Target peptides produced from simple carbon sources by genetically modified fungal microorganisms.

Abstract

This invention relates to genetically modified fungal microorganisms for producing target proteins in the presence of a simple carbon source and to a method for producing said target proteins.

Description

Technical Field

[0001] This invention relates to genetically modified fungal microorganisms for producing target proteins in the presence of a simple carbon source and to a method for producing said target proteins. Background Technology

[0002] The industrial-scale production of target proteins constitutes a major commercial challenge and represents a considerable market. For example, enzymes have long been used industrially in various applications across different sectors, such as agri-food, papermaking, textiles, biofuel production, and detergents. They are particularly widely used in the agri-food industry, for instance, in animal nutrition, where they compensate for the lack or insufficiency of enzymes in animals capable of hydrolyzing specific chemical bonds or degrading certain complex substrates. Therefore, they can release molecules that are easily absorbed by animals or beneficial to their digestive health, break down complex molecular networks, thereby increasing the availability of nutrients by endogenous enzymes in animals, degrade anti-nutritional factors in feed to limit their effects, degrade soluble polysaccharides to reduce the viscosity of digestible boluses, and ultimately improve feed digestibility.

[0003] Some of these proteins, crucial for improving animal nutrition, are not produced by mammals, or are produced in insufficient quantities. This is the case for glycosidases, particularly xylanases and arabinofuranosaccharides, which are essential enzymes for degrading hemicellulose, a major component of plant cell walls. In fact, since diets used in animal nutrition are rich in plant fiber, providing exogenous glycosidases in feed is often advantageous, especially for livestock, to optimize their utilization of the fiber portion of the feed, thereby improving their growth performance. The same is true for certain phytases or proteases that constitute target proteins in animal foods (e.g., as zootechnical additives) and human foods (e.g., for use in juice preparation, where they enable the release of larger quantities of juice and / or the acquisition of juice of improved quality, or for use in bread dough preparation, where they allow the dough to have greater extensibility and elasticity).

[0004] These proteins can be naturally produced by many organisms, such as plants, algae, gastropods, arthropods, yeast, fungi, bacteria, and protozoa. Their production levels depend on the expression levels of the genes encoding these proteins, which are precisely controlled by activation and repression mechanisms, particularly dependent on the carbon sources present in their environment. Specifically, the presence of simple carbon sources (such as glucose, sucrose, or glycerol) triggers catabolite repression mechanisms (Adnan et al., 2017; Galinier, 2018). This mechanism is highly conserved in microorganisms, enabling them to conserve energy by preventing the synthesis of excess proteins under given conditions. In other words, this phenomenon allows microorganisms to preferentially use certain carbon sources. When simple carbon sources are no longer available, and therefore when catabolite repression is deactivated, non-preferred carbon sources (such as complex carbon sources) trigger mechanisms that induce the production of enzymes involved in their degradation and catabolism, thus making them available to the microorganisms. For example, fungi will grow in the first stage by first assimilating rapidly metabolizable carbon sources, and then in the second stage by assimilating non-preferred carbon sources.

[0005] Mechanisms of gene expression repression and induction involve numerous transcription factors, essential proteins that interact with upstream regulatory sequences and RNA polymerases. The activation or repression of these transcription factors is itself dependent on the environment in which the microorganism resides, particularly the carbon sources present.

[0006] In filamentous fungi, some transcription factors, such as XlnR, AraR, ClrA, ClrB, ManR, and AraA, have been described as positive regulators of gene expression encoding certain target proteins (such as glycosidases), while CreA and ACEI are the major negative regulators identified.

[0007] On an industrial scale, enzymes are produced primarily through microbial fermentation in solid or liquid media, in the presence of suitable substrates, using so-called batch, fed-batch, or continuous processes. Among these microorganisms, filamentous fungi are particularly well-suited to secreting large quantities of enzymes (Wösten, 2019). One of the main challenges of this production is ensuring its economic viability. Therefore, inexpensive and readily available carbon sources are preferred, such as those available in soluble forms like glucose, sucrose, or glycerol. However, as previously mentioned, in most microorganisms, these simple carbon sources induce catabolite repression mechanisms, leading to repressor gene expression and thus preventing the transcription and / or translation of the target protein. Another major challenge is that the production of these target proteins can be regulated (e.g., according to changes in customer demand). Currently, for target proteins such as enzymes, there is no production solution that can rapidly and efficiently regulate the production of the target protein while maintaining economic viability.

[0008] In conclusion, it is clear that new technological solutions are needed to effectively produce target proteins, especially on an industrial scale, which can be tailored to market demand and do not suffer from the drawbacks of existing technologies, such as high production costs, long production times, and / or dependence on the presence of suitable carbon sources in the culture medium. Summary of the Invention

[0009] The inventors have unexpectedly and surprisingly developed genetically modified microorganisms, particularly genetically modified filamentous fungi, which, compared with parental microorganisms (also referred to independently as: natural or wild microorganisms or strains), have an enhanced ability to produce target proteins from simple carbon sources, said ability being independent of catabolite repression mechanisms.

[0010] One object of the present invention is to enable the industrial-scale, tunable production of one or more target proteins from a simple carbon source using genetically modified microorganisms, particularly fungal microorganisms. In other words, the object of the present invention is to provide a production platform for one or more target proteins, the produced proteins of which can be tuned over time (e.g., depending on changes in customer demand at a given time).

[0011] Therefore, this invention relates to a genetically modified fungal microorganism for producing at least one target protein in the presence of a simple carbon source, the genome of which comprises:

[0012] a) At least:

[0013] i. An inactivated version of the gene consisting of the nucleotide sequence shown in SEQ ID NO: 1.

[0014] ii. A nucleotide sequence encoding an inactivated version of the peptide sequence shown in SEQ ID NO: 2, or

[0015] iii. A nucleotide sequence encoding an inactivated version of the peptide sequence SEQ ID NO: 2 that has at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with the peptide sequence.

[0016] b) At least one of the following additional copies:

[0017] i. A gene encoding the transcription factor XlnR and consisting of the nucleotide sequence shown in SEQ ID NO: 3.

[0018] ii. The nucleotide sequence encoding the transcription factor XlnR, as shown in SEQ ID NO: 4, or

[0019] iii. The nucleotide sequence encoding the transcription factor XlnR shown in SEQ ID NO: 4, having at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with the peptide sequence, and

[0020] c) At least one of the following additional copies:

[0021] i. A gene encoding the peptide sequence of the target protein, wherein the gene is placed under the control of a promoter regulated by the transcription factor XlnR, or

[0022] ii. A nucleotide sequence encoding a peptide sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the peptide sequence of the target protein, said nucleotide sequence being placed under the control of a promoter regulated by the transcription factor XlnR.

[0023] This modification of the fungal genome leads to:

[0024] a) The protein does not produce the corresponding protein or the expressed protein is inactivated (i.e., a non-functional protein), and the protein participates in the catabolic repression mechanism naturally present in fungal microorganisms.

[0025] b) Production of transcription factors that are no longer repressed by naturally occurring catabolites, and

[0026] c) Increased production of the target protein, regulated by transcription factors that are no longer inhibited by catabolites.

[0027] Genetically modified fungal microorganisms according to the present invention or implemented in the use or method according to the present invention are genetically modified to bypass the catabolic repression mechanism naturally present in fungal microorganisms, for example in filamentous fungi, according to which microorganisms “conserve their energy” by ceasing the synthesis of complex proteins (such as enzymes) involved in the degradation of more complex substrates if a simple carbon source (such as glucose) is present in the culture medium.

[0028] According to the present invention, the fungal microorganism does not naturally possess:

[0029] -The ability to produce the target protein when the target protein is a heterologous protein, or

[0030] -The ability to produce the target protein from a simple carbon source, assuming the target protein is a homologous protein.

[0031] The genetic modifications made were intended to give it this ability from simple carbon sources.

[0032] Therefore, the present invention has various advantages, in particular:

[0033] - The industrial benefits of producing target proteins without affecting the viability and morphology of the microorganisms (e.g., producing enzymes for use as animal feed additives, particularly for livestock, or for improving food, such as juice or bread dough).

[0034] -The economic benefits of producing homologous or heterologous target proteins by using simple, inexpensive and soluble carbon sources, which can vary over time to meet changing needs;

[0035] - This target protein is efficiently bioproduced at industrial yields using microorganisms.

[0036] In the context of this invention, the term "genetically modified microorganism" refers to a microorganism according to the invention that is not found in nature but is modified by introducing new genetic elements and / or by deleting and / or modifying endogenous genetic elements of the microorganism. Such modification can be produced by genome editing techniques, in vivo evolution, genome recombination, directed or random mutagenesis, or any other strain modification known to those skilled in the art.

[0037] In the context of this invention, the terms “inactivation,” “alteration,” and “deletion” are used interchangeably and refer to the non-production of the corresponding protein or the production of a non-functional corresponding protein, for example, by implementing gene silencing techniques corresponding to transcriptional 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 frameshifting resulting in the introduction of an early stop codon into the sequence encoding the protein, or by deleting a gene encoding a transcription factor that regulates the promoter controlling the expression of the gene encoding the protein, and / or by deleting the gene encoding the protein. In this sense, the expressions “inactivated version of a gene” and “inactivated version of a peptide sequence” refer to the complete or partial loss of functional activity of the mentioned gene and protein. These inactivations can be achieved, in particular, by the various genetic methods described above (e.g., introducing mutations, deleting key regions of a gene or protein, or interrupting its expression through interference mechanisms). For example, an inactivated version of a given gene can result in a nucleotide sequence that can no longer be transcribed or translated, or produce truncated or altered proteins that cannot perform their usual biological functions. For example, an inactive version of a peptide sequence can cause a protein to no longer interact with its usual molecular chaperones, no longer catalyze necessary enzymatic reactions, or no longer be localized in the appropriate cellular compartment to perform its function. For instance, due to mutations, an inactive enzyme may lack its active site, thereby preventing any substrate binding and rendering the enzymatic reaction impossible.

[0038] In the context of this invention, the terms "identity" and "homology" are used interchangeably to refer to the sequence identity between two polypeptides. Two molecules are homologous or identical at that position when one position in each of two compared sequences is occupied by the same amino acid monomer subunit. The percentage of identity between two sequences is a function of the number of corresponding positions shared by the two sequences and corresponds to that number divided by the number of compared positions and multiplied by 100. For example, if six out of ten positions in two paired sequences are the same, then the two sequences are 60% identical. Typically, the maximum homology / identity is obtained by aligning two sequences for comparison.

[0039] The term "coding" is used interchangeably and refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) as a template for the synthesis of other polymers having a defined amino acid sequence, and the resulting biological properties. Thus, if the transcription of a gene and the translation of the corresponding mRNA into a protein produce a protein in a cell or other biological system, then the gene encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is typically described in sequence listings and databases) and the non-coding strand (which serves as a template for the transcription of the gene or cDNA) can be referred to as encoding that protein or other product of the gene or cDNA.

[0040] In the context of this invention, the statement "the genome contains at least one additional copy" means that, regardless of whether the genome of the parent microorganism originally contained a copy of the gene mentioned, one or more additional copies of that gene have been introduced into the genome through recombination, transfection, or any other genetic modification technique. In other words, if the microorganism's genome did not originally contain such a gene, then it will therefore include at least one copy of the introduced gene. Conversely, if the genome already contains one copy, then it will have at least two copies. These additional copies may be identical to or modified relative to the gene's natural sequence, and their presence is intended to increase the expression of the encoded protein, thereby enhancing a specific biological function or conferring new properties to the microorganism.

[0041] In the context of this invention, the expression "gene under promoter control" or "nucleotide sequence under promoter control" refers to the control exerted by a promoter (also called a promoter sequence) on the transcription of said gene or said nucleotide sequence. This transcription is carried out according to known mechanisms, particularly initiated by the binding of an RNA polymerase to the nucleotide sequence of a promoter located upstream of said gene or said nucleotide sequence.

[0042] In the context of this invention, the terms "target protein(s)" and "protein(s) of interest" are used interchangeably and sometimes in combination, referring to proteins produced by genetically modified microorganisms according to the invention. These proteins include oligopeptides (composed of 2 to several tens of amino acids) and more complex polypeptides (composed of several tens or more amino acids), such as enzymes.

[0043] In the context of this invention, the term "simple carbon source" refers to a nutrient that provides the carbon necessary for the production of the target protein according to the invention by the genetically modified microorganisms. This so-called "simple" carbon source is a low-molecular-weight small molecule, that is, having a molecular weight of less than or equal to 500 g / mol, such as a monosaccharide, disaccharide, or polyol. According to the invention, "simple carbon source" is contrasted with "complex carbon source," which corresponds to a high-molecular-weight macromolecule, that is, a molecular weight greater than 500 g / mol.

[0044] In the context of this invention, the term "inducing substrate" refers to a carbon source that induces the production of the target protein according to the invention. Furthermore, this inducing carbon source is not the sole carbon source for producing the protein.

[0045] Preferably, the present invention relates to a fungal microorganism for the genetic modification of at least one target protein in the presence of a simple carbon source as defined above, which has the following technical features, alone or in combination:

[0046] - A copy of the gene encoding transcription factor XlnR or the nucleotide sequence encoding transcription factor XlnR as shown in SEQ ID NO: 4, or a copy of the nucleotide sequence encoding a peptide sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the transcription factor XlnR as shown in SEQ ID NO: 4, is placed under the control of its natural promoter or constitutive promoter.

[0047] The genome of the microorganism further comprises at least one additional copy of: i) a gene encoding the transcription factor XlnR and consisting of the nucleotide sequence shown in SEQ ID NO: 3; ii) a nucleotide sequence encoding the transcription factor XlnR shown in the peptide sequence SEQ ID NO: 4; or iii) a nucleotide sequence encoding the peptide sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the transcription factor XlnR shown in the peptide sequence SEQ ID NO: 4.

[0048] - The constitutive promoter is selected from all constitutive promoters known to those skilled in the art. For example, it can be selected from pGPD, ptef1, penoA, or ppkiA.

[0049] - The promoter regulated by the transcription factor XlnR is selected from all promoters known to those skilled in the art, including those containing the transcription factor XlnR binding motif. For example, it can be selected from one of the promoters of pXynB, pAbf62b, pXynC, or genes encoding arabinofuranyl esterase B3, β-xylosidase GH3, or acetylxylan esterase.

[0050] -The target protein is either a homologous protein or a heterologous protein.

[0051] The target protein is either arabinofuranyl glycosidase (TvAbf51a) from *Talaromyces versatilis* or glucose oxidase from *Aspergillus niger*.

[0052] - The genetically modified fungal microorganisms are selected from the class Ascomycetes.

[0053] - The genetically modified fungal microorganisms are selected from the group consisting of either the class Eurotiomycetes or the class Sordariomycetes.

[0054] - The genetically modified fungal microorganisms are selected from the group consisting of Trichocomaceae, Aspergillaceae, or Hypocreaceae.

[0055] - The genetically modified fungal microorganisms are selected from the group consisting of the genera *Talaromyces*, *Aspergillus*, *Trichoderma*, or *Penicillium*.

[0056] - The genetically modified fungal microorganisms are selected from the group consisting of various basket-shaped fungi, Talamomyces cellulolyticus, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, or Penicillium chrysogenum.

[0057] The genetically modified fungal microorganisms according to the invention are 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 monosaccharides and / or disaccharides and / or polyols, advantageously composed of monosaccharides and / or disaccharides and / or polyols, preferably glucose, sucrose, lactose, glycerol, or mixtures thereof, more preferably glucose.

[0058] The modification of the fungal microbial genome according to the present invention unexpectedly makes it possible to induce the production of target proteins under the regulation of a given transcription factor, which is typically subjected to a catabolic repression mechanism in the presence of a simple carbon source.

[0059] The genetically modified fungal microorganisms of the present invention induce the production of target proteins in the presence of a second simple or complex substrate, referred to as an "inducing substrate" (which differs from a simple carbon source as defined in the present invention, particularly in relation to the function and / or activity of the microorganisms of the present invention), such as xylose, xylooligosaccharides, xylan, hemicellulose, cellulose, or mixtures thereof, said inducing substrate being present in a small proportion in the culture medium, i.e., a proportion lower than that of the simple carbon source. Advantageously, the proportion of the inducing substrate is not replenished in the culture medium after being consumed by the microorganisms of the present invention.

[0060] To achieve high enzyme production capacity, it is advantageous to provide the genetically modified fungal microorganisms according to the invention with rapidly absorbable carbon sources (i.e., simple, particularly soluble carbon sources, such as glucose and / or sucrose) and inducing substrates (e.g., xylose), which allow the expression of the target protein according to the invention and its secretion into the culture medium.

[0061] The genetically modified fungal microorganisms, particularly filamentous fungi, according to the present invention can produce target proteins through aerobic fermentation in solid or liquid culture media.

[0062] The present invention also relates to a method for producing at least one target protein, comprising the following steps:

[0063] a) Prepare a culture medium containing at least one simple carbon source, an inducing substrate for the production of the target protein, at least one nitrogen source, and all minerals and nutrients necessary for the growth of fungal microorganisms that produce biomass and protein.

[0064] b) Inoculate the culture medium with a genetically modified fungal microbial strain according to one of the variants of the present invention described above.

[0065] c) Control and maintain the temperature of the culture medium between 25°C and 34°C, the pH value between 3.5 and 5, and the oxygen partial pressure (PO2) greater than 30%; and

[0066] d) Add a simple carbon source to the culture medium periodically for at least 12 hours.

[0067] Preferably, the present invention relates to a method for producing at least one target protein as defined above, which has the following technical features, alone or in combination:

[0068] The inducing substrate is selected from the group consisting of xylose, xylose polymers, cellulose, hemicellulose, xylan, and mixtures thereof.

[0069] The simple carbon source comprises monosaccharides and / or disaccharides and / or polyols, preferably glucose, sucrose, lactose, glycerol, or mixtures thereof, more preferably glucose.

[0070] - The simple carbon source is present in the culture medium at a concentration higher than that of the inducing substrate.

[0071] -The method further includes a step e) of recovering the culture supernatant containing the target protein. Attached Figure Description

[0072] [ Figure 1 During fermentation for 72 hours in a medium containing glucose as the sole simple carbon source, the arabinofuranosylase activity of two strains (TR 3889 and TR 3885) according to the invention and a deregulated wild-type strain (IMI 378536 Δ) was determined by ion chromatography (high-performance anion exchange chromatography).

[0073] B / During fermentation for 72 hours in a medium containing glucose as the sole simple carbon source, the arabinofurase activity of the three strains (TR 3885, TR 4232 and TR 4184), the deregulated wild-type strain (IMI 378536 Δ), and the unregulated parental wild-type strain (IMI 378536 (WT)) according to the invention was determined using PNP-ABF (p-nitrophenyl-arabinofuranoside).

[0074] In both cases, after fermentation for 72 hours in a medium containing glucose at a concentration of 11 g / L, the arabinofuranosidase activity (expressed as U / mL culture supernatant) was determined in the medium.

[0075] [ Figure 2 Proteomic analysis of the secretory group of strain TR 3885 obtained at the end of fermentation.

[0076] Mass spectrometry was used to perform proteomic analysis of the secretome to determine the abundance of each protein secreted by the strain during fermentation.

[0077] The invention is illustrated in a non-limiting manner by way of the following examples. Detailed Implementation

[0078] Example 1: An example of a filamentous fungal strain according to the present invention

[0079] 1. Diverse basket-shaped bacteria strains TR 3889 and TR 3885

[0080] The diverse *Basilaria* strains TR 3889 and TR 3885 were obtained through genetic modification of the diverse *Basilaria* strain IMI 378536. The genome modifications for these two strains were performed as follows.

[0081] 1.1. Deregulation of the production strain

[0082] Using genome editing tools, a mutation was introduced into the gene of *Bacillus divaricata* strain IMI 378536, consisting of the nucleotide sequence SEQ ID NO: 1, to produce the insertion of a frameshift and stop codon, resulting in the production of a truncated, nonfunctional peptide. This produced a deregulated *Bacillus divaricata* strain IMI 378536 Δ, having a mutant gene consisting of the nucleotide sequence SEQ ID NO: 12, which is a nucleotide sequence of at least one inactivated version of the native peptide sequence shown in SEQ ID NO: 2, and the inactivated version consisting of the peptide sequence SEQ ID NO: 13.

[0083] 1.2. Constructing the Expression Box

[0084] - Cloning the gene encoding the transcription factor XlnR (sequence SEQ ID NO: 4) (sequence SEQ ID NO: 3):

[0085] The gene encoding the transcription factor XlnR was cloned under the control of its own promoter or constitutive promoter (pGPD) and its own terminator. Expression cassette 1, generated in the presence of a constitutive promoter (pGPD), consists of the nucleotide sequence shown in sequence ID NO: 5. Expression cassette 2, generated in the presence of the natural promoter of XlnR, consists of the nucleotide sequence shown in sequence ID NO: 9.

[0086] - Cloning the gene encoding the target protein:

[0087] Under the control of the promoter (pXynB) of gene XynB and its own terminator, the gene encoding arabinofuranyl glycosidase TvAbf51a (sequence SEQ ID NO: 7) was cloned (sequence SEQ ID NO: 6). The resulting expression cassette 3 consists of the nucleotide sequence shown in sequence ID NO: 8.

[0088] Under the control of the promoter (pabf62b) and its own terminator, the gene encoding arabinofuranyl glycosidase TvAbf51a (sequence SEQ ID NO: 7) (sequence SEQ ID NO: 6) was cloned. The resulting expression cassette 4 consists of the nucleotide sequence shown in sequence ID NO: 10.

[0089] Under the control of the promoter (pXynC) of gene XynC and its own terminator, the gene encoding arabinofuranyl glycosidase TvAbf51a (sequence SEQ ID NO: 7) was cloned (sequence SEQ ID NO: 6). The resulting expression cassette 5 consists of the nucleotide sequence shown in sequence ID NO: 11.

[0090] 1.3. Co-transformation of various basket-shaped bacteria IMI 378536 Δ and selection of transformants:

[0091] The diverse basket bacterium strains IMI378536 Δ were co-transformed with expression cassettes 1, 2, 3, 4 and 5 (as well as selection markers) to deregulated genes, resulting in multiple copies of the two genes being randomly integrated into the genome.

[0092] Transformants were then screened based on their arabinofuranase activity during fermentation using glucose as a simple carbon source. At the end of the sequential screening steps, four transformants were selected: TR 3889, TR 3885, TR 4184, and TR 4232. More specifically, transformant TR 3889 was obtained by co-transformation with expression cassettes 1 and 3, transformant TR 3885 by co-transformation with expression cassettes 2 and 3, transformant TR 4184 by co-transformation with expression cassettes 2 and 4, and transformant TR 4232 by co-transformation with expression cassettes 2 and 5.

[0093] Example 2: Production of the target enzyme (arabinofuranyl glycosidase TvAbf51)

[0094] 1. Cultivating fungi in a fermenter

[0095] Prior to fermentation, fungi according to the invention, namely strains *Bambusa multiplex* TR 3889 and TR 3885, are precultured in conical flasks to produce sufficient biomass for inoculation of the fermenter. The preculture medium consists of a simple carbon source (glucose) and a nitrogen nutrient source (corn steep liquor).

[0096] The pre-incubation period lasts approximately 25 hours.

[0097] At the end of the pre-culture, the fermenter was inoculated with the pre-culture at a rate of 5% (v / v).

[0098] 2. Fermentation

[0099] The production of the target enzyme, arabinofuranosylase, was carried out in a 2L fermenter. The culture medium used for fermentation consisted of a single simple carbon source (glucose), an inducer (cellulose), and several nitrogen sources (ammonium sulfate, diammonium hydrogen phosphate, corn stalk liquor), minerals (CaCl2, KH2PO4, MgSO4), and trace elements (MnSO4, FeSO4, ZnSO4).

[0100] Among the main culture parameters that were controlled, the temperature was 30℃, the pH was 4, the stirring frequency varied from 500 to 950 rpm, and the aeration rate was 75 L / h.

[0101] After approximately 24 hours of growth, glucose is fed continuously at a rate of 1 to 2 grams of glucose per liter per hour.

[0102] The culture continues for a total duration of approximately 72 hours.

[0103] 3.A. The activity of arabinofuranosylase TvAbf51 during fermentation was monitored by ion chromatography (high-performance anion exchange chromatography).

[0104] After culturing in a fermenter for 72 hours, biomass was separated from the supernatant by centrifugation. The arabinofuranase activity of the culture supernatant was evaluated by hydrolyzing the arabinoxylan substrate followed by anion-exchange chromatography to determine the released arabinose. A diluted solution of the fermentation supernatant was incubated at 50°C for 15 minutes in the presence of wheat arabinoxylan. The arabinofuranase hydrolyzes the glycosidic bonds between branched arabinofuranyl glycosides and the main xylan chain. This results in the release of arabinose monomers, which were then separated by ion chromatography (high-performance anion exchange chromatography) on a CarboPac PA10 column and quantified by amperometric detection.

[0105] The results show Figure 1 In A, it was demonstrated that during glucose-fed culture, the mutant strains of the present invention (i.e., *Bacteroides diversicolor* TR 3889 and TR 3885) produced arabinofuranosylase significantly higher than the deregulated parental strains (i.e., *Bacteroides diversicolor* IMI 378536 Δ); the latter produced less because of the lack of copies of two genes integrated into the genome of the mutant strains. In fact, the deregulated parental strains did not benefit from the increased presence of transcription factor XlnR, as they were no longer affected by the glucose-induced catabolite repression mechanism associated with the increased presence of the gene encoding arabinofuranosylase (whose expression is positively regulated by transcription factor XlnR).

[0106] 3.B. The activity of arabinofuranoside TvAbf51 during fermentation was monitored using PNP-ABF (p-nitrophenyl-arabinofuranoside).

[0107] After culturing in a fermenter for 72 hours, biomass was separated from the supernatant by centrifugation. The arabinofurase activity of the culture supernatant was evaluated by hydrolyzing a substrate composed of 4-nitrophenyl α-L-arabinofuranylglycoside. A diluted solution of the fermentation supernatant was incubated at 40°C for 12 minutes in the presence of 4-nitrophenyl α-L-arabinofuranylglycoside, and the reaction was terminated by adding sodium carbonate solution. The arabinofurase was able to hydrolyze the glycosidic bonds in 4-nitrophenyl α-L-arabinofuranylglycoside. This resulted in the release of p-nitrophenol, which turned yellow in alkaline medium and could be measured spectrophotometrically at 405 nm.

[0108] The results show Figure 1 In section B, it was demonstrated that during glucose-fed culture, the mutant strains of the present invention (i.e., *Bacteroides diversicolor* TR 3885, TR 4232, and TR 4184) produced significantly higher arabinofuranosylase production than the deregulated parental strains (i.e., *Bacteroides diversicolor* IMI 378536 Δ), and the non-deregulated parental strains (i.e., *Bacteroides diversicolor* IMI 378536 (WT) corresponding to the natural strain) produced no arabinofuranosylase at all; the latter did not produce protein because, in the presence of a simple carbon source, the naturally occurring catabolism repression mechanism in fungal microorganisms lacks a bypass, while the lower production in the deregulated parental strain is due to the absence of two gene copies integrated into the mutant strain's genome. In fact, the deregulated parental strain IMI 378536 Δ did not benefit from the increased presence of transcription factor XlnR, and was no longer affected by the glucose-induced catabolism repression mechanism associated with the increased presence of the gene encoding arabinofuranosylase (whose expression is positively regulated by transcription factor XlnR). Finally, very high production was also observed for two other mutant strains of the present invention, TR 4232 and TR 4184, which confirms the effect of the microorganisms of the present invention on other promoters regulated by transcription factor XlnR (i.e., promoters pXynC and pabf62b, respectively), and thus confirms this effect on all promoters known to include transcription factor XlnR binding motifs.

[0109] 4. Proteomic analysis of the secretory group of strain TR 3885 obtained at the end of fermentation.

[0110] The results of the proteomic analysis showed that Figure 2 The production of the enzyme mixture was confirmed, with arabinofuranosylase TvAbf51 being the most abundant enzyme (22% relative abundance).

[0111] 5. Conclusion

[0112] These data clearly demonstrate that the genetically modified filamentous fungus according to the present invention 1) alters the gene consisting of the nucleotide sequence SEQ ID NO: 1, 2) enhances the expression of the transcription factor XnlR, and 3) integrates one or more copies of the gene encoding arabinofuranase TvAbf51a, placing it under the control of one of the promoters known to be regulated by XnlR, thus ensuring the efficient production of arabinofuranase TvAbf51a in the presence of a simple and inexpensive carbon source (i.e., glucose). This conclusion was also observed using two different assay techniques, thus confirming the accuracy of the reported effects of the genetically modified fungal microorganism according to the present invention, regardless of the assay methods used. Furthermore, the viability or colony morphology of this microorganism remains unchanged. In other words, the present invention ensures the efficient growth of the microorganism without disrupting amino acid transport and nitrogen assimilation. Moreover, it is feasible to modify this microorganism to produce one or more other target proteins, and makes it possible to provide a true protein production platform through which target proteins can be intentionally produced.

Claims

1. A genetically modified fungal microorganism belonging to a diverse basketbacterial species, for producing at least one target protein in the presence of a simple carbon source, the genome of which comprises: a) At least: i. An inactivated version of the gene consisting of the nucleotide sequence shown in SEQ ID NO:

1. ii. The nucleotide sequence of an inactivated version of the peptide sequence shown in SEQ ID NO: 2, or iii. A nucleotide sequence encoding an inactivated version of the peptide sequence having at least 65% identity with the peptide sequence of SEQ ID NO:

2. b) At least one of the following additional copies: i. A gene encoding the transcription factor XlnR and consisting of the nucleotide sequence shown in SEQ ID NO:

3. ii. The nucleotide sequence encoding the transcription factor XlnR, as shown in SEQ ID NO: 4, or iii. The nucleotide sequence encoding the transcription factor XlnR shown in SEQ ID NO: 4, having at least 65% identity with the peptide sequence, and c) At least one of the following additional copies: i. A gene encoding the peptide sequence of the target protein, wherein the gene is placed under the control of a promoter regulated by the transcription factor XlnR, or ii. A nucleotide sequence encoding a peptide sequence having at least 65% identity with the peptide sequence of the target protein, wherein the nucleotide sequence is under the control of a promoter regulated by the transcription factor XlnR.

2. The genetically modified fungal microorganism according to claim 1, wherein a copy of the gene encoding transcription factor XlnR or a copy of the nucleotide sequence encoding the transcription factor XlnR shown in SEQ ID NO: 4, or a copy of the nucleotide sequence encoding the peptide sequence having at least 65% identity with the transcription factor XlnR shown in SEQ ID NO: 4, is placed under the control of its natural promoter or constitutive promoter.

3. The genetically modified fungal microorganism according to claim 1 or 2, wherein its genome further comprises at least one additional copy of the following: i. A gene encoding the transcription factor XlnR and consisting of the nucleotide sequence shown in SEQ ID NO:

3. ii. The nucleotide sequence encoding the transcription factor XlnR, as shown in SEQ ID NO: 4, or iii. A nucleotide sequence encoding the peptide sequence having at least 65% identity with the transcription factor XlnR shown in SEQ ID NO:

4.

4. The genetically modified fungal microorganism according to any one of claims 2 or 3, wherein the constitutive promoter is selected from pGPD, ptef1, penoA or ppkiA.

5. The genetically modified fungal microorganism according to any one of the preceding claims, wherein the promoter regulated by the transcription factor XlnR is selected from one of the promoters of the gene encoding arabinofuranosylase B3, β-xylosidase GH3 or acetylxylan esterase.

6. A genetically modified fungal microorganism according to any one of the preceding claims, wherein the target protein is a homologous protein or a heterologous protein.

7. The genetically modified fungal microorganism according to the preceding claims, wherein the target protein is arabinofuranosidase (TvAbf51a) of *Aspergillus niger* or glucose oxidase of *Aspergillus niger*.

8. A method for producing at least one target protein, comprising the following steps: a) Prepare a culture medium containing at least one simple carbon source, an inducing substrate for the production of the target protein, at least one nitrogen source, and all minerals and nutrients necessary for the growth of fungal microorganisms that produce biomass and protein. b) Inoculate the culture medium with a genetically modified fungal microbial strain belonging to a diverse basket-shaped fungal species according to any one of claims 1 to 7. c) Control and maintain the temperature of the culture medium between 25°C and 34°C, the pH of the culture medium between 3.5 and 5, and the partial pressure of oxygen (PO2) greater than 30%; and d) Add the simple carbon source to the culture medium periodically for at least 12 hours.

9. The method according to the preceding claims, wherein the inducing substrate is selected from the group consisting of xylose, xylose polymers, cellulose, hemicellulose, xylan, and mixtures thereof.

10. The method according to any one of claims 8 or 9, wherein the simple carbon source comprises monosaccharides and / or disaccharides and / or polyols, preferably glucose, sucrose, lactose, glycerol or mixtures thereof, more preferably glucose.

11. The method according to any one of claims 8 to 10, wherein the simple carbon source is present in the culture medium at a concentration higher than that of the inducing substrate.

12. The method according to any one of claims 8 to 11, wherein the target protein is arabinofuranylase TvAbf51a.

13. The method according to any one of claims 8 to 12, further comprising step e) recovering the culture supernatant containing the target protein.

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