Modified filamentous fungi for the production of exogenous proteins

Abstract

The present invention relates to a genetically modified Ascomycota filamentous fungus, particularly the species Thermothelomyces heterothallica, having reduced activity or expression of KEX2 and / or ALP7, which is capable of producing increased amounts and stability of exogenous proteins.

Description

Technical Field

[0001] The present invention relates to the production of exogenous proteins in genetically engineered Ascomycota filamentous fungi, particularly Thermothelomyces heterothallica (formerly Myceliophthora thermophila), in which the expression or activity of KEX2 and / or ALP7 protease is reduced. Genetically engineered Ascomycota filamentous fungi are used for the robust production of highly stable proteins.

Background Art

[0002] Recombinant protein production The expression and purification of recombinant proteins with functional post-translational protein modifications such as glycosylation or phosphorylation can only be achieved using eukaryotic expression systems. Eukaryotic protein expression systems, including mammalian cells, plants, and fungi, have become essential for the production of functional eukaryotic proteins.

[0003] Wild-type Thermothelomyces heterothallica (Th. heterothallica) C1 (recently renamed from Myceliophthora thermophila and the scientific name renamed from Chrysosporium lucknowense) is a thermotolerant Ascomycota filamentous fungus that produces high levels of cellulase and is attractive for the production of these and other proteins on a commercial scale.

[0004] For example, the applicants of the present invention, U.S. Patent Nos. 8,268,585 and 8,871,493, disclose a transformation system in the field of filamentous fungal hosts for expressing and secreting heterologous proteins or polypeptides. They also disclose a process for producing large quantities of polypeptides or proteins in an economical manner. The system includes transformed or transfected strains of the genus Chrysosporium, more specifically Chrysosporium lucknowense and its mutants or derivatives. Transformants including Chrysosporium coding sequences and Chrysosporium gene expression regulatory sequences are also disclosed.

[0005] Wild-type C1 was deposited on August 29, 1996, under the designation VKM F-3500D, in accordance with the Budapest Convention. High-cellulase (HC) and low-cellulase (LC) strains, such as those described in U.S. Patent No. 8,268,585, have also been deposited.

[0006] In recent years, the applicant of this application has shown that filamentous fungi, particularly Th. heterothallica, are very suitable for the production of secondary metabolites. International (PCT) application number PCT / IB2020 / 051015 discloses that Th. heterothallica can produce cannabinoids and their precursors, in particular can produce cannabigerol acid (CBGA) and / or cannabigerovalic acid (CBGVA), including tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabidivaric acid (CBDVA), and their products, as well as its use for producing the precursors and cannabinoids.

[0007] Landowski et al.'s international patent publication WO / 2015 / 004241 discloses a method useful for producing multiple protease-deficient filamentous fungal cells and heterologous proteins.

[0008] coronavirus Coronaviruses (CoV) are the largest group of viruses belonging to the order Nidovirales, which includes the families Coronaviridae, Arteriviridae, and Roniviridae. The Coronaviridae family includes one of the two subfamilies of Coronaviridae, the other being the Torovirinae subfamily. Coronaviruses are associated with illnesses ranging from the common cold to more serious conditions such as severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA coronavirus that causes coronavirus disease 2019 (COVID-19). Coronaviruses are zoonotic, meaning they are transmitted between animals and humans. Common signs of coronavirus infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. High concentrations of cytokines have been recorded in the plasma of severely ill patients infected with COVID-19. In more severe cases, infection can cause pneumonia, respiratory inflammation, severe acute respiratory syndrome, renal failure, and death. Recombinant production of viral proteins may be used as a potent vaccine. The coronavirus spike protein is being considered as a primary target for vaccine development.

[0009] There remains a need for expression systems for the mass production of proteins that can be used in the pharmaceutical industry in an efficient and cost-effective manner. In particular, there is a need for improved and robust expression systems that can produce stable antibodies and viral antigens for vaccines. [Overview of the Initiative]

[0010] The present invention provides a genetically modified Ascomycete filamentous fungus in which the expression of the proteases KEX2 and / or ALP7 is reduced, enabling the production of large quantities of highly stable proteins.

[0011] In particular, the present invention provides Thermothelomyces heterothallica strain C1 as a representative Ascomycete filamentous fungus genetically modified to enhance the production of exogenous proteins. In some embodiments, the fungi disclosed herein have been modified to be deficient in 14 proteases, including KEX2 and ALP7.

[0012] Surprisingly, the present invention demonstrates that Th. heterothallica, a representative filamentous fungus of the Ascomycota phylum, can be genetically modified to significantly enhance the expression and stability of exogenous proteins expressed by Th. heterothallica cells compared to previously disclosed fungal strains. The present invention also demonstrates that the deficiency of specific proteases, including KEX2 or ALP7, significantly enhances the stability of the expressed proteins.

[0013] It is further disclosed that a combination of KEX2 and ALP7 deficiencies significantly increases the stability and quantity of expressed exogenous proteins.

[0014] Conveniently, the genetically modified Ascomycetes filamentous fungi of the present invention are, in some embodiments, designed to produce secretory proteins with reduced expression of secretory proteases. Preventing the secretion of expressed proteins and the fragmentation of proteins in the culture medium simplifies the purification procedure and increases protein yield.

[0015] The representative Th. heterothallica C1 strain of this invention was designed for the production of the target protein by disrupting genes encoding proteases naturally expressed by the fungus. Surprisingly, the deletion of as many as 13 or 14 proteases did not hinder the growth and proliferation rate of the fungus, but at least maintained, and even increased, the growth rate, enabling the mass production of exogenous proteins.

[0016] Some Th. heterothallica C1 strains developed by the applicants of this invention are less sensitive to feedback inhibition by glucose and other fermentable sugars present in the growth medium as carbon sources than conventional yeast strains and most other Ascomycete filamentous fungal hosts, and therefore can tolerate high supply rates of carbon sources, leading to high yield production by this fungus.

[0017] In addition, some Th. heterothallica C1 strains developed by the applicants of this invention can be grown in liquid media with significantly reduced medium viscosity in fermenters compared to most other Ascomycete filamentous fungal species. Low-viscosity cultures of Th. heterothallica C1 are comparable to those of S. cerevisiae and other yeast species. The low viscosity may be due to morphological changes in the strains, from those with long, highly intertwined hyphae in the parent strain(s) to shorter, less intertwined hyphae in the developed strain(s). Low medium viscosity is highly advantageous for large-scale industrial production.

[0018] According to one embodiment, the present invention provides a genetically modified filamentous fungus to produce a target protein, the genetically modified filamentous fungus comprising at least one cell having reduced expression of KEX2 and / or ALP7 and / or protease activity, the at least one cell comprising at least one exogenous polynucleotide encoding the target protein.

[0019] According to some embodiments, ALP7 comprises an amino acid sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% identity with the amino acid sequence of Thermothelomyces heterothallica ALP7. According to certain embodiments, Thermothelomyces heterothallica ALP7 comprises the amino acids of SEQ ID NO: 13.

[0020] According to some embodiments, KEX2 comprises an amino acid sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% identity with the amino acid sequence of Thermothelomyces heterothallica KEX2. According to certain embodiments, Thermothelomyces heterothallica KEX2 comprises the amino acids of SEQ ID NO: 14.

[0021] According to some embodiments, the modified filamentous fungus comprises at least one cell in which the expression and / or activity of KEX2 and ALP7 are reduced.

[0022] According to some embodiments, the modified filamentous fungus comprises at least one cell in which the expression and / or activity of at least one additional protease is reduced.

[0023] According to some embodiments, the modified filamentous fungus comprises at least one cell in which the expression and / or activity of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 proteases is reduced. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the modified filamentous fungus comprises at least one cell in which the expression and / or activity of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 proteases is reduced. Each possibility represents a separate embodiment of the invention.

[0024] According to some embodiments, at least one additional protease is selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4. Each possibility represents a separate embodiment of the invention.

[0025] According to some embodiments, at least one additional protease is selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALP5, ALP6, SRP3, SRP5, and SRP8.

[0026] According to some embodiments, in at least one cell, the expression and / or activity of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteases selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALP5, ALP6, SRP3, SRP5, SRP8, and SRP10 is reduced.

[0027] According to some embodiments, the modified filamentous fungus comprises at least one cell in which the expression and / or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4 and ALP7 is reduced. According to some embodiments, the modified filamentous fungus comprises at least one cell in which the expression and / or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4 and KEX2 is reduced. According to some embodiments, the modified filamentous fungus further comprises at least one cell in which the expression and / or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALP7 and KEX2 is reduced. <>

[0028] According to some embodiments, the filamentous fungus is further modified to produce proteins having N-glycans similar to those of human, companion animal and other mammalian proteins. According to some embodiments, the filamentous fungus comprises a deletion or disruption of the alg3 gene such that the fungus does not produce a functional alpha-1,3-mannosyltransferase. According to some additional or alternative embodiments, the filamentous fungus comprises a deletion or disruption of the alg11 gene such that the fungus does not produce a functional alpha-1,2-mannosyltransferase. According to yet further additional or alternative embodiments, the filamentous fungus is modified to overexpress flippase. Overexpression of flippase can be obtained by overexpressing the endogenous flippase of the fungus or by expression of a heterologous flippase.

[0029] According to certain additional or alternative embodiments, the filamentous fungus further comprises the expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2). In certain embodiments, GNT1 comprises a heterologous Golgi localization signal.

[0030] According to some embodiments, the protein of interest is selected from the group consisting of antigens, antibodies, enzymes, vaccines, and structural proteins.

[0031] According to some embodiments, the protein of interest is a secreted protein. According to certain embodiments, the protein of interest has a leader or signal peptide. According to other embodiments, the protein of interest is an intracellular protein.

[0032] According to some embodiments, the protein of interest comprises two or more repeat sequences of a protein or protein fragment.

[0033] In some embodiments, the protein of interest is fused to the tag. In some embodiments, the tag is a C-terminal or N-terminal tag. In some embodiments, the tag is selected from the group consisting of chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), FLAG-tag, Spytag, C-tag, ALFA-tag, V5-tag, Myc-tag, HA-tag, spot-tag, T7-tag, NE-tag, and poly(His)-tag. In some embodiments, the tag is a Spytag. In some embodiments, the tag is a C-tag.

[0034] In some embodiments, the protein of interest is an antibody or a fragment thereof. In certain embodiments, the antibody is IgG4 or IgG1. In additional embodiments, the antibody is a bispecific or multispecific antibody. In certain embodiments, the antibody or a fragment thereof is a neutralizing antibody against coronavirus.

[0035] According to some embodiments, the protein of interest is antikalin.

[0036] In some embodiments, the protein of interest is an FC-fusion protein.

[0037] In some embodiments, the protein of interest is an antigen.

[0038] In some embodiments, the protein of interest is a component of an infectious agent. In some embodiments, the protein of interest is a component of a fungus, bacterium, or virus. In some embodiments, the protein of interest is a viral component.

[0039] In some embodiments, the viral component is that of an epidemic virus. In certain representative embodiments, the viral component is that of a coronavirus, influenza virus, hepatitis B virus, hepatitis C virus, papillomavirus, HIV, HTLV-1, or EBV.

[0040] According to some embodiments, the protein of interest is an influenza virus protein. According to certain embodiments, the protein of interest is hemagglutinin (HA) or a fragment thereof. According to certain embodiments, the protein of interest comprises a transmembrane domain (TMD) of hemagglutinin. According to certain embodiments, the protein of interest is that of influenza subtype H1N1.

[0041] In some embodiments, the produced hemagglutinin protein is secreted.

[0042] In some embodiments, the viral component is that of a coronavirus. In certain current representative embodiments, the coronavirus is SARS-CoV-2 (COVID-19).

[0043] According to some embodiments, the protein of interest is a spike protein. According to some embodiments, the protein of interest comprises a receptor-binding domain (RBD) sequence or a fragment thereof of the SARS-CoV-2 spike protein. According to some embodiments, the protein of interest comprises the RBD of the SARS-CoV-2 spike protein. According to some embodiments, the protein of interest consists of the RBD of the SARS-CoV-2 spike protein. According to certain embodiments, the protein of interest comprises a receptor-binding motif (RBM) of the SARS-CoV-2 spike protein. According to certain embodiments, the RBD or a fragment thereof is fused to a Spytag. According to certain embodiments, the protein of interest comprises two, three, or four repeats of RBDs or fragments thereof. According to additional embodiments, the protein of interest is a nucleocapsid. According to some embodiments, the protein of interest is the S2 fragment of the SARS-CoV-2 spike protein.

[0044] In some embodiments, the protein of interest is a viral antigen fused to an Fc fragment. In one particular embodiment, the Fc is fused to the N-terminus of the antigen. In another embodiment, the Fc is fused to the C-terminus of the antigen.

[0045] According to some embodiments, the target protein is Fc-RBD. According to other embodiments, the target protein is RBD-Fc.

[0046] According to some embodiments, the target protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, and SEQ ID NO: 57.

[0047] In some embodiments, the protein of interest is insulin. In additional embodiments, the protein of interest is fibrinogen.

[0048] In some embodiments, the protein of interest is a therapeutic protein.

[0049] According to some embodiments, the protein of interest is a vaccine protein antigen derived from Rift Valley fever virus (RVFV).

[0050] According to some embodiments, the protein of interest is a fusion protein composed of two different antigens. According to some embodiments, the protein of interest is a fusion protein composed of two components of different viral antigens. According to certain embodiments, the viral antigens are those of coronavirus and influenza virus.

[0051] In some embodiments, the viral antigen is fused to the MHCII target sequence. In certain embodiments, the viral antigen and the MHCII target sequence are linked via a linker.

[0052] In some embodiments, the tag is a site-directed fluorescently labeled peptide / protein.

[0053] According to some embodiments, genetically modified Ascomycetes produce increased amounts of exogenous protein compared to the amount produced by the corresponding non-genetically modified parent Ascomycetes cultured under similar conditions. According to certain embodiments, genetically modified Ascomycetes can produce at least twice as much exogenous protein compared to their parent strain.

[0054] According to some embodiments, genetically modified Ascomycetes can increase the amount of secreted exogenous proteins in the growth medium by at least 1.5, 2, 5, or 10 times compared to their parent Ascomycetes. According to certain embodiments, the secreted proteins are intact proteins.

[0055] According to some embodiments, genetically modified Ascomycetes can increase the amount of intracellular and extracellular proteins in fungal cells by at least 1.5, 2, 5, or 10 times compared to their parent Ascomycetes.

[0056] In some embodiments, exogenous proteins produced by genetically modified Ascomycetes exhibit improved stability compared to the corresponding proteins produced by their parent Ascomycetes strains cultured under similar conditions.

[0057] According to some embodiments, genetically modified Ascomycetes grow at a faster rate compared to the corresponding parent Ascomycete strains cultured under similar conditions.

[0058] The polynucleotides encoding the target protein can form part of a DNA construct or expression vector.

[0059] According to some embodiments, at least one exogenous polynucleotide is a DNA construct or expression vector further comprising at least one regulatory element that is operable in the Ascomycete filamentous fungus. According to certain embodiments, the regulatory element is selected from the group consisting of regulatory elements endogenous to the fungus and regulatory elements heterologous to the fungus.

[0060] According to some embodiments, filamentous fungi of the Ascomycota phylum belong to genera within the subphylum Pezizomycotina.

[0061] In some embodiments, filamentous fungi of the Ascomycota phylum belong to genera selected from the group consisting of Thermothelomyces, Myceliophthora, Trichoderma, Aspergillus, Penicillium, Rasamsonia, Chrysosporium, Corynascus, Fusarium, Neurospora, and Talaromyces.

[0062] According to some embodiments, the Ascomycota filamentous fungi include Thermothelomyces heterothallica (also expressed as Myceliophthora thermophila), Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, and Trichoderma viride. The species are selected from the group consisting of *Viride*, *Rasamsonia emersonii*, *Penicillium chrysogenum*, *Penicillium verrucosum*, *Sporotrichum thermophile*, *Corynascus fumimontanus*, *Corynascus thermophilus*, *Chrysosporium lucknowense*, *Fusarium graminearum*, *Fusarium venenatum*, *Neurospora crassa*, and *Talaromyces piniphilus*.

[0063] According to some embodiments, the Ascomycetes filamentous fungus is a strain of Thermothelomyces heterothallica containing an rDNA sequence that has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%, identity with the nucleic acid sequence described in Sequence ID No. 20.

[0064] According to some embodiments, the filamentous fungus of the Ascomycota phylum is Thermothelomyces heterothallica C1.

[0065] According to one embodiment, the present invention provides a method for producing a fungus capable of producing a target protein, the method comprising designing the fungus to inhibit or reduce the expression and / or activity of KEX2 and / or ALP7.

[0066] According to some embodiments, the method includes transforming at least one fungal cell with at least one exogenous polynucleotide.

[0067] In an additional aspect, the present invention provides a method for producing a fungus capable of producing a protein of interest, the method comprising transforming at least one cell of a fungus with at least one exogenous polynucleotide, the at least one cell having reduced expression of KEX2 and / or ALP7 and / or protease activity.

[0068] According to some embodiments, the method involves transforming at least one fungal cell with at least two exogenous polynucleotides encoding different proteins.

[0069] According to some embodiments, the method further comprises designing a fungus in which the expression and / or activity of at least one protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4 is inhibited or reduced in at least one cell.

[0070] According to a particular embodiment, the method further comprises designing a fungus in which the expression and / or activity of at least two different proteases selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4 is inhibited or reduced.

[0071] According to some embodiments, inhibiting protease expression includes deleting or disrupting the endogenous gene encoding the protease.

[0072] According to some embodiments, filamentous fungi of the Ascomycota phylum belong to the genera within the subphylum Pezizomycotina.

[0073] In some embodiments, filamentous fungi of the Ascomycota phylum are genera selected from the group consisting of Thermothelomyces, Myceliophthora, Trichoderma, Aspergillus, Penicillium, Rasamsonia, Chrysosporium, Corinascus, Fusarium, Neurospora, and Talaromyces.

[0074] According to some embodiments, the Ascomycetes phylum filamentous fungi include Thermothelomyces heterothallica or Myceliophthora thermophila, Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, and Trichoderma viride. The species are selected from the group consisting of *Viride*, *Rasamsonia emersonii*, *Penicillium chrysogenum*, *Penicillium verrucosum*, *Sporotrichum thermophile*, *Corynascus fumimontanus*, *Corynascus thermophilus*, *Chrysosporium lucknowense*, *Fusarium graminearum*, *Fusarium venenatum*, *Neurospora crassa*, and *Talaromyces piniphilus*.

[0075] According to some embodiments, the Ascomycetes filamentous fungus is a strain of Thermothelomyces heterothallica containing an rDNA sequence that has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%, identity with the nucleic acid sequence described in Sequence ID No. 20.

[0076] According to some embodiments, the filamentous fungus of the Ascomycota phylum is Thermothelomyces heterothallica C1.

[0077] In a further embodiment, the present invention provides a method for producing at least one target protein, the method comprising culturing a genetically modified fungus as described herein in a suitable medium; and recovering at least one protein product.

[0078] According to some embodiments, the recovery step includes recovering proteins from the growth medium, from the fungal mass, or both.

[0079] In some embodiments, the protein is recovered from the growth medium. In certain embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the protein is secreted.

[0080] According to some embodiments, the culture medium comprises a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose, glycerol, and any combination thereof.

[0081] According to a particular embodiment, culturing a genetically modified fungus in a suitable medium provides the production of the desired protein in an increased amount compared to the amount produced by the corresponding non-genetically modified parent fungal strain cultured under similar conditions.

[0082] According to a particular embodiment, the corresponding parent fungus is of the same species as the genetically modified fungus. According to some embodiments, the corresponding parent fungus is isogenic to the genetically modified fungus.

[0083] In another embodiment, the present invention provides a protein of interest produced by any of the methods described herein.

[0084] According to one embodiment, the present invention provides a target protein provided by a method comprising culturing a genetically modified fungus as described herein in a suitable culture medium; and recovering the target protein.

[0085] The target protein is as described above.

[0086] According to some embodiments, the protein of interest is a coronavirus antigen. According to some embodiments, the protein of interest is a coronavirus spike protein. According to certain embodiments, the protein of interest comprises a coronavirus RBD sequence or a fragment thereof. According to some embodiments, the protein of interest comprises a receptor-binding motif (RBM) sequence of a coronavirus spike protein.

[0087] The present invention further provides compositions comprising two or more different proteins of interest produced by any of the methods described herein.

[0088] According to a particular embodiment, the composition comprises at least two different coronavirus antigens, the antigens comprising sequences of different coronavirus variants.

[0089] It should be clearly understood that the scope of the present invention includes homologs, analogues, variants and derivatives comprising shorter and longer polypeptides, proteins and polynucleotides, as well as polypeptides, proteins and polynucleotide analogues having one or more amino acid or nucleic acid substitutions as known in the art, and that these variants and modifications must be such as to preserve the activity of the proteins or enzymes described herein.

[0090] It should be understood that any combination of each aspect and embodiment disclosed herein is expressly encompassed within the disclosure of the present invention.

[0091] Other objects, features, and advantages of the present invention will become apparent from the following specification and drawings. [Brief explanation of the drawing]

[0092] [Figure 1] Figure 1 shows Western blotting using C-tag detection from 24-well plate cultures of C1 metamorphogens that produce either an RBD-C-tag (left panel) or an RBD-Spytag-C-tag (right panel). [Figure 2] Production of RBD-C-tag and RBD-Spytag-C-tag in C1 protease-deficient strains lacking 12-14 protease genes. The highest production of RBD protein was observed in DNL155 and DNL159 strains lacking kex2. One of three parallel clones of both RBD-C-tag and RBD-Spytag-C-tag showed insufficient proliferation and therefore lower protein levels. [Figure 3]Figures 3A-3B. C-tag affinity purification of RBD-C-tag from bioreactor culture of strain C1 M4169. Stained SDS gel (Figure 3A) and Western blotting (Figure 3B) analyses of samples from different purification steps are shown. Start = initial sample after clarification, in gel, 1:5 dilution; Flow start = flow-through at the start of sample loading, in gel, 1:5 dilution; Flow end = flow-through at the end of sample loading, in gel, 1:5 dilution; Fr4~F9 = eluted fractions. Note that the movement of the eluted sample before dialysis is not normal due to the high MgCl2 concentration. [Figure 4] A brief explanation of the C1 system. [Figure 5] Figure 5 shows spike experiments using antibodies in different protease-deficient strains. C1 protease-deficient strains were cultured in 24-well culture plates for 4 days. For the spike experiment, antibodies were incubated in the culture supernatant, and samples were taken from the samples at different time intervals (0 hours, 3 hours, o / n, and o / 2n) and analyzed by Western blotting. Separate antibodies were used for the detection of heavy and light chains. 270 ng of mAb was loaded into each lane. Control - 200 ng. [Figure 6] Figure 6 shows spike experiments using antibodies in different 13× protease-deficient strains. C1 protease-deficient strains were cultured in 24-well culture plates for 4 days. For the spike experiment, antibodies were incubated in the culture supernatant, and samples were taken at different time intervals (0 hours, 3 hours, o / n, and o / 2n) and analyzed by Western blotting. 270 ng of mAb was loaded into each lane. Control - 200 ng. [Figure 7] Figure 7 shows spike experiments using fibrinogen in different 13× protease-deficient strains. C1 protease-deficient strains were cultured in 24-well culture plates for 4 days. For the spike experiment, fibrinogen protein was incubated in the culture supernatant, and samples were taken from the samples at different time intervals (0 hours, 3 hours, o / n, and o / 2n) and analyzed by Western blotting. Polyclonal anti-fibrinogen antibodies (all fibrinogen chains) were used for detection. 240 ng of fibrinogen was loaded into each lane. Control - 200 ng. [Figure 8] Figure 8 shows spike experiments using Fc-FGF21 in different 13× protease-deficient strains. C1 protease-deficient strains were cultured in 24-well culture plates for 4 days. For the spike experiment, Fc-FGF21 was incubated in the culture supernatant, and samples were taken from the samples at different time intervals (0 hours, 4 hours, o / n, and o / 2n) and analyzed by Western blotting. Two antibodies (anti-Fc and anti-FGF21) were used for detection. 240 ng of Fc-FGF21 was loaded into each lane. Control - 200 ng. [Figure 9] Figure 9 shows mAb spiking (left panel) and expression (right panel) in 12× protease versus 13× protease-deficient strains, as indicated. [Figure 10] Figure 10 shows mAb expression in 12× and 13× protease-deficient strains. mAb expression constructs were transformed into 13× protease-deficient strains with kex2 deficiency. Transformants were grown in 24-well plates, and the generated mAbs were analyzed by Western blotting. The same mAb expression in the parental 12× protease-deficient and 13× Δalp7-deficient strains is shown as a control. [Figure 11] Figure 11 shows the production of RVFV antigen protein under the BGL promoter by the 14× protease-deficient strains dnl155 and 13× protease-deficient strains, as indicated. [Figure 12] Figures 12A-12B. Figures 12A-12B show that conjugation of RBD-Spytag and RBD-SpyTag with SpyCatcher HBsAg VLP produces trimers and / or dimers. Figure 12A - Western blot. Figure 12B - SDS-PAGE. [Figure 13]Figures 13A-13F. Figures 13A-13F show the binding of solublely conjugated RBD to hACE-2 by indirect ELISA. Figure 13A - Schematic diagram of the binding of anti-RBD CR3022 antibody to RBD-ST:SC-HBsAg VLP particles and detection by labeled goat α-human IgG-Ap. Figure 13B - Detection of different batches of RBD with and without VLP particles. Figures 13C-13D - Schematic diagrams of the binding of RBD-ST:SC-HBsAg VLP to hACE (13C) and control (13D). Figures 13E-13F. ELISA results of the binding of hACE to VLP-RBD with conjugated protein (13E) or VLP alone (13F). [Figure 14] Figures 14A-14B. Western blotting analysis of C1 transformants producing RBD-Fc (Figure 14A) or Fc-RBD (Figure 14B) fusion proteins. The parent strain used for production is shown. Strain DNL155 is shown as a negative control. Lanes numbered 1-12 correspond to individual transformants. [Figure 15] Western blotting using C-tag detection from 24-well plate cultures of C1 transformants producing recombinant antigen αMHCII-Cal07 under the control of either the endogenous C1 bgl8 promoter or the synthetic AnSES promoter in transformants derived from DNL155 and M3599 strains. Gel migration of the target protein is consistent with its predicted size of 87 kDa. In addition, the 70 kDa endogenous C1 background protein reacts with antibodies present in all DNL155-derived parental samples. [Figure 16]Figures 16A-16C. C-tag affinity purification of αMHCII-CalO7 from bioreactor culture of strain C1 M4540. Stained SDS gel (Figure 16A) and Western blot (Figure 16B) analyses of samples from different purification steps are shown. Loading = Initial sample after clarification, in gel, 1:10 dilution; Flow-through = Flow-through at the start of sample loading, in gel, 1:10 dilution; Wash = Flow-through during column washing. Note that the migration of eluted samples before dialysis is not normal due to the high MgCl2 concentration. Figure 16C - Stained SDS-PAGE gel and Western blot analyses of αMHCII-CalO7 samples after dialysis compared to a reference protein. [Figure 17] Western blotting results from 24-well plate cultures of C1 transformants producing RBD mutants. Yellow indicates overlapping signals from both anti-RBD (red signal) and anti-C-tag (green signal) detectors. UK is RBD_B.1.1.7-UK, SA is RBD_B.1.351-SA, and BR is RBD_1.1.28.1(P.1)-BR. Samples labeled Wuhan are from the M4169 C1 strain producing Wuhan RBD (Example 4). [Modes for carrying out the invention]

[0093] The present invention provides an alternative and highly efficient system for producing large quantities of protein. The system of the present invention is in part based on the filamentous fungus Thermothelomyces heterothallica C1 and in particular its strains, which have been previously developed as a natural biological factory for the production of protein and secondary metabolites. These strains exhibit high growth rates while maintaining low culture viscosity and are therefore very suitable for continuous growth in fermentation cultures at volumes of approximately 100,000 to 150,000 liters or more. In some embodiments, the present invention provides genetically modified fungi in which the expression and / or activity of KEX2 and / or ALP7 are reduced.

[0094] definition As defined herein, Ascomycetes refer to any fungal strain belonging to the Pezizomycotina subphylum. The Pezizomycotina subphylum includes the following groups: Genus: The genus Thermothelomyces (including species heterothallica and thermophila), The genus Myceliophthora (including the species lutea and an unnamed species), The genus Corynascus (including the species fumimontanus), The genus Neurospora (including the species crassa), The order Sordariales, which includes the red crass fungi; Genus: The genus Fusarium (including species graminearum and venenatum), The genus Trichoderma (including the species *Trichoderma* reesei, *Trichoderma* harzianum, *Trichoderma* longibrachiatum, and *Trichoderma* viride) The order Hypocreales, which includes the order Hypocreales; Genus: The genus Chrysosporium (including the species Chrysosporium lucknowense) The order Onygenales, which includes this order; Genus: The genus Rasamsonia (including the species emersonii), The genus Penicillium (including the species verrucosum), The genus Aspergillus (including the species funiculosus, nidulans, niger, and oryzae), The genus Talaromyces (including the species piniphilus (formerly Penicillium funiculosum)) The order Eurotiales, which includes Aspergillaceae; This includes, but is not limited to, the following:

[0095] It should be understood that the above list is not definitive and provides an incomplete list of industrially relevant filamentous ascomycete species.

[0096] While there may be filamentous ascomycete species outside the subphylum Pezizomycotina, this group includes the most commonly known non-filamental industrially relevant genera such as Saccharomyces, Komagataella (including the former Pichia pastoris), Kluyveromyces, or the subphylum Taphrinomycotina, but does not include the subphylum Saccharomycotina, which includes some other commonly known non-filamental industrially relevant genera such as Schizosaccharomyces.

[0097] All of the above taxonomic categories are defined according to the NCBI Taxonomy Browser (ncbi.nlm.nih.gov / taxonomy) as of the patent application date.

[0098] It is important to understand that fungal taxonomy is constantly evolving, and the naming and hierarchical positions of taxa may change in the future. However, those skilled in the art will be able to clearly determine whether a particular fungal strain belongs to one of the groups defined above.

[0099] According to a particular embodiment, the filamentous fungus genus is selected from the group consisting of Myceliophthora, Thermothelomyces, Aspergillus, Penicillium, Trichoderma, Rasamsonia, Chrysosporium, Corinascus, Fusarium, Neurospora, Talaromyces, and others.According to some embodiments, the fungi include Myceliophthora thermophila, Thermothelomyces thermophila (formerly M. thermophila), Thermothelomyces heterothallica (formerly M. thermophila and heterothallica), Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, and Penicillium chrysogenum. Chrysogenum), Penicillium verrucosum, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma viride, Chrysosporium lucknowense, Rasamsonia emersonii, Sporotrichum thermophile, Corinascus fumimontanus, Corinascus thermophilus, Fusarium graminearum The group is selected from the species *graminearum*, *Fusarium venenatum*, *Neurospora crassa*, and *Talaromyces piniphilus*.

[0100] In particular, the present invention provides Thermothelomyces heterothallica strain C1 as a model of Ascomycete filamentous fungi capable of producing large quantities of stable protein.

[0101] The terms "Thermothelomyces" and its species "Thermothelomyces heterothallica and thermophila" are used most broadly herein, as is well known in the art. Descriptions of the genus and species can be found, for example, in Marin-Felix Y (2015, Mycologica 107(3):619-632 doi.org / 10.3852 / 14-228) and van den Brink J et al. (2012, Fungal Diversity 52(1):197-207). As used herein, "C1" or "Thermothelomyces heterothallica C1" or "Th. heterothallica C1," or simply C1, all refer to Thermothelomyces heterothallica strain C1.

[0102] It should be noted that the authors mentioned above (Marin-Felix et al., 2015) proposed a division of the genus Myceliophthora based on differences in optimal growth temperature, conidial morphology, and details of the sexual reproductive cycle. According to the proposed criteria, C1 clearly belongs to a newly established genus Thermothelomyces, which includes the former heat-tolerant Myceliophthora species rather than the Myceliophthora species that remain non-heat-tolerant. Since C1 can form ascospores with some other Thermothelomyces (formerly Myceliophthora) strains having the opposite mating type, C1 is best classified as Th. heterothallica strain C1 rather than Th. thermophila C1.

[0103] Furthermore, it is necessary to understand that fungal taxonomy has constantly changed in the past, and therefore, the current names listed above may have been preceded by a variety of older names that are now considered synonyms, other than Myceliophthora thermophila (van Oorschot, 1977. Persoonia 9(3):403). For example, Thermothelomyces heterothallica (Marin-Felix et al., 2015. Mycologica, 3:619-63) is synonymous with Corinascus heterotchallicus, Thielavia heterothallica, Chrysosporium lucknowense, thermophile, and Sporotrichium thermophile (Alpinis 1963. Nova Hedwigia 5:74).

[0104] It is important to understand more clearly that the present invention encompasses any strain containing a ribosomal DNA (rDNA) sequence exhibiting 99% or greater homology to Sequence ID No. 20, and all such strains are considered to be congenerally related to Thermothelomyces heterothallica.

[0105] In particular, the term Th. heterothallica strain C1 encompasses genetically modified sublines derived from wild-type strains that have been mutated using random or indicated approaches, for example, by UV mutagenesis, or by deleting one or more endogenous genes. For example, a C1 strain may refer to a wild-type strain modified to delete one or more genes encoding endogenous proteases. For example, C1 strains encompassed by this invention include strain UV18-25, deposit number VKM F-3631 D; strain NG7C-19, deposit number VKM F-3633 D; and strain UV13-6, deposit number VKM F-3632 D. Furthermore, C1 strains that can be used in accordance with the teachings of the present invention include HC strain UV18-100f, deposit number CBS141147; HC strain UV18-100f, deposit number CBS141143; LC strain W1L#100I, deposit number CBS141153; and LC strain W1L#100I, deposit number CBS141149, as well as their derivatives.

[0106] It is important to understand that the teachings of the present invention encompass mutants, derivatives, offspring, and clones of the Th. heterothallica C1 strain, insofar as these derivatives, offspring, and clones can produce at least one protein product in accordance with the teachings of the present invention when genetically modified according to the teachings of the present invention.

[0107] It is important to understand that the term “derivative” in relation to fungal strains encompasses any parent fungal strain having modifications that positively affect product yield, efficiency, or efficacy, or affect any traits that improve the fungal derivative as a tool for producing a desired protein. As used herein, the term “offspring” refers to unmodified or partially modified offspring from a parent fungal strain, such as cell-to-cell. The term “parent strain” refers to a corresponding fungal strain in which the expression or activity of a particular protease according to the present invention is reduced.

[0108] According to one aspect of the present invention, a genetically modified filamentous fungus for producing a target protein is provided, the genetically modified filamentous fungus comprising at least one cell having reduced or absent expression and / or activity of proteases KEX2 and / or ALP7, and the filamentous fungus comprising at least one cell comprising at least one exogenous polynucleotide encoding the target protein.

[0109] According to one aspect of the present invention, a genetically modified filamentous fungus for producing a heterologous protein is provided, the genetically modified filamentous fungus comprising at least one cell in which the expression and / or activity of KEX2 and at least one additional protease is reduced or absent, the filamentous fungus comprising at least one cell comprising at least one exogenous polynucleotide encoding a heterologous protein.

[0110] According to one aspect of the present invention, a genetically modified filamentous fungus for producing a heterologous protein is provided, the genetically modified filamentous fungus comprising at least one cell in which the expression and / or activity of ALP7 and at least one additional protease is reduced or absent, the filamentous fungus comprising at least one cell comprising at least one exogenous polynucleotide encoding a heterologous protein.

[0111] According to one aspect of the present invention, a genetically modified filamentous fungus for producing heterologous proteins is provided, the genetically modified filamentous fungus comprising at least one cell in which the expression and / or activity of the proteases ALP7, KEX2 and at least one additional proteases are reduced or absent, the filamentous fungus comprising at least one cell comprising at least one exogenous polynucleotide encoding a heterologous protein.

[0112] In some embodiments, at least one cell has reduced or absent expression and / or activity of 13 proteases, one of which is KEX2. In some embodiments, at least one cell has reduced or absent expression and / or activity of 13 proteases, one of which is ALP7. In some embodiments, at least one cell has reduced or absent expression and / or activity of 14 proteases, including KEX2 and ALP7.

[0113] The terms “protein” and “polypeptide” are used interchangeably herein and refer to polymers of amino acids, not products of a specific length; therefore, peptides, oligopeptides, and polypeptides are included within this definition.

[0114] As used herein, the term “target protein” refers to a protein that is desirable to be expressed at high levels in filamentous fungi. Such proteins include, but are not limited to, antibodies, enzymes, substrate-binding proteins, structural proteins, and antigens.

[0115] According to some embodiments, the Ascomycetes filamentous fungi include at least one cell in which the expression and / or activity of KEX2 and at least one or more proteases are reduced or absent.

[0116] According to a particular embodiment, the Ascomycete filamentous fungus includes at least one cell in which the expression and / or activity of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 proteases is reduced or absent.

[0117] According to some embodiments, genetically modified filamentous fungi do not express KEX2. According to some embodiments, genetically modified filamentous fungi do not express ALP7.

[0118] According to some embodiments, genetically modified filamentous fungi do not express ALP1. According to some embodiments, genetically modified filamentous fungi do not express PEP4. According to some embodiments, genetically modified filamentous fungi do not express ALP2. According to some embodiments, genetically modified filamentous fungi do not express PRT1. According to some embodiments, genetically modified filamentous fungi do not express SRP1. According to some embodiments, genetically modified filamentous fungi do not express ALP3. According to some embodiments, genetically modified filamentous fungi do not express PEP1. According to some embodiments, genetically modified filamentous fungi do not express MTP2. According to some embodiments, genetically modified filamentous fungi do not express PEP5. According to some embodiments, genetically modified filamentous fungi do not express MTP4. According to some embodiments, genetically modified filamentous fungi do not express PEP6. According to some embodiments, genetically modified filamentous fungi do not express ALP4.

[0119] According to a particular embodiment, the Ascomycetes filamentous fungus comprises at least one cell in which the expression and / or activity of at least one additional protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4 is reduced or absent. Each possibility represents a distinct embodiment of the present invention.

[0120] According to one aspect of the present invention, a genetically modified Ascomycete filamentous fungus for producing a target protein is provided, the genetically modified filamentous fungus comprising at least one cell containing an exogenous polynucleotide encoding the target protein, the genetically modified Ascomycete filamentous fungus either does not express or expresses in reduced amounts KEX2 and / or ALP7, as well as at least one additional protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4.

[0121] According to some embodiments, filamentous fungi do not express KEX2, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4, or express them in reduced amounts.

[0122] According to some embodiments, filamentous fungi do not express ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4, or express them in reduced amounts.

[0123] According to some embodiments, filamentous fungi do not express KEX2, ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4, or express them in reduced amounts.

[0124] According to one embodiment, the present invention provides a genetically modified Ascomycete filamentous fungus for producing viral antigens, the genetically modified Ascomycete filamentous fungus comprising at least one cell containing an exogenous polynucleotide encoding a viral antigen, the genetically modified Ascomycete filamentous fungus either does not express or expresses reduced amounts of KEX2, ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4.

[0125] In some embodiments, the viral antigen is a vaccine antigen protein derived from Rift Valley fever virus (RVFV).

[0126] According to one embodiment, the present invention provides a genetically modified Ascomycete filamentous fungus for producing the receptor-binding domain (RBD) of the SARS-CoV-2 spike domain, wherein the genetically modified Ascomycete filamentous fungus comprises at least one cell containing an exogenous polynucleotide encoding the RBD, and the genetically modified Ascomycete filamentous fungus either does not express or expresses reduced amounts of KEX2, ALP7, ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTB2, PEP5, MTP4, PEP6, and ALP4.

[0127] The kex2 gene, also known as qds1, srb1, and vmn45, encodes the KEX2 or KEXIN protease. The KEX2 protease is a serine peptidase. The amino acid sequence of Thermothelomyces heterothallica KEX2 is shown in SEQ ID NO: 14.

[0128] According to some embodiments, KEX2 contains an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14.

[0129] The amino acid sequence for Thermothelomyces heterothallica ALP7 is shown in Sequence ID No. 13.

[0130] According to some embodiments, ALP7 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 13.

[0131] The alp1 gene encodes alkaline protease 1. ALP1 is a secreted alkaline protease that enables the assimilation of proteinaceous substrates. The amino acid sequence of Thermothelomyces heterothallica ALP1 is shown in Sequence ID No. 1.

[0132] According to some embodiments, ALP1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1.

[0133] The pep4 gene (also known as pho9, pra1, yscA) is an aspartate peptidase. The amino acid sequence of Thermothelomyces heterothallica PEP4 is shown in Sequence ID No. 2.

[0134] According to some embodiments, PEP4 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2.

[0135] The amino acid sequence for Thermothelomyces heterothallica ALP2 is shown in Sequence ID No. 3.

[0136] According to some embodiments, ALP2 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3.

[0137] The amino acid sequence for Thermothelomyces heterothallica PRT1 is shown in Sequence ID No. 4.

[0138] According to some embodiments, PRT1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4.

[0139] The amino acid sequence for Thermothelomyces heterothallica SRP1 is shown in Sequence ID No. 5.

[0140] According to some embodiments, SRP1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 5.

[0141] The amino acid sequence for Thermothelomyces heterothallica ALP3 is shown in Sequence ID No. 6.

[0142] According to some embodiments, ALP3 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 6.

[0143] The amino acid sequence for Thermothelomyces heterothallica PEP1 is shown in Sequence ID No. 7.

[0144] According to some embodiments, PEP1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 7.

[0145] The amino acid sequence for Thermothelomyces heterothallica MTP2 is shown in Sequence ID No. 8.

[0146] According to some embodiments, MTP2 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 8.

[0147] The amino acid sequence for Thermothelomyces heterothallica PEP5 is shown in Sequence ID No. 9.

[0148] According to some embodiments, PEP5 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 9.

[0149] The amino acid sequence for Thermothelomyces heterothallica MTP4 is shown in Sequence ID No. 10.

[0150] According to some embodiments, MTP4 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10.

[0151] The amino acid sequence for Thermothelomyces heterothallica PEP6 is shown in Sequence ID No. 11.

[0152] According to some embodiments, PEP6 contains an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11.

[0153] The amino acid sequence for Thermothelomyces heterothallica ALP4 is shown in Sequence ID No. 12.

[0154] According to some embodiments, ALP4 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12.

[0155] The amino acid sequence for Thermothelomyces heterothallica ALP5 is shown in Sequence ID No. 15.

[0156] According to some embodiments, ALP5 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15.

[0157] The amino acid sequence for Thermothelomyces heterothallica ALP6 is shown in Sequence ID No. 16.

[0158] According to some embodiments, ALP6 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 16.

[0159] The amino acid sequence for Thermothelomyces heterothallica SRP3 is shown in Sequence ID No. 17.

[0160] According to some embodiments, SRP3 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 17.

[0161] The amino acid sequence of Thermothelomyces heterothallica SRP5 is shown in Sequence ID No. 18.

[0162] According to some embodiments, SRP5 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 18.

[0163] The amino acid sequence for Thermothelomyces heterothallica SRP8 is shown in Sequence ID No. 19.

[0164] According to some embodiments, SRP8 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 19.

[0165] In some embodiments, the protein of interest is fused to the tag. In some embodiments, the tag is a C-terminal or N-terminal tag. In some embodiments, the tag is selected from the group consisting of chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), FLAG-tag, Spytag, C-tag, ALFA-tag, V5-tag, Myc-tag, HA-tag, spot-tag, T7-tag, NE-tag, and poly(His)-tag. In some embodiments, the tag is a Spytag. In some embodiments, the tag is a C-tag.

[0166] As used herein, the term “tag” typically refers in the art to an amino acid sequence that is fused to or contained within another amino acid sequence for the following purposes: a) to facilitate the purification of an entire amino acid sequence or polypeptide; b) to improve the expression of an entire amino acid sequence or peptide; and / or c) to facilitate the detection of an entire amino acid sequence or polypeptide.

[0167] The term "C-tag" is well known in this art and refers to the four amino acid affinity tag: EPEA (glutamic acid-proline-glutamic acid-alanine), which can be fused to the C-terminus of any recombinant protein. When used for purification purposes, the tag provides high affinity and selectivity.

[0168] The term "Spytag" is well known in this technical field and refers to a short-chain peptide covalently bound to the SpyCatcher protein. The Spytag sequence is Ala-His-Ile-Val-Met-Val-Asp-Ala-Tyr-Lys-Pro-Thr-Lys.

[0169] The term "Strep-tag" is used herein as is known in the art and refers to a method that enables the purification and detection of proteins by affinity chromatography. The method is based on Strep-Tactin binding.

[0170] The term "glutathione S-transferase (GST)" is used herein as is known in the art and is based on the strong binding affinity of the GST protein to glutathione (GSH). GST tags are often used to isolate and purify proteins containing GST fusion proteins. The tag has an amino acid length of 220.

[0171] The term "FLAG-tag" is used herein as is known in the art and refers to a polypeptide protein tag that can be attached to a protein using recombinant DNA technology. It is one of the most specific tags and is an artificial antigen for which specific high-affinity monoclonal antibodies have been developed and which can therefore be used for protein purification by affinity chromatography.

[0172] The term "ALFA-tag" refers to an epitope tag that is specifically recognized by a nanobody, which can be used herein as is known in the art and for detection and purification.

[0173] V5 tags are short-chain peptide tags for protein detection and purification. V5 tags can be fused / cloned to recombinant proteins and can be detected in ELISA, flow cytometry, immunoprecipitation, immunofluorescence, and Western blotting using antibodies and nanobodies.

[0174] The term "Myc-tag" is used herein as is known in the art and refers to a short-chain peptide tag derived from the c-myc gene that can be recognized by a specific antibody.

[0175] "HA-tag" is used herein as is known in the art and refers to a peptide derived from the human influenza hemagglutinin (HA) molecule corresponding to amino acids 98-106. This tag is used to facilitate the detection, isolation, and purification of the protein of interest.

[0176] "Spot tags" are 12-amino acid peptide tags recognized by single-domain antibody nanobodies (sdAbs). The tags can be used in a wide variety of applications, including immunoprecipitation, affinity purification, immunofluorescence, and super-resolution microscopy.

[0177] The term "T7 tag" is used herein as is known in the art and refers to an epitope tag consisting of 11 residue peptides encoded from the leader sequence of the T7 bacteriophage gene 10.

[0178] The term "NE-tag" is used herein as is known in the art and refers to a synthetic peptide tag (NE-tag) designed as an epitope tag for the detection, quantification, and purification of recombinant proteins. This peptide tag consists of 18 hydrophilic amino acids.

[0179] The term "poly(His) tag" or "polyhistidine tag" is known in the art and refers to an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N-terminus or C-terminus of the protein. It is also known as a hexahistidine tag, a 6×His- tag, and a His6 tag. Short-chain peptides can be bound by metal ions such as divalent nickel or cobalt.

[0180] According to some embodiments, the filamentous fungi are further modified to produce proteins having N-glycans similar to those of human, companion animal, and other mammalian proteins. According to some embodiments, the filamentous fungi include deletion or disruption of the alg3 gene so that the fungus does not produce functional alpha-1,3-mannosyltransferase. According to some embodiments, the filamentous fungi include deletion or disruption of the alg11 gene so that the fungus does not produce functional alpha-1,2-mannosyltransferase. According to some embodiments, the filamentous fungi include overexpression of endogenous flippase or expression of heterologous flippase.

[0181] According to certain embodiments, the filamentous fungus further comprises the expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2). In certain embodiments, GNT1 comprises heterologous Golgi localization signals. In some embodiments, heterologous GNT1 and GNT2 are of animal origin.

[0182] According to some embodiments, the protein of interest is an antigen. According to some embodiments, the protein of interest is a spike protein. According to some embodiments, the protein of interest comprises a receptor-binding domain (RBD) sequence or a fragment thereof of the SARS-CoV-2 spike protein. According to some embodiments, the protein of interest is the RBD of the SARS-CoV-2 spike protein. According to certain embodiments, the protein of interest comprises a receptor-binding motif (RBM) of the SARS-CoV-2 spike protein. According to some embodiments, the protein of interest comprises a glycoprotein-binding domain (GBD) sequence of the SARS-CoV-2 S protein. According to certain embodiments, the RBD or a fragment thereof is fused to a Spytag. According to certain embodiments, the RBD or a fragment thereof is fused to a C-tag. According to additional embodiments, the RBD is fused to the Fc of an antibody. According to certain embodiments, the protein of interest comprises two, three, or four repeats of RBDs or fragments thereof.

[0183] Coronavirus antigen sequences can be manipulated according to any known or discovered variant of coronavirus. For example, the sequences can be manipulated according to those described in Rambaut et al. nCoV-2019 Genomic Epidemiology, December 2020 (https: / / virological.org / t / preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations / 563), Tegally, H. et al. 2020 (https: / / www.medrxiv.org / content / 10.1101 / 2020.12.21.2024 8640v1), or Faria NR, et al. 2020 (https: / / virological.org / t / genomic-characterisation-of-an-emergentsars-cov-2-lineage-in-manaus-preliminary-findings / 586). The present invention encompasses amino acid sequences that are substantially homologous to an amino acid sequence based on any one of the sequences identified in this application. The terms “sequence identity” and “sequence homology” are considered synonymous herein.

[0184] There are many established algorithms available for aligning two amino acid sequences. Typically, one sequence acts as a reference sequence and can be compared to the test sequence. Sequence comparison algorithms calculate the sequence identity percentage of the test sequence(s) to the reference sequence based on specified program parameters. Alignment of amino acid sequences for comparison can be performed using, for example, computer-implemented algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA), or the BLAST and BLAST2.0 algorithms.

[0185] In comparison, identity may exist across a region of the sequence that is at least 10 amino acid residue lengths (e.g., at least 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 685 amino acid residue lengths, e.g., up to the full length of the reference sequence). Each possibility represents a distinct embodiment of the present invention.

[0186] As used herein, the term “exogenous” refers to polynucleotides or proteins that are not naturally expressed within fungi (e.g., heterologous polynucleotides from different species). Exogenous polynucleotides can be introduced into fungi in a stable or transient manner to produce ribonucleic acid (RNA) molecules and / or polypeptide molecules.

[0187] As used herein, the term "heterogeneous" includes sequences inserted into fungi that are not found naturally in the fungi.

[0188] The terms “DNA construct,” “expression vector,” “expression construct,” and “expression cassette” are used to refer to artificially assembled or isolated nucleic acid molecules that contain a nucleic acid sequence encoding a protein of interest and are assembled to functionally express the protein of interest in target host cells. An expression vector typically contains a suitable regulatory sequence operably bound to the nucleic acid sequence encoding the protein of interest. An expression vector may further contain a nucleic acid sequence encoding a marker of choice.

[0189] The terms “polynucleotide,” “nucleic acid sequence,” and “nucleotide sequence” are used herein to refer to deoxyribonucleotides (DNA), ribonucleotides (RNA), and their modified polymers in the form of distinct fragments or components of a larger construct. A nucleic acid sequence may be a coding sequence, i.e., a sequence that codes for an end product in a cell, such as a protein.

[0190] A sequence that is "homologous" to a reference sequence (e.g., a nucleic acid sequence and an amino acid sequence) is referred herein to a percentage of identity between sequences, where the percentage of identity is at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%. Each possibility represents a distinct embodiment of the present invention. Homologous nucleic acid sequences include mutations related to codon usage frequency and genetic coding denaturation.

[0191] The nucleic acid sequences encoding the proteins of the present invention can be optimized for expression. Examples of such sequence modifications include, but are not limited to, altered G / C content for a closer approach, which is typically found in filamentous fungi.

[0192] The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment to approach codon usage frequency within a target organism, and / or the modification of a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon in the native sequence (e.g., one or about one, two, three, four, five, ten, fifteen, twenty, twenty-five, fifty or more codons) with a codon more frequently or most frequently used in the host cell gene while maintaining the native amino acid sequence. Different species exhibit a particular bias for certain amino acids with respect to certain codons. Codon bias (differences in codon usage frequency between organisms) is often thought to correlate with the efficiency of messenger RNA (mRNA) translation and, among other things, depend on the characteristics of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be adjusted to achieve optimal gene expression in a particular organism based on codon optimization. Thus, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a naturally occurring gene has been modified to utilize codons that are statistically favorable or statistically advantageous within the organism.

[0193] Sequence identity can be determined using nucleotide / amino acid sequence comparison algorithms, as is known in the art.

[0194] The term "coding sequence" is used herein to refer to a sequence of nucleotides that encode a functional polypeptide, beginning with a start codon (ATG) and a stop codon (TAA, TGA, TAA), and containing any number of codons except a stop codon.

[0195] Any coding or amino acid sequence listed herein also includes incomplete sequences in which one, two, three, four, five, ten, fifteen, twenty, twenty-five, fifty, or more codons or amino acids are deleted from any part of the sequence. Incomplete versions of coding or amino sequences can be identified using nucleotide / amino acid sequence comparison algorithms, as is known in the art.

[0196] Any coding sequences or amino acid sequences listed herein also include fused sequences that include other sequences in addition to the coding sequences provided herein or sequences as defined above. Fused sequences may be sequences such as those disclosed herein or other sequences. Fused coding sequences or amino sequences can be identified using nucleotide / amino acid sequence comparison algorithms, as is known in the art.

[0197] DNA sequences are assembled into expression cassettes, selection cassettes, and further DNA constructs and / or expression vectors by conventional molecular biological approaches utilizing restriction endonucleases and ligases, Gibson assembly, or yeast recombination. These can also be synthesized by DNA synthesis service providers. As is known in the art, several different techniques can achieve the same results.

[0198] DNA sequences are assembled into expression cassettes that combine a 5' regulatory region (promoter), a coding sequence, and a 3' regulatory region (terminator), as described below herein and as known in the art. Any combination of these three sequences can form a functional expression cassette.

[0199] The terminator list includes: Th. heterothallica gene encoding the protein of unknown nature G2QF75 (XP_003664349); polyubiquitin homolog (G2QHM8, XP_003664133); protein of unknown nature (G2QIA5, XP_003664731); beta-glucosidase (G2QD93, XP_003662704); elongation factor 1-alpha (G2Q129, XP_003660173); chitinase (G2QDD4, XP_003663544); phosphoglycerate kinase (PGK) (Uniprot This includes, but is not limited to, G2QLD8, glyceraldehyde 3-phosphate dehydrogenase (GPD) (G2QPQ8), phosphofructokinase (PFK) (G2Q605); or triose phosphate isomerase (TPI) (G2QBR0); actin (ACT) (G2Q7Q5); cbh1 (GenBank AX284115) or β-glucosidase 1bgl1 (XM_003662656). Exogenous terminators include those of Aspergillus nidulans gpdA terminator.

[0200] The 5' regulatory region (promoter) is actually defined as a stretch of up to 2000 base pairs preceding the start codon of the coding sequence of the gene they regulate, however, the preceding region is non-coding.

[0201] The 3' regulatory region (terminator) is actually defined as a stretch of up to 300 base pairs downstream from the stop codon in the coding sequence of a gene, however, the region that follows is non-coding.

[0202] DNA sequences are also assembled into selective marker cassettes, which are expression cassettes that encode genes that offer selective advantages when the coding sequence is present in the transformant. Such advantages may include the use of novel carbon or nitrogen sources, or resistance to toxic substances.

[0203] The protease deletions disclosed herein can be carried out as are known in the art. In some embodiments, the deletion is performed by transformation of a suitable DNA construct. The DNA construct used for targeted transformation consists of (a) a suitable vector that allows the maintenance of the DNA construct in a particular host, (b) zero, one or more expression cassettes in any direction, (c) a selective marker cassette in any direction, and (d) a sequence identical to a selective stretch of the target genomic DNA (also called the targeting group). These components are arranged such that two targeting groups encompass any expression cassette and selective marker cassette, so that when homologous recombination occurs between the targeting group and two identical regions in the genomic DNA, the sequence between the targeting groups of the DNA construct is inserted into the chromosome, replacing a sequence originally present on the chromosome. Using this principle, a gene can be knocked out of or inserted into the genome. By arranging an array downstream of the selection marker cassette that is identical to the array immediately upstream of the selection marker cassette, it is possible to recycle the markers, as is known in the art.

[0204] The term "regulatory sequence" refers to DNA sequences that control the expression (transcription) of coding sequences such as promoters, enhancers, and terminators.

[0205] The term “promoter” refers to a regulatory DNA sequence that controls or directs the transcription of another DNA sequence in vivo or in vitro. Typically, promoters are located in the 5' region of the transcribed sequence (i.e., preceding, upstream). Promoters may be entirely of natural origin, composed of different elements derived from different naturally occurring promoters, or even contain synthetic nucleotide segments. Promoters can be constitutive (i.e., promoter activation is not regulated by inducers, and therefore the rate of transcription is constant) or inductive (i.e., promoter activation is regulated by inducers or environmental conditions). Promoters may also restrict transcription to a particular developmental stage of an organism or to a particular morphologically distinct region. In most cases, the precise boundaries of regulatory sequences are not fully defined, and in some cases, cannot be fully defined, and therefore, some variant DNA sequences may have identical promoter activity.

[0206] The term "terminator" refers to another regulatory DNA sequence that modulates a transcriptional terminator. The terminator sequence is operably bound to the 3' end of the nucleic acid sequence being transcribed.

[0207] The terms "C1 promoter" and "C1 terminator" refer to promoter and terminator sequences suitable for use in C1, i.e., sequences that can direct gene expression in C1.

[0208] However, as is known to those skilled in the art, the selection of promoters and terminators may not be important, but similar results can be obtained using a wide variety of promoters and terminators that provide similar or identical gene expression.

[0209] The term "operatably bound" means that the selected nucleic acid sequence is adjacent to a regulatory element (promoter, enhancer, and / or terminator) that allows the regulatory element to regulate the expression of the selected nucleic acid sequence.

[0210] This invention discloses the production of a target protein using the genetically modified strain Th. heterothallica C1. Other species of filamentous fungi that share an endogenous similar pathway for precursor production, as described herein, can also be used.

[0211] According to certain embodiments, the polynucleotides of the present invention are designed based on the amino acid sequence of a protein produced by utilizing the codon usage frequency of a filamentous fungus. According to certain embodiments, the filamentous fungus belongs to the subphylum Pezizomycotina. According to some embodiments, the filamentous fungus belongs to a group selected from the orders Sordariales, Hypocreales, Onygenales, and Eurotiales, including genera and species as described in the “Definitions” section above. According to certain representative embodiments, the fungus is Th. heterothallica. According to these embodiments, the polynucleotides of the present invention are polynucleotides or homologs identified in Th. heterothallica. According to certain current representative embodiments, the fungus is Th. heterothallica C1.

[0212] According to a particular representative embodiment, strain Th. heterothallica C1 is a derivative of strain UV18-#100.

[0213] Each DNA construct or expression vector or multiple identical constructs contain regulatory elements that control the transcription of polynucleotides within at least one fungal cell. These regulatory elements may be endogenous to the fungus, particularly Th. heterothallica C1, or exogenous to that fungus.

[0214] According to a particular embodiment, the regulatory elements are selected from a group consisting of 5' regulatory elements (collectively called promoters) and 3' regulatory elements (collectively called terminators), but these nucleotide sequences may include additional regulatory elements that are not strictly classified as promoter or terminator sequences.

[0215] According to certain embodiments, the DNA construct or expression vector comprises at least one promoter operably bound to at least one polynucleotide containing a coding sequence, which is operably bound to at least one terminator. According to certain embodiments, the promoter is an endogenous promoter of a fungus, particularly Th. heterothallica. According to additional or alternative embodiments, the promoter is heterogeneous to a fungus, particularly Th. heterothallica. According to certain embodiments, the terminator is an endogenous terminator of a fungus, particularly Th. heterothallica. According to additional or alternative embodiments, the terminator is heterogeneous to a fungus, particularly Th. heterothallica.

[0216] According to a particular representative embodiment, the DNA construct essentially includes synthetic regulatory elements called "synthetic expression systems" (SES), as described in International (PCT) application publication number WO2017 / 144777.

[0217] According to certain embodiments, the polynucleotide is stably integrated into at least one chromosomal locus in at least one cell of a genetically modified fungus. According to certain embodiments, the polynucleotide is stably integrated into a defined site on a fungal chromosome. According to certain embodiments, the polynucleotide is stably integrated into a random site on a chromosome. According to certain embodiments, the polynucleotide may be incorporated in a targeted or random manner as one, two or more copies to one, two or more chromosomal loci.

[0218] According to a particular alternative embodiment, the polynucleotide is transiently expressed using an extrachromosomal expression vector, such as those known to those skilled in the art.

[0219] According to a particular embodiment, culturing a genetically modified fungus in a suitable medium provides the production of the target protein in an increased amount compared to the amount produced by the corresponding parent fungus cultured under similar conditions.

[0220] According to certain representative embodiments, the present invention provides a genetically modified Th. heterothallica C1 fungus capable of producing the protein of interest. According to these embodiments, such a genetically modified Th. heterothallica C1 fungus comprises at least one cell in which the expression and / or activity of KEX2 and / or ALP7, as well as at least one additional protease, are reduced.

[0221] According to certain embodiments, a suitable culture medium for genetically modified fungi comprises a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose, and glycerol. According to some embodiments, the carbon source is provided from waste from ethanol production or other biological productions from lignocellulose biomass containing high molecular weight carbohydrates such as starch, sugar beet and sugarcane, molasses containing fermentable sugars, starch, cellulose and hemicellulose.

[0222] According to a particular current representative embodiment, the fungus is Th. heterothallica C1. According to a particular embodiment, the strain of Th. heterothallica C1 is selected from the group consisting of strain UV18-25, deposit number VKM F-3631 D; strain NG7C-19, deposit number VKM F-3633 D; and strain UV13-6, deposit number VKM F-3632 D. Additional strains that can be used are HC strain UV18-100f, deposit number CBS141147; HC strain UV18-100f, deposit number CBS141143; LC strain W1L#100I, deposit number CBS141153; and LC strain W1L#100I, deposit number CBS141149, as well as their derivatives. Each possibility represents a distinct embodiment of the present invention.

[0223] In another aspect, the present invention provides a method for generating a fungus capable of producing an exogenous protein of interest, the method comprising transforming at least one cell of the fungus with at least one polynucleotide encoding the protein of interest, the at least one cell of the fungus having reduced expression and / or activity of KEX2 and / or ALP7 and at least one additional protease.

[0224] According to some embodiments, the method further comprises deleting, inhibiting, or reducing the expression of KEX2 or ALP7. According to some embodiments, the method further comprises deleting, inhibiting, or reducing the expression of at least one protease selected from the group consisting of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, and ALP4.

[0225] As described herein, the terms “reduced expression” or “inhibited expression” of proteins, particularly proteases, are used interchangeably and include, but are not limited to, the deletion or disruption of the gene encoding the protein.

[0226] As described herein, the terms “reduced activity” or “inhibited activity” of proteins, particularly proteases, are used interchangeably and further include post-translational modifications that reduce or eliminate the activity of a protein.

[0227] Any method known in the art for transforming filamentous fungi with polynucleotides encoding a target protein can be used in accordance with the teachings of this invention.

[0228] Fungi and polynucleotides are as described herein.

[0229] In yet another aspect, the present invention provides a method for producing an exogenous protein, comprising culturing a genetically modified fungus, particularly the Th. heterothallica C1 fungus of the present invention, in a suitable medium; and recovering the protein product.

[0230] According to certain embodiments, the method involves culturing genetically modified fungi, such as those described herein, each expressing a different protein of interest. According to certain embodiments, the fungi express antigens of different coronavirus variants.

[0231] According to a particular embodiment, the culture medium comprises a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose, and glycerol. According to a particular embodiment, the carbon source is waste obtained from ethanol production or other biological production from lignocellulose biomass containing high molecular weight carbohydrates such as starch, sugar beet and sugarcane, molasses containing fermentable sugars, starch, cellulose and hemicellulose.

[0232] According to some embodiments, exogenous proteins are purified from fungal growth media.

[0233] According to other embodiments, the exogenous protein is extracted from the fungal mass. Any method known in the art for extracting and purifying proteins from plant tissue can be used.

[0234] In a further embodiment, the present invention provides an exogenous protein produced by a genetically modified fungus, particularly the genetically modified Th. heterothallica C1 of the present invention.

[0235] In some embodiments, the exogenous protein product is a coronavirus antigen. In some embodiments, the antigen is the complete spike protein of the coronavirus. In certain embodiments, the antigen comprises an RBD sequence of the coronavirus spike protein or a fragment thereof. In certain embodiments, the RBD or fragment thereof is fused directly or indirectly to a spytag. In certain embodiments, the antigen is bound to a spy catcher.

[0236] The following embodiments are presented to more fully illustrate some embodiments of the present invention. However, they should not be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0237] Examples Example 1 :C1 alp7 gene deficiency The C1 alp7 protease gene was deleted from a C1 protease-deficient strain that previously lacked 12 proteases. A deletion cassette for alp7 was constructed in two parts on two separate plasmids. The marker fragments of these two plasmids overlapped, and this region was to undergo homologous recombination in C1 between plasmids, simultaneously with the recombination of the 5' and 3' facile region fragments with genomic DNA on both sides of the alp7 gene. Recombination between the selected marker fragments makes the marker gene functional and allows the transformant to grow under selection. The deletion cassette also contains a direct repeat sequence of the 5' facile region for the removal of the pyr4 marker. The sequences of the deletion construct plasmids are shown in SEQ ID NOs. 21 and 22. The 5' arm plasmid pMYT0936 contained the alp7 5' facile region fragment for integration (positions 9-1,025 in SEQ ID NOs. 21) and half of the pyr4 marker (positions 1,033-2,812 in SEQ ID NOs. 21). The 3' arm plasmid pMYT0937 contained the remaining half of the pyr4 marker (positions 17–1273 of SEQ ID NO: 22), a direct repeat sequence (positions 1282–1781 of SEQ ID NO: 22), and an alp7 3' adjacent region fragment for integration (positions 1790–2759 of SEQ ID NO: 22).

[0238] alp7 facile region fragments and direct repeats were amplified from C1 genomic DNA and cloned into a pyr4 marker-containing backbone vector originating from the pSR426 plasmid by Gibson cloning using the NEBbuilder® HiFi DNA Assembly Kit (New England Biolabs) according to the manufacturer's instructions. Both portions of the deletion construct were excised from the plasmid and co-transformed into C1 strain DNL146 with 12 deletions of the protease gene using the protoplast / PEG method as described in Visser, VJ, et al. (Industrial Biotechnology 2011, 7, 214-223).

[0239] Transformed colonies grown on pyr4 selective medium plates were streaked again in the same selective medium. Accurate identification of transformants was performed by PCR. Mycelium from transformant streaks was dissolved in 20 mM NaOH and incubated at 100°C to lyse the cells. 1-2 μl of this solution was used as a template for PCR using the Phire Plant PCR Kit (Thermo Fisher). The oligonucleotide primers used in this PCR are shown in Table 1. Integration of the deletion construct into the alp7 locus was demonstrated by two PCR reactions. Integration at the 5' end of the gene was demonstrated by a reaction using the primers shown as SEQ ID NOs. 25 and 26. A 1233 bp fragment was amplified, indicating successful integration into the alp7 locus. Integration at the 3' end of alp7 was demonstrated using the primers shown as SEQ ID NOs. 27 and 28. A 1748 bp fragment was amplified, indicating successful integration into the alp7 locus. Loss of the alp7 gene was demonstrated by reactions using the primers described in SEQ ID NOs. 29 and 30. None of the 569 bp fragments were amplified, indicating a deletion of the alp7 gene.

[0240] [Table 1]

[0241] Transformants that were positive for both integration PCR reactions and positive for alp7 orf loss were further analyzed by quantitative PCR using primers indicated as SEQ ID NOs. 31 and 32, showing that the alp7 gene was completely absent from the tested transformants. One C1 transformant clone that was positive for integration of the deficient cassette to the alp7 locus and negative for qPCR testing for the presence of the alp7 gene was stored at -80°C and designated strain number DNL150.

[0242] Example 2: C1 kex2 gene deletion The C1 kex2 protease gene was deleted from a C1 protease-deficient strain that had previously lacked 12 proteases. The deletion cassette for kex2 was constructed in two parts on two separate plasmids and functions upon transformation to C1 in a similar manner to the alp7 deletion cassette (described above). The deletion cassette also contains a direct repeat sequence of the 5' faciesta region for the removal of the pyr4 marker. The sequences of the deletion construct plasmids are shown in SEQ ID NOs. 23 and 24. The 5' arm plasmid pMYT0997 contained the kex2 5' faciesta region fragment for integration (positions 9-1,058 in SEQ ID NOs. 23) and half of the pyr4 marker (positions 1,033-2,812 in SEQ ID NOs. 23). The 3' arm plasmid pMYT0998 contained the remaining half of the pyr4 marker (positions 17–1273 of SEQ ID NO: 24), a direct repeat sequence (positions 1281–1782 of SEQ ID NO: 24), and a kex2 3' adjacent region fragment for integration (positions 1791–2690 of SEQ ID NO: 24).

[0243] Fragments and direct repeats of the kex2 faciesta region were amplified from C1 genomic DNA and cloned into a pyr4 marker-containing backbone vector originating from the pSR426 plasmid by Gibson cloning using NEBbuilder® HiFi DNA assembly (New England Biolabs), according to the manufacturer's instructions. Both portions of the deletion construct were excised from the plasmid and co-transformed into C1 strain DNL146, which has 12 deletions of protease genes as previously described by Visser, VJ, et al. (ibid.).

[0244] Transformed colonies grown on pyr4 selective medium plates were streaked again in the same selective medium. Accurate identification of transformants was performed by PCR. Mycelium from transformant streaks was dissolved in 20 mM NaOH and incubated at 100°C to lyse the cells. 1-2 μl of this solution was used as a template for PCR using the Phire Plant PCR Kit (Thermo Fisher). The oligonucleotide primers used in this PCR are shown in Table 2. Integration of the deletion construct into the kex2 locus was demonstrated by two PCR reactions. Integration at the 5' end of the gene was demonstrated by a reaction using the primers shown as SEQ ID NOs. 33 and 34. An 1187 bp fragment was amplified, indicating successful integration into the kex2 locus. Integration at the 3' end of kex2 was demonstrated using the primers shown as SEQ ID NOs. 35 and 36. An 1849 bp fragment was amplified, indicating successful integration into the kex2 locus. Loss of the kex2 gene was demonstrated by reactions using primers shown as SEQ ID NOs. 37 and 38. None of the 510 bp fragments were amplified, indicating a defect in the kex2 gene.

[0245] [Table 2]

[0246] Transformants that were positive for both integration PCR reactions and positive for kex2 ORF loss were further analyzed by quantitative PCR using primers indicated as SEQ ID NOs. 39 and 40, and SEQ ID NOs. 41 and 42, demonstrating that the kex2 gene was completely absent from the tested transformants. One C1 transformant clone that was positive for integration of the deficient cassette to the kex2 locus and negative for qPCR testing for the presence of the kex2 gene was stored at -80°C and designated strain number DNL152.

[0247] Example 3: Combination deletion of C1 alp7 and C1 kex2 genes The generation of the C1 strain, which is deficient in both the alp7 and kex2 genes, was performed by deleting the kex2 gene from the DNL150 strain, which was previously deficient in the alp7 gene and 12 other protease genes. Prior to the deletion of the kex2 gene, the pyr4 marker in the DNL150 strain was removed to use the same deletion cassette for the kex2 gene deletion, as described in the generation of the DNL152 strain above.

[0248] The removal of the pyr4 selective marker using the deletion cassette described in the generation of DNL150 above is based on two features: a) the functional pyr4 gene converts 5-fluoroorotic acid (5-FOA) to 5-fluorouracil, a toxic metabolite, so that clones lacking the functional pyr4 gene can grow in the presence of 5-FOA; and b) under 5-FOA selective pressure, the direct repeat sequence of the deletion construct allows the clone to remove the pyr4 selective marker through an allogeneic recombination event between the 5' adjacency region and the direct repeat. If the recombination is successful, the complete pyr4 marker is looped out, and the correct clone can grow in the presence of 5-FOA.

[0249] The removal of the pyr4 marker from DNL150 was performed according to the following protocol: A small portion of fresh mycelium from the plate was suspended in 0.9% NaCl, 0.025% Tween20 solution. Dilutions of the suspension were prepared. Various amounts of mycelial suspension were spread onto plates containing 5-fluoroorotic acid (5-FOA) (5-FOA plate medium components: 7 mM KCl, 11 mM KH2PO4, 0.1% glucose, 10 mM uracil, 10 mM uridine, 2 mM MgSO4, 10 mM proline, trace element solution (1000×: 174 mM EDTA, 76 mM ZnSO4·7H2O, 178 mM H3BO3, 25 mM MnSO4·H2O, 18 mM FeSO4·7H2O, 7.1 mM CoCl2·6H2O, 6.4 mM CuSO4·5H2O, 6.2 mM Na2MoO4·2H2O), 4 mM 5-fluoroorotic acid, 20 g / l agar granules, pH 6.0). The plates were incubated at +35°C until colonies appeared. Colonies grown on 5-FOA agar plates were streaked again on the same selective medium. Growth on the 5-FOA selective medium was poor, and the streaks did not develop into clear streaks, so mycelium from the weak streaks was transferred to a non-selective medium (medium components: 7mM KCl, 55mM KH2PO4, 1.0% glucose, 670mM sucrose, 0.6% yeast extract, 35mM (NH4)2SO4, 2mM MgSO4, 10mM uracil, 10mM uridine, trace element solution (1000×: 174mM EDTA, 76mM ZnSO4·7H2O, 178mM H3BO3, 25mM MnSO4·H2O, 18mM FeSO4·7H2O, 7.1mM CoCl2·6H2O, 6.4mM CuSO4·5H2O, 6.2mM The cells were re-streaked on Na2MoO4·2H2O, 16 g / l agar granules, pH 6.5, and good growth was obtained. Streaks that grew efficiently on non-selective medium plates were then streaked onto pyr4 selective medium plates without uracil and uridine for phenotypic testing.In phenotypic studies, clones that successfully removed pyr4 could not be grown on a medium without uracil and uridine (medium components: 7 mM KCl, 11 mM KH2PO4, 1.0% glucose, 670 mM sucrose, 35 mM (NH4)2SO4, 2 mM MgSO4, trace element solution (1000×: 174 mM EDTA, 76 mM ZnSO4·7H2O, 178 mM H3BO3, 25 mM MnSO4·H2O, 18 mM FeSO4·7H2O, 7.1 mM CoCl2·6H2O, 6.4 mM CuSO4·5H2O, 6.2 mM Na2MoO4·2H2O), 15 g / l agar granules, pH 6.5). Clones that did not grow in the phenotypic test plate were analyzed for pyr4 removal by quantitative PCR using the primers indicated as SEQ ID NOs. 43 and 44. The oligonucleotide primers used in the qPCR reaction are shown in Table 3.

[0250] [Table 3]

[0251] One DNL150pyr4 loop-out clone, which failed to propagate in phenotypic testing and showed a negative result for the pyr4 gene by quantitative PCR, was stored at -80°C and designated as strain DNL151.

[0252] The kex2 protease was deleted from the C1 strain DNL151 using the same deletion cassette and a transformation method as described in the generation of DNL152 above. Accurate integration of kex2 and identification of the deletion by PCR reaction were performed as described in the generation of DNL152 above. One C1 transformant clone that was positive for integration of the deletion cassette into the kex2 locus and negative for the presence of the kex2 gene by qPCR was stored at -80°C and designated as strain DNL155 (Δalp1Δalp2Δpep4Δprt1Δsrp1Δalp3Δpep1Δmtp2Δpep5Δmtp4Δpep6Δalp4Δalp7Δkex2).

[0253] Example 4: Expression of SARS-CoV-2 RBD in protease-deficient C1 strain The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was expressed in a protease-deficient C1 strain. The first construct contained a sequence encoding the C1 endogenous CBH1 signal sequence, residues 333–527 of the SARS-CoV-2-derived spike protein, a Gly-Ser linker, and a C-tag and MssI restriction enzyme site adjacent to the recombination sequence into the C1 expression vector. The fragment was synthesized by GenScript (USA) and is shown as SEQ ID NO: 45 (RBD-C-tag amino acid sequence containing the signal sequence and Gly / Ser linker between the RBD and the C-tag). The gene's codon utilization frequency was optimized for expression in Thermothelomyces heterothallica. Synthetic fragments were amplified by PCR from a GenScript plasmid and cloned into the PacI site of the C1 expression vector pMYT1055 under the endogenous C1 bgl8 promoter and C1 chi1 terminator using the Gibson Assembly (NEBbuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs) method. The exact sequence of the construct was confirmed by sequencing the fragment inserted into the plasmid. The plasmid with the exact sequence was designated plasmid number pMYT1142 (SEQ ID NO: 46). The second construct had the same sequence as pMYT1142, plus a Gly-Ser-linker and a Spytag between the RBD domain and the C-terminus of the Gly-Ser-linker and the C-tag. This sequence is shown as SEQ ID NO: 47 (RBD-Spytag-C-tag amino acid sequence containing a signal sequence and Gly / Ser-linker between the Spytag and the C-tag). The second construct was built into a pMYT1055 expression vector in the same manner as pMYT1142, and the plasmid with the exact sequence was designated plasmid number pMYT1143 (sequence number 48).

[0254] The expression vector pMYT1142 and mock vector partner pMYT1140, necessary for the completion of the hygromycin resistance marker gene and its integration into the bgl8 locus, were digested with MssI and co-transformed into the DNL155 strain lacking 14 protease genes. Transformation was performed using the protoplast / PEG method (Visser, VJ, et al., ibid.), and transformants were selected for the nia1+ phenotype and hygromycin resistance. Transformants were streaked onto a selection medium plate and inoculated from the streak into liquid medium in a 24-well plate. The culture medium components were glucose 5 (g / L), yeast extract 1, (NH4)2SO4 4.6, MgSO4·7H2O 0.49, KH2PO4 7.48, and EDTA 45 (mg / L), ZnSO4·7H2O 1 9.8, MnSO4·4H2O 3.87, CoCl2·6H2O 1.44, CuSO4·5H2O 1.44, Na2MoO4·2H2O 1.35, FeSO4·7H2O 4.5, H3BO 4 9.9, D-biotin 0.004, 50 U / ml penicillin, and 0.05 mg streptomycin. 24-well plates were incubated at 35°C for 4 days with shaking at 800 RPM. The culture supernatant was collected and analyzed by Western blotting performed using a standard method with the primary detection agent Capture Select biotin anti-C-tag conjugate (Thermo Fisher) and the secondary agent IRDye800CW streptavidin (Li-Cor). Western blotting (Figure 1) showed strong signals of the predicted size in many RBD-C-tag and RBD-Spytag-C-tag transformants, indicating that both of these proteins were produced at C1.

[0255] Transformants producing the RBD-C-tagged protein were purified by single colony plating. The purified clones were validated by PCR for accurate integration of the expression cassette and by qPCR for clonal purity. One validated transformant producing the RBD-C-tag was stored at -80°C and designated as strain M4169.

[0256] The expression vector pMYT1143, which carries the RBD-Spytag-C-tagged version, was co-transformed with the mock vector partner pMYT1140. The transformants were analyzed from 24-well plate cultures (Figure 1), and pMYT1142 was purified by single colony plating using the same method as described above. After PCR validation, one C1 transformant clone producing the RBD-Spytag-C-tagged version was stored at -80°C and designated as strain number M4173.

[0257] Plasmids pMYT1142 and pMYT1143 were used to transform other C1 protease-deficient strains besides DNL155, and production levels were compared in different protease-deficient strains. The protease gene deficiencies in these strains are listed in Table 4. RBD-producing plasmids pMYT1142 and pMYT1143 were used to transform four other protease-deficient strains: 1) DNL145, which is deficient in 12 proteases; 2) DNL150, which is deficient in 13 proteases; 3) DNL159, a parallel clone of DNL155; and 4) DNL157, which is deficient in 14 proteases but has a complete kex2 gene. Transformation, analysis of transformants, single-colony purification, and PCR analysis were performed in the same manner as described above to generate strains M4169 and M4173. Several validated production strains were obtained for the production of both RBD-C-tag and RBD-Spytag-C-tag in all four protease-deficient strains. Three parallel transformants from these novel production strains, along with M4169 and M4173, in DNL145, DNL150, DNL157, and DNL159, as well as two other parallel clones of both of these strains, were cultured in liquid medium in 24-well plates at 35°C for 4 days with shaking at 800 RPM. The culture supernatant was collected and analyzed in Coomassie-stained SDS gel using methods known in the art. The highest production of RBD protein was observed in the kex2-deficient DNL155 and DNL159 strains (Figure 2).

[0258] [Table 4]

[0259] C1 strain M4169, which produces RBD-C-tagged protein, was cultured in a 2 L bioreactor using a fed-batch process in a medium containing yeast extract as an organic nitrogen source and glucose as a carbon source. The culture was carried out at 38°C for 5 days. After the culture was completed, the mycelium was removed by centrifugation at 4000 g for 20 minutes, and phenylmethylsulfonyl fluoride was added at a concentration of 1-2 mM to inhibit the protease activity in the resulting liquid culture supernatant. The supernatant was stored at -80°C. For RBD purification by C-tag affinity chromatography, 100 ml of liquid medium was thawed on ice, and after thawing, the sample was clarified by centrifugation at +4°C for 3 × 20 minutes at 20000 g, followed by filtration through a 0.45 μM filter. 90 ml of clear supernatant was diluted to a final volume of 200 ml with 1 × PBS (12 mM Na₂HPO₄·2H₂O, 3 mM NaH₂PO₄·H₂O, 150 mM NaCl pH 7.3). C-tag affinity purification was performed on a 10 ml column of packed CaptureSelect C-tag XL resin (Thermo Fisher) attached to an AKTA Start protein purification system (Cytiva) at a flow rate of 2.5 ml / min. Before loading the sample, the column was initially equilibrated with 5 column volumes (CV) of 1 × PBS. After loading the sample, the column was washed with 15 CV of 1 × PBS, and then eluted in 3 ml fractional volumes using a one-step gradient of 5 CV of 20 mM Tris-HCl, 2 MgCl₂, and 1 mM EDTA pH 7.5. The amount of eluted RBD was quantified by combining UV tracing of the elution peak with the Unicorn 1.0 software included in the AKTA Start system. An extinction coefficient of 1.498 was used to calculate the RBD-C-tag amount, and 1.450 was used to calculate the RBD-Spytag-C-tag amount. After elution, the column was regenerated with 5 CV of 0.1 M glycine at pH 2.3 and washed with 1×PBS until pH 7.3 was reached. The eluted fraction containing protein was pooled for the dialysis step, and the elution buffer was replaced with 1×PBS buffer. The pooled fraction was filled into a 12 ml dialysis cassette, and the dialysis cassette was dialyzed with 1.5 L of 1×PBS for 1 hour at +4°C while agitated on a magnetic stirrer.After 1 hour, the 1×PBS was replaced with fresh buffer, and dialysis was continued under the same conditions for 2 hours. Finally, the 1×PBS was replaced with fresh buffer, and dialysis was continued overnight. The concentration of the dialyzed RBD was determined using a Nanodrop spectrophotometer to measure absorbance at 280 nm, with an extinction coefficient of 1.498 for RBD-C-tags and 1.450 for RBD-Spytag-C-tags. Aliquots of the RBD prepare were stored at -80°C. Affinity purification of RBD-C-tags from M4169 fermentation is shown as an example in Figures 3A and 3B. SARS-CoV-2 spike RBD antibody, rabbit polyclonal antiserum (SinoBiologicals), and goat anti-rabbit IRDye680RD (Li-Cor) were used for Western blotting detection.

[0260] Example 5 Protein expression and stability in different strains of Thermothelomyces heterothallica C1 Different strains of Thermothelomyces heterothallica C1 are shown in Figure 4.

[0261] The stability of proteins and antibodies was investigated using spike experiments. Target proteins were added to the culture supernatant of fungal strains and incubated. Samples were collected at different time points and analyzed using Western blotting. As shown in Figures 5 and 6, ALP7 deficiency had a positive effect on antibody stability.

[0262] Figure 7 shows the spike experiment using fibrinogen. Improved stability was observed in the KEX2-deficient strain.

[0263] Figure 8 shows the spike experiment using Fc-FGF21. Improved stability was observed in KEX2 and SRP10-deficient strains.

[0264] Figure 9 shows spike experiments and mAb expression in protease-deficient strains. Improved stability and protein amounts were observed in the 13×ALP7 protease-deficient strain compared to the 12× and 13×SRP10 protease-deficient strains. When the same mAb was expressed in the 13×ALP7 protease-deficient strain, even more complete mAbs were produced.

[0265] Figure 10 shows mAb expression in 13× protease-deficient strains with either kex2 or alp7 deficiency. The 27 kDa degradation fragment (marked with an arrow) was not observed in the KEX2-deficient strain compared to the 12× parental strain. In addition, the 37 kDa degradation fragment was not produced in the 13×ALP7-deficient strain compared to the 12× protease-deficient parental strain.

[0266] Example 6 : RVFV expression in 14× protease-deficient strains The vaccine antigen protein derived from Rift Valley fever virus was expressed as a fusion protein with the Spycatcher domain from the same expression vector in the 13× protease-deficient strain DNL150 and the 14× protease-deficient strain DNL155 with kex2 deficiency.

[0267] Strains transformed with the RVFV antigen expression vector were grown in 24-well plates, and antigen production was analyzed by Western blotting using an antibody against the RVFV antigen.

[0268] As shown in Figure 11, the transformants of the 14× protease-deficient strain DNL155 showed high expression of RVFV. The expression level was much higher than that of the 13× protease-deficient strain (DNL150).

[0269] Example 7 : Expression and functionality of RBD-Spytag in 14× protease-deficient strains Figures 12A and 12B show the structure of the receptor-binding domain of the SARS-CoV-2 spike protein fused to Spytag in a 14× protease-deficient strain of Thermothelomyces heterothallica C1. The potential of using the produced protein as a vaccine was investigated by conjugating the protein to a SpyCatcher recombinant hepatitis B surface antigen (HBsAg)-virus-like particle (VLP) vaccine. Two batches (#2 and #4) of C1 RBD-Spytag were examined. The stability of the protein and conjugate was examined using SDS-PAGE gel, and the latter was analyzed by Western blotting using mouse anti-HBsAg antibody (Ab 1) and goat anti-mouse IgG-Ap (Ab 2). As shown in Figures 12A and 12B, RBD-Spytag effectively conjugated to SpyCatcher HBsAg VLP. Importantly, RBD proteins with or without the conjugated Spycatcher were able to produce dimers / trimers. Dimerization and trimerization of recombinant RBDs are expected to simulate the native structure of coronavirus RBS and lead to the production of efficient vaccines.

[0270] Next, the binding of RBD-Spytag to the human ACE-2 protein was investigated using the CR3022 antibody. As shown in Figures 13A–13F, the CR3022 antibody was able to bind to RBD presented on VLC particles. In addition, indirect ELISA was used to show that the conjugated RBD bound to hACE-2 but not to VLC particles. In summary, the results indicate that the produced RBD fused to Spytag is accurately assembled, presented on VLC particles, and therefore usable as a vaccine.

[0271] Example 8: Production of Fc fusion protein of SARS-CoV-2 receptor-binding domain in C1 Two potential coronavirus SARS-CoV-2 vaccine proteins were produced in which the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was fused to the IgG1 antibody Fc domain at either the N-terminus or C-terminus, using C1. A 40 bp encoding DNA fragment duplicates the C1 bgl8 promoter, the C1 CBH1 signal sequence, a coding region of either the RBD-Fc or Fc-RBD amino acid sequence (shown as SEQ ID NOs. 49 and 51; the sequences include the signal sequence and linker between the RBD and Fc), a stop codon, and either the bgl8 or chi1 terminator of C1. The protein-coding region of the DNA fragment is shown as SEQ ID NOs. 50 and 52. The DNA fragment with duplication to the chi1 terminator was cloned into the 5' arm of the expression construct (plasmid pMYT1055), and the fragment with duplication to the bgl8 terminator was cloned into the 3' arm of the expression construct (plasmid pMYT1056). Cloning was performed using the Gibson assembly method with the NEBuilder® HiFi DNA Assembly Kit (New England Biolab) according to the manufacturer's instructions. The resulting expression plasmids were named pMYT1302 (RBD-Fc5' arm), pMYT1303 (RBD-Fc3' arm), pMYT1304 (Fc-RBD5' arm), and pMYT1305 (Fc-RBD3' arm).

[0272] To construct RBD-Fc-producing C1 strains, the expression plasmids pMYT1302 and pMYT1303 were used to transform three different C1 strains: DNL155 (Δalp1Δalp2Δpep4Δprt1Δsrp1Δalp3Δpep1Δmtp2Δpep5Δmtp4Δpep6Δalp4Δalp7Δkex2), DNL157 (Δalp1Δalp2Δpep4Δprt1Δsrp1Δalp3Δpep1Δmtp2Δpep5Δmtp4Δpep6Δalp4Δalp7Δsrp10), and a glycoengineered strain, M3599 (Δalp1Δalp2Δpep4Δprt1Δsrp1Δalp3Δpep1Δmtp2Δalp6Δsrp7), which has 10 protease deficiencies. During transformation, the 5' and 3' arms of the expression construct are integrated into the bgl8 locus, and the hygromycin resistance gene duplication fragments of the two arms are recombined with each other to form a final expression construct with two expression cassettes at the bgl8 locus. Transformation was performed as described in Visser, VJ, et al. (ibid.). Transformants were selected for hygromycin resistance and screened for RBD-Fc protein production using 24-well plate culture and Western blotting. Western blotting was performed using the standard method with a 1:10000 dilution of anti-human IgGF(c) goat polyclonal antibody-IRDye700DX conjugate (Licor). Signal detection was performed using a Licor Odyssey fluorometer. The results show that only a small number of RBD-Fc proteins produced in strain M3599, which have 10 protease deficiencies, are of full length (calculated molecular weight 49.4 kDa). In strains M155 and M157, most RBD-Fc is not degraded by proteases and is produced as a complete product (Figure 14A). These strains have protease deficiencies of either alp7 (DNL157) or both alp7 and kex2 (DNL155). Production levels in DNL155 are significantly higher than in DNL157. In conclusion, deficiencies of alp7 and kex2 have a beneficial effect on RBD-Fc production.

[0273] To generate a strain expressing the Fc-RBD fusion protein, the DNL155 strain was transformed with plasmids pMYT1304 and pMYT1305 together, as described above for constructing an RBD-Fc-producing strain. Transformants were analyzed for Fc-RBD by Western blotting from 24-well plate cultures as described above (Figure 14B). Several transformants producing good levels of Fc-RBD protein were detected. The majority of the products were complete.

[0274] Example 9 Vaccination of mice using the SARS-CoV-2 RBD antigen The SARS-CoV-2 spike protein produced in Example 4 was tested for use as a vaccine. SARS-CoV-2 RBD antigen was injected into K18 hACE2 transgenic mice. Two groups of transgenic mice were vaccinated with 20 μg of RBD formulated in alhydrogel. Prime vaccinations were given on day 1 ("prime") and day 21 ("boost"). On day 42, the mice received 2000 PFU of SARS-CoV-2. Serum studies revealed that the antigen produced high-titer neutralizing antibodies. Two days after SARS exposure, all control mice died, but 13 of the 14 vaccinated mice survived with little weight loss.

[0275] Example 10 Expression of αMHCII-Cal07 recombinant antigen in protease-deficient C1 strain The recombinant antigen αMHCII-Cal07, consisting of the MHCII-targeting domain and HA antigen of influenza strain A / California / 07 / 2009 (subtype H1N1), was expressed in a protease-deficient C1 strain. The expression construct included a sequence encoding the C1 endogenous CBH1 signaling sequence, an MHCII-specific target unit, a 20-aa linker, residues 18-541 of the HA protein derived from influenza strain A / California / 07 / 2009, and a recombinant sequence, along with a C-tag adjacent to the C1 expression vector and the MssI restriction enzyme recognition site. Fragments were synthesized using GenScript (USA). The gene codon utilization frequency was optimized for expression in Thermothelomyces heterothallicus. The synthesized fragment was released from the GenScript plasmid by digestion with the restriction enzyme MssI and cloned into the PacI site of the C1 expression vector pMYT1055 under the endogenous C1 bgl8 promoter and C1 chi1 terminator using the Gibson Assembly method (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs). The exact sequence of the construct was confirmed by sequencing the fragment inserted into the plasmid. The plasmid with the exact sequence was designated plasmid number pMYT1242.

[0276] In the second example, the synthesized fragment was amplified by PCR from a GenScript plasmid and cloned into the PacI site of the C1 expression vector pMYT0987 under the conditions of a synthetic AnSES promoter and an endogenous C1 chi1 terminator using Gibson assembly. The exact sequence of the construct was confirmed by sequencing the fragment inserted into the plasmid. The plasmid with the exact sequence was assigned plasmid number pMYT1243.

[0277] The expression vector pMYT1242 and mock vector partner pMYT1140, necessary for the completion of the hygromycin resistance marker gene and its integration into the bgl8 locus, were digested with MssI and co-transformed into the DNL155 strain, which lacks 14 protease genes, and the M3599 strain, which lacks 10 protease genes. The proteases lacking in the aforementioned strains are listed in Table 5. Transformation was performed using the protoplast / PEG method (Visser, VJ, et al., ibid.), and transformants were selected for the nia1+ phenotype and hygromycin resistance. Transformants were streaked onto a selection medium plate and inoculated from the streak into liquid medium in a 24-well plate. The culture medium components were glucose 5 (g / L), yeast extract 1, (NH4)2SO4 4.6, MgSO4·7H2O 0.49, KH2PO4 7.48, and EDTA 45 (mg / L), ZnSO4·7H2O 1 9.8, MnSO4·4H2O 3.87, CoCl2·6H2O 1.44, CuSO4·5H2O 1.44, Na2MoO4·2H2O 1.35, FeSO4·7H2O 4.5, H3BO 4 9.9, D-biotin 0.004, 50 U / ml penicillin, and 0.05 mg streptomycin. 24-well plates were incubated at 35°C for 4 days with shaking at 800 RPM. The culture supernatant was collected and analyzed by Western blotting performed using a standard method with the primary detection agent Capture Select biotin anti-C-tag conjugate (Thermo Fisher) and the secondary agent IRDye680CW streptavidin (Li-Cor). Western analysis (Figure 15) showed a strong signal of the predicted size (87 kDa) in many αMHCII-Cal07 transformants derived from the DNL155 strain, confirming that the protein was produced at C1. However, the product of the predicted size could not be detected in any of the transformants derived from M3599. The additional proteases present in the M3599 transformants compared to the DNL155 transformants caused proteolysis of the product.

[0278] Transformants producing the αMHCII-Cal07 protein were purified by single colony plating. The purified clones were validated by PCR for accurate integration of the expression cassette and by qPCR for clonal purity. One validated transformant producing αMHCII-Cal07 was stored as a glycerol stock at -80°C and designated strain number M4540.

[0279] C1 strain DNL155 was further co-transformed with the MssI digestion expression vector pMYT1243, which holds an αMHCII-CalO7 construct controlled by a synthetic AnSES promoter, together with mock vector partner pMYT1141. The transformants were analyzed from 24-well plate cultures (Figure 15), and pMYT1242 was purified by single colony plating in the same manner as described above. After PCR validation, one C1 transformant clone producing αMHCII-CalO7 was stored at -80°C and designated strain number M4543.

[0280] Furthermore, the C1 strain M4621, which has deficiencies in 14 protease genes and the alg3 gene encoding dolichol-P-Man-dependent alpha(1-3)mannosyltransferase, was prepared using the same method as described above for DNL155. MssSimultaneous transformation was performed with the I digestion expression vector pMYT1243 and the mock vector pMYT1141. Deletion of the alg3 gene caused changes in the structure of N-glycans bound to glycoproteins, resulting in a shift to smaller N-glycan species with fewer mannose residues. The transformants obtained after this transformation were cultured in liquid medium in 24-well plates at 35°C for 4 days with shaking at 800 RPM. The culture supernatant was collected and analyzed by Western blotting performed using a standard method with the primary detection agent Capture Select biotin anti-C-tag conjugate (Thermo Fisher) and mouse monoclonal antibody 29E3 elevated against the HA antigen of influenza strain A / California / 07 / 2009 (Manicassamy et al., 2010; PLoS Pathog 6(1):e1000745.doi:10.1371 / journal.ppat.1000745), and the secondary agents IRDye680RD streptavidin (Li-Cor) and IRDye800CW goat anti-mouse IgG secondary antibody (Li-Cor). Western analysis showed the presence of signals of the expected size (87 kDa) for many transformants, confirming that the protein was produced in transformants derived from M4621 (data not shown).

[0281] [Table 5]

[0282] Strain M4540 of strain C1, which produces recombinant αMHCII-CalO7 protein, was cultured in a 0.25 L bioreactor using a fed-batch process in a medium containing yeast extract as the organic nitrogen source and glucose as the carbon source. The culture was carried out at 38°C for 7 days. After the completion of the culture, the fermentation broth was stored at -80°C. For the purification of αMHCII-CalO7 by C-tag affinity chromatography, 50 ml of liquid medium was thawed on ice, and after thawing, the sample was clarified by centrifugation at 20000 × g at +4°C for 3 × 20 minutes, followed by filtration through a 0.45 μM filter. 33 ml of the clear supernatant was diluted to a final volume of 100 ml with 1 × PBS / 0.5 M NaCl (12 mM Na2HPO4·2H2O, 3 mM NaH2PO4·H2O, 650 mM NaCl pH 7.3). C-tag affinity purification was performed using a 1 ml CaptureSelect C-tag XL column (Thermo Fisher) attached to an AKTA Start protein purification system (Cytiva) at a flow rate of 1 ml / min. Before loading the sample, the column was initially equilibrated with 5 column volumes (CV) of 1 × PBS / 0.5 M NaCl. After loading the sample, the column was washed with 15 CV of 1 × PBS / 0.5 M NaCl, and then eluted in 1 ml fractional volumes using a one-step gradient of 10 CV of 20 mM Tris-HCl, 2 M MgCl2, and 1 mM EDTA pH 7.5. The amount of eluted αMHCII-CalO7 was quantified by combining UV tracing of the elution peak with the Unicorn 1.0 software included in the AKTA Start system. An extinction coefficient of 1.7 was used to calculate the amount of αMHCII-CalO7. After elution, the column was regenerated with 5 CV of 0.1 M glycine pH 2.3 and washed with 1×PBS until pH 7.3 was reached. The eluted fraction containing protein was pooled for the dialysis step, and the elution buffer was replaced with 1×PBS buffer. The pooled fraction was loaded into a 12 ml dialysis cassette, and the cassette was dialyzed with 1.5 L of 1×PBS for 1 hour at +4°C while being agitated on a magnetic stirrer. After 1 hour, the 1×PBS was replaced with fresh buffer, and dialysis was continued for 2 hours under the same conditions. Finally, the 1×PBS was replaced, and dialysis was continued overnight.The concentration of dialyzed αMHCII-CalO7 was determined using a Nanodrop spectrophotometer measuring absorbance at 280 nm, with an extinction coefficient of 1.7. Aliquots of the RBD prepare were stored at -80°C. Affinity purification of αMHCII-CalO7 from the M4540 fermentation supernatant is shown as an example in Figures 16A to 16C. Western blotting was performed using the primary drug CaptureSelect biotin anti-C-tag conjugate (Thermo Fisher) and mouse monoclonal antibody 29E3 elevated against influenza HA antigen, and the secondary drugs IRDye680RD streptavidin (Li-Cor) and IRDye800CW goat anti-mouse IgG secondary antibody (Li-Cor).

[0283] Example 11 Expression of SARS-CoV-2 RBD mutant in 14x protease-deficient C1 strain Three mutants of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were expressed in the protease-deficient C1 strain DNL155. The three mutants were: 1) RBD_B.1.1.7-UK with the N501Y mutation, 2) RBD_B.1.351-SA with the K417N, E484K, and N501Y mutations, and 3) RBD_1.1.28.1(P.1)-BR with the K417T, E484K, and N501Y mutations. Fragments of each mutant were synthesized using GenScript (USA), and the optimized sequence of the Wuhan RBD (as in pMYT1142 Example 4) was used as a base where the mutant amino acids most frequently substituted codons at C1. The design of the synthesized fragment was similar to the C-tagged Wuhan RBD (used in pMYT1142 Example 4), except that the linker between the RBD mutant and the C-tagged cell was 3 amino acids long, compared to 5 amino acids long in the Wuhan RBD-C-tagged cell. The mutant RBD was expressed as two gene copies in C1, and due to dual copy expression at the same genomic locus, two plasmid constructs (5' arm and 3' arm), each containing one gene copy, were created for each mutant. In C1 cells, recombination between the selective marker fragments within the 5' arm and 3' arm plasmids made the marker gene functional, allowing the transformants to grow under selection. For the 5' arm plasmid, the synthesized fragment was amplified from the GenScript plasmid by PCR and cloned into the PacI site of the C1 expression vector pMYT1055 under the endogenous C1 bgl8 promoter and C1 chi1 terminator using the Gibson assembly method (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs). The exact sequence of the construct was confirmed by sequencing of the fragment inserted into the plasmid. Plasmids with the exact sequences were designated pMYT1572 for RBD_B.1.1.7-UK, pMYT1574 for RBD_B.1.351-SA, and pMYT1576 for RBD_1.1.28.1(P.1)-BR.For the 3' arm plasmids, the synthesized fragments of the GenScript plasmid were excised with MssI restriction enzyme and cloned to the PacI site of the C1 expression vector pMYT1056 under the endogenous C1 bgl8 promoter and C1 bgl8 terminator using the Gibson assembly method (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs). Plasmids with the exact sequences were designated pMYT1573 for RBD_B.1.1.7-UK, pMYT1575 for RBD_B.1.351-SA, and pMYT1577 for RBD_1.1.28.1(P.1)-BR.

[0284] Due to dual copy expression, both the 5' and 3' arm plasmids were digested with MssI, and plasmids containing the same mutant gene were co-transformed into the DNL155 strain, which lacks 14 protease genes. Wuhan RBD production was tested in several C1 protease-deficient strains (Example 4), and DNL155 was selected as the host strain because production was highest in DNL155 and DNL159 strains, both 14x protease-deficient strains with kex2 deficiency. Transformation and transformant screening were performed in 24-well cultures as in Wuhan RBD (Example 4), except that the culture supernatant was analyzed by Western blotting using two primary detection agents simultaneously: SARS-CoV-2 (2019-nCoV) spike RBD antibody, rabbit polyclonal antiserum (SinoBiologicals Cat.No.40592-T62), and Capture Select biotin anti-C-tag conjugate (Thermo Fisher). The secondary detection agents were goat anti-rabbit IRDye680RD (Li-Cor) and IRDye800CW streptavidin (Li-Cor). Figure 17 shows an example of Western blotting results using at least one positive transformant for each RBD variant. Strong signals of the expected size were detected with both primary antibodies, and the production level of the variant RBD-C-tagged protein appeared to be comparable to that of the M4169 control strain producing the Wuhan RBD-C-tagged protein.

[0285] The RBD_B.1.1.7-UK amino acid sequence is shown in SEQ ID NO: 53, and the DNA sequence is shown in SEQ ID NO: 54. The sequence includes a signal sequence, a Gly / Ser linker, and a C-tag.

[0286] The RBD_B.1.351-SA amino acid sequence is shown in SEQ ID NO: 55, and the DNA sequence is shown in SEQ ID NO: 56. The sequence includes a signal sequence, a Gly / Ser linker, and a C-tag.

[0287] The RBD_1.1.28.1(P.1)-BR amino acid sequence is shown in SEQ ID NO: 57, and the DNA sequence is shown in SEQ ID NO: 58. The sequence includes a signal sequence, a Gly / Ser linker, and a C-tag.

[0288] The foregoing description of specific embodiments has sufficiently revealed the general nature of the present invention so that others can, by applying current knowledge, readily modify and / or adapt it for various applications such as specific embodiments without undue experimentation and without departing from the general concept, and thus such adaptations and modifications should be understood to be within the meaning and scope of the equivalents of the disclosed embodiments and are intended to be understood. It should be understood that the expressions and terms used herein are for the purpose of description and not of limitation. Means, materials, and steps for performing various disclosed functions can take a variety of alternative forms without departing from the present invention.

Claims

1. A genetically modified Ascomycete filamentous fungus for producing a target exogenous protein, comprising at least one cell in which the expression and / or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4 and KEX2 proteases are reduced, The at least one of the cells contains an exogenous polynucleotide encoding an antibody, The antibody is the target exogenous protein, Genetically modified Ascomycetes filamentous fungi.

2. The genetically modified Ascomycete filamentous fungus according to Claim 1, wherein the expression and / or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, ALP7, and KEX2 are reduced.

3. A genetically modified ascomycete filamentous fungus according to any one of claims 1 to 2, wherein KEX2 contains an amino acid sequence having at least 95% identity with the amino acid sequence of Thermothelomyces heterothallica KEX2, and the Thermothelomyces heterothallica KEX2 contains the amino acids of SEQ ID NO:

14.

4. A genetically modified ascomycete filamentous fungus according to any one of claims 2 to 3, wherein ALP7 comprises an amino acid sequence having at least 95% identity with the amino acid sequence of Thermothelomyces heterothallica ALP7, and the Thermothelomyces heterothallica ALP7 comprises the amino acids of SEQ ID NO:

13.

5. The genetically modified Ascomycete filamentous fungus according to any one of claims 1 to 4, wherein the genetically modified Ascomycete filamentous fungus produces the target protein in an increased amount compared to the amount produced by the non-genetically modified Ascomycete filamentous fungus strain cultured under similar conditions.

6. The genetically modified Ascomycete filamentous fungus according to any one of claims 1 to 5, wherein the protein produced by the genetically modified Ascomycete filamentous fungus has improved stability compared to the protein produced by a non-genetically modified Ascomycete filamentous fungus strain cultured under similar conditions.

7. The genetically modified ascomycete fungus according to any one of claims 1 to 6, wherein the ascomycete fungus is a genus within the subphylum Pezizomycotina.

8. The genetically modified ascomycete filamentous fungus according to claim 7, wherein the ascomycete filamentous fungus is Thermothelomyces heterothallica C1.

9. A method for producing fungi capable of producing antibodies, The method comprises transforming at least one cell of the fungus with at least one exogenous polynucleotide encoding the antibody, The expression and / or activity of the ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4 and KEX2 proteases is reduced in the cell of the fungus. method.

10. The method according to claim 9, further comprising designing the fungus to inhibit the expression and / or protease activity of ALP7.

11. A method for producing at least one target protein, comprising culturing a genetically modified Ascomycete filamentous fungus according to any one of claims 1 to 8 in a suitable medium; and recovering the produced target protein.

12. A genetically modified Ascomycete filamentous fungus for producing a target exogenous protein, comprising at least one cell in which the expression and / or activity of ALP1, PEP4, ALP2, PRT1, SRP1, ALP3, PEP1, MTP2, PEP5, MTP4, PEP6, ALP4, and KEX2 are reduced, The at least one of the cells contains an exogenous polynucleotide encoding the protein of interest. Genetically modified Ascomycetes filamentous fungi.

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