Protein-hyperproducing fungal 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 and to the method for genetic modification.

Description

Description Title: 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 protein secretion has been modified, by overexpression or inhibition. The invention also relates to the various uses of a strain thus modified, as well as the genetic modification method making it possible to obtain a strain according to the invention. Background to the invention

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

[0003] Various methods for increasing said protein production have 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 (especially endogenous ones). These enzymes, for example cellulases, are 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 bio-sourced products derived from sugars from (ligno)cellulosic biomass.

[0005] In order to improve the production of second-generation biofuels or bio-based products, it has already been considered to improve the production of cellulases, 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 fungal strains in order to modify their production capacity. Thus, patent EP3397768 describes the overexpression of the TrAZFI gene in a strain in order 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 produces high amounts of protein.

[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 genetically modifying 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 fungal strain according to the invention, as defined above or a strain not expressing or having an expression reduced at least one protein encoded by 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 pretreatment of 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 coded 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 of an appropriate 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 bio-sourced 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 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. The present invention thus relates to a variant strain of fungus, wherein 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 reduction in the expression of at least one protein encoded by a 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, 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%, preferably at least 98% or 99%, and 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, in particular at least 85%, at least 90%, more particularly at least 95%, preferably 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, 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%, preferably at least 98% or 99%, and 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, in particular at least 85%, at least 90%, more particularly at least 95%, preferably 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, 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 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 (vps1). 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, 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 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, the 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 the 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 identity between two sequences. For example, according to the invention, the percentage identity of a given sequence with respect to SEQ ID NO: 1 or SEQ ID NO: 5 is understood to mean the percentage 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 that 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 that regulates amino acid biosynthesis. The Fungi DB 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, 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 in a 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 ID120583 gene, said copy being expressed constitutively or inducibly. It therefore comprises in this case at least two copies of the ID120583 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 ID 43599 gene, said gene being under the control of 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. A "constitutive promoter" means 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. An "inducible promoter" means 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 (e.g. for the promoters pcbhl , pcbh2, pegll ), or xylose, xylobiose, cellobiose, sophorose, xylooligosaccharides or xylan (e.g. for the promoters pxynl , pxyn2).

[0040] According to a preferred embodiment, the constitutive promoter a) is chosen from: - le promoteur gpd de 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 / 1475-2859-1 1 -84). Ce promoteur a pour séquence SEQ ID NO : 13. - le promoteur gpd d’ Aspergillus nidulans (Penttilâ 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), - le promoteur cDNA1 de Trichoderma reesei (Nakari T, Alatalo E. et Pentillâ M: Isolation of Trichoderma reesei genes highly expressed on glucose-containing media: characterization of the tef1 gene encoding translation elongation factor 1 a Gene 1993 : 313-318), et - the tef1 promoter of Trichoderma reesei (Nakari-Setâlâ T, Penttilâ M., Production of Trichoderma reesei cellulases 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 cbh1 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 cDNA1 terminator of Trichoderma reesei, - the TrpC terminator of Trichoderma reesei, - the cbh1 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 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 can 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 protein activity targeting the vacuole or other cellular compartments (vps1), and with gene ID 122457 the protein has a multiprotein binding factor activity (mbf1).

[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 genes ID 43599 and ID 35768 have been invalidated.

[0052] Any gene invalidation technique may be used to obtain a strain according to the invention. By way of non-limiting examples, invalidation may 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, etc.

[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 foreign nucleic acid fragments can be inserted, with vectors being used to introduce foreign DNA into a host cell. Examples of vectors are plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacteriophage P1-derived artificial chromosomes (PACs), 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 preferably made up of 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 The knockout cassette may be operably 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 fungus 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 include, for example, strain Rut-C30 (deposited under ATCC number 56765), strain NG14 (deposited under ATCC number 56767) or strain QM9414 (deposited under ATCC number 26921).According to a preferred embodiment, it is the strain Rut-C30 or its descendants (i.e. all strains which are derived from or originate 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 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, 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 selected 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 selected 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 stated, all techniques can be used to overexpress or knock out 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 culturing a mushroom strain 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 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.

[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 growth phase of a fungal strain 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 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, residues obtained after ethanolic fermentation of the monomeric sugars of enzymatic hydrolysates of cellulosic biomass, a crude extract of water-soluble pentoses originating from the pretreatment of a biomass cellulosic, 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 (biofuel, food, cosmetic or pharmaceutical production, 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" is understood more particularly to mean enzymes chosen from endoglucanases, exoglucanases and glucosidases, and more particularly p-glucosidase. The term "cellulase" refers more particularly to an enzyme adapted to the hydrolysis of cellulose and allowing the microorganisms (such as Trichoderma reesei) 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 reesei) that produce them to use xylan as a carbon source, 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 or lignocellulosic substrates, 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 culture step, in a medium of culture 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 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 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.

[0102] 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 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.

[0103] In a sixth aspect, the invention relates to a process 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 producing 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] Sugars (or sweet juice, 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 process 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 biochemically, 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 is, 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 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, 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 more particularly understood 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 an 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 Cs-Ce 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 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.

[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 amended, 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 not intended in any way to limit it. Fig. 1

[0133] [Fig. 1 ] 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 secreted protein yield 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 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] [Table 1] List of references cited in this patent application

[0137] Muhammad Adnan, Xuekun Ma, Stefan Olsson, Juan Wang, Gang Liu. Promoter regulation 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 differences in transcriptional responses to secretion 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] Strain Rut-C30 was transformed, for each of the genes ID 65290 (Acpd ), 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 knockout cassettes are shown 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 purification of transformants, the resulting clones are subcultured onto a new plate (1 clone per plate) containing selective medium. Once these clones have sporulated, the spores are plated again onto selective medium. Isolated clones that appear on this plate are then kept. Each isolated clone is then cultured in rich liquid medium to obtain mycelium from which DNA is extracted.

[0145] Integration of the knockout cassette can be validated by PCR or Southern blotting. 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 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] Transformed strains, as indicated in Example 1, are analyzed.

[0149] First, the effect of each deletion on growth was determined (Figure 1). Rut-C30 and 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 invalidating each of the genes ID 65290, ID 120583, ID 43599, ID 35768, ID 80200 or ID 122457 on protein production was then studied. Transformants were grown on BTCA (Butanetetracarboxylic acid) medium containing 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 Figure 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 used: the mycelium was recovered by passing 6 mL of sample under pressure through 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 Figure 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, wherein: - 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.

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 35769.

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: - 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, - 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, - 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: 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, - 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, - 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 overexpression of at least one gene chosen 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 chosen 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 reduced expression of a protein encoded by a 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 pretreatment of 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) and of an appropriate substrate, 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 strain of a mushroom 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 method of producing a biofuel from cellulosic or lignocellulosic substrates according to claim 12, comprising: - i) a step of pretreatment of 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) 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.

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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