Production of glycosidases from simple carbon sources by genetically modified fungal microorganisms

Abstract

The present invention relates to a genetically modified fungal microorganism for the production of a glycosidase (EC 3.2. 1) in the presence of a simple carbon source, as well as to a method for the production of a glycosidase (EC 3.2. 1).

Description

Technical Field

[0001] The present invention relates to genetically modified fungal microorganisms for producing glycosidases (EC 3.2.1) in the presence of a simple carbon source, and methods for producing glycosidases (EC 3.2.1). Background Art

[0002] Enzymes have long been used in industry for a variety of applications in various fields, such as agri-food, papermaking, textiles, biofuels and detergent production. They are particularly widely used in animal nutrition, where they compensate for the lack or deficiency of enzymes in the animal body that are able to hydrolyze specific chemical bonds or degrade certain complex substrates. Thus, they can release molecules that are easily absorbed by the animal or that are beneficial to its digestive health; deconstruct complex molecular networks, thereby increasing the accessibility of nutrients to the animal's endogenous enzymes; degrade anti-nutritional factors contained in the feed, thereby limiting their effects; degrade soluble polysaccharides, thereby reducing the viscosity of the digestive bolus, and ultimately improving the digestibility of the feed.

[0003] In addition to phytases and proteases, glycosidases (also called glycoside hydrolases or glycosyl hydrolases) are one of the enzymes mainly used in animal feed. They catalyze the hydrolysis of glycosidic bonds present in complex sugars, i.e. high molecular weight macromolecules such as cellulose, hemicellulose, pectin and starch.

[0004] The glycosidase group includes xylanases, arabinofuranosidases, amylases, cellulases, glucanases and pectinases.

[0005] Endo-β-1,4-xylanases or xylanases (EC 3.2.1.8) are primarily responsible for the hydrolysis of β-1,4 bonds present in the backbone of xylan, a non-starch polysaccharide that is very abundant in nature and is the main component of hemicellulose present in plant cell walls. In other words, they catalyze the hydrolysis of (1→4)-β-D-xylosidic bonds in xylan to release oligosaccharides.

[0006] α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the hydrolysis of L-arabinose bonds. In particular, they catalyze the hydrolysis of the non-reducing terminal residues α-L-1,2-, α-L-1,3-, and α-L-1,5-arabinofuranosyl of a variety of oligosaccharides and polysaccharides, including arabinoxylans, the main components of plant cell walls, thereby releasing arabinose molecules and facilitating access of xylanases to the xylan backbone.

[0007] Glycosidases (particularly xylanases and arabinofuranosidases) are essential enzymes for the degradation of hemicellulose, one of the main components of plant cell walls. However, mammals cannot produce these enzymes, or at least cannot produce enough for effective hydrolysis. Since the diet for animal nutrition is rich in plant fiber, it is generally advantageous to provide exogenous glycosidases in the feed, particularly for livestock, in order to optimize the utilization of the fiber portion of the feed by the animal, and thereby improve the growth performance of these livestock.

[0008] Glycosidases can be produced naturally by many organisms, such as plants, algae, gastropods, arthropods, yeast, fungi, bacteria and protozoa. Their production level will depend on the expression level of the genes encoding these proteins, which are very precisely controlled by activation and repression mechanisms that are particularly dependent on the carbon sources present in their environment. In particular, the presence of simple carbon sources (such as glucose, sucrose or glycerol) will trigger the catabolite repression mechanism (Adnan et al., 2017; Galinier, 2018). This mechanism is highly conserved in microorganisms and allows them to save energy by preventing them from synthesizing excess 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 the catabolite repression is relieved, non-preferential carbon sources (such as complex carbon sources) will trigger mechanisms to induce the production of enzymes involved in their degradation and catabolism, allowing them to be used by microorganisms. For example, fungi will grow first by absorbing rapidly metabolizable carbon sources in the first stage, and then by absorbing non-preferential carbon sources in the second stage.

[0009] The mechanisms of repression and induction of gene expression involve a number of transcription factors, essential proteins that interact with regulatory sequences upstream of the gene and with RNA polymerase. The activation or repression of these transcription factors themselves depends on the environment in which the microorganism is located, in particular the carbon source present.

[0010] 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, whereas CreA and ACEI are the major identified negative regulators.

[0011] On an industrial scale, enzymes are mainly produced by microbial fermentation in the presence of suitable substrates in solid or liquid media by so-called batch, fed-batch or continuous processes. Among these microorganisms, filamentous fungi are particularly adapted to secrete large amounts of enzymes ( 2019). One of the main challenges of such production is that it is economically profitable. Therefore, it is advantageous to use carbon sources that are cheap and easily available, such as those in soluble forms like glucose and sucrose or glycerol. However, these carbon sources induce catabolite repression mechanisms in most organisms that have the effect of inhibiting gene expression, especially those encoding glycosidases such as cellulases and hemicellulases.

[0012] The scheme that is usually provided is to allow the production of enzymes in the presence of a simple carbon source, that is, the production strain is genetically modified to inactivate the catabolite inhibition mechanism. In general, this consists of the following: inactivating the transcription factor creA (the main element of catabolite inhibition), or inactivating the expression of the corresponding coding gene. However, studies have shown that the complete and / or partial deletion of the creA gene can cause harmful effects, such as the severe reduction of viability or the change of colony morphology in Aspergillus nidulans, the development of aerial hyphae and spore formation in Trichoderma reesei. Therefore, the specific area of ​​() CreA protein seems to be necessary for growth, and the CreA protein is a highly conserved protein in fungi (different species of Aspergillus (Aspergillus), Trichoderma (Trichoderma)), which establishes the important role of CreA in amino acid transport and nitrogen absorption.

[0013] As can be clearly seen from the above, there is a clear need to develop new technical solutions that allow the efficient production of glycosidases, especially on an industrial scale, without the disadvantages of the prior art, such as high production costs, long production time and / or dependence on the presence of a suitable carbon source in the culture medium. Summary of the invention

[0014] The inventors have unexpectedly and surprisingly developed genetically modified microorganisms, in particular genetically modified filamentous fungi, which have an increased ability to produce enzymes involved in the degradation of complex carbon sources (such as glycosidases (EC 3.2.1)) from simple carbon sources compared to parent microorganisms or microorganisms of the prior art and independent of catabolite repression mechanisms.

[0015] The object of the present invention is to produce high titers of glycosidases, especially hemicellulases, especially xylanases and / or arabinofuranosidases from simple carbon sources using genetically modified microorganisms, especially fungal microorganisms.

[0016] Thus, 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, the modification of whose genome comprises, advantageously, consists of, the inactivation, alteration or deletion of the expression of the following genes:

[0017] - A gene consisting of the nucleotide sequence shown in SEQ ID NO: 1;

[0018] - a nucleotide sequence encoding the peptide sequence shown in SEQ ID NO: 2; or

[0019] - a sequence that is at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identical to the peptide sequence of SEQ ID NO:2.

[0020] Such modifications result in the non-production of the corresponding protein, or the production of a non-functional protein, or the production of a truncated and therefore non-functional protein.

[0021] The genetically modified fungal microorganism according to the invention or implemented in the use or method according to the invention is genetically modified to circumvent the catabolite repression mechanism naturally occurring in fungal microorganisms (for example in filamentous fungi), according to which the microorganism "preserves its strength" by stopping the synthesis of enzymes involved in the degradation of more complex substrates (such as glycosidases) if a simple carbon source such as glucose is present in the culture medium.

[0022] According to the present invention, the fungal microorganism does not naturally have the ability to produce glycosidases from simple carbon sources, and the genetic modification performed is aimed at conferring such ability.

[0023] Therefore, the present invention has various advantages, in particular:

[0024] - industrial interest in producing enzymes for feeding animals, especially livestock, without changing the viability and morphology of the microorganisms;

[0025] - Economic benefits through the use of simple carbon sources that are cheap and soluble;

[0026] -Efficient bioproduction of glycosidases by microorganisms in industrial quantities.

[0027] In the context of the present invention, the expression "genetically modified microorganism" means that the microorganism according to the present invention is not found in nature and is modified by introducing new genetic elements and / or by deleting and / or modifying endogenous genetic elements of the microorganism. Such modifications can be produced by genome editing techniques, in vivo evolution, genome recombination, site-directed or random mutagenesis or any other strain engineering techniques known to those skilled in the art.

[0028] In the context of the present invention, the terms "inactivation", "alteration" and "deletion" are used interchangeably and refer to the non-production of the corresponding protein or the production of the corresponding non-functional protein, which is obtained in the following ways: for example, by implementing gene silencing techniques corresponding to transcriptional gene silencing or post-transcriptional gene silencing, by introducing mutations into the promoter sequence controlling the expression of the gene encoding the protein and / or into the sequence encoding the protein, by shifting the reading frame leading to the introduction of early stop codons into the sequence encoding the protein, or by deleting the gene encoding the promoter controlling the expression of the gene encoding the protein and / or the gene encoding the protein.

[0029] For the purposes of the present invention, the terms "identical" and "homologous" are used interchangeably to refer to sequence identity between two polypeptides. When a certain position in each of the two sequences being compared is occupied by the same amino acid monomer subunit, then the two molecules are homologous or identical at that position. The percent identity between the 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 positions in the two paired sequences are identical, then the two sequences are 60% identical. Typically, comparison is performed by aligning the two sequences to produce the greatest homology / identity.

[0030] The terms "encode" or "encode for" are used interchangeably and refer to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) to serve as a template for the synthesis of other polymers having a defined amino acid sequence, and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the 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 is usually described in sequence listings and databases, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be said to encode a protein or other product of the gene or cDNA.

[0031] In the context of the present invention, the expression "simple carbon source" refers to the nutrients that provide the necessary carbon for producing enzymes according to the present invention. This so-called "simple" carbon source is a molecule of small size and low molecular weight, that is to say that the molecular weight is less than or equal to 500g / mol, such as monosaccharides, disaccharides or polyols. According to the present invention, "simple carbon source" is opposite to "complex carbon source", and "complex carbon source" corresponds to large size and higher molecular weight molecules, that is to say that the molecular weight is greater than 500g / mol.

[0032] In the context of the present invention, the expression "inducing substrate" refers to an inducing carbon source for producing the enzyme according to the present invention. In addition, this inducing carbon source is not the entire carbon source for producing the enzyme.

[0033] Preferably, the subject of the present invention is a genetically modified fungal microorganism for producing a glycosidase (EC 3.2.1) in the presence of a simple carbon source as defined, which has the following technical characteristics alone or in combination:

[0034] - the fungal microorganism is selected from the class Ascomycetes;

[0035] - the fungal microorganism is selected from the group consisting of Eurotiomycetes or Sordariomycetes;

[0036] - the fungal microorganism is selected from the group consisting of Trichocomaceae, Aspergillaceae or Hypocreaceae;

[0037] - the fungal microorganism is selected from the group consisting of Talaromyces, Aspergillus, Trichoderma or Penicillium;

[0038] - the fungal microorganism is selected from the group consisting of Talaromyces versatilis, Talaromyces cellulolyticus, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei or Penicillium chrysogenum;

[0039] - the fungal microorganism is a filamentous fungus;

[0040] - the fungal microorganism is a Talaromyces diversiformis;

[0041] - the fungal microorganism has a "wild" genotype, that is to say it is the microorganism in its natural / reference form, consisting of the standard alleles present at a given locus;

[0042] - the fungal microorganism has a "mutated" or "mutant" genotype, that is to say it is a microorganism that is not in its natural / reference form, consisting of the presence of an allele at a given locus that differs from the standard allele.

[0043] The fungal microorganism according to genetic modification of the present invention can grow in the presence of a simple carbon source, which is advantageously used as the sole carbon source. The simple carbon source according to the present invention comprises (advantageously, consisting of): monosaccharides and / or disaccharides and / or polyols, preferably glucose, sucrose, lactose, glycerine or their mixture, more preferably glucose.

[0044] The modification of the genome of the fungal microorganism according to the invention unexpectedly enables the induction of 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 simple carbon sources, generally known as activators of the catabolite repression mechanism.

[0045] The genetically modified fungal microorganism of the present invention induces the production of glycosidase (EC 3.2.1) in the presence of a second simple or complex substrate called "inducing substrate" (this inducing substrate is different from the simple carbon source in the sense of the present invention, especially in terms of the function and / or activity of the growth of the microorganism of the present invention), such as xylose, oligoxylose, xylan, hemicellulose, cellulose or a mixture thereof, and the inducing substrate is 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 the inducing substrate is not renewed in the culture medium after being consumed by the microorganism of the present invention.

[0046] In order to obtain good enzyme productivity, it is necessary to provide a rapidly assimilable carbon source (i.e. a simple carbon source, in particular a soluble one, such as glucose and / or sucrose) and an inducing substrate (e.g. xylose) for the growth of the genetically modified fungal microorganism according to the present invention, which allows the expression and secretion of the glycosidase according to the present invention into the culture medium.

[0047] The genetically modified fungal microorganisms according to the present invention, in particular filamentous fungi, can produce glycosidases (EC 3.2.1) by aerobic fermentation in solid or liquid culture medium.

[0048] The method for producing glycosidase according to the present invention comprises the following steps:

[0049] (a) preparing a culture medium containing at least one simple carbon source as described above, an inducing substrate for producing glycosidase, at least one nitrogen source, and all minerals and nutrients necessary for the growth of the fungal microorganism of the present invention for producing biomass and protein;

[0050] (b) inoculating a fermentation medium with the genetically modified fungal microorganism strain according to the present invention;

[0051] (c) controlling and maintaining the temperature of the fermentation medium at a value between 25°C and 34°C, the pH value of the fermentation medium at a value between 3.5 and 5, and the oxygen partial pressure (PO 2 ) is greater than 30%; and

[0052] (d) fermenting by supplying a simple carbon source 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 12 hours to 300 hours, in particular 24 hours to 300 hours.

[0053] Preferably, the subject of the present invention is a method for producing a glycosidase (EC 3.2.1) as defined above, which has the following technical features, alone or in combination:

[0054] - The glycosidase (EC 3.2.1) is a hemicellulase, in particular a xylanase and / or an arabinofuranosidase

[0055] - the simple carbon source comprises monosaccharides and / or disaccharides and / or polyols, preferably glucose, sucrose, lactose, glycerol or a mixture thereof, more preferably glucose;

[0056] - the simple carbon source consists of monosaccharides and / or disaccharides and / or polyols, preferably glucose, sucrose, lactose, glycerol or a mixture thereof, more preferably glucose;

[0057] - the inducing substrate is selected from the group consisting of xylose, xylose polymers, cellulose, hemicellulose, xylan and mixtures thereof;

[0058] - in the culture medium, the concentration of the simple carbon source is greater than the concentration of the inducing substrate;

[0059] - in the culture medium, the concentration of the simple carbon source is equal to the concentration of the inducing substrate;

[0060] - in the culture medium, the concentration of the simple carbon source is strictly greater than the concentration of the inducing substrate;

[0061] - the concentration of the simple carbon source contained in the culture medium is 10-60 g / L culture medium, preferably 10-30 g / L;

[0062] - the concentration of the induction substrate contained in the culture medium is 5-60 g / L culture medium, preferably 5-30 g / L;

[0063] - the at least one nitrogen source is, for example, ammonium sulfate, ammonium phosphate or a mixture thereof;

[0064] - the at least one nitrogen source is contained in the culture medium in a concentration of, for example, 0.1-5%, advantageously 0.1-2% of the total mass of the culture medium;

[0065] - the minerals included in the culture medium according to step (a) are, for example, ammonium sulfate, advantageously in a concentration ranging from 0.5% to 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;

[0066] - the nutrients contained in the culture medium according to step (a) are, for example, cell growth factors;

[0067] - the supply of the simple carbon source according to step (d) is carried out in a continuous, semi-continuous or batch manner; and

[0068] - The method of the present invention further comprises a step (e) aimed at recovering the culture supernatant containing the glycosidase, said recovery being a continuous or discontinuous extraction;

[0069] - The method of the present invention further comprises step (e) performed by centrifugation or ultrafiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] [ Figure 1 ]: Xylanase activity of the strain according to the invention and the wild-type strain during 72 hours of fermentation in a medium comprising glucose as the only simple carbon source.

[0071] Xylanase activity (expressed as U DNS / g culture supernatant) was determined in the fermentation medium after 0 h, 48 h and 72 h of fermentation in a medium comprising glucose at a concentration of 11 g / L of culture medium.

[0072] [ Figure 2 ]: Arabinofuranosidase activity of the strain according to the present invention and the wild-type strain during 72 hours of fermentation in a medium comprising glucose as the only simple carbon source.

[0073] Arabinofuranosidase activity (expressed as U / mL culture supernatant) was determined in the fermentation medium after 72 h of fermentation in a medium containing glucose at a concentration of 11 g / L of medium. DETAILED DESCRIPTION

[0074] The following examples illustrate the invention in a non-limiting manner.

[0075] Example

[0076] Example 1: Examples of filamentous fungal strains according to the present invention

[0077] 1. Talaromyces diversicolor strain Tr3864

[0078] The T. diversiformis strain Tr3864 was obtained by genetically modifying the T. diversiformis strain IMI 378536. A mutation was introduced into the gene consisting of the nucleotide sequence SEQ ID NO: 1 by a genome editing tool, thereby generating a frame shift and introducing a stop codon, resulting in the production of a truncated and non-functional peptide.

[0079] Example 2: Evaluation of the production of glycosidase (EC 3.2.1) by the diverse Talaromyces Tr3864 strain cultured in the presence of glucose

[0080] 1. Growing fungi in fermenters

[0081] Before fermentation, the fungus according to the present invention, i.e., Talaromyces diversiformis Tr3864 strain, was pre-cultured in a conical flask to produce enough biomass for inoculation of the fermenter. The pre-culture medium consisted of a simple carbon source (glucose) and a nitrogen-containing nutrient source (corn steep liquor).

[0082] The duration of the pre-incubation was approximately 25 hours.

[0083] At the end of the preculture, the fermenter was inoculated with the preculture at a rate of 5% (v / v).

[0084] The fermentation medium consists of a single source of simple carbon (glucose), an inducer (a mixture of cellulose and hemicellulose), a variety of nitrogen sources (ammonium sulfate, diammonium phosphate, corn steep liquor), minerals (CaCl 2 , KH 2 PO 4 MgSO 4 ) and trace elements (MnSO 4 、FeSO 4 、ZnSO 4 )composition.

[0085] Among the main culture parameters adjusted, the temperature was 30°C, the pH was 4, the agitation was varied between 500 and 950 rpm, and the aeration was 75 L / h.

[0086] After about 24 hours of growth, glucose feeding was implemented. This feeding was continuous at a rate of 1-2 g glucose / hour / liter.

[0087] The culture lasted for a total duration of approximately 72 hours.

[0088] 2. Monitoring of Xylanase Activity During Fermentation

[0089] After 48 hours and 72 hours of cultivation in a fermenter, biomass was separated from the supernatant by centrifugation. The xylanase activity of the culture supernatant was assessed by dinitrosalicylic acid (DNS) assay. The method consists of incubating a diluted solution of the fermentation supernatant for 10 minutes at 50°C in the presence of beechwood xylan. Xylanase cuts xylan chains (composed of xylose) into shorter chains or oligosaccharides, thereby releasing reducing ends or reducing xylose. At 95°C and alkaline pH conditions, it was assayed by DNS, which binds to reducing sugars, showing a yellow-orange color proportional to the amount of reducing sugars, and can be measured by a spectrophotometer at 540nm. Each measurement was carried out in duplicate. Quantification was performed using a standard range of xylose (a reducing sugar).

[0090] The results are shown in Figure 1 and showed that during the cultivation process supplied with glucose, the mutant strain of the present invention (i.e., T. diversifolia Tr3864) had significantly higher xylanase production than the parent strain (i.e., T. diversifolia IMI 378536); the latter's production was almost zero due to the glucose-induced catabolite repression mechanism.

[0091] 3. Monitoring of Arabinofuranosidase Activity during Fermentation

[0092] After 72 hours of cultivation in the fermenter, the biomass was separated from the supernatant by centrifugation. The culture supernatant was evaluated for arabinofuranosidase activity by hydrolysis of the arabinoxylan substrate followed by determination of the released arabinose by anion exchange chromatography. A diluted solution of the fermentation supernatant was incubated at 50°C for 15 minutes in the presence of wheat xylan. Arabinofuranosidase is able to hydrolyze the glycosidic bonds between the branched arabinofuranosides and the main xylan chain. This results in the release of arabinose monomers, which are then separated by ion chromatography (high pressure anion exchange chromatography) on a CarboPac PA10 column and quantified by amperometric detection.

[0093] The results are shown in Figure 2 and showed that during the cultivation process supplied with glucose, the production of arabinofuranosidase of the mutant strain of the present invention (i.e., T. diversifolia Tr3864) was significantly higher than that of the parent strain (i.e., T. diversifolia IMI 378536); the latter's production was almost zero due to the glucose-induced catabolite repression mechanism.

[0094] 4. in conclusion

[0095] It is clear from these data that the genetically modified filamentous fungi according to the present invention alter the gene consisting of the nucleotide sequence SEQ ID NO: 1, ensuring the efficient production of xylanase (EC 3.2.1.8) and arabinofuranosidase (EC 3.2.1.55) in the presence of an inexpensive simple carbon source (i.e. glucose). In addition, there is no change in the viability or colony morphology of this microorganism. In other words, the present invention ensures the efficient growth of microorganisms without disrupting amino acid transport and nitrogen absorption.

Claims

1. A genetically modified fungal microorganism for producing a glycosidase (EC 3.2.1) in the presence of a simple carbon source, the genome of which comprises the inactivation of the expression of: - A gene consisting of the nucleotide sequence shown in SEQ ID NO: 1; - a nucleotide sequence encoding the peptide sequence shown in SEQ ID NO: 2; or - a sequence that has at least 65% identity to the peptide sequence of SEQ ID NO:

2.

2. The fungal microorganism according to claim 1, characterized in that It is selected from the class Ascomycetes.

3. The fungal microorganism according to any one of claims 1 or 2, characterized in that It is selected from the group consisting of Eurotium or Sphaerotheca.

4. The fungal microorganism according to any one of the preceding claims, characterized in that It is selected from the group consisting of Trichodermaceae, Aspergillusaceae or Hypocreaceae.

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

6. The fungal microorganism according to any one of the preceding claims, characterized in that It is selected from the group consisting of Talaromyces species, cellulose degrading Talaromyces species, Aspergillus niger species, Aspergillus oryzae species, Aspergillus nidulans species, Trichoderma reesei species or Penicillium chrysogenum species.

7. The fungal microorganism according to any one of the preceding claims, characterized in that It has a wild genotype or a mutant genotype.

8. A method for producing a glycosidase (EC 3.2.1), comprising the following steps: (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 and all minerals and nutrients necessary for the growth of the fungal microorganism for the production of biomass and protein; (b) inoculating a fermentation medium with the genetically modified fungal microorganism strain according to any one of claims 1 to 5; (c) controlling and maintaining the temperature of the fermentation medium at a value between 25°C and 34°C, the pH value of the fermentation medium at a value between 3.5 and 5, and the oxygen partial pressure (PO2) greater than 30%; and (d) Fermentation was carried out by supplying a simple carbon source for at least 12 hours.

9. The method according to claim 8, characterized in that The glycosidase (EC 3.2.1) is a hemicellulase.

10. The method according to claim 8 or 9, characterized in that: The glycosidase (EC 3.2.1) is a xylanase and / or an arabinofuranosidase.

11. The method according to any one of claims 8 to 10, characterized in that The simple carbon source comprises monosaccharides and / or disaccharides and / or polyols, preferably glucose, sucrose, lactose, glycerol or a mixture thereof, more preferably glucose.

12. The 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, xylose polymers, cellulose, hemicellulose, xylan, and mixtures thereof.

13. The 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 higher concentration than the inducing substrate.

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

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