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

Abstract

The present invention relates to a genetically modified fungal microorganism for producing a target protein in the presence of a simple carbon source and to a method for producing the target protein.

Description

[0001] DESCRIPTION

[0002] TITLE: PRODUCTION OF PEPTIDES OF INTEREST FROM A SIMPLE CARBON SOURCE BY A GENETICALLY MODIFIED FUNGAL MICROORGANISM

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a genetically modified fungal microorganism for the production of a target protein in the presence of a simple carbon source and a method for producing said target protein.

[0005] STATE OF THE ART

[0006] The production of proteins of interest on an industrial scale is a major commercial challenge and represents a considerable market. For example, enzymes have long been used industrially for a variety of applications in diverse fields such as food processing, papermaking, textiles, the production of biofuels, detergents, etc. They are particularly widely used in the food industry, for example 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 that are easily assimilated by the animal or molecules that are 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.

[0007] Some of these proteins essential for improving animal nutrition are not produced by mammals or are produced in insufficient quantities. This is the case, for example, of glycosidases, in particular xylanases and arabinofuranosidases, which are enzymes essential for the degradation of hemicelluloses, one of the main constituents of cell walls in plants. Indeed, 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.This is also the case for certain phytases or proteases which constitute proteins of interest in animal feed (for example as a zootechnical additive) as well as in human food (for example for their use in the preparation of fruit juices, where they allow a greater quantity of juice to be released and / or a better quality juice to be obtained, or in the preparation of bread doughs, where they allow greater extensibility and malleability of the doughs).

[0008] These proteins 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, an 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 need to synthesize 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.

[0009] 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.

[0010] In filamentous fungi, several transcription factors, such as XlnR, AraR, ClrA, ClrB, Man R, and AraA, have been described as positive regulators of the expression of genes encoding certain proteins of interest, such as glycosidases, while CreA and ACEI are the main negative regulators identified.

[0011] 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, as explained previously, these simple carbon sources induce a catabolic repression mechanism in most microorganisms which has the effect of repressing gene expression, thus preventing the transcription and / or translation of the proteins of interest.Another major challenge is that the production of these proteins of interest can be modulated on a customized basis (for example, depending on the evolution of a customer's demands). Currently, no solution for the production of proteins of interest, such as enzymes, allows for rapid and efficient adjustment of the protein of interest produced, while remaining economically profitable.

[0012] It is clear from the above that there is a clear need to develop new technical solutions allowing efficient production of proteins of interest, in particular on an industrial scale, which can be adapted according to market needs and which do not have the disadvantages of the prior art, such as costly, long production and / or production dependent on the presence in the culture medium of a suitable carbon source.

[0013] DESCRIPTION OF THE INVENTION

[0014] The inventors have unexpectedly and surprisingly developed genetically modified microorganisms, in particular genetically modified filamentous fungi, having an increased capacity to produce proteins of interest from a simple carbon source, independently of the catabolic repression mechanism, in comparison with the parent microorganisms (also called independently: native or wild microorganisms or strains). An objective of the present invention is to enable the scalable production, on an industrial scale, of one or more targeted proteins of interest, from a simple carbon source using a genetically modified microorganism, in particular a fungal microorganism.In other words, the objective of the invention is to propose a platform for producing one or more proteins of interest, the proteins produced being able to be modulated over time (for example depending on the evolution of a customer's demands, at a given moment).

[0015] Thus, the present invention relates to a genetically modified fungal microorganism for the production of 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 presented by the sequence SEQ ID NO: 1, ii. a nucleotide sequence encoding an inactivated version of the peptide sequence presented by the sequence SEQ ID NO: 2, or iii. a nucleotide sequence encoding an inactivated version of a peptide 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, b) at least one additional copy: i. of a gene encoding the transcription factor XlnR and consisting of the nucleotide sequence presented by the sequence SEQ ID NO: 3, ii. a nucleotide sequence encoding the transcription factor XlnR presented by the peptide sequence SEQ ID NO: 4, or iii.of 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 transcription factor XlnR presented by the sequence SEQ ID NO: 4, and c) at least one additional copy: i. of a gene encoding the peptide sequence of said target protein, said gene being placed under the control of a promoter regulated by the transcription factor XlnR, or ii. of 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 said target protein, said nucleotide sequence being placed under the control of a promoter regulated by the transcription factor XlnR.

[0016] This modification of the genome of the fungal microorganism results in: a) the absence of production of the corresponding protein or the inactivation of the expressed protein (i.e. a non-functional protein), said protein being involved in the catabolite repression mechanism naturally present in fungal microorganisms, b) the production of a transcription factor no longer subject to the effects of the catabolite repression naturally present, and c) the increased production of the target protein, this production being regulated by the transcription factor no longer subject to the effects of the catabolite repression.

[0017] 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 complex proteins, such as enzymes, involved in the degradation of more complex substrates.

[0018] According to the invention, the fungal microorganism does not naturally have: a capacity to produce the target protein, in the case of a heterologous protein, or a capacity to produce the target protein, in the case of a homologous protein, from a simple carbon source, and the genetic modifications made are intended to confer this capacity on it from a simple carbon source.

[0019] The present invention therefore has various advantages, in particular:

[0020] - an industrial interest for the production of proteins of interest (for example the production of enzymes used as a zootechnical additive in animal feed, in particular farm animals, or used in the improvement of foodstuffs, such as fruit juices or bread dough), without altering the viability and morphology of the microorganism;

[0021] - an economic interest via the use of a simple, inexpensive and soluble carbon source to produce a target protein of interest, homologous or heterologous, which can be different over time depending on the evolution of needs;

[0022] - efficient bio-production of this protein of interest by microorganisms at industrial yields.

[0023] 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.

[0024] 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 which controls the expression of the gene coding for said protein and / or into the sequence coding for said protein,by the shift of the reading frame at the origin of the introduction of an early stop codon in the sequence coding for the protein or by the deletion of the gene coding for the transcription factor which regulates the promoter controlling the expression of the gene coding for said protein and / or by the deletion of the gene coding for said protein. In this sense, the expressions "an inactivated version of a gene" and "an inactivated version of a peptide sequence" mean the total or partial absence of the functional activity of the gene and protein concerned. These inactivations can be obtained in particular by the various genetic approaches mentioned above (such as the introduction of mutations, the deletion of critical regions of the gene or protein, or the interruption of its expression by interference mechanisms). For example, an inactivated version of a given gene can result in a nucleotide sequence which can no longer be transcribed or translated,or producing a truncated or altered protein, unable to perform its usual biological function. For example, an inactivated version of a peptide sequence may result in a protein no longer interacting with its usual molecular partners, no longer catalyzing an essential enzymatic reaction, or no longer being localized in the appropriate cellular compartment to perform its function. For example, an inactivated enzyme may lack its active site due to a mutation, thus preventing any substrate from binding and making the enzymatic reaction impossible.

[0025] For the purposes of the invention, the terms "identity" and "homology" 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 monomeric amino acid subunit, then the molecules are homologous or identical for that position. The percentage identity between two sequences is a function of the number of matching 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.

[0026] 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.

[0027] In the context of the present invention, the expression "genome comprises at least one additional copy" means that, regardless of whether or not the genome of the parent microorganism contains a copy of the gene in question, one or more additional copies of this gene have been introduced into the genome, whether by recombination, transfection or any other genetic modification technique. In other words, if the genome of the microorganism does not initially contain one, it will therefore contain at least one copy of the introduced gene. On the other hand, if the genome already contains one copy, it will then have at least two copies. These additional copies may be identical or modified compared to the native sequence of the gene, and their presence is intended to increase the expression of the encoded protein, thus aiming to reinforce a particular biological function, or to confer a new property on the microorganism.

[0028] In the context of the present invention, the expressions "a gene placed under the control of a promoter" or "a nucleotide sequence placed under the control of a promoter" refer to the control exercised by the promoter (also called promoter sequence) on the transcription of said gene or said nucleotide sequence. This transcription is carried out according to known mechanisms and is in particular initiated by the attachment of RNA polymerase to the nucleotide sequence of the promoter, which is located upstream of said gene or said nucleotide sequence.

[0029] In the context of the present invention, the expressions "target protein(s)" and "protein(s) of interest" are used interchangeably, sometimes combined, and refer to the proteins that are to be produced via the genetically modified microorganism according to the invention. These proteins include both oligopeptides (composed of 2 to a few dozen amino acids) and more complex polypeptides (composed of a few dozen amino acids or more), such as enzymes.

[0030] In the context of the present invention, the expression "simple carbon source" designates a nutrient providing the carbon necessary for the genetically modified microorganism according to the invention to produce the proteins of interest 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.

[0031] In the context of the present invention, the expression "inducing substrate" designates an inducing carbon source for the production of the proteins of interest according to the invention. Furthermore, this inducing carbon source is not the total carbon source for the production of said proteins.

[0032] Preferably, the subject of the present invention is a genetically modified fungal microorganism for the production of at least one target protein in the presence of a simple carbon source as defined above and having the following technical characteristics, taken alone or in combination:

[0033] - the copy of the gene coding for the transcription factor XlnR or the nucleotide sequence coding for the transcription factor XlnR presented by the sequence SEQ ID NO: 4 or the nucleotide sequence coding for the peptide sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the transcription factor XlnR presented by the sequence SEQ ID NO: 4 is placed under the control of its native promoter or a constitutive promoter,

[0034] - the genome of the microorganism further comprises at least one additional copy i) of the gene encoding the transcription factor XlnR and consisting of the nucleotide sequence presented by the sequence SEQ ID NO: 3, ii) of the nucleotide sequence encoding the transcription factor XlnR presented by the peptide sequence SEQ ID NO: 4 or iii) of the nucleotide sequence encoding said peptide sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the transcription factor XlnR presented by the peptide sequence SEQ ID NO: 4,

[0035] - the constitutive promoter is chosen from all the constitutive promoters known to those skilled in the art. For example, it may be chosen from pGPD, ptefl, penoA or ppkiA,

[0036] - the promoter regulated by the transcription factor XlnR is chosen from all the promoters known to those skilled in the art as comprising a motif for binding the transcription factor XlnR. For example, it may be chosen from pXynB, pAbf62b, pXynC or one of the promoters of the genes encoding arabinofuranosidase B3, beta-xylosidase GH3 or acetylxylan esterase,

[0037] - the target protein is a homologous protein or a heterologous protein,

[0038] - the target protein is arabinofuranosidase from Talaromyces versatilis (TvAbf51a) or Glucose oxidase from Aspergillus niger,

[0039] - the genetically modified fungal microorganism is selected from Ascomycetes,

[0040] - the genetically modified fungal microorganism is selected from the group consisting of the classes Eurotiomycetes or Sordariomycetes, - the genetically modified fungal microorganism is selected from the group consisting of the families Trichocomaceae, Aspergillaceae or Hypocreaceae,

[0041] - the genetically modified fungal microorganism is selected from the group consisting of the genera Talaromyces, Aspergillus, Trichoderma or Penicillium,

[0042] - the genetically modified 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 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.

[0044] The modification of the genome of the fungal microorganism according to the invention unexpectedly makes it possible to induce production of target proteins under the regulation of a given transcription factor which normally undergoes the effects of the catabolic repression mechanism implemented in the presence of a simple carbon source.

[0045] The genetically modified fungal microorganism of the invention induces a production of target proteins 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, that is to say 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.

[0046] In order to obtain good enzyme productivity, it is advantageous 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 target proteins according to the invention and their secretion into the culture medium.

[0047] The genetically modified fungal microorganism according to the invention, in particular a filamentous fungus, can be used for the production of target proteins by aerobic fermentation in a solid or liquid medium.

[0048] The invention also relates to a method for producing at least one target protein comprising the following steps: a) preparation of a culture medium containing at least one simple carbon source, an inducing substrate for the production of said target protein, at least one nitrogen source, as well as all the minerals and nutrients necessary for the growth of the fungal microorganism for the production of biomass and proteins, b) seeding the culture medium with a strain of genetically modified fungal microorganism according to one of the variants of the invention previously described, c) controlling and maintaining the temperature of the culture medium at a value between 25°C and 34°C, the pH of the culture medium at a value between 3.5 and 5 and an oxygen partial pressure (PO2) greater than 30%; and d) regular addition of the simple carbon source to the culture medium for at least 12 hours.

[0049] Preferably, the subject of the present invention is a method for producing at least one target protein as defined above having the following technical characteristics, taken alone or in combination:

[0050] - the inducing substrate is chosen from the group consisting of xylose, a xylose polymer, cellulose, hemicelluloses, xylan and their mixtures,

[0051] - 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,

[0052] - the simple carbon source is present in the culture medium at a concentration higher than that of the inducing substrate,

[0053] - the method further comprises a step e) of recovering the culture supernatant containing said target protein. Figures:

[0054] [Fig. 1]: A / Arabinofuranosidase activity measured by ion chromatography (High Pressure Anion Exchange Chromatography) of two strains according to the invention (TR 3889 and TR 3885) and of a deregulated wild strain (IMI 378536 A) during 72 hours of fermentation in culture medium comprising glucose as the only source of simple carbon.

[0055] B / Arabinofuranosidase activity assayed with PNP-ABF (para-nitrophenyl-arabinofuranoside) of three strains according to the invention (TR 3885, TR 4232 and TR 4184), of a deregulated wild strain (IMI 378536 A) and of a non-deregulated parent wild strain (IMI 378536 (WT)) during 72 hours of fermentation in culture medium comprising glucose as the sole source of simple carbon.

[0056] In both cases, arabinofuranosidase activity (expressed in U / mL of culture supernatant) was determined in the culture medium after 72 hours of fermentation in a medium comprising glucose at a concentration of 11 g / L of culture medium.

[0057] [Fig. 2]: Proteomic analysis of the secretome of strain TR 3885 obtained at the end of fermentation.

[0058] Proteomic analysis of the secretome was performed by mass spectrometry in order to give the abundance of each of the proteins secreted during fermentation of the strain.

[0059] The present invention is illustrated in a non-limiting manner by the following examples.

[0060] EXAMPLES OF ACHIEVEMENT

[0061] Example 1: Examples of filamentous fungal strains according to the invention

[0062] 1. Talaromvces versatilis strains TR 3889 and TR 3885

[0063] T. versatilis strains TR 3889 and TR 3885 were obtained by genetic modification of T. versatilis strain IMI 378536. Genome modifications of these two strains were carried out as follows.

[0064] 1.1. Deregulation of the production strain

[0065] Mutations were introduced into the gene of the T. versatilis strain IMI 378536 consisting of the nucleotide sequence SEQ ID NO: 1, by genome editing tools, so as to create a frameshift and the introduction of a stop codon resulting in the production of a truncated and non-functional peptide. A deregulated T. versatilis strain IM I 378536 A is thus obtained, having the mutated gene consisting of the nucleotide sequence SEQ ID NO: 12 encoding at least one inactivated version of the native peptide sequence presented by the sequence SEQ ID NO: 2, said inactivated version consisting of the peptide sequence SEQ ID NO: 13.

[0066] 1.2. Construction of expression cassettes

[0067] Cloning of the gene (sequence SEQ ID NO: 3) encoding the transcription factor XlnR (sequence SEQ ID NO: 4):

[0068] The gene encoding the transcription factor XlnR was cloned under the control of either its own promoter or a constitutive promoter (pGPD) and its own terminator. Expression cassette 1 thus created in the presence of the constitutive promoter (pGPD) consists of the nucleotide sequence presented by the sequence ID NO: 5. Expression cassette 2 thus created in the presence of the native XlnR promoter consists of the nucleotide sequence presented by the sequence ID NO: 9.

[0069] Cloning of the gene encoding the protein of interest:

[0070] The gene (sequence SEQ ID NO: 6) encoding the arabinofuranosidase TvAbf51a (sequence SEQ ID NO: 7) was cloned under the control of the XynB gene promoter (pXynB) and its own terminator. The expression cassette 3 thus created consists of the nucleotide sequence presented by the sequence ID NO: 8.

[0071] The gene (sequence SEQ ID NO: 6) encoding the arabinofuranosidase TvAbf51a (sequence SEQ ID NO: 7) was cloned under the control of the promoter of the abf62b gene (pabf62b) and its own terminator. The expression cassette 4 thus created consists of the nucleotide sequence presented by sequence ID NO: 10.

[0072] The gene (sequence SEQ ID NO: 6) encoding the arabinofuranosidase TvAbf51a (sequence SEQ ID NO: 7) was cloned under the control of the XynC gene promoter (pXynC) and its own terminator. The expression cassette 5 thus created consists of the nucleotide sequence presented by sequence ID NO: 11.

[0073] 1.3. Co-transformation of T. versatilis IMI 378536 A and selection of transformants: The deregulated T. versatilis strain IMI 378536 A was co-transformed with the expression cassettes 1, 2, 3, 4 and 5 described above (as well as a selection marker), which led to the random integration into the genome of multiple copies of the 2 genes.

[0074] The resulting transformants were then screened based on their arabinofuranosidase activity during fermentation using glucose as a simple carbon source. After successive screening steps, the four transformants TR 3889, TR 3885, TR 4184 and TR 4232 were selected. Specifically, transformant TR 3889 resulted from co-transformation by expression cassettes 1 and 3, transformant TR 3885 resulted from co-transformation by expression cassettes 2 and 3, transformant TR 4184 resulted from co-transformation by expression cassettes 2 and 4, while transformant TR 4232 resulted from co-transformation by expression cassettes 2 and 5.

[0075] Example 2: Production of the enzyme of interest (arabinofuranosidase TvAbf51)

[0076] 1. Cultivating the mushroom in a fermenter

[0077] Prior to fermentation, the fungi according to the invention, i.e. T. versatilis strains TR 3889 and TR 3885, were 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).

[0078] The preculture duration is approximately 25 hours.

[0079] At the end of this preculture, the fermenter is inoculated with the preculture at a rate of 5% (v / v).

[0080] 2. Fermentation

[0081] The production of the target enzyme, arabinofuranosidase, is carried out in a 2L fermenter. The culture medium for fermentation consists of a single simple carbon source (glucose), an inducer (cellulose), several nitrogen sources (ammonium sulfate, diammonium phosphate, Corn Steep Liquor), minerals (CaCI2, KH2PO4, MgSO4), and trace elements (MnSO4, FeSO4, ZnSO4).

[0082] 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. After approximately 24 hours of growth, a glucose supply is implemented. This supply is continuous, at a rate of between 1 and 2 grams of glucose / hour / Liter.

[0083] The culture is continued for a total duration of approximately 72 hours.

[0084] 3.A Monitoring of arabinofuranosidase TvAbf51 activity during fermentation by ion chromatography (High Pressure Anion Exchange Chromatography)

[0085] 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 arabinoxylan 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.

[0086] The results are represented by Figure 1A and show a production of arabinofuranosidase during the glucose-fed culture that is very significantly higher for the mutant strains of the invention (i.e. T. versatilis TR 3889 and TR 3885) than for the deregulated parent strain (i.e. T. versatilis IMI 378536 A); the production being lower for the latter given the absence of copies of the two genes incorporated into the genome of the mutant strains. Indeed, the deregulated parent strain does not benefit from the advantages of an increased presence of XlnR transcription factors, no longer undergoing the effects of the catabolic repression mechanisms induced by glucose, associated with an increased presence of the gene coding for arabinofuranosidase, the expression of the latter being positively regulated by the XlnR transcription factors.

[0087] 3.B Monitoring of arabinofuranosidase activity TvAbf51 during fermentation by PNP-ABF Ipara-nitrophénvl-arabinofuranoside}

[0088] 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 a substrate consisting of 4-nitrophenyl aL-arabinofuranoside. A diluted solution of the fermentation supernatant was incubated in the presence of 4-nitrophenyl aL-arabinofuranoside for 12 minutes at 40°C, then the reaction was stopped by the addition of a sodium carbonate solution. Arabinofuranosidase is capable of hydrolyzing the osidic bond within 4-nitrophenyl aL-arabinofuranoside. This results in the release of paranitrophenol, which adopts a yellow color in basic medium, measurable by spectrophotometer at 405 nm.

[0089] The results are represented by Figure 1B and show a significantly higher production of arabinofuranosidase during the glucose-fed culture for the mutant strains of the invention (i.e. T. versatilis TR 3885, TR 4232 and TR 4184) than for the deregulated parent strain (i.e. T. versatilis IMI 378536 A), as well as the complete absence of production for the non-deregulated parent strain (i.e. T. versatilis IMI 378536 (WT) corresponding to the native strain); the production of the latter was zero given the absence of bypassing the catabolite repression mechanism naturally occurring in fungal microorganisms in the presence of a simple carbon source, while the lower production of the deregulated parent strain is explained by the absence of copies of the two genes incorporated into the genome of the mutant strains.Indeed, the deregulated parent strain IMI 378536 A does not benefit from the advantages of an increased presence of XlnR transcription factors, no longer undergoing the effects of glucose-induced catabolite repression mechanisms, associated with an increased presence of the gene coding for arabinofuranosidase, the expression of the latter being positively regulated by the XlnR transcription factors. Finally, the very significant production also observed for the two additional mutant strains of the invention TR 4232 and TR 4184 validates the effect associated with the microorganism according to the invention for other promoters regulated by the XlnR transcription factor (in this case, respectively, the pXynC and pabf62b promoters) and, consequently, validates this effect for all promoters known to contain a XlnR transcription factor binding motif.

[0090] 4. Proteomic analysis of the secretome of strain TR 3885 obtained at the end of fermentation.

[0091] The results of this proteomic analysis are represented in Figure 2 and confirm the production of an enzyme cocktail in which the arabinofuranosidase TvAbf51 is the most abundant enzyme (22% relative abundance).

[0092] 5. Conclusion It is clear from these data that a filamentous fungus genetically modified according to the invention to 1 / alter the gene consisting of the nucleotide sequence SEQ ID NO: 1, 2 / promote the expression of XnIR transcription factors and 3 / integrate one or more copies of a gene encoding the arabinofuranosidase TvAbf51a placed under the control of one of the promoters known to be regulated by XnIR, ensures efficient production of TvAbf51a arabinofuranosidases, in the presence of a simple (i.e. glucose) and inexpensive carbon source. This conclusion could also be observed via two different assay techniques, thus confirming the veracity of the effect announced for the genetically modified fungal microorganism according to the invention, independently of the assay method used. In addition, this microorganism does not show any alteration in its viability or colony morphology.

[0093] In other words, the present invention ensures efficient growth of the microorganism without disrupting amino acid transport and nitrogen assimilation. Furthermore, the adaptation of this microorganism for the purpose of producing one or more other protein(s) of interest is feasible and makes it possible to offer a real protein production platform via which it is possible to produce proteins of interest on purpose.

Claims

CLAIMS 1. Genetically modified fungal microorganism, belonging to the species Talaromyces versatilis, for the production of 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 presented by the sequence SEQ ID NO: 1, ii. a nucleotide sequence encoding an inactivated version of the peptide sequence presented by the sequence SEQ ID NO: 2, or iii. a nucleotide sequence encoding an inactivated version of a peptide sequence having at least 65% identity with the peptide sequence of sequence SEQ ID NO: 2, b) at least one additional copy: i. of a gene encoding the transcription factor XlnR and consisting of the nucleotide sequence presented by the sequence SEQ ID NO: 3, ii. a nucleotide sequence encoding the transcription factor XlnR presented by the peptide sequence SEQ ID NO: 4, or iii.of a nucleotide sequence encoding a peptide sequence having at least 65% identity with the transcription factor XlnR presented by the sequence SEQ ID NO: 4, and c) at least one additional copy: i. of a gene encoding the peptide sequence of said target protein, said gene being placed under the control of a promoter regulated by the transcription factor XlnR, or ii. of a nucleotide sequence encoding a peptide sequence having at least 65% identity with the peptide sequence of said target protein, said nucleotide sequence being placed under the control of a promoter regulated by the transcription factor XlnR.

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

3. Genetically modified fungal microorganism according to one of claims 1 or 2, the genome of which further comprises at least one additional copy: i. of the gene encoding the transcription factor XlnR and consisting of the nucleotide sequence presented by the sequence SEQ ID NO: 3, ii. of the nucleotide sequence encoding the transcription factor XlnR presented by the peptide sequence SEQ ID NO: 4 or iii. of the nucleotide sequence encoding said peptide sequence having at least 65% identity with the transcription factor XlnR presented by the peptide sequence SEQ ID NO:

4.

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

5. Genetically modified fungal microorganism according to any one of the preceding claims, in which the promoter regulated by the transcription factor XlnR is chosen from pXynB, pAbf62b, pXynC or one of the promoters of the genes encoding arabinofuranosidase B3, beta-xylosidase GH3 or acetylxylan esterase.

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

7. Genetically modified fungal microorganism according to the preceding claim, wherein the target protein is arabinofuranosidase from Talaromyces versatilis (TvAbf51a) or Glucose oxidase from Aspergillus niger.

8. Method for producing at least one target protein comprising the following steps: a) preparing a culture medium containing at least one simple carbon source, an inducing substrate for the production of said target protein, at least one nitrogen source, as well as all the minerals and nutrients necessary for the growth of a fungal microorganism for the production of biomass and proteins, b) seeding the culture medium with a strain of a genetically modified fungal microorganism, belonging to the species Talaromyces versatilis, according to one of claims 1 to 7, c) controlling and maintaining the temperature of the culture medium at a value between 25°C and 34°C, the pH of the culture medium at a value between 3.5 and 5 and a partial pressure of oxygen (PO2) greater than 30%; and d) regularly adding the simple carbon source to the culture medium for at least 12 hours.

9. Method according to the preceding claim, in which the inducing substrate is chosen from the group consisting of xylose, a xylose polymer, cellulose, hemicelluloses, xylan and mixtures thereof.

10. Method according to one of claims 8 or 9, in which 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.

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

12. A method according to any one of claims 8 to 11, wherein said target protein is arabinofuranosidase TvAbf51a.

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

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