Production of glycosidases from a simple carbon source by a genetically modified fungal microorganism

Abstract

The present invention relates to a genetically modified fungal microorganism for producing glycosidases (EC 3.2.1) in the presence of a simple carbon source as well as to a method for producing glycosidases (EC 3.2.1).

Description

[0001] TITLE: PRODUCTION OF GLYCOSIDASES FROM A SIMPLE CARBON SOURCE BY A GENETICALLY MODIFIED FUNGAL MICROORGANISM.

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a genetically modified fungal microorganism for the production of glycosidases (EC 3.2.1) in the presence of a simple carbon source and a method for producing glycosidases (EC 3.2.1).

[0004] STATE OF THE ART

[0005] Enzymes have long been used industrially for a variety of applications in diverse fields such as agri-food, papermaking, textiles, biofuel and detergent production, etc. They are particularly widely used in animal nutrition, where they compensate for the absence or insufficiency in animals of enzymes capable of hydrolyzing particular chemical bonds or degrading certain complex substrates. They thus make it possible to release molecules easily assimilated by the animal or molecules beneficial to its digestive health, to deconstruct complex molecular meshes, thus improving the accessibility of nutrients to the animal's endogenous enzymes, to degrade antinutritional factors contained in food, thus limiting their action, to degrade soluble polysaccharides, thus reducing the viscosity of the digestive bolus, and, ultimately, to improve the digestibility of food.

[0006] Besides phytases and proteases, glycosidases (also called glycoside hydrolases or glycosyl hydrolases) are one of the most widely used groups of enzymes in animal feed. They catalyze the hydrolysis of glycosidic bonds present in complex sugars, i.e., in large molecules with a high molecular weight such as cellulose, hemicelluloses, pectins, and starch.

[0007] The group of glycosidases includes xylanases, arabinofuranosidases, amylases, cellulases, glucanases and pectinases.

[0008] Endo-pl,4-xylanases or xylanases (EC 3.2.1.8) are primarily responsible for the hydrolysis of p-1,4 bonds present in the main chain of xylans, which are non-starch polysaccharides that are very abundant in nature and are the main components of hemicelluloses present in plant cell walls. In other words, they catalyze the hydrolysis of (l->4)-pD-xylosidic bonds in xylans to release oligosaccharides.

[0009] α-Arabinofuranosidases (EC 3.2.1.55) are the enzymes involved in the hydrolysis of L-arabinose bonds. In particular, they catalyze the hydrolysis of the terminal non-reducing αL-1,2-, αL-1,3-, and αL1,5-arabinofuranosyl residues of various oligosaccharides and polysaccharides, including arabinoxylans, which are major components of plant cell walls, thereby releasing arabinose molecules and facilitating the access of xylanases to the xylan main chain.

[0010] Glycosidases, particularly xylanases and arabinofuranosidases, are essential enzymes for the degradation of hemicelluloses, one of the main constituents of cell walls in plants. However, mammals are not able to produce these enzymes or at least not in sufficient quantities for efficient hydrolysis. Since diets used in animal nutrition are rich in plant fibers, it is generally advantageous to provide exogenous glycosidases in the feed, particularly for livestock, in order to optimize the use of the fiber portion of the feed by the animal and thus improve the growth performance of these livestock.

[0011] Glycosidases can be produced naturally by many organisms such as plants, algae, gastropods, arthropods, yeasts, fungi, bacteria, and protozoa. Their level of production will depend on the level of expression of the genes encoding these proteins, expression controlled very precisely by activation and repression mechanisms dependent, among other things, on the carbon source(s) present in their environment. In particular, the presence of a simple carbon source(s), such as glucose, sucrose, or glycerol, will trigger a catabolic repression mechanism (Adnan et al., 2017; Galinier, 2018). This mechanism, highly conserved among microorganisms, allows them to save energy by avoiding the synthesis of superfluous proteins under given conditions.In other words, this phenomenon allows microorganisms to preferentially use certain carbon sources. When the simple carbon source is no longer available, and therefore when catabolic repression is lifted, non-preferential carbon sources, such as complex carbon sources, will trigger mechanisms to induce the production of enzymes involved in their degradation and catabolism, thus allowing their use by the microorganism. For example, a fungus will grow in a first phase by first assimilating rapidly metabolizable carbon sources, then in a second phase by assimilating non-preferential carbon sources.

[0012] The mechanisms of repression and induction of gene expression involve numerous transcription factors, essential proteins that interact with regulatory sequences upstream of genes and with RNA polymerase. The activation or inhibition of these transcription factors is itself dependent on the environment in which the microorganism is found, in particular the carbon sources present.

[0013] In filamentous fungi several transcription factors, such as XlnR, AraR, ClrA, ClrB, ManR, and AraA, have been described as positive regulators of the expression of genes encoding glycosidases, while CreA and ACEI are the main negative regulators identified.

[0014] On an industrial scale, enzymes are mainly produced by fermentation of microorganisms, by so-called batch, fed batch or continuous processes, in solid or liquid media, in the presence of appropriate substrates. Among these microorganisms, filamentous fungi are particularly adapted to the secretion of high quantities of enzymes (Wôsten, 2019). One of the major challenges of this production is that it is economically profitable. Therefore, the use of inexpensive and easy-to-use carbon sources, for example those in a soluble form, such as glucose and sucrose or glycerol, is favored. However, these carbon sources induce in most organisms the mechanisms of catabolic repression which have the effect of repressing the expression of genes, in particular those encoding glycosidases such as cellulases and hemicellulases.

[0015] The solution generally adopted to enable the production of enzymes in the presence of a simple carbon source is to genetically modify the production strain in order to disable the catabolite repression mechanisms. Generally, this consists of deactivating the transcription factor creA, which is a major element in catabolite repression, or of deactivating the expression of the corresponding coding gene. However, studies show that a complete and / or partial deletion of the creA gene induces deleterious effects such as a severe reduction in viability in Aspergillus nidulans or an alteration in colony morphology, aerial hyphal development and sporulation in Trichoderma reesei.It therefore appears that a specific region of the CreA protein, which is a highly conserved protein in fungi (different species of Aspergillus, Trichoderma), appears to be essential for growth, establishing an important role for CreA in amino acid transport and nitrogen assimilation.

[0016] It is clear from the above that there is a clear need to develop new technical solutions allowing efficient production of glycosidases, particularly on an industrial scale, which do not have the drawbacks of the prior art such as costly, long production and / or production dependent on the presence of a suitable carbon source in the culture medium.

[0017] DESCRIPTION OF THE INVENTION

[0018] The inventors have unexpectedly and surprisingly developed genetically modified microorganisms, in particular genetically modified filamentous fungi, having an increased capacity to produce enzymes involved in the degradation of a complex carbon source such as glycosidases (EC 3.2.1), from a simple carbon source, independently of the catabolic repression mechanism, this in comparison with the parent microorganisms or microorganisms of the state of the art.

[0019] An objective of the present invention is to produce a high titer of glycosidases, in particular hemicellulases, in particular xylanases and / or arabinofuranosidases, from a simple carbon source using a genetically modified microorganism, in particular a fungal microorganism.

[0020] Thus, the present invention relates to a genetically modified fungal microorganism for the production of glycosidases (EC 3.2.1) in the presence of a simple carbon source, a modification of the genome of which comprises, advantageously consists of, the inactivation, alteration or deletion of the expression:

[0021] - of the gene consisting of the nucleotide sequence presented by the sequence SEQ ID NO: 1;

[0022] - a nucleotide sequence encoding the peptide sequence presented by the sequence SEQ. ID NO: 2; or

[0023] - a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the peptide sequence of sequence SEQ ID NO: 2.

[0024] This modification results in the absence of production of the corresponding protein or the production of a non-functional protein or the production of a truncated and therefore non-functional protein.

[0025] A genetically modified fungal microorganism according to the invention or implemented in a use or method according to the invention is genetically modified to circumvent the catabolite repression mechanism naturally present in fungal microorganisms, for example in filamentous fungi, according to which, if a simple carbon source such as glucose is present in the medium, the microorganism "saves its strength" by stopping the synthesis of enzymes, such as glycosidases, involved in the degradation of more complex substrates.

[0026] According to the invention, the fungal microorganism does not naturally have the capacity to produce glycosidases from a simple carbon source and the genetic modifications made are intended to confer this capacity on it.

[0027] The present invention therefore has various advantages, in particular:

[0028] - an industrial interest in the production of enzymes involved in animal feeding, particularly livestock, without altering the viability and morphology of the microorganism;

[0029] - an economic interest through the use of a simple, inexpensive and soluble carbon source;

[0030] - efficient bio-production of glycosidases by microorganisms at industrial yields.

[0031] In the context of the invention, the expression "genetically modified microorganism" means that the microorganism according to the invention is not found in nature and is modified by the introduction of new genetic elements and / or by deletion and / or modification of the endogenous genetic elements of the microorganism. This modification can be generated by genome editing techniques, in vivo evolution, genomic recombination, site-directed or random mutagenesis or any other strain engineering technique known to those skilled in the art.

[0032] In the context of the invention, the terms "inactivation" and "alteration" and "deletion" are used interchangeably and refer to the absence of production of the corresponding protein or the production of a corresponding non-functional protein obtained, for example, by implementing the gene silencing technique corresponding to transcriptional gene silencing or post-transcriptional gene silencing, by introducing a mutation into the promoter sequence that controls the expression of the gene coding for said protein and / or into the coding sequence for said protein, by shifting the reading frame causing the introduction of an early stop codon into the coding sequence for the protein or by deleting the gene coding for the promoter that controls the expression of the gene coding for said protein and / or the gene coding for said protein.

[0033] For the purposes of the invention, the terms "identical" and "homologous" are used interchangeably and refer to the sequence identity between two polypeptides. When a position in each of the two sequences being compared is occupied by the same amino acid monomeric subunit, then the molecules are homologous or identical for that position. The percentage identity between two sequences is a function of the number of corresponding positions shared by the two sequences, and is that number divided by the number of positions being compared and multiplied by 100. For example, if 6 out of 10 of the positions in two paired sequences are identical, then the two sequences are 60% identical. Typically, the comparison is performed by aligning the two sequences to give maximum homology / identity.

[0034] The terms "coding" or "coding for," "code" or "codes for" are used interchangeably and refer to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers having a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is generally described in sequence listings and databases, and the non-coding strand, used as a template for transcription of a gene or cDNA, may be designated as coding for the protein or other product of that gene or cDNA.

[0035] In the context of the present invention, the expression "simple carbon source" designates a nutrient providing the carbon necessary for the production of enzymes according to the invention. This so-called "simple" carbon source is a molecule of small size and low molecular weight, i.e. a molecular weight less than or equal to 500 g / mol, such as a monosaccharide, a diholoside or a polyol. According to the invention, a "simple carbon source" is opposed to a "complex carbon source" which corresponds to a molecule of large size and higher molecular weight, i.e. a molecular weight greater than 500 g / mol.

[0036] In the context of the present invention, the expression "inducing substrate" designates an inducing carbon source for the production of enzymes according to the invention. Furthermore, this inducing carbon source is not the total carbon source for the production of said enzymes.

[0037] Preferably, the subject of the present invention is a genetically modified fungal microorganism for the production of glycosidases (EC 3.2.1) in the presence of a simple carbon source as defined having the following technical characteristics, taken alone or in combination:

[0038] - the fungal microorganism is selected from Ascomycetes;

[0039] - the fungal microorganism is selected from the group consisting of the class of Eurotiomycetes or Sordariomycetes;

[0040] - the fungal microorganism is selected from the group consisting of the Trichocomaceae, Aspergillaceae or Hypocreaceae families;

[0041] - the fungal microorganism is selected from the group consisting of the genus Talaromyces, Aspergillus, Trichoderma or Penicillium;

[0042] - the fungal microorganism is selected from the group consisting of the species Talaromyces versatilis, Talaromyces cellulolyticus, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei or Penicillium chrysogenum;

[0043] - the fungal microorganism is a filamentous fungus;

[0044] - the fungal microorganism is Talaromyces versatilis;

[0045] - the fungal microorganism has a “wild” genotype, i.e. it is a microorganism in its natural / reference form, which consists of the presence of a standard allele at a given locus;

[0046] - the fungal microorganism has a "mutated" or "mutant" genotype, i.e. it is a microorganism that is not in its natural / reference form, which consists of the presence of an allele different from the standard allele at a given locus. The genetically modified fungal microorganism according to the invention is capable of growing in the presence of a simple carbon source, advantageously, as the sole carbon source. The simple carbon source according to the invention comprises, advantageously consists of, a monosaccharide and / or a diholoside and / or a polyol, preferably glucose, sucrose, lactose, glycerol or mixtures thereof, more preferably glucose.

[0047] The modification of the genome of the fungal microorganism according to the invention unexpectedly makes it possible to induce the production of enzymes involved in the degradation of complex sugars, such as glycosidases (EC 3.2.1), in particular hemicellulases, in particular xylanases and / or arabinofuranosidases, in the presence of a simple carbon source generally known as an activator of catabolic repression mechanisms.

[0048] The genetically modified fungal microorganism of the invention induces a production of glycosidases (EC 3.2.1) in the presence of a second simple or complex substrate called "inducing substrate" (the inducing substrate is different from a simple carbon source within the meaning of the invention, in particular in terms of function and / or activity with regard to the growth of the microorganism of the invention), such as xylose, oligoxyloses, xylans, hemicelluloses, cellulose or mixtures thereof, said inducing substrate being present in the culture medium in a small proportion, i.e. a proportion lower than that of the simple carbon source. Advantageously, the proportion of inducing substrate is not renewed in the culture medium after its consumption by the microorganism of the invention.

[0049] In order to obtain good enzyme productivity, it is necessary to provide a rapidly assimilable carbon source for the growth of the genetically modified fungal microorganism according to the invention (i.e. a simple carbon source, in particular soluble, such as glucose and / or sucrose) and an inducing substrate (e.g. xylose) which allows the expression of the glycosidases according to the invention and secretion into the culture medium.

[0050] The genetically modified fungal microorganism according to the invention, in particular a filamentous fungus, can be used for the production of glycosidases (EC 3.2.1) by aerobic fermentation in a solid or liquid medium.

[0051] The method for producing glycosidases according to the invention comprises the following steps:

[0052] (a) preparing a culture medium containing at least one simple carbon source, an inducing substrate for the production of glycosidases, at least one nitrogen source, as well as all the minerals and nutrients necessary for the growth of the fungal microorganism of the invention, as described above, for the production of biomass and proteins;

[0053] (b) inoculating the fermentation medium with a strain of genetically modified fungal microorganism according to the invention;

[0054] (c) controlling and maintaining the temperature of the fermentation medium at a value between 25°C and 34°C, the pH of the fermentation medium at a value between 3.5 and 5 and a partial pressure of oxygen (PO2) greater than 30%; and

[0055] (d) carrying out the fermentation for at least 12 hours, advantageously at least 24 hours, or even at least 48 hours or at least 72 hours, preferably at least 96 hours, preferably between 12 hours and 300 hours, in particular between 24 hours and 300 hours, by feeding with a simple carbon source.

[0056] Preferably, the subject of the present invention is a process for producing glycosidases (EC 3.2.1) as defined above having the following technical characteristics, taken alone or in combination:

[0057] - glycosidases (EC 3.2.1) are hemicellulases, in particular xylanases and / or arabinofuranosidases

[0058] - the simple carbon source comprises a monosaccharide and / or a diholoside and / or a polyol, preferably glucose, sucrose, lactose, glycerol or mixtures thereof, more preferably glucose;

[0059] - the simple carbon source consists of a monosaccharide and / or a diholoside and / or a polyol, preferably glucose, sucrose, lactose, glycerol or mixtures thereof, more preferably glucose;

[0060] - the inducing substrate is selected from the group consisting of xylose, a xylose polymer, cellulose, hemicellulose, xylan and mixtures thereof;

[0061] - in the culture medium, the simple carbon source is present in a concentration higher than that of the inducing substrate;

[0062] - in the culture medium, the simple carbon source is present in a concentration equal to that of the inducing substrate;

[0063] - in the culture medium, the simple carbon source is present in a concentration strictly higher than that of the inducing substrate;

[0064] - the simple carbon source is included in the culture medium at a concentration of between 10 and 60 g / L of culture medium, preferably between 10 and 30 g / L;

[0065] - the inducing substrate is included in the culture medium at a concentration of between 5 and 60 g / L of culture medium, preferably between 5 and 30 g / L;

[0066] - the at least one nitrogen source is, for example, ammonium sulfate, ammonium phosphate or their mixture;

[0067] - the at least one source of nitrogen is included in the culture medium at a concentration of, for example, between 0.1 and 5% of the total mass of the culture medium, advantageously between 0.1 and 2% of the total mass of the culture medium;

[0068] - the minerals included in the culture medium according to step (a) are, for example, ammonium sulfate, advantageously in a concentration of between 0.5 and 3% of the total mass of the culture medium; potassium phosphate; calcium chloride; magnesium sulfate; copper sulfate; iron sulfate; manganese sulfate; zinc sulfate and mixtures thereof;

[0069] - the nutrients included in the culture medium according to step (a) are, for example, cell growth factors;

[0070] - the feeding with a simple carbon source according to step (d) is carried out in continuous, semi-continuous or batch mode; and

[0071] - the method of the invention further comprises a step (e) aimed at recovering the culture supernatant containing the glycosidases, said recovery being a continuous or non-continuous withdrawal;

[0072] - the method of the invention further comprises a step (e) carried out by centrifugation or ultrafiltration.

[0073] Figures:

[0074] [Fig. 1]: Xylanase activity of a strain according to the invention and of a wild strain during 72 hours of fermentation in culture medium comprising glucose as the only source of simple carbon.

[0075] Xylanase activity (expressed as U DNS / g of culture supernatant) was determined in the fermentation medium after 0 hours, 48 ​​hours and 72 hours of fermentation in a medium comprising glucose at a concentration of 11 g / L of culture medium.

[0076] [Fig. 2]: Arabinofuranosidase activity of a strain according to the invention and of a wild strain during 72 hours of fermentation in culture medium comprising glucose as the only source of simple carbon.

[0077] Arabinofuranosidase activity (expressed in U / mL of culture supernatant) was determined in the fermentation medium after 72 hours of fermentation in a medium comprising glucose at a concentration of 11 g / L of culture medium

[0078] The present invention is illustrated in a non-limiting manner by the following examples.

[0079] EXAMPLES OF ACHIEVEMENT filamentous according to the invention

[0080] 1. Strain Versatilis Tr3864

[0081] The Talaromyces versatilis Tr3864 strain was obtained by genetic modification of the Talaromyces versatilis IMI 378536 strain. Mutations were introduced into the gene consisting of the nucleotide sequence SEQ. ID NO: 1 by genome editing tools in order to create a frameshift and the introduction of a stop codon resulting in the production of a truncated and non-functional peptide. the strain versatilis Tr3864 grown in the presence of

[0082] 1. Culture of the in fermenter

[0083] Prior to fermentation, the fungus according to the invention, i.e. strain Talaromyces versatilis Tr3864, is pre-cultured in an Erlenmeyer flask in order to produce sufficient biomass for inoculation of the fermenter. The pre-culture medium consists of a simple carbon source (glucose) and a nitrogenous nutrient source (Corn Steep Liquor).

[0084] The preculture duration is approximately 25 hours.

[0085] At the end of this preculture, the fermenter is inoculated with the preculture at a rate of 5% (v / v).

[0086] The fermentation medium consists of a single simple carbon source (glucose), an inducer (a mixture of cellulose and hemicelluloses), several nitrogen sources (ammonium sulfate, diammonium phosphate, Corn Steep Liquor), minerals (CaCL, KH2PO4, MgSCU), and trace elements (MnSCU, FeSCU, ZnSCU).

[0087] Among the main culture parameters that are regulated, the temperature is 30°C, the pH is 4, the agitation varies between 500 and 950 rpm, and the aeration is 75 L / h.

[0088] After approximately 24 hours of growth, glucose feeding is implemented. This feeding is continuous, at a rate of between 1 and 2 grams of glucose / hour / Liter.

[0089] The culture is continued for a total duration of approximately 72 hours.

[0090] 2. Monitoring xylanase activity during fermentation

[0091] After 48 hours and 72 hours of culture in a fermenter, the biomass was separated from the supernatant by centrifugation. The xylanase activity of the culture supernatant was assessed by the dinitrosalicylic acid (DNS) assay method. This method consists of incubating a diluted solution of the fermentation supernatant in the presence of beech xylan for 10 minutes at 50°C. Xylanase cuts the xylan chain (composed of xyloses) into shorter chains or oligosaccharides and thus releases reducing ends or reducing xyloses. These are assayed by DNS at 95°C and at alkaline pH, DNS binds to reducing oses, revealing a yellow-orange color proportional to the quantity of reduced oses and measurable by spectrophotometer at 540 nm. Each measurement is carried out in duplicate. Quantification is carried out using a standard range of xylose, a reducing sugar.

[0092] The results are represented by Figure 1 and show a production of xylanases during the culture supplied with glucose which is very significantly higher for the mutant strain of the invention (i.e. T. versatilis Tr3864) than for the parent strain (i.e. T. versatilis IM I 378536); the production being for the latter almost zero due to the catabolic repression mechanisms induced by glucose.

[0093] 3. Monitoring of arabinofuranosidase activity during fermentation

[0094] After 72 hours of culture in a fermenter, the biomass was separated from the supernatant by centrifugation. The arabinofuranosidase activity of the culture supernatant was evaluated by hydrolysis of an arabinoxylan substrate followed by determination of the released arabinose by anion exchange chromatography. A diluted solution of the fermentation supernatant was incubated in the presence of wheat xylan for 15 minutes at 50°C. Arabinofuranosidase is capable of hydrolyzing the osidic bonds between branched arabinofuranosides and the xylan main chain. This results in the release of arabinose monomers which are then separated by ion chromatography (High Pressure Anion Exchange Chromatography) on a CarboPac PAIO column and quantified by amperometric detection. The results are represented by Figure 2 and show a production of arabinofuranosidases during the glucose-fed culture that is very significantly higher for the mutant strain of the invention (i.e. T.versatilis Tr3864) than for the parent strain (i.e. T. versatilis IMI 378536); production being for the latter almost zero due to the mechanisms of catabolic repression induced by glucose. 4. Conclusion.

[0095] It is clear from these data that a filamentous fungus genetically modified according to the invention to alter the gene consisting of the nucleotide sequence SEQ. ID NO: 1 ensures efficient production of xylanases (EC 3.2.1.8) and arabinofuranosidases (EC 3.2.1.55) in the presence of a simple carbon source (i.e. glucose), which is inexpensive. In addition, this microorganism does not show any alteration in its viability or colony morphology.

[0096] In other words, the present invention ensures efficient growth of the microorganism without disrupting amino acid transport and nitrogen assimilation.

Claims

DEMANDS 1. Genetically modified fungal microorganism for the production of glycosidases (EC 3.2.1) in the presence of a single carbon source, whose genome includes the inactivation of expression: - of the gene consisting of the nucleotide sequence presented by the sequence SEQ ID NO: 1; - of a nucleotide sequence encoding the peptide sequence presented by the SEQ sequence. ID NO: 2; or - of a sequence showing at least 65% identity with the peptide sequence of sequence SEQ ID NO:

2.

2. Fungal microorganism according to claim 1, characterized in that it is selected from among the Ascomycetes.

3. Fungal microorganism according to one of claims 1 or 2, characterized in that it is selected from the group constituted by the class of Eurotiomycetes or Sordariomycetes.

4. Fungal microorganism according to any one of the preceding claims, characterized in that it is selected from the group consisting of the family Trichocomaceae, Aspergillaceae or Hypocreaceae.

5. Fungal microorganism according to any one of the preceding claims, characterized in that it is selected from the group consisting of the genera Talaromyces, Aspergillus, Trichoderma or Penicillium.

6. Fungal microorganism according to any one of the preceding claims, characterized in that it is selected from the group consisting of the species Talaromyces versatilis, Talaromyces cellulolyticus, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei or Penicillium chrysogenum.

7. Fungal microorganism according to any one of the preceding claims, characterized in that it has a wild-type genotype or a mutated genotype.

8. Process for the production of glycosidases (EC 3.2.1) comprising the following steps: (a) prepare a culture medium containing at least one simple carbon source, an inducing substrate for the production of glycosidases, at least one nitrogen source, and all the minerals and nutrients necessary for the growth of the fungal microorganism for the production of biomass and protein; (b) inoculate the fermentation medium with a strain of genetically modified fungal microorganism according to any one of claims 1 to 5; (c) control and maintain the temperature of the fermentation medium between 25°C and 34°C, the pH of the fermentation medium between 3.5 and 5, and a partial pressure of oxygen (PO2) above 30%; and (d) conduct the fermentation for at least 12h by feeding with a simple carbon source.

9. A method according to claim 8, characterized in that the glycosidases (EC 3.2.1) are hemicellulases.

10. A process according to claim 8 or 9, characterized in that the glycosidases (EC 3.2.1) are xylanases and / or arabinofuranosidases.

11. A process according to any one of claims 8 to 10, characterized in that the simple carbon source comprises a monosaccharide and / or a disaccharide and / or a polyol, preferably glucose, sucrose, lactose, glycerol or mixtures thereof, more preferably glucose.

12. A method according to any one of claims 8 to 11, characterized in that the culture medium further comprises an inducing substrate selected from the group consisting of xylose, a xylose polymer, cellulose, hemicellulose, xylan and mixtures thereof.

13. A method according to any one of claims 8 to 12, characterized in that the simple carbon source is present in the culture medium at a concentration higher than that of the inducing substrate.

14. A method according to any one of claims 8 to 13, characterized in that it further comprises a step (e) for recovering the culture supernatant containing the glycosidases.

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