HYPERPRODUCTIVE PROTEIN MUSHROOM STRAIN

Abstract

The invention relates to a protein-hyperproducing fungal strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which the expression of at least one protein encoded by a gene selected from ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 and ID 122457, or an orthologous gene, is modified. The invention also relates to the various uses of this strain, as well as the method of genetic modification. (no figure)

Description

Title of the invention: PROTEIN-HYPERPRODUCING MUSHROOM STRAIN

[0001] The present invention relates to a strain of fungus belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola in which the expression of at least one gene impacting the secretion of proteins has been modified, by overexpression or inhibition. The invention also relates to the various uses of a strain thus modified, as well as the method of genetic modification making it possible to obtain a strain according to the invention. Context of the invention

[0002] Fungal strains, particularly filamentous ones, such as the Trichoderma reesei fungus, are today mainly used for the production of proteins (whether endogenous or exogenous (heterologous)).

[0003] Various methods for increasing said protein production have thus already been studied. For example, international application PCT / EP2016 / 069101 describes fungal strains deficient in certain regulatory proteins, which are correlated with reduced protease activity, in order to improve the production of heterologous proteins by said strains. Application US2022 / 0064228 also describes strains deficient in certain transcription factors in order to improve the production of heterologous proteins by said strains. On the contrary, other applications, for example international application PCT / US2 022 / 035655 or PCT / US2022 / 079686 have already described the overexpression of certain genes to increase the production of heterologous proteins by fungi.

[0004] In addition to exogenous proteins, fungi are particularly useful for producing enzymes (particularly endogenous ones). These enzymes, for example cellulases, are in fact used to hydrolyze cellulosic or lignocellulosic biomass into simple sugars. The enzymes produced by filamentous fungi are therefore useful in the production of second-generation biofuels or even bio-sourced products derived from sugars from (ligno)cellulosic biomass.

[0005] In order to improve the production of second-generation biofuels or bio-sourced products, it has already been considered to improve the production of cellulases, as for example in patent EP448430 which describes an optimized industrial production of cellulases by Trichoderma reesei. In addition to the optimization of cellulase production processes, it has also been considered to genetically modify the fungal strains in order to modify their production capacity. Thus, the patent EP3397768 describes the overexpression of the TrAZFl gene in a strain to significantly increase the production of cellulolytic proteins.

[0006] Fermentation processes and strains useful for improving the production of cellulolytic enzymes by filamentous fungi, and thereby of bio-sourced products but also second-generation biofuels, are therefore already described in the prior art.

[0007] The fungal strain Trichoderma reeseï Rut-C30 (Eveleigh and Montenecourt, 1979) is one of the reference strains that is hyper-producing of proteins.

[0008] The present invention is based here on the unexpected results of the inventors who have highlighted new genes impacting the secretion of proteins by Trichoderma fungi, in particular Trichoderma reesei. The inventors have in fact shown that the overexpression and / or inhibition of at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 and / or ID 122457 made it possible to obtain a strain hyperproducing proteins of interest, compared to a parent strain in which the expression of at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 and / or ID 122457 has not been modified (neither overexpressed nor invalidated). Brief description of the invention

[0009] The present invention therefore relates to a strain of fungus belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified, and / or - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene is increased.

[0010] The present invention also relates to a method for genetic modification of a fungal strain according to the invention, comprising: - a step of overexpression of at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768, and ID 80200, or an orthologous gene, or - a step of invalidating at least one gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene.

[0011] The present invention also relates to a method for producing mushroom biomass, comprising a step of culturing a fungal strain according to the invention, as defined above or a strain not expressing or having reduced expression of at least one protein encoded by a gene ID 122457, or an orthologous gene, in a culture medium comprising a suitable substrate.

[0012] The present invention also relates to a method for producing proteins of interest, in particular enzymes, comprising a step of culturing a strain of fungus according to the invention, as defined above or a strain not expressing or having reduced expression of at least one protein encoded by a gene ID 122457, or an orthologous gene, in a culture medium comprising a suitable substrate.

[0013] The present invention further relates to a method for producing bio-sourced products from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to the invention, as defined above or a strain not expressing or having reduced expression of at least one protein encoded by a gene ID 122457, or an orthologous gene.

[0014] The present invention also relates to a process for producing a sweet juice or sugars from cellulosic or lignocellulosic substrates, comprising: - i) a step of pretreating a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - ii) a step of producing cellulolytic enzymes by a fungal strain according to the invention, as defined above or a strain not expressing or having reduced expression of at least one protein encoded by a gene ID 122457, or an orthologous gene, - iii) a step of enzymatic hydrolysis of the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step ii) and a suitable substrate, in order to obtain a hydrolysate.

[0015] The present invention also relates to a process for producing biofuel or alcohol from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to the invention, as defined above, or a strain not expressing or having reduced expression of at least one protein encoded by a gene ID 122457, or an orthologous gene.

[0016] The present invention also relates to various uses of the strain according to the invention, as defined above or a strain not expressing or having reduced expression of at least one protein encoded by a gene ID 122457, or an orthologous gene, for the production of proteins of interest, for the hydrolysis of cellulose or lignocellulose into glucose, for the production of biosourced products from cellulosic or lignocellulosic substrates, for the production of biofuel from cellulosic or lignocellulosic substrates, for the production of alcohol from cellulosic or lignocellulosic substrates or for the production of a sweet juice or sugars from cellulosic or lignocellulosic substrates. Statement of the invention

[0017] In a first aspect, the present invention thus relates to a strain of fungus belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified and / or the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased. The present invention thus relates to a variant strain of fungus, in which the expression of at least one protein encoded by a gene selected from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified and / or the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, compared to a parent strain.In some embodiments, the present invention also relates to a fungal strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, not expressing or having reduced expression of at least one protein encoded by a gene ID 122457, or an orthologous gene, i.e., a variant fungal strain, in which the expression of at least one protein encoded by a gene ID 122457, or an orthologous gene, is deficient or reduced compared to a parent strain. According to one embodiment, the invention relates to a strain of fungus belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which the expression of at least one protein encoded by a gene selected from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified.

[0018] According to the invention, the term "variant strain" means a strain genetically modified with respect to a parent strain. According to the invention, the term "parent strain" thus means a strain from which the variant strain is derived or originated, and in which the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is not modified and / or the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is not increased and / or the expression of at least one protein encoded by a gene ID 122457, or an orthologous gene, is not deficient or reduced.The strain according to the invention thus corresponds to a variant strain derived from a parent strain, said variant strain having improved production of proteins of interest compared to the parent strain and said variant strain comprising at least one genetic modification corresponding to the modification of the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, and / or the increase in the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, and / or the deficiency or the . decreased expression of at least one protein encoded by gene ID 122457, or an orthologous gene.

[0019] The expression "improved production compared to the parent strain" or "the expression of at least one protein is increased" means that the variant strain has a production higher than that of the parent strain, and in particular an improved / increased production of at least 5% compared to that of the parent strain. According to the invention, "improved production of at least 5%" means all values ​​between 5% and 100%, and in particular 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%.

[0020] According to the invention, the gene ID 65290 corresponds to a gene represented by SEQ ID NO: 1 or SEQ ID NO: 23, or a gene having at least 80% identity with SEQ ID NO: 1 or SEQ ID NO: 23, in particular at least 85%, at least 90%, more particularly at least 95%, preferentially at least 98% or 99%, and the protein encoded by the gene ID 65290 is represented by SEQ ID NO: 5 or a protein having at least 80% identity with SEQ ID NO: 5, in particular at least 85%, at least 90%, more particularly at least 95%, preferentially at least 98% or 99%. Preferably, gene ID 65290 corresponds to a gene represented by SEQ ID NO: 1 or SEQ ID NO: 23 and encodes a protein represented by SEQ ID NO: 5. Gene ID 65290 encodes a transcription factor that regulates amino acid biosynthesis and plays other regulatory roles under several stress conditions.The protein represented by SEQ ID NO: 5 or a protein having at least 80% identity with SEQ ID NO: 5 thus corresponds to a transcription factor that regulates amino acid biosynthesis. It has been found to be upregulated in the fungus Trichoderma reesei Rut-C30 during secretion stress (Arvas et al.).

[0021] According to the invention, the gene ID 120583 corresponds to a gene represented by SEQ ID NO: 2 or SEQ ID NO: 22 or a gene having at least 80% identity with SEQ ID NO: 2 or SEQ ID NO: 22, in particular at least 85%, at least 90%, more particularly at least 95%, preferentially at least 98% or 99%, and the protein encoded by the gene ID 120583 is represented by SEQ ID NO: 6 or a protein having at least 80% identity with SEQ ID NO: 6, in particular at least 85%, at least 90%, more particularly at least 95%, preferentially at least 98% or 99%. Preferably, gene ID 120583 corresponds to a gene represented by SEQ ID NO: 2 or SEQ ID NO: 22 and encodes a protein represented by SEQ ID NO: 6. Gene ID 120583 encodes an F-box protein. The protein represented by SEQ ID NO: 6 or a protein having at least 80% identity with SEQ ID NO: 6 thus corresponds to an F-box protein.

[0022] According to the invention, the gene ID 43599 corresponds to a gene represented by SEQ ID NO: 3 or SEQ ID NO: 24 or a gene having at least 80% identity with SEQ ID NO: 3 or SEQ ID NO: 24, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%, and the protein encoded by the gene ID 43599 is represented by SEQ ID NO: 7 or a protein having at least 80% identity with SEQ ID NO: 7, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%. Preferably, gene ID 43599 corresponds to a gene represented by SEQ ID NO: 3 or SEQ ID NO: 24 and encodes a protein represented by SEQ ID NO: 7. Gene ID 43599 encodes a protein whose putative function is the targeting of proteins to the vacuole or other cellular compartments (vpsl).The protein represented by SEQ ID NO: 7 or a protein having at least 80% identity with SEQ ID NO: 7 thus corresponds to a protein whose putative function is the targeting of proteins to the vacuole or other cellular compartments.

[0023] According to the invention, the gene ID 35768 corresponds to a gene represented by SEQ ID NO: 4 or a gene having at least 80% identity with SEQ ID NO: 4, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%, and the protein encoded by the gene ID 35768 is represented by SEQ ID NO: 8 or a protein having at least 80% identity with SEQ ID NO: 8, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%. Preferably, gene ID 35768 corresponds to a gene represented by SEQ ID NO: 4 and encodes a protein represented by SEQ ID NO: 8. Gene ID 35768 encodes a putative protein.

[0024] According to the invention, the gene ID 80200 corresponds to a gene represented by SEQ ID NO: 9 or a gene having at least 80% identity with SEQ ID NO: 9, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%, and the protein encoded by the gene ID 80200 is represented by SEQ ID NO: 10 or a protein having at least 80% identity with SEQ ID NO: 10, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%. Preferably, gene ID 80200 corresponds to a gene represented by SEQ ID NO: 9 and encodes a protein represented by SEQ ID NO: 10. Gene ID 80200 encodes a transcription factor with similarity to the SND1 co-activator in humans. The protein represented by SEQ ID NO: 10 or a protein having at least 80% identity with SEQ ID NO: 10 thus corresponds to a transcription factor with similarity to the SND1 co-activator in humans.

[0025] According to the invention, gene ID 122457 corresponds to a gene represented by SEQ ID NO: 11 or SEQ ID NO: 21 or a gene having at least 80% identity with SEQ ID NO: 11 or SEQ ID NO: 21, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%, and the protein encoded by gene ID 122457 is represented by SEQ ID NO: 12 or a protein having at least 80% identity with SEQ ID NO: 12, in particular at least 85%, at least 90%, more particularly at least 95%, preferably at least 98% or 99%. Preferably, gene ID 122457 corresponds to a gene represented by SEQ ID NO: 11 or SEQ ID NO: 21 and encodes a protein represented by SEQ ID NO: 12. Gene ID 122457 encodes a multiprotein binding factor (MBF1). The protein represented by SEQ ID NO: 12 or a protein having at least 80% identity with SEQ ID NO: 12 thus corresponds to a multiprotein binding factor (MBF1).

[0026] According to the invention, the term "at least 80%" means all values ​​between 80% and 100%, in particular the values ​​of 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 a percentage of identity between two sequences. For example, according to the invention, the percentage of identity of a given sequence with respect to SEQ ID NO: 1 or SEQ ID NO: 5 is understood to mean the percentage of identity over the total length of the sequences. The percentage thus corresponds to the number of identical nucleotides / residues between this given sequence and SEQ ID NO: 1 or 5 divided by the number of nucleotides or residues in the longer of the two sequences.

[0027] According to the invention, an orthologous gene is understood to mean a gene which has the same function as gene ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 or ID 122457 in the species Trichoderma reesei. For example, an orthologous gene of gene ID 65290 also codes for a transcription factor which regulates the biosynthesis of amino acids. The FungiDB database can for example be used to identify an ortholog. According to one embodiment, in the fungal strain according to the invention, the expression of at least one gene selected from ID 65290, ID 120583, ID 43599 and ID 35768 is modified. This means that the expression of the gene is increased, decreased or extinguished compared to a reference strain (in particular the parent strain of the variant strain according to the invention).

[0028] According to one embodiment, in the fungal strain according to the invention at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed. According to this embodiment, the strain according to the invention thus has an expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 increased compared to a reference strain.

[0029] According to a preferred embodiment, in the fungal strain according to the invention at least one gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is overexpressed. According to this embodiment, the strain according to the invention thus has an expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 increased compared to a reference strain.

[0030] “Overexpression of a gene” means that the strain according to the invention has a higher expression of said gene compared to a parent strain (e.g. which has not been genetically modified to increase the expression of the gene). Preferably, the strain according to the invention has a higher expression of said gene compared to a Rut-C30 strain.

[0031] All techniques for overexpressing a gene can be used to obtain a strain according to the invention.

[0032] According to one embodiment, the overexpression of a gene means the addition of at least one copy of said gene into the genome of the fungal strain and / or the expression of said gene under the control of a strong promoter. The expression of a gene under the control of a strong promoter may mean the expression of the gene under the control of a constitutive promoter and / or the expression of the gene under the control of an inducible promoter.

[0033] According to one embodiment, the fungal strain according to the invention comprises at least the endogenous ID 65290 gene, said gene being under the control of a strong promoter. In this case, the endogenous ID 65290 gene is present in the native genome of the strain, and the promoter has been modified, mutated or replaced, to be constitutive or inducible. According to another embodiment, the fungal strain according to the invention comprises, in addition to the ID 65290 gene present in its genome, an additional copy of the ID 65290 gene, said copy being expressed constitutively or inducibly. It therefore comprises in this case at least two copies of the ID 65290 gene, one of these copies being expressed constitutively or inducibly.

[0034] According to one embodiment, the fungal strain according to the invention comprises at least the endogenous ID 120583 gene, said gene being under the control of a strong promoter. In this case, the endogenous ID 120583 gene is present in the native genome of the strain, and the promoter has been modified, mutated or replaced, to be constitutive or inducible. According to another embodiment, the fungal strain according to the invention comprises, in addition to the ID 120583 gene present in its genome, an additional copy of the ID 120583 gene, said copy being expressed constitutively or inducibly. It therefore comprises in this case at least two copies of the ID 120583 gene, one of these copies being expressed constitutively or inducibly.

[0035] According to one embodiment, the fungal strain according to the invention comprises at least the endogenous gene ID 43599, said gene being dependent on a strong promoter. In this case, the endogenous ID 43599 gene is present in the native genome of the strain, and the promoter has been modified, mutated or replaced, to be constitutive or inducible. According to another embodiment, the fungal strain according to the invention comprises, in addition to the ID 43599 gene present in its genome, an additional copy of the ID 43599 gene, said copy being expressed constitutively or inducibly. It therefore comprises in this case at least two copies of the ID 43599 gene, one of these copies being expressed constitutively or inducibly.

[0036] According to one embodiment, the fungal strain according to the invention comprises at least the endogenous ID 35768 gene, said gene being under the control of a strong promoter. In this case, the endogenous ID 357768 gene is present in the native genome of the strain, and the promoter has been modified, mutated or replaced, to be constitutive or inducible. According to another embodiment, the fungal strain according to the invention comprises, in addition to the ID 35768 gene present in its genome, an additional copy of the ID 35768 gene, said copy being expressed constitutively or inducibly. It therefore comprises in this case at least two copies of the ID 35768 gene, one of these copies being expressed constitutively or inducibly.

[0037] According to one embodiment, the fungal strain according to the invention comprises at least the endogenous ID 80200 gene, said gene being under the control of a strong promoter. In this case, the endogenous ID 80200 gene is present in the native genome of the strain, and the promoter has been modified, mutated or replaced, to be constitutive or inducible. According to another embodiment, the fungal strain according to the invention comprises, in addition to the ID 80200 gene present in its genome, an additional copy of the ID 80200 gene, said copy being expressed constitutively or inducibly. It therefore comprises in this case at least two copies of the ID 80200 gene, one of these copies being expressed constitutively or inducibly.

[0038] According to one embodiment, the overexpression of the gene is carried out by introducing an (expression) cassette comprising said gene into the reference genome. Preferably, this cassette comprises (a) at least one constitutive or inducible promoter, (b) a gene of sequence SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 4, and / or SEQ ID NO: 9, or a gene having at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 4, and / or SEQ ID NO: 9, and (c) optionally a terminator.

[0039] The constitutive or inducible promoter a) is a strong promoter. By "constitutive promoter" is meant a promoter that is not inducible. This constitutive promoter expresses the gene all the time; thus, a constant and strong production of proteins takes place. This promoter can come from Trichoderma reesei, but also from Aspergillus nidulans. By "inducible promoter" is meant a promoter that is inducible in the presence of an inducer. Said inducer is for example an inducing substrate, and is preferably chosen from cellulose, cellooligosaccharides, lactose, sophorose, cellobiose or sorbose (eg for the promoters pcbhl, pcbh2, pegll), or xylose, xylobiose, cellobiose, sophorose, xylooligosaccharides or xylan (eg for the promoters pxynl, pxyn2).

[0040] According to a preferred embodiment, the constitutive promoter a) is chosen from: - the gpd promoter of Trichoderma reesei (Li J. et al (2012), Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters, Microbial cell factories, 11(1), 84. doi:10.1186 / 1475-2859-ll-84). This promoter has the sequence SEQ ID NO: 13. - the gpd promoter (TAspergillus nidulans (Penttila M. et al (1987), A versatile transformation System for the cellulolytic filamentous fungus Trichoderma reesei, Gene, 61(2), 155-64; http: / / www.ncbi.nlm.nih.gov / pubmed / 3127274), - the cDNAl promoter of Trichoderma reesei (Nakari T, Alatalo E. and Pentillâ M: Isolation of Trichoderma reesei genes highly expressed on glucose-containing media: characterization of the tefl gene encoding translation elongation factor la Gene 1993: 313-318), and - the tefl promoter of Trichoderma rec.sAlNakaiLSctala T, Penttila M., Production of Trichoderma reesei celluloses on glucose-containing media, Applied and Environmental Microbiology. 1995;61(10):3650-3655).

[0041] According to a preferred embodiment, the inducible promoter a) is the cbhl promoter of Trichoderma reesei, or the pcbhl, pcbh2, pegll, pxynl or pxyn2 promoter (Adnan et al.).

[0042] When a terminator is used, it can be chosen from: - the cDNAl terminator of Trichoderma reesei, - the TrpC terminator of Trichoderma reesei, - the cbhl terminator of Trichoderma reesei, - or the gpd terminator of Trichoderma reesei, of sequence SEQ ID NO: 14.

[0043] The overexpression of at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is preferably carried out by introducing a DNA fragment comprising at least one gene ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 with a constitutive or inducible promoter, optionally a terminator and optionally a selection marker. A cassette as mentioned above may also be introduced into the cell by a vector, such as a plasmid, comprising the cassette.

[0044] According to one embodiment, in the fungal strain according to the invention, at least the genes ID 65290 and ID 120583 are overexpressed.

[0045] According to one embodiment, in the fungal strain according to the invention, at least the genes ID 43599 and ID 35768 are overexpressed.

[0046] According to one embodiment, in the fungal strain according to the invention at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated. This also means that in the strain according to the invention at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768, ID 122457 is invalidated and / or that a protein encoded by at least one of these genes is not expressed, has reduced or deficient activity. More particularly, the genome of the strain according to the invention has thus been modified, in particular so that at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 is no longer expressed.

[0047] Thus, according to one embodiment, in the strain according to the invention, at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768, and ID 122457 is not functional, and / or the protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 is not produced, is not functional, or has reduced activity / function.

[0048] According to a preferred embodiment, in the strain according to the invention, at least one gene chosen from ID 43599 and ID 35768 is not functional, and / or the protein encoded by a gene chosen from ID 43599 and ID 35768 is not produced, is not functional, or has reduced activity / function.

[0049] According to the invention, the term "functional gene" is understood to mean in particular a gene which makes it possible to produce a functional protein, and the term "functional protein" is understood to mean a protein which has a function or an activity. For example, with gene ID 65290 the protein has a regulatory factor activity, with gene ID 43599 the protein has a vacuole or other cellular compartment targeting protein (vpsl) activity, and with gene ID 122457 the protein has a multiprotein binding factor (mbfl) activity.

[0050] According to one embodiment, in the fungal strain according to the invention, at least the genes ID 65290 and ID 120583 have been invalidated.

[0051] According to one embodiment, in the fungal strain according to the invention, at least the genes ID 43599 and ID 35768 have been invalidated.

[0052] All gene invalidation techniques can be used to obtain a strain according to the invention. By way of non-limiting examples, invalidation can be carried out by mutagenesis (PCR mutagenesis, oligonucleotide-directed mutagenesis, insertion mutagenesis, etc.), homologous recombination, RNA interference, using meganucleases or zinc finger nucleases, TALEN (Transcription activator-like effector nucleases), or CRISPR / cas technology,...

[0053] According to one embodiment, the strain according to the invention thus comprises a deletion of all or part of at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768, and ID 122457. For example, all or part of the region coding for the DNA binding domain, the activation domain of a transcription factor, the coding region and / or a control sequence necessary for the expression of the coding region are deleted.

[0054] According to one embodiment, the invalidation of the gene can be carried out using an invalidation cassette. For example, this has for example: (1) a region upstream of the target region, (2) a selection marker, and (3) a region downstream of the target region.

[0055] In the case of the present invention, the "target region" means the region which makes it possible to invalidate the gene ID 65290, ID 120583, ID 43599, ID 35768, and / or ID 122457, for example the sequence of the gene or the promoter sequence of this gene. The regions upstream and downstream of the target region are two recombination elements, one at each end of the gene, and serve to precisely target the sequence to be invalidated.

[0056] Said invalidation cassette can be introduced into the cell by a vector, such as a plasmid, comprising the cassette.

[0057] The term "vector" means any DNA sequence into which it is possible to insert fragments of foreign nucleic acid, the vectors allowing the introduction of foreign DNA into a host cell. Examples of vectors are plasmids, cosmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacteriophage PI-derived artificial chromosomes (PAC), and virus-derived vectors.

[0058] The vector according to the invention may comprise a selection marker. The term "selection marker" means a gene whose expression confers on the cells containing it a characteristic enabling them to be selected. The use of a selection marker makes it possible in particular to identify cells which have integrated a genetic modification compared to those which have not. This is, for example, an antibiotic resistance gene, such as the gene for resistance to the antibiotic hygromycin hph.

[0059] More specifically, according to the invention, the invalidation cassette is preferentially constituted by a resistance gene placed under the control of a promoter and a terminator, with upstream and downstream the 5' and 3' flanking regions of the target region. According to the invention, said invalidation cassette may be operationally linked to a promoter, terminator, or other sequence necessary for its expression in a host cell.

[0060] According to one embodiment, in the fungal strain according to the invention: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0061] According to a preferred embodiment, in the fungal strain according to the invention: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 43599, ID 35768 and ID 122457 has been invalidated.

[0062] According to a preferred embodiment, in the fungal strain according to the invention: - at least one gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is overexpressed, and / or - at least one gene chosen from ID 43599 and ID 35768 has been invalidated.

[0063] The mushroom according to the invention belongs to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola. As examples, the mushroom species according to the invention are: Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus japonicus, Aspergillus terreus, Aspergillus fumigatus, Aspergillus aculeatus, Aspergillus awamori, Fusarium oxysporum, Penicillium oxalicum, Penicillium funiculosum, Penecillium chrysogenum, Humicola insolens, Trichoderma viride, Trichoderma harzianum, Chrysosporium lucknowense, Talaromyces emersonii.

[0064] According to a preferred embodiment, the strain according to the invention belongs to the genus Trichoderma. According to a preferred embodiment, it is a fungus belonging to the species Trichoderma reesei. More particularly, the parent strain is thus understood to be a strain originating or derived from the natural isolate QM6a (deposited under the number ATCC 13631). Indeed, the strain QM6a is the strain from which all industrial strains are developed, and this is why all industrial strains are said to be derived from QM6a (here it is a direct / first generation descendant of QM6a) or derived from QM6a (here it is a second generation or later generation descendant of QM6a). Strains derived from QM6a are, for example, strain Rut-C30 (deposited under number ATCC 56765), strain NG14 (deposited under number ATCC 56767) or strain QM9414 (deposited under number ATCC 26921). According to a preferred embodiment, it is the strain Rut-C30 or its descendants (i.e., all strains that are descended from or derived from Rut-C30).

[0065] In a second aspect, the invention also relates to a method of genetically modifying a fungal strain according to the invention, as mentioned above, comprising: - a step of overexpression of at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200, or an orthologous gene, and / or - a step of invalidating at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457, or an orthologous gene.

[0066] The invention also relates to a method for genetic modification of a fungal strain according to the invention, as mentioned above, comprising: - a step of overexpression of at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200, and / or - a step of invalidating at least one gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768.

[0067] According to a preferred embodiment, said method of genetic modification of a fungal strain according to the invention, as mentioned above, comprises: - a step of overexpression of at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200, and / or - a step of invalidating at least one gene chosen from ID 43599 and ID 35768.

[0068] According to a preferred embodiment, said method of genetic modification of a fungal strain according to the invention, as mentioned above, comprises: - a step of overexpression of at least one gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, and / or - a step of invalidating at least one gene chosen from ID 43599 and ID 35768.

[0069] According to one embodiment, said method further comprises a step of obtaining a strain in which at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed and / or a step of obtaining a strain in which at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0070] The method of genetic modification of a strain according to the invention thus makes it possible to obtain a hyperproductive fungal strain compared to the parent fungal strain. This is why the strain according to the invention can be considered as a variant of the parent fungal strain.

[0071] As previously indicated, all techniques can be used to overexpress or invalidate the gene(s) of interest.

[0072] In a third aspect, the present invention also relates to a method for producing mushroom biomass, comprising a step of cultivating a strain of mushroom according to the invention, in a culture medium comprising a suitable substrate. This step thus allows the growth of the mushroom strain according to the invention. Any substrate allowing the growth of the mushroom strain can be used.

[0073] According to one embodiment, the present invention thus relates to a method for producing mushroom biomass, comprising a step of culturing, in a culture medium comprising a suitable substrate, a strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified, - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, and / or - at least the protein encoded by a gene ID 122457, or an orthologous gene, is not produced or has reduced expression, preferably is not produced.

[0074] The term "the expression of at least one protein is reduced" means that the variant strain according to the invention has an expression / production lower than that of the parent strain, and in particular a production reduced by at least 5% compared to that of the parent strain. According to the invention, a "production reduced by at least 5%" means all values ​​between 5% and 100%, and in particular 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%.

[0075] According to a preferred embodiment, the present invention relates to a method for producing mushroom biomass, comprising a step of culturing, in a culture medium comprising a suitable substrate, a strain belonging to the genus Trichoderma in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is modified, and / or - the expression of at least one protein encoded by a gene ID 80200 is increased.

[0076] According to a preferred embodiment, said method for producing mushroom biomass comprises a step of culturing, in a culture medium comprising a suitable substrate, a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0077] According to a preferred embodiment, said method for producing mushroom biomass comprises a step of culturing, in a culture medium comprising a suitable substrate, a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably chosen from ID 65290, ID 120583, ID 43599 and ID 35768, and / or - at least one gene selected from ID 43599, ID 35768 and ID 122457 has been invalidated, preferably selected from ID 43599 and ID 35768.

[0078] According to a preferred embodiment, said method for producing mushroom biomass comprises a step of culturing, in a culture medium comprising a suitable substrate, a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 43599 and ID 35768 has been invalidated.

[0079] In a fourth aspect, the present invention also relates to a method for producing proteins of interest, in particular enzymes, comprising a step of culturing a fungal strain according to the invention, in a culture medium comprising a suitable substrate. The invention thus relates to the use of a fungal strain according to the invention, for the production of proteins of interest.

[0080] According to one embodiment, the present invention thus relates to a method for producing proteins of interest, comprising a step of culturing, in a culture medium comprising an appropriate substrate, a strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified, - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, and / or - the protein encoded by a gene ID 122457, or an orthologous gene, is not produced or has reduced expression, preferably is not produced.

[0081] According to one embodiment, the present invention thus relates to a method for producing proteins of interest, comprising a step of culturing, in a culture medium comprising an appropriate substrate, a strain belonging to the genus Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is modified, and / or - the expression of at least one protein encoded by a gene ID 80200 is increased.

[0082] According to one embodiment, the present invention thus relates to a method for producing proteins of interest, comprising a step of culturing, in a culture medium comprising an appropriate substrate, a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0083] According to a preferred embodiment, said method for producing proteins of interest comprises a step of culturing, in a culture medium comprising an appropriate substrate, a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably chosen from ID 65290, ID 120583, ID 43599 and ID 35768, and / or - at least one gene selected from ID 43599, ID 35768 and ID 122457 has been invalidated, preferably selected from ID 43599 and ID 35768.

[0084] According to one embodiment, the present invention relates to a method for producing proteins of interest, comprising a step of culturing, in a culture medium comprising a suitable substrate, a strain belonging to the genus Trichoderma, in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 43599 and ID 35768 has been invalidated.

[0085] According to one embodiment, said method for producing proteins of interest comprises: - (i) a step of culturing a strain of fungus according to the invention, in a culture medium comprising a suitable substrate, - (ii) a step of obtaining a protein of interest, and optionally a step of purifying said protein of interest from the culture medium.

[0086] According to one embodiment, said method for producing proteins of interest comprises: - (i) a step of culturing a fungal strain according to the invention, in a culture medium comprising a suitable substrate, for the production and secretion of the protein of interest, - (ii) a step of obtaining a protein of interest, and optionally a step of purifying said protein of interest from the culture medium.

[0087] Advantageously, said method thus comprises a phase of growth of a strain of fungus according to the invention, then a phase of production of proteins. of interest by said strain. Even more preferably, said growth phase is carried out in the presence of a growth substrate and said phase of production of proteins of interest is carried out in the presence of an inducing substrate. The growth substrate and the inducing substrate are preferably carbon substrates.

[0088] Thus, the carbon growth substrate is preferably chosen from lactose, glucose, xylose, residues obtained after ethanolic fermentation, monomeric sugars from enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses originating from the pretreatment of a cellulosic biomass.

[0089] The inducing carbon substrate is thus preferably chosen from lactose, cellobiose, cellulose (including cellulose oligosaccharides or partially hydrolyzed cellulose), glucose, xylose (including xylose oligosaccharides), sophorose, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, a crude extract of water-soluble pentoses originating from the pretreatment of a cellulosic biomass, or a mixture of these substrates. Even more preferably, the inducing carbon substrate is chosen from lactose, glucose, xylose, a lactose and cellulose mixture, a glucose and cellulose mixture, a glucose and lactose mixture, or a lactose and xylose mixture.

[0090] According to the invention, the proteins of interest are all the proteins that can be produced by a fungus, naturally or by genetic modification (for example after transformation using an appropriate vector). The protein of interest can be endogenous or exogenous. The protein of interest can be a native protein (as found in nature), a chimeric protein, a synthesized protein or a variant of a native protein, in particular having improved and / or modified biological properties. According to one embodiment, the sequence of the gene of interest can comprise appropriate additional elements such as, for example, a sequence coding for a secretion signal. The proteins of interest can be proteins useful in any type of industry (the production of biofuel, food, cosmetics or pharmaceuticals, etc.)

[0091] Advantageously, the proteins of interest according to the invention are enzymes, in particular cellulolytic enzymes such as cellulases, hemicellulases or xylanases. Preferably, the enzymes are cellulases. According to the invention, the term "cellulases" means more particularly enzymes chosen from endoglucanases, exoglucanases and glucosidases, and more particularly [3-glucosidase. The term "cellulase" refers more particularly to an enzyme adapted to the hydrolysis of cellulose and allowing the microorganisms (such as Trichoderma reeseï) which produce them to use cellulose as a carbon source, by hydrolyzing this polymer into simple sugars (glucose). The term "xylanase" refers more specifically to an enzyme adapted to the hydrolysis of cellulose and allowing the microorganisms (such as Trichoderma reeseï) that produce them to use xylan as a carbon source, by hydrolyzing this polymer into sugars such as xylose.

[0092] According to a preferred embodiment, the culture medium comprising a substrate suitable for the production of proteins of interest is chosen from glucose or a mixture of cellulose and lactose.

[0093] According to one embodiment, said method for producing proteins of interest comprises a step of culturing a strain belonging to the genus Trichoderma in which the expression of at least one protein encoded by a gene ID 80200 is increased, in a culture medium comprising glucose as a suitable substrate.

[0094] According to one embodiment, said method for producing proteins of interest comprises a step of culturing a strain belonging to the genus Trichoderma in which at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, in a culture medium comprising glucose as a suitable substrate.

[0095] According to one embodiment, said method for producing proteins of interest comprises a step of culturing a strain belonging to the genus Trichoderma in which at least the protein encoded by a gene ID 122457 is not produced or has reduced expression, preferably is not produced, in a culture medium comprising as a suitable substrate a mixture of cellulose and lactose.

[0096] According to one embodiment, said method for producing proteins of interest comprises a step of culturing a strain belonging to the genus Trichoderma in which at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been disabled, preferably a gene chosen from ID 43599 and ID 35768, in a culture medium comprising as a suitable substrate a mixture of cellulose and lactose.

[0097] In a fifth aspect, the present invention also relates to a method for producing bio-sourced products from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to the invention. The invention therefore also relates to the use of a fungal strain according to the invention, for the production of bio-sourced products from cellulosic or lignocellulosic substrates.

[0098] According to one embodiment, the present invention thus relates to a method for producing bio-sourced products from cellulosic substrates or lignocellulosic, comprising a step of culturing, in a culture medium comprising a suitable substrate, a strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified, - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, and / or - the protein encoded by a gene ID 122457, or an orthologous gene, is not produced or has reduced expression, preferably is not produced.

[0099] According to one embodiment, the present invention thus relates to a method for producing bio-sourced products from cellulosic or lignocellulosic substrates, comprising a step of culturing, in a culture medium comprising an appropriate substrate, a strain belonging to the genus Trichoderma in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is modified, and / or - the expression of at least one protein encoded by a gene ID 80200 is increased.

[0100] According to one embodiment, said method for producing bio-sourced products from cellulosic or lignocellulosic substrates comprises a step of culturing, in a culture medium comprising an appropriate substrate, a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0101] According to a preferred embodiment, said method for producing bio-sourced products from cellulosic or lignocellulosic substrates comprises a step of culturing, in a culture medium comprising an appropriate substrate, a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably chosen from ID 65290, ID 120583, ID 43599 and ID 35768, and / or - at least one gene selected from ID 43599, ID 35768 and ID 122457 has been invalidated, preferably selected from ID 43599 and ID 35768.

[0102] According to one embodiment, said method for producing bio-sourced products from cellulosic or lignocellulosic substrates comprises a cultivation step, in a culture medium comprising a suitable substrate, of a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 43599 and ID 35768 has been invalidated.

[0103] In a sixth aspect, the invention relates to a method for producing a sweet juice or sugar from cellulosic or lignocellulosic substrates, comprising: i) a step of pretreating a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, ii) a step of production of cellulolytic enzymes by a strain according to the invention, iii) a step of enzymatic hydrolysis of the pretreated substrate obtained in step i), in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolysate.

[0104] According to one embodiment, in said method for producing a sweet juice or sugar from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified, - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, and / or - the protein encoded by a gene ID 122457, or an orthologous gene, is not produced or has reduced expression, preferably is not produced.

[0105] According to one embodiment, in said method for producing a sweet juice or sugar from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is modified, and / or - the expression of at least one protein encoded by a gene ID 80200 is increased.

[0106] According to one embodiment, in said method for producing a sweet juice or sugars from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0107] According to one embodiment, in said method for producing a sweet juice or sugars from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably chosen from ID 65290, ID 120583, ID 43599 and ID 35768, and / or - at least one gene selected from ID 43599, ID 35768 and ID 122457 has been invalidated, preferably selected from ID 43599 and ID 35768.

[0108] According to one embodiment, in said method for producing a sweet juice or sugars from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 43599 and ID 35768 has been invalidated.

[0109] The sugars (or sweet juice, the sugars generally being in the form of an aqueous solution) are thus obtained at the end of the hydrolysis step. Preferably, these are sugars with 5 carbons (called C5 sugars) and / or sugars with 6 carbons (called C6 sugars).

[0110] The method according to the invention thus makes it possible to treat (ligno)cellulosic biomass to produce so-called second generation (2G) sweet juices. These sweet juices can be used to produce other products by biochemical means, in particular by fermentation (for example alcohols such as ethanol, butanol), or other molecules, for example solvents such as acetone, etc.), or be recovered as such, after possible separation / treatment steps, for example in the chemical / food industry.

[0111] According to one embodiment, said method for producing sugars (sweet juice) may also comprise one or more separation steps. This thus makes it possible to separate, in particular by distillation, the different products of interest diluted in water.

[0112] In a seventh aspect, the present invention relates to a method for producing biofuel, from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to the invention. The invention thus relates to the use of a fungal strain according to the invention, for the production of biofuel, such as an alcohol, from cellulosic or lignocellulosic substrates.

[0113] According to one embodiment, said method for producing a biofuel from cellulosic or lignocellulosic substrates according to the invention comprises: - i) a step of pretreatment of a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - ii) a step of production of cellulolytic enzymes by a strain according to the invention, - iii) a step of enzymatic hydrolysis of the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step ii) and of an appropriate substrate, in order to obtain a hydrolysate, - iv) a step of alcoholic fermentation of the hydrolyzate obtained, step iv) being optionally carried out simultaneously with step iii). - v) a separation step, in particular by distillation.

[0114] According to one embodiment, in said process for producing a biofuel from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified, - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, and / or - the protein encoded by a gene ID 122457, or an orthologous gene, is not produced or has reduced expression, preferably is not produced.

[0115] According to one embodiment, in said method for producing a biofuel from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is modified, and / or - the expression of at least one protein encoded by a gene ID 80200 is increased.

[0116] According to one embodiment, in said method for producing a biofuel from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0117] According to a preferred embodiment, in said process for producing a biofuel from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably chosen from ID 65290, ID 120583, ID 43599 and ID 35768, and / or - at least one gene selected from ID 43599, ID 35768 and ID 122457 has been invalidated, preferably selected from ID 43599 and ID 35768.

[0118] According to one embodiment, in said method for producing a biofuel from cellulosic or lignocellulosic substrates, the step of producing cellulolytic enzymes is carried out by a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 43599 and ID 35768 has been invalidated.

[0119] According to the invention, the term "biofuel" is understood more particularly to mean a second-generation biofuel, i.e. derived from non-food resources. According to the invention, the term "biofuel" can also be defined as any product resulting from the transformation of biomass and which can be used for energy purposes. On the one hand, and without wishing to be limited, examples include biogas, products which can be incorporated (possibly after further transformation) into a fuel or be a fuel in their own right, such as alcohols (ethanol, butanol and / or isopropanol depending on the type of fermentation organism used), solvents (acetone), acids (butyric), lipids and their derivatives (short- or long-chain fatty acids, fatty acid esters), as well as hydrogen. Preferably, the biofuel according to the invention is an alcohol, for example ethanol, butanol and / or isopropanol.More preferably, the biofuel according to the invention is ethanol. In another embodiment, the biofuel is biogas. In another embodiment, the product is a molecule of interest to the chemical industry, such as, for example, another alcohol such as 1,2-propane diol, 1,3-propane diol, 1,4-butane diol, 2,3-butane diol, organic acids such as acetic, propionic, acrylic, butyric, succinic, malic, fumaric, citric, itaconic acid, or hydroxy acids such as glycolic, hydroxypropionic, or lactic acid. Preferably, the alcohol is, for example, ethanol, butanol, isopropanol, 1,2-propane diol, 1,3-propane diol, 1,4-butane diol, and / or 2,3-butane diol. More preferably, it is ethanol. The alcohol thus obtained can also be used as a solvent or as an intermediate product in the chemical industry.

[0120] According to one embodiment, in said process for producing a biofuel from cellulosic or lignocellulosic substrates, step iv) is carried out simultaneously with step iii). This is typically the case in so-called “SSF” (Simultaneous Saccharification and Fermentation) production processes.

[0121] According to a particular embodiment, the step of pretreating a cellulosic or lignocellulosic substrate is a step of suspending said cellulosic or lignocellulosic substrate in aqueous phase. Preferably, the step of pretreating a cellulosic or lignocellulosic substrate is a steam explosion under acidic conditions or under non-acidic conditions.

[0122] According to a particular embodiment, the hydrolyzate obtained in step iii) is a hydrolyzate containing C5-C6 sugars, in particular glucose.

[0123] According to a particular embodiment, the step of alcoholic fermentation of the hydrolyzate obtained is a step of fermentation, in the presence of a fermentative organism, of the glucose from the hydrolyzate so as to produce a fermentation must. A fermentative organism is for example a yeast.

[0124] According to a particular embodiment, the separation step is a separation of the biofuel and the fermentation must, in particular by distillation.

[0125] In an eighth aspect, the invention also relates to the use of a fungal strain according to the invention, (i) for the production of proteins of interest, more particularly enzymes, in particular cellulolytic enzymes such as cellulases, (ii) for the hydrolysis of cellulose or lignocellulose into glucose, (iii) for the production of biosourced products from cellulosic or lignocellulosic substrates, (iv) for the production of biofuel from cellulosic or lignocellulosic substrates, (v) for the production of alcohol from cellulosic or lignocellulosic substrates, and / or (vi) for the production of a sweet juice or sugars from cellulosic or lignocellulosic substrates.

[0126] According to one embodiment, the invention thus relates to the use of a strain belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, is modified, - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, and / or - the protein encoded by a gene ID 122457, or an orthologous gene, is not produced or has reduced expression, preferably is not produced.

[0127] According to one embodiment, the invention thus relates to the use of a strain belonging to the genus Trichoderma in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is modified, and / or - the expression of at least one protein encoded by a gene ID 80200 is increased.

[0128] According to one embodiment, the invention relates to the use of a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 122457 has been invalidated.

[0129] According to a preferred embodiment, the invention relates to the use of a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably chosen from ID 65290, ID 120583, ID 43599 and ID 35768, and / or - at least one gene selected from ID 43599, ID 35768 and ID 122457 has been invalidated, preferably selected from ID 43599 and ID 35768.

[0130] According to one embodiment, the invention relates to the use of a strain belonging to the genus Trichoderma in which: - at least one gene chosen from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, and / or - at least one gene selected from ID 43599 and ID 35768 has been invalidated.

[0131] In the present description, the definitions and preferences indicated in one aspect apply mutatis mutandis to the other aspects. For example, all the definitions and preferences indicated in the first aspect of the invention above also apply to the second, third, fourth, fifth, sixth, seventh and eighth aspects. Brief description of the Figures

[0132] Other characteristics, details and advantages of the invention will appear on reading the attached Figures and the examples which illustrate the invention and are in no way intended to limit it. Fig.l

[0133] [Fig.l] represents the growth of a representative clone of each mutant and the parent strain Rut-C30 on minimal medium containing either 2% glucose, 2% glucose and 5 mM DTT (dithiotreitol), 2% lactose, or 2% lactose and 5 mM DTT. The photos show the mycelia after 5 days of culture. Fig. 2

[0134] [Fig.2] represents the concentration of proteins secreted by the parent strain Rut-C30 and the mutated strains at day 5 and day 7 of a flask culture on a BTCA medium containing as carbon source either 2% glucose or 2% of a cellulose and lactose mixture. A Student's test (T-test) was used to calculate the significant differences between the values ​​of the Rut-C30 strain and those of the mutants (*p < 0.05, **p < 0.01). Fig. 3

[0135] [Fig.3] represents the yield of secreted proteins relative to the biomass of the parent strain Rut-C30 and the mutated strains at day 7 of a flask culture on BTCA medium containing 2% glucose as carbon source. A Student's test (T-test) was used to calculate the significant differences between the values ​​of the Rut-C30 strain and those of the mutants (*p < 0.05, **p < 0.01, ***p < 0.001). Sequences of the present invention

[0136] [Tables 1] Name of the sé quence Sequence SEQI atgatgaacg ctgcagatgt cgatttggac gacttcaccg tctttgaagg aggtgcctcc acggccttct cttc DNO tccggc tgtcgcttcc agcttcgact tcagcagcgg cgcctcgagc gcagccttca agtccctc a gtccctc 1 : caggaccttc tggtccagga gcccttcatg tcggcgccca actctgcggc cctgactgcc ttgacgtctc cttcgctgta caatggcagt cccgacttcg attcctttga cgtctctccc aactttggca ctgctgaatt tgacaacggt cctagcccc cctggcct ttggcct tgggc cggtccctca cgagcttgtc aacccaagcg agtctcccgt ccagaagcc t ttggaatcag agcccatgta ccgatcgtct tcttcaggca gtggcagtgg cagcggcaag aagaagcctg cgagtggttc gcccacgagc ggcactcgcc actctccgtc gccgcgcgc cccctgccc cccatcattg ttgaagacgc caacgacaca gtggccatga agcgagcccg caataccttg g tgctcgaa agtcgcgtga gcgaaaggct cagcgattcg aagagctgga ggaccgtatt gcgaagctcg ag gccgagcg tgatcag aggctcgtgttca ttgacggagt aa SEQI atgctgagcctcatcaccaacaccctccccggcggcgccatcgcgcacctcaccgagttcgcc DNOtccgagcgctacttcgagaagctcaaccagctgcacgcgagccgccagcaggcggaatctgccgactcgggcg : 2 cctcgacaacgacgacaacgacgacagctgccgccaccgccaacgcacaggcgacgtcgaggttcatcctgcc gctgcgagaagcaaaggttccggacgtggacgccattaggcccagcgactcgaagcagaacagacgattcttcg gcggcatccgacaaaaggtcgccctcaagcacaccaggagcgatgcggacgcgaccgctgtggctctacccat cagagcctcgacggattcgacgcgaaaaagcgtcgccttcgatcaactattcttcgggctgccgaccgagcttcaa ttccaaatcatctcctctctcccattgacagatattcttaacctccgattagcctcgaaatcatggcacgcgcgcatcac cctcaacgagacgttcatcgtccgacatctactacgggaccacatacccgcctatgccactcgcctgtaccccgtgc cggacaactccgagatcaacttccactacttgtgtagcttgtggcatcggctacatgtcgcggcgaagctttcatacc taatgtgcgaatggatcaccaaagacatcttcctacgacagacggaagcccagcggctggcattcgcaccacagc acgagcgcatgcgccgacgattgatacccctcctattcaccgttttccacttcttcgagatgtataggaagctgcacct ggagtacattgcagaaaacggacacggcctgacgaaggagccgtacacggtgaacccgatcgagaagcgcatc atggacatgtacgacgaccggacgttgttgagggtccacgaagtcttccccctcgtcatttcgtccttttgccgccgc ctgcgaccgccgacgtacgttggtcgcgtggagcgatcgcttcgtggttacctgagggagcggccgccggacga ggtccatgtcgccatcttgtgccttggcggccttcgtcaggtcgagagactatgggagaccaagggctacaacaac cgccgggctgctgttgacacctggtacgcctctttgaccaggccgcccgccaatgaggcttcctccaaaggccgg ctgttgaagagctttggacgaaagaagtcaaccagcagcgatcggcccccgcaccggtcatcgttcagcgacagc ggccgcaggagtccgtggggatcggtggactataccgcctccgggatggagatgccgtacagcgtcttcaacac cagcctctcggccgatgcgcccatggcgcctctggagcgcgaccaggccaaggatctgctaggggacctgccat cgctgcagagcatctggctaaccacagccgaggccatgattctggatcgcaagattgtgaatcggccgcaggaca ttaagcggaaccagcaggtcatgctggagctgatccgcgaggacggcatggaggaggaggacgaatggtgcta cggccgccagatctctgactcggtcagaccaccgatttcagccctccatgacgacgcggattaa SEQI DNO : 3atgagcggttctggctcttcgcttacccagacgggtggcatcaccgatcccgccctgatccagctcgtcaacaagctt caggatgttt SEQI DNO : 4 atgtccgaagacgagattcagtcgtctcgatttgccgaggcatgcaagttgcgagtcgttgagcaggaactcaacac ggcgacac SEQI DNO : 5 MMNAADVDLDDFTVFEGGASTAFSSPAVASSFDFSSGASSAASNLGTVS PQDLLVQEPFMSAPNSAALTALTSPSLYNGSPDFDSFDVSPNFGTAEFDN GPSDPWYPLFPSADTVPHELVNPSESPVQKPLESEPMYRSSSSGSGSGSG KKKPASGSPTSGTRHSSVSGVNSRRRDKPLPPIIVEDANDTVAMKRARNT LAARKSRERKAQRFEELEDRIAKLEAERDHWKRIALSQSGVLTE SEQI DNO : 6 MAEPHHQQHPPRRVAMDPPHLTEFASERYFEKLNQLHASRQQAESADS GASTTTTTTTAAATANAQATSRFILPLREAKVPDVDAIRPSDSKQNRRFF GGIRQKVALKHTRSDADATAVALPIRASTDSTRKSVAFDQLFFGLPTELQ FQIISSLPLTDILNLRLASKSWHARITLNETFIVRHLLRDHIPAYATRLYPV PDNSEINFHYLCSLWHRLHVAAKLSYLMCEWITKDIFLRQTEAQRLAFA PQHERMRRRLIPLLFTVFHFFEMYRKLHLEYIAENGHGLTKEPYTVNPIE KRIMDMYDDRTLLRVHEVFPLVISSFCRRLRPPTYVGRVERSLRGYLRER PPDEVHVAILCLGGLRQVERLWETKGYNNRRAAVDTWYASLTRPPANE ASSKGRLLKSFGRKKSTSSDRPPHRSSFSDSGRRSPWGSVDYTASGMEM PYSVFNTSLSADAPMAPLERDQAKDLLGDLPSLQSIWLTTAEAMILDRKIVNRPQDIKRNQQVMLELIREDGMEEEDEWCYGRQISDSVRPPISALHDD AD SEQI DNO MSGSGSSLTQTGGITDPALIQLVNKLQDVFATVGVNNPIDLPQIAVVGSQ SSGKSSVLENIVGRDFLPRGTGIVTRRPLVLQLINRPAQTNGVSHEDIEAG : 7 ADKAANPDEWGEFLHMPGQKFYDFSKIREEISRETEAKVGRNAGISAAP INLRIYSPNVLTLTLVDLPGLTKVPVGDQPRDIERQIRDMVLKYISKSNAI ILAVTAANIDLANSDGLKLAREVDPEGQRTIGVLTKVDLMEEGTDVIDIL SNRIIPLRLGYVPVVNRGQRDIDNKKAIGAALEAEKNFFENHAAYRNKSS YCGTPYLARKLNLILMMHIKQTLPDIKARISSSLQKYSAELESLGPSMLG NSSNIVLNIITEFTNEWRTVLDGNNTELSSTELSGGARISFVFHELYANGV KALDPFDVVKDLDIRTYLYNSSGPSPALFVGTTAFEKQQIKRMEDPS LKCVSLVYDELVRILSQLLSKQLYRRYPALKEKMHSTVVSFFKKAMEPT NKLVRDLVSMEACYINTAHPDFLNGHRAMAIVNERHNPSRPVQVDPKT GKALPSSTPARAASPTVPDADGSSNGGFFGSFFAAKNKKKAAMEPPPP SLKASGTLSEREVIEVEVIKLLISSYYINIVKRTMTDMVPKAIMFNLVQLT KDGMQRELENMYKTDSIDDLLRESDFTIRRRKECQQMVESLGKASEIV SQVQ SEQI DNO MSEDEIQSSRFAEACKLRVVEQELNTGDTLVFVDFPDLSRRRSRQTDCY GVPYSSQKLRVHSQKLLATGSAKFAEMLSPTYQFRIKRRKKLTKDLPEG : 8 VKYVLDLTPPSEGDDLVSQMTQLSLTPGIIKWWAASGLHKVTNWLVSG HDDVCSCGRQHIPGWGAPRQDGTGQEQDGERSVNGGATLFDNKKPGG NTDDTVVTAPTPQDLLSKKEHGINQPYEIPDYRNIPDYCPIRHCNSIIRLLMMIEGQDIMLDSANRVWTLVAVAKIFDCTSIIRDRVAQWIMFSDNTKFIE VLPEEALQIGFDLELVQVTQCAFRILVNEPALKEAATKAPPGGSSPGESK PDLDWVTVFGRRLGNLDDELHNIVQHAARALIERVSVIPGIFRDRETLDN WMVDEWAKLRKLEALLQQQSDEASTKASKSLSSLLMAMRRSISTAFFR LSVAATTEGKQALVSMDEDRATYVLPEDFKLLEDILPKLNIHQKFLCPFV YQQMSEMWHSDPSQSKWIAMAYQESPVSIKSNMDLTVDDVQRAVTAN PEMASQPSWAMFMDPGEGPLRVKNPIVDLEALNRQIYDAVSPYINLWLR YDIEPALNMTRHLLLTLNNNEMKFLPLWAGGCNDGTGGVFQEELPPAY WGPSGPGPAYHTGITIPSASSTSGSLVDGFSTFNVRGSTVRGSTVVGSVD VQDGASTVYAPNHVIADDVSIASESFDVDGSESYQEARFAVPAEHQDMG HAVDMLVESVDSDDDTASDTTDRVMISGSAMETGADDLDEDEDMSFDE SEQI atgtccaagaccttcttcggcaatgtcaagagtgtcttgagcggcgacactctggtcctcaccagcgccaacaatcccgcggccga; DNO : 9 SEQI DNO MSKTFFGNVKSVLSGDTLVLTSANNPAAERTFSLAYVSAPHLKREGDEP FAFQSREYLRNLVVGKPVQCTVLYTIPTTGREFGTAQLKDGTLLPDELV : 10 KAGWVKVREDAGRKEESEELLDRLEKLRALESEAKGASKGLWSGTDGT IEVQNDLGGPEFLTQWKGKTVDGIVERVLSGDRLLVRLLLSDKKHVQPL TLLAGIRTPSTERTLPSTGATQPAEEYGNEAKAFVESRLLQRQVKVEIVG ASAQGQLIASVIHPRGNIAEFLLQEGLARCNDFHSTMLGEKMAPLRAE KQAQAKKLHRHHVAKDAGTNEMVVTKIIGADTIMVKGKNDNTEK RISFSSIRGPRTNEPSESPFRDEAKEFVRSRLIGKHVKVSVDGTKPASEGFE ARDVATVTEKGKNIGLALVEAGLASVIRHRKDDTDRAPNYDELLAAQE KAKEEKKGMWSGKPQKAKQYLDLSENTQKAKIMLATLQRQKKVPAIV DFCKAGSRFTILIPRENVKLTLVLGGIRAPRADGQGGEPFGKEALDL ANRRCNQRDCEVDIHDMDKVGGFIGSLYIGRENFAKVLVEEGLASVHA YSAEXNAAELFAAEKRAKEARKGMWHDYDPSQEENAEEESGEADA PEAEVTLDKKPADYRDVIITSIDGNGKLKIQEIGKGTAALESLMSDFRKF HIDSKNNKPLEAPKTGEFVSAKFSADDQWYRARVRANDRTAKMSEVI YVDYGNTEKVPWSSLRSLDQSQFGVQRLKAIDASLSFVQLPTGAHY FSEAIAFIADLTEGRRLVGNFDYVDSKENVSYITLYDTKADGSLPGPNDS INKEIVASGYGMVPKKLKSWERSKAFESYLKHLREVESQAKQDRLYGDIGMTEW SEQIGDINOD atgctaacctcgactgggatcgccaccaagattggaagccgtgttcgggtcgcgcttcggaccgcgaga ccgtcatccgaggaaagagtgcgctgaacgctgcccagcgagccggtgccattgccacagagaagaagtac : 11 tctactgccaacctcggccggcggtactgagggccacgactcacaaaggtcgatcgctccgacgacatcatcaagcccaagacggtcggaaaggaagtcggaaaggccattgagcagggccgccagaagtttgagcccaccatgaccc aggccgagctcggcaagaagattggcgagaccgctgccacagtcgcctcgtacgagcgcggtactgccacgcc tgaccagaacatcctctccaagatggagcgggtgctcaacgtcaagctgcggggtgccaacattggtgccccccg cttgggccccaagaagaaataa SEQI DNO MSNLDWDSATKIGSRVRGPGASDRETVIRGKSALNAAQRAGAAIATEK KYSTANSAGGTEGQRLTKVDRSDDIIKPKTVGKEVGKAIEQGRQKFEPT : 12 MTQAELGKKIGETAATVASYERGTATPDQNILSKMERVLNVKLRGANIG APRLGPKKK SEQI DNO gacgcagaagaaggaaatcgccccgccggttccgtaaaaaaaatatgagcgcagggacaagcacagccttggc cctgggcctagccttgcgccttgtttgatgcaatcggcgacatgtcgaatgctgtaattttttttgtttaggttccccttttc : 13 cttttgtgttaataataattctcgaagggcgctgattttgaaatttgtcggtgagagccaaacggatatacaggcgcgg ctgatgaataatgatgaatcgagctgacttgatgctgtatgtacaatattgactgcgaggaccatcaggtgttgtatgg atggaatcattctgtaaccaccaaggtgcatgcatcataaggtattctcctcagctcaccaacaacgaacgatggcc atgttagtaaaggcaccgtgatggcaagatagaaccactattgcatctgcgcttcccacgcacagtacgtcaatgta acgtcaaagccgccctcccgtaacctcgcccgttgttgctccccccgattgcctcaatcacatagtacctacctatgcattatggcgcctcaacccacccccccagattgagagctaccttacatcaatatggccagcacctcttcggcgataca tactcgccaccccagccggggcgattgtgtgtactaggtaggctcgtactataccagcaggagaggtgctgcttgg caatcgtgctcagctgttaggttgtacttgtatggtacttgtaaggtggtcatgcagttgctaaggtacctagggaggg attcaacgagccctgcttccaatgtccatctggataggatggcggctggcggggccgaagctgggaactcgccaa cagtcatatgtaatagctcaagttgatgataccgttttgccaggattaggatgcgagaagcagcatgaatgtcgctca tccgatgccgcatcaccgttgtgtcagaaacgaccaagctaagcaactaaggtaccttaccgtccactatctcaggt aaccaggtactaccagctaccctacctgccgtgcctacctgctttagtattaatctttccacctccctcctcaatcttcttt tccctcctctcctcttttttttttcttcctcctcttcttctccataaccattcctaacaacatcgacattctctcctaatcaccag cctcgcaaatcctcaggttagtattactactactacaatcatcaccacgatgctccgcccgacgatgcggcttctgttc gcctgcccctcctctcactcgtgcccttgacgagctaccccgccagactctcctgcgtcaccaatttttttccctattta cccctcctccctctctccctctcgtttcttcctaacaaacaaccaccaccaaaatctctttggaagctcacgactcacg caagctcaattcgcagatacaaa SEQI DNO : 14 tctagaaagaaggattacctctaaacaagtgtacctgtgcattctgggtaaacgactcataggagagttgtaaaaaag tttcggccggcgtattgggtgttacggagcattcactaggcaaccatgcatccttactattgtataccatcttagtagga atgatttcgaggtttatacctacgatgaatgtgtgtcctgtaggcttgagagttcaaggaagaaacatgcaattatcttt gcgaacccagggctggtgacggaattttcatagtcaagctatcagagtaaagaagaggagcatgtcaaagtacaattagagacaaatatatagtcgcgtggagccaagagcggattcctcagtctcgtaggtctcttgacgaccgttgatctg cttgatctcgtctcccgaaaatgaaaatagctctgctaagctattcttctcttcgccggagcctgaaggcgttactagg ttgcagtcaatgcattaatgcattgcagatgagctgtatctggaagaggtaaacccgaaaacgcgttttattcttgttga catggagctattaaatcactagaaggcactctttgctgcttggacaaatgaacgtatcttatcgagatcctgaacacca tttgtctcaactccgg SEQI DNO : 15 ccttcgggccacaacattgacatttttgctattcgttcaccactgagtagcaagacgcggcaatatggacagtcgattc lacgagccg SEQI DNO : 16 aaaacaactccctgaagccgcctagtgccttttccaagtcaggctctcttccatgcgagtcctcctaaatccatcacag :cataaactc SEQI DNO : 17 ccctgtctattcctcaaccgactgccggcacgacggcatcgtgatgccccaggttgaggcgttgggaggagtttgtg >tgcaccgt£ SEQI DNO : 18 aacttcattttggctcggtaaattcccagatatcttgccatttctttctccgtctcccgctcaggtatgtatcaaccccgcgj ftgctccaga SEQI DNO : 19 gccttcacacgccttacaagaaatcctgcgccgccgcatccgatagaagtcgctcacattcacctgcttggacttgga tttacacgaa SEQI DNO : 20 ccaatcaaatacttgtaatgccttaggtaagccgcgggactctagcacattccatgagcggcgatgcgacgccccac ggaggcgc< SEQI DNO atgtctaacctcgactgggattccgccaccaagattggaagccgtgttcgcggtcctggcgcttcggaccgcgaga ccgtcatccgaggaaagagtgcgctgaacgctgcccagcgagccggtgctgccattgccacagagaagaagtac : 21 tctactgccaactcggtatgttcgagcgcctgtcaaggtttttgacgcccaagtatcaacgagccagagctaaccctg aaatttgtttaggccggcggtactgagggccagcgactcacaaaggtcgatcgctccgacgacatcatcaagccc aagacggtcggaaaggaagtcggaaaggccattgagcagggccgccagaagtttgagcccaccatgacccagg ccgagctcggcaagaagattggcgagaccgctgccacagtcgcctcgtacgagcgcggtactgccacgcctgac cagaacatcctctccaagatggagcgggtgctcaacgtcaagctgcggggtgccaacattggtgccccccgcttg ggccccaagaagaaataa SEQI DNO atggctgagcctcatcaccaacaacaccctccccggcgcgtcgccatggatccgccgcacctcaccgagttcgcc tccgagcgctacttcgagaagctcaaccagctgcacgcgagccgccagcaggcggaatctgccgactcgggcg : 22 cctcgacaacgacgacaacgacgacagctgccgccaccgccaacgcacaggcgacgtcgaggttcatcctgcc gctgcgagaagcaaaggttccggacgtggacgccattaggcccagcgactcgaagcagaacagacgattcttcg gcggcatccgacaaaaggtcgccctcaagcacaccaggagcgatgcggacgcgaccgctgtggctctacccatcagagcctcgacggattcgacgcgaaaaaggtttgtcatgatgcatcaaccccaagaagagcaaggaagaagaa ggcttggaatcaatccggatcccctctgcgaaccgccatttccagtagatacacagctgctaactgcgtcttccctct agcgtcgccttcgatcaactattcttcgggctgccgaccgagcttcaattccaaatcatctcctctctcccattgacag atattcttaacctccgattagcctcgaaatcatggcacgcgcgcatcaccctcaacgagacgttcatcgtccgacatc tactacgggaccacatacccgcctatgccactcgcctgtaccccgtgccggacaactccgagatcaacttccacta cttgtgtagcttgtggcatcggctacatgtcgcggcgaagctttcatacctaatgtgcgaatggatcaccaaagacat cttcctacgacagacggaagcccagcggctggcattcgcaccacagcacgagcgcatgcgccgacgattgatac ccctcctattcaccgttttccacttcttcgagatgtataggaagctgcacctggagtacattgcagaaaacggacacg gcctgacgaaggagccgtacacggtgaacccgatcgagaagcgcatcatggacatgtacgacgaccggacgtt gttgagggtccacgaagtcttccccctcgtcatttcgtccttttgccgccgcctgcgaccgccgacgtacgttggtcg cgtggagcgatcgcttcgtggttacctgagggagcggccgccggacgaggtccatgtcgccatcttgtgccttgg cggccttcgtcaggtcgagagactatgggagaccaagggctacaacaaccgccgggctgctgttgacacctggtacgcctctttgaccaggccgcccgccaatgaggcttcctccaaaggccggctgttgaagagctttggacgaaaga agtcaaccagcagcgatcggcccccgcaccggtcatcgttcagcgacagcggccgcaggagtccgtggggatc ggtggactataccg cctccgggatggagatgccgtacagcgtcttcaacaccagcctctcggccgatgcgcccatggcgcctctggagc gcgaccaggccaaggatctgctagggg acctgccatcgctgcagagcatctggctaaccacagccgaggccatgattctggatcgcaagattgtgaatcggcc gcaggacattaagcggaaccagcagg tcatgctggagctgatccgcgaggacggcatggaggaggaggacgaatggtgctacggccgccagatctctgac tcggtcagaccaccgatttcagccctcc atgacgacgcggattaa SEQI atgatgaacgctgcaggttcgtgccactcttcgaactcgtctctcctgatcacccttgctcatacccctcaacagatg DNO : 23 tcgatttggacgacttcaccgtctttgaaggaggtgcctccacggctctctctctcggctgcgctcgccttcga cttcagcagcggcgcctcgagcgcgcttcaaaccttggcactgtgtctccccaggaccttggtccaggagccc ttcatgtcggcgcccaactctgcggccctgactgccttgacgtctcttcgctgtacaatggcagtcccgacttcgatt cctttgacgtctctcccaacttggcactgctgaatttgacaacggtcctagcgacccctggtatccttgttcccttg cggacacggtccctcacgagcttgtcaacccaagcgagtctccgtccagaagcctttggaatcagagcccatgta ccgatcgtcttcttcaggcagtggcgtggcgcggcaagaagaagcctggagtggttcgcccacgagcggca ctcgccactctcgtctcgggtcaacctcgccgccgagacaagcccctgcccccatcattgttgaagacgcc aacgacacagtggccatgaagcgagcccgcaataccttggctgctcgaaagtcggtgagcgaaaggctcagcg attcgaagagctggaccgtattgcgaagctcgaggccgagcgtgatcactggaagaggattgcactgtcgca atctggtgtttttgacggagta SEQINO : 24 atgagcggttctggctcttcgcttacccagacgggtggcatcaccgatcccgccctgatccagtgagcctcgactgc tctctcacgc List of references cited in the present patent application

[0137] Muhammad Adnan, Xuekun Ma, Stefan Olsson, Juan Wang, Gang Liu. Promoter régulation and genetic engineering strategies for enhanced cellulase expression in Trichoderma reesei, Microbiological Research, Volume 259, 2022

[0138] Arvas, Mikko; Pakula, Tiina; Lanthaler, Karin; Saloheimo, Markku; Valkonen, Mari; Suortti, Tapani et al. (2006) Common features and interesting différences in transcriptional responses to sécrétion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae. In : BMC genomics, vol. 7, p. 32. DOI: 10.1186 / 1471-2164 -7-32

[0139] Eveleigh DE and Montenecourt BS (1979) Increasing yields of extracellular enzymes. Adv Appl Microbiol 25, p. 57-74 Exemples

[0140] Exemple 1 : Invalidation des gènes dans la souche hyper-productrice RutC30 de T. reesei

[0141] The Rut-C30 strain was transformed, for each of the genes ID 65290 (Acpcl), ID 120583 (Aexol), ID 43599 (Avpsl), ID 35768 (ApProt), ID 80200 (Asdnl), and / or ID 122457 (Ambfl), with a knockout cassette containing a hygromycin resistance gene and the flanking regions (200 bp) of each gene ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 and / or ID 122457. Integration of the cassettes by homologous recombination was facilitated by the use of CRISPR-Cas9 technology.

[0142] The invalidation cassettes are illustrated in SEQ ID NO: 15 (ID 35768), SEQ ID NO: 16 (ID 43599), SEQ ID NO: 17 (ID 65290), SEQ ID NO: 18 (ID 80200), SEQ ID NO: 19 (ID 120583) and SEQ ID NO: 20 (ID 122457).

[0143] The guide RNA targeting the gene to be deleted (SEQ ID NO: 1 or SEQ ID NO: 23, SEQ ID NO: 2 or SEQ ID NO: 22, SEQ ID NO: 3 or SEQ ID NO: 24, SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 21) and the Cas9 protein are assembled in vitro.

[0144] For the purification of transformants, the clones obtained are subcultured onto a new dish (1 clone per dish) containing selective medium. Once these clones have sporulated, the spores are plated again onto selective medium. Isolated clones that appear on this dish are then preserved. Each isolated clone is then cultured in a rich liquid medium to obtain mycelium from which DNA is extracted.

[0145] Integration of the knockout cassette can be validated by PCR or Southern Blot. The absence of off-target integration is also confirmed by qPCR

[0146] The transformation of the strain can also be carried out according to the techniques described in example 1 of international application PCT / FR2020 / 052282. Any other techniques allowing the invalidation of the gene ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 and / or ID 122457, in particular those cited previously, can also be used instead of the CRISPR-Cas9 tool.

[0147] Example 2: Phenotype of Rut-C30 strains invalidated for a gene chosen from ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 or ID 122457

[0148] The transformed strains, as indicated in Example 1, are analyzed.

[0149] First, the effect of each deletion on growth was determined ([Fig.l]). Rut-C30 and the mutants were placed on solid minimal medium containing glucose or lactose as a cellulase-inducing substrate in the presence or absence of 5 mM DTT.

[0150] The results show that the growth of Rut-C30 and the mutants was faster on glucose than on lactose. However, the radial growth of all mutants was clearly reduced compared to that of the parental strain Rut-C30 on both substrates, especially on lactose.

[0151] The impact of invalidation of each of the genes ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 or ID 122457 on protein production was then studied. The transformants were cultured on a BTCA (Butanetetracarboxylic acid) medium comprising glucose or a mixture of cellulose and lactose as a carbon source. The supernatant was used to perform protein assays (Bradford assay) after 5 and 7 days of culture.

[0152] The results are presented in [Fig.2].

[0153] The dosage of the biomass produced was also analyzed, for example by filtration of the mycelium and determination of the weight after drying. The following method was used here: the mycelium was recovered by passing 6 mL of sample under pressure over a cellulose filter previously dried at 105 °C for 24 h. After placing the cellulose filter carrying the mycelia at 105 °C for 24 h, the dry weight of the biomass was determined by calculating the difference between the mass of the cellulose filter and the dried mycelia vs. the cellulose filter alone.

[0154] The results are presented in [Fig.3] and show the yield after 7 days of culture on glucose substrate (batch mode).

Claims

Claims

1. A strain of fungus belonging to the genus Trichoderma, Aspergillus, Fusarium, Penicillium, Talaromyces, Chrysosporium or Humicola, preferably Trichoderma, in which: - the expression of at least one protein encoded by a gene chosen from ID 65290, ID 120583, ID 43599 and ID 35768 is modified, or an orthologous gene, and / or - the expression of at least one protein encoded by a gene ID 80200, or an orthologous gene, is increased, and in which: - the gene ID 65290 corresponds to a gene represented by SEQ ID NO: 1 or SEQ ID NO: 23, or a gene having at least 80% identity with SEQ ID NO: 1 or SEQ ID NO: 23, - the gene ID 120583 corresponds to a gene represented by SEQ ID NO: 2 or SEQ ID NO: 22, or a gene having at least 80% identity with SEQ ID NO: 2 or SEQ ID NO: 22, - gene ID 43599 corresponds to a gene represented by SEQ ID NO: 3 or SEQ ID NO: 24, or a gene having at least 80% identity with SEQ ID NO: 3 or SEQ ID NO: 24,- gene ID 35768 corresponds to a gene represented by SEQ ID NO: 4 or a gene having at least 80% identity with SEQ ID NO: 4, - gene ID 80200 corresponds to a gene represented by SEQ ID NO: 9 or a gene having at least 80% identity with SEQ ID NO: 9.,

2. The fungal strain of claim 1, wherein the expression of at least one gene selected from ID 65290, ID 120583, ID 43599 and ID 35768 is altered.

3. A fungal strain according to claim 1 or 2, wherein at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200 is overexpressed, preferably ID 65290, ID 120583, ID 43599 and ID 35768.

4. A fungal strain according to claim 1 or 2, wherein at least one gene selected from ID 65290, ID 120583, ID 43599 and ID 35768 has been disabled, preferably ID 43599 and ID 35768.

5. A fungal strain according to any preceding claim, wherein: - the protein encoded by gene ID 65290 is represented by SEQ ID NO: 5 or a protein having at least 80% identity with SEQ ID NO: 5, - the protein encoded by gene ID 120583 is represented by SEQ ID NO: 6 or a protein having at least 80% identity with SEQ ID NO: 6, - the protein encoded by gene ID 43599 is represented by SEQ ID NO: 7 or a protein having at least 80% identity with SEQ ID NO: 7, - the protein encoded by gene ID 35768 is represented by SEQ ID NO: 8 or a protein having at least 80% identity with SEQ ID NO: 8, - the protein encoded by gene ID 80200 is represented by SEQ ID NO: 10 or a protein having at least 80% identity with SEQ ID NO:

10.

6. A fungal strain according to any preceding claim, wherein said fungus belongs to the species Trichoderma reesei.

7. A method of genetically modifying a fungal strain according to any one of claims 1 to 6, comprising: - a step of overexpressing at least one gene selected from ID 65290, ID 120583, ID 43599, ID 35768 and ID 80200, or an orthologous gene, preferably ID 65290, ID 120583, ID 43599 and ID 35768, or - a step of invalidating at least one gene selected from ID 65290, ID 120583, ID 43599 and ID 35768, or an orthologous gene, preferably ID 43599 and ID 35768.

8. A method of producing mushroom biomass, comprising a step of culturing a mushroom strain according to any one of claims 1 to 6, or a strain not expressing or having reduced expression of a protein encoded by gene ID 122457, or an orthologous gene, in a culture medium comprising a suitable substrate.

9. A method for producing proteins of interest, in particular enzymes, comprising a step of culturing a fungal strain in a culture medium comprising a suitable substrate, said strain being a strain according to any one of claims 1 to 6, or a strain not expressing or having a reduced expression of a protein encoded by gene ID 122457, or an orthologous gene.

10. A method for producing bio-sourced products from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to any one of claims 1 to 6, or a strain not expressing or having reduced expression of a protein encoded by a gene ID 122457, or an orthologous gene.

11. A process for producing a sweet juice or sugars from cellulosic or lignocellulosic substrates, comprising: - i) a step of pretreating a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - ii) a step of producing cellulolytic enzymes by a strain according to any one of claims 1 to 6, or a strain not expressing or having reduced expression of a protein encoded by a gene ID 122457, or an orthologous gene, - iii) a step of enzymatic hydrolysis of the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolysate.

12. A method of producing a biofuel from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to any one of claims 1 to 6, or a strain not expressing or having reduced expression of a protein encoded by a gene ID 122457, or an orthologous gene.

13. A process for producing a biofuel from cellulosic or lignocellulosic substrates according to claim 12, comprising: - i) a step of pretreating a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - ii) a step of producing cellulolytic enzymes by a strain according to any one of claims 1 to 6, or a strain not expressing or having reduced expression of a protein encoded by a gene ID 122457, or an orthologous gene, - iii) a step of enzymatic hydrolysis of the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolysate, - iv) a step of alcoholic fermentation of the hydrolyzate obtained, step iv) being optionally carried out simultaneously with step iii). - v) a separation step, in particular by distillation.

14. Use of a fungal strain according to any one of claims 1 to 5, or a strain not expressing or having reduced expression of a protein encoded by a gene ID 122457, or an orthologous gene, (i) for the production of proteins of interest, more particularly enzymes, in particular cellulolytic enzymes such as cellulases, (ii) for the hydrolysis of cellulose or lignocellulose to glucose, (iii) for the production of bio-sourced products from cellulosic or lignocellulosic substrates, (iv) for the production of biofuel from cellulosic or lignocellulosic substrates, (v) for the production of alcohol from cellulosic or lignocellulosic substrates, and / or (vi) for the production of a sweet juice or sugars from cellulosic or lignocellulosic substrates.

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