinckii NCIMB 8052,

or. A instead of G at position 1465 of the Cbei_4691 gene (SEQ ID NO: 21), which comprises positions 5449160 to 5450662 of the genome of C. beijerinckii NCIMB 8052,

v. A instead of G at position 997 of the Cbei_4699 gene (SEQ ID NO: 22), which comprises positions 5462404 to 5464674 of the genome of C. beijerinckii

NCIMB 8052,

w. C instead of T at position 101 of the Cbei_4761 gene (SEQ ID NO: 23), which comprises positions 5555079 to 5556296 of the genome of C. beijerinckii NCIMB 8052,

x. A instead of G at position 27 of the Cbei_4865 gene (SEQ ID NO: 24), which comprises positions 5699972 to 5701330 of the genome of C. beijerinckii NCIMB 8052, and

Y. A instead of G at position 708 of the Cbei_4918 gene (SEQ ID NO: 25), which comprises positions 5785244 to 5787250 of the genome of C. beijerinckii

NCIMB 8052. From now on this strain will be referred to as a "BP25 mutant", and 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 Científic Universitat de Valencia, Professor Agustín 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", as used herein, refers to any mutant strain of the species C. beijerinckii produced, obtainable or obtained by the method of mutant generation described above in the present invention, preferably to mutants BP1 1, BP31 and BP25. 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. In a preferred embodiment, the solvent is butanol, that is, the main component of the solvent mixture obtained is butanol. In a more preferred embodiment, the solvents are acetone, butanol and ethanol (ABE), that is, the solvents produced comprise a mixture of these three. The production of any combination or proportion of acetone, butanol and / or ethanol by using the mutants of the invention is included within the scope of the present invention.

In another preferred embodiment, 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, termentative activity and maintenance in cultivation of C. beijerinckii strains. Examples of these culture media are, for example, but not limited to, P2, TGY, PT or TYA, preferably TYA. Preferably, 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. Preferably, 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 In a preferred embodiment, 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. More preferably, 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. In another preferred embodiment, 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.

When glucose is 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.

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. Preferably, 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. In a more preferred embodiment, the carbon source is hydrolyzed from urban organic waste. 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. Preferably, the "idolized urban organic waste" is the product resulting from the enzymatic hydrolysis of the organic part of the solid urban waste.

In another preferred embodiment, the vegetable biomass hydrolyzate is corn straw hydrolyzate. When 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. Preferably, 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. Preferably, in the present invention, 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.

In another preferred embodiment, 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

 b. recover the solvents produced by the strain from the culture medium.

The fermentation of step (a) is an anaerobic fermentation, that is, it is carried out in the absence of oxygen. To ensure anaerobiosis, it is possible, for example, but not limited to this method, to gasify the culture medium with N _2. 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.

During fermentation can be monitored and controlled process conditions, such as volume, 0 ₂ dissolved, J-, pH, concentration of the producing strain and / or carbohydrate concentration in the environment, and can be adding or deleting quantities of producing strain, of agents that control the pH, of culture medium and / or of carbohydrates, to maintain the pH and the desired concentrations of each of these elements during the fermentation process. Thus, preferably, the fermentation of 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. These suitable means are, for example, valves and pipes or pipes connected from the bioreactor, where the fermentation reaction is taking place, to one or several storage tanks or, in the case of the supply of the producing strain, to one or more culture tanks where adequate biomass of the growing production strain is maintained. The bioreactor where the fermentation is taking place can also have coupled systems or devices for controlling the reaction volume, 0 ₂ , pH and / or J- of the fermentation medium. In a preferred embodiment of the method for the production of solvents of the invention, the fermentation of 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. More preferably, the pH during the fermentation stage is maintained at or above 5, even more preferably the pH is between 5.5 and 6.

In another preferred embodiment of this method, the solvent produced is butanol. In a more preferred embodiment, the solvents produced are acetone, butanol and ethanol (ABE).

In another preferred embodiment, 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.

In another preferred embodiment, 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.

Preferably, the carbon source is hydrolyzed from urban organic solid waste. In a more preferred embodiment, the vegetable biomass hydrolyzate is corn straw hydrolyzate.

In another preferred embodiment, 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.

In another preferred embodiment, the concentration of corn straw hydrolyzate in the culture medium employed in this method of the invention is 16% (v / v) or less. In another preferred embodiment, 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

One of the problems associated with ABE fermentation by C. beijerinckii is butanol toxicity in the culture. This toxicity requires the continuous elimination of toxic products during the fermentation process to achieve maximum solvent production. Thus, 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.

The term "recovery", as used in 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.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES

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. A saturated culture was used to inoculate a bottle at an OD ₆₆₀ = 0.1, and the culture was incubated for about 5-6 hours until an OD ₆₆₀ = 1 was reached. At this time, 1 ml of culture was collected and centrifuged, the supernatant being decanted. The collected cells were treated with different concentrations of EMS for 1 hour. After treatment, the cells were washed twice with TYA medium, to finally grow in fresh medium (TYA-glucose) for two hours, and allow the recovery of the cells. After this time the cells were seeded (after diluting the culture properly) to determine the number of CFUs in the culture. This data (CFUs / ml) was represented according to the amount of EMS used, giving rise to the graph presented in this figure. As can be seen, 15 μΙ of EMS causes a mortality of 90%, and this was the amount of EMS that was used in the different mutagenesis tests. FIG. 2. Selection of mutants in purple Bromocresol plates. A culture of C. beijerínckii was treated with the EMS mutagen and, after recovery of the culture, the cells were conveniently diluted and seeded on TYA-glucose plates. The cells were grown for two days, to ensure colonies with enough biomass to re-sow in purple Bromocresol plates. Mutants that less acidify the medium during growth form colonies lacking the characteristic yellow halo showing a colony of C. beijerínckii (both strain 8052 and BA101). The figure shows an example of one of the Purple Bromocresol plates used in the selection, and it is indicated, marked with an asterisk, a mutant that shows a low acidity phenotype.

FIG. 3. Butanol production by low acid mutants. A) Production profile of two low acid mutants (BP25 and BP31) and their corresponding parental strain (BA101, wt) in TYA-glucose medium. The culture was performed in anaerobiosis, in the bottle, without pH control. The metabolites (glucose, butanol and butyrate) analyzed in the culture medium are plotted as a function of time for the three strains studied. As can be seen in the figure, the two mutants, BP25 and BP31, produce more butanol and consume more glucose than their parental strain. B) Production profile of strain NCIMB 8052 and the low acid mutant isolated from it, BP1 1. The fermentations are carried out in TYA-glucose medium, anaerobiosis and in the bottle without pH control. Under these conditions, the parental strain does not produce butanol, while the isolated mutant is capable of producing it. FIG. 4. Production of butanol from ground corn (corn mash). A) The mutant BP25 and its parental strain BA101 produce butanol from ground and clarified corn. The fermentations were carried out in 1 I volume reactors, with 500 ml of TYA medium with 25% clarified ground corn as a source of sugars. The pH was maintained above 5.5 during fermentation with diluted NH ₄ ⁺ . The production of mutant butanol is higher in the first 24 hours, increasing productivity. Production at the end of fermentation is similar for both strains. 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. As can be seen in the figure, 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. As shown in the figure, 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). An analysis of other sugars present in the sample (B) indicates that xylose is consumed by both strains in a similar way, while differences in the consumption of cellobiose and arabinose are observed. 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%. When the hydrolyzate reaches a concentration of 25% it is lethal to the cells. B) Analysis of the sugars present in the corn straw hydrolyzate. The representative sugar concentrations present in the TYA medium with 16% corn straw hydrolyzate are shown, both at the beginning of fermentation (tO), and at 72 hours (t72). Duplicates of a fermentation of C. beijerinckii BA101 (wt) are shown.

FIG. 10. Butanol production using corn straw hydrolyzate as a carbon source. The butanol production of strain BA101 (wt), and of the two low acid mutants BP25 and BP31, was tested in a medium containing the salts present in TYA and 16% corn straw hydrolyzate. In the figure it is appreciated that 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. A saturated culture was used to inoculate the media to be tested at an OD ₆₆₀ = 0.1. Butanol production was followed over time, and, as shown in A, the two strains can use arabinose to produce butanol. However, when we have glucose and arabinose in the culture medium, 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. Strains BA101, BP1 1, BP25 and BP31 were analyzed for their ability to produce butanol in a TYA medium with arabinose as a carbon source. A saturated culture was used to inoculate the media to be tested at an OD ₆₆₀ = 0.1. 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. A culture in saturated TY A-glucose was used to inoculate the desired medium at an OD ₆₆₀ = 0.1. 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.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the high yields of the mutants developed in the present invention in the production of ABE from different carbon sources.

Example 1. Generation and selection of C. beijerinckii mutants.

The mutants were generated by mutagenesis with the chemical ethyl methanesulfonate (EMS). In order to carry out the mutagenesis, the appropriate EMS concentration was first defined to achieve a good proportion of mutagenesis / cell death in Clostridium beijerinckii. As observed in Fig. 1, the concentration of EMS for Clostridium beijerinckii mutagenesis was set at 15 μΙ 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), 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. After growth in anaerobiosis for 5 or 6 hours, when the culture has reached an optical density at 660 nm (OD ₆₆₀ ) of 1, one milliliter of culture is collected, centrifuged and treated with 15 μΙ of EMS for one hour. After treatment 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.

For the isolation of mutants, a selection method was used to identify cells that reduce the pH less than the parental. The reasoning for obtaining butanol over-producing mutants based on the low acidity of the culture is as follows: 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). For the isolation of 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.

Two strains of Clostrídium beijerínckii, C. beijerínckii NCIBM 8052 (8052; collection strain), and C. beikjerínckii BA101 (BA101) were used as parental strains in this selection. 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. 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.

Example 2. Production of butanol from glucose with the generated mutants.

To analyze the capacity to produce butanol of the different mutants, the cultures were started in a 50 ml bottle, in anaerobiosis, from a saturated culture, to an OD ₆₆₀ = 0.1. 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. Once the samples were collected, they were centrifuged to separate the cells (13000 rpms, 10 minutes), and the supernatant was taken to determine glucose, butyrate and butanol by high pressure liquid chromatography (HPLC; GE Healthcare) . The method of Chromatography uses a column of type Aminex HPX-87H (Biorad), of a size of 300 X 7.8 mm, with a column temperature of 40 ^e C and a flow of 0.6 ml / min. The injection volume is 20 μΙ, and the chromatography time is 40 minutes. 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 . For quantification 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). Under the conditions used in this test, 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.

Example 3. Production of butanol from ground corn with the generated mutants.

In addition to the production in TYA-glucose, the production of butanol in a medium containing ground corn (corn mash) as a carbon source was analyzed. The medium contained the salts present in TYA together with 25% ground corn, treated with amylase to increase soluble glucose, and clarified (centrifuged). In this case, the cultivation of the different strains was done in 1 liter thermostats, with 500 ml of work volume, and under conditions in which the pH was controlled above 5.5. Stirring was set at 50 rpm, and the temperature at 37 ^e C. The culture was started using 25 ml of a saturated culture (grown for about 16 hours). As seen in Figure 4, all strains can use ground and clarified corn as a carbon source, and the mutants produce significantly more butanol in the first 24 hours than their corresponding parental strains, thereby increasing the productivity of the process. . In addition, and as seen in Figure 4, the production of the BP1 1 mutant at 72 hours is significantly higher than the production of its parental strain. Example 4. Production of butanol from hydrolyzate of urban waste with the generated mutants.

The ability to produce butanol was also analyzed when different waste products are used as a carbon source. As waste products, an urban solid waste hydrolyzate (waste syrup) and a vegetable biomass hydrolyzate (corn straw or corn stover) were used.

Solid urban waste hydrolyzate is the resulting product after enzymatic hydrolysis of the organic part of urban solid waste. Using this syrup, 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. beijerínckii BA101 cannot grow in a medium containing 10% waste syrup, while concentrations of up to 5% waste syrup allow the growth of BA101 ( Figure 5). Under these conditions, the butanol production of strain BA101 was analyzed, observing that with 5% of municipal solid waste syrup it can produce butanol with a very high productivity in relation to glucose consumption (which is consumed almost entirely in first 50 hours of fermentation; Figure 6).

For the analysis of the low acidity mutants, a culture medium was used that contained only the salts present in the TYA and 5% urban solid waste syrup (composition of the medium in example 8). The data (Figure 7A) shows that the BP25 mutant produces butanol more quickly when using this substrate as a carbon source, while the BP31 mutant produces the same butanol as the parental strain (Figure 7B).

The sugar composition of this medium was analyzed by chromatography (HPLC), using an Aminex HPX-87H column (300 x 7.8 mm). 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.

The ability to ferment syrup of solid urban waste from the mutant BP1 1 was also analyzed in a medium containing TYA and a lower concentration of syrup of urban solid waste. The experiments were performed in the bottle, without pH control. Under these conditions the parental strain of mutant BP1 1 (strain 8052) does not produce butanol, as we have already described. That is why the capacity to produce butanol of this mutant was compared with that of the BA101 line. As seen in Figure 8A, mutant BP1 1 produces butanol using this complex substrate as a carbon source, and produces it with better titres than strain BA101. In this figure it can be seen that the glucose present in this medium was consumed in the first 24 hours, and that both strains consume it equally. At the same time other sugars present in the sample were analyzed, and, as seen in Figure 8B, the xylose is consumed completely in the first 24 hours by both strains. However, there are differences in the consumption of cellobiose and arabinose, which the mutant BP1 1 seems to consume more efficiently (Figure 8B).

Example 5. Production of butanol from corn straw hydrolyzate with the generated mutants.

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. Using this substrate as a carbon source (composition of the medium in example 8), the viability of strain BA101 was analyzed. A saturated culture of BA101 (cultivated for 16 hours) was used to inoculate (OD ₆₆₀ = 0.1) 50ml of TYA with different concentrations of corn straw hydrolyzate (0%, 16% and 25%). Viability was analyzed by determining CFUs / ml, as already described. The results show that BA101 cannot grow when the medium contains 25% corn straw hydrolyzate. However, growth is adequate when the substrate concentration is decreased to 16% (Figure 9A). The sugars present in this medium after fermentation were analyzed, and it is again observed that BA101 consumes C6 and C5 sugars during growth (Figure 9B). Finally, the butanol production of the different mutants was analyzed in a medium with 16% corn straw hydrolyzate. The results (Figure 10) show that all strains tested produce butanol using this carbon source, and that the BP25 mutant produces more butanol than the parental strain.

Example 6. Production of butanol from C5 sugars with the generated mutants. The results obtained in fermentations with complex substrates showed that C. beijerínckii strains can consume the C5 present in these substrates. To analyze the ability of different strains to consume C5 sugars, 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. To determine the production of butanol and the consumption of C5 sugars after fermentation, liquid chromatography (HPLC) was used, with the method described above for the determination of butanol. The retention time of arabinose in this method is 10.1 minutes, while xylose is identified at 9.2 minutes of chromatography.

Arabinose intake

The results show that, when fermentation occurs in a medium containing only arabinose (TYA + arabinose), both BA101 and BP25 can use arabinose as a carbon source, and produce butanol from it (Figure 1 1 A).

Analyzing the behavior in a medium that contains both glucose and arabinose (and both in limiting concentrations to analyze the pattern of sugar consumption, and determine if C6 and C5 are consumed simultaneously or consecutively), it was observed that arabinose consumption does not occur until that glucose has been completely consumed (Figure 1 1 B), and its use is not very efficient (only the mutant BP25 consumes it, and not completely), showing a glucose inhibition in the consumption of arabinose. The use of arabinose by the mutant BP1 1 was more efficient than that of strain BA101 in the complex substrate of urban solid waste syrup (Figure 8), so that the production of butanol from this sugar of this mutant was also analyzed. The tests were carried out in 50 ml bottles, without pH control. Under these conditions, the parental strain of the mutant BP1 1 does not produce butanol, which were included as controls to assess the production of the mutant BP1 1, strains BA101, BP25 and BP31. As seen in Figure 12, 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.

Xylose Consumption

In the case of C5 xylose sugar, when present as the only source of carbon in the medium, the results show that the tested lines (BA101, BP25 and BP31) consume xylose and produce butanol from this sugar (Figure 13A). It is interesting that when xylose is in the medium together with glucose (both in limiting concentrations), the cells can consume both sugars simultaneously, and completely in the first 30 hours of fermentation (Figure 13B). Under these conditions, it is observed that the production of butanol in the first 24 hours is better for low acid mutants than for the parental strain, while the production at the end of fermentation is very similar in the three strains.

Example 7. Analysis of the genomic DNA of the three mutants generated.

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. It has been previously described that the intracellular decrease of this molecule results in a less response to stress, and greater tolerance to butanol of the cells. An increase in butanol tolerance could lead to increased production, or to deregulation of processes that result in faster butanol production. Another interesting mutation is the one that appears in the Cbei_2475 gene. This gene encodes a transcriptional regulator of the tetR type, and is located at 5 'of a gene coding for an extrusion pump. This mutation could lead to a greater tolerance to butanol, and, therefore, to greater production.

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. In addition, two mutated genes (Cbei_4691 and Cbei_4699) were found whose function is the maintenance of the cell wall and the synthesis of peptidoglycan. Mutations in these genes can lead to an increase in butanol tolerance, which can result in increased production. Finally, several genes involved in purine metabolism appear mutated in this lineage. The concentration of intracellular ATP is important for the production of butanol, and it has been reported that an increase in intracellular ATP leads to increased butanol production.

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.

TYA:

 Glucose: 60 g / l

 Yeast Extract: 2 g / l

Tryptone Bacto: 6 g / l

 Ammonium Acetate: 3 g / l

KH ₂ P0 ₄ : 0.5 g / l

FeS0 ₄ .7H ₂ 0: 0.01 g / l

PH adjustment to 6.5 and autoclave at 1 10 ^e C

MgS0 ₄ (1 M): 1.22 ml per liter

TYA-plates:

 Glucose: 60 g / l

 Yeast Extract: 2 g / l

Tryptone Bacto: 6 g / l

 Ammonium Acetate: 3 g / l

KH ₂ P0 ₄ : 0.5 g / l

FeS04.7H ₂ 0: 0.01 g / l

 Bacto agar: 15 g / l

PH adjustment to 6.5 and autoclave at 1 10 ^e C

MgS0 ₄ (1 M): 1.22 ml per liter

Bromocresol Purple Plates:

 Glucose: 60 g / l

Yeast Extract: 2 g / l

 Tryptone Bacto: 6 g / l

 Ammonium Acetate: 3 g / l

KH ₂ P0 ₄ : 0.5 g / l

FeS04.7H ₂ 0: 0.01 g / l

Bromocresol Purple: 0.4 g / l

Bacto agar: 15 g / l PH adjustment to 6.5 and autoclave at 1 10 ^e C

MgS0 ₄ (1 M): 1.22 ml per liter

TYA (-):

Yeast Extract: 2 g / l

Tryptone Bacto: 6 g / l

Ammonium Acetate: 3 g / l

KH ₂ P0 ₄ 0.5 g / l

FeS04.7H ₂ 0: 0.01 g / l

NaCI: 35 g / l

 Bacto-agar: 15 g / l

PH adjustment to 6.5 and autoclave at 1 10 ^e C

MgS0 ₄ (1 M): 1.22 ml per liter TYA salts + 5% hydrolyzed urban waste:

Ammonium Acetate: 3 g / l

KH ₂ P0 ₄ : 0.5 g / l

FeS04.7H ₂ 0: 0.01 g / l

 NaCI: 35 g / l

Bacto-agar: 15 g / l

 Urban waste hydrolyzate: 50 ml / l

PH adjustment to 6.5 and autoclave at 1 10 ^e C

MgS0 ₄ (1 M): 1, 22 mi per liter TYA salts + 16% hydrolysed maize straw:

Ammonium Acetate: 3 g / l

KH ₂ P0 ₄ : 0.5 g / l

FeS04.7H ₂ 0: 0.01 g / l

 NaCI: 35 g / l

Bacto-agar: 15 g / l

 Corn straw hydrolyzate: 160 ml / l

PH adjustment to 6.5 and autoclave at 1 10 ^e C

MgS0 ₄ (1 M): 1.22 ml per liter

Claims

one . A mutant strain of Clostridium beijerinckii characterized in that it comprises in its genome the following mutations with respect to the genome of the parental strain Clostridium beijerinckii NCIMB 8052: a. A instead of G at position 235 of the Cbei_0083 gene,

 b. T instead of C at position 151 of the Cbei_0769 gene,

 C. A instead of G at position 54 of the Cbei_R0027 gene,

 d. A instead of G at position 186 of the Cbei_0123 gene,

 and. A instead of G at position 201 of the Cbei_0196 gene,

 F. A instead of G at position 871 of the Cbei_0316 gene,

 g- T instead of C at position 731 of the Cbei_1206 gene,

 h. T instead of C at position 437 of the Cbei_1472 gene,

 i. C instead of T at position 968 of the CbeM 854 gene,

 j- A instead of C at position 139 of the Cbei_1935 gene,

 k. T instead of G at position 31 of the Cbei_1975 gene,

 I. T instead of G at position 21 13 of the Cbei_3078 gene,

 m. G instead of A at position 88 of the Cbei_3625 gene,

 n. T instead of C at position 160 of the Cbei_3757 gene,

 or. A instead of G at position 325 of the Cbei_4026 gene,

 P- A instead of G at position 1079 of the Cbei_4207 gene,

 q- T instead of G at position 628 of the Cbei_4256 gene,

 r. G deletion at position 672 of the Cbei_4308 gene,

 s. T instead of G at position 1766 of the Cbei_4400 gene,

 t. A instead of G at position 1732 of the Cbei_4548 gene,

 or. A instead of G at position 1465 of the Cbei_4691 gene,

 v. A instead of G at position 997 of the Cbei_4699 gene,

 w. C instead of T at position 101 of the Cbei_4761 gene,

 x. A instead of G at position 27 of the Cbei_4865 gene, and

 Y. A instead of G at position 708 of the Cbei_4918 gene.

2. A strain according to claim 1, which is the mutant strain of Clostridium beijerinckii deposited in the Spanish Type Culture Collection under the accession number CECT 9306.

3. Use of the strain according to any of claims 1 or 2 for the production of solvents.

4. Use of the strain according to claim 3, wherein the solvent is butanol.

5. Use of the strain according to any of claims 3 or 4, wherein the solvents are acetone, butanol and ethanol (ABE).

6. Use of the strain according to any of claims 3 to 5, wherein the production of solvents is carried out by means of an anaerobic fermentation process in the presence of a culture medium comprising a carbon source.

7. Use of the strain according to claim 6, wherein the carbon source comprises glucose, cellobiose, xylose and / or arabinose.

8. Use of the strain according to claim 7, wherein the carbon source comprises glucose and arabinose.

9. Use of the strain according to any of claims 6 to 8, wherein the carbon source is selected from the list consisting of: glucose, ground corn, urban organic waste hydrolyzate, or vegetable biomass hydrolyzate.

10. Use of the strain according to claim 9, wherein the vegetable biomass hydrolyzate is corn straw hydrolyzate.

eleven . Use of the strain according to any one of claims 6 to 10, wherein the culture medium further comprises yeast extract, tryptone, ammonium acetate, magnesium acetate, iron sulfate, potassium phosphate and / or magnesium sulfate.

12. Method for the production of solvents comprising the following steps: a. fermenting the strain according to any one of claims 1 or 2 in the presence of a culture medium comprising a carbon source, and

b. recover the solvents produced by the strain from the culture medium.

13. Method according to claim 12, wherein the solvent is butanol.

14. Method according to any of claims 12 or 13, wherein the solvents are acetone, butanol and ethanol (ABE).

15. Method according to any of claims 12 to 14, wherein the carbon source comprises glucose, cellobiose, xylose and / or arabinose.

16. Method according to claim 15, wherein the carbon source comprises glucose and arabinose.

17. Method according to any of claims 12 to 16, wherein the carbon source is selected from the list consisting of: glucose, ground corn, urban organic solid waste hydrolyzate, or vegetable biomass hydrolyzate.

18. Method according to claim 17, wherein the vegetable biomass hydrolyzate is corn straw hydrolyzate.

19. Method according to any of claims 17 or 18, wherein the concentration of hydrolyzate of urban organic solid wastes in the culture medium is 5% (v / v) or less.

20. Method according to claim 18, wherein the concentration of corn straw hydrolyzate in the culture medium is 16% (v / v) or less.

twenty-one . Method according to any of claims 12 to 20, wherein the culture medium further comprises yeast extract, tryptone, ammonium acetate, magnesium acetate, iron sulfate, potassium phosphate and / or magnesium sulfate.

22. Method according to any of claims 12 to 21, wherein the fermentation step (a) is carried out at a temperature of between 30 and 40 ^and C, for a time of between 30 and 275 hours, preferably under stirring.