Multicopy insertion of a gene of interest into the genome of a fungus

Abstract

The invention relates to a method for inserting multiple copies of a gene of interest into the genome of a fungus, said method comprising a step of transforming said fungus using a vector comprising an expression cassette, said cassette comprising: (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest, and (3) a terminator, except for the endogenous TEF1 terminator of the fungal species. The invention also relates to fungi obtained by such a method, and the various uses of the strains thus modified. (no figure).

Description

Title of the invention: Multicopy insertion of a gene of interest into the genome of a fungus

[0001] The present invention relates to a method for inserting several copies of a gene of interest into the genome of a fungus. The present invention also relates to the fungal strains thus obtained, and the various uses of such a modified fungal strain. Background to the invention

[0002] Fungal strains are used for the production of proteins of interest. For example, fungal strains belonging to the genus Trichoderma, in particular Trichoderma reesei, are today mainly used for the production of enzymes. These enzymes, for example cellulases, are in fact used to hydrolyze cellulosic or lignocellulosic biomass into simple sugars. The enzymes produced by filamentous fungi are therefore useful in the production of second-generation biofuels or even bio-sourced products derived from sugars from (ligno)cellulosic biomass.

[0003] In order to improve the production of second-generation biofuels or even bio-sourced products, it has been considered 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 in a fed-batch protocol (feeding without racking) using a feed solution containing lactose as a sugar inducing protein production. This fermentation process comprises two stages: a first stage of growth of the fungus in the presence of an excess of carbon source and a second stage of production of enzymes thanks to the addition of an inducer in the medium with an optimized flow rate (fed-batch mode). These stages are carried out in a liquid medium in bioreactors under agitation and in the presence of oxygen because the fungus is strictly aerobic. Another example of an optimized process for producing cellulases is described in patent EP 2 744 899 B1.

[0005] In addition to optimizing cellulase production processes, it has also been considered to genetically modify fungal strains in order to modify their production capacity. Thus, international application PCT / FR2016 / 053601 describes the overexpression of the TrAZFl gene in a strain in order to improve its capacity to produce a protein of interest, in particular a cellulolytic enzyme.

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

[0007] Fungi can also be used to produce proteins useful in other types of industries, such as the food, cosmetic or pharmaceutical industries (see for example Rantasalo, A., Vitikainen, M., Paasikallio, T. et al. Novel genetic tools that enable highly pure protein production in Trichoderma reesei. Sci Rep 9, 5032 (2019) or patent EP3405580).

[0008] The TEF1 promoter is known as a strong promoter in fungi (Gao S, Zhou H, Zhou J, Chen J. Promoter-Library-Based Pathway Optimization for Efficient (2S)-Naringenin Production from p-Coumaric Acid in Saccharomyces ce-revisiae. J Agric Food Chem. 2020 Jun 24;68(25):6884-6891. doi: 10.1021 / acs.jafc.0c01130. Epub 2020 Jun 11).

[0009] The use of this TEF1 promoter has also been described or suggested to promote the expression of genes of interest (see for example US20080044878, US9732338, US6011147, WO2018067068, US8071298, or more Kitamoto , N. , Matsui , J. , Kawai , Y. et al Utilization of the TEFl-a gene (TEF1) promoter for expression of polygalac-turonase genes, pgaA and pgaB , in Aspergillus oryzae Appl Microbiol Biotechnol 50 , 85–92 ( 1998 ); J Microbiol 2017 Jul;55(7):538-544 Epub 2017 Umemura M, Dao LV, Okuda T, Terai G. Evaluation and application of constitutive promoters for cutinase production by Aspergillus oryzae as a platform for fungal secondary metabolite production Fungal Biol Biotechnol 2020 Mar 23;7:3;A vector set for systematic metabolic engineering in Saccharomyces cerevisiae. Yeast. 2011 Feb;28(2): 123-36. Epub 2010 Oct 8; Tiina Nakari, Edward Alatalo and Merja E. Penttila, Isolation of Trichoderma reesei genes highly expressed on glucose containing media: characterization of the TEF1 gene encoding translation elongation factor 1 a, Gene, 136(1993)313-318, 1993 Elsevier Science Publishers BV; Partow S, Siewers V, Bjpm S, Nielsen J, Maury J. Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast. 2010 Nov;27(l1):955-64). .

[0010] Also, multicopy integration of expression vectors into the genome of a fungus has already been described, see for example EP0778348 or WO1991002803.

[0011] Genetic engineering techniques, notably involving the TEF1 promoter and the expression of genes of interest, have therefore already been described in the prior art.

[0012] Furthermore, to target an insertion at a well-defined locus, it is usually necessary for the transgene to be flanked by sequences upstream and downstream of the target locus. The size (number of base pairs) of these flanking regions depends on the organisms. For those having the capacity to carry out homologous recombination, these flanking sequences can be short, for example in the case of Saccharomyces cerevisiae, about twenty base pairs are enough to target a locus (Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004 Aug;21(l 1):947-62). On the other hand, in other fungi such as Trichoderma reesei these flanking sequences must be 1000 base pairs and even under these conditions, the probability that the insertion will be made at the desired site is low.

[0013] Gene editing techniques allow the size of flanking regions to be reduced to 200 base pairs and increase the probability of insertion at the site.

[0014] Nevertheless, there is still a need for new methods allowing the improved expression of genes of interest and / or methods for producing proteins of interest, which are as efficient as possible, in particular in Trichoderma reesei.

[0015] The present invention is based here on the results of the inventors showing that the TEF1 promoter can be used for the multicopy insertion of a gene of interest. Each inserted functional copy leads to an increase in the expression of said gene of interest, thus increasing the production capacity of the protein of interest. The results of the present invention also show that it is possible to successfully carry out, in particular in Trichoderma reesei, the insertion at the target locus (the 5' region of the TEF1 promoter) by using a single flanking region of the insertion site in the expression cassette according to the invention (instead of the usual two). Brief description of the invention

[0016] The present invention thus relates to a method for inserting several copies of a gene of interest into the genome of a fungus, said method comprising a step of transforming said fungus using a vector comprising an expression cassette, said cassette comprising: - a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, - a gene of interest, - a terminator, except for the endogenous TEF1 terminator of the fungal species.

[0017] The invention also relates to the use of an expression cassette for inserting several copies of a gene of interest into the genome of a fungus, said expression cassette comprising: - a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, - a gene of interest, - a terminator, except for the endogenous TEF1 terminator of the fungal species.

[0018] The invention also relates to a method for producing a protein of interest comprising the following steps: - (i) a step of transforming a fungus using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest coding for said protein of interest and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (ii) a step of culturing the fungus transformed in step (i) under conditions allowing the production of the protein of interest.

[0019] The invention also relates to a fungal strain comprising in its genome several copies of a gene of interest under the control of a TEF1 promoter identical to the endogenous TEF1 promoter of the genetically modified fungal species. Statement of the invention

[0020] In a first aspect, the invention relates to a method for inserting several copies of a gene of interest into the genome of a fungus, said method comprising a step of transforming said fungus using a vector comprising an expression cassette, said cassette comprising: - a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, - a gene of interest, - a terminator, except for the endogenous TEF1 terminator of the fungal species.

[0021] According to the invention, the term "TEF1" refers to Translation Elongation Factor 1. This is a GTPase-type protein involved in protein biosynthesis. In Trichoderma reesei, the TEF1 protein, encoded by the TEF1 gene, is represented by SEQ ID NO: 2. The expression of the TEF1 gene is essentially constitutive (Tiina Nakari, Edward Alatalo and Merja E. Penttila, Isolation of Trichoderma reesei genes highly expressed on glucose-containing media: characterization of the TEF1 gene encoding translation elongation factor 1 a, Gene, 136(1993)313-318, 1993 Elsevier Science Publishers BV). More particularly, according to the invention, the term "TEF1" refers to "TEF1 alpha".

[0022] According to the invention, the TEF1 promoter means the promoter under the control of which the TEF1 gene is naturally dependent, in a non-genetically modified organism. Typically, in Trichoderma reesei, the TEF1 gene which codes for the TEF1 protein of SEQ ID NO: 2 is dependent on the TEF1 promoter represented by the sequence SEQ ID NO: 1. According to one embodiment, in the method according to the invention, the TEF1 promoter is represented by SEQ ID NO: 1 or a sequence having at least 60% identity with SEQ ID NO: 1, preferably at least 80%. According to the invention, the expression "at least 60% identity with SEQ ID NO: 1" means all values ​​between 60% and 100%, in particular the values ​​of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, preferably at least 80%, at least 85%, at least 90%, at least 95%, even more particularly at least 98%, at least 99%. A person skilled in the art knows how to calculate a percentage of identity between two sequences.For example, according to the invention, the percentage identity of a given sequence with respect to SEQ ID NO: 1 is understood to be the percentage identity over the total length of the sequences. The percentage thus corresponds to the number of nucleotides (residues where applicable) identical between this given sequence and SEQ ID NO: 1 divided by the number of nucleotides (residues where applicable) in the longer of the two sequences. According to one embodiment, in the method according to the invention, the TEF1 promoter can also be understood to be a sequence having less than 60% identity with SEQ ID NO: 1 while retaining an identical function.This includes in particular orthologous promoters of the TEF1 promoter in fungi other than Trichoderma, in particular Trichoderma reesei, which have sequences very far from SEQ ID NO: 1, although ensuring the same function as the TEF1 promoter in Trichoderma reesei (namely the TEF1 promoter of the TEF1 gene coding for a translation elongation factor 1, more particularly translation elongation factor 1 alpha). This may also be the case if the promoter is mutated (on nucleotides not essential to its promoter function). According to one embodiment, the TEF1 promoter may also be understood to mean the promoter under the control of which is a gene coding for an orthologous protein or variant of the TEF1 protein represented by SEQ ID NO: 2.Thus, the TEF1 promoter can also be understood to mean the promoter of a gene coding for a protein of SEQ ID NO: 2 or a protein having at least 80% identity with SEQ ID NO: 2, preferably at least 85%, at least 90%, at least 95%, at least 98%, at least 99%. The expression "at least 80%" represents all values ​​between 80% and 100%, i.e. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.

[0023] According to the invention, a promoter represented by a sequence having at least 60% identity with SEQ ID NO: 1, preferably represents a variant promoter or an orthologous promoter in a fungus other than Trichoderma reesei.

[0024] In the expression cassette according to the invention, a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species is used. The term “endogenous TEF1 promoter of the fungal species” also refers to the “native TEF1 promoter” of the fungal species. Typically, the TEF1 promoter in Trichoderma reesei being represented by SEQ ID NO: 1, then it is a promoter of SEQ ID NO: 1 which must be used in the expression cassette intended to be integrated when the host fungus is Trichoderma reesei. In other words, the TEF1 promoter which must be used, in the expression cassette according to the invention, must be identical to the TEF1 promoter naturally present in the fungal species which will be transformed. For example, if the host fungus used is Saccharomyces cerevisiae, then the TEF1 promoter to be used in the expression cassette is the endogenous / native TEF1 promoter of Saccharomyces cerevisiae.The term "TEF1 promoter identical to the endogenous TEF1 promoter" means that the sequence of the TEF1 promoter used is strictly identical to that of the endogenous promoter, but also that the sequence of the TEF1 promoter used may vary at the level of certain nucleotides (not essential for the function of the promoter).

[0025] The TEF1 promoter present in a fungal species can easily be determined, for example using databases such as FungiDB, by sequencing the fungal genome or any other appropriate technique. According to one embodiment, the method according to the invention can thus comprise, before the transformation step, an optional step of determining the sequence of the TEF1 promoter naturally present in the fungus intended to be transformed.

[0026] According to the invention, the term "multiple copies" means at least two copies. According to a preferred embodiment, between 2 and 15 copies of the gene of interest are inserted into the genome of the transformed fungus, preferably between 2 and 11 copies. Between 2 and 15 copies is of course understood to mean all values ​​between 2 and 15, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0027] The transformation step means any technique allowing the integration of the expression cassette into the genome of the fungus. Such a technique is well known to those skilled in the art and is understood, for example, to be biolistics (Te'o VS, Bergquist PL, Nevalainen KM.,(2002). Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor System. J Microbiol Methods. 51(3):393-9) or the protoplast method (Penttilâ M, Nevalainen H, Râttô M, Salminen E, Knowles J., (1987). A versatile transformation System for the cellulolytic filamentous fungus Trichoderma reesei. Gene. 61(2):155-64).

[0028] According to the invention, a “vector” means any DNA sequence into which it is possible to insert fragments of foreign nucleic acid, the vectors making it possible to introduce foreign DNA into a host cell (here the fungus). Examples of vectors are plasmids, cosmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) and bacteriophage PI-derived artificial chromosomes (PAC), virus-derived vectors. The vector according to the invention is an integrative vector (preferably an integrative plasmid), allowing the integration of the expression cassette into the fungal genome. According to a particular embodiment, the vector does not comprise a fungal origin of replication.

[0029] According to the invention, an "expression cassette" comprises a DNA fragment (the gene of interest) placed under the control of the elements necessary for its expression. Said DNA fragment is thus placed under the control of the TEFL promoter. According to the invention, the expression cassette comprises at least (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest, and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species.

[0030] According to the invention, the terminator used in the expression cassette according to the invention is a terminator different from the endogenous TEF1 terminator of the fungal species (i.e. the native TEF1 terminator of the fungal species). According to the invention, a TEF1 terminator of another fungus can therefore be used, provided that it is a TEF1 terminator different from the endogenous TEF1 terminator of the transformed fungal species (this is the case if the TEF1 terminator has a percentage identity of less than or equal to 75% with respect to the sequence of the endogenous TEF1 terminator of the transformed fungal species). According to another particular embodiment, the terminator is different from an endogenous TEF1 terminator of a fungal species different from the transformed fungal species.According to a particular embodiment, the terminator is a terminator other than a TEFL terminator. In other words, it is not a terminator called a TEF1 terminator in a species. For example, a CBH1 terminator can be used.

[0031] According to a particular embodiment, the integrative vector according to the invention (preferably a plasmid) comprises an expression cassette as mentioned above and a selection marker. According to a particular embodiment, the integrative vector according to the invention comprises an expression cassette as mentioned above, a non-fungal (for example bacterial) origin of replication and a selection marker. The expression "selection marker" means a gene whose expression confers on the cells which contain it a characteristic allowing them to be selected. The selection marker is a marker compatible with the host. The use of a selection marker makes it possible to identify the fungi which have integrated a genetic modification compared to those which have not integrated it. This is for example an antibiotic resistance gene, in particular the gene for resistance to the antibiotic hygromycin hph.

[0032] Fungi naturally possess a copy of the TEF1 gene, under the control of the TEFL promoter. According to the invention, the multicopy insertion is thus carried out so that the expression of the endogenous TEF1 gene of the transformed fungal species is maintained. In other words, the endogenous TEF1 coding sequence and TEF1 promoter are functional and exhibit wild-type expression. According to one embodiment, in the method for inserting several copies of a gene of interest into the genome of a fungus, the insertion is carried out upstream of the endogenous TEF1 promoter in the genome of said fungus. Indeed, as indicated below, fungi naturally possess a copy of the TEF1 gene, under the control of the TEFL promoter. According to this embodiment, the multicopy insertion is therefore carried out upstream of the endogenous TEF1 promoter of the transformed fungal species. The insertion is thus carried out 5' of the endogenous TEF1 promoter.Preferably, in the method according to the invention, the insertion is thus carried out between 750 and 1250 base pairs, preferably approximately 1000 base pairs, relative to the translation initiation site of the gene coding for TEFL Preferably, in the method according to the invention, the insertion is thus carried out between 750 and 1250 base pairs, preferably approximately 1000 base pairs, relative to the translation initiation site of the gene coding for the TEF1 protein of SEQ ID NO: 2 or a protein having at least 80% identity with SEQ ID NO: 2.

[0033] The choice of the TEF1 promoter and the location of the multicopy insertion of the cassette as defined according to the invention, have several advantages. (1) The TEF1 promoter has the advantage of being a strong promoter, thus allowing a large number of transcripts from the same carbon source. (2) Just like the TEF1 gene, the genes of interest under the control of the TEF1 promoter thus have a continuous, essentially constitutive expression, and little subject to repression systems. (3) The sequence of the TEF1 promoter also corresponds to the only flanking region necessary to target the TEFL locus. According to an embodiment according to the invention, the expression cassette according to the invention thus comprises a single flanking region to target the target locus of the insertion (the 5' region of the TEF1 promoter).

[0034] According to one embodiment of the invention, the endogenous TEF1 gene under the control of the endogenous TEF1 promoter is conserved in the transformed genome of said fungus.

[0035] Among the fungi that can be used according to the invention, mention may be made of certain fungi from the phylum Ascomycetes (Ascomycota), Basidiomycetes (Basidiomycota) and Zygomycetes (Zygomycota). In a preferred aspect, according to the invention, the fungus is a filamentous fungus. According to one embodiment lization, the filamentous fungus is chosen from the classes of orbiliomycetes, pezizomycetes, dothideomycetes, eurotiomycetes, lecanoromycetes, leotiomycetes, sordariomycetes and saccharomycetes, preferably sorda-riomycetes. Advantageously, the fungus according to the invention belongs to the genus Trichoderma, and more particularly to the species Trichoderma reesei.

[0036] According to the invention, a “gene of interest” means any gene whose production is desired. Preferably, the gene of interest codes for a protein of interest. According to the invention, the proteins of interest are all proteins that can be produced by a fungus, naturally (such as enzymes) or by genetic modification (for example after transformation using an appropriate vector). The protein of interest may be endogenous or exogenous. The protein of interest may be a native protein (as found in nature), a chimeric protein, a synthesized protein or a variant of a native protein, in particular having improved and / or modified biological properties. According to one embodiment, the sequence of the gene of interest may comprise appropriate additional elements such as, for example, a sequence coding for a secretion signal.The proteins of interest can be proteins useful to any type of industry (biofuel, food, cosmetic or pharmaceutical production, etc.).

[0037] According to one embodiment, in the method according to the invention, the gene of interest codes for an enzyme, in particular a cellulolytic enzyme such as a cellulase or a hemicellulase. Preferably, the enzymes are cellulases. According to the invention, the term "cellulases" is understood more particularly to mean enzymes chosen from endoglucanases, exoglucanases and glucosidases, and more particularly [3-glucosidase. The term "cellulase" refers more particularly to an enzyme adapted to the hydrolysis of cellulose and allowing the microorganisms (such as Trichoderma reesei) which produce them to use cellulose as a carbon source, by hydrolyzing this polymer into simple sugars (glucose).

[0038] According to one embodiment, the method according to the invention comprises a step of selecting the transformed strain, after the transformation step.

[0039] The definitions and embodiments relating to the first aspect of the invention 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 and seventh aspects of the invention described below.

[0040] In a second aspect, the invention also relates to the use of an expression cassette for inserting several copies of a gene of interest into the genome of a fungus, said expression cassette comprising: - a TEF1 promoter identical to the endogenous TEF1 promoter of the species mushroom, - a gene of interest, - a terminator, except for the endogenous TEF1 terminator of the fungal species.

[0041] In a third aspect, the invention also relates to a method for producing a biomass comprising the following steps: - (i) a step of transforming a fungus using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (ii) a step of culturing the fungus, in an appropriate culture medium.

[0042] According to a particular embodiment, said method for producing a biomass comprises a step of selecting a transformed fungus between the transformation step and the cultivation step.

[0043] According to one embodiment, the culture medium may comprise one or more substrates necessary for the growth of the fungus.

[0044] In a fourth aspect, the invention also relates to a method for producing a protein of interest comprising the following steps: - (i) a step of transforming a fungus using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest coding for said protein of interest and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (ii) a step of culturing the fungus transformed in step (i) under conditions allowing the production of the protein of interest.

[0045] According to a particular embodiment, said method for producing a protein of interest comprises a step of selecting a transformed fungus between the transformation step and the culture step.

[0046] The culture step means a culture in an appropriate medium. This is a medium suitable for the survival of the transformed fungus and / or for the growth of the fungus and / or for the production of the protein of interest, or even its secretion. It may be a medium suitable for the culture of the fungus, supplemented as needed to become a medium suitable for the production and / or secretion of the protein of interest.

[0047] Advantageously, more particularly when the gene of interest codes for a cellulolytic enzyme, said method for producing a protein of interest also comprises a phase of growth of a transformed fungal strain according to the invention, then a phase of growth and production of proteins of interest. Even more preferably, said growth phase is carried out in the presence of a growth substrate and said growth and production phase of proteins of interest is carried out in the presence of an inducing substrate. The growth substrate and the inducing substrate are preferably carbon substrates.

[0048] Thus, the carbon growth substrate is preferably chosen from lactose, glucose, xylose, residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses originating from the pretreatment of a cellulosic biomass.

[0049] The inducing carbon substrate is thus preferably chosen from lactose, cellobiose, sophorose, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses originating from the pretreatment of a cellulosic biomass.

[0050] According to one embodiment, the method for producing a protein of interest may comprise a step of secreting said protein of interest. The secretion may be consecutive or concomitant with the production of the protein of interest.

[0051] In a fifth aspect, the present invention relates to a process for producing sugar from cellulosic or lignocellulosic substrates, comprising the following steps: - (i) a step of pretreatment of a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - (ii) a step of using a fungus transformed using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest coding for a cellulolytic enzyme and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (iii) a step of enzymatic hydrolysis of the pretreated substrate obtained in step i), in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolysate.

[0052] The sugars (or sweet juice) are thus obtained at the end of the hydrolysis step. Preferably, they are C5-C6 sugars.

[0053] According to one embodiment, said sugar production process may also comprise one or more separation steps. This thus makes it possible to separate, in particular by distillation, the different products of interest diluted in water.

[0054] In a sixth aspect, the present invention relates to a process for producing an alcohol from cellulosic or lignocellulosic substrates, comprising the following steps: - (i) a step of pretreatment of a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - (ii) a step of using a fungus transformed using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest coding for a cellulolytic enzyme and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (iii) a step of enzymatic hydrolysis of the pretreated substrate obtained in step i), in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolysate, - (iv) a step of alcoholic fermentation of the hydrolyzate obtained, - (v) a separation step, in particular by distillation.

[0055] The alcohol thus obtained is for example used as a biofuel. The alcohol thus obtained can also be used as a solvent or as an intermediate product in the chemical industry.

[0056] According to the invention, the term "biofuel" is understood more particularly to mean a second-generation biofuel, i.e. derived from non-food resources. According to the invention, the term "biofuel" can also be defined as any product resulting from the transformation of biomass and which can be used for energy purposes. On the one hand, and without wishing to be limited, examples include biogases, products which can be incorporated (possibly after further transformation) into a fuel or be a fuel in their own right, such as alcohols (ethanol, butanol and / or isopropanol depending on the type of fermentation organism used), solvents (acetone), acids (butyric), lipids and their derivatives (short- or long-chain fatty acids, fatty acid esters), as well as hydrogen. The biofuel is thus an alcohol or a biogas.

[0057] Preferably, the alcohol is, for example, ethanol, butanol, isopropanol, 1,2-propane diol, 1,3-propane diol, 1,4-butane diol, and / or 2,3-butane diol. More preferably, it is ethanol.

[0058] According to a preferred embodiment, said steps iii) and iv) are carried out simultaneously. This is typically the case in so-called “SSF” (Simultaneous Saccharification and Fermentation) production processes.

[0059] According to a particular embodiment, the step of pretreatment of a cellulosic or lignocellulosic substrate is a step of suspending said cellulosic or lignocellulosic substrate in aqueous phase.

[0060] According to a particular embodiment, the hydrolyzate obtained in step iii) is a hydrolyzate containing C5-C6 sugars, in particular glucose.

[0061] According to a particular embodiment, the alcoholic fermentation step of the hydrolyzate obtained is a fermentation step, in the presence of a fer- supplementary, glucose from the hydrolyzate in order to produce a fermentation must. A fermentative organism is, for example, a yeast.

[0062] According to a particular embodiment, the separation step is a separation of the biofuel and the fermentation must, in particular by distillation.

[0063] According to an even more preferred embodiment, the cellulosic or lignocellulosic substrate to be hydrolyzed is suspended in an aqueous phase at a rate of 6 to 40% of dry matter, preferably 20 to 30%. The pH is adjusted between 4 and 5.5, preferably between 4.8 and 5.2 and the temperature between 40°C and 60°C, preferably between 45°C and 50°C. The hydrolysis reaction is started by adding enzymes acting on the pretreated substrate. The quantity of enzymes usually used is 10 to 30 mg of excreted proteins 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, in particular glucose. The sugar solution is separated from the non-hydrolyzed solid fraction, mainly consisting of lignin, by filtration or centrifugation and then treated in a fermentation unit.

[0064] According to another even more preferred embodiment, when the hydrolysis and fermentation step are carried out jointly, the enzymes and the fermentative organism are added simultaneously and then incubated at a temperature between 30°C and 35°C to produce a fermentation must. 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 fermentative organism (for example a yeast) converts the glucose into the final product according to an SSF (Simultaneous Saccharification and Fermentation) process known to those skilled in the art. Depending on the metabolic and hydrolytic capacities of the fermentative organism, the proper conduct of the operation may require the addition of a greater or lesser quantity of exogenous cellulolytic mixture.

[0065] In a seventh aspect, the invention also relates to a strain as obtained by the method of the invention. The invention also relates to a fungal strain comprising in its genome several copies of a gene of interest under the control of a TEF1 promoter identical to the endogenous TEF1 promoter of the genetically modified fungal species.

[0066] The sequences of the present invention are described in Table 1.

[0067] [Tables 1] SEQID NO : 1 (promoteu rTEFl chez Tri-choderma reeseï) AAAGCGGCGAATGACTCTTCGGGGTTGGCCAGAAACAACGACG ACCACTGAGGGATCGGGACGATGTACATCTACTGCGATATGCC GGGCTCGAATGTGACTGCACACACCACACACACACACACACAC GGACACATCTGAGCAGTACTATCAAAGCGGTGGGAGTGCATCT GCACTGGGCACCCTTACTGTAGGTAGGAAGGATACCTAAGGTA GAGGGCTCGCTGCTGCAAGTTGATATGACGGCCACCAATCGGA AGGGTGTCGTATCTCACATCACCAGCGTCAGGACACGCTTCAC AAAATTCCCACAAGTTCCAGTTGAAATGACCCGTAGGAGAGCG CCTCGAGGGACAGAATGTACAGTACTATACTACCTACCTTAGAT GCCATCACCCCTTCGCAGGCCATGGAATTTCTGGCAAGTCGCAC CCTTCAGCCATGCCTGTCCGCCATCATCATACCAGATCGTGTCG TGGCGGGCACTTGGGAAGGACGAGCTCCAACCGCCGCCGCTGA TTGGCCTGATCCGCCACAGCCACTGGCGCTCGCGGCGAGGAAA AAAAAAAATCTTGCGGGTGTGCAGCAGCATTGAGTGGAAAGCG GGAGCCGCTGCACGACGCTGATTCGTCCGGGCTGCCTCGTTAG GGCTAGACCAGCAGGCGGGCGCCGCTTATTTTGCCCCTCCTCAG GCTTTTTTTCACAAGCCCCGTTTTGCTGCCCCGCGCGTGGCAGA AAAAAAAAATACAGCACGAGCCACTGGCACTTGCACCCCTCCA CCCCTCCGCCGACCACCTTTTAAATCTCCTCCTCCCACCATCTGC TTTCGAAATTTTCTCTCTTCTGCTCTTCGTCTCGCATACCCGGTT CAAGCATCCGATCTGCGAATTTGTATGTGCTGCCTCTCCCTCTGACCTTCTGGTCTGGTGATACCATCCCTCCCTCAGTTTGGATCATC GCCTTATTCTTCTTCCCTCTTCTGCATCTGCTTCCTGCTCGTTTG AGGAACATCGCCAGCTGACTCTGCTTGCCTCGCAGCGATCTAGT CAAGAACAACACAGCTCTCACGCTACATCACACAAACCGTCAA A SEQID (NOTE : NOTE TX) MAPQDYDDDDFVDQYDEDLGEEEDELSPEDKIAMQKGTEEVRKA LGEEASKVTVAQIQEALWHYYYDIDKSVAYLTRTFIAPPPKA AQKPAPKKNGALSFASGASFKPRIPPNFFDDMPWLNIPRD RETTFIEPPRLCAPGGKKKKKKKKKKKKKKKKKKKAPKA NPDSLEGEVTKLALSDANQENTGTKSKIQRRNSSTDSSSPPQPQQK QGQNIPTASTDNSLGERVTLPTATESKTDHIIPQAPQPPVMSRSHAPS AFAQALFESDSEADQRHRRDVFAMPYTQSSLFVASVFSEPSPDDIV LAARAKATPKTPKKKKKKKKKKKGKGKVKVKVKV GIDVIKEFEQSASKKHASFVVVGHVDAGKSTLMGRLLLELKLVQE RTVDKYRRQAEKMGKTSFALAWVFDQRSEERDRGVTIDIATNHFE TDSTNFTILDAPGHRDFVPNMIAGASQADFAVLVIDASTGAYEKGL KGQTKEHVLLLRSLGVQRIVVAVNKLEAVGWSQERFQEISEEISGF LTGLGFQEKSIKFIPISGLNGDNIVKRSEDEACSWYTGPTLIEGLEAS VPSTVRSLQKPFRMAISEIFRSQQGTTTLAGRIDSGTIQVGDALLVQ PSGESAHVRSIMLDTETRDWAVAGQNVSIGLAGIDPVHVKVGDIV CHTKDPINVGDTFTMKAMAFAHLMPMPVDLHRGRLHAAGQIQSI AALLDKATGEVIKKKPKVVQPGSVARVLIKLDTKVPLEAGQRVVI RTGGETIAAGLLE

[0068] Sequences of the invention

[0069] Other characteristics, details and advantages of the invention will appear on reading the attached Figures and the examples which aim only to illustrate the invention, without limiting it. Brief description of the Figures Fig.l

[0070] [fig.l] represents the construction of the plasmid used in Example 1. Fig. 2

[0071] [fig-2] represents the fluorescence intensity per unit size of Tri- spores choderma reesei germinating. [Fig.2] shows the results of 8 transformants: E1, E2, E3, E4, E5, E6, E8 and E9 and those of 3 non-transformed strains: CL847-1, CL847-2 and CL847-3. Fig. 3

[0072] [fig.3] represents the TEF promoter (TEF1) upstream of the TEF1 gene in the genome of a mushroom. Fig. 4

[0073] [fig-4] represents an illustration of the integration in the transformant El. 7 copies of the EGFP gene of interest have been integrated. The triple indication “(1) the TEF1 promoter, (2) the EGFP gene of interest and (3) the CBH1 terminator” illustrates that the cassette has been correctly integrated and that the EGFP gene is functional. Fig. 5

[0074] [fig.5] represents an illustration of the integration in the E3 transformant. lOcopies of the EGFP gene of interest have been integrated. The triple indication “(1) the TEF1 promoter, (2) the EGFP gene of interest and (3) the CBH1 terminator” illustrates only the cases where the cassette has been correctly integrated and the EGFP gene is functional.

[0075] Examples

[0076] Example 1: Construction of a vector containing an expression cassette according to the invention

[0077] The plasmid of the following construction was obtained: - an origin of replication of Escherichia coli, - a selection marker comprising a fungal “cpc” and bacterial “IM7” promoter followed by the Hygromycin resistance gene (hph) and the fungal “TrpC” terminator, - an expression cassette comprising (1) a TEF1 promoter, (2) followed by the EGFP gene expressing a green fluorescent protein and (3) the CBH1 terminator.

[0078] This vector can be obtained by any conventional method, well known to those skilled in the art. The construction of this plasmid is presented in [fig.l]. Those skilled in the art can also easily obtain a vector (of plasmid type) comprising an expression cassette according to the invention and a selection marker making it possible to select the transformed fungi.

[0079] Example 2: Transformation of a fungus with the expression vector of Example 1

[0080] The plasmid of Example 1 was used to transform the strain of Trichoderma reesei called CL847 (Durand H, Clanet M, Tiraby G. Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microb Technol 1988, 10:341-346; Durand et al, 1984, Proc. Colloque SFM "Génétique des microorganismes industriels". Paris. H. HESLOT Ed, pp 39-50).

[0081] Transformants were selected based on their ability to grow on a medium containing Hygromycin.

[0082] The subculturing revealed different macroscopies on the dish between the transformants. Only those showing, after 3 subculturings, growth at confluence of the perimeter of the Petri dishes of 9 cm, and displaying a dense, green and hydrophobic spore carpet were preserved.

[0083] At the end of these 3 selection steps, eight transformants were kept and used for characterization by flow cytometry. The study of fluorescence made it possible to highlight eight levels of fluorescence expression ([fig.2]).

[0084] The fluorescence intensity per unit size of germinating Trichoderma reesei spores is shown in [fig.2]. These measurements were carried out using a cytometer.

[0085] The genomic study of four of the eight transformants studied by cytometry made it possible to highlight the insertion site and the copy number of the plasmid. In the four cases studied, all the insertions were made upstream of the TEFL promoter. The native and constitutive promoter of the TEF1 gene is conserved.

[0086] [Fig.3] illustrates the TEF1 gene under the control of the native and constitutive promoter of the TEF1 gene in the fungal genome.

[0087] [Fig.4] shows an illustration of the integration in the EL 7 transformer copies of the gene of interest EGFP were integrated

[0088] [Fig.5] represents an illustration of the integration in the E3 transformant. 10 copies of the gene of interest EGFP were integrated.

Claims

Claims

1. Method for inserting several copies of a gene of interest into the genome of a fungus, said method comprising a step of transforming said fungus using a vector comprising an expression cassette, said cassette comprising: - a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, - a gene of interest, - a terminator, with the exception of the endogenous TEF1 terminator of the fungal species.

2. The method of claim 1, wherein the insertion is carried out upstream of the endogenous TEF1 promoter in the genome of said fungus, preferably the insertion is carried out between 750 and 1250 bp relative to the translation initiation site of the gene coding for TEF1, the TEF1 protein preferably being represented by SEQ ID NO: 2 or a protein having at least 80% identity with SEQ ID NO:

2.

3. A method according to any preceding claim, wherein the endogenous TEF1 gene under the control of the endogenous TEF1 promoter are retained in the transformed genome of said fungus.

4. Method according to any one of the preceding claims, in which the TEF1 promoter is represented by: - ​​SEQ ID NO: 1 or a sequence having at least 60% identity with SEQ ID NO: 1, preferably at least 80%, or - the promoter of a gene coding for a protein of SEQ ID NO: 2 or a protein having at least 80% identity with SEQ ID NO:

2.

5. A method according to any one of the preceding claims, wherein the fungus is selected from the classes Orbiliomycetes, Pezizomycetes, Dothideomycetes, Eurotiomycetes, Leca-noromycetes, Leotiomycetes, Sordariomycetes and Saccha-romycetes.

6. A method according to any one of the preceding claims, wherein the fungus belongs to the genus Trichoderma, and more particularly to the species Trichoderma reesei.

7. A method according to any preceding claim, wherein the gene of interest codes for an enzyme, in particular a cellulolytic enzyme such as a cellulase or a hemicellulase.

8. Use of an expression cassette to insert multiple copies of a gene of interest in the genome of a fungus, said expression cassette comprising: - a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, - a gene of interest, - a terminator, with the exception of the endogenous TEF1 terminator of the fungal species.

9. A method for producing a protein of interest comprising the following steps: - (i) a step of transforming a fungus using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest coding for said protein of interest and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (ii) a step of culturing the fungus transformed in step (i) under conditions allowing the production of the protein of interest.

10. A method for producing a biomass comprising the following steps: - (i) a step of transforming a fungus using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (ii) a step of culturing the fungus in a suitable culture medium.

11. A method for producing sugar from cellulosic or lignocellulosic substrates, comprising the following steps: - (i) a step of pretreating a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - (ii) a step of using a fungus transformed using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest coding for a cellulolytic enzyme and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (iii) a step of enzymatic hydrolysis of the pretreated substrate obtained in step i), in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolysate.

12. A process for producing an alcohol from cellulosic or lignocellulosic substrates, comprising the following steps: - (i) a step of pretreatment of a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, - (ii) a step of using a fungus transformed using a vector comprising an expression cassette comprising (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the fungal species, (2) a gene of interest coding for a cellulolytic enzyme and (3) a terminator, with the exception of the endogenous TEF1 terminator of the fungal species, - (iii) a step of enzymatic hydrolysis of the pretreated substrate obtained in step i), in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolysate, - (iv) a step of alcoholic fermentation of the hydrolyzate obtained, - (v) a separation step, in particular by distillation, steps (iii) or (iv) possibly being carried out simultaneously.

13. A fungal strain comprising in its genome several copies of a gene of interest under the control of a TEF1 promoter identical to the endogenous TEF1 promoter of the genetically modified fungal species.

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