METHOD FOR PRODUCING PROTEINS, SUGARS, AND ALCOHOL BY A STRAIN OF TRICHODERMA FUNGUS IN WHICH THE CEL1A GENE IS INVALID

Abstract

The invention relates to the various uses of a fungal strain belonging to the genus Trichoderma in which the cel1a gene is inactivated. In particular, the present invention relates to a method for producing proteins using a fungal strain belonging to the genus Trichoderma in which the cel1a gene is inactivated, comprising at least two steps: - a first step of batch growth in the presence of at least one carbon growth substrate, and - a second step of fed-batch protein production in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition. (no figure)

Description

Title of the invention: METHOD FOR PRODUCING PROTEINS, SUGARS, ALCOHOL, BY A STRAIN OF TRICHODERMA FUNGUS IN WHICH THE CEL1A GENE IS INVALID

[0001] The present invention relates to a method for producing proteins using (i) a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, and (ii) an optimized inducing composition. Context of the invention

[0002] Fungal strains belonging to the genus Trichoderma, particularly Trichoderma reesei, are now primarily used for enzyme production. These enzymes, such as cellulases, are used to hydrolyze cellulosic or lignocellulosic biomass into simple sugars. Enzymes produced by filamentous fungi are therefore useful in the production of second-generation biofuels or bio-based products derived from sugars obtained from (ligno)cellulosic biomass.

[0003] In order to improve the production of second generation biofuels or bio-based products, it has therefore been envisaged to improve the production of cellulases.

[0004] For example, patent EP 448 430 B1 describes an optimized industrial production of cellulases by Trichoderma reesei. This production is carried out using a fed-batch protocol (feeding without withdrawal) with a feed solution containing lactose as the sugar that induces protein production. This fermentation process comprises two steps: a first step of fungal growth in the presence of an excess of carbon source and a second step of enzyme production through the addition of an inducer to the medium at an optimized flow rate (fed-batch mode). These steps are carried out in a liquid medium in bioreactors under agitation and in the presence of oxygen, as the fungus is a strict aerobe. Another example of an optimized cellulase production process is described in patent EP 2 744 899 B1.

[0005] Traditionally, particularly in the laboratory, the feed solution used in the second step contains only lactose. However, industrially, lactose is too expensive to use alone in the feed solution. Various solutions have therefore been considered to reduce the amount of lactose required.

[0006] For example, Jourdier et al. showed that it was possible to replace some of the lactose with other, less expensive sugars such as glucose and / or purified xylose (see Jourdier et al. Biotechnology for Biofuels 2013, 6:79). In this study, the The authors analyzed the impact of glucose and / or xylose on enzyme secretion by a hyperproducing strain of Trichoderma reesei (strain CL847). This study concludes that in the industrial strain CL847, which originates from the model hyperproducing strain RutC30, induction capacity is positively correlated with the lactose content in the feed solution: the higher the lactose content, the better the induction.

[0007] International application PCT / FR2011 / 000350 also describes a process for the production of cellulolytic and / or hemicellulolytic enzymes using during the production phase a composition comprising 40 to 65% by weight of glucose, 21 to 25% by weight of lactose and 10 to 39% by weight of xylose in combination with a Trichoderma reesei fungus knocked out for catabolic repression by glucose.

[0008] Modified strains of Trichoderma reesei, in which regulatory elements of the promoter sequences of the xylanases genes xyn1 and xyn2 have been inserted into the cellulase promoters, have also been described in international application PCT / FR2016 / 050950. Thus, in said modified strains, the genes are inducible by their own inducing substrates, such as lactose, cellobiose, or cellulose, but also by the inducing substrates of the xylanases, such as xylan, xylose, ...

[0009] Fermentation processes and strains useful for improving the production of cellulolytic enzymes by filamentous fungi, and thereby of bio-based products but also of second-generation biofuels, are therefore already described in the prior art.

[0010] Nevertheless, there is still a need for new processes for the production of proteins, in particular cellulolytic enzymes, which are as efficient as possible, allowing in particular sufficient production while using as little lactose as possible in order to reduce costs.

[0011] Trichoderma reesei strains in which the cella gene has been inactivated have been described in the prior art. For example, Zhou et al. (Differential Involvement of fi-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulose Genes by Cellulose and Cellobiose. Eukaryotic Cell, 11 (11) 1371-1381 (2012)) and Xu et al. (Intracellular fi-Glucosidases CELla and CELlb Are Essential for Cellulase Induction on Lactose in Trichoderma reesei, Eukaryotic Cell 13 (8), pp. 1001-1013 (2014)) described the inactivation of the cella gene in the parent strain Trichoderma reesei TU-6 (ATCC MYA-256). Xu et al. concludes in particular that the CELla protein is an intracellular [3-glucosidase that is essential in lactose induction.

[0012] On the contrary, the present invention is based on the unexpected results of the inventors who demonstrated that it was possible to induce the production of proteins using an inducing composition containing only about 5 to 30% by weight of lactose relative to the total sugar content in said composition, using a strain of Trichoderma fungus, in particular Trichoderma reesei, in which the genome has been modified in order to invalidate the cella gene.

[0013] The inventors have shown in particular that the inactivation of the cella gene in a strain of Trichoderma reesei made it possible to divide by 10 the amount of lactose used in the production phase, while maintaining an equivalent specific productivity compared to a reference strain.

[0014] The inventors have shown in particular that the strain invalidated for cella, although it does not produce proteins when fed pure glucose or pure lactose, produces proteins correctly when fed glucose / lactose mixtures, with an optimal range for lactose contents in the feeding solution between 5% and 30% by weight of the cumulative sugars, with a maximum for contents between 10% and 20% by weight.

[0015] In particular, the inventors have shown that feed solutions having lactose contents comprising between 10 and 15% by weight of the cumulative sugars give an induction equivalent to pure lactose in the parent strain (in which the cella gene has not been invalidated), with specific protein production rates of the order of 20 ± 2 mg protein / g biomass / h. Brief description of the invention

[0016] The present invention thus relates to a method for producing proteins by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, comprising at least two steps: - a first growth stage in batch phase in the presence of at least one carbon-based growth substrate, and - a second stage of protein production in fed-batch phase in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition.

[0017] The present invention also relates to the use of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition for the production of proteins of interest by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated.

[0018] The present invention also relates to a process for producing sugars from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition. In this aspect, the invention therefore relates to the use of a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, for the hydrolysis of cellulose or lignocellulose into sugar.

[0019] The present invention also relates to a method 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 genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition. In this aspect, the invention therefore relates to the use of a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, for the production of bio-based products from cellulosic or lignocellulosic substrates.

[0020] The present invention also relates to a method for producing biofuel / alcohol from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition. In this aspect, the invention therefore relates to the use of a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, for the production of biofuel / alcohol from cellulosic or lignocellulosic substrates.

[0021] The present invention finally relates to a strain of fungus belonging to the species Trichoderma reeseL said strain being derived from the strain as filed under reference ATCC 56765 and said strain comprising an invalidation of the cella gene. Description of the invention

[0022] In a first aspect, the invention thus relates to a method for producing proteins by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, comprising at least two steps: - a first growth stage in batch phase in the presence of at least one carbon-based growth substrate, and - a second stage of protein production in fed-batch phase in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition.

[0023] According to the invention, the cella gene corresponds to the gene represented by SEQ ID NO: 1 or a gene having at least 80% identity with SEQ ID NO: 1. The cella gene is also named TRIREDRAFT_120749 in the Trichoderma reesei reference genome (https: / / www.uniprot.org / uniprot / G0RD31). This gene encodes a CELla protein belonging to family 1 of glycoside hydrolases. More specifically, CELla is an intracellular β3-glucosidase, also known as bgl2 or bglll (M. Saloheimo, J. Kuja-Panula, E. Ylôsmaki, et al. (2002) Enzymatic Properties and Intracellular Localization of the Novel Trichoderma reesei β3-Glucosidase BGLII (CellA). Applied and Environmental Microbiology, 68 (9) 4546-4553). The CELla protein is represented by SEQ ID NO: 2.

[0024] The cella gene is the reference gene in Trichoderma reesei. A gene having at least 80% identity thus represents a variant of this gene or an orthologous gene in another Trichoderma species. According to the invention, the expression "at least 80% identity with SEQ ID NO: 1" 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%, preferably at least 90%, at least 95%, and even more particularly at least 98%, at least 99%. A person skilled in the art can 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 means the percentage of identity over the total length of the sequences.The percentage thus corresponds to the number of identical nucleotides (residues if any) between this given sequence and SEQ ID NO: 1 divided by the number of nucleotides (residues if any) in the longer of the two sequences.

[0025] When the CEL1A protein is encoded by an orthologous gene or a variant of the cell gene, said protein may be represented by a protein having at least 80% identity with SEQ ID NO: 2, in particular at least 90%, at least 95%, preferably at least 98% or at least 99%.

[0026] According to a preferred embodiment of the invention, the fungus belongs to the species Trichoderma reesei. According to this embodiment, the cella gene is preferably represented by SEQ ID NO: 1 or a sequence having at least 98% identity with SEQ ID NO: 1. Most preferably, the cella gene is represented by SEQ ID NO: 1. The parent strain of Trichoderma reesei may in particular be the QM6a strain (deposited under number ATCC 13631), or a strain from the natural isolate QM6a (in particular obtained by random or directed mutagenesis), such as the Rut-C30 strain (deposited under number ATCC 56765), the strain deposited under number CNCM 1-5221 (deposited on August 3, 2017 with the CNCM, National Collection of Microorganism Cultures of the Pasteur Institute, located at 25 rue du Docteur Roux, F-75724 Paris cedex 15), the NG14 strain (deposited under number ATCC 56767) or the QM9414 strain (deposited under number ATCC 26921).

[0027] According to the invention, proteins are all proteins that can be produced by a fungus, either naturally or by genetic modification (for example, after transformation using a suitable vector).

[0028] 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 belonging to the glycoside hydrolase family, for example, selected from endoglucanases, exoglucanases, and glucosidases. Glycoside hydrolases are notably grouped under the nomenclature "EC 3.2.1." 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).The production of cellulases by a strain according to the invention, in particular Trichoderma reesei, can be determined by any techniques customary for a person skilled in the art, or by the techniques described in patents EP 448 430 B1 or EP2 744 899 Bl. .

[0029] The expression "the lactose content representing approximately between 5 and 30% by weight of the total sugar content" means that during the fed-batch step, between 70 and 95% by weight of the sugar(s) supplied by said composition are sugar(s) other than lactose. The total sugar content thus corresponds to 100% by weight of the sugar content of the composition.

[0030] According to one embodiment, the process according to the invention is carried out in a stirred and aerated bioreactor. More particularly, the stirring speed is controlled, notably to maintain a dissolved oxygen concentration above 40% of the saturation concentration. For example, in the laboratory, the stirring speed is generally controlled between 400 and 1200 rpm.

[0031] According to one embodiment, in said process the temperature is controlled during the first and second steps, in particular between 20 and 35°C. More specifically, The temperature is controlled at 27°C during the first stage and at 25°C during the second stage.

[0032] According to one embodiment, in said process, the pH is controlled. More particularly, the pH is controlled at 4.0, notably by automatic addition of a 5N ammonia solution.

[0033] According to one embodiment, the first phase continues until the carbon growth substrate is exhausted. This first stage generally lasts between approximately 18 and 48 hours, in particular 24 to 36 hours.

[0034] According to one embodiment, the second phase takes place over approximately 70 to 240 hours, in particular 70 hours.

[0035] According to a preferred embodiment, in said process, the first phase lasts between 18 and 48 hours, in particular for 24 to 36 hours, and the second phase takes place for about 70 to 240 hours, in particular 70 hours.

[0036] According to one embodiment, the process according to the invention makes it possible to obtain a productivity of between 15 and 20 mg protein / g biomass / h, in particular between 16 and 19 mg protein / g biomass / h. The ability of a sugar solution to induce growth on a strain is assessed by measuring the specific rate of protein production (also called "specific productivity"), expressed in mg protein / g biomass / h, during a culture with optimized feed rate of the sugar solution. It is known to those skilled in the art that the proteins produced by Trichoderma reesei are mainly enzymes, including cellulases. A correlation between total secreted proteins and cellulases can be established because in Trichoderma reesei, the main exoglucanases (CBHI, CBHII) and endoglucanases (EGI, EGII) can represent up to 90% of the total amount of secreted proteins (see, for example, Markov, AV, Gusakov, AV, Kondratyeva, EG, Okunev, ON, Bekkarevich, AO)., and Sinitsyn, AP (2005). New Effective Method for Analysis of the Component Composition of Enzyme Complexes from Trichoderma reesei. Biochemistry (Moscow) 70, 657-663). Thus, the productivity of enzymes or cellulases is therefore equivalent to the productivity of proteins.

[0037] The expression "a fungal strain belonging to the genus Trichoderma in which the cella gene is inactivated" means that the genome of the strains used in the present invention has been modified so that the cella gene is no longer expressed (or the variant gene or the orthologous gene, as the case may be). Thus, in the strains used in the present invention, the CELla protein is not produced, or a non-functional CELla is produced (i.e., the protein is no longer biologically active). In other words, the genome of the strain according to the invention is modified (or has been modified) so that the CELla protein is not synthesized or is synthesized in a non-functional form. Preferably, the CELla protein is not produced / synthesized. Gene inactivation is well known to those skilled in the art. According to one embodiment, the cella gene was inactivated in the strain by mutagenesis or homologous recombination, notably using an inactivation cassette such as represented by SEQ ID NO: 3.

[0038] The present invention thus relates to the use of a variant strain of the Trichoderma fungus in which the cella gene has been inactivated. 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 cella gene has not been inactivated. The strain according to the invention thus corresponds to a variant strain derived from a parent strain, said variant strain comprising at least one genetic modification corresponding to the inactivation of the cella gene compared to the parent strain.

[0039] During the "batch" growth stage, it is necessary to provide a rapidly assimilable carbon source for the growth of the Trichodermü fungus. According to the invention, the "carbon growth substrate" is preferably chosen from lactose, glucose, xylose, liquid residues obtained after ethanolic fermentation (optionally obtained after ethanolic fermentation and then distillation) of monomeric sugars from enzymatic hydrolysates of cellulosic biomass, a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass, an enzymatic hydrolysate of lignocellulose (i.e., before fermentation), and / or a hydrolysate of starchy biomass.Preferably, the substrate is chosen from glucose, xylose, liquid residues obtained after ethanolic fermentation (optionally obtained after ethanolic fermentation followed by distillation of monomeric sugars from enzymatic hydrolysates of cellulosic biomass), a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass, an enzymatic hydrolysate of lignocellulose (i.e., before fermentation), and / or a hydrolysate of starchy biomass. Very preferably, the substrate is glucose.

[0040] According to the invention, the concentration of carbonaceous growth substrate is in particular between 10 and 80 g / L, in particular between 15 and 40 g / L.

[0041] The composition used in the second protein production step of the process according to the invention may also be called a "feed solution" or "sugar feed solution." This composition comprises at least two sugars: lactose and another sugar. In one aspect of the invention, this composition comprises at least three different sugars: lactose and two other sugars.

[0042] Lactose is an inducing substrate, meaning that it allows the expression of proteins, particularly cellulases, in the culture medium. Preferably, in said composition, said second sugar is not an inducing sugar but is nevertheless a carbon substrate.

[0043] According to the invention, the sugar concentration of the composition used during the fed-batch step is between 200 and 500 g / L, in particular 250 g / L.

[0044] More specifically, the second sugar is supplied via a glucose and / or xylose solution (preferably purified), a starch biomass hydrolysate, an enzymatic hydrolysate of lignocellulosic biomass, a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass, and / or the liquid residues obtained after ethanolic fermentation (optionally obtained after ethanolic fermentation followed by distillation) of the monomeric sugars from the enzymatic hydrolysates of cellulosic biomass. The present invention thus makes it possible to use industrial sugar solutions, provided they are supplemented with lactose, to induce protein production.

[0045] Since these solutions mainly contain glucose and xylose, two sugars that do not induce protein production in Trichoderma reesei, it is necessary to supplement them with lactose so that the resulting mixture provides a sufficient level of induction. According to the invention, the composition comprising at least lactose and a second sugar can therefore also be called an inducing composition.According to a particular embodiment, the feed composition / solution used in the fed-batch step thus corresponds to a solution of glucose and / or xylose (preferably purified), a starch biomass hydrolysate, an enzymatic hydrolysate of lignocellulosic biomass, a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass and / or the liquid residues obtained after ethanolic fermentation (optionally obtained after ethanolic fermentation and then distillation) of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, to which lactose is added, so that the lactose content in said composition represents approximately between 5 and 30% by weight of the total sugar content.

[0046] Typically, a starchy biomass hydrolysate mainly contains glucose.

[0047] Typically, an enzymatic hydrolysate of lignocellulosic biomass mainly contains glucose and xylose.

[0048] Typically, a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass contains mainly xylose.

[0049] Typically, the liquid residues obtained after ethanolic fermentation (optionally obtained after ethanolic fermentation followed by distillation) of monomeric sugars from enzymatic hydrolysates of cellulosic biomass contain mainly non-fermentable sugars (arabinose, mannose, galactose) and residues of fermentable sugars (glucose and xylose).

[0050] More specifically, according to a preferred embodiment, the second sugar is selected from glucose and / or xylose, preferably glucose. According to one embodiment, said composition comprises only lactose and glucose as sugars, OR only lactose and xylose, OR only lactose, glucose, and xylose.

[0051] According to the invention, the percentages of lactose, glucose, and / or xylose in the composition are calculated relative to the total weight content of the sugars in said composition. Typically, the total sugar content is that used in the fed-batch mode. The content of each sugar in the composition / solution is, for example, measured by high-performance liquid chromatography (HPLC), optionally after concentration by evaporation. Then, purified lactose or glucose powder is dissolved, or a concentrated glucose solution is mixed. The new contents of each sugar are then remeasured by HPLC in the final mixture.

[0052] According to one embodiment, the composition during the fed-batch step is supplied continuously, in a flow-limiting manner. This means that the sugar concentration in the culture medium is controlled to maintain a residual sugar concentration close to zero. Preferably, the sugar concentration in the culture medium is less than 1 g / L during this phase, in particular less than 0.5 g / L, and more specifically less than 0.1 g / L. This promotes the induction and production of proteins.

[0053] Since the Trichoderma fungus, particularly Trichoderma reesei, is capable of consuming all the sugars present in said inducing composition, the calculation of the optimized flow rate of said composition (feeding solution for the fed-batch mode) must be based not on the lactose concentration of the solution but on the summation of the concentrations of all the sugars present in the solution. In one embodiment, it is the mass flow rate (in g sugars / h) that is optimized and not the total sugar concentration in the feeding solution (in g sugars / L). The volumetric flow rate of the feeding solution (in L / h) can thus be adjusted to provide the correct mass flow rate of sugars regardless of the sugar concentration in the feeding solution.According to one embodiment, said composition in the production step (fed-batch) is supplied at a flow rate of between 0.8 and 8 mLsoiution / Lmiiieu / h, preferably between 1 and 3 mLsoiution / Lmiiieu / h. The term "mLsoiution" here represents the volume of said composition (feeding solution for the fed-batch mode) and the term "Lmiiieu" here represents the volume of the bioreactor / fermenter.

[0054] According to the invention, the expression "approximately between 5 and 30% by weight of the total sugar content in said composition" represents all values ​​between 5 and 30, that is, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. More specifically, the term "approximately" means that values ​​very slightly below 5 (for example, between 4.5 and 5) are included in the 5-30% range. It also means that values ​​very slightly above 30 (for example, between 30 and 30.5) are included in the 5-30% range.

[0055] According to a particular embodiment, in the protein production process according to the invention, said composition comprises, with respect to the total sugar content in said composition: - approximately between 70-95% by weight of glucose and / or xylose, and - approximately between 5-30% by weight of lactose.

[0056] The expression "approximately between 70-95%" means that values ​​very slightly below 70 (for example, between 69.5 and 70) are included in the 70-95% range. It also means that values ​​very slightly above 95 (for example, between 95 and 95.5) are included in the 70-95% range.

[0057] According to a further more particular embodiment, in the protein production process according to the invention, said composition comprises, with respect to the total sugar content in said composition: - between 70-95% by weight of glucose, and - between 5-30% by weight of lactose.

[0058] Even more preferably, in said protein production process according to the invention, the lactose content in said composition represents approximately between 10 and 20% by weight, in particular between 10 and 20% by weight, of the total sugar content in said composition.

[0059] The expression "approximately between 10 and 20%" means that values ​​very slightly below 10 (for example, between 9.5 and 10) are included in the 10-20% range. It also means that values ​​very slightly above 20 (for example, between 20 and 20.5) are included in the 10-20% range.

[0060] According to a further particular embodiment, in the protein production process according to the invention, said composition comprises: - approximately 80-90% by weight of glucose and / or xylose, and - approximately 10-20% by weight of lactose, and more particularly - approximately 80-90% by weight of glucose, and - approximately 10-20% by weight of lactose.

[0061] Even more preferably, in said protein production process according to the invention, the lactose content in said composition represents approximately between 10 and 15% by weight, in particular between 10 and 15% by weight, of the total sugar content in said composition.

[0062] The expression "approximately between 10 and 15%" means that values ​​very slightly below 10 (for example, between 9.5 and 10) are included in the 10-15% range. It also means that values ​​very slightly above 15 (for example, between 15 and 15.5) are included in the 10-15% range.

[0063] According to a further more particular embodiment, in the protein production process according to the invention, said composition comprises: - approximately 85-90% by weight of glucose and / or xylose, and - approximately 10-15% by weight of lactose, and more specifically - approximately 85-90% by weight of glucose, and - approximately 10-15% by weight of lactose.

[0064] According to one embodiment, the substrates are sterilized. Thus, according to one embodiment, the carbon-based growth substrate is introduced into the bioreactor before sterilization. According to another embodiment, the carbon-based growth substrate is sterilized separately and then introduced into the bioreactor after sterilization. According to either of these alternatives, in one embodiment, the sugar composition used during the fed-batch step is sterilized separately and then introduced into the bioreactor after sterilization.

[0065] In a second aspect, the invention relates to the use of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition for the production of proteins of interest by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated.

[0066] In a third aspect, the invention relates to a process for producing sugars from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition. In other words, the invention relates to the use of a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, for the hydrolysis of cellulose or lignocellulose into sugar.

[0067] In a fourth aspect, the invention 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 genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition. In other words, the invention relates to the use of a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, for the production of bio-based products from cellulosic or lignocellulosic substrates.

[0068] According to the invention, the term "bio-based products" refers more particularly to molecules of interest to the chemical industry, such as organic acids like acetic, propionic, acrylic, butyric, succinic, malic, fumaric, citric, itaconic acid, or hydroxy acids like glycolic, hydroxypropionic, or lactic acid.

[0069] In a fifth aspect, the invention relates to a process for producing a biofuel / alcohol, in particular ethanol, from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition. In other words, the invention relates to the use of a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, for the production of biofuel / alcohol from cellulosic or lignocellulosic substrates.

[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 resulting from the transformation of biomass and capable of being used for energy purposes. Examples include, but are not limited to, biogas, products that 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 fermentative organism used), solvents (acetone), acids (butyric acid), 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, isopropanol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, and / or or 2,3-butane diol. More preferably, the biofuel according to the invention is ethanol. In another embodiment, the biofuel is biogas.

[0071] According to a particular embodiment, said process for producing a biofuel or alcohol from cellulosic or lignocellulosic substrates comprises the following steps: - 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 of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition, - iii) an enzymatic hydrolysis step of the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step ii) and a suitable substrate, in order to obtain a hydrolysate, - iv) an alcoholic fermentation step of the hydrolysate obtained,. - v) a separation step, in particular by distillation.

[0072] According to another particular embodiment, said process for producing a biofuel or alcohol from cellulosic or lignocellulosic substrates comprises the following steps: - 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 of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition, - iii) an enzymatic hydrolysis step of the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step ii) and a suitable substrate, in order to obtain a hydrolysate, - 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 Fermentation) production processes.

[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 or alcohol and the fermentation must, in particular by distillation.

[0077] According to a further preferred embodiment, the pretreated 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 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 sixth aspect, the invention relates to a strain of fungus belonging to the species Trichoderma reesei, said strain being derived from the strain as filed under reference ATCC 56765 (Rut-C30) and said strain comprising a cella gene inactivation. The invention thus also relates to a RutC30 strain whose genome has been modified to inactivate the cella gene. This variant strain of RutC30 is therefore a strain in which the CELla protein is not produced, or a non-functional CELla protein is produced.

[0080] According to one embodiment, such a strain is obtained by a genetic modification process of a Rut-C30 fungal strain, comprising a cella gene knockout step. This cella gene knockout step is carried out in particular by mutagenesis, by homologous recombination or more preferably using a knockout cassette represented by SEQ ID NO: 3.

[0081] Mutagenesis is a technique commonly used in genetic engineering. It aims to intentionally introduce mutations into DNA in order to create genetically modified genes. According to the invention, mutagenesis refers more specifically to site-directed mutagenesis. Site-directed mutagenesis allows the introduction of identified mutations into a specific gene. To do this, the DNA of interest (here, the cella gene) containing the mutations is synthesized and then introduced into the cell to be mutated, typically using a vector, where the DNA repair mechanism integrates it into the genome.

[0082] Homologous recombination is a technique commonly used in genetic engineering which consists of an exchange between DNA molecules, typically using a vector.

[0083] The term “vector” refers to any DNA sequence into which fragments of foreign nucleic acid can be inserted, vectors enabling the introduction of foreign DNA into a host cell. Examples of vectors include plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and PI-derived bacteriophage artificial chromosomes (PACs), and virus-derived vectors. The vector according to the invention allows the introduction of a mutation or deletion.

[0084] In a preferred embodiment, said invalidation cassette comprises three DNA fragments:

[0085] (1) a region upstream of the target gene,

[0086] (2) a selection marker, and

[0087] (3) a region downstream of the target gene.

[0088] In the case of the present invention, the "target gene" means the cella gene. The upstream and downstream regions of the target gene are two recombination elements, one at each end of the gene, and are necessary to precisely target the sequence to be inactivated.

[0089] According to the invention, the region upstream of the target gene (i.e. the 5' sequence upstream of the cella gene) is notably represented by the sequence of SEQ ID NO: 4.

[0090] According to the invention, the region downstream of the target gene (i.e. the 3' sequence downstream of the cella gene) is notably represented by the SEQ ID NO sequence: 5.

[0091] The expression "selection marker" refers to a gene whose expression confers on the cells that contain it a characteristic enabling them to be selected. The use of a selection marker makes it possible to identify cells that have incorporated a genetic modification compared to those that have not. This is, for example, a gene for antibiotic resistance, in particular the gene for resistance to the antibiotic hygromycin hph, as represented by the SEQ ID NO sequence: 6.

[0092] More specifically, according to the invention, the inactivation cassette preferably consists of a resistance gene placed under the control of a promoter and a terminator, with the 5' and 3' flanking regions of the cella gene upstream and downstream. According to the invention, said inactivation cassette can be operationally linked to a promoter, a terminator, or any other sequence necessary for its expression in a host cell.

[0093] The inactivation cassette can be amplified using conventional techniques well known to those skilled in the art, typically by a method chosen from conventional cloning, PCR fusion, or in vivo PCR cloning. Preferably, this inactivation cassette is amplified by PCR, in particular using the sequences represented by SEQ ID NO: 9 and SEQ ID NO: 11. The inactivation cassette is then introduced by recombination into a Trichoderma strain, in particular Trichoderma reesei, which does not express a gene of the selection marker. After culture, the variant / mutant strains incorporating the inactivation cassette are selected based on the expression or absence of the selection marker; the clones that have been transformed express said selection marker. These are, in particular, the strains to be used according to the invention. Preferably, mutant strains are identified using SEQ ID NO: 8 and SEQ ID NO: 13 primers.These genetic recombination techniques are well known to those skilled in the art.

[0094] In this description, the definitions, embodiments, 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, and sixth aspects. Brief description of the Figures

[0095] Other features, details and advantages of the invention will become apparent from reading the accompanying Figures. Fig. 1

[0096] [Fig. 1] represents the pRS426-Acella-hph plasmid which was used to obtain a strain in which the cella gene was inactivated. The positions of the different fragments of the inactivation cassette and of the oligonucleotides used for the construction are indicated. Fig. 2

[0097] [Fig.2] represents the optimization of the lactose / glucose mixture composition used as a feeding solution for the protein production phase by Trichoderma reesei RutC30-Acella. Fig. 3

[0098] [Fig.3] represents the comparison of the specific productivity in bioreactor of RutC30 and RutC30-Acella strains when fed-batch with sugar solutions of different compositions. Sequences of the present invention

[0099] [Tables 1] Sequence Name nce SEQID NO : 1 atgttgcccaaggactttcagtgggggttcgccacggctgcctaccagatcgagggcgcc gtcgaccaggacggccgcggccccagcatctgggacacgttctgcgcgcagcccggcaag atcgccgacggctcgtcgggcgtgacggcgtgcgactcgtacaaccgcacggccgaggac attgcgctgctcaagtcgctcggggccaagagctaccgcttctccatctcgtggtcgcgc atcatccccgagggcggccgcggcgatgccgtcaaccaggcgggcatcgaccactacgtc aagttcgtcgacgacctgctcgacgccggcatcacgcccttcatcaccctcttccactgg gacctgcccgagggcctgcatcagcggtacggggggctgctgaaccgcaccgagttcccg ctcgactttgaaaactacgcccgcgtcatgttcagggcgctgcccaaggtgcgcaactgg atcaccttcaacgagccgctgtgctcggccatcccgggctacggctccggcaccttcgcc cccggccggcagagcacctcggagccgtggaccgtcggccacaacatcctcgtcgcccac ggccgcgccgtcaaggcgtaccgcgacgacttcaagcccgccagcggcgacggccagatc ggcatcgtcctcaacggcgacttcacctacccctgggacgccgccgacccggccgacaag gaggcggccgagcggcgcctcgagttcttcacggcctggttcgcggatcccatctacttg ggcgactacccggcgtcgatgcgcaagcagctgggcgaccggctgccgacctttacgccc gaggagcgcgccctcgtccacggctccaacgacttttacggcatgaaccactacacgtcc aactacatccgccaccgcagctcgcccgcctccgccgacgacaccgtcggcaacgtcgacgtgctcttcaccaacaagcagggcaactgcatcggccccgagacgcagtccccctggctg cgcccctgtgccgccggcttccgcgacttcctggtgtggatcagcaagaggtacggctac ccgcccatctacgtgacggagaacggcacgagcatcaagggcgagagcgacttgcccaag gagaagattctcgaagatgacttcagggtcaagtactataacgagtacatccgtgccatg gttaccgccgtggagctggacggggtcaacgtcaaggggtactttgcctggtcgctcatg gacaactttgagtgggcggacggctacgtgacgaggtttggggttacgtatgtggattat gagaatgggcagaagcggttccccaagaagagcgcaaagagcttgaagccgctgtttgac gagctgattgcggcggcgtga SEQID NO : 2 MLPKDFQWGFATAAYQIEGAVDQDGRGPSIWDTFCAQPGKIA DGSSGVTACDSYNRTAEDIALLKSLGAKSYRFSISWSRIIPEGGR GDAVNQAGIDHYVKFVDDLLDAGITPFITLFHWDLPEGLHQRYGG LLNRTEFPLDFENYARVMFRALPKVRNWITFNEPLCSAIPGYGSG TFAPGRQSTSEPWTVGHNILVAHGRAVKAYRDDFKPASGDGQ IGIVLNGDFTYPWDAADPADKEAAERRLEFFTAWFADPIYLGDYP ASMRKQLGDRLPTFTPEERALVHGSNDFYGMNHYTSNYIRHRSSP ASADDTVGNVDVLFTNKQGNCIGPETQSPWLRPCAAGFRDFLVWI SKRYGYPPIYVTENGTSIKGESDLPKEKILEDDFRVKYYNEYIRA MVTAVELDGVNVKGYFAWSLMDNFEWADGYVTRFGVTYVDYE NGQKRFPKKSAKSLKPLFDELIAAA SEQID GTGACCGAACAGCAGCAGCAGCAGCAGCAGGTGCCGACGACG NO : 3 ACGGAGGCGAAGCGGAGCGCCGCGCTGCGCCAGTCGTTTGGC GTGCCCTGGTTCGAGACGCTGATTGAGGGCTCGCGCCTGGGC AGCATGCGCCGGAGCTACGGGGCGCAGCGGTCGCGCGACGGG CAGGCGAGCATCGAGTGGGAGATTGTCGAGTTCTCGGACGGC GGCGGGGAGATGGACTTGGGCGAGGCGGACGACGCGGCGCTG CAGCAGCTGGGCAAGAGGAAGCATCACGAGGTTTGACCGGGG AGGGGGCCAAATAGCTTTGGGTATTACACACACACGTGTGTG TGTGTGTGTGAGAGGTGCAAGTGAGGTGGCGAGTGAGTTGAC TGAGTAACTGAGTGGCTGAGTGAGCGAGTGATGTGTAACAAC AACAGCTGTCGGAAAACAAAAGGGGAACCGATACGCCGTTCG TGGTTCAGGGGTAATGTGTGTGTCTGGCATGGTTGGCGTGGTCC GGTCTGTATATGAGCTGTCGCTTCTGTATCGTTTGTCTTCTTGTT GCAATTCTTTTTTTTCTTGTCCTCGGTCAGGCGTTGTTGTGCAGA CAGTGGTTGGGGAGGGAGGCGGTTGTCTGTCCACCTTGCACC TTTTTCATCATTTTGCCTTTAGGCGTAACTCGATAGCAGAGG CTATATAGAGAGATCCAGATGGGAATCGACGCAAAACCTTTT GGAGTCACCACTCGGGACGCTTGTGCCTTTCGTCTTTATTAT GCATAAATGCCACCTCCAGCCGGCGTGCAGCGTATACTTGTA CACTTCAGACTTGTAGTAGTAGGTAGTACTGTATGTGACTAG GTACTATTCATCCGCAAAGTGTTGTCAAGGAACATGCGGCTTATAAGAGTAGCACTCGGCTGGAATATGGCGTTTGATCCTCGC AATCCAATTCCCCTTTGCATGATTACATGGCAACTGTCCCAC GTGCTGTCTCGGCTGAATCTATCCGTCGGCTTACCAGAAAGAAA CGAAGCCGGACAGAATAGCCAGATTTCCATACCTTCAACTCC TTTGTTTCTGTCTCCTATTCCCGCCATTCCCCGATGCCTCGAACT ACTGGCAGCAAAAATCTCCCCGTCTTCTCACCTTTCGACCAC TCCTCCTCCTCCGATCCTCTCTTTCCCCTCTCAACGTCCCTCAGC GTACCATCAAAGTGATAATCAGCAGCAGCAGCGGCAGCAGCT CTTTCGGAGTATCACCGTGTCACACCCTCTCCCCCCATCGCC GGGCTAAGCTGCTTGGAGCCGTCCGGATCGCCCGCCTCCGCT TCTCCAGCTTCCCCATTTACGTTGAGGTGCGGCTAGCCTTGCCTT GTGACTCGCCTTTTCTGCTTGTATCGGCCAGGGGGGGGTTTG GTTAGGTGGTTGGCTGGTTGGCTGGTTGGTTAGGATACTGTA GAAAAGGGATCCGAGAGCTACCTTACATCAATATGGCCAGCACCT CTTCGGCGATACATACTCGCCACCCCAGCCGGGGCGATTGTGTGT ACTAGGTAGGCTCGTACTATACCAGCAGGAGAGGTGCTGCTTGGC AATCGTGCTCAGCTGTTAGGTTGTACTTGTATGGTACTTGTAAGG TGGTCATGCAGTTGCTAAGGTACCTAGGGAGGGATTCAACGAGCC CTGCTTCCAATGTCCATCTGGATAGGATGGCGGCTGGCGGGGCCG AAGCTGGGAACTCGCCAACAGTCATATGTAATAGCTCAAGTTGAT GATACCGTTTTGCCAGGATTAGGATGCGAGAAGCAGCATGAATGT CGCTCATCCGATGCCGCATCACCGTTGTGTCAGAAACGACCAAGC TAAGCAACTAAGGTACCTTACCGTCCACTATCTCAGGTAACCAGG TACTACCAGCTACCCTACCTGCCGTGCCTACCTGCTTTAGTATTA ATCTTTCCACCTCCCTCCTCAATCTTCTTTTCCCTCCTCTCCTCTTT TTirTTTCirccTCCTCTTCTTCTCCATAACCAirccTAACAACATCG ACATTCTCTCCTAATCACCAGCCTCGCAAATCCTCAGGTTAGTATTAC TACTACTACAATCATCACCACGATGCTCCGCCCGACGATGCGGCT TCTGTTCGCCTGCCCCTCCTCTCACTCGTGCCCTTGACGAGCTAC CCCGCCAGACTCTCCTGCGTCACCAATTTTTTTCCCTATTTACCC CTCCTCCCTCTCTCCCTCTCGTTTCTTCCTAACAAACAACCACCA CCAAAATCTCTTTGGAAGCTCACGACTCACGCAAGCTCAATTCGC AGATACAAATCTAGAATGAAAAAGCCTGAACTCACCGCGACGTCT GTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTG ATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGA TGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGC CGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGA GAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTT GCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGT CGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGAC GAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACAC TACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTA TCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGC GCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGA AGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCT GACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGG CGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCT GGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCG AGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGG CGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGG TTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCG ACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAA TCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAG TACTCGCCGATAGTGGAAACCGACGCCCAGCACTCGTCCGAGGG CAAAGGAATAATGCATGTGCTGTGTTCCTCCAGAATGGGGCCCAGA AGGGCGTCGAGCATTGTCTATGAATGCAAAAAATAGTAAATA AATAGTAATTCTGGCCATGACGAATAGGCCAATCTGCTCCACTTGA CTATCCTTGTGAACTGTTATCGTTATGTATGTCGAACCCTTGACTGCCCATT CAAACAATTGTAAGGAATAGCTACAAGTTAGTTCCACGTTTGCACGTTTGTACGTTATTTGGAAAAGCGTTGCCATCAC ATGCTCACAGTCACTTGGGCTTACGATCATGTTTGCGATCTTTCGG TAAGAATACACAGAGTAACGATTATACATCCATCGCTTTCTATGATTA GGTACTCAGACAACACATGGGAAACAAGATAACCATCGCATGCAA GGTCGATTCCAATCATGATCTGACTGGGGTATTCCATCTAAGCC ATAGTACCCTCGAGCCCATCATCGTCTGCGCCATGGCGCTCATG GCGGACGGGGGCATGGATGATGGCATGGATGATGGCATGGAT GATGGCATGGACGATGGCATGGACGATGGCTGGGGCCCGCCA CCGCCCTTAACGGGACCGTAGATGGCCTCGTCGTCCTCGTG TCATCTGTATCATCGTCGTCTTCGTCCTTTTTCGCTTTTGGG TGCTTCGCGGGAGACGCATATTCTTTGCGACTTCGTCCGCG CTGAAGCCGTAGATGCTCTTGAGGTAGCGCATGAACTCGGG TACGCATCCGACACGATTTGCACATGCAAGTGCGTCAACTCG ACCTTGGCATCCTCATCCCTCACGCCCTGCTTCTTCTTCTTTCCTTCTTCTCCCACGTTGGTGCTGCTGATGCTCCGATTCTGGGCG TCAAACTCGGCCATGGCCAGCGCCGTCTGGCACGCGTTGCGA ATCTGCCGGCCGTTCCAGCGCATGTTTTCGTGCTTCTTCCAG TATGCGGTGGCGTACTTGAGAATGTCCTTTTCGTGGATGTCGAT TTCGCGGCCCTTGTCGCGGAAGCGCTGCTTGATGATGCGCAG GTTGAGGCGGAAGATTTCGCGCGTGGACGATTTGTCGAGGGG CGGGTAGTAGAGGGAGATGTGGATGCGGGAGGTGAAGGCCTC GTCAAAGTCGCCGATGCGGTTGGTCGTGAGGAAGAGGATGCC TGCGTAGTATTCGAGGACGCGGAGGAAGACTTGAGTTGACGA GAGTTTAGCTGATTCTGAGTAGGGGGTAGCGCACAGATAGGG GTAAAGGGGTTGGAGGTCAACTTACCTGCTACGAGGCCGTTG CGGACAAAGTTCTTGGGGGAGCGCTGGGCGAGAAAGACGTCT GCTTCGTCGAGGAGGAGGATGCAGCCCCATCGGTTGGCCAGG CTAAAGTTTCTCTCCAGGGCCGCTTCTACTTCACTGGCAGTT GCACCAAGATCTCCTGCAAAAGGAGTTGTTGTGTACATGCTTTA GCCTATATGAATCTTGTAGATTACTTGGACACAGGAAGTGAC AGTGCACATACCGCAGGTGATTTGGAACAAAGGCTTGTTGAA GCGTTCGGCAACACCCTCTATAACATCCATGTCAGCACATGT ACGAGGAGGGTTGGCATGATTCTCTGAACTTACCAGCAGTTG TTGTCTTTCCCACGCCTGGACTTCCATGTAACAGAAGAATGA GGCCCTTCCCTGTATAAAGAAGTCAGTTGATTCGCGCTCAAA AAGCCTTGTCAAAAAGGGGAATACCTTTGCCTCGAATAATGT CCACCTCTTCATTATCGCTAACCCTCGCCTCCTTGTTCCGAAAGT GCTGGTCAACCAAACAATACACAATGTCCTTGTGCTGCTTCG GGAGCACCAGCTGGTCAAACGCCGTCTGCTCTTTTGAGCCCT CGCTGACGGGACTGAGATACTTGAGATCGAGCTTCGCTATCC AGCAAGTCAGCACACACACTCTTCAACGCAGTAAAAAAGAG AAGAAAAAAGGGGGGGGATAAGTGACGAACCC SEQID ACGATGATACCGACGCCGCCGTGACCGAACAGCAGCAGCAG NO : 4 CAGCAGCAGGTGCCGACGACGACGGAGGCGAAGCGGAGCGCC GCGCTGCGCCAGTCGTTTGGCGTGCCCTGGTTCGAGACGCTG ATTGAGGGCTCGCGCCTGGGCAGCATGCGCCGGAGCTACGGG GCGCAGCGGTCGCGCGACGGGCAGGCGAGCATCGAGTGGGAG ATTGTCGAGTTCTCGGACGGCGGCGGGGAGATGGACTTGGGC GAGGCGGACGACGCGGCGCTGCAGCAGCTGGGCAAGAGGAAG CATCACGAGGTTTGACCGGGGAGGGGGCCAAATAGCTTTGGG TATTACACACACACGTGTGTGTGTGTGTGTGAGAGGTGCAAG TGAGGTGGCGAGTGAGTTGACTGAGTAACTGAGTGGCTGAGT GAGCGAGTGATGTGTAACAACAACAGCTGTCGGAAAACAAAAGGGGAACCGATACGCCGTTCGTGGTTCAGGGGTAATGTGTGT GTCTGGCATGGTTGGCGTGGTCCGGTCTGTATATGAGCTGTCGC TTCTGTATCGTTTGTCTTCTTGTTGCAATTCTTTTTTTTCTTGTC CTCGGTCAGGCGTTGTTGTGCAGACAGTGGTTGGGGAGGGAG GCGGTTGTCTGTCCACCTTGCACCTTTTTCATCATTTTGCCTTTA GGCGTAACTCGATAGCAGAGGCTATATAGAGAGATCCAGATG GGAATCGACGCAAAACCTTTTGGAGTCACCACTCGGGACGCT TGTGCCTTTCGTCTTTATTATGCATAAATGCCACCTCCAGCC GGCGTGCAGCGTATACTTGTACACTTCAGACTTGTAGTAGTA GGTAGTACTGTATGTGACTAGGTACTATTCATCCGCAAAGTG TTGTCAAGGAACATGCGGCTTATAAGAGTAGCACTCGGCTGG AATATGGCGTTTGATCCTCGCAATCCAATTCCCCTTTGCATG ATTACATGGCAACTGTCCCACGTGCTGTCTCGGCTGAATCTA TCCGTCGGCTTACCAGAAAGAAACGAAGCCGGACAGAATAG CCAGATTTCCATACCTTCAACTCCTTTGTTTCTGTCTCCTATTCC CGCCATTCCCCGATGCCTCGAACTACTGGCAGCAAAAATCTC CCCGTCTTCTCACCTTTCGACCACTCCTCCTCCTCCGATCCTCTC TTTCCCCTCTCAACGTCCCTCAGCGTACCATCAAAGTGATAA TCAGCAGCAGCAGCGGCAGCAGCTCTTTCGGAGTATCACCGT GTCACACCCTCTCCCCCCATCGCCGGGCTAAGCTGCTTGGAG CCGTCCGGATCGCCCGCCTCCGCTTCTCCAGCTTCCCCATTT ACGTTGAGGTGCGGCTAGCCTTGCCTTGTGACTCGCCTTTTC TGCTTGTATCGGCCAGGGGGGGGTTTGGTTAGGTGGTTGGCT GGTTGGCTGGTTGGTTAGGATACTGTAGAAAAG SEQID CCCATCATCGTCTGCGCCATGGCGCTCATGGCGGACGGGGGC NO : 5 ATGGATGATGGCATGGATGATGGCATGGATGATGGCATGGAC GATGGCATGGACGATGGCTGGGGCCCGCCACCGCCCTTAACG GGACCGTAGATGGCCTCGTCGTCGCTCGTGTCATCTGTATCA TCGTCGTCGTCTTCGTCCTTTTTCGCTTTTGGGTGCTTCGCGCGG AGACGCATATTCTTTGCGACTTCGTCCGCGCTGAAGCCGTAG ATGCTCTTGAGGTAGCGCATGAACTCGAGGTACGCATCCGAC ACGATTTGCACATGCAAGTGCGTCAACTCGACCTTGGCATCC TCATCCCTCACGCCCTGCTTCTTCTTCTTTCCTTCTTCTCCCACG TTGGTGCTGCTGATGCTCCGATTCTGGGCGTCAAACTCGGCC ATGGCCAGCGCCGTCTGGCACGCGTTGCGAATCTGCCGGCCG TTCCAGCGCATGTTTTCGTGCTTCTTCCAGTATGCGGTGGCGTACTTGAGAATGTCCTTTTCGTGGATGTCGATTTCGCGGCCCTTGTC GCGGAAGCGCTGCTTGATGCGCAGGTTGAGGCGGAAGAT TTCGCGCGTGGACGATTTGTCGAGGGGCGGGTAGTAGAGGGA GATGTGGATGCGGGAGGTGAAGGCCCGTCAAAGTCGCGAT GCGGTTGGTCGTGAGGGAAGGAGGATGCCTCGTAGTATTCCGAG GACGCGGAAGACTTGAGTTGACGAGAGTTTAGCTGATTC TGAGTAGGGGGTAGCGCACAGATAGGGGTGAAAGGGGTTGGAG GTCAACTTACCTGCTACGAGGCCGTTGCGGACAAAGTTCTTG GGGGAGCGCTGGCGGAGAAAGACGTCTGCTTCGTCGAGGAGG AGGATGCAGCCCCATCGGTTGGCCAGGCTAAAGTTTCTTCTCTC AGGGCCGCTTCTACTTCACTGGCAGTTGCACCAAGATCTCCT GCAAAAAGGAGTGTGTGTGACATGCTTTAGCCTATATGAATCTT GTAGATTACTTGGACACAGAAGTGCAGTGCACATACCACAGATCCT GGTGATTTGGAACAAAGGCTTGTTGAAGCGTTCGGCAACACC CTCTATAACATCCATGTCAGCACATGTACGAGGAGGGTTGGC ATGATTCTCTGAACTTACCAGCAGTTGTTGTCTTTCCCACGCCTG GACTTCCATGTAACAGAAGAATGAGGCCCTTCCCTGTATAAA GAAGTCAGTTGATTCGCGCTCAAAAAGCCTTGTCAAAAAGGG GAATACCTTTGCCTCGAATAATGTCCACCTCTTCATTATCGC TAACCCTCGCCTCCTTGTTCCGAAAGTGCTGGTCAACCAAAC AATACACAATGTCCTTGTGCTGCTTCGGGAGCACCAGCTGGT CAAACGCCGTCTGCTCTTTTGAGCCCTCGCTGACGGGACTGA GATACTTGAGATCGAGCTTCGCTATCCAGCAAGTCAGCACAC ACACTCTTCAACGCAGTAAAAAAGAGAAGAAAAAAGGGGGG GGATAAGTGACGAACCCCATTTACGGCTCCTCAACACA SEQID GGATCCGAGAGCTACCTTACATCAATATGGCCAGCACCTCTT NO : 6 CGGCGATACATACTCGCCACCCCAGCCGGGGCGATTGTGTGT ACTAGGTAGGCTCGTACTATACCAGCAGGAGAGGTGCTGCTT GGCAATCGTGCTCAGCTGTTAGGTTGTACTTGTATGGTACTT GTAAGGTGGTCATGCAGTTGCTAAGGTACCTAGGGAGGGATT CAACGAGCCCTGCTTCCAATGTCCATCTGGATAGGATGGCGG CTGGCGGGGCCGAAGCTGGGAACTCGCCAACAGTCATATGTA ATAGCTCAAGTTGATGATACCGTTTTGCCAGGATTAGGATGC GAGAAGCAGCATGAATGTCGCTCATCCGATGCCGCATCACCG TTGTGTCAGAAACGACCAAGCTAAGCAACTAAGGTACCTTAC CGTCCACTATCTCAGGTAACCAGGTACTACCAGCTACCCTAC CTGCCGTGCCTACCTGCTTTAGTATTAATCTTTCCACCTCCCTCCTCAATCTTCTTTTCCCTCCTCTCCTCTTTTTTTTTTCTTCCTCC TCTTCTTCTCCATAACCATTCCTAACAACATCGACATTCTCTCCT AATCACCAGCCTCGCAAATCCTCAGGTTAGTATTACTACTAC TACAATCATCACCACGATGCTCCGCCCGACGATGCGGCTTCT GTTCGCCTGCCCCTCCTCTCACTCGTGCCCTTGACGAGCTAC CCCGCCAGACTCTCCTGCGTCACCAATTTTTTTCCCTATTTACCC CTCCTCCCTCTCTCCCTCTCGTTTCTTCCTAACAAACAACCACCA CCAAAATCTCTTTGGAAGCTCACGACTCACGCAAGCTCAATT CGCAGATACAAATCTAGAATGAAAAAGCCTGAACTCACCGCG ACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTC TCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCT TTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGT AAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTA TCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCT TGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTC CCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAAC CGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGA TGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGG CCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCG TGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTG GCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCA GGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGA AGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGT CCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGA GCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACA TCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGA CGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGC CGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAAC TCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTT GGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCG GGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCG TCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAA ACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAATGCA TGTGCTGTGTTCCTCAGAATGGGCCCCAGAAGGGCGTCGAGC ATTGTCTATGAATGCAAACAAAAATAGTAAATAAATAGTAAT TCTGGCCATGACGAATAGAGCCAATCTGCTCCACTTGACTATCC TTGTGACTGTATCGTATGTCGAACCCTTGACTGCCCATTCAA ACAATTGTAAAGGAATATGAGCTACAAGTTATGTCTCACGTTTGCGTGCGAGCCCGTTTGTACGTTATTTTGAGAAAGCGTTGC CATCACATGCTCACAGTCACTTGGCTTACGATCATGTTTGCG ATCTTTCGGTAAGAATACACAGAGTAACGATTATACATCCAT CGCTTTCTATGATTAGGTACTCAGACAACACATGGGAAACAA GATAACCATCGCATGCAAGGTCGATTCCAATCATGATCTGGA CTGGGGTATTCCATCTAAGCCATAGTACCCTCGAG SEQID NO : 7 GGATCCGAGAGCTACCTTAC SEQID NO : 8 CTCGAGGGTACTATGGCTTA SEQID GTAACGCCAGGGTTTTCCCAGTCACGACGACGATGATACCGA NO : 9 CGCCGCC SEQID NO : 10 CATATTGATGTAAGGTAGCTCTCGGATCCCTTTTCTACAGTA TCCTAACCA SEQID NO : 11 TATTCCATCTAAGCCATAGTACCCTCGAGCCCATCATCGTCT GCGCCA SEQID NO : 12 GCGGATAACAATTTCACACAGGAAACAGCTGTGTTGAGGAGC CGTAAATG SEQID NO : 13 ATCACCACGAAGCTTTGTCT Bibliographie des exemples

[0100] Hartl, Lukas; Kubicek, Christian P.; Seiboth, Bernhard (2007): Induction of the gai pathway and cellulase genes involves no transcriptional inducer function of the galactokinase in Hypocrea jecorina. In The Journal of Biological Chemistry 282 (25), pp. 18654-18659. DOI: 10.1074 / jbc.M700955200.

[0101] Christianson, T. W.; Sikorski, R. S.; Dante, M.; Shero, J. H.; Hieter, P. (1992): Multifunctional yeast high-copy-number shuttle vectors. In Gene 110 (1), pp. 119-122.

[0102] Montenecourt, B. S.; Eveleigh, D. E. (1977) Semiquantitative Plate Assay for Détermination of Cellulase Production by Trichoderma viride. In : Applied and environmental microbiology, vol. 33, ri 1, p. 178-183

[0103] Schiestl, Robert H.; Gietz, R. Daniel (1989): High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. In Current Genetics 16 (5), pp. 339-346. DOI: 10.1007 / BF00340712. Exemples

[0104] Example 1: Cella gene knockout in a hyperproducing strain

[0105] For the construction of the cella knockout cassette containing the hygromycin B resistance gene (hph), the flanking regions (1 kb) of cella were amplified from T. reesei genomic DNA using the Phusion polymerase (Thermo Fisher Scientific) and the following oligonucleotides: cella-5F and cella-5R; cella-3F and cella-3R (see Table 2). The hph marker was amplified from the pLHhphl plasmid using the oligonucleotides hphF and hphR (Hartl et al., 2007). The oligonucleotides used for amplification of the flanking regions overlap the different fragments of the construct ([Fig. 1]). The deletion cassette will be assembled into the pRS426 plasmid by homologous recombination during passage through yeast. Yeast transformation was performed using the method described by Schiestl and Gietz (1989).The ATCC 208405 yeast strain was transformed with the two flanking regions, the hph marker and the pRS426 plasmid previously digested by EcoRI and Xbal (Christianson et al., 1992) for . The plasmid pRSA-26-Acella-hph was introduced and amplified in thermocompetent NEB 10-beta E. coli bacteria (New England Biolabs). The oligonucleotides cella-3F and cella-5R were used to amplify the inactivation cassette from the pRS4-26-Acella-hph plasmid. The cassette was purified using the QIAquick PCR purification kit (QIAGEN). The strain used for transformation was the hyperproducing strain RutC30 (Montenecourt and Eveleigh, 1977). The strain was transformed by the protoplast method (Penttila et al. (1987)) using 1 pg of purified cassette. Integration of the hph cassette was verified by PCR using the oligonucleotides cella_ch and hphR to obtain a PCR product only if the cassette was correctly integrated. Three independent clones were isolated and analyzed as biological replicates. The sequences and primer names are presented in Tables 1 and 2.The strain invalidated for the cella gene is named RutC30-Acella.

[0106] [Tables2] Name of primers Sequences corresponds to primer hphF SEQ ID NO: 7 hphR SEQ ID NO: 8 cella-5F SEQ ID NO: 9 cella-5R SEQ ID NO: 10 cella-3F SEQID NO: 11 cella-3R SEQ ID NO: 12 cella_ch SEQ ID NO: 13

[0107] Primer sequences of the present invention

[0108] Example 2: Vial culture protocol

[0109] The cultures in fed flasks are carried out in Erlenmeyer flasks of 8 cm in diameter, containing 60mL of culture medium, inoculated with spores of the desired strain stored in cryotubes, and incubated at 150 rpm and 30°C in an Infors Multitron incubator.

[0110] The culture medium has the following final composition: - 1 mL / L H3PO4 85% - 2.8 g / L of (NH4)2SO4 - 0.3 g / L of MgSO4,7H2O - 0.15 g / L of CaCl2,2H2O - 1 mL / L of trace element solution (FeSO4: 5 g / L, MnSO4: 1.4 g / L, ZnSO4: 1.4 g / L, CoCl2: 3.7 g / L) - 8.0 g / L of dipotassium phthalate - 1.5 g / L of cornsteep - 12.5 g / L of glucose - pH adjusted to 5.4 with 30% sodium hydroxide - Sterilization 20 min at 121°C (glucose is sterilized separately from other compounds)

[0111] The first growth phase is carried out for 48 hours until exhaustion of the glucose, which leads to acidification of the medium to a pH of approximately 3.5. The pH is then raised to approximately 4.3 by adding sodium hydroxide.

[0112] The second production phase is carried out over 48 hours by feeding 0.3 mL / h of a "fed-batch" solution containing: - 50 g / L of sugars (pure lactose, or lactose + glucose mixtures) - 5.6 g / L of urea (added from a stock of 250 g / L sterilized by filtration) which makes it possible to mimic the ammonia that would be supplied by the regulation of pH during a culture in a bioreactor.

[0113] Regular 2 mL samples are taken to monitor pH, residual glucose, and protein concentration. At the end of each of the two phases, a larger sample (approximately 10 mL) is taken to measure the fungal concentration in the culture medium (by filtration and drying with a 1.2 µm filter).

[0114] To judge the level of induction of a sugar composition on the strain, the specific rate of production (in mg protein / g biomass / h) is calculated by relating the protein productivity (in mg protein S / L / h) to the mushroom concentration (g biomass / L).

[0115] Example 3: Optimization of the fed-batch composition for the induction of the RutC30-Acella strain

[0116] The RutC30-Acella strain was cultured according to the protocol described in Example 2 to measure the specific protein productivity of the strain when fed-batch with different glucose / lactose mixtures. The glucose / lactose mixtures tested ranged from 99% glucose / 1% lactose to 50% glucose / 50% lactose. The % here refers to the total amount of sugars present in the solution.

[0117] The specific productivity is shown [Fig.2] after normalization by the maximum value that was measured. Maximum productivity is observed with the 15% lactose / 85% glucose mixture, optimal in the lactose content range between 10% and 20%, and good in the lactose content range between 5% and 30%.

[0118] Example 4: Bioreactor culture protocol

[0119] Bioreactor cultures are carried out in 10 cm diameter fermenters containing 800 mL of culture medium, inoculated at 10% v / v from a preculture. Agitation is performed by a Rushton turbine and a pitch- A 5 cm diameter blade is used. The stirring speed is controlled between 400 and 1200 rpm to maintain a dissolved oxygen concentration above 40% of the saturation concentration. The temperature is controlled at 27°C during the first phase and then at 25°C during the second phase. The pH is controlled throughout the culture at 4.0 by automatic addition of a 5N ammonia solution.

[0120] Preculture is carried out in 19 cm diameter flasks containing 250 mL of the same culture medium, buffered with 5 g / L dipotassium phthalate and initially adjusted to pH 5.0 with sodium hydroxide. The precultures are inoculated with spores of the desired strain stored in cryotubes and incubated at 150 rpm and 30°C in an Infors Multitron incubator.

[0121] The culture medium has the following final composition: - 3 mL / L of 85% orthophosphoric acid - 0.25 mL / L of 96% sulfuric acid - 1.66 g / L of potassium hydroxide (KOH) crystals - 2.8 g / L of (NH4)2SO4 - 0.6 g / L of MgSO4,7H2O - 0.6 g / L of CaCl2,2H2O - 0.12 g / L of Na2HPO4,12H2O - 1 mL / L of trace element solution (FeSO4: 5 g / L, MnSO4: 1.4 g / L, ZnSO4: 1.4 g / L, CoCl2: 3.7 g / L) - 1 g / L of comsteep - 20 g / L of glucose - Sterilization for 20 min at 121°C (glucose is sterilized separately from other compounds) - Adjustment of pH to 4.0 with the ammonia solution used for pH control

[0122] The first phase (batch growth on glucose) is carried out for 27 to 30 h (until glucose is exhausted), then the 2nd phase (protein production in fed-batch mode) is carried out for 70h by feeding a sugar solution at 250g / L containing either pure lactose or a mixture of lactose and glucose.

[0123] Regular samples of approximately 15 mL are taken for: - monitor residual glucose - measure the mushroom concentration (by filtration then drying on 1.2 µm filters) - measure protein concentration (Biorad DC-Protein Assay kit using BSA as a standard).

[0124] Example 5: Comparison of the performance of RutC30 and RutC30-Acella strains

[0125] To more precisely measure the specific rate of protein production of the RutC30-Acella strain when fed with the different optimal solutions (identified in Example 3), and to compare with the reference strain RutC30, cultures in bioreactors were carried out with these 2 strains and with different feeding solutions for the fed-batch phase, according to the protocol described in Example 4. The results are presented in [Fig.3].

[0126] The wild-type RutC30 strain fed with a pure lactose solution has a specific productivity of 20 to 22 mg protein / g biomass / h. Feeding this same strain with a solution containing a mixture of 10% lactose and 90% glucose results in a specific productivity approximately 2.5 times lower, around 8 mg protein / g biomass / h. This strategy would therefore be of no industrial interest because the additional cost associated with lower productivity would not be offset by the savings achieved by using less lactose.

[0127] The RutC30-Acella strain fed with a pure lactose solution has a very low specific productivity, around 3 mg protein / g biomass / h, because it is no longer able to assimilate large quantities of lactose. In contrast, when fed with a solution containing a glucose / lactose mixture with 10 to 20% lactose in the mixture, the RutC30-Acella strain has a specific productivity between 16 and 19 mg protein / g biomass / h, which is almost as good as the RutC30 strain fed with pure lactose. With 10% or 15% lactose in the mixture, a specific productivity equivalent (not significantly different) to the RutC30 control fed with pure lactose was observed.

Claims

Demands

1. A process for producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, comprising at least two steps: - a first step of growth in batch phase in the presence of at least one carbon growth substrate, and - a second step of production of cellulolytic enzymes in fed-batch phase in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition.

2. A method for producing cellulolytic enzymes according to claim 1, wherein the fungus belongs to the species Trichoderma reesei.

3. A process for producing cellulolytic enzymes according to any one of claims 1 or 2, said strain being derived from the strain as filed under reference ATCC 56765.

4. A method for producing cellulolytic enzymes according to any one of claims 1-3, wherein the cellulolytic enzymes are cellulases or hemicellulases.

5. A method for producing cellulolytic enzymes according to any one of claims 1-4, wherein the second sugar is chosen from glucose or xylose, preferably glucose.

6. A process for producing cellulolytic enzymes according to any one of claims 1-5, wherein the lactose content in said composition represents approximately between 10 and 20% by weight of the total sugar content in said composition.

7. A process for producing cellulolytic enzymes according to any one of claims 1-6, wherein said composition comprises: - about 70-95% by weight of glucose and / or xylose, and - about 5-30% by weight of lactose.

8. A process for producing cellulolytic enzymes according to any one of claims 1-7, wherein said composition comprises: - approximately 80-90% by weight of glucose and / or xylose, and - approximately 10-20% by weight of lactose, and more specifically - approximately between 80-90% by weight of glucose, and - approximately between 10-20% by weight of lactose.

9. A process for producing cellulolytic enzymes according to any one of claims 1-8, wherein said carbon growth substrate is selected from lactose, glucose, xylose, liquid residues obtained after ethanolic fermentation, optionally obtained after ethanolic fermentation and then distillation, monomeric sugars from enzymatic hydrolysates of cellulosic biomass, a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass, an enzymatic hydrolysate of lignocellulose, and / or a hydrolysate of starchy biomass.

10. A method for producing cellulolytic enzymes according to any one of claims 1-9, wherein the cella gene has been inactivated in said strain by mutagenesis or homologous recombination, in particular using an inactivation cassette such as represented by SEQ ID NO:

3.

11. A process for producing sugars from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition.

12. 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 genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition.

13. A process for producing alcohol, in particular ethanol, from cellulosic or lignocellulosic substrates, comprising a step of producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the gene This is invalidated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition.

14. A process for producing an alcohol from cellulosic or lignocellulosic substrates according to claim 13, comprising: - i) a pretreatment step of a cellulosic or lignocellulosic substrate to obtain a pretreated substrate, - ii) a step of producing cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated, in the presence of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing approximately between 5 and 30% by weight of the total sugar content in said composition, - iii) an enzymatic hydrolysis step of the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step ii) and a suitable substrate, to obtain a hydrolysate, - iv) an alcoholic fermentation step of the hydrolysate obtained, step iv) being optionally carried out simultaneously with step iii).- v) a separation step, in particular by distillation.

15. Use of a composition comprising at least lactose and a second sugar, the lactose content in said composition representing about between 5 and 30% by weight of the total sugar content in said composition, for the production of cellulolytic enzymes by a strain of fungus belonging to the genus Trichoderma in which the cella gene is inactivated.

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