Fungal strains with reduced viscosity

Abstract

The present invention relates to a fungal strain with reduced viscosity, wherein the ID78713 (GEL3) gene has been disabled. The invention also relates to different uses of the strain and methods for genetic modification.

Description

Technical Field

[0001] The present invention relates to fungal strains, in particular filamentous fungal strains, having reduced viscosity, wherein the ID78713 (GEL3) gene has been disabled. The invention also relates to different uses of the strains and genetic modification methods capable of obtaining the strains according to the invention. Background Art

[0002] Fungal strains, particularly filamentous fungi, such as Trichoderma reesei, are currently primarily used for the production of enzymes. These enzymes, such as cellulolytic enzymes, are actually used to hydrolyze cellulose or lignocellulosic biomass into monosaccharides. Therefore, the enzymes produced by filamentous fungi can be used for the production chain of second-generation biofuels or biologically derived products from (ligno) cellulose biomass sugars.

[0003] To improve the production of second generation biofuels or bio-derived products, improvements in the production of cellulolytic enzymes have been contemplated.

[0004] For example, patent EP 448430B1 has described the optimization industry production of carrying out cellulose hydrolase by Trichoderma reesei.By using the feed solution containing lactose as the sugar that induces cellulose hydrolase to produce, this production is carried out with batch feeding scheme (supply without extraction).This fermentation process comprises two steps: the first step is the fungal growth under the existence of excessive carbon source, and the second step is to produce enzyme by adding inducer with optimization flow velocity in substratum.These steps are carried out in the bioreactor of stirring and in the presence of oxygen in liquid medium, because fungi are obligate aerobes.Another example of the cellulose hydrolase production method of optimization has been described in patent EP 2744899B1.

[0005] In addition to optimizing cellulolytic enzyme production methods, genetically modifying fungal strains to alter their production capacity is also contemplated. Thus, patent application EP 18197010.4 describes a Trichoderma reesei strain that is a high producer of cellulolytic enzymes.

[0006] Fermentation methods and strains useful for improving the production of cellulolytic enzymes by filamentous fungi, resulting in bioderived products, as well as second generation biofuels, have been described in the prior art.

[0007] However, the morphology of industrial filamentous fungi has significant consequences for the design and implementation of fermentation processes, as it increases the viscosity of the culture medium. Viscosity therefore has a negative impact on oxygen transfer and limits the maximum concentration of fungi that can be targeted at the end of the growth step (thus, limiting the amount of cellulolytic enzymes that can be produced).

[0008] To alleviate this problem, stirring the fermenter at a higher speed was first conceived. However, increasing agitation is not an ideal solution because it leads to increased energy consumption and increased shear stress on the mycelium, which also has a negative impact on the production of cellulolytic enzymes.

[0009] Modifications of the genome of filamentous fungi to alter their viscosity have also been contemplated. These modifications primarily consist of null genes, where the absence of proteins or enzymes produced by these genes results in altered cellular function (Bodie and Pratt 2012; Dodge, Virag and Ward 2012).

[0010] Thus, in application PCT / US2012 / 034379, Bodie and Pratt conceived of nulling the mpg1 gene to alter the viscosity of fungal strains. Additional nulls (in the sfb3, seb1, gas1, crz1 or tps2 genes) were also conceived.

[0011] For their part, Dodge, Virag and Ward, in application PCT / US2011 / 049164, conceived a null sfb3 gene.

[0012] Fungal strains that have been genetically modified to alter their viscosity compared to a non-mutated strain (ie the parent strain) have been described in the prior art. However, it should be noted that the prior art does not mention a specific improvement in viscosity values ​​compared to the parent strain.

[0013] Furthermore, there is always a need for new, less sticky strains. Additionally, there is always a need for new, better performing strains that are able to grow the fungus better than the parent strain.

[0014] Summary of the Invention

[0015] The present invention is therefore based on the inventors' unexpected results, which demonstrate that invalidating the ID78713 (GEL3) gene in a fungal strain (particularly belonging to the class Sordaria) can yield a strain with a significantly reduced viscosity compared to the parent strain in which the ID78713 (GEL3) gene is not invalidated. The inventors have in fact demonstrated that invalidating the ID78713 (GEL3) gene alters the viscosity of the fungus, particularly filamentous fungi. Thus, using this strain makes it possible to reduce the viscosity of the fermentation slurry (must) at equal concentrations of fungi (i.e., at the same concentration of fungi in the slurry), thereby enabling the cost of the method for fermenting filamentous fungi. This can also improve the yield of the method by achieving a higher fungus concentration. In fact, the enzyme volumetric yield is proportional to the fungus concentration and the specific rate of enzyme production. Therefore, strains with lower viscosity make it possible to achieve higher fungus concentrations and improve the enzyme yield of the method.

[0016] Thus, the inventors of the present invention have demonstrated for the first time that the (GEL3) gene can influence the phenotypic viscosity of fungal strains. In fact, the ID78713 (GEL3) gene encodes a protein from glycoside hydrolase family 72, in particular a 1,3-β-glucanosyltransferase. The document Mouyna et al., Microbiology (1998), 144, 3171-3180 describes the acquisition of a mutant of Aspergillus fumigatus (a fungus belonging to the class Eurotiomycetes) in which the gene encoding the 1,3-β-glucanosyltransferase BGT1 was disabled; however, the authors concluded that the variant strain did not exhibit a different phenotype compared to the parent strain.

[0017] Therefore, the present invention relates to fungal strains in which the ID78713 (GEL3) gene has been nullified.

[0018] The present invention also relates to a method for genetically modifying the fungal strain according to the invention, comprising the step of nullifying the ID78713 (GEL3) gene.

[0019] The present invention also relates to a method for producing fungal biomass comprising the step of cultivating the fungal strain according to the present invention in a culture medium comprising a suitable substrate.

[0020] The present invention also relates to a method for producing a target protein, in particular an enzyme, comprising the step of cultivating the fungal strain according to the present invention in a culture medium comprising a suitable substrate.

[0021] The present invention also relates to a method for producing a bioderived product from a cellulosic or lignocellulosic substrate comprising the step of producing a cellulolytic enzyme using the fungal strain according to the invention.

[0022] The present invention also relates to a method for producing biofuel from a cellulosic or lignocellulosic substrate comprising the step of producing a cellulolytic enzyme using the fungal strain according to the present invention.

[0023] The present invention also relates to different uses of the strain according to the invention for producing a target protein, for hydrolyzing cellulose or lignocellulose to glucose, for producing products of biosource from cellulosic or lignocellulosic substrates, or for producing biofuels.

[0024] Finally, the present invention relates to the use of the fungal strain according to the invention for improving the properties of compatible strains, in particular industrial strains. Detailed Description of the Invention

[0026] Thus, in a first aspect, the present invention relates to a fungal strain in which the ID78713 (GEL3) gene has been invalidated. Thus, in the strain according to the invention, the ID78713 (GEL3) gene is no longer functional. This also means that in the strain according to the invention, the ID78713 (GEL3) gene has been invalidated. Thus, the present invention relates to a variant strain of a fungus in which the ID78713 (GEL3) gene has been invalidated. In other words, this means, for example, that in the strain according to the invention, no protein corresponding to the ID78713 (GEL3) gene is produced. Alternatively, a protein corresponding to the ID78713 (GEL3) gene may be produced, but it is not functional.

[0027] According to the present invention, the term "variant strain" is understood to refer to a strain that has been genetically modified compared to a parent strain. Thus, according to the present invention, the term "parent strain" is understood to refer to a strain from which a variant strain evolved or was derived, and in which the ID78713 (GEL3) gene has not been nullified. Thus, the strain according to the present invention corresponds to a variant strain derived from a parent strain, said variant strain having reduced viscosity compared to the parent strain, and said variant strain comprises at least one genetic modification corresponding to the nullification of the ID78713 (GEL3) gene.

[0028] According to the present invention, the term "functional gene" is understood to mean in particular a gene which is able to produce a functional protein.

[0029] According to the present invention, the term "functional protein" is understood to mean in particular a protein having the following activity: for example, in the case of the protein encoded by the ID78713 (GEL3) gene, glycoside hydrolase activity.

[0030] According to the present invention, the fungal strain has reduced viscosity compared to the parent strain in which the ID78713 (GEL3) gene is not nullified. According to a preferred embodiment, the viscosity of the fungal strain is at least 3 times lower than that of the parent strain, more particularly at least 8 or 10 times lower.

[0031] The expression "reduced viscosity compared to the parent strain" means that the viscosity of the variant strain is lower than that of the parent strain. Those skilled in the art know that for the same fungal concentration in the fermentation slurry, the viscosity of the variant strain according to the present invention should be compared with the viscosity of the parent strain.

[0032] According to the invention, the viscosity is preferably measured by the method described in Example 2 (also described in Hardy et al., Rheologie, Vol. 27, 43-48 (2015)) or by using the following parameters (referred to as Test A, for example):

[0033] - The shaft (rotor) used is an impeller with a diameter of 38 mm, a height of 32 mm, a pitch of 29 mm and a belt width of 8 mm;

[0034] - a cup (stator) with an inner diameter of 45 mm;

[0035] - The vertical space between the rotor and stator is 500 μm; and the following scheme:

[0036] -Fill the cup with 70 mL of the medium whose viscosity is to be measured;

[0037] - Pass 4s at 27℃ -1 to 100s -1 , then 100s -1 to 4s -1 The viscosity is measured by logarithmic shear rate sweep;

[0038] - Optionally, measurements are performed in duplicate.

[0039] Preferably, the viscosity according to the present invention is determined by using a TA Instruments AR 2000 rheometer, in particular by measuring the viscosity of the product at a temperature of 27° C. for 4 to 100 s. -1 logarithmic shear rate scan, even more preferably by using a bidirectional scan (from 4s -1 to 100s -1 And then from 100s -1 to 4s -1 According to one embodiment, when the fungus concentration is about 35 g / L, the 5s -1 The strain according to the present invention has a viscosity of approximately 1.5 Pa.s, measured at 27°C using a TA Instruments AR 2000 rheometer with a logarithmic shear rate scan. "Fungus concentration" is understood to mean the concentration in the culture medium in which the viscosity is measured. Typically, the culture medium corresponds to the fermentation slurry. According to the present invention, the term "approximately" means that these values ​​should not be considered strict values. Thus, "approximately 1.5 Pa.s" is understood to mean a value between 1.45 Pa.s and 1.55 Pa.s, and "approximately 35 g / L" is understood to mean a value between 34.5 g / L and 35.5 g / L.

[0040] According to the present invention, and in another specific embodiment, the strain according to the present invention has a viscosity that is at least 50% lower than that of the parent strain. According to the present invention, the term "at least 50%" refers to all values ​​between 50% and 100%, in particular the values ​​55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Preferably, the strain according to the present invention has a viscosity that is at least 65% lower than that of the parent strain. Those skilled in the art will know how to calculate the percentage reduction. This rate of reduction can be calculated, for example, according to the formula: ((final value - initial value) / initial value) * 100.

[0041] The GEL3 gene (also known as ID78713 in the Trichoderma reesei reference genome, see https: / / www.uniprot.org / uniprot / G0RL27) encodes a protein belonging to glycoside hydrolase family 72. For example, these enzymes are β-1,3-glucanosyltransferases involved in fungal cell wall biogenesis. According to the present invention, the term ID78713 is preferred over the term GEL3.

[0042] According to the present invention, the ID78713 (GEL3) gene is represented by SEQ ID NO: 2, but may also correspond to a variant or orthologous gene of the gene. Preferably, the ID78713 (GEL3) gene is represented by SEQ ID NO: 2 only.

[0043] According to the present invention, "gene variants or orthologous genes" are understood to mean genes that also encode proteins belonging to glycoside hydrolase family 72. Variants of the ID78713 (GEL3) gene or orthologous genes of the ID78713 (GEL3) gene are typically represented by sequences that are at least 80% identical to the gene of SEQ ID NO: 2. Thus, variants of the ID78713 (GEL3) gene or orthologous genes of the ID78713 (GEL3) gene correspond to genes derived from the sequence shown in SEQ ID NO: 2. More specifically, variants of the ID78713 (GEL3) gene or orthologous genes of the ID78713 (GEL3) gene are represented by sequences that are at least 90%, in particular at least 95%, preferably at least 98% or 99% identical to the SEQ ID NO: 2 gene.

[0044] The ID78713 (GEL3) gene is represented by SEQ ID NO: 2, and the protein encoded by the ID78713 (GEL3) gene is represented by SEQ ID NO: 3. Therefore, variants of the ID78713 (GEL3) gene or orthologous genes of the ID78713 (GEL3) gene encode the protein of SEQ ID NO: 3, or encode a sequence having at least 80%, particularly at least 90%, more particularly at least 95%, 98% or 99% identity with said SEQ ID NO: 3.

[0045] According to the present invention, the term "at least 80%" is intended to include all values ​​from 80% to 100%, in particular the values ​​80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. A person skilled in the art knows how to calculate the percentage identity between two sequences. For example, according to the present invention, the percentage identity of a given sequence compared to SEQ ID NO: 2 or SEQ ID NO: 3 is understood to refer to the percentage identity over the entire length of the sequence. Thus, this percentage corresponds to the number of identical nucleotides / residues between the given sequence and SEQ ID NO: 2 or 3 divided by the number of nucleotides or residues in the longer of the two sequences.

[0046] Thus, in one embodiment, in the strain according to the invention, the null ID78713 (GEL3) gene corresponds to the gene shown in SEQ ID NO: 2 or a gene that is at least 80% identical, in particular at least 90%, more particularly at least 95% identical to the gene shown in SEQ ID NO: 2. In an even more preferred embodiment, in the strain according to the invention, the null ID78713 (GEL3) gene corresponds to the gene shown in SEQ ID NO: 2. According to this last embodiment, the strain according to the invention thus comprises a deletion of the gene encoding the protein shown in SEQ ID NO: 3.

[0047] In a preferred aspect, in the strain according to the invention, the ID78713 (GEL3) gene has been nullified by mutagenesis or by homologous recombination. According to the invention, nullification comprises deletion of all or part of the ID78713 (GEL3) gene. Preferably, in the strain according to the invention, the ID78713 (GEL3) gene has been nullified using a nulling cassette. Still more preferably, the nulling cassette is represented by SEQ ID NO: 1 and is particularly useful for fungi belonging to the species Trichoderma reesei.

[0048] Mutagenesis is a commonly used technique in genetic engineering. Its purpose is to actively introduce mutations into DNA to produce genetically modified genes. According to the present invention, mutagenesis should be more specifically understood as designated site mutagenesis. In fact, site-directed mutagenesis can introduce identified mutations into specific genes. For this reason, a target DNA (here, the ID78713 (GEL3) gene) containing a mutation is synthesized and then introduced into the cell to be mutated, typically by using a vector, wherein the DNA repair mechanism is integrated into the genome.

[0049] Homologous recombination is a commonly used technique in genetic engineering that consists of the exchange of DNA molecules, typically through the use of vectors.

[0050] The term "vector" is understood to mean any DNA sequence into which an exogenous nucleic acid fragment can be inserted, which vector is capable of introducing exogenous DNA into a host cell. Examples of vectors are plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), phage P1-derived artificial chromosomes (PACs), and virus-derived vectors. The vector according to the present invention enables the introduction of mutations or deletions.

[0051] In a preferred embodiment, the null cassette comprises three DNA fragments:

[0052] (1) The region upstream of the target gene,

[0053] (2) selection markers, and

[0054] (3) The region downstream of the target gene.

[0055] In the context of the present invention, "target gene" is understood to mean the ID78713 (GEL3) gene. The regions upstream and downstream of the target gene are two recombination elements, one at each end of the gene, and are necessary for precise targeting of the sequence to be nullified.

[0056] According to the present invention, the region upstream of the target gene (ie, the sequence 5′ upstream of the ID78713 (GEL3) gene) is specifically represented by the sequence SEQ ID NO:10.

[0057] According to the present invention, the region downstream of the target gene (ie, the sequence 3′ downstream of the ID78713 (GEL3) gene) is specifically represented by the sequence SEQ ID NO:12.

[0058] The expression "selectable marker" is understood to mean a gene whose expression confers on cells containing it a characteristic that allows them to be selected. Indeed, the use of a selectable marker allows the identification of cells that have integrated the genetic modification, compared to cells that have not integrated it. For example, it is an antibiotic resistance gene, in particular the hygromycin antibiotic resistance gene hph, as shown in SEQ ID NO: 11.

[0059] More specifically, according to the present invention, the null cassette preferably consists of a resistance gene placed under the control of a promoter and a terminator, with flanking regions 5' and 3' upstream and downstream of the ID78713 (GEL3) gene. Even more preferably, according to the present invention, the null cassette consists of the hygromycin antibiotic resistance gene hph placed under the control of the GPDa promoter and the TRPc terminator (Punt and van den Hondel, 1992), with flanking regions 5' and 3' upstream and downstream of the ID78713 (GEL3) gene. According to the present invention, the null cassette can be operably linked to a promoter, a terminator, or any other sequence required for expression in a host cell.

[0060] The null cassette can be amplified according to conventional techniques well known to those skilled in the art, typically by a method selected from standard cloning, fusion PCR or in vivo PCR cloning. Preferably, the null cassette is amplified by PCR, in particular by using the sequences shown in SEQ ID NO:4 and SEQ ID NO:5. The null cassette is then introduced into a strain that does not express the selectable marker gene, in particular a strain of Trichoderma reesei, by recombination. The selectable marker gene relevant to the practice of the present invention can be easily identified by those skilled in the art. After cultivation, variant / mutant strains into which the null cassette has been introduced are selected based on expression or non-expression of the selectable marker; the transformed clones are clones that express the selectable marker. These are strains according to the present invention. Preferably, mutant strains are identified by using primers according to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. These genetic recombination techniques are well known to those skilled in the art. In a preferred aspect, according to the present invention, the fungus is a filamentous fungus. Even more preferably, the filamentous fungus is selected from the following classes: Orbiliaceae, Pezizomycetes, Dothideomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, Sordariomycetes, and Saccharomycetes. Advantageously, the filamentous fungus according to the present invention belongs to the class of Orbiliaceae, in particular to the genus Trichoderma, more particularly to the species Trichoderma reesei.

[0061] According to the present invention, when the fungus belongs to the species Trichoderma reesei, the parent strain of Trichoderma reesei may be strain QM6a (deposited as ATCC 13631), or a strain derived from the natural isolate QM6a (particularly obtained by random or directed mutagenesis), such as strain Rut-C30 (deposited as ATCC 56765), strain deposited as CNCM I-5221 (deposited on August 3, 2017 at the National Collection of Cultures and Microorganisms (CNCM, Collection Nationale de Cultures de Microorganismes), of the Pasteur Institute, 25 rue du Docteur Roux, F-75724 Paris cedex 15), strain NG14 (deposited as ATCC 56767) or strain QM9414 (deposited as ATCC 26921). Typically, the strain according to the present invention exhibits a low viscosity phenotype while still maintaining its ability to produce the target protein.

[0062] In a second aspect, the present invention also relates to a method for genetically modifying a fungal strain such as the one described above, comprising a step of deactivating the ID78713 (GEL3) gene. Thus, the method for genetically modifying a strain according to the present invention allows for obtaining fungal strains that are less viscous than the parent fungal strain. This is why the strain according to the present invention can be considered a variant of the parent fungal strain. Thus, during its growth, at the same biomass concentration and temperature, for a given shear rate, the fungal strain according to the present invention produces a lower viscosity than the parent strain.

[0063] Preferably, in the genetic modification method according to the present invention, the step of nullifying the ID78713 (GEL3) gene is performed using a mutagenesis or homologous recombination step. Still more preferably, in the genetic modification method according to the present invention, the step of nullifying the ID78713 (GEL3) gene is performed in a fungus belonging to the species Trichoderma reesei using a null cassette such as described above, in particular a null cassette as shown in SEQ ID NO: 1.

[0064] In a third aspect, the present invention also relates to a method for producing fungal biomass, comprising the step of culturing a fungal strain according to the present invention in a culture medium containing a suitable substrate. Thus, this step enables the growth of the fungal strain according to the present invention. Suitable substrates for growing fungal strains are known to those skilled in the art.

[0065] In a fourth aspect, the present invention also relates to a method for producing a target protein, in particular an enzyme, comprising the step of culturing the fungal strain according to the invention in a culture medium containing a suitable substrate. Therefore, the present invention relates to the use of the fungal strain according to the invention for producing a target protein.

[0066] Thus, advantageously, the method comprises a growth phase in which the fungal strain according to the invention is grown, followed by a phase in which the strain grows and produces the target protein. Still more preferably, the growth phase is carried out in the presence of a growth substrate, and the growth and production phase of the target protein is carried out in the presence of an induction substrate. The growth substrate and the induction substrate are preferably carbon substrates.

[0067] Thus, the carbon growth substrate is preferably selected from lactose, glucose, xylose, residues obtained after ethanol fermentation of monosaccharides of enzymatic hydrolysates of cellulosic biomass, and / or crude extracts of water-soluble pentoses from pretreatment of cellulosic biomass.

[0068] Therefore, the inducing carbon substrate is preferably selected from the group consisting of lactose, cellobiose, sophorose, residues obtained after ethanol fermentation of monosaccharides of enzymatic hydrolysates of cellulosic biomass, and / or crude extracts of water-soluble pentoses from pretreatment of cellulosic biomass.

[0069] According to the present invention, target proteins are all proteins that can be produced by fungi, naturally or by genetic modification (eg by following transformation with a suitable vector).

[0070] Advantageously, the target protein according to the present invention is an enzyme, particularly a cellulolytic enzyme (cellulolyticenzymes), such as a cellulose hydrolase (cellulases) or a hemicellulose hydrolase. Preferably, the enzyme is a cellulose hydrolase. According to the present invention, the term "cellulose hydrolase" should be understood to refer more specifically to an enzyme selected from endoglucanases, exoglucanases and beta-glucosidases, and more specifically, to beta-glucosidases. The term "cellulose hydrolase" more specifically refers to an enzyme that is suitable for hydrolyzing cellulose and enables the microorganism (e.g., Trichoderma reesei) that produces them to use cellulose as a carbon source by hydrolyzing the polymer into monosaccharides (glucose). By bacterial strains according to the present invention, particularly Trichoderma reesei producing cellulose hydrolase can be determined by any conventional technique available to those skilled in the art, or by the technology described in patent EP 448430B1 or EP2744899B1.

[0071] A correlation between total secreted proteins and cellulose hydrolases can be obtained because in Trichoderma reesei, the major exoglucanases (CBHI, CBHII) and endoglucanases (EGI, EGII) can account for up to 90% of the total secreted proteins (see, e.g., Markov, AV, Gusakov, AV, Kondratyeva, EG, Okunev, ON, Bekkarevich, AO, and Sinitsyn, AP (2005). New Effective Method for Analysis of the Component Composition of Enzyme Complexes from Trichodermareesei. Biochemistry (Moscow) 70, 657-663).

[0072] In a fifth aspect, the present invention also relates to a method for producing a bioderived product from a cellulosic or lignocellulosic substrate, comprising the step of producing a cellulolytic enzyme using a fungal strain according to the invention. Thus, the present invention also relates to a method for producing a bioderived product from a cellulosic or lignocellulosic substrate using a fungal strain according to the invention.

[0073] In a sixth aspect, the present invention relates to a method for producing biofuel from a cellulosic or lignocellulosic substrate, comprising the step of producing a cellulolytic enzyme using a fungal strain according to the invention. Accordingly, the present invention also relates to the use of a fungal strain according to the invention for producing biofuel from a cellulosic or lignocellulosic substrate.

[0074] According to the present invention, term " biofuel " should be understood as referring to second generation biofuel more specifically, the biofuel that promptly is derived from non-food resources.According to the present invention, term " biofuel " also can be defined as being derived from biomass conversion and can be used for any product of energy purpose.First, do not wish to be subject to any restriction, can enumerate for example biogas, can be mixed (possibly after conversion subsequently) in fuel or can be the product of fully mature fuel, such as alcohol (ethanol, butanols and / or isopropanol, depend on the type of the fermentation organism that uses), solvent (acetone), acid (butyric acid), lipid and their derivative (short-chain or long-chain fatty acid, fatty acid ester), and hydrogen.Preferably, biofuel according to the present invention is alcohol, for example ethanol, butanols and / or isopropanol.More preferably, biofuel according to the present invention is ethanol.In another embodiment, biofuel is biogas. In another embodiment, the product is a target molecule for the chemical industry, for example another alcohol such as 1,2-propylene glycol, 1,3-propylene glycol, 1,4-butanediol, 2,3-butanediol; an organic acid such as acetic acid, propionic acid, acrylic acid, butyric acid, succinic acid, malic acid, fumaric acid, citric acid, itaconic acid, or a hydroxy acid such as glycolic acid, hydroxypropionic acid, or lactic acid.

[0075] In a preferred aspect, the method for producing biofuel from a cellulosic or lignocellulosic substrate according to the present invention comprises:

[0076] i) a step of pretreating a cellulosic or lignocellulosic substrate to obtain a pretreated substrate, ii) a step of producing a cellulolytic enzyme using the strain according to the invention,

[0077] iii) a step of enzymatically hydrolyzing the pretreated substrate obtained in step i) in the presence of the cellulolytic enzyme obtained in step ii) to obtain a hydrolyzate,

[0078] iv) a step of alcoholic fermentation of the hydrolyzate obtained,

[0079] v) a separation step, in particular by distillation.

[0080] In an even more preferred aspect, the method according to the invention for producing a biofuel from a cellulosic or lignocellulosic substrate comprises:

[0081] i) a step of pretreating a cellulosic or lignocellulosic substrate to obtain a pretreated substrate, ii) a step of producing a cellulolytic enzyme using the strain according to the invention,

[0082] iii) a step of enzymatically hydrolyzing the pretreated substrate obtained in step i) in the presence of the cellulolytic enzyme obtained in step ii) to obtain a hydrolyzate,

[0083] iv) a step of alcoholic fermentation of the hydrolyzate obtained,

[0084] v) a separation step, in particular by distillation.

[0085] Said steps iii) and iv) are carried out simultaneously. This is typically the case in the production process known as "SSF" (simultaneous saccharification and fermentation).

[0086] According to a particular embodiment, the step of pre-treating the cellulosic or lignocellulosic substrate is a step of suspending said cellulosic or lignocellulosic substrate in an aqueous phase.

[0087] According to a particular embodiment, the hydrolysate obtained in step iii) is a glucose-containing hydrolysate.

[0088] According to a particular embodiment, the step of alcoholic fermentation of the hydrolysate obtained is a step of fermenting the glucose produced from the hydrolysate in the presence of a fermenting organism to produce a fermentation syrup. For example, the fermenting organism is yeast.

[0089] According to a particular embodiment, the separation step is the separation of biofuel and fermentation slurry, in particular by distillation.

[0090] According to an even more preferred embodiment, the cellulosic or lignocellulosic substrate to be hydrolyzed is suspended in an aqueous phase at 6-40% dry matter, preferably 20-30% dry matter. The pH is adjusted to 4-5.5, preferably 4.8-5.2, and the temperature is adjusted to 40°C-60°C, preferably 45°C-50°C. The hydrolysis reaction is initiated by adding enzymes that act on the pretreated substrate. The amount of enzyme typically used is 10-30 mg of secreted protein per gram of pretreated substrate or less. The reaction typically lasts 15 to 48 hours. The reaction is monitored by measuring the released sugars, particularly glucose. The sugar solution is separated from the unhydrolyzed solid fraction, which essentially consists of lignin, by filtration or centrifugation and then processed in a fermentation unit.

[0091] According to another even more preferred embodiment, when the hydrolysis and fermentation steps are performed simultaneously, the enzymes and the fermenting organism are added simultaneously, followed by incubation at a temperature of 30° C. to 35° C. to produce a fermentation slurry. According to this embodiment, the cellulose present in the pretreated substrate is converted to glucose, and at the same time, in the same reactor, the fermenting organism (e.g., yeast) converts the glucose into the end product according to the SSF (simultaneous saccharification and fermentation) method known to those skilled in the art. Depending on the metabolic and hydrolytic capacity of the fermenting organism, the successful completion of the operation may require the addition of a greater or lesser amount of exogenous cellulose mixture.

[0092] In a seventh aspect, the present invention also relates to the use of a fungal strain according to the present invention for hydrolyzing cellulose or lignocellulose to glucose.

[0093] In an eighth aspect, the present invention also relates to the use of a fungal strain according to the invention for improving the performance of compatible strains, in particular industrial strains.

[0094] In this specification, the definitions and preferences indicated in one aspect apply to the other aspects. For example, all definitions and preferences indicated in the first aspect of the present invention above also apply to the second, third, fourth, fifth, sixth, seventh and eighth aspects.

[0095] BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Other features, details and advantages of the invention will appear on reading the accompanying drawings.

[0097] Figure 1

[0098] [ Figure 1 ] indicates that different strains cultured in shake flasks were -1 The apparent viscosity is measured at a shear rate of .

[0099] Figure 2

[0100] [ Figure 2 ] indicates the different strains cultured in bioreactors (RutC30 and TR3126-ΔGEL3) at 5s -1 Apparent viscosity measured at shear rate.

[0101] Figure 3

[0102] [ Figure 3 ] indicates the different strains cultured in bioreactors (CL847 and CL847-ΔGEL3) at 5s -1 Apparent viscosity measured at shear rate.

[0103] Sequences of the present invention

[0104] [Table 1]

[0105]

[0106]

[0107]

[0108]

[0109] References cited in this patent application

[0110] Durand H,Clanet M,Tiraby G.Genetic improvement of Trichoderma reeseifor large scale cellulase production.Enzyme Microb Technol 1988,10:341–346.

[0111] Guangtao Z,Hartl L,Schuster A,Polak S,Schmoll M,Wang T,Seidl V,SeibothB.,(2009).Gene targeting in a nonhomologous end joining deficientHypocrea jecorina.J Biotechnol.139(2):146-51.

[0112] Montenecourt,B.S.;Eveleigh,D.E.(1977)Semiquantitative Plate Assay forDetermination of Cellulase Production by Trichoderma viride.In:Applied andenvironmental microbiology,vol.33,No.1,p.178–183

[0113] M,Nevalainen H, Salminen E,Knowles J.,(1987).Aversatile transformation system for the cellulolytic filamentous fungusTrichoderma reesei.Gene.61(2):155-64.

[0114] Punt,P.J.;van den Hondel,C.A.,(1992)Transformation of filamentousfungi based on hygromycin B and phleomycin resistance markers.In:Methods inenzymology,vol.216,p.447–457.

[0115] Te'o VS, Bergquist PL, Nevalainen KM., (2002). Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system. JMicrobiol Methods.51(3):393-9. DETAILED DESCRIPTION

[0116] Example 1a: Inactivation of the ID78713 (GEL3) gene in a high-yielding strain

[0117] The null cassette for ID78713 (GEL3) consists of the hygromycin antibiotic resistance gene hph under the control of the GPDa promoter and the TRPc terminator (Punt and van den Hondel, 1999), with 5' and 3' flanking regions upstream and downstream of the ID78713 (GEL3) gene. The sequence is shown in SEQ ID NO: 1. A DNA cassette was synthesized and inserted into the pEX-A plasmid (e.g., available from Addgene). After amplification and extraction of the plasmid, the null cassette was amplified by PCR (polymerase chain reaction) using primers p61 and p62 (see Table 2 below).

[0118] [Table 2]

[0119] Primer name Sequence corresponding to the primer P61 SEQ ID NO:4 P62 SEQ ID NO:5 P78 SEQ ID NO:6 P79 SEQ ID NO:7 P91 SEQ ID NO:8 P92 SEQ ID NO:9

[0120] Primer sequences of the present invention

[0121] The strain used for transformation was the high-producing strain RutC30 (Montenecourt and Eveleigh, 1977), in which the gene KU70 (ID 63200) was rendered ineffective by replacing the coding sequence with the gene encoding the selectable marker AmdS ( et al., 1987). Inactivation of this gene promotes homologous recombination (Guangtao et al., 2009). This strain was designated TR3126.

[0122] Transformation of the TR3126 strain with the cassette represented by SEQ ID NO: 1 was performed by Biolistique (Te'o et al., 2002) using 5 μg of the purified cassette. Integration at the null cassette locus was verified by PCR using primers upstream of the cassette (p91) and in the hph gene (p78) (5' verification) and downstream of the cassette (p92) and in the hph gene (p79) (3' verification). The strain thus obtained, which was null for the ID 78713 (GEL3) gene, was designated TR3126-ΔGEL3.

[0123] Example 1b: Invalidation of the ID78713 (GEL3) gene in a high-yielding strain

[0124] A second strain was tested: strain CL847 (Durand H et al.).

[0125] Transformation of CL847 strain was performed by protoplast method ( M et al., doi: 10.1016 / 0378-1119(87)90110-7). Similar to Example 1a, integration at the null cassette locus was verified. The strain null for the ID78713 (GEL3) gene obtained in this way was designated CL847-ΔGEL3.

[0126] Example 2: Method for measuring viscosity

[0127] For rheological measurements, the shaft (rotor) used was a large stainless steel impeller with a diameter of 38 mm, a height of 32 mm, a pitch of 29 mm, and a ribbon width of 8 mm. This impeller was used with a cup (stator) having an inner diameter of 45 mm and a vertical spacing of 500 μm between the rotor and stator. After calibration, the impeller resembled a Couette cylinder with a radius of 14 mm.

[0128] The cup is filled with 70 mL of fermentation slurry collected from a reactor (shake flask, for example as described in Example 3, or a bioreactor). Viscosity measurements are made at a temperature of 27°C over a 4 s -1 to 100s -1 The logarithmic shear rate scan was performed. This range corresponds to the average shear rate expected on an industrial scale. The scan was a bidirectional scan (from 4s -1 to 100s -1 , then from 100s -1 to 4s -1 ). Rheological measurements were performed in duplicate.

[0129] All measurements were performed on a TA Instruments AR 2000 rheometer.

[0130] Example 3: Shake flask culture protocol

[0131] Shake flask cultures were performed in 19 cm diameter Fernbach flasks containing 400 mL of medium, inoculated with spores of the different strains from frozen vials, and incubated in an Infors Multitron incubator at 150 rpm and 30°C.

[0132] The culture medium had the following final composition:

[0133] -5.6g / L(NH4)2SO4

[0134] -4.4g / L K2HPO4

[0135] -0.3g / LMgSO4,7H2O

[0136] -0.15g / L CaCl2,2H2O

[0137] -1mL / L trace element solution (FeSO4: 5g / L, MnSO4: 1.4g / L, ZnSO4: 1.4g / L, CoCl2: 3.7g / l)

[0138] -5.85g / L BTCA (butanetetracarboxylic acid)

[0139] -3.0g / L KOH, crystals

[0140] -1.5g / L corn steep liquor (e.g. Roquette )

[0141] -30g / L glucose.

[0142] The pH of the medium was adjusted to 6.0 with 30% sodium hydroxide.

[0143] The compounds were sterilized at 121°C for 20 minutes (glucose was sterilized separately from the other compounds).

[0144] A regular 2 mL sample was taken to monitor residual glucose. Then, when the residual glucose was less than 5 g / L (corresponding to a fungal concentration of the order of 10 g / L), a 100 mL sample was taken to accurately measure the fungal concentration (by filtration and subsequent drying on a 1.2 μm filter) and the juice viscosity (according to the method described in Example 2).

[0145] Example 4: Comparison of viscosity of shake flask cultures

[0146] The two strains were cultured in duplicate according to the method described in Example 3, and the viscosity of the fermentation slurry was characterized according to the method described in Example 2:

[0147] The two strains tested were:

[0148] - TR3126-ΔGEL3 strain showing null for the ID78713 (GEL3) gene;

[0149] - Parent strain TR3126 used as a high viscosity control

[0150] Fungal concentration measurements showed that in all cultures performed, the concentrations were of the same order of magnitude, approximately 10 g / L of fungi in suspension (see Table 3 below).

[0151] [Table 3]

[0152]

[0153] Fungal concentration of tested strains (experiments performed in duplicate)

[0154] Viscosity measurement (eg Figure 1 As shown in Figure 5, the inactivation of the ID78713 (GEL3) gene resulted in a sharp decrease in viscosity. -1 At shear rates of 1.5 Å, the viscosity achieved by the TR3126-ΔGEL3 strain was approximately 8 to 10 times lower than that of the viscous control (TR3126).

[0155] Example 5: Cultivation protocol in a bioreactor

[0156] Bioreactor cultivation was carried out in a fermenter with a diameter of 16 cm containing 2 L of medium inoculated at 10% v / v from a preculture prepared according to the protocol described in Example 3. Agitation was provided by a Rayneri turbine with a diameter of 8 cm at a fixed speed of 1000 rpm. The temperature was controlled at 27° C. and the pH was controlled at 4.8 by the automatic addition of a 5N ammonia solution.

[0157] The culture medium had the following final composition:

[0158] -3mL / L 85% orthophosphoric acid

[0159] -0.25mL / L 96% sulfuric acid

[0160] -1.66g / L potassium hydroxide KOH, crystals

[0161] -2.8g / L(NH4)2SO4

[0162] -0.6g / L MgSO4,7H2O

[0163] -0.6g / L CaCl2,2H2O

[0164] -0.12g / L Na2HPO4,12H2O

[0165] -1mL / L trace element solution (FeSO4: 5g / L, MnSO4: 1.4g / L, ZnSO4: 1.4g / L, CoCl2: 3.7g / l)

[0166] -1g / L corn steep liquor (e.g. Roquette )

[0167] -80g / L glucose

[0168] The compounds were sterilized at 121°C for 20 minutes (glucose was sterilized separately from the other compounds).

[0169] The pH of the medium was adjusted and then controlled at 4.8 with an ammonia solution for pH checking.

[0170] Routine samples of approximately 100 mL were taken: (i) to monitor residual glucose, (ii) to accurately measure the fungal concentration (by filtration and subsequent drying on a 1.2 μm filter), and (iii) to measure the viscosity of the slurry (according to the method described in Example 2).

[0171] Example 6: Comparison of viscosity in bioreactor cultures

[0172] Both strains were cultured in duplicate according to the method described in Example 5 and the viscosity of the fermentation slurries was characterized according to the method described in Example 2 (for different concentrations of fungi in the slurry):

[0173] -TR3126-ΔGEL3 strain showing null expression of the ID78713 (GEL3) gene

[0174] - Reference strain Rut-C30 (used as a high viscosity control)

[0175] Characterization of viscosity at different fungal concentrations revealed a substantial advantage conferred by nulling the ID78713 (GEL3) gene (see Figure 2 ),in:

[0176] - For example, in shake flasks, when the fungal concentration is about 10 g / L, the viscosity is about 10 times lower;

[0177] - When the concentration is about 25g / L, the viscosity is about 3 times lower;

[0178] The viscosity of the strain null for -ID78713(GEL3) (TR3126-ΔGEL3) at 35 g / L was of the same order of magnitude as the viscosity of the wild-type strain (Rut-C30) at 15 g / L.

[0179] Therefore, the culture of the ID78713(GEL3)-null strain at 35 g / L does not require more energy for stirring than the culture of the wild-type strain at 15 g / L, which enables the productivity of the culture to be improved at the same energy consumption.

[0180] Example 7: Comparison of viscosity of two other strains in bioreactor culture

[0181] Both strains were cultivated according to the method described in Example 5 and the viscosity of the fermentation slurries was characterized according to the method described in Example 2 (for different concentrations of fungi in the slurry):

[0182] -CL847-ΔGEL3 strain showing null expression of the ID78713 (GEL3) gene

[0183] - Parent strain CL847 (used as control)

[0184] Viscosity characterization at different fungal concentrations again showed that in strain CL847, the ID78713 (GEL3) gene was ineffective at fungal concentrations of 12-14 g / L (see Figure 3 ) confers the advantage of approximately 3 times lower viscosity.

Claims

1. A fungal strain belonging to the species Trichoderma reesei, wherein the ID78713 gene has been disabled and / or the protein corresponding to the ID78713 gene is not produced or is not functional, and wherein: - the ID78713 gene corresponds to the gene shown by SEQ ID NO: 2, -The protein corresponding to the ID78713 gene is represented by SEQ ID NO:

3.

2. The fungal strain according to claim 1, which has reduced viscosity compared to a parent strain in which the ID78713 gene is not nullified.

3. The fungal strain according to any one of claims 1 or 2, wherein the ID78713 gene has been abolished by site-directed mutagenesis or by homologous recombination.

4. A method for genetically modifying the fungal strain according to any one of claims 1 to 3, comprising the step of deactivating the ID78713 gene.

5. The method for genetically modifying a fungal strain according to claim 4, wherein the step of nullifying the ID78713 gene is performed by site-directed mutagenesis or homologous recombination.

6. A method for producing fungal biomass, comprising the step of cultivating the fungal strain according to any one of claims 1 to 3 in a culture medium comprising a suitable substrate.

7. A method for producing a target protein, comprising the step of culturing the fungal strain according to any one of claims 1 to 3 in a culture medium containing a suitable substrate.

8. A method for producing a bio-derived product from a lignocellulosic substrate comprising the step of producing a cellulolytic enzyme using a fungal strain according to any one of claims 1 to 3.

9. A method for producing biofuel from lignocellulosic substrates comprising the step of producing cellulolytic enzymes using the fungal strain according to any one of claims 1 to 3.

10. The method of claim 9 for producing biofuel from a lignocellulosic substrate, comprising: - (i) a step of pretreating the lignocellulosic substrate to obtain a pretreated substrate, -(ii) a step of producing a cellulolytic enzyme using the strain according to any one of claims 1 to 3, (iii) a step of enzymatically hydrolyzing the pretreated substrate in the presence of the cellulolytic enzyme obtained in step (ii) and a suitable substrate to obtain a hydrolysate, - (iv) a step of alcoholic fermentation of the hydrolysate obtained, which is carried out simultaneously with step (iii), - (v) separation step.

11. Use of the fungal strain according to any one of claims 1 to 3 for producing a target protein.

12. Use of the fungal strain according to any one of claims 1 to 3 for hydrolyzing lignocellulose to glucose.

13. Use of a fungal strain according to any one of claims 1 to 3 for producing bioderived products from lignocellulosic substrates.

14. Use of a fungal strain according to any one of claims 1 to 3 for the production of biofuels from lignocellulosic substrates.

Images