NON-ANTIBIOTIC SELECTION MARKER FOR FILAMENTOUS FUNGI

Abstract

The present invention relates to a strain of filamentous fungus belonging to the Ascomycetes division, excluding fungi belonging to the Saccharomycetes class, in which a gene encoding an arginine permease (CAN1), or a variant or orthologous gene, has been inactivated. The invention also relates to the various uses of this strain, as well as the genetic modification process for obtaining a strain according to the invention. The invention further relates to the use of L-canavanine as a selection marker for transformants in which a gene encoding an arginine permease (CAN1), or a variant or orthologous gene, has been inactivated. (no figure)

Description

Title of the invention: NON-ANTIBIOTIC SELECTION MARKER FOR FILAMENTOUS FUNGI

[0001] The present invention relates to a strain of filamentous fungus belonging to the Ascomycetes division, excluding fungi belonging to the Saccharomycetes class, in which a gene encoding an arginine permease (CAN1), in particular gene ID 18487637 (TRI67806), or a variant or orthologous gene, has been inactivated. The invention also relates to the various uses of this strain, as well as the genetic modification process for obtaining a strain according to the invention. The invention also relates to the use of L-canavanine as a marker for selecting transformants in which said gene encoding an arginine permease CAN1, or a variant or orthologous gene, has been inactivated. Background of the invention

[0002] Filamentous fungal strains, such as the fungus Trichoderma reesei, are now increasingly used for the production of proteins of interest.

[0003] The development of industrial fungal strains requires the use of selection markers to select transformed strains carrying the genetic mark of interest. Transformed strains are typically selected using marker genes such as an antibiotic resistance gene. However, the use of such antibiotic markers is sometimes prohibited or undesirable.

[0004] The auxotrophic marker URA can be used instead of an antibiotic marker to screen transformed fungal strains, as can the dominant marker amdS. However, these have drawbacks: the use of the URA gene as a selection marker is recyclable, but this leads to an accumulation of invalid gene copies, and the amdS marker is not practically recyclable.

[0005] There is therefore a need for a new non-antibiotic selection marker.

[0006] L-canavanine, the guanidinooxy structural analog of L-arginine, is a non-protein amino acid synthesized by higher plants. It was first isolated in 1932 from the kidney bean (Kitagawa et al.), and led to a better understanding of the biological effects and mode of action of non-protein amino acids that act as analogs of protein amino acids. Arginyl-tRNA synthetases in many L-canavanine-deficient species utilize and incorporate it into the nascent polypeptide chain. The production of L-canavanine-containing proteins disrupts critical reactions in RNA and DNA metabolism and protein synthesis. L-canavanine also affects L-canavanine disrupts the regulatory and catalytic reactions of arginine metabolism and alters essential biochemical reactions. It becomes a potent arginine antimetabolite in a wide range of species. These deleterious properties of L-canavanine make it a highly toxic secondary plant constituent that likely functions as an allelochnic agent, deterring the feeding activity of phytophagous insects and other herbivores.

[0007] As early as 1946, L-canavanine was successfully used as an antimetabolite to limit growth in bacteria (Volcani et al.), and then in 1948 in fungi (Horowitz and SRB, 1948). Its first use in the yeast Saccharomyces cerevisiae (SRB, 1955) was reported in 1954. The study of yeast made it possible to understand the mechanism of action of L-canavanine (Grenson et al.). L-canavanine is imported from the culture medium into the cell via the arginine permease, whose gene is designated CAN1. Upon entering the cytoplasm, L-canavanine is incorporated into nascent proteins, rendering them non-functional and leading to cell death. Inactivation of the permease by mutation prevents L-canavanine from entering the cell, thus rendering it resistant. In 1979, L-canavanine was used for the first time in yeast as a selection marker (Broach et al.), by inactivating the CAN1 gene.In 1986, the complete nucleic acid sequence of the CAN1 gene (Broach et al.; Ahmad and Bussey) was published.

[0008] The CAN1 gene has already been used as a selection marker in yeast (Broach et al.; Wong et al.) but never in other fungi, and even less so in fungi belonging to the genus Trichoderma.

[0009] The present invention is thus based on the results of the Inventors who showed that L-canavanine could be used as a selection marker for strains of fungus belonging to the division Ascomycetes, excluding a fungus belonging to the class Saccharomycetes, in which the gene ID 18487637 (CAN1), or a variant or an orthologous gene or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been invalidated. Brief description of the invention

[0010] The present invention therefore relates to a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - the gene ID 18487637 (CAN1), or a variant or orthologous gene, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog protein, has been invalidated.

[0011] The present invention also relates to a method for genetically modifying a strain of fungus according to the invention, belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, comprising an invalidation step: - of the gene ID 18487637 (CAN1), or a variant or an orthologous gene, or - of the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein.

[0012] The present invention also relates to the use of L-canavanine as a selection marker for a fungal strain belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - gene ID 18487637 (CAN1), or a variant or orthologous gene, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog protein, has been invalidated.

[0013] The present invention also relates to a method for selecting a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - gene ID 18487637 (CAN1), or a variant or orthologous gene, has been invalidated, or - The gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been invalidated. including: - (i) contacting a strain with a culture medium containing L-canavanine, and - (ii) the selection of the strain if it is capable of growing in the culture medium.

[0014] The present invention also relates to a method for producing mushroom biomass, comprising a cultivation step, in a culture medium comprising a suitable substrate, of a fungal strain belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - gene ID 18487637 (CAN1), or a variant or orthologous gene, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog protein, has been invalidated.

[0015] The present invention also relates to a method for producing proteins of interest, in particular enzymes, comprising a culture step, in a medium of culture comprising a suitable substrate, of a strain of fungus belonging to the division Ascomycetes, excluding a fungus belonging to the class Saccharomycetes, in which: - gene ID 18487637 (CANly or a variant or an ortholog gene has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog protein, has been invalidated.

[0016] The present invention further relates to a process for producing bio-based products from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - gene ID 18487637 (CAN1), or a variant or orthologous gene, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog protein, has been invalidated.

[0017] The present invention also relates to a process for producing a sugary juice or sugars from cellulosic or lignocellulosic substrates comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - gene ID 18487637 (CAN1), or a variant or orthologous gene, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog protein, has been invalidated.

[0018] The present invention also relates to a process for producing a biofuel from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - gene ID 18487637 (CAN1), or a variant or orthologous gene, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog protein, has been invalidated.

[0019] The present invention also relates to various uses of the strain according to the invention, belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, wherein the gene ID 18487637 (CAN1), or a variant or orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has has been invalidated-, for the production of biomass, for the production of proteins of interest, for the hydrolysis of cellulose or lignocellulose into glucose, for the production of bio-based products from cellulosic or lignocellulosic substrates, or for the production of a sweet juice or sugars from cellulosic or lignocellulosic substrates, or for the production of biofuel from cellulosic or lignocellulosic substrates. Description of the invention

[0020] In a first aspect, the present invention relates to a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which (a) the gene ID 18487637 (CAN1), or a variant, or an ortholog of this gene, or (b) the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog of this gene, has been inactivated. Thus, in the strain according to the invention, the gene ID 18487637 (CAN1), or a variant or an ortholog, or the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an ortholog is never expressed or is no longer functional. This also means that in the strain according to the invention the gene ID 18487637 (CAN1), or a variant or an orthologous gene or the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein is invalidated.The present invention thus relates to a variant strain of fungus, in which (a) the gene ID 18487637 (CAN1), or a variant or orthologous gene, has been inactivated, or (b) the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein has been inactivated. In other words, this means, for example, that in the strain according to the invention, the protein corresponding to the gene ID 18487637 (CAN1), or a variant or orthologous gene, or a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein is not produced. Alternatively, the protein corresponding to gene ID 18487637 (CAN1) or a variant or an orthologous gene or a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein can be produced, but it is not functional. The strain according to the invention is thus a strain with a deficient arginine permease.

[0021] According to the invention, the term "variant strain" means a strain that is genetically modified compared to a parent strain. According to the invention, the term "parent strain" means a strain from which the variant strain is derived, and in which the gene ID 18487637 (CAN1), or a variant or orthologous gene, has not been inactivated, or in which the gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein has not been invalidated. The strain according to the invention thus corresponds to a variant strain derived from a parent strain, said variant strain being able to grow in a minimal medium comprising L-canavanine while the parent strain is not able to grow in a minimal medium comprising L-canavanine.

[0022] According to the invention, the term "functional gene" means in particular a gene which enables the production of a functional protein.

[0023] According to the invention, the term "functional protein" means in particular a protein which has an activity, for example with the protein encoded by the gene ID 18487637 (CANT), or a variant or an orthologous gene or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, it is an arginine permease activity.

[0024] According to the invention, a fungal strain is understood to be a fungus belonging to the division Ascomycetes, excluding a fungus belonging to the class Saccharomycetes. In one embodiment, the fungal strain according to the invention is understood to be a fungus belonging to the division Ascomycetes, excluding a fungus belonging to the class Saccharomycetes or the class Eurotiomycetes. In another embodiment, the fungal strain according to the invention is understood to be a fungus belonging to the division Ascomycetes, excluding a fungus belonging to the class Saccharomycetes or the class Eurotiomycetes or to the order Sordariales. Preferably, it is a strain belonging to the order Hypocreales, in particular to the genus Trichoderma, and more especially to the species Trichoderma reesei.

[0025] When the fungus belongs to the species Trichoderma reesei, according to the invention, the parent strain of Trichoderma reesei may be strain QM6a (filed under number ATCC 13631), or a strain derived from the natural isolate QM6a (in particular obtained by random or directed mutagenesis), such as strain Rut-C30 (filed under number ATCC 56765), strain NG14 (filed under number ATCC 56767), or strain QM9414 (filed under number ATCC 26921). Typically, the strains according to the invention can be selected in a medium containing L-canavanine while retaining their ability to produce proteins of interest.

[0026] The CAN1 gene, which is annotated as gene ID 18487637 in the Trichoderma reesei reference genome (TRIREDRAFT_67806 or TRI67806), codes for a protein belonging to the arginine permease family. These permeases allow the import of L-canavanine into cells. In such a case, the L-canavanine would then be incorporated into the synthesized proteins, rendering them non-functional and leading to cell death. In the strains according to the invention, L-canavanine cannot be incorporated and therefore does not lead to cell death.

[0027] According to the invention, the gene ID 18487637 (CANP) is represented by the SEQ ID NO: 1, but may also correspond to a variant of this gene or to an orthologous gene. Preferably, the gene ID 18487637 (CANP) is only represented by the SEQ ID NO: 1.

[0028] According to the invention, "a gene variant or an orthologous gene" means a gene that also codes for a protein that belongs to the arginine permease family. The variant of gene ID 18487637 (CAN1) or an ortholog of gene ID 18487637 (CANP) is preferably represented by a sequence having at least 80% identity with the gene of SEQ ID NO: 1. The variant of gene ID 18487637 (CANP) or an ortholog of gene ID 18487637 (CANP) thus corresponds to a gene derived from the sequence represented by SEQ ID NO: 1. More specifically, the variant of gene ID 18487637 (CAN1) or an ortholog of gene ID 18487637 (CAN1) is represented by a sequence having at least 90% identity with the gene of SEQ ID NO: 1, in particular at least 95%, preferably at least 98% or 99%.

[0029] According to the invention, the protein of SEQ ID NO: 2 corresponds to an arginine permease. The terms "variant protein, or orthologous protein" thus refer to proteins also having this arginine permease activity. Preferably, a variant protein of a protein represented by SEQ ID NO: 2, or an orthologous protein of a protein represented by SEQ ID NO: 2, corresponds to a protein having at least 80% identity with said SEQ ID NO: 2, in particular at least 90%, more particularly at least 95%, 98% or 99%.

[0030] The gene ID 18487637 (CAN1) is represented by the SEQ ID NO: 1, and the protein encoded by the gene ID 18487637 (CAN1) is represented by the SEQ ID NO: 2. Thus, the variant of the gene ID 18487637 (CAN1) or an ortholog of the gene ID 18487637 (CAN1) can code either for the protein of SEQ ID NO: 2, or for a protein having at least 80% identity with said SEQ ID NO: 2, in particular at least 90%, more particularly at least 95%, 98% or 99%.

[0031] According to the invention, the term "at least 80%" means all values ​​between 80% and 100%, including 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%. Those skilled in the art know 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: 2 means the percentage of identity with respect to 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 2 divided by the number of nucleotides or residues in the longer of the two sequences.

[0032] Thus, in one embodiment, in said strain according to the invention, the ID gene The invalidated gene ID 18487637 (CANÏ) corresponds to a gene represented by SEQ ID NO: 1 or to a gene having at least 80% identity with the gene of SEQ ID NO: 1, in particular at least 90% and more particularly at least 95%. In a further preferred embodiment, in said strain according to the invention, the invalidated gene ID 18487637 (CAMP) corresponds to a gene represented by SEQ ID NO: 1. According to this latter embodiment, the strain according to the invention thus comprises a deletion of the gene encoding the protein represented by SEQ ID NO: 2.

[0033] According to one embodiment, said strain of the invention thus corresponds to a strain in which: - the gene ID 18487637 (CANl) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2, has been invalidated.

[0034] According to a preferred embodiment, the invention relates to a strain of filamentous fungus belonging to the order Hypocreales, in particular to the genus Trichoderma, and more particularly to the species Trichoderma reesei, in which: - the gene ID 18487637 (CANl) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1, has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2, has been invalidated.

[0035] According to the invention, the gene ID 18487637 (CANP), or a variant or orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been inactivated. All gene inactivation techniques can be used to obtain a strain according to the invention. By way of non-limiting examples, inactivation can be achieved through PCR mutagenesis, oligonucleotide-directed mutagenesis, RNA interference, using meganucleases or zinc finger nucleases, TALENs (Transcription activator-like effector nucleases), or CRISPR / Cas technology, etc. Preferably, the inactivation of gene ID 18487637 (CANP), or a variant or orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, is achieved through genome editing. Such genome editing can be performed with or without a repair template.

[0036] According to one embodiment, the invalidation is a deletion of all or part of the gene ID 18487637 (CANP), or a variant or an orthologous gene or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein. Preferably, the invalidation is a deletion of all or part of the gene ID 18487637 (CANP represented by SEQ ID NO: 1) or a deletion of all or part of the gene encoding a protein represented by SEQ ID NO: 2.

[0037] In a second aspect, the invention also relates to a method for genetically modifying a fungal strain belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, comprising a step of inactivating the ID gene 18487637 (CAMP) or a variant or an orthologous gene or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein. The genetic modification method according to the invention thus makes it possible to obtain strains that can be selected in a medium containing L-canavanine compared to the parent fungal strain.

[0038] According to one embodiment, the invention thus relates to a method for genetically modifying a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, comprising an inactivation step: - of the gene ID 18487637 (CANl) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1, or - of the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2.

[0039] In a third aspect, the present invention also relates to the use of L-canavanine as a selection marker of a fungal strain according to the invention belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which the ID gene 18487637 (CANP), or a variant or an orthologous gene or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been invalidated.

[0040] According to one embodiment, the invention thus relates to the use of L-canavanine as a selection marker for a fungal strain belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - the gene ID 18487637 (CANl) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1 has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2 has been invalidated.

[0041] Preferably, the invention relates to the use of L-canavanine as a selection marker for a strain in which the gene ID 18487637 (CANP or a gene encoding a protein represented by SEQ ID NO: 2 has been inactivated.

[0042] According to the invention, L-canavanine refers to the non-proteinogenic amino acid represented by the semi-developed formula: (NH2)2C=NO-CH2-CH2-CH(NH2)-COOH, or by the following chemical formula: [Chem 1] o This also applies to L-canavanine in salt form.

[0043] The expression "selection marker" here means that L-canavanine allows the selection of strains that have integrated a genetic modification over those that have not integrated it (in this case the invalidation of the gene ID 18487637 (CAN1), or a variant or an orthologous gene or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein).

[0044] Preferably, L-canavanine is present in a culture medium for a fungal strain. Preferably, the culture medium is arginine-free, and optionally also alanine-free, aspartate-free, and / or glutamate-free. The culture medium may thus be arginine-free, alanine-free, aspartate-free, and / or glutamate-free. In a preferred embodiment, L-canavanine is present in a minimal culture medium for a fungal strain, in particular a minimal medium that is arginine-free, and optionally also alanine-free, aspartate-free, and / or glutamate-free. In another preferred embodiment, L-canavanine is present in a synthetic culture medium for a fungal strain that is arginine-free, and optionally also alanine-free, aspartate-free, and / or glutamate-free.

[0045] According to the invention, a "minimal medium" is understood to be a medium containing only the elements necessary for the growth of the fungal strain, and a "synthetic medium" is understood to be a medium in which the quantity and quality of each element present in the medium is well defined. The synthetic medium may include elements not necessary for the growth of the microorganism. The minimal and synthetic media are contrasted with a "rich medium," which does not contain only the elements necessary for the growth of the fungal strain, and in which the quantity and quality of the elements are not well defined. A minimal medium for Trichoderma is described, for example, by Penttilâ et al.

[0046] According to one embodiment, L-canavanine is used at a concentration between 50 and 200 pg / mL in the culture medium.

[0047] In a fourth aspect, the present invention also relates to a method for selecting a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which the gene ID 18487637 (CAN1), or a variant or an orthologous gene or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been invalidated, including: - (i) contacting a strain with a culture medium containing L-canavanine, and - (ii) the selection of the strain if it is capable of growing in the culture medium.

[0048] According to one embodiment, the present invention thus relates to a method for selecting a strain of fungus, belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - the gene ID 18487637 (CANl) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1 has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2, has been invalidated, including: - (i) contacting a strain with a culture medium containing L-canavanine, and - (ii) the selection of the strain if it is able to grow in the minimum culture medium or a synthetic medium free of arginine, optionally also free of alanine, aspartate and / or glutamate.

[0049] According to one embodiment, the process comprises, prior to step (i), an inactivation step of gene ID 18487637 (CAN1), or a variant or orthologous gene, or of a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, in a fungal strain belonging to the division Ascomycetes, excluding a fungus belonging to the class Saccharomycetes. According to this embodiment, the fungus preferably belongs to the order Hypocreales.

[0050] According to a preferred embodiment, the present invention thus relates to a method for selecting a strain in which the gene ID 18487637 (CAMP) represented by SEQ ID NO: 1 or the gene encoding a protein represented by SEQ ID NO: 2, has been inactivated, comprising: - an inactivation step of the gene ID 18487637 (CANl) represented by SEQ ID NO: 1 or of the gene encoding a protein represented by SEQ ID NO: 2 in a strain, - bringing said strain into contact with a culture medium containing L-canavanine, and - the selection of the strain if it is able to grow in the culture medium, in particular the minimum culture medium or said synthetic culture medium free of arginine, optionally also free of alanine, aspartate and / or glutamate.

[0051] The selection process according to the invention thus makes it possible to select the correctly transformed strains.

[0052] In a fifth aspect, the present invention also relates to a method for producing biomass from fungi belonging to the Ascomycetes division, excluding fungi belonging to the Saccharomycetes class, comprising a step of culturing, in a culture medium comprising a suitable substrate, a fungal strain according to the invention, in which the gene ID 18487637 (CAN1), or a variant or an orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been inactivated. This step thus enables the growth of the fungal strain according to the invention, enabling in particular the production of biomass. Those skilled in the art know the substrates suitable for the growth of fungal strains.

[0053] According to one embodiment, the present invention thus relates to a method for producing biomass of fungi belonging to the Ascomycetes division, excluding fungi belonging to the Saccharomycetes class, comprising a step of cultivating, in a culture medium comprising a suitable substrate, a strain of fungus according to the invention, in which: - the gene ID 18487637 (CAN1) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1 has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2 has been invalidated.

[0054] In a sixth aspect, the present invention also relates to a method for producing proteins of interest, in particular enzymes, comprising a step of culturing, in a culture medium comprising a suitable substrate, a fungal strain according to the invention belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which the gene ID 18487637 (CAN1), or a variant or an orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein has been inactivated. The invention thus relates to the use of a fungal strain according to the invention for the production of proteins of interest.

[0055] According to one embodiment, the present invention thus relates to a process for producing proteins of interest, in particular enzymes, comprising a step of culturing, in a culture medium comprising a suitable substrate, a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - the gene ID 18487637 (CAN1) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1 has been invalidated, or - the gene coding for a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the SEQ ID NO protein: 2 has been invalidated.

[0056] Advantageously, the process thus comprises a growth phase of a fungal strain according to the invention, followed by a growth and protein production phase of interest by said strain. More preferably, the growth phase is carried out in the presence of a growth substrate, and the growth and protein production phase is carried out in the presence of an inducing substrate. The growth substrate and the inducing substrate are preferably carbon substrates.

[0057] Thus, the carbon substrate for growth is preferably chosen from lactose, glucose, xylose, residues obtained after ethanolic fermentation of monomeric sugars from enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass.

[0058] The inducing carbon substrate is thus preferably chosen from lactose, cellobiose, sophorose, residues obtained after ethanolic fermentation of monomeric sugars from enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass.

[0059] According to the invention, the proteins of interest are all proteins that can be produced by a fungus, either naturally or by genetic modification (for example after transformation using a suitable vector).

[0060] Advantageously, the proteins of interest according to the invention are enzymes, in particular cellulolytic enzymes such as cellulases or hemicellulases. Preferably, the enzymes are cellulases. According to the invention, the term "cellulases" refers more particularly to enzymes selected from endoglucanases, exoglucanases, and glucosidases, and more particularly 3-glucosidase. The term "cellulase" refers more particularly to an enzyme adapted for the hydrolysis of cellulose and enabling the microorganisms (such as Trichoderma reesei) that produce it to use cellulose as a carbon source by hydrolyzing this polymer into simple sugars (glucose). According to one embodiment, the invention thus relates to the use of a strain of fungus according to the invention, for the production of proteins of interest, more particularly enzymes, in particular cellulolytic enzymes such as cellulases.

[0061] In a seventh aspect, the present invention also relates to a process for producing bio-based products from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus according to the invention belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in in which the gene ID 18487637 (CANI), or a variant, or an orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been invalidated. The invention therefore also relates to the use of a fungal strain according to the invention for the production of bio-based products from cellulosic or lignocellulosic substrates.

[0062] According to one embodiment, the present invention thus relates to a process for producing bio-based products from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes, by a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - the gene ID 18487637 (CANI) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1 has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2 has been invalidated.

[0063] In an eighth aspect, the present invention also relates to a method for producing sugar juice or sugars from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to the invention belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which the ID gene 18487637 (CANI), or a variant or orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been inactivated. The invention therefore also relates to the use of a fungal strain according to the invention for the production of sugar juice or sugars from cellulosic or lignocellulosic substrates.

[0064] According to one embodiment, the present invention thus relates to a process for producing a sugar juice or sugars from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - the gene ID 18487637 (CANI) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1 has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2 has been invalidated.

[0065] In a preferred aspect, the process for producing a sweet juice or sugars from cellulosic or lignocellulosic substrates according to the invention comprises: (i) a pretreatment step 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) an enzymatic hydrolysis step 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.

[0066] The sugars (or sugar juice, the sugars generally being in 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).

[0067] The process according to the invention thus makes it possible to treat (ligno)cellulosic biomass to produce so-called second-generation (2G) sugar juices. These sugar 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 used as is, after possible separation / treatment steps, for example in the chemical / food industry.

[0068] In a ninth aspect, the present invention relates to a process for producing biofuel from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a fungal strain according to the invention belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which the gene ID 18487637 (CAN1), or a variant or orthologous gene, or a gene encoding a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been inactivated. The invention thus relates to the use of a fungal strain according to the invention for the production of biofuel from cellulosic or lignocellulosic substrates.

[0069] According to one embodiment, the present invention relates to a process for producing a biofuel from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus according to the invention belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, in which: - the gene ID 18487637 (CAN1) represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO: 1 has been invalidated, or - the gene encoding a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2 has been invalidated.

[0070] According to the invention, the term "biofuel" refers more particularly to a second-generation biofuel, that is, one derived from non-food resources. According to the invention, the term "biofuel" can also be defined as any product obtained from the transformation of biomass and which can be used for purposes Energy. On the one hand, and without being limited to this, examples include biogas, products that can be incorporated (possibly after further processing) into a fuel or be a fuel in their own right, such as alcohols (ethanol, butanol, and / or isopropanol depending on the type of fermentative 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 another alcohol like 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 2,3-butanediol, organic acids like acetic, propionic, acrylic, butyric, succinic, malic, fumaric, citric, itaconic acid, or hydroxyacids like glycolic, hydroxypropionic, or lactic acid.

[0071] In a preferred aspect, the process for producing a biofuel from cellulosic or lignocellulosic substrates according to the invention comprises: (i) a pretreatment step 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 the invention, iii) an enzymatic hydrolysis step 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, iv) an alcoholic fermentation step of the hydrolysate obtained, v) a separation step, in particular by distillation.

[0072] In an even more preferred aspect, the process for producing a biofuel from cellulosic or lignocellulosic substrates, according to the invention, comprises: i) a pretreatment step 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 the invention iii) an enzymatic hydrolysis step 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, iv) an alcoholic fermentation step of the hydrolysate obtained, v) a separation step, in particular by distillation, said steps iii) and iv) being carried out simultaneously. This is typically the case in so-called "SSF" (Simultaneous Saccharification and Fer- mentation).

[0073] According to a particular embodiment, the pretreatment step of a cellulosic or lignocellulosic substrate is a step of suspending said cellulosic or lignocellulosic substrate in aqueous phase.

[0074] According to a particular embodiment, the hydrolysate obtained in step iii) is a hydrolysate containing glucose.

[0075] According to a particular embodiment, the alcoholic fermentation step of the hydrolysate obtained is a fermentation step, in the presence of a fermentative organism, of the glucose from the hydrolysate so as to produce a fermentation must. A fermentative organism is, for example, yeast.

[0076] According to a particular embodiment, the separation step is a separation of the biofuel and the fermentation must, in particular by distillation.

[0077] According to a further preferred embodiment, the cellulosic or lignocellulosic substrate to be hydrolyzed is suspended in aqueous solution at a concentration of 6 to 40% dry matter, preferably 20 to 30%. The pH is adjusted to between 4 and 5.5, preferably between 4.8 and 5.2, and the temperature to between 40°C and 60°C, preferably between 45°C and 50°C. The hydrolysis reaction is initiated by adding enzymes that act on the pretreated substrate. The quantity of enzymes typically used is 10 to 30 mg of protein excreted per gram of pretreated substrate or less. The reaction generally lasts from 15 to 48 hours. The reaction is monitored by measuring the sugars released, particularly glucose. The sugar solution is separated from the non-hydrolyzed solid fraction, mainly consisting of lignin, by filtration or centrifugation and then processed in a fermentation unit.

[0078] According to another, even more preferred embodiment, when the hydrolysis and fermentation steps are carried out jointly, the enzymes and the fermenting organism are added simultaneously and then incubated at a temperature between 30°C and 35°C to produce a fermentation broth. According to this embodiment, the cellulose present in the pre-treated substrate is converted into glucose, and at the same time, in the same reactor, the fermenting organism (for example, yeast) converts the glucose into the final product according to a Simultaneous Saccharification and Fermentation (SSF) process known to those skilled in the art. Depending on the metabolic and hydrolytic capacities of the fermenting organism, the proper execution of the operation may require the addition of a greater or lesser quantity of exogenous cellulolytic mixture.

[0079] In a tenth aspect, the invention also relates to the use of a strain of fungus according to the invention belonging to the Ascomycetes division, excluding a fungus belonging to the Saccharomycetes class, for the hydrolysis of cellulose or lignocellulose into glucose.

[0080] In this description, the definitions and preferences indicated in an 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, eighth, ninth and tenth aspects. Brief description of the Figures

[0081] Other features, details and advantages of the invention will become apparent from reading the accompanying Figures. Fig.l

[0082] [Fig. 1] shows the sensitivity test of Trichoderma spores in PDA medium (left column, without L-canavanine; right column, with 200 pg / mL of L-canavanine) and minimal medium (MM) (left column, without L-canavanine; right column, with 50 pg / mL of L-canavanine). The numbers represent the number of spores spread on each medium. Fig. 2

[0083] [Fig.2] represents the diagram of the repair matrix used in Example 2.

[0084] MR5' and MR3' represent the two Ikb flanks with sequences identical to the two Ikb regions flanking the target gene, here ID 18487637 (67806-TrF1047C); Prom cpc represents the CPC promoter; IM7 represents the IM7 promoter; Term TrpC represents the TrPC terminator; and Hygromycine represents the hygromycin resistance gene. The term "Cas9 437" refers to the CRISPR-Cas9 tool used.

[0085] Sequences of the present invention

[0086] [Tables 1] Nom de la séquence Séquence SEQID NO : 1 ATGACGGACGAGAAGCTCGTCCACGGCATCAGCGACAAGCCCG CCGGCGATGTCTCCAACCCGGACGTCGAGACGGGCCAGACCGAC GAGGGCCTGAAGCGCGAGCTGCACTCGCGGCACCTGCAGTTCAT CGCCATCGGCGCGGCCCTCGGCACGGGCCTGTTCCTGGGCATCG GCGGCGCCCTGTCCAAGGCCGGCCCCGGGTCGCTCTTCATCTCGT TCCTGTTCCTCGGCTCCGTCGTCTTCTCCATCATGACGGCGCTGG GCGAGATGGCGTCGTACATCCCCGTGACGGGCGCCTTCACCGTG TACGCCTCGCGCTTCATCGACCCGACGCTGGGTTTCGCCATGGGC TGGATCTACTGGTTCAGCTGGGTCATCACCATCGGGCTGGAGCT GACGGCCAGCGGGCTCATCATCCAGTACTGGACCGAGTCGGTCA GCATCGGCGTCTGGATCGCCATCTTCTGGGTCGTCTTCACGGCCG CGCAGTTCCTGCCCACCTTCGTCTTTGGCGAGATGGAGGCGTACC TGTCGAGCATCAAGGTCGTCACCATTGTCGGCTTCGTCATCTTCG GCATCTGCGTCAACGCTGGCGTCGGCGACCAGGGCTACATTGGC TTCAAGTACTGGCGCGAGCCGGGCGCCTTTGCCGAGTACATGGT CGACGGCGCCATTGGCAAGTTTGTCGGCTTCTGGACGGTGCTCG TGACGGCGGGCTTCAGCTTCCAGGGCACGGAGCTGGTCGTCGTC GGCGCGGGCGAGACGGCCAACCCGCGCACGGCGATCCCGTCGG CCATCCGCTGGACCTTTTGGGCCGTCTTTTTGCTCTTCAACTGCA CCGTCTTCTTCGTCGGCATCAACGTCCCCTACGACAACCCGGACC TGCAAAACGGCTCGACCAACGCGTCCGCATCGCCGCTCGTCATCGTCGCCAAGCTGGCCGGCATCCAGGCGCTGCCGTCCATCATCAA CGCCGTCCTGCTGACCGCCGTCCTGTCCGCCGCCAACTCGGACAT CTACTCGAGCAGCCGCATCCTCGTCGCCATGGCCGGCGAGGGCC ACGCGCCCGCCTTCTTCAAGCGCACCAACAAGTACGGCACCCCC TACTACGCCGTCGCCTGCTGCTCCGCCTTTGGCCTGCTGGCCTTT CTCAACCTCAGCAACAACGGCACCGTCGTCTTCAACTGGCTGCT CAACATCACCGCCGTCGCCGGCTTCATCAGCTGGGCCCTCATCA ACCTGTGCCACATCCGCTTCATGCGCGTCCTCAAGTACCAGAAC ATCCCCCGCGACGAGCTGCCCTACAAGGCCCCCTTCCAGCCCTA CCTGTCCTGGTTCGGCTTCTTCTTCAACGTCCTCATCCTGCTGACC AACGGCTTCACCGTCTTCATCGACTGGGACACGAGCACCTTTTTC GCCTGCTACGTCAGCGTCATGCTCTTTATCGTGCTGTACGTCGGC CACAAGCTTGTCTACAGGACAAAGATTATTCCCGTGTCCGAGGT GGACATTTCCAAGGGCCGGTCAGGGCAGTAA SEQID MTDEKLVHGISDKPAGDVSNPDVETGQTDEGLKRELHSRHLQFIAI NO GAALGTGLFLGIGGALSKAGPGSLFISFLGSVVFSIMTALGEMAS YIPVTGAFTVYASRFIDPTLGFAMGWIYWFSWVITIGLELTASGLIIQ YWTESVSIGVWIAIFWVVFTAAQFLPTFVFGEMEAYLSSIKVVTIVG FVIFGICVNAGVGDQGYIGFKYWREPGAFAEYMVDGAIGKFVGFW TVLVTAGFSFQGTELVVVGAGETANPRTAIPSAIRWTFWAVFLLFN CTVFFVGINVPYDNPDLQNGSTNASPLVIVAKLAGIQALPSIINAV LLTAVLSAANSDIYSSSRILVAMAGHAPAFFKRTNKYGTPYYAV ACCSAFGLLAFLNLSNNGTVVFNWLLNITAVAGFISWALINLCHIRF MRVLKYQNIPRDELPYKAPFQPYLSWFGFFFNVLILLTNGFTVFIDW DTSTFFACYVSVMLFIVLYVGHKLVYRTKIIPVSEVDISKGRSGQ SEQID NO : 3 CATCCAGTACTGGACCGAGT CGG SEQID TTCTTTTCTTCTTGTCTGTTGCTTTTTGCGCTCTTTTTTTGCTCT NO : 4 CAAGGCCTCGAGGCTTATCAGCCAGCCAAATGGCGATTGGGATA CAAGCGCTAGAGAAGGCCGCTAGAAGGATGCAGAGAAGGGCGA AAAGAGTCGTGCTTGTTGTAGTCGAGCTTGTTTAGGTCAGGGGG AGAGGATCTCTGTAGGTACCTATTACAGTACCGCCCTTACGAT GACGACTATGGTAATGTTTGATTTCGAATTGCTTCTTGTTGATGTGTTGTTGTTGTTGTTGTATCTTGACGACGTCTCGAGGTCAACTGAAG TTGTTGGAGGTGCCCTGGCCAAGAGTCTCCGTGTCATCGGCATC AACGCCTTACGAGTGACGCATTCCAGAGAGAACATGGCATGGCT TTGCCTCGTCTCCATGGCTTGACCGGCTTTGCTGTGCCGAGGACT GAAGTAGCCTAGTCTCCATCAGAGTCTTCATCGATGGGGCTTAT ATGGGGCGGCAAGTGAGGGTCTCGCCAGCAGGTGATAAGCCAG CGTGTATGTAGCTGTTCTTAGTGTAGCAGCCCGACAATAAGGTA ACGTTGATGTGTCCGAGTTTTTATTATTGGAAGGGCAATGTGACG CTCTCTAGTCTCTACTCTAGTATGTATATAGATTGAGCGCCCCAG AGGTTGTCGAGCCCTTTTCCGCCTTGTTTTTCTTCCGTCTTGCGGA TGGTTTTGGTTGCTTTGCGGGCGATATAGAGAGAGGTCTTGCCTT TCGCCCCCTCCTTGTCCGGAAGCGTCGTTATATATACTACTCTTC gTTTTTCTCTCAATTgCCACACCgACgAgCCgATTTCgATTgAAT TTTTTCTCTTCTGTCTTTCTTTCGCTCTCÀÀTCTTGÀÀÀGTTTGÀC TCATCTCTCTCTCTCTCTCTTCTGTTCAATCCGTCGCGCGGCCCTT CAGACGACCAAACGATGGCCGACGACGAAAAGACCCCGACGCC TCCCCCCGTCGGCCCCCTCGGTGGGGCAGTGCTAGTGTGTGTACC GACCCGCAGGATTGGTGCTTTGCCCAGAGCTCTACAGAATAGCG CGCGCATCCATATGTTAGTTCTGCAATTTTCTTGTATCGGTGCTG TGACTCATACTTCCCCCTTTGGCTGGCCTTGCGGCAACCAATAAG AACGCACAGTGAAATCTTGCGGGTGGGGAGTGCATGGCGCCTGC ATTGGCTTGGGGACGCGCACTGTCGCACACTTCCATCTGACCTTT CAGAAGGGTTTCGTGGTGGGCAAGGACCAACCGGTTGCGCGGCC GTGCGTGGGTGCCTCGCCCGGCACTGCCAGGGCCACTGCAGTGG CAGTTTGCTGCCTGATACAAAATCCTTCCCTCCGCCCAGTTTTCC CTCTTTGACCTTCCTTTCTCTTCTCTGCAACCAAATCCACCCTATC AAACCAAAACAGTGTCTCGACCGAGGTATCAACCTGAATCAGCA ACATCGTAGCCAGCATTTGTCTCCGTCTCTGCAGAACCAGCGAG TTGCAAACATTATCCAGGCAACAGGGCACCAACTCACTTCTTCG GCTTTCACCAATCGGTACAGCTCTTCTCAGAACTCGCGTCCGCAA CAGTTCTACGCTTCCTCAGCACCTTCTTCAGCTTCAATCCTGAAC ACTCAGAACCGCGCACAGCAGCGCCCTCCTGTTCCCTTGTTTCCC AAAAGTACCGGTAGTATTTCGCACGGAAAGCAGGGCAACACGT AAGTGATATCTACTAGTTGTGACCGGCGCCTAGTGTTGACAATT AATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTA TAGGAGGGCCACCATGTCGACTACTAACCTTCTTCTCTTTCCTACAGCTGAGATCACCGGTAGGAGGGCCATCATGAAAAAGCCTGAA CTCACCGCGACGTCTGTCGCGAAGTTTCTGATCGAAAAGTTCGA CAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTC GTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGG GTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTAT CGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGA CATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCC GTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTG CCCGCTGTTCTGCAACCCGTCGCGGAGCTCATGGATGCGATCGC TGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGAC CGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGC GCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGA CGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGA TGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCAC GCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCAT AACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCC AATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTT GTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGA GCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTG GTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATG ATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCC GGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCG CGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGT GGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAACCGG AGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCAT TTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATAGCTCC ATGTCAACAAGAATAAAACGCGTTTCGGGTTTACCTCTTCCAGA TACAGCTCATCTGCAATGCATTAATGCATTGGACCTCGCAACCCT AGTACGCCCTTCAGGCTCCGGCGAAGCAGAAGAATAGCTTAGCA GAGTCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACG GTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGCTCC ACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTC TTCTTTACTCTGATAGCTTGACTATGAAAATTCCGTCACCAGCCC CTGGGTTCGCAAAGATAATTGCACTGTTTCTTCCTTGAACTCTCA AGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAAAAT CATTCCTACTAAGATGGGTATACAATAGTAACCATGCATGGTTG CCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTT TTTACAACTCTCCTATGAGTCGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTCATGAGAAAGAAGGAAGGAAG GAAGAAAAGAGAACACTTTGAGGAGATGAGACGACTGTACAA GTGTTTGAGTGTTGTGTGTATGCATGTTGGGAATAGCGAGTAAT GGCTGTATTTTTTGAATATCTCTGATTTTTTGTTCATATCATTCA TGCTGTACGAGTGTCATGATCTACCGATAGTAATTGATACCCCTT TTTTGTGAGAAATTGGAGAGCTGGAGTCGCGTGTTCTTATGGGA GAATTTCCCTCACTTGGCACTGTCACATGTCAGACATGCAGG AACAGCGTATTCCTTTTTGTGGGCAATCGGATTGAATCTGTTT GGACAAAAAAAAAAAAAAATAGAACCACGACAATTAGCCTCA ATGGAGCACACACACGATACCCACGTTGCTCGCTCCAGCTCTCCAG ATCGACGATTTGATGTCTTCAGTGGAGAGAAACAGAAACGGG ATCAACGCATAAGATGCTCGTCAGCGTGGCTGCTGCTTAGAGGCCA GACAAACAAACCTTGAAAATCAATGTCGACTTTCTTCTGCCCC CCTTGAACCCGAGTCGTTATATCCGAGAGACGTGCAGACGTTC AGAACATTACAGAACAATACAGAACCTCCCAAAACAGCAAAAGG CCACGCGACACCCAACAACACGAACAGTGCTACATTGCCAAAGA GCTAAACTCGATAGGGGAAACGCAGCAGGTCGAGGCAGCAAG ATTCACATGCAAGGCCAATCACGGCGCTTCAAGCAGCAACGGAT TCCAATTGATAAGCCCCACGAACGGACATTTTTAGCTTGTTA AACCCAGTAAAAGCTGCTTGGGCCGTTGAATTTACCACATGTTA CTTAAAGGTCGGTCCTGTGTGTGTGTGACGGGCTGCGTGGGT GGTGTGTACTATAGCTTCAGCTTCTTCCTCTTTCCCCCCCTTTACA ACCTTTTTTTGCCTTTTTTGATAACTTGTCCTCACATCAGCATTGG CATCGTTGTAA SEQID TTCTTTTCTTCTTGTCTGTTGCTTTGTTGCTTTGCT NO : CAAGGCCTCGAGGCTTCTAGCAGCCGCAAAAATGGGTGTAGAGC CAAGCGCTAGAGAAGGCCGCTAGAAGGATGCAGAGAAGGGCGA AAAGAGTCGTGCTTGTTGTAGTCGAGCTTGTTTAGGTCAGGGGG AGAGGATCTCTGTAGGTACCTATTACAGTACTACGCCCTTACGAT GACGACTATGGTAATGTTTGATTTCGAATTGCTTCTTGTTGATGT TGTTGTTGTTGTTGTATCTTGACGACGTCTCGAGGTCAACTGAAG TTGTTGGAGGTGCCCTGGCCAAGAGTCTCCGTGTCATCGGCATC AACGCCTTACGAGTGAGTGAGGCATTCCAGAGAGAGAGAGATGGCTGCT TTGCCTCGTCTCCATGGCTTGACCGGCTTTGCTGTGCCGAGGACT GAAGTAGCCTAGTCTCCATCAGAGTCTTCATCGATGGGGCTTAT ATGGGGCGGCAAGTGAGGGTCTCGCCAGCAGGTGATAAGCCAG CGTGTATGTAGCTGTTCTTAGTGTAGCAGCCCGACAATAAGGTA ACGTTGATGTGTCCGAGTTTTTATTTGGAAGGGCAATGTGACGCTCTCTAGTCTCTACTCTAGTATGTATATAGATTGAGCGCCCCAG AGGTTGTCGAGCCCTTTTCCGCCTTGTTTTTCTTCCGTCTTGCGGA TGGTTTTGGTTGCTTTGCGGGCGATATAGAGAGAGGTCTTGCCTT TCGCCCCCTCCTTTGTCCGGAAGCGTCGTTATATATACTACTCTTC gTTTTTCTCTCAATTgCCACACCgACgAgCCgATTTCgATTgAAT TTTTTCTCTTCTGTCTTTCTTGCTCTCÀÀTCTTGÀÀÀGTTTGÀC TCÀTCTCTCTCTCTCCTCTTCTGTTCÀÀTCCGTCGCGCGGCCCCTT CÀGÀCGÀCCÀÀÀCGÀTGGCCGÀCGÀCGÀÀÀÀGÀCCCCGÀCGCC TCCCCCCGTCGGCCCCCTC SEQID GGTGGGGCÀGTGCTÀGTGTGTGTÀCCGÀCCCGCÀGGÀTTGGTGC NO: 6 TTTGCCÀGÀGCTCTÀCÀGÀTÀGCGCGCÀTCCÀTÀTGTTÀG TTCTGCAATTTTCTGTÀTCGGTGCTGTGAGTCATAGTTCCCCTT TGGCTGGCCTTGCGGCÀÀCCÀÀTÀGÀCGCÀCÀGTGÀÀTCTT GCGGTGGGÀGTGCÀTGGCGCTGCATTGGCTTGGCTGGGGÀCGCG AGTGTCGCAGAGTTCCÀTCTGAGCTTTCÀGAAGGGTTTCGTGGTG GGCAAGGACCAACCGGTTGCGCGGCCGTGCGTGGGTGCCTCGCC CGGCACTGCCAGGGCCACTGCAGTGGCAGTTTGCTGCCTGATAC AAAATCCTTCCCTCCGCCCAGTTTTCCCTCTTTGACCTTCCTTTCT CTTCTCTGCAACCAAATCCACCCTATCAAACCAAAACAGTGTCTC GACCGAGGTATCAACCTGAATCAGCAACATCGTAGCCAGCATTT GTCTCCGTCTCTGCAGAACCAGCGAGTTGCAAACATTATCCAGG CAACAGGGCACCAACTCACTTCTTCGGCTTTCACCAATCGGTAC AGCTCTTCTCAGAACTCGCGTCCGCAACAGTTCTACGCTTCCTCA GCACCTTCTTCAGCTTCAATCCTGAACACTCAGAACCGCGCACA GCAGCGCCCTCCTGTTCCCTTGTTTCCCAAAAGTACCGGTAGTAT TTCGCACGGAAAGCAGGGCAACAC SEQID GTAAGTGATATCTACTAGTTGTGACCGGCGCCTAGTGTTGACAA NO : 7 TTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACT ATAGGAGGGCCACCATGTCGACTACTAACCTTCTTCTCTTTCCTA CAGCTGAGATCACCGGTAGGAGGGCCATC SEQID ATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGCGAAGTTTCT NO : 8 GATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGG AGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGT GGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAA AGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGAT TCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCT ATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAACCCGTCGCGGAGCT CATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGT TCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGG CGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGG CAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGC TCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCC GGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACG GACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGAT GTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGA GGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAG CGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTA TATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGA CGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACG CAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATC GCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGT ACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGG CAAAGGAA SEQID CCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGT NO : 9 TCATTTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATAGC TCCATGTCAACAAGAATAAAACGCGTTTCGGGTTTACCTCTTCCA GATACAGCTCATCTGCAATGCATTAATGCATTGGACCTCGCAAC CCTAGTACGCCCTTCAGGCTCCGGCGAAGCAGAAGAATAGCTTA GCAGAGTCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCA ACGGTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGC TCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTC CTCTTCTTTACTCTGATAGCTTGACTATGAAAATTCCGTCACCAG CCCCTGGGTTCGCAAAGATAATTGCACTGTTTCTTCCTTGAACTC TCAAGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAA AATCATTCCTACTAAGATGGGTATACAATAGTAACCATGCATGG TTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAAC TTTTTTACAACTCTCCTATgAgTCgTTTACCCAgAATgCACAggT ÀCÀCTTGTTTÀGÀGGTÀÀTCCTTCTTTCÀTG SEQID ÀGÀÀÀGÀÀGÀGGÀÀÀGGÀÀGÀÀÀÀÀGÀGÀÀCÀCTTTGÀGGÀG NO : 10 ÀTGÀGÀCGÀCTGTÀCÀÀGTGTTTGÀGTGTTGTGTGTÀTGCÀTGTT GGGÀÀTÀGCGÀGTÀÀTGGCTGTÀTTTTTGÀÀTÀTCTCTGÀTTTTT TTTGTTCÀTÀTCÀTTCÀTGCTGTÀCGÀGTGTCÀTGÀTCTÀCCGÀT ÀGTÀÀTTGÀTÀCCCCTTTTTTGTGÀGÀÀÀTTGGÀGÀGCTGGÀGTCGCGTGTTCTTÀTGGGÀGAATTTCCCTCAGTTGGCAGTGTCGAT GTCÀGÀCÀTGCÀGGÀGÀCÀGCGTÀTTCCTTTTGTTGGGCÀ ATCGGATTGAATCTGTTGGACAAAAAAAAAAAAAAATAGAAC CÀCGÀCÀTÀGCCTCÀTGGÀGCÀCÀCÀCGCTT CGCCTCÀGCTCTCCAGGÀTCGAGGÀTTTGÀTGTCÀTTTCÀGTGÀG ÀGGÀÀCÀGGGÀTCÀCGGÀTÀGCGCTTGTCÀGCGT GGCTGCTTÀGÀGCCÀGÀCÀCÀCÀCÀCÀCÀCÀCÀCÀCÀCÀCÀCÀTGTC GAGTTTCTTCTCTCCCTTGAACCGÀGTCGTTÀTÀTCCGÀGÀGÀGÀCGTGCÀGÀC CCÀÀÀÀCÀGCÀÀÀGGCCÀCGCGÀCÀCCÀÀCÀÀCÀCGÀÀCÀG TGCTÀCÀTTGCCÀÀGÀGCTÀÀCTCGÀTÀGGGGÀÀCGC ÀGGTCGÀGGCÀGCÀGÀTTCÀCÀTGCÀGGCCÀÀTCÀCGGCGCT TCÀGCÀGCÀCGGÀTTCCÀÀTTGÀTÀGÀGCCCCÀCGÀÀCGGÀ CAttttAgcttgttAAacccAgtAAAgctgcttgggccgttgA ÀTTTÀCCÀCÀTGTTÀCTTÀÀGGTCGGTCCTGTÀTGTGTGTG CGGGCTGCGTGGGTGGTGTGTACTATAGCTTCAGCTTCTTCCTCT TTCCCCCCCTTTACAACCTTTTTTGCCTTTTTTGATAACTTGTCC TCACATCAGCATTGGCATCGTTGTAA List of references cited in this patent application

[0087] Kitagawa, M., and Yamada, H., Studies on the diamino acid, canavanin (II). J. Biochem. (Tokyo) 16, 339-349 (1932).

[0088] Benjamin E. Volcani, Esmond E. Snell - THE EFFECTS OF CANAVANINE, ARGININE, AND RELATED COMPOUNDS ON THE GROWTH OF BACTERIA -J. Biol. Chem. 174: 893-901

[0089] Horowitz (N. H.), SRB (A. M.), Growth inhibition of Neurospora by canavanine and its reversai. Journ. biol. Cbem., 1948, 174, 371

[0090] SRB, A. M., 1954 Effect antibiotique de la canavanine sur la levure. Academie de Sciences Paris Compt. Rend. 239: 447-448. - ,1955 Spontaneous and chemically-induced mutations giving rise to canavanine-resistance in yeast. Compt. Rend. Lab Carlesberg, Ser. Physiol. 23: 363

[0091] Grenson M, Mousset M, Wiame JM, Bechet J. - Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. I. Evidence for a spécifie arginine -transporting System. Biochim Biophys Acta 127(2):325-38

[0092] Broach JR, Strathem JN, Hicks JB. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(l):121-33. doi: 10.1016 / 0378-1119(79)90012-x

[0093] Ahmad M, Bussey H. Yeast arginine permease: nucléotide sequence of the CAN1 gene. Curr Genet. 1986; 10(8):587-92. doi: 10.1007 / BF00418125. PMID: 3327612.

[0094] Wong L, Holdridge B, Engel J, Xu P. Genetic Tools for Streamlined and Accelerated Pathway Engineering in Yarrowia lipolytica. Methods Mol Biol. 2019;1927:155-177. doi: 10.1007 / 978-1-4939-9142-6_11

[0095] M. Penttilâ, H. Nevalainen, M. Ratto, E. Salminen, J. Knowles. A versatile transformation System for the cellulolytic filamentous fungus Trichoderma reesei. Gene, 61 (1987), pp. 155-164, https: / / doi.org / 10.1016 / 0378-l 119(87)90110-7 Exemples

[0096] Exemple 1 : Test de sensibilité à la L-canavanine des souches de Trichoderma reesei

[0097] This test consists of verifying whether the growth of Trichoderma reesei is altered in culture on a medium containing L-canavanine, in order to validate the use of L-canavanine as a selection marker.

[0098] Varying quantities of Trichoderma reesei spores, from a fresh culture on PDA (Potato Dextrose Agar), are spread on two different media: - a rich medium called PDA containing in particular arginine, in which L-canavanine at a concentration of 200 qg / mL is added. - a minimal medium used for the culture of Trichoderma without arginine in which L-canavanine at a concentration of 50 qg / mL is added.

[0099] Spore culture is observed and the results are presented in [Fig. 1]. The results are observed at 4 days for the rich medium and at 8 days for the minimal medium.

[0100] Spores grow normally on PDA medium (which is a rich medium). On PDA medium supplemented by the addition of L-canavanine at 200 qg / mL, the spores are not affected by the presence of L-canavanine. Therefore, the rich medium does not allow for the observation of growth inhibition following the addition of the antimetabolite.

[0101] On minimal medium, spores grow more slowly than on PDA medium. This result is expected. On minimal medium supplemented by the addition of L-canavanine at 50 µg / mL, growth is very different from the control on minimal medium (MM) alone. The thalli are very small and fragile, and the higher the L-canavanine concentration, the greater the growth inhibition. MM medium, a minimal medium notably lacking arginine, allows for the observation of a significant effect on thallus growth following the addition of L-canavanine, even at a low concentration (50 µg / mL).

[0102] This result confirms that the addition of L-canavanine to a minimal medium, in particular devoid of arginine, does indeed have a deleterious effect on the growth of Trichoderma reesei.

[0103] The addition of L-canavanine can thus be used as a selection marker, provided that strains sensitive or not sensitive to the addition of L-canavanine in the medium are distinguished.

[0104] Example 2: Transformation and selection of transformed strains using L-canavanine

[0105] The aim of this experiment is to invalidate the ID gene 18487637 of Trichoderma reesei, and to verify whether its invalidation allows the selection of TRI67806 transformants (the strains in which the ID gene 18487637 has been invalidated) from the TRI67806+ strains (the strains in which the ID gene 18487637 has not been invalidated).

[0106] The invalidation is performed using the CRISPR-Cas9 tool, with the following components: - A nuclease: Alt-R™ Sp HiFi Cas9 Nuclease V3 - A guide: Alt-R™ CRISPR-Cas9 sgRNA (represented by SEQ ID NO: 3, CATC-CAGTACTGGACCGAGT CGG) A repair matrix (SEQ ID NO: 4) is also used. This is composed of: - A selection marker: a Hygromycin cassette composed of [CPC Promoter] (SEQ ID NO: 6), IM7 Promoter (SEQ ID NO: 7), Hygromycin Resistance Gene (SEQ ID NO: 9), TrpC Terminator (SEQ ID NO: 9)], - From two Ikb flanks with sequences identical to the two Ikb regions flanking the targeted gene, here ID 18487637 (SEQ ID NO: 5 and SEQ ID NO: 10).

[0107] All other techniques allowing the invalidation of the ID gene 18487637, in particular those mentioned above, can also be used instead of the CRISPR-Cas9 tool.

[0108] The protocol and the result of the transformation are presented in Table 2.

[0109] [Tables2] Composition Sublayer Toplayer Number of strains (protoplasts) Control Regeneration 50 pL of strains (1 / 1000 dilution) + 50 pL of CTS50 PDA medium + 0.2 M sucrose PDA medium + 0.8 M sucrose 77 Control - Sensitivity Hy-gromycin 50 pL of strains + 50 pL of CTS50 Minimum Medium + 0.2 M sucrose + 100 mg / mL Hy-gromycin Minimum Medium + 0.8 M sucrose + 100 mg / mL Hy-gromycin 0 Control - Sensitivity L-canavanine 50 pL of strains + 50 pL of CTS50 Minimum Medium + 0.2 M sucrose + 100 mg / mL L-canavanine Minimum Medium + 0.8 M sucrose + 100 mg / mL L-canavanine Transformation: 50 pL of strains + 43 pL of CTS50 + 5 pL of RNP complex + 2 pL of repair matrix. Minimum Medium + 0.2 M sucrose + 100 mg / mL L-canavanine. Minimum Medium + 0.8 M sucrose + 100 mg / mL L-canavanine. 142

[0110] Protocol and result

[0111] CTS50 corresponds to a dilution solution comprising 50 mM of CaC12; 0.1M of Tris-HCl; 0.4M of Sucrose, pH 7.5.

[0112] The regeneration control allows the toxicity of the transformation protocol to be assessed.

[0113] The protoplasts produced are not subjected to mutagenic agents (the complex CRISPR-Cas9 RNP (nuclease and SEQ ID NO guide: 3) and repair matrix (as indicated above), nor to growth on a selective medium. Its study, reported and compared to other transformations, provides information on the quality of the transformation protocol.

[0114] Sensitivity controls allow the number of false positives on the selection medium to be evaluated.

[0115] The protoplasts produced are not subjected to mutagenic agents (the CRISPR-Cas9 RNP complex (nuclease and SEQ ID NO guide: 3) and the repair template as described above), but are cultured on the selective medium set up to select for genetic mutants. These controls allow for the evaluation of the false positive rate, i.e., the number of spontaneous mutants that grow on the screen. For both sensitivity controls and within the framework of the transformation described above, no protoplasts were able to grow on a medium containing hygromycin or L-canavanine.

[0116] Transformation

[0117] The protoplasts produced are modified so that the mutation in the ID 18487637 gene, which transports arginine and its antimetabolite, is no longer functional. Thus, the transport of arginine and its antimetabolite from the culture medium into the cell is no longer possible. The mutation at the ID 18487637 site relies on the action of the RNP complex (CRISPR-Cas9 tool: nuclease and SEQ ID NO: 3 guide as indicated above) and on the insertion of the repair template (SEQ ID NO: 4) in place of the ID 18487637 gene.

[0118] By selecting transformants on minimal medium (MM) + L-canavanine, only those carrying a deleterious mutation in gene ID 18487637 are able to grow on a medium containing the antimetabolite. The L-canavanine sensitivity control shows no thallus, confirming that the transformants correspond to the expected genetic mutation (inactivation of gene ID 18487637 by its removal by the repair matrix). 142 transformants were obtained.

Claims

Claims

1. A strain of filamentous fungus belonging to the division Ascomycetes, excluding a fungus belonging to the class Saccharomycetes, in which: - gene ID 18487637, or a variant or an orthologous gene, has been invalidated, or - a gene coding for a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein, has been invalidated, said gene ID 18487637, or a variant or an orthologous gene, corresponding to a gene represented by SEQ ID NO: 1, or a gene having at least 80% identity with the gene of SEQ ID NO:

1.

2. Strain of fungus according to the preceding claim, in which the fungus belongs to the order Hypocreales, in particular to the genus Trichoderma, and even more particularly to the species Trichoderma reesei.

3. A fungal strain according to any one of claims 1 to 2, wherein gene ID 18487637, or a variant or orthologous gene, corresponds to a gene having at least 90% identity with the gene of SEQ ID NO: 1, more particularly at least 95%.

4. A fungal strain according to any one of claims 1 to 2, wherein the gene coding for a protein represented by SEQ ID NO: 2, or a variant protein, or an orthologous protein corresponds to a gene coding for a protein represented by SEQ ID NO: 2, or a protein having at least 80% identity with the protein of SEQ ID NO: 2, in particular at least 90% and more particularly at least 95%.

5. A method of genetically modifying a fungal strain according to any one of claims 1 to 4, comprising a step of invalidating said gene, a variant or an orthologous gene.

6. Use of L-canavanine as a strain selection marker according to any one of claims 1 to 4.

7. Use of L-canavanine according to claim 6, in the presence of a minimal medium or a synthetic medium devoid of arginine, optionally also devoid of alanine, aspartate and / or glutamate.

8. A method of selecting a strain according to any one of claims 1 to 4 comprising: - bringing a strain into contact with a culture medium containing L-canavanine, - selecting the strain if it is capable of growing in the culture medium.

9. A method of producing mushroom biomass, comprising a step of culturing a mushroom strain according to any one of claims 1 to 4 in a culture medium comprising a suitable substrate.

10. Method for producing proteins of interest, in particular enzymes, comprising a step of culturing a fungal strain according to any one of claims 1 to 4 in a culture medium comprising a suitable substrate.

11. Process 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 4.

12. 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 production of cellulolytic enzymes by a strain according to any one of claims 1 to 4, - 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.

13. 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 4.

14. Process for producing a biofuel from cellulosic or lignocellulosic substrates according to claim 13, comprising: - 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 any one of claims 1 to 4, - 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.

15. Use of a fungal strain according to any one of claims 1 to 4, for the hydrolysis of cellulose or lignocellulose into glucose, for the production of bio-sourced products, biofuel, sweet juice or sugars, from cellulosic or lignocellulosic substrates, and / or for the production of proteins of interest, more particularly enzymes, in particular cellulolytic enzymes such as cellulases.

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